Nematode Control

FIELD OF THE INVENTION

The present invention relates to the field of double-stranded RNA (dsRNA) mediated gene silencing in nematode species. More particularly, the present invention relates to genetic constructs designed for the expression of dsRNA corresponding to novel target genes. These constructs are particularly useful in dsRNA-mediated nematode pest control. The invention further relates to methods for controlling nematodes, methods for preventing nematode infestation and methods for down-regulating gene expression in nematodes using dsRNA. The invention also relates to transgenic plants resistant to nematode infestation.

BACKGROUND OF THE INVENTION

Nematodes comprise a large phylum of animals that include plant and animal parasites as well as many free living species (Maggenti, A. (1981). General Nematology. (New-York: Springer-Verlag)). Plant parasitic nematodes reduce annual U.S. agricultural production by more than $5 billion. Plant parasitic nematodes are obligate parasites, obtaining nutrition only from the cytoplasm of the living plant cells. Some plant parasitic nematodes are ectoparasites, living outside the host. Other species spend much of their lives inside roots as migratory or sedentary endoparasites. It is the sedentary endoparasites of the family Heteroderidae that cause the most economic damage worldwide.

The Heteroderidae can be divided into two groups: the cyst nematodes, which include the genera Heterodera and Globodera; and the root-knot nematodes—genus Meloidogyne. The life cycle of cyst and root-knot nematodes passes through a series of four juvenile stages, separated by molts, during which the cuticle is replaced. The infective stage is the motile, second-stage juvenile that penetrates the plant root to establish a permanent feeding site. After feeding is initiated, the nematode becomes sedentary and then undergoes three molts during development to the adult stage. Interactions between nematodes and their plant host are complex, generally last more than a month and result in major morphological and developmental changes in both organisms.

Root-knot nematodes (RKN: Meloidogyne spp.), so-called for the characteristic root galls or knots formed on the root, infect thousands of plant species and cause severe losses in yield of many food and fiber crops through the world (Mai, W. F. (1985). “Plant parasitic nematodes: Their threat to agriculture” In: An Advanced Treatise on Meloidogyne, Vol. 1, J. N. Sasser and C. C. Carter, eds. (Raleigh: North Carolina State University Graphics), pp. 11-17).

The root knot nematode Meloidogyne incognita (hereinafter M. incognita) is widespread in tropical and subtropical regions and can reproduce on more than 700 host plants including most cultivated crops and ornamentals (Sasser, J. N., (1980) Root-knot nematodes: A global menace to crop production, Plant Disease, 64: 36-41). M. incognita causes significant damage to cotton as a single pest problem and as part of the Fusarium wilt disease complex. M. incognita also causes varying degrees of damage to tomato, potato and tobacco.

Plants infected with root-knot nematodes are at a greater risk of contracting other diseases that are detrimental to its health. Fusarium oxysporum f. sp. Vasinfectum and the root-knot nematode, Meloidogyne incognita, have been associated with “frenching” or Fusarium wilt of cotton Gossypium hirsutum. Giant cells form in the host roots as the juveniles feed and as females mature they cause the roots to split and crack providing an entry into the host for the fungus. The fungus moves through the plant into the vascular system which becomes clogged reducing the transportation of water and nutrients through the xylem tissue. This in turn causes the host plant to loose its vigor and wilt.

Nematicides are widely used in M. incognita management but may, however, be less effective when nematodes are embedded in plant tissue. Because of the broad host range, crop rotation is frequently not feasible. Natural host-plant resistance genes to M. incognita occur in plant genera, such as clovers, cotton, peach, peanut, pineapple, corn, sweet potato, tobacco and tomatoes, and may be incorporated into another plant to protect it against M. incognita. However, there are many crops for which appropriate resistance loci are not available and it is uncertain that nematode resistance genes, even if it is possible to transfer them to a different species or line, will function effectively in the new host plant. Furthermore, acquisition of virulence resulting in a breakdown of the resistance offered by such a gene by M. incognita may shorten the effective utility of this approach.

To effectively control M. incognita infestation, novel strategies to engineer synthetic resistance in plants are becoming imperative.

An alternative biological agent is dsRNA. Over the last few years, downregulation of genes (also referred to as “gene silencing”) in multicellular organisms by means of RNA interference or “RNAi” has become a well-established technique.

RNA interference or “RNAi” is a process of sequence-specific down-regulation of gene expression (also referred to as “gene silencing” or “RNA-mediated gene silencing”) initiated by double-stranded RNA (dsRNA) that is complementary in sequence to a region of the target gene to be down-regulated (Fire, A. Trends Genet. Vol. 15, 358-363, 1999; Sharp, P. A. Genes Dev. Vol. 15, 485-490, 2001).

Over the last few years, down-regulation of target genes in multicellular organisms by means of RNA interference (RNAi) has become a well established technique. Reference may be made to International applications WO 99/32619 (Carnegie Institution) and WO 00/01846 (by applicant).

DsRNA gene silencing finds application in many different areas, such as for example dsRNA mediated gene silencing in clinical applications (WO2004/001013) and in plants. In plants, dsRNA constructs useful for gene silencing have also been designed to be cleaved and to be processed into short interfering RNAs (siRNAs).

RNAi has been proposed as a means of protecting plants against plant parasitic nematodes, i.e. by expressing in the plant (e.g. in the entire plant, or in a part, tissue or cell of a plant) one or more nucleotide sequences that form a dsRNA fragment that corresponds to a target gene in the plant parasitic nematode that is essential for its growth, reproduction and/or survival. Reference may be made to the International application WO 00/01846 (by applicant) and U.S. Pat. No. 6,506,559 (based on WO 99/32619). Experiments using particular genes have been reported to show some reduction in nematode reproduction (WO04/005485). However, complete efficacy was not observed.

The understanding of the biology of miRNAs and siRNAs has opened up new methods of gene silencing in the pharmaceutical and agricultural fields. Key to the application of this technique to the problem of nematode infection of plants is the observation that when nematodes ingest dsRNA molecules, these may be absorbed by the digestive tract. Silencing of genes with sequences homologous to the RNA molecules then occurs. This has been demonstrated in the model nematode C. elegans. Not only can RNA molecules be ingested, but feeding the nematodes E. coli cells harboring recombinant constructs producing such RNA molecules results in absorption of the E. coli-produced RNA molecules and silencing of homologous genes (WO 00/01846). This, combined with the availability of the complete genome sequence of C. elegans, has made possible high throughput functional genomics programs, testing the phenotypic effect of silencing essentially every gene from the genome one by one. This makes possible the assembly of a list of genes which, when silenced, result in death or lack of reproduction of the worm.

With plant parasitic nematodes such experiments are more difficult since these are obligate parasites, meaning that they normally will feed only in its natural state, that is, when infecting a susceptible plant root.

The present invention provides target genes and constructs useful in the dsRNA-mediated nematode pest control, especially the control of Meloidogyne pests.

DESCRIPTION OF THE INVENTION

The present inventors have shown that using certain chemical treatments root-knot nematodes are able to ingest molecules in vitro such as FITC and dsRNA (outside of the root). Absorption of RNA molecules from the digestive tract leading to silencing of target genes opens up a possible new strategy for M. incognita control.

The present invention describes a novel non-compound, non-protein based approach for the control of nematode crop pests. The active ingredient is a nucleic acid, a double-stranded ribonucleic acid (dsRNA), which can be used as a nematicidal formulation, for example, as a foliar spray. In another embodiment, the dsRNA can be expressed constitutively in the host plant to protect the plant against infesting nematodes. The sequence of the dsRNA corresponds to part or whole of an essential nematode gene and causes downregulation of the nematode target via RNA interference (RNAi). As a result of the downregulation of mRNA, the dsRNA prevents expression of the target nematode protein and hence causes death, growth arrest or sterility of the nematode.

In a first embodiment, the present invention thus relates to a double stranded ribonucleotide sequence produced from the expression of a polynucleotide sequence comprising a nucleic acid sequence selected from the group comprising:

(i) sequences represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof,

(ii) sequences which are at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98% or 99% identical to a sequence represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof, and

(iii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of the sequences represented by SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof,

or wherein said nucleic acid sequence is an orthologue of a gene comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 61 to 662, or a complement thereof,

wherein ingestion of said ribonucleotide sequence by a pathogenic pest inhibits the growth of said pest. In preferred aspects of the invention, this pathogenic pest is a nematode pest. The double stranded RNA(s) is (are) used in several aspects of the invention.

The methods of the invention can find practical application in any area of technology where it is desirable to inhibit viability, growth, development or reproduction of the nematode, or to decrease pathogenicity or infectivity of the nematode. The methods of the invention further find practical application where it is desirable to specifically down-regulate expression of one or more target genes in a nematode. Particularly useful practical applications include, but are not limited to, (1) protecting plants against plant pathogenic nematodes; (2) pharmaceutical or veterinary use in humans and animals (for example to control, treat or prevent nematode infections in humans); (3) protecting materials against damage caused by nematodes; (4) protecting perishable materials (such as foodstuffs, seed, etc.) against damage caused by nematodes; (5) functional genomics in nematodes to elucidate the gene function of nematode target genes and generally any application wherein nematodes need to be controlled and/or wherein damage caused by nematodes needs to be prevented.

In accordance with one embodiment the invention relates to a method for controlling nematode growth in or on a cell or an organism or for preventing nematode infestation of a cell or an organism susceptible to nematode infection, comprising contacting nematodes with a double-stranded RNA, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of a nematode target gene, whereby the double-stranded RNA is taken up by the nematode and thereby controls growth or prevents infestation.

The expression “double-stranded RNA”, “double stranded RNA” and “double stranded ribonucleotide sequence” are used interchangeable herein.

“Controlling pests” as used in the present invention means killing pests, or preventing pests to develop, or to grow or preventing pests to infect or infest. Controlling pests as used herein also encompasses controlling pest progeny (development of eggs). Controlling pests as used herein also encompasses inhibiting viability, growth, development or reproduction of the pest, or to decrease pathogenicity or infectivity of the pest. The compounds and/or compositions described herein, may be used to keep an organism healthy and may be used curatively, preventively or systematically to control pests or to avoid pest growth or development or infection or infestation. Particular pests envisaged in the present invention are plant pathogenic nematode pests. “Controlling nematodes” as used herein thus also encompasses controlling nematode progeny (such as development of eggs). Controlling nematodes as used herein also encompasses inhibiting viability, growth, development or reproduction of the nematode, or decreasing pathogenicity or infectivity of the nematode. In the present invention, controlling nematodes may inhibit a biological activity in a nematode, resulting in one or more of the following attributes: reduction in feeding by the nematode, reduction in viability of the nematode, death of the nematode, inhibition of differentiation and development of the nematode, absence of or reduced capacity for sexual reproduction by the nematode, muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antennae formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, haemolymph synthesis, haemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration, apoptosis, and any component of a eukaryotic cells' cytoskeletal structure, such as, for example, actins and tubulins. The compounds and/or compositions described herein, may be used to keep an organism healthy and may be used curatively, preventively or systematically to control a nematode or to avoid nematode growth or development or infection or infestation. Thus, the invention may allow previously susceptible organisms to develop resistance against infestation by the nematode organism.

The expression “to at least part of” as used herein means that the nucleotide sequence is fully complementary to the nucleotide sequence of the target over more than two nucleotides, for instance over at least 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 contiguous nucleotides.

According to a further embodiment, the invention relates to a method for down-regulating expression of a target gene in a nematode, comprising contacting said nematode with a double-stranded RNA, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of the nematode target gene to be down-regulated, whereby the double-stranded RNA is taken up into the nematode and thereby down-regulates expression of the nematode target gene.

Preferably, the present invention extends to methods as described herein, wherein said nematode target gene comprises a sequence which is selected from the group comprising:

(i) sequences which are at least 75%, at least 80% or 85% identical, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% and still more preferably at least 99% identical to a sequence represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof, and

(ii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof, or wherein said target gene is an orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 61 to 662, or the complement thereof.

Whenever the term “a” is used within the context of “a target gene”, this means “at least one” target gene. The same applies for “a” target organism meaning “at least one” target organism, and “a” RNA molecule or host cell meaning “at least one” RNA molecule or host cell. This is also detailed further below.

According to one embodiment, the methods of the invention rely on uptake by the nematode of double-stranded RNA present outside of the nematode (e.g. by feeding) and does not require expression of double-stranded RNA within cells of the nematode. In addition, the present invention also encompasses methods as described above wherein the nematode is contacted with a composition comprising the double-stranded RNA.

The methods of the invention are applicable to all nematodes that are susceptible to gene silencing by RNA interference and that are capable of internalising double-stranded RNA from their immediate environment.

In terms of “susceptible organisms”, which benefit from the present invention, any organism which is susceptible to pest infestation is included.

In preferred, but non-limiting, embodiments of the invention the nematode is chosen from the group consisting of:

(1) a nematode which is a plant pathogenic nematode, such as but not limited to Root Knot Nematodes (Meloidogyne spp.) in rice (e.g. M. incognita, M. javanica or M. graminicola), in soybean (e.g. M. incognita or M. arenaria), in cotton (e.g. M. incognita), in potato (e.g. M. chitwoodi or M. hapla), in tomato (e.g. M. chitwoodi), in tobacco (e.g. M. incognita, M. javanica or M. arenaria), and in corn (e.g. M. incognita); Cyst Nematodes (Heterodera spp.) in rice (e.g. H. oryzae), in soybean (e.g. H. glycines) and in corn (e.g. H. zeae); Cyst nematodes (Globodera spp.) in potato (e.g. G. pallida or G. rostochiensis); Reniform Nematodes (Rotylenchulus spp.) in cotton (e.g. R. reniformis); Root lesion nematodes (Pratylenchus spp.) in banana (e.g. P. coffeae or P. goodeyi); Burrowing Nematodes (Radopholus spp.) in banana (e.g. R. similis); Other rice damaging nematodes such as rice root nematode (Hirschmaniella spp., e.g. H. oryzae);

(2) a nematode capable of infesting humans such as, but not limited to: Enterobius vermicularis, the pinworm that causes enterobiasis; Ascaris lumbridoides, the large intestinal roundworm that causes ascariasis; Necator and Ancylostoma, two types of hookworms that cause ancylostomiasis; Trichuris trichiura, the whipworm that causes trichuriasis; Strongyloides stercoralis that causes strongyloidiasis; and Trichonella spirae that causes trichinosis; Brugia malayi and Wuchereria bancrofti, the filarial nematodes associated with the worm infections known as lymphatic filariasis and its gross manifestation, elephantiasis, and Onchocerca volvulus that causes river blindness. Transfer of nematodes to humans may also occur through blood-feeding mosquitoes which have fed upon infected animals or humans;

(3) a nematode capable of infesting animals such as, but not limited to: dogs (Hookworms e.g. Ancylostoma caninum or Uncinaria stenocephala, Ascarids e.g. Toxocara canis or Toxascaris leonina, or Whipworms e.g. Trichuris vulpis), cats (Hookworms e.g. Ancylostoma tubaeforme, Ascarids e.g. Toxocara cati), fish (herring worms or cod worms e.g. Anisakid, or tapeworm e.g. Diphyllobothrium), sheep (Wire worms e.g. Haemonchus contortus) and cattle (Gastro-intestinal worms e.g. Ostertagia ostertagi, Cooperia oncophora);

(4) a nematode that causes unwanted damage to substrates or materials, such as nematodes that attack foodstuffs, seeds, wood, paint, plastic, clothing etc. Examples of such nematodes include but are not limited to Meloidogyne spp. (e.g. M. incognita, M. javanica, M. arenaria, M. graminicola, M. chitwoodi or M. hapla); Heterodera spp. (e.g. H. oryzae, H. glycines, H. zeae or H. schachtii); Globodera spp. (e.g. G. pallida or G. rostochiensis); Ditylenchus spp. (e.g. D. dipsaci, D. destructor or D. angustus); Belonolaimus spp.; Rotylenchulus spp. (e.g. R. reniformis); Pratylenchus spp. (e.g. P. coffeae, P. goodeyi or P. zeae); Radopholus spp. (e.g. R. Similis); Hirschmaniella spp. (e.g. H. oryzae); Aphelenchoides spp. (e.g. A. besseyi); Criconemoides spp.; Longidorus spp.; Helicotylenchus spp.; Hoplolaimus spp.; Xiphinema spp.; Paratrichodorus spp. (e.g. P. minor); Tylenchorhynchus spp;

(5) virus transmitting nematodes (e.g. Longidorus macrosoma: transmits prunus necrotic ring spot virus, Xiphinema americanum: transmits tobacco ring spot virus, Paratrichadorus teres: transmits pea early browning virus, or Trichodorus similis: transmits tobacco rattle virus).

Preferably plants may benefit from the present invention by protection from infestation by plant pathogenic organisms.

In a preferred embodiment the susceptible organism is a plant and the pest is a plant pathogenic nematode. In this embodiment the nematode is contacted with the RNA molecule by expressing the dsRNA molecule in a plant, plant part, plant cell or plant seed that is infested with or susceptible to infestation with the plant pathogenic pest.

According to one embodiment, the methods of the invention rely on a GMO approach wherein the double-stranded RNA is expressed by a cell or an organism infested with or susceptible to infestation by pathogenic pests, preferably plant pathogenic nematode pests. Preferably, said cell is a plant cell or said organism is a plant.

In this context the term “plant” encompasses any plant material that it is desired to treat to prevent or reduce nematode growth and/or nematode infestation. This includes, inter alia, whole plants, seedlings, propagation or reproductive material such as seeds, cuttings, grafts, explants, etc. and also plant cell and tissue cultures. The plant material should express, or have the capability to express, the RNA molecule comprising at least one nucleotide sequence that is the RNA complement of or that represents the RNA equivalent of at least part of the nucleotide sequence of the sense strand of at least one target gene of the pest organism, such that the RNA molecule is taken up by a pest upon plant-pest interaction, said RNA molecule being capable of inhibiting the target gene or down-regulating expression of the target gene by RNA interference.

The invention provides for use of a plant, plant part, plant cell or seed as defined herein for down regulation of expression of a nematode target gene. In more detailed terms, the invention provides for use of a host cell as defined herein and/or an RNA molecule comprising a nucleotide sequence that is the RNA complement of or that represents the RNA equivalent of at least part of the nucleotide sequence of a target gene from a nematode organism, as produced by transcription of a nucleic acid molecule in a plant, plant part, plant cell or seed, for instance in the manufacture of a commodity product, for down regulation of expression of a target gene. Suitable target genes and target organisms in respect of the invention are discussed below in further detail.

Nematodes of the invention are chosen from the group consisting of but not limited to: Meloidogyne spp. (e.g. M. incognita, M. javanica, M. graminicola, M. arenaria, M. chitwoodi, M. hapla or M. paranaensis); Heterodera spp. (e.g. H. oryzae, H. glycines, H. zeae or H. schachtii); Globodera spp. (e.g. G. pallida or G. rostochiensis); Rotylenchulus spp. (e.g. R. reniformis); Pratylenchus spp. (e.g. P. coffeae, P. Zeae or P. goodeyi); Radopholus spp. (e.g. R. similis); Hirschmaniella spp. (e.g. H. oryzae); Ancylostoma spp. (e.g. A. caninum, A. ceylanicum, A. duodenale or A. tubaeforme); Anisakid; Aphelenchoides spp. (e.g. A. Besseyi); Ascarids; Ascaris spp., (e.g. A. suum or A. lumbridoides); Belonolaimus spp.; Brugia spp. (e.g. B. malayi or B. pahangi); Bursaphelenchus spp. Caenorhabditis spp. (e.g. C. elegans, C. briggsae or C. remanei); Clostridium spp. (e.g. C. acetobutylicum); Cooperia spp. (e.g. C. oncophora); Criconemoides spp.; Cyathostomum spp. (e.g. C. catinatum, C. coronatum or C. pateratum); Cylicocyclus spp. (e.g. C. insigne, C. nassatus or C. radiatus); Cylicostephanus spp. (e.g. C. goldi or C. longibursatus); Diphyllobothrium; Dirofilaria spp. (e.g. D. immitis); Ditylenchus spp. (e.g. D. dipsaci, D. destructor or D. Angustus); Enterobius spp. (e.g. E. vermicularis); Haemonchus spp. (e.g. H. contortus); Helicotylenchus spp.; Hoplolaimus spp.; Litomosoides spp. (e.g. L. sigmodontis); Longidorus spp. (e.g. L. macrosoma); Necator spp. (e.g. N. americanus); Nippostrongylus spp. (e.g. N. brasiliensis); Onchocerca spp. (e.g. O. volvulus); Ostertagia spp. (e.g. O. ostertagi); Parastrongyloides spp. (e.g. P. trichosun); Paratrichodorus spp. (e.g. P. minor or P. teres); Parelaphostrongylus spp. (e.g. P. tenuis); Scutellonerna. spp.; Strongyloides spp. (e.g. S. Ratti or S. stercoralis); Teladorsagia spp. (e.g. T. circumcincta); Toxascaris spp. (e.g. T. leonina); Toxocara spp. (e.g. T. canis or T. cati); Trichinella spp. (e.g. T. britovi, T. spiralis or T. spirae); Trichodorus spp. (e.g. T. similis);Trichuris spp. (e.g. T. muris, T. vulpis or T. trichiura); Tylenchulus spp.; Tylenchorhynchus spp.; Uncinaria spp. (e.g. U. stenocephala); Wuchereria spp. (e.g. W. bancrofti); and Xiphinema spp. (e.g. X. Index or X. americanum).

Preferred plant pathogenic nematodes according to the invention include but are not limited to Root Knot Nematodes (Meloidogyne spp.) in rice (e.g. M. incognita, M. javanica or M. graminicola), in soybean (e.g. M. incognita and M. arenaria), in cotton (e.g. M. incognita), in potato (e.g. M. chitwoodi (columbia root-knot nematode), causing small, raised swellings on potato tuber surface, dark specks in the potato flesh and reduced potato quality, or M. hapla (northern root knot nematode)), in tomato (e.g. M. chitwoodi, M. incognita causing root galls), in corn (e.g. M. incognita, causing stunting and chlorosis, numerous root galls and proliferation of fibrous roots), in tobacco, sugarcane, banana and vegetables (e.g. M. javanica) and in peanut (e.g. M. arenaria); Cyst Nematodes (Heterodera spp.) in rice (e.g. H. oryzae), in soybean (e.g. H. glycines, causing yield loss, stunting and chlorosis), in corn (e.g. H. zeae, causing stunting, pale color and narrow leaves) and in sugarbeet (e.g. H. schachtii, causing stunting and yellowing of plants, misshapen and excess fibrous roots); Cyst nematodes (Globodera spp.) in potato, tomato and other Solanum species (e.g. G. pallida (white potato cyst nematode) or G. rostochiensis (golden nematode or yellow potato cyst nematode), causing root damage, poor growth, yellowing and wilting); Stem and bulb nematodes (Ditylenchus spp.) in potato (e.g. D. dipsaci, causing tubers rot; leaves and stems swell and become distorted, or D. destructor (potato rot nematode or potato tuber nematode), causing potato dry rot; tuber rot) and in rice (D. angustus); Sting nematodes (Belonolaimus spp.) in soybean and in corn, causing severe trim of the roots of growing plants or seedlings; Reniform Nematodes (Rotylenchulus spp.) in cotton, maize, cowpea and black gram and banana (e.g. R. reniformis); Root lesion nematodes (Pratylenchus spp.) in banana (e.g. P. coffeae or P. goodeyi), in rice (e.g. P. zeae) and in corn (causing severe pruning of the roots, resulting in stunting, as well as reduction in stalk diameter, stalk and root weights); Burrowing Nematodes (Radopholus spp.) in banana (e.g. R. similis, causing rhizome rot, pepper slow wilt); Other rice damaging nematodes such as rice root nematode (Hirschmaniella spp., e.g. H. oryzae) and rice white tip nematode (Aphelenchoides spp., e.g. A. besseyi); Other corn damaging nematodes such as ring nematodes (Criconemoides spp.), needle nematodes (Longidorus spp.), spiral nematodes (Helicotylenchus spp., causing mild stunting and reduced yields), lance nematodes (Hoplolaimus spp., causing stunting), dagger nematodes (Xiphinema spp.), stubby-root nematodes (Paratrichodorus spp., causing stunting, chlorosis, and reduced yields; e.g. P. minor feeds largely on the root tips, stopping terminal growth of the rootlets, and resulting in the stubby-root effect) and stunt nematodes (Tylenchorhynchus spp., causing stunting).

Preferred plant pathogenic nematodes according to the invention include but are not limited to Meloidogyne spp. (e.g. M. incognita, M. javanica, M. graminicola, M. arenaria, M. chitwoodi, M. hapla or M. paranaensis); Heterodera spp. (e.g. H. oryzae, H. glycines, H. zeae or H. schachtii); Globodera spp. (e.g. G. paffida or G. rostochiensis); Rotylenchulus spp. (e.g. R. reniformis); Pratylenchus spp. (e.g. P. coffeae, P. Zeae or P. goodeyi); Radopholus spp. (e.g. R. similis); Hirschmaniella spp. (e.g. H. oryzae); Aphelenchoides spp. (e.g. A. besseyi); Belonolaimus spp.; Criconemoides spp.; Ditylenchus spp. (e.g. D. dipsaci, D. destructor or D. Angustus); Helicotylenchus spp.; Hoplolaimus spp.; Longidorus spp. (e.g. L. macrosoma); Paratrichodorus spp. (e.g. P. minor or P. teres); Tylenchorhynchus spp.; Xiphinema spp. (e.g. X. Index or X. americanum).

In most preferred embodiments of the invention, the nematode may belong to the family of the Heteroderidae, encompassing the genera Heterodera and Globodera, including the plant pathogenic nematodes Meloidogyne spp. (e.g. M. incognita, M. javanica, M. graminicola, M. arenaria, M. chitwoodi, M. hapla or M. paranaensis) and Heterodera spp. (e.g. H. oryzae, H. glycines, H. zeae or H. schachtii).

The present invention also relates to a method for producing a plant resistant to a plant pathogenic nematode, comprising:

(a) transforming a plant cell with a recombinant construct comprising at least one regulatory sequence operably linked to a sequence complementary to at least part of

- (1) a nucleotide sequence of a target nematode gene selected from the group consisting of: (i) sequences which are at least 75%, at least 80% or 85% identical, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% and still more preferably at least 99% identical to a sequence represented by any of SEQ ID Nos 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof, (ii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID Nos 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof, and (iii) sequences comprising a sense strand comprising a nucleotide sequence of (i) and an antisense strand comprising the complement of said nucleotide sequence of (i), wherein the transcript encoded by said nucleotide sequence is capable of forming a double-stranded RNA, or
- (2) a nucleotide sequence which is a nematode orthologue of a gene comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 95 to 963, or the complement thereof;

(b) regenerating a plant from the transformed plant cell; and

(c) growing the transformed plant under conditions suitable for the expression of the recombinant construct, said grown transformed plant resistant to plant pathogenic nematodes compared to an untransformed plant. According to still other embodiments, in the methods of the invention, the double-stranded RNA is expressed from a recombinant construct, which construct comprises at least one regulatory sequence operably linked to said nucleotide sequence which is complementary to at least part of said nucleotide sequence of said nematode target gene to be down-regulated.

Preferred plants according to the invention include but are not limited to alfalfa, apple, apricot, artichoke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, carrot, cassaya, cauliflower, a cereal, celery, cherry, citrus, clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, figs, grape, grapefruit, groundnuts, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, nut oat, okra, onion, orange, an ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, soy, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, tangerine, tea, tobacco, tomato, a vine, watermelon, wheat, yams, zucchini, Brassica and Arabidopsis; preferably rice, cotton, potato, tomato, corn, banana or soybean.

According to one preferred embodiment the plant is a rice plant. According to another embodiment the plant is a cotton plant. According to another embodiment the plant is a tomato plant. According to another embodiment the plant is a soybean plant. According to another embodiment, the plant is a potato plant. According to another embodiment, the plant is a corn plant. According to another embodiment, the plant is a banana plant.

The term “nematode” encompasses nematodes of all types and at all stages of development, including but not limited to eggs, free living pre-parasitic J2, parasitic J2, parasitic J3, young adults (male and female J4) and mature adults (male and female).

According to one embodiment of the present invention, the nematode which is contacted with the dsRNA is a plant pathogenic nematode in a life stage outside a plant cell, for example in the form of eggs or pre-parasitic J2. According to another embodiment of the present invention, the nematode which is contacted with the dsRNA is a plant pathogenic nematode in a life stage inside a plant cell, for example a pathogenic form of eggs, parasitic J2, parasitic J3, young adults (male and female J4) or mature adults (male and female).

The present invention relates to any gene of interest in the nematode (which may be referred to herein as the “target gene”) that can be down-regulated.

The terms “down-regulation of gene expression” and “inhibition of gene expression” are used interchangeably and refer to a measurable or observable reduction in gene expression or a complete abolition of detectable gene expression, at the level of protein product and/or mRNA product from the target gene. Preferably the down-regulation does not substantially directly inhibit the expression of other genes of the nematode. The down-regulation effect of the dsRNA on gene expression may be calculated as being at least 30%, 40%, 50%, 60%, preferably 70%, 80% or even more preferably 90% or 95% when compared with normal gene expression. Depending on the nature of the target gene, down-regulation or inhibition of gene expression in cells of a nematode can be confirmed by phenotypic analysis of the cell or the whole nematode or by measurement of mRNA or protein expression using molecular techniques such as RNA solution hybridization, PCR, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, or fluorescence activated cell analysis (FACS).

The “target gene” may be essentially any gene that is desirable to be inhibited because it interferes with growth or pathogenicity or infectivity of the nematode. For instance if the method of the invention is to be used to prevent nematode growth and/or infestation then it is preferred to select a target gene which is essential for viability, growth, development or reproduction of the nematode, or any gene that is involved with pathogenicity or infectivity of the nematode, such that specific inhibition of the target gene leads to a lethal phenotype or decreases or stops nematode infestation.

According to one non-limiting embodiment, the target gene is such that when its expression is down-regulated or inhibited using the method of the invention, the nematode is killed, or the reproduction or growth of the nematode is stopped or retarded. This type of target genes is considered to be essential for the viability of the nematode and is referred to as essential genes. Therefore, the present invention encompasses a method as described herein, wherein the target gene is an essential gene.

According to a further non-limiting embodiment, the target gene is such that when it is down-regulated using the method of the invention, the infestation or infection by the nematode, the damage caused by the nematode, and/or the ability of the nematode to infest or infect host organisms and/or cause such damage, is reduced. The terms “infest” and “infect” or “infestation” and “infection” are generally used interchangeably throughout. This type of target genes is considered to be involved in the pathogenicity or infectivity of the nematode. Therefore, the present invention extends to methods as described herein, wherein the target gene is involved in the pathogenicity or infectivity of the nematode. The advantage of choosing the latter type of target gene is that the nematode is blocked to infect further plants or plant parts and to form further generations.

According to one embodiment, target genes are conserved genes or nematode -specific genes.

In addition, any suitable double-stranded RNA fragment capable of directing RNAi or RNA-mediated gene silencing or inhibition of a nematode target gene may be used in the methods of the invention.

In the methods of the present invention, dsRNA is used to inhibit growth or to interfere with the pathogenicity or infectivity of the nematode.

The invention thus relates to isolated double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of a target gene of a nematode. The target gene may be any of the target genes described herein, or a part thereof that exerts the same function.

According to one embodiment of the present invention, an isolated double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a nematode target gene, wherein said target gene comprises a sequence which is selected from the group comprising: (i) sequences which are at least 75%, at least 80% or 85% identical, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% and still more preferably at least 99% identical to a sequence represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof, and (ii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement, or wherein said target gene is an orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 61 to 662, or the complement thereof.

Depending on the assay used to measure gene silencing, the growth inhibition can be quantified as being greater than about 5%, 10%, more preferably about 20%, 25%, 33%, 50%, 60%, 75%, 80%, most preferably about 90%, 95%, or about 99% as compared to a pest organism that has been treated with control dsRNA.

According to another embodiment of the present invention, an isolated double-stranded RNA is provided, wherein at least one of said annealed complementary strands comprises the RNA equivalent of at least one of the nucleotide sequences represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the RNA equivalent of a fragment of at least 17 basepairs in length thereof, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 basepairs in length thereof. According to another embodiment of the present invention, an isolated double-stranded RNA is provided comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a nematode target, for use as a medicament. According to another embodiment of the present invention the use as a medicament is provided for the isolated double-stranded RNA as described above.

If the method of the invention is used for specifically controlling growth or infestation of specific nematodes in or on a host cell or host organism, it is preferred that the double-stranded RNA does not share any significant homology with any host gene, or at least not with any essential gene of the host. In this context, it is preferred that the double-stranded RNA shows less than 30%, more preferably less that 20%, more preferably less than 10%, and even more preferably less than 5% nucleic acid sequence identity with any gene of the host cell. % sequence identity should be calculated across the full length of the double-stranded RNA region. If genomic sequence data is available for the host organism one may cross-check sequence identity with the double-stranded RNA using standard bioinformatics tools. In one embodiment, there is no sequence identity between the dsRNA and a host sequences over 21 contiguous nucleotides, meaning that in this context, it is preferred that 21 contiguous base pairs of the dsRNA do not occur in the genome of the host organism. In another embodiment, there is less than about 10% or less than about 12.5% sequence identity over 24 contiguous nucleotides of the dsRNA with any nucleotide sequence from a host species.

The double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which corresponds to a target nucleotide sequence of the target gene to be down-regulated. The other strand of the double-stranded RNA is able to base-pair with the first strand.

The expression “target region” or “target nucleotide sequence” of the target nematode gene may be any suitable region or nucleotide sequence of the gene. The target region should comprise at least 17, at least 18 or at least 19 consecutive nucleotides of the target gene, more preferably at least 20 or at least 21 nucleotide and still more preferably at least 22, 23 or 24 nucleotides of the target gene.

It is preferred that (at least part of) the double-stranded RNA will share 100% sequence identity with the target region of the nematode target gene. However, it will be appreciated that 100% sequence identity over the whole length of the double-stranded region is not essential for functional RNA inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for RNA inhibition. The terms “corresponding to” or “complementary to” are used herein interchangeable, and when these terms are used to refer to sequence correspondence between the double-stranded RNA and the target region of the target gene, they are to be interpreted accordingly, i.e. as not absolutely requiring 100% sequence identity. However, the % sequence identity between the double-stranded RNA and the target region will generally be at least 80% or 85% identical, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% and still more preferably at least 99%.

The term “complementary” as used herein relates to both DNA-DNA complementarity as to DNA-RNA complementarity. In analogy herewith, the term “RNA equivalent” substantially means that in the DNA sequence(s), the base “T” may be replaced by the corresponding base “U” normally present in ribonucleic acids.

Although the dsRNA contains a sequence which corresponds to the target region of the target gene it is not absolutely essential for the whole of the dsRNA to correspond to the sequence of the target region. For example, the dsRNA may contain short non-target regions flanking the target-specific sequence, provided that such sequences do not affect performance of the dsRNA in RNA inhibition to a material extent.

The dsRNA may contain one or more substitute bases in order to optimise performance in RNAi. It will be apparent to the skilled reader how to vary each of the bases of the dsRNA in turn and test the activity of the resulting siRNAs (e.g. in a suitable in vitro test system) in order to optimise the performance of a given dsRNA.

The dsRNA may further contain DNA bases, non-natural bases or non-natural backbone linkages or modifications of the sugar-phosphate backbone, for example to enhance stability during storage or enhance resistance to degradation by nucleases.

It has been previously reported that the formation of short interfering RNAs (siRNAs) of about 21 by is desirable for effective gene silencing. However, in applications of applicant it has been shown that the minimum length of dsRNA preferably is at least about 80-100 by in order to be efficiently taken up by certain pest organisms. There are indications that in invertebrates such as the free living nematode C. elegans or the plant parasitic nematode Meloidogyne incognita, these longer fragments are more effective in gene silencing, possibly due to a more efficient uptake of these long dsRNA by the invertebrate.

It has also recently been suggested that synthetic RNA duplexes consisting of either 27-mer blunt or short hairpin (sh) RNAs with 29 by stems and 2-nt 3′ overhangs are more potent inducers of RNA interference than conventional 21-mer siRNAs. Thus, molecules based upon the targets identified above and being either 27-mer blunt or short hairpin (sh) RNA's with 29-bp stems and 2-nt 3′ overhangs are also included within the scope of the invention.

Therefore, in one embodiment, the double-stranded RNA fragment (or region) will itself preferably be at least 17 by in length, preferably 18 or 19 bp in length, more preferably at least 20 bp, more preferably at least 21 bp, or at least 22 bp, or at least 23 bp, or at least 24 bp, 25 bp, 26 by or at least 27 by in length. The expressions “double-stranded RNA fragment” or “double-stranded RNA region” refer to a small entity of the double-stranded RNA corresponding with (part of) the target gene.

Generally, the double-stranded RNA is preferably between about 17-1500 bp, even more preferably between about 80-1000 by and most preferably between about 17-27 by or between about 80-250 bp; such as double-stranded RNA regions of about 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 27 bp, 50 bp, 80 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 900 bp, 100 bp, 1100 bp, 1200 bp, 1300 bp, 1400 by or 1500 bp.

The upper limit on the length of the double-stranded RNA may be dependent on i) the requirement for the dsRNA to be taken up by the nematode and ii) the requirement for the dsRNA to be processed within the cell into fragments that direct RNAi. The chosen length may also be influenced by the method of synthesis of the RNA and the mode of delivery of the RNA to the cell. Preferably the double-stranded RNA to be used in the methods of the invention will be less than 10,000 by in length, more preferably 1000 by or less more preferably 500 by or less, more preferably 300 by or less, more preferably 100 by or less. For any given target gene and nematode, the optimum length of the dsRNA for effective inhibition may be determined by experiment.

The double-stranded RNA may be fully or partially double-stranded. Partially double-stranded RNAs may include short single-stranded overhangs at one or both ends of the double-stranded portion, provided that the RNA is still capable of being taken up by nematodes and directing RNAi. The double-stranded RNA may also contain internal non-complementary regions.

The methods of the invention can encompass the simultaneous or sequential provision of two or more different double-stranded RNAs or RNA constructs to the same nematode, so as to achieve down-regulation or inhibition of multiple target genes or to achieve a more potent inhibition of a single target gene.

Alternatively, multiple targets are hit by the provision of one double-stranded RNA that hits multiple target sequences, and a single target is more efficiently inhibited by the presence of more than one copy of the double-stranded RNA fragment corresponding to the target gene. Thus, in one embodiment of the invention, the double-stranded RNA construct comprises multiple dsRNA regions, at least one strand of each dsRNA region comprising a nucleotide sequence that is complementary to at least part of a target nucleotide sequence of a nematode target gene. According to the invention, the dsRNA regions in the RNA construct may be complementary to the same or to different target genes and/or the dsRNA regions may be complementary to targets from the same or from different nematode species. Use of such dsRNA constructs in a plant host cell, thus establishes a more potent resistance to a single or to multiple nematode species in the plant.

The terms “hit”, “hits” and “hitting” are alternative wordings to indicate that at least one of the strands of the dsRNA is complementary to, and as such may bind to, the target gene or nucleotide sequence.

In one embodiment, the double-stranded RNA region comprises multiple copies of the nucleotide sequence that is complementary to the target gene. Alternatively, the dsRNA hits more than one target sequence of the same target gene. The invention thus encompasses isolated double-stranded RNA constructs comprising at least two copies of said nucleotide sequence complementary to at least part of a nucleotide sequence of a nematode target. In another embodiment the invention relates to an isolated double stranded RNA construct comprising at least two copies of the RNA equivalent of at least one of the nucleotide sequences represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or comprising at least two copies of the RNA equivalent of a fragment of at least 17 basepairs in length thereof, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 basepairs in length thereof. Preferably, said isolated double stranded RNA construct comprises at least two copies of the RNA equivalent of the nucleotide sequence as represented in SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 or 59, or a fragment of at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 basepairs in length thereof.

The term “multiple” in the context of the present invention means at least two, at least three, at least four, at least five, at least six, etc.

The expressions “a further target gene” or “at least one other target gene” mean for instance a second, a third or a fourth, etc. target gene.

DsRNA that hits more than one of the above-mentioned targets, or a combination of different dsRNA against different of the above mentioned targets are developed and used in the methods of the present invention.

Accordingly the invention relates to an isolated double stranded RNA or RNA construct comprising the RNA equivalents of at least two nucleotide sequences independently chosen from the sequences represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or fragments thereof of at least 17 basepairs in length, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 basepairs in length thereof.

Accordingly, the present invention extends to methods as described herein, wherein the dsRNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of an nematode target gene, and which comprises the RNA equivalents of at least two nucleotide sequences independently chosen from each other. In one embodiment, the dsRNA comprises the RNA equivalents of at least two, preferably at least three, four or five, nucleotide sequences independently chosen from the sequences represented by any of SEQ ID Nos 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or fragments thereof of at least 17 basepairs in length, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 basepairs in length thereof.

The at least two nucleotide sequences may be derived from the target genes herein described. According to one preferred embodiment the dsRNA hits at least one target gene that is essential for viability, growth, development or reproduction of the nematode and hits at least one gene involved in pathogenicity or infectivity as described hereinabove. Alternatively, the dsRNA hits multiple genes of the same category, for example, the dsRNA hits at least 2 essential genes or at least 2 genes involved in the same cellular function. According to a further embodiment, the dsRNA hits at least 2 target genes, which target genes are involved in a different cellular function. For example the dsRNA hits two or more genes involved in protein synthesis (e.g. ribosome subunits), intracellular protein transport, nuclear mRNA splicing, or involved in one of the functions described in Table 1.

The dsRNA regions (or fragments) in the double-stranded RNA may be combined as follows:

- a) when multiple dsRNA regions targeting a single target gene are combined, they may be combined in the original order (ie the order in which the regions appear in the target gene) in the RNA construct,
- b) alternatively, the original order of the fragments may be ignored so that they are scrambled and combined randomly or deliberately in any order into the double-stranded RNA construct,
- c) alternatively, one single fragment may be repeated several times, for example from 1 to 10 times, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, in the ds RNA construct, or
- d) the dsRNA regions (targeting a single or different target genes) may be combined in the sense or antisense orientation.

In addition, the target gene(s) to be combined may be chosen from one or more of the following categories of genes:

- e) “essential” genes or “pathogenicity genes” as described above encompass genes that are vital for one or more target nematodes and result in a lethal or severe (e.g. feeding, reproduction, growth) phenotype when silenced. The choice of a strong lethal target gene results in a potent RNAi effect. In the RNA constructs of the invention, multiple dsRNA regions targeting the same or different (very effective) lethal genes can be combined to further increase the potency, efficacy or speed of the RNAi effect in nematode control.
- f) “weak” genes encompass target genes with a particularly interesting function in one of the cellular pathways described herein, but which result in a weak phenotypic effect when silenced independently. In the RNA constructs of the invention, multiple dsRNA regions targeting a single or different weak gene(s) may be combined to obtain a stronger RNAi effect.
- g) “nematode specific” genes encompass genes that have no substantial homologous counterpart in non-nematode organisms as can be determined by bioinformatics homology searches, for example by BLAST searches. The choice of a nematode specific target gene results in a species specific RNAi effect, with no effect or no substantial (adverse) effect in non-target organisms.
- h)“conserved genes” encompass genes that are conserved (at the amino acid level) between the target organism and non-target organism(s). To reduce possible effects on non-target species, such effective but conserved genes are analysed and target sequences from the variable regions of these conserved genes are chosen to be targeted by the dsRNA regions in the RNA construct. Here, conservation is assessed at the level of the nucleic acid sequence. Such variable regions thus encompass the least conserved sections, at the level of the nucleic acid sequence, of the conserved target gene(s).
- i) “conserved pathway” genes encompass genes that are involved in the same biological pathway or cellular process, or encompass genes that have the same functionality in different nematode species resulting in a specific and potent RNAi effect and more efficient nematode control;
- j) alternatively, the RNA constructs according to the present invention target multiple genes from different biological pathways, resulting in a broad cellular RNAi effect and more efficient nematode control.

According to the invention, all double-stranded RNA regions comprise at least one strand that is complementary to at least part or a portion of the nucleotide sequence of any of the target genes herein described. According to the invention, there is provided an isolated double-stranded RNA or RNA construct, which comprises at least one additional dsRNA region, at least one strand thereof comprising a nucleotide sequence which is complementary to at least part of a nucleotide sequence of at least one other nematode target gene.

However, provided that one of the double-stranded RNA regions comprises at least one strand that is complementary to a portion of the nucleotide sequence of any one of the target genes herein described, the other double-stranded RNA regions may comprise at least one strand that is complementary to a portion of any other nematode target gene (including known target genes).

The invention also relates to an isolated double-stranded RNA or RNA construct comprising at least two nucleotide sequences chosen from the group of sequences represented in any of SEQ ID NOs 29, 51 or 54. In a preferred embodiment, these double-stranded RNA or RNA construct(s) comprise a nucleotide sequence as represented in SEQ ID NO 59.

According to yet another embodiment of the present invention, there is provided an isolated double-stranded RNA or RNA construct, further comprising at least one additional sequence and optionally a linker. In one embodiment, the additional sequence is chosen from the group comprising (i) a sequence facilitating large-scale production of the dsRNA construct; (ii) a sequence effecting an increase or decrease in the stability of the dsRNA; (iii) a sequence allowing the binding of proteins or other molecules to facilitate uptake of the RNA construct by nematodes; (iv) a sequence which is an aptamer that binds to a receptor or to a molecule on the surface or in the cytoplasm of a nematode to facilitate uptake, endocytosis and/or transcytosis by the nematode; or (v) one or more additional sequences to catalyze processing of dsRNA regions. In one embodiment, the linker is a conditionally self-cleaving RNA sequence, preferably a pH sensitive linker or a hydrophobic sensitive linker. In one embodiment, the linker is an intron. In one embodiment of the present invention, there is provided an isolated double-stranded RNA or RNA construct for use as a medicament.

In one embodiment, the multiple dsRNA regions of the double-stranded RNA construct are connected by one or more linkers. In another embodiment, the linker is present at a site in the RNA construct, separating the dsRNA regions from another region of interest. Different linker types for the dsRNA constructs are provided by the present invention.

In another embodiment, the multiple dsRNA regions of the double-stranded RNA construct are connected without linkers.

In a particular embodiment of the invention, the linkers may be used to disconnect smaller dsRNA regions in the pest organism. Advantageously, in this situation the linker sequence may promote division of a long dsRNA into smaller dsRNA regions under particular circumstances, resulting in the release of separate dsRNA regions under these circumstances and leading to more efficient gene silencing by these smaller dsRNA regions. Examples of suitable conditionally self-cleaving linkers are RNA sequences that are self-cleaving at high pH conditions. Suitable examples of such RNA sequences are described by Borda et al. (Nucleic Acids Res. 2003 May 15; 31(10):2595-600), which document is incorporated herein by reference. This sequence originates from the catalytic core of the hammerhead ribozyme HH16.

In another aspect of the invention, a linker is located at a site in the RNA construct, separating the dsRNA regions from another, e.g. the additional, sequence of interest, which preferably provides some additional function to the RNA construct.

In one particular embodiment of the invention, the dsRNA constructs of the present invention are provided with an aptamer to facilitate uptake of the dsRNA by the nematode. The aptamer is designed to bind a substance which is taken up by the nematode. Such substances may be from a nematode or plant origin. One specific example of an aptamer, is an aptamer that binds to a transmembrane protein, for example a transmembrane protein of a nematode. Alternatively, the aptamer may bind a (plant) metabolite or nutrient which is taken up by the nematode.

Alternatively, the linkers are self-cleaving in the endosomes. This may be advantageous when the constructs of the present invention are taken up by the nematode via endocytosis or transcytosis, and are therefore compartmentalized in the endosomes of the nematode species. The endosomes may have a low pH environment, leading to cleavage of the linker.

The above mentioned linkers that are self-cleaving in hydrophobic conditions are particularly useful in dsRNA constructs of the present invention when used to be transferred from one cell to another via the transit in a cell wall, for example when crossing the cell wall of a nematode pest organism.

An intron may also be used as a linker. An “intron” as used herein may be any non-coding RNA sequence of a messenger RNA. Particular suitable intron sequences for the constructs of the present invention are (1) U-rich (35-45%); (2) have an average length of 100 by (varying between about 50 and about 500 bp) which base pairs may be randomly chosen or may be based on known intron sequences; (3) start at the 5′ end with -AG:GT- or -CG:GT- and/or (4) have at their 3′ end -AG:GC- or -AG:AA.

A non-complementary RNA sequence, ranging from about 1 base pair to about 10,000 base pairs, may also be used as a linker.

Without wishing to be bound by any particular theory or mechanism, it is thought that long double-stranded RNAs are taken up by the nematode from their immediate environment. Double-stranded RNAs taken up into the gut and transferred to the gut epithelial cells are then processed within the cell into short double-stranded RNAs, called small interfering RNAs (siRNAs), by the action of an endogenous endonuclease. The resulting siRNAs then mediate RNAi via formation of a multi-component RNase complex termed the RISC or RNA interfering silencing complex.

In order to achieve down-regulation of a target gene within a nematode cell the double-stranded RNA added to the exterior of the cell wall may be any dsRNA or dsRNA construct that can be taken up into the cell and then processed within the cell into siRNAs, which then mediate RNAi, or the RNA added to the exterior of the cell could itself be an siRNA that can be taken up into the cell and thereby direct RNAi.

siRNAs are generally short double-stranded RNAs having a length in the range of from 19 to 25 base pairs, or from 20 to 24 base pairs. In preferred embodiments siRNAs having 19, 20, 21, 22, 23, 24 or 25 base pairs, and in particular 21 or 22 base pairs, corresponding to the target gene to be down-regulated may be used. However, the invention is not intended to be limited to the use of such siRNAs.

siRNAs may include single-stranded overhangs at one or both ends, flanking the double-stranded portion. In a particularly preferred embodiment the siRNA may contain 3′ overhanging nucleotides, preferably two 3′ overhanging thymidines (dTdT) or uridines (UU). 3′ TT or UU overhangs may be included in the siRNA if the sequence of the target gene immediately upstream of the sequence included in double-stranded part of the dsRNA is AA. This allows the TT or UU overhang in the siRNA to hybridise to the target gene. Although a 3′ TT or UU overhang may also be included at the other end of the siRNA it is not essential for the target sequence downstream of the sequence included in double-stranded part of the siRNA to have AA. In this context, siRNAs which are RNA/DNA chimeras are also contemplated. These chimeras include, for example, the siRNAs comprising a double-stranded RNA with 3′ overhangs of DNA bases (e.g. dTdT), as discussed above, and also double-stranded RNAs which are polynucleotides in which one or more of the RNA bases or ribonucleotides, or even all of the ribonucleotides on an entire strand, are replaced with DNA bases or deoxynucleotides.

The dsRNA may be formed from two separate (sense and antisense) RNA strands that are annealed together by (non-covalent) basepairing. Alternatively, the dsRNA may have a foldback stem-loop or hairpin structure, wherein the two annealed strands of the dsRNA are covalently linked. In this embodiment the sense and antisense stands of the dsRNA are formed from different regions of single polynucleotide molecule that is at least partially self-complementary. RNAs having this structure are convenient if the dsRNA is to be synthesised by expression in vivo, for example in a host cell or organism as discussed below, or by in vitro transcription. The precise nature and sequence of the “loop” linking the two RNA strands is generally not material to the invention, except that it should not impair the ability of the double-stranded part of the molecule to mediate RNAi. The features of “hairpin” or “stem-loop” RNAs for use in RNAi are generally known in the art (see for example WO 99/53050, in the name of CSIRO, the contents of which are incorporated herein by reference). In other embodiments of the invention, the loop structure may comprise linker sequences or additional sequences as described above.

The double-stranded RNA or construct may be prepared in a manner known per se. For example, double-stranded RNAs may be synthesised in vitro using chemical or enzymatic RNA synthesis techniques well known in the art. In one approach the two separate RNA strands may be synthesised separately and then annealed to form double-strands. In a further embodiment, double-stranded RNAs or constructs may be synthesised by intracellular expression in a host cell or organism from a suitable expression vector. This approach is discussed in further detail below.

The amount of double-stranded RNA with which the nematode is contacted is such that specific down-regulation of the one or more target genes is achieved; such an amount is an effective amount for down-regulating genes. The RNA may be introduced in an amount which allows delivery of at least one copy per cell. However, in certain embodiments higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded RNA may yield more effective inhibition. For any given nematode gene target the optimum amount of dsRNA for effective inhibition may be determined by routine experimentation.

The nematode can be contacted with the double-stranded RNA in any suitable manner, permitting direct uptake of the double-stranded RNA by the nematode. For example, the nematode can be contacted with the double-stranded RNA in pure or substantially pure form, for example an aqueous solution containing the dsRNA. In this embodiment, the nematode may be simply “soaked” with an aqueous solution comprising the double-stranded RNA. In a further embodiment the nematode can be contacted with the double-stranded RNA by spraying the nematode with a liquid composition comprising the double-stranded RNA.

Alternatively, the double-stranded RNA may be linked to a food component of the nematodes, such as a food component for a pathogenic nematode, in order to increase uptake of the dsRNA by the nematode.

In other embodiments the nematode may be contacted with a composition containing the double-stranded RNA. The composition may, in addition to the dsRNA, contain further excipients, diluents or carriers. Preferred features of such compositions are discussed in more detail below.

The double-stranded RNA may also be incorporated in the medium in which the nematode grows or in or on a material or substrate that is infested by the nematode or impregnated in a substrate or material susceptible to infestation by nematode.

In a further aspect of the invention, said double-stranded RNA may be expressed by a prokaryotic (for instance but not limited to a bacterial) or eukaryotic (for instance but not limited to a yeast or a plant) host cell or host organism and the prokaryotic or eukaryotic cell is taken up or eaten by the nematode species.

As illustrated in the examples, bacteria can be engineered to produce any of the dsRNA or dsRNA constructs of the invention. These bacteria can be eaten by the nematode species. When taken up, the dsRNA can initiate an RNAi response, leading to the degradation of the target mRNA and weakening or killing of the feeding nematode.

According to these embodiments, any bacterium or yeast cell that is capable of expressing dsRNA or dsRNA constructs can be used. The bacterium is chosen from the group comprising Gram-negative and Gram-positive bacteria, such as, but not limited to, Escherichia spp. (e.g. E. coli), Bacillus spp. (e.g. B. thuringiensis), Rhizobium spp., Lactobacilllus spp., Lactococcus spp., etc. The yeast may be chosen from the group comprising Saccharomyces spp., etc.

Some bacteria have a very close interaction with the host plant, such as, but not limited to, symbiotic Rhizobium with the Legminosea (for example Soy). Such recombinant bacteria could be mixed with the seeds (for instance as a coating) and used as soil improvers.

Accordingly, the present invention also encompasses a cell comprising any of the nucleotide sequences or recombinant DNA constructs described herein. The invention further encompasses prokaryotic cells (such as, but not limited to, gram-positive and gram-negative bacterial cells) and eukaryotic cells (such as, but not limited to, yeast cells or plant cells). Preferably said cell is a bacterial cell or a yeast cell or an algal cell.

In other embodiments the nematode may be contacted with a composition as described further herein. The composition may, in addition to the dsRNA or DNA contain further excipients, diluents or carriers. Preferred features of such compositions are discussed in more detail below.

Alternatively, dsRNA producing bacteria or yeast cells can be sprayed directly onto the crops.

Thus, as described above, the invention provides a host cell comprising an RNA construct and/or a DNA construct and/or an expression construct of the invention. Preferably, the host cell is a bacterial or yeast cell, but may be a virus for example. A virus may be utilised which specifically infects nematodes. This ensures safety for mammals, especially humans, since the virus will not infect the mammal, so no unwanted RNAi effect will occur.

The bacterial cell or yeast cell preferably should be inactivated before being utilised as a biological pesticide, for instance when the agent is to be used in an environment where contact with humans or other mammals is likely (such as a kitchen). Inactivation may be achieved by any means, such as by heat treatment, phenol or formaldehyde treatment for example, or by mechanical treatment.

Possible applications include intensive greenhouse cultures, for instance crops that are less interesting from a GMO point of view, as well as broader field crops such as soy.

This approach has several advantages, eg: since the problem of possible dicing by a plant host is not present, it allows the delivery of large dsRNA fragments into the gut lumen of the feeding pest; the use of bacteria as nematicides does not involve the generation of transgenic crops, especially for certain crops where transgenic variants are difficult to obtain; there is a broad and flexible application in that different crops can be simultaneously treated on the same field and/or different pests can be simultaneously targeted, for instance by combining different bacteria producing distinct dsRNAs.

The amount of targeted RNA which is taken up, preferably by ingestion, by the target organism is such that specific down-regulation of the one or more target genes is achieved. When the RNA is expressed by a bacterial or fungal host cell, an amount may be applied which allows delivery of at least one copy per host cell. However, in certain embodiments higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell of the target organism) of RNA may yield more effective inhibition. For any given target gene and target organism the optimum amount of the targeted RNA molecules for effective inhibition may be determined by routine experimentation.

The target organism can be contacted with the host cell expressing the RNA molecule in any suitable manner, to permit ingestion by the target organism. Preferably, the host cells expressing the dsRNA may be linked to a food component of the target organisms in order to increase uptake of the dsRNA by the target organism. The host cells expressing the dsRNA may also be incorporated in the medium in which the target organism grows or in or on a material or substrate that is infested by a pest organism or impregnated in a substrate or material susceptible to infestation by a pest organism.

In alternative embodiments, a suitable extract derived from the host cells expressing the RNA molecule may be utilised in order to achieve down regulation of a target gene in a target organism. Here, the extracts may be derived by any suitable means of lysis of the host cells expressing the RNA molecules. For example, techniques such as sonication, French press, freeze-thaw and lysozyme treatment (see Sambrook and Russell—Molecular Cloning: A laboratory manual—third edition and the references provided therein in table 15-4) may be utilised in order to prepare a crude host cell extract (lysate). Further purification of the extract may be carried out as appropriate provided the ability of the extract to mediate targeted down regulation of target gene expression is not adversely affected. Affinity purification may be utilised for example. It may also be appropriate to add certain components to the extract, to prevent degradation of the RNA molecules. For example, RNase inhibitors may be added to the extracts derived from the host cells expressing the RNA. In one example, the target organism can be contacted with the host cell expressing the RNA in pure or substantially pure form, for example an aqueous solution containing the cell extract. In this embodiment, the target organism, especially pest organisms such as nematodes may be simply “soaked” with an aqueous solution comprising the host cell extract. In a further embodiment the nematode can be contacted with the host cells expressing the RNA molecule by spraying the nematode, or the soil containing the nematodes with a liquid composition comprising the cell extract.

Another aspect of the present invention are target nucleotide sequences of the nematode target genes herein disclosed. Such target nucleotide sequences are particularly important to design the dsRNA constructs according to the present invention. Such target nucleotide sequences are preferably at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 nucleotides in length. Non-limiting examples of preferred target nucleotide sequences are given in the examples.

According to one embodiment, the present invention provides an isolated nucleotide sequence encoding a double-stranded RNA or double-stranded RNA construct as described herein.

According to a more specific embodiment, the present invention relates to an isolated nucleic acid sequence consisting of a sequence represented by any of SEQ ID NOs 61 to 662, or a fragment of at least 17 preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 nucleotides thereof.

According to yet another embodiment, the present invention provides nematode target genes, which comprise a sequence as herein represented by SEQ ID No 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or a fragment thereof of at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 nucleotides thereof, and which target genes can be used in the methods of the present invention.

According to a more specific embodiment, the present invention relates to an isolated nucleic acid sequence consisting of a sequence represented by any of SEQ ID Nos 7, 13, 19, 25, 30, 34, 38, 42, 47, 48, 58 or 60, or the complement thereof, or a fragment of at least 17 preferably at least 18, 19, or 21, more preferably at least 22, 23 or 24 nucleotides thereof, or the complement thereof.

A person skilled in the art will recognize that homologues of these target genes can be found and that these homologues are also useful in the methods of the present invention.

Protein, or nucleotide sequences are likely to be homologous if they show a “significant” level of sequence similarity or more preferably sequence identity. Truly homologous sequences are related by divergence from a common ancestor gene. Sequence homologues can be of two types: (i) where homologues exist in different species they are known as orthologues. e.g. the a-globin genes in mouse and human are orthologues.(ii) paralogues are homologous genes in within a single species. e.g. the α- and β-globin genes in mouse are paralogues.

Preferred homologues are genes comprising a sequence which is at least about 85% or 87.5%, still more preferably about 90%, still more preferably at least about 95% and most preferably at least about 99% identical to a sequence selected from the group of sequences represented by SEQ ID Nos 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 or 59, or the complement thereof. Methods for determining sequence identity are routine in the art and include use of the Blast software and EMBOSS software (The European Molecular Biology Open. Software Suite (2000), Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp 276-277). The term “identity” as used herein refers to the relationship between sequences at the nucleotide level. The expression “% identical” is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions or deletions as compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not comprise insertions or deletions. The reference window is chosen from between at least 10 contiguous nucleotides to about 50, about 100 or to about 150 nucleotides, preferably between about 50 and 150 nucleotides. “% identity” is then calculated by determining the number of nucleotides that are identical between the sequences in the window, dividing the number of identical nucleotides by the number of nucleotides in the window and multiplying by 100.

Other homologues are genes which are alleles of a gene comprising a sequence as represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 and 59. Further preferred homologues are genes comprising at least one single nucleotide polymorphism (SNP) compared to a gene comprising a sequence as represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 and 59. Further preferred homologues are genes comprising at least one single nucleotide polymorphism (SNP) compared to a gene comprising a sequence as represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 and 59.

According to another embodiment, the invention encompasses target genes which are nematode orthologues of a gene comprising a nucleotide sequence as represented in any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 and 59. By way of example, orthologues may comprise a nucleotide sequence as represented in any of SEQ ID NOs 61 to 614, or a fragment of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides thereof. The invention thus encompasses any of the methods described herein for controlling nematode growth on a cell or an organism, or for preventing nematode infestation of a cell or an organism susceptible to nematode infection, comprising contacting nematodes with a double-stranded RNA, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of a target gene comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 61 to 514, whereby the double-stranded RNA is taken up by the nematode and thereby controls growth or prevents infestation. The invention also relates to nematode-resistant transgenic plants comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 61 to 514. A non-limiting list of nematode orthologues genes of sequences comprising at least a fragment of 17 by of one of the sequences of the invention is given in Table 6.

According to another embodiment, the invention encompasses target genes which are insect or arachnida orthologues of a gene comprising a nucleotide sequence as represented in any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 and 59. By way of example, orthologues may comprise a nucleotide sequence as represented in any of SEQ ID NOs 515 to 629, or a fragment of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides thereof. According to another aspect, the invention thus encompasses any of the methods described herein for controlling insect or arachnida growth on a cell or an organism, or for preventing insect or arachnida infestation of a cell or an organism susceptible to insect or arachnida infection, comprising contacting insects with a double-stranded RNA, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of a target gene comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 515 to 629, whereby the double-stranded RNA is taken up by the insect or arachnida and thereby controls growth or prevents infestation. The invention also relates to insect-resistant or arachnida-resistant transgenic plants comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 515 to 629. A non-limiting list of insect or arachnida orthologues genes of sequences comprising at least a fragment of 17 by of one of the sequences of the invention is given in Table 7.

According to another embodiment, the invention encompasses target genes which are fungal orthologues of a gene comprising a nucleotide sequence as represented in any of SEQ ID Nos 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 and 59. By way of example, orthologues may comprise a nucleotide sequence as represented in any of SEQ ID NOs 625 to 662, or a fragment of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides thereof. According to another aspect, the invention thus encompasses any of the methods described herein for controlling fungal growth on a cell or an organism, or for preventing fungal infestation of a cell or an organism susceptible to fungal infection, comprising contacting fungal cells with a double-stranded RNA, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of a target gene comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 625 to 662, whereby the double-stranded RNA is taken up by the fungus and thereby controls growth or prevents infestation. The invention also relates to fungal-resistant transgenic plants comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 625 to 662. A non-limiting list of fungal orthologues genes of sequences comprising at least a fragment of 17 by of one of the sequences of the invention is given in Table 8.

In one preferred embodiment of the invention the dsRNA may be expressed by (e.g. transcribed within) a host cell or host organism, the host cell or organism being an organism susceptible or vulnerable to infestation by a nematode. In this embodiment RNAi-mediated gene silencing of one or more target genes in the nematode may be used as a mechanism to control growth of the nematode in or on the host organism and/or to prevent or reduce nematode infestation of the host organism. Thus, expression of the double-stranded RNA within cells of the host organism may confer resistance to a particular nematode or to a class or family of nematodes. In case the dsRNA hits more than one nematode target gene (or hits a target gene of more than one nematode), expression of the double-stranded RNA within cells of the host organism may confer resistance to more than one nematode or more than one class of nematodes.

In a preferred embodiment the host organism is a plant and the nematode is a plant pathogenic nematode. In this embodiment the nematode is contacted with the double-stranded RNA by expressing the double-stranded RNA in a plant or plant cell that is infested with or susceptible to infestation with the plant pathogenic nematode.

Therefore, in a further aspect the invention provides a plant, preferably atransgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell, which expresses or is capable of expressing at least one double-stranded RNA, wherein said double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a nematode target gene, and wherein said double-stranded RNA is taken up by the nematode upon plant-nematode interaction, said double-stranded RNA being capable of inhibiting the target gene or down-regulating expression of the target gene by RNA interference. The target gene may be any of the target genes herein described, for instance a target gene that is essential for the viability, growth, development or reproduction of the nematode.

In this embodiment the nematode can be any nematode, but is preferably a plant pathogenic nematode. Preferred plant pathogenic nematodes include, but are not limited to, those listed above.

A plant to be used in the methods of the invention, or a transgenic plant according to the invention encompasses any plant, but is preferably a plant that is susceptible to infestation by a plant pathogenic nematode, including but not limited to the following plants: alfalfa, apple, apricot, artichoke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, carrot, cassaya, cauliflower, a cereal, celery, cherry, citrus, clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, figs, grape, grapefruit, groundnuts, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, nut oat, okra, onion, orange, an ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, soy, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, tangerine, tea, tobacco, tomato, a vine, watermelon, wheat, yams, zucchini, Brassica and Arabidopsis.

Accordingly, the present invention extends to methods as described herein wherein the plant is rice, soybean, cotton, potato, tomato, corn, banana, tobacco, sugarcane, sugarbeet, maize, cowpea and black gram wheat, oats, sorghum, barley, Brassica and Arabidopsis. Most preferably the plant is rice, soybean, cotton, potato, tomato, corn or banana.

In one embodiment the present invention extends to methods as described herein, wherein the plant is rice and the target gene is a gene from a nematode selected from the group consisting of: Meloidogyne spp. (e.g. M. incognita, M. javanica or M. graminicola), Hirschmaniella spp. (e.g. H. oryzae), Aphelenchoides spp. (e.g. A. besseyi), Heterodera spp. (e.g. H. oryzae), Ditylenchus spp. (e.g. D. angustus) or Pratylenchus spp. (e.g. P. zeae).

In another embodiment the present invention extends to methods as described herein, wherein the plant is corn and the target gene is a gene from a nematode selected from the group consisting of: Meloidogyne spp. (e.g. M. incognita), Heterodera spp. (e.g. H. zeae), Criconemoides spp., Longidorus spp., Helicotylenchus spp., Hoplolaimus spp., Xiphinema spp., Paratrichodorus spp. (e.g. P. minor), Tylenchorhynchus spp., Belonolaimus spp. or Pratylenchus spp.

In another embodiment the present invention extends to methods as described herein, wherein the plant is cotton and the target gene is a gene from a nematode selected from the group consisting of: Rotylenchulus spp. (e.g. R. reniformis) or Meloidogyne spp. (e.g. M. incognita).

In another embodiment the present invention extends to methods as described herein, wherein the plant is potato and the target gene is a gene from a nematode selected from the group consisting of: Meloidogyne spp. (e.g. M. chitwoodi or M. hapla), Globodera spp. (e.g. G. pallida and G. rostochiensis) or Ditylenchus spp. (e.g. D. dipsaci or D. destructor).

In another embodiment the present invention extends to methods as described herein, wherein the plant is banana and the target gene is a gene from a nematode selected from the group consisting of: Meloidogyne spp. (e.g. M. javanica), Rotylenchulus spp. (e.g. R. reniformis); Pratylenchus spp. (e.g. P. coffeae and P. goodey) or Radopholus spp. (e.g. R. similis).

In another embodiment the present invention extends to methods as described herein, wherein the plant is tomato and the target gene is a gene from a nematode selected from the group consisting of: Meloidogyne spp. (e.g. M. chitwoodi and M. incognita) or Globodera spp. (e.g. G. pallida and G. rostochiensis).

In another embodiment the present invention extends to methods as described herein, wherein the plant is soybean and the target gene is a gene from a nematode selected from the group consisting of: Meloidogyne spp. (e.g. M. incognita and M. arenaria), Heterodera spp. (e.g. H. glycines) or Belonolaimus spp.

In a specific embodiment the plant is rice and the nematode is Meloidogyne spp. (e.g. M. incognita, M. javanica or M. graminicola). In another embodiment the plant is soybean and the nematode is Meloidogyne spp. (e.g. M. incognita and M. arenaria). In another embodiment the plant is cotton and the nematode is Meloidogyne spp. (e.g. M. incognita) causing root knots. In another embodiment the plant is potato and the nematode is Meloidogyne spp. (e.g. M. chitwoodi) causing e.g. small, raised swellings on potato tuber surface, dark specks in the potato flesh and reduced potato quality. In another embodiment the plant is potato and the nematode is Meloidogyne spp. (e.g. M. hapla). In another embodiment the plant is tomato and the nematode is Meloidogyne spp. (e.g. M. chitwoodi) causing e.g. root galls. In another embodiment the plant is corn and the nematode is Meloidogyne spp. (e.g. M. incognita) causing e.g. stunting and chlorosis, numerous root galls and proliferation of fibrous roots. In another embodiment the plant is tobacco, sugarcane or banana and the nematode is Meloidogyne spp. (e.g. M. javanica). In another embodiment the plant is peanut and the nematode is Meloidogyne spp. (e.g. M. arenaria). In another specific embodiment the plant is rice and the nematode is Heterodera spp. (e.g. H. oryzae). In another embodiment the plant is soybean and the nematode is Heterodera spp. (e.g. H. glycines) causing e.g. yield loss, stunting and chlorosis. In another embodiment the plant is corn and the nematode is Heterodera spp. (e.g. H. zeae) causing e.g. stunting, pale color and narrow leaves. In another embodiment the plant is sugarbeet and the nematode is Heterodera spp. (e.g. H. schachtii) causing e.g. stunting and yellowing of plants, misshapen and excess fibrous roots. In another specific embodiment the plant is potato, tomato or another Solanum species and the nematode is Globodera spp. (e.g. G. pallida or G. rostochiensis) causing e.g. root damage, poor growth, yellowing and wilting. In another embodiment the plant is potato and the nematode is Ditylenchus spp. (e.g. D. dipsaci, causing e.g. tubers rot, whereby leaves and stems swell and become distorted, or D. destructor, causing e.g. potato dry rot). In another embodiment the plant is rice and the nematode is Ditylenchus spp. (e.g. D. angustus). In another embodiment the plant is soybean and the nematode is Belonolaimus spp., causing e.g. severe trim of the roots of growing plants or seedlings. In another embodiment the plant is corn and the nematode is Belonolaimus spp., causing e.g. severe trim of the roots of growing plants or seedlings. In another embodiment the plant is cotton, maize, cowpea, black gram or banana and the nematode is Rotylenchulus spp. (e.g. R. reniformis). In another embodiment the plant is banana and the nematode is Pratylenchus spp. (e.g. P. coffeae or P. goodeyi). In another embodiment the plant is rice and the nematode is Pratylenchus spp. (e.g. P. zeae). In another embodiment the plant is corn and the nematode is Pratylenchus spp., causing e.g. severe pruning of the roots, resulting in stunting, as well as reduction in stalk diameter, stalk and root weights. In another embodiment the plant is banana and the nematode is Radopholus spp. (e.g. R. similis) causing e.g. rhizome rot, pepper slow wilt. In another embodiment the plant is rice and the nematode is Hirschmaniella spp. (e.g. H. oryzae). In another embodiment the plant is rice and the nematode is Aphelenchoides spp. (e.g. A. besseyi). In another embodiment the plant is corn and the nematode is Criconemoides spp. In another embodiment the plant is corn and the nematode is Longidorus spp. In another embodiment the plant is corn and the nematode is Helicotylenchus spp. causing e.g. mild stunting and reduced yields. In another embodiment the plant is corn and the nematode is Hoplolaimus spp. causing e.g. stunting. In another embodiment the plant is corn and the nematode is Xiphinema spp. In another embodiment the plant is corn and the nematode is Paratrichodorus spp. causing e.g. stunting, chlorosis, and reduced yields; e.g. P. minor feeds largely on the root tips, stopping terminal growth of the rootlets, and resulting in the stubby-root effect. In another embodiment the plant is corn and the nematode is Tylenchorhynchus spp. causing e.g. stunting.

In another embodiment the present invention extends to methods as described herein, wherein the plant is rice, cotton, potato, tomato, corn, tobacco or soybean and wherein said target gene is a gene coding for a nematode ortholgue, preferably a Meloidogyne incognita orthologue, of a protein selected from the group of proteins whose function is given in Table 1.

Preferred transgenic plants (or reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell) are plants (or reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell) wherein said nematode target gene comprises a sequence which is selected from the group comprising: (i) sequences which are at least 75%, at least 80% or 85% identical, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% and still more preferably at least 99% identical to a sequence represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof, and (ii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662, or the complement thereof, or wherein said nematode target gene is a nematode orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 61 to 662, or the complement thereof.

Transgenic plants according to the invention extend to all plant species specifically described above being resistant to the respective nematode species as specifically described above.

In one embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a rice plant or reproductive or propagation material for a rice plant or a cultured rice plant cell, wherein the target gene is a gene from a nematode selected from the group consisting of Meloidogyne spp., Hirschmaniella spp., Aphelenchoides spp., Heterodera spp., Ditylenchus spp. and Pratylenchus spp.

In another embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a corn plant or reproductive or propagation material for a corn plant or a cultured corn plant cell, wherein the target gene is a gene from a nematode selected from the group consisting of Meloidogyne spp., Heterodera spp., Criconemoides spp., Longidorus spp., Helicotylenchus spp., Hoplolaimus spp., Xiphinema spp., Paratrichodorus spp., Tylenchorhynchus spp., Belonolaimus spp. and Pratylenchus spp.

In another embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a cotton plant or reproductive or propagation material for a cotton plant or a cultured cotton plant cell, wherein the target gene is a gene from a nematode selected from the group consisting of Meloidogyne spp., and Rotylenchulus spp.

In another embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a potato plant or reproductive or propagation material for a potato plant or a cultured potato plant cell, wherein the target gene is a gene from a nematode selected from the group consisting of Meloidogyne spp., Globodera spp. and Ditylenchus spp.

In another embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a banana plant or reproductive or propagation material for a banana plant or a cultured banana plant cell, wherein the target gene is a gene from a nematode selected from the group consisting of Meloidogyne spp., Rotylenchulus spp., Pratylenchus spp. and Radopholus spp.

In another embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a tomato plant or reproductive or propagation material for a tomato plant or a cultured tomato plant cell, wherein the target gene is a gene from a nematode selected from the group consisting of Meloidogyne spp. and Globodera spp.

In another embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a soybean plant or reproductive or propagation material for a soybean plant or a cultured soybean plant cell, wherein the target gene is a gene from a nematode selected from the group consisting of Meloidogyne spp., Belonolaimus spp. and Heterodera spp.

In yet another embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a rice, cotton, potato, tomato, corn, tobacco or soybean plant or reproductive or propagation material coding for a Meloidogyne incognita orthologue of a protein selected from the group of proteins whose function is given in Table 1.

The present invention also encompasses transgenic plants (or reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell) which express or are capable of expressing at least one of the nucleotides of the invention, for instance at least one of the nucleotide sequences represented in any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 or 59, or the complement thereof, or comprising a fragment thereof comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 nucleotides.

The plant may be provided in a form wherein it is actively expressing (transcribing) the double-stranded RNA in one or more cells, cell types or tissues. Alternatively, the plant may be “capable of expressing”, meaning that it is transformed with a transgene which encodes the desired dsRNA but that the transgene is not active in the plant when (and in the form in which) the plant is supplied.

Therefore, according to another embodiment, a recombinant DNA construct is provided comprising the nucleotide sequence encoding the dsRNA or dsRNA construct according to the present invention operably linked to at least one regulatory sequence. Preferably, the regulatory sequence is selected from the group comprising constitutive promoters or tissue specific promoters as described in the invention.

The target gene may be any target gene herein described. Preferably the regulatory element is a regulatory element that is active in a plant cell. More preferably, the regulatory element is originating from a plant.

The term “regulatory sequence” is to be taken in a broad context and refers to a regulatory nucleic acid capable of effecting expression of the sequences to which it is operably linked.

Encompassed by the aforementioned term are promoters and nucleic acids or synthetic fusion molecules or derivatives thereof which activate or enhance expression of a nucleic acid, so called activators or enhancers. The term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.

A promoter according to the invention may be a constitutive or an inducible promoter. Preferred promoters are inducible promoters to allow tight control of expression of the RNA molecules. Promoters inducible through use of an appropriate chemical, such as IPTG are preferred. Alternatively, the transgene encoding the RNA molecule is placed under the control of a strong constitutive promoter. Preferably, any promoter which is used will direct strong expression of the RNA. The nature of the promoter utilised may, in part, be determined by the specific host cell utilised to produce the RNA. In one embodiment, the regulatory sequence comprises a bacteriophage promoter, such as a T7, T3, SV40 or SP6 promoter, most preferably a T7 promoter. In yet other embodiments of the present invention, other promoters useful for the expression of RNA are used and include, but are not limited to, promoters from an RNA Pol I, an RNA Pol II or an RNA Pol III polymerase. These promoters are typically used for in vitro-production of dsRNA, which dsRNA is then included in an anti-nematicidal agent, for example in an anti-nematicidal liquid, spray or powder.

Other promoters derived from yeast or viral genes may also be utilised as appropriate.

In an alternative embodiment, the regulatory sequence comprises a promoter selected from the well known tac, trc and lac promoters. Inducible promoters suitable for use with bacterial hosts include β-lactamase promoter, E. coli λ phage PL and PR promoters, and E. coli galactose promoter, arabinose promoter and alkaline phosphatase promoter. Therefore, the present invention also encompasses a method for generating any of the RNA molecules or RNA constructs of the invention. This method comprises the steps of introducing (e.g. by transformation, transfection or injection) an isolated nucleic acid or a recombinant (DNA) construct of the invention in a host cell of the invention under conditions that allow transcription of said nucleic acid or recombinant (DNA) construct to produce the RNA which acts to down regulate a target gene of interest (when the host cell is ingested by the target organism or when a host cell or extract derived therefrom is taken up by the target organism).

By way of example, the transgene nucleotide sequence encoding the double-stranded RNA could be placed under the control of an inducible or growth or developmental stage-specific promoter which permits transcription of the dsRNA to be turned on, by the addition of the inducer for an inducible promoter or when the particular stage of growth or development is reached.

Alternatively, the transgene encoding the double-stranded RNA is placed under the control of a strong constitutive promoter such as any selected from the group comprising the CaMV35S promoter, doubled CaMV35S promoter, ubiquitin promoter, actin promoter, rubisco promoter, GOS2 promoter, Figwort mosaic viruse (FMV) 34S promoter, cassaya vein mosaic virus (CvMv) promoter, Laccase promoter, Stawberry virus 2 (SBV2) promoter.

Alternatively, the transgene encoding the double-stranded RNA is placed under the control of a tissue specific promoter. Tissue specific promoters are advantageous in that they limit the expression of the foreign gene to the area where its activity is required, reducing the risk of obtaining gene products which are undesired or lethal to other tissues. As used herein “tissue specific” includes root, tuber, vascular tissue, mesophyl tissue, stem, stamen, fruit, seed or leaf specific promoters. Examples of tissue specific promoters include any selected from the group comprising root specific promoters of genes encoding PsMTA Class III Chitinase, photosynthetic tissue-specific promoters such as promoters of cab1 and cab2, rbcS, gapA, gapB and ST-LS1 proteins, JAS promoters, chalcone synthase promoter, pyk10 promoter (Nitz, I., et al. 2001 Plant Science, 161:337-346), TUB-1 promoter (Lilley, C. J., et al., 2004 Plant Biotechnology Journal 2:3-12), ARSK1 promoter (Lilley, C. J., et al., 2004 Plant Biotechnology Journal 2:3-12), RPL16A promoter (Lilley, C. J., et al., 2004 Plant Biotechnology Journal 2:3-12).

Furthermore, when using the methods of the present invention for developing transgenic plants resistant against nematodes, it might be beneficial to place the nucleic acid encoding the double-stranded RNA according to the present invention under the control of a nematode inducible promoter such as TobRB7 (Opperman, C. H., et al., 1994 Science 263:221-223), phosphoglycerate mutase (PGM) of Arabidopsis (Mazarei, M., et al., 2003 Plant Molecular Biology 53:513-530), auxin promoter (Mazarei, M., et al., 2003 Plant Molecular Biology 53:513-530), ABI3 promoter (De Meutter, J., et al., 2005 Molecular Plant Pathology 6:321-326), endo-1,4-β glucanase (Cel1) promoter (Mitchum, M. G., et al., 2004 Molecular Plant Pathology 5:175-181), copper amine oxidase (atao1) promoter (Møller, S. G., et al., 1998 Physiological and Molecular Plant Pathology 53: 73-79), Late embryogenesis abundant (LEA) promoter (De Meutter, J., et al., 2005 Molecular Plant Pathology 6:321-326), pyk20 promoter (Puzio, P. S., et al., 2000 Plant Science 157: 245-255).

Therefore, the present invention also encompasses a method for generating any of the double-stranded RNA or RNA constructs of the invention. This method comprises the steps of (a) contacting an isolated nucleic acid or a recombinant DNA construct of the invention with cell-free components; or (b) introducing (e.g. by transformation, transfection or injection) an isolated nucleic acid or a recombinant DNA construct of the invention in a cell, under conditions that allow transcription of said nucleic acid or recombinant DNA construct to produce the dsRNA or RNA construct.

In one embodiment of the present invention, there is provided a recombinant DNA construct as described herein for use as a medicament.

Accordingly, the present invention also encompasses a cell comprising any of the nucleotide sequences or recombinant DNA constructs described herein. The invention further encompasses prokaryotic cells (such as, but not limited to, gram-positive and gram-negative bacterial cells) or eukaryotic cells (such as, but not limited to, yeast cells or plant cells). Preferably said cell is a bacterial cell or a plant cell.

Optionally, one or more transcription termination sequences may also be incorporated in the recombinant construct of the invention. The term “transcription termination sequence” encompasses a control sequence at the end of a transcriptional unit, which signals 3′ processing and poly-adenylation of a primary transcript and termination of transcription. The transcription termination sequence is useful to prevent read through transcription such that the RNA molecule is accurately produced in or by the host cell. In one embodiment, the terminator comprises a T7, T3, SV40 or SP6 terminator, preferably a T7 terminator. Other terminators derived from yeast or viral genes may also be utilised as appropriate.

Additional regulatory elements, such as transcriptional or translational enhancers, may be incorporated in the expression construct.

The recombinant constructs of the invention may further include an origin of replication which is required for maintenance and/or replication in a specific cell type. One example is when an expression construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule) in a cell. Preferred origins of replication include, but are not limited to, f1-ori and colE1 ori.

The recombinant construct may optionally comprise a selectable marker gene. As used herein, the term “selectable marker gene” includes any gene, which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells, which are transfected or transformed, with an expression construct of the invention. Examples of suitable selectable markers include resistance genes against ampicillin (Ampr), tetracycline (Tcr), kanamycin (Kanr), phosphinothricin, and chloramphenicol (CAT) gene. Other suitable marker genes provide a metabolic trait, for example manA. Visual marker genes may also be used and include for example beta-glucuronidase (GUS), luciferase and Green Fluorescent Protein (GFP).

Plants that have been stably transformed with a transgene encoding the dsRNA may be supplied as seed, reproductive material, propagation material or cell culture material which does not actively express the dsRNA but has the capability to do so.

Accordingly, the present invention encompasses a plant (e.g. a rice plant), or a seed (e.g. a rice seed), or a cell (e.g. a bacterial or plant cell), comprising any of the nucleotide sequences encoding the dsRNA or dsRNA construct as described herein. Preferably the plant (e.g. a rice plant), or seed (e.g. a rice seed), or cell (e.g. a bacterial or plant cell) comprises at least one double-stranded RNA, at least one double-stranded RNA construct, at least one nucleotide sequence, at least one recombinant DNA construct or at least one plant cell as described herein. The present invention also encompasses a plant (e.g. a alfalfa, apple, apricot, artichoke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, carrot, cassaya, cauliflower, a cereal, celery, cherry, citrus, clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, figs, grape, grapefruit, groundnuts, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, nut oat, okra, onion, orange, an ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, soy, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, tangerine, tea, tobacco, tomato, a vine, watermelon, wheat, yams, zucchini, Brassica or Arabidopsis plant; preferably a potato, rice, corn, cotton, potato, banana, tomato or soybean plant), or a seed or tuber (e.g. a a alfalfa, apple, apricot, artichoke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, carrot, cassaya, cauliflower, a cereal, celery, cherry, citrus, clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, figs, grape, grapefruit, groundnuts, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, nut oat, okra, onion, orange, an ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, soy, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, tangerine, tea, tobacco, tomato, a vine, watermelon, wheat, yams, zucchini, Brassica or Arabidopsis plant; preferably a potato, rice corn, cotton or tomato seed or tuber), or a cell (e.g. a bacterial or plant cell), comprising any of the dsRNA or dsRNA constructs described herein. Preferably, these plants or seeds or cells comprise a recombinant construct wherein the nucleotide sequence encoding the dsRNA or dsRNA construct according to the present invention is operably linked to at least one regulatory element as described above. Preferably, the seed is from a plant as described herein. Preferably the plant, seed or cell is rice, cotton, potato, tomato, corn, banana, tobacco or soybean.

General techniques for expression of exogenous double-stranded RNA in plants for the purposes of RNAi are known in the art (see Baulcombe D, 2004, Nature. 431(7006):356-63. RNA silencing in plants, the contents of which are incorporated herein by reference). More particularly, methods for expression of double-stranded RNA in plants for the purposes of down-regulating gene expression in plant pests such as nematodes are also known in the art. Similar methods can be applied in an analogous manner in order to express double-stranded RNA in plants for the purposes of down-regulating expression of a target gene in a plant pathogenic nematode. In order to achieve this effect it is necessary only for the plant to express (transcribe) the double-stranded RNA in a part of the plant which will come into direct contact with the nematode, such that the double-stranded RNA can be taken up by the nematode. Depending on the nature of the nematode and its relationship with the host plant, expression of the dsRNA could occur within a cell or tissue of a plant within which the nematode is also present during its life cycle, or the RNA may be secreted into a space between cells, such as the apoplast, that is occupied by the nematode during its life cycle. Furthermore, the dsRNA may be located in the plant cell, for example in the cytosol, or in the plant cell organelles such as a chloroplast, mitochondrion, vacuole or endoplastic reticulum.

Alternatively, the dsRNA may be secreted by the plant cell and by the plant to the exterior of the plant. As such, the dsRNA may form a protective layer on the surface of the plant.

In a further aspect, the invention also provides combinations of methods and compositions for preventing or protecting plants from pest infestation. For instance, one means provides using the plant transgenic approach combining methods using expression of dsRNA molecules and methods using expression of such Bt nematicidal or insecticidal proteins.

Therefore the invention also relates to a method or a plant cell or plant described herein, wherein said plant cell or plant expressing said RNA molecule comprises or expresses a pesticidal agent selected from the group consisting of a patatin, a Bacillus thuringiensis insecticidal protein, a Bacillus thuringiensis nematicidal protein a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphearicus insecticidal protein. Preferably said Bacillus thuringiensis insecticidal protein is selected from the group consisting of a Cry1, a Cry3, a TIC851, a CryET170, a Cry22, a binary insecticidal protein CryET33 and CryET34, a binary insecticidal protein CryET80 and CryET76, a binary insecticidal protein TIC100 and TIC101, and a binary insecticidal protein PS149B1.

In a further embodiment, the invention relates to a composition for controlling nematode growth and/or preventing or reducing nematode infestation, comprising at least one double-stranded RNA, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a nematode target gene and optionally further comprising at least one suitable carrier, excipient or diluent. The target gene may be any target gene described herein. Preferably the nematode target gene is essential for the viability, growth, development or reproduction of the nematode.

In another aspect the invention relates to a composition as described above, wherein the nematode target gene comprises a sequence which is at least 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, 98% or 99% identical to a sequence selected from the group of sequences represented by any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 and 59, and sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57 or 59, or the complement thereof, or wherein said nematode target gene is a nematode orthologue of a gene comprising any of SEQ ID Nos 61 to 662.

The present invention further relates to a composition comprising at least one double-stranded RNA, at least one double-stranded RNA construct, at least one nucleotide sequence and/or at least one recombinant DNA construct as described herein, optionally further comprising at least one suitable carrier, excipient or diluent. In one embodiment, the composition comprises at least one double-stranded RNA as described herein or at least one double-stranded RNA construct as described herein and further at least one suitable carrier, excipient or diluent. In another embodiment, the composition comprises at least one nucleotide sequence as described herein and/or at least one recombinant DNA construct as described herein and further at least one suitable carrier, excipient or diluent.

The composition may contain further components which serve to stabilise the dsRNA and/or prevent degradation of the dsRNA during prolonged storage of the composition.

The composition may still further contain components which enhance or promote uptake of the dsRNA by the nematode. These may include, for example, chemical agents which generally promote the uptake of RNA into cells e.g. lipofectamine etc.

The composition may be in any suitable physical form for application to nematodes, to substrates, to cells (e.g. plant cells), or to organisms infected by or susceptible to infection by nematodes. In terms of “susceptible organisms” which benefit from the present invention, any organism which is susceptible to pest infestation is included. Further to plants as described in more detail before, pests of many different organisms, for example animals such as humans, domestic animals (such as pets like cats, dogs etc) and livestock (including sheep, cows, pigs, chickens etc.) are envisaged.

The invention also relates to a spray comprising at least one composition or comprising at least one host cell as described herein, and further at least one adjuvant and optionally at least one surfactant

The effectiveness of a pesticide may depend on the effectiveness of the spray application. Adjuvants can minimize or eliminate many spray application problems associated with pesticide stability, solubility, incompatibility, suspension, foaming, drift, evaporation, volatilization, degradation, adherence, penetration, surface tension, and coverage. Adjuvants are designed to perform specific functions, including wetting, spreading, sticking, reducing evaporation, reducing volatilization, buffering, emulsifying, dispersing, reducing spray drift, and reducing foaming. No single adjuvant can perform all these functions, but different compatible adjuvants often can be combined to perform multiple functions simultaneously. These chemicals, also called wetting agents and spreaders, physically alter the surface tension of a spray droplet. For a pesticide to perform its function properly, a spray droplet must be able to wet the foliage and spread out evenly over a leaf. Surfactants enlarge the area of pesticide coverage, thereby increasing the pest's exposure to the chemical. Surfactants are particularly important when applying a pesticide to waxy or hairy leaves. Without proper wetting and spreading, spray droplets often run off or fail to adequately cover these surfaces. Too much surfactant, however, can cause excessive runoff or deposit loss, thus reducing pesticide efficacy. Pesticide formulations often contain surfactants to improve the suspension of the pesticide's active ingredient. This is especially true for emulsifiable concentrate (EC) formulations.

As used herein the term “adjuvant” means any nonpesticide material added to a pesticide product or pesticide spray mixture to improve the mixing and stability of the products in the spray tank and the application. As further used herein the term “surfactant” means a chemical that modifies surface tension. Surfactants can influence the wetting and spreading of liquids, and can modify the dispersion, suspension, or precipitation of a pesticide in water. There are nonionic surfactants (no electrical charge), anionic surfactants (negative charge), and cationic surfactants (positive charge)

In particular embodiments the host cells comprised in the spray are inactivated, for instance by heat inactivation or mechanical disruption (as discussed in greater detail herein).

The nature of the excipients and the physical form of the composition may vary depending upon the nature of the substrate that it is desired to treat. For example, the composition may be a liquid that is brushed or sprayed onto or imprinted into the material or substrate to be treated, or a coating or powder that is applied to the material or substrate to be treated. Thus, in one embodiment, the composition is in the form of a coating on a suitable surface which adheres to, and is eventually ingested by an nematode which comes into contact with the coating.

According to a preferred embodiment, the substrate is a plant or crop to be treated against nematode pest infestation. The composition is then internalized or eaten by the nematode, from where it can mediate RNA interference, thus controlling the nematode. The spray is preferably a pressurized/aerosolized spray or a pump spray. The particles may be of suitable size such that they adhere to the substrate to be treated or to the nematode, for example to the outer bodywall, of the nematode and may be absorbed therefrom.

In one embodiment, the composition is in the form of a bait. The bait is designed to lure the nematode to come into contact with the composition. Upon coming into contact therewith, the composition is then internalised by the nematode, by ingestion for example and mediates RNAi to thus kill the nematode. Said bait may comprise a food substance, such as a protein based food, for example fish meal. Boric acid may also be used as a bait. The bait may depend on the species being targeted. An attractant may also be used. The attractant may be a pheromone, such as a male or female pheromone for example. The attractant acts to lure the nematode to the bait, and may be targeted for a particular nematode or may attract a whole range of nematodes. The bait may be in any suitable form, such as a solid, paste, pellet or powdered form.

Additionally, compositions which come into contact with the nematodes may remain on the cuticle of the nematode.

The baits may be provided in a suitable “housing” or “trap”. Such housings and traps are commercially available and existing traps may be adapted to include the compositions of the invention. Any housing or trap which may attract a nematode to enter it is included within the scope of the invention. A trap is distinguished from a housing because the nematode can not readily leave a trap following entry, whereas a housing acts as a “feeding station” which provides the nematode with a preferred environment in which they can feed and feel safe from predators.

Accordingly, in a further aspect the invention provides a housing or trap for nematodes which contains a composition of the invention, which may incorporate any of the features of the composition described herein.

It is contemplated that the “composition” of the invention may be supplied as a “kit-of-parts” comprising the double-stranded RNA in one container and a suitable diluent or carrier for the RNA in a separate container. The invention also relates to supply of the double-stranded RNA alone without any further components. In these embodiments the dsRNA may be supplied in a concentrated form, such as a concentrated aqueous solution. It may even be supplied in frozen form or in freeze-dried or lyophilised form. The latter may be more stable for long term storage and may be de-frosted and/or reconstituted with a suitable diluent immediately prior to use.

The present invention further relates to the medical use or the use as a medicament of any of the double-stranded RNAs, double-stranded RNA constructs, nucleotide sequences, recombinant DNA constructs, hairpin sequences or compositions described herein. In one specific embodiment, the composition is a pharmaceutical or veterinary composition for treating or preventing nematode disease or infections of humans or animals, respectively. Such compositions will comprise at least one double-stranded RNA or RNA construct, or nucleotide sequence or recombinant DNA construct encoding the double-stranded RNA or RNA construct, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which corresponds to a target nucleotide sequence of a nematode target gene that causes the disease or infection, and at least one carrier, excipient or diluent suitable for pharmaceutical use.

In another embodiment the compositions described herein are used as a nematicide for a plant or for propagation or reproductive material of a plant. In yet another embodiment, the compositions described herein are used for controlling nematode growth. In yet another embodiment, the compositions described herein are used for preventing nematode infestation of plants susceptible of nematode infection.

The composition may be a composition suitable for topical use, such as application on the skin of an animal or human, for example as liquid composition to be applied to the skin as drops, gel, aerosol, or by brushing, or a spray, cream, ointment, etc. for topical application or as transdermal patches.

Alternatively, the nematode dsRNA is produced by bacteria (e.g. lactobacillus) which can be included in food and which functions as an oral vaccine against the nematode infection.

Other conventional pharmaceutical dosage forms may also be produced, including tablets, capsules, pessaries, transdermal patches, suppositories, etc. The chosen form will depend upon the nature of the target nematode and hence the nature of the disease it is desired to treat.

Preferred target human pathogenic and animal pathogenic nematodes include, but are not limited to the following:

In humans: Enterobius verraicularis, Ascaris lumbridoides, Necator, Ancylostoma, Trichuris trichiura, Strongyloides stercoralis, Trichonella spirae, Brugia malayi, Wuchereria bancrofti or Onchocerca volvulus.

In animals: Hookworms e.g. Ancylostoma caninum, Ancylostoma tubaeforme or Uncinaria stenocephala; Ascarids e.g. Toxocara canis, Toxocara cati or Toxascaris leonine; Whipworms e.g. Trichuris vulpis; herring worms or cod worms e.g. Anisakid; or tapeworm e.g. Diphyllobothrium.

In one specific embodiment, the composition may be a coating that can be applied to a substrate in order to protect the substrate from infestation by a nematode and/or to prevent arrest or reduce nematode growth on the substrate and thereby prevent damage caused by the nematode. In this embodiment, the composition can be used to protect any substrate or material that is susceptible to infestation by or damage caused by a nematode, for example foodstuffs and other perishable materials, and substrates such as wood. Preferred target nematode species for this embodiment include, but are not limited to, the following: Meloidogyne spp. e.g. M. incognita, M. javanica, M. Parenaria, M. graminicola, M. chitwoodi or M. hapla; Heterodera spp. e.g. H. oryzae, H. glycines, H. zeae or H. schachtii; Globodera spp. e.g. G. pallida or G. rostochiensis; Ditylenchus spp. e.g. D. dipsaci, D. destructor or D. angustus; Belonolaimus spp.; Rotylenchulus spp. e.g. R. reniformis; Pratylenchus spp. e.g. P. coffeae, P. goodeyi or P. zeae; Radopholus spp. e.g. R. Similis; Hirschmaniella spp. e.g. H. oryzae; Aphelenchoides spp. e.g. A. besseyi; Criconemoides spp.; Longidorus spp.; Helicotylenchus spp.; Hoplolaimus spp.; Xiphinema spp.; Paratrichodorus spp. e.g. P. minor; Tylenchorhynchus spp.

The nature of the excipients and the physical form of the composition may vary depending upon the nature of the substrate that is desired to treat. For example, the composition may be a liquid that is brushed or sprayed onto or imprinted into the material or substrate to be treated, or a coating that is applied to the material or substrate to be treated.

The present invention further encompasses a method for treating and/or preventing nematode infestation on a substrate comprising applying an effective amount of any of the compositions described herein to said substrate.

The invention further encompasses a method for treating and/or preventing a nematode disease or condition, comprising administering to a subject in need of such treatment and/or prevention, any of the compositions as herein described, said composition comprising at least one double-stranded RNA or double-stranded RNA construct comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a nematode target gene that causes the nematode disease or condition. Therapies are being developed based on RNAi in human and in animals; dsRNA of the target genes can be used as vaccines if the targets work against a range of nematode species.

In another embodiment of the invention the compositions are used as a nematicide for a plant or for propagation or reproductive material of a plant, such as on seeds. As an example, the composition can be used as a nematicide by spraying or applying it on plant tissue or spraying or mixing it on the soil before or after emergence of the plantlets.

In a more specific embodiment, the invention relates to the use of a spray comprising at least one host cell or at least one host cell (e.g. a bacterial or a yeast) expressing a dsRNA of the invention, or a virus encoding a dsRNA described herein, or to any of the compositions comprising the same, for controlling nematode growth; for preventing nematode infestation of plants susceptible to nematode infection; or for treating nematode infection of plants. Preferably said host cell comprises at least one of the sequences represented by any of SEQ ID NOs SEQ ID NOs 1, 5, 6, 8, 12, 14, 18, 20, 24, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 52, 54, 55, 57, 59, or any of SEQ ID NOs 61 to 662or a fragment thereof of at least 17 contiguous nucleotides.

In a further aspect, the invention also provides combinations of methods and compositions for preventing or protecting plants from pest infestation. For instance, one means provides using a combination of the transgenic approach with methods using double stranded RNA molecules and compositions with one or more Bt insecticidal proteins or chemical (organic) compounds that are toxic to the target pest. Another means provides using the transgenic approach combining methods using expression of double stranded RNA molecules in bacteria or yeast and expression of such Bt insecticidal proteins in the same or in distinct bacteria or yeast. According to these approaches, for instance, a nematode can be targeted or killed using the RNAi-based method or technology, while an insect can be targeted or killed using the Bt insecticide or the chemical (organic) insecticide.

Therefore the invention also relates to any of the compositions, sprays or methods for treating plants described herein, wherein said composition comprises a bacterial cell or yeast expressing said RNA molecule and further comprises a pesticidal agent or comprises a bacterial cell or yeast cell comprising or expressing a pesticidal agent (the bacterial or yeast cell can be the same or different from the first ones mentioned), said pesticidal agent selected from the group consisting of a chemical (organic) insecticide, a patatin, a Bacillus thuringiensis insecticidal protein, a Bacillus thuringiensis nematicidal protein a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphearicus insecticidal protein. Preferably said Bacillus thuringiensis insecticidal protein is selected from the group consisting of a Cry1, a Cry3, a TIC851, a CryET170, a Cry22, a binary insecticidal protein CryET33 and CryET34, a binary insecticidal protein CryET80 and CryET76, a binary insecticidal protein TIC100 and TIC101, and a binary insecticidal protein PS149B1.

The spray can be used in a greenhouse or on the field. Typical application rates for bacteria-containing biopestides (e.g. as an emulsifiable suspension) amount to 25-100 liters/ha (10-40 liters/acre) for water based sprays: comprising about 2.5-5 liter of formulated product (emulsifiable suspension) per hectare with the formulated product including about 25% (v/v) of ‘bacterial cells’ plus 75% (v/v) ‘other ingredients’. The amount of bacterial cells are measured in units, e.g. one unit is defined as 10⁹bacterial cells in 1 ml. Depending on the crop density per hectare and the leaf surface per plant, one liter of formulated product comprises between 0.001 and 10000 units of bacteria, preferably at least 0.001, 0.003, 0.005, 0.007, 0.01, 0.03, 0.05, 0.07, 0.1, 0.3, 0.5, 0.7, more preferably at least 1, 3, 5, 7, 10, 30, 50, 70, 100, 300, 500, 700, or more preferably at least 1000, 3000, 5000, 7000 or 10000 units of bacteria.

For instance, typical plant density for potato crop plants is approximately 4.5 plants per square meter or 45,000 plants per hectare (planting in rows with spacing between rows at 75 cm and spacing between plants within rows at 30 cm). The present invention thus relates to a spray comprising at least 0.001, 0.003, 0.005, 0.007, 0.01, 0.03, 0.05, 0.07, 0.1, 0.3, 0.5, 0.7, more preferably at least 1, 3, 5, 7, 10, 30, 50, 70, 100, 300, 500, 700, or more preferably at least 1000, 3000, 5000, 7000 or 10000 units of bacteria expressing at least one of the dsRNA molecules or dsRNA constructs described herein.

In yet another embodiment, the present invention provides a method for treating and/or preventing nematode growth and/or nematode infestation of a plant or propagation or reproductive material of a plant, comprising applying an effective amount of any of the compositions herein described to a plant or to propagation or reproductive material of a plant.

In another embodiment the invention relates to the use of any double-stranded RNA or RNA construct, or nucleotide sequence or recombinant DNA construct encoding the double-stranded RNA or RNA construct described herein, or cell or to any of the compositions comprising the same, used for controlling nematode growth; for preventing nematode infestation of plants susceptible to nematode infection; or for treating nematode infection of plants. Specific plants to be treated for nematode infections caused by specific nematode species are as described earlier and are encompassed by the said use.

The invention further relates to a kit comprising at least one double-stranded RNA, or double-stranded RNA construct, or nucleotide sequence, or recombinant DNA construct, or cell, or composition as described earlier for treating nematode infection in plants. The kit may be supplied with suitable instructions for use. The instructions may be printed on suitable packaging in which the other components are supplied or may be provided as a separate entity, which may be in the form of a sheet or leaflet for example. The instructions may be rolled or folded for example when in a stored state and may then be unrolled and unfolded to direct use of the remaining components of the kit.

According to a still further embodiment, the present invention extends to a method for increasing plant yield comprising introducing in a plant any of the nucleotide sequences or recombinant DNA constructs as herein described in an expressible format. Plants encompassed by this method are as described earlier (e.g. alfalfa, apple, apricot, artichoke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, carrot, cassaya, cauliflower, a cereal, celery, cherry, citrus, clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, figs, grape, grapefruit, groundnuts, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, nut oat, okra, onion, orange, an ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, soy, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, tangerine, tea, tobacco, tomato, a vine, watermelon, wheat, yams, zucchini, Brassica and Arabidopsis; preferably rice, cotton, potato, tomato, corn, banana or soybean). Preferably, said plant is rice, cotton, potato, tomato, corn, banana, tobacco or soybean.

In one specific embodiment, the method of the invention may also be used as a tool for experimental research, particularly in the field of functional genomics. Targeted down-regulation of nematode genes by RNAi can be used in in vitro or in vivo assays in order to study gene function, in an analogous approach to that which has been described in the art for the nematode C. elegans and also Drosophila melanogaster. Assays based on targeted down-regulation of specific nematode genes, leading to a measurable phenotype may also form the basis of compound screens for novel anti-nematode agents.

BRIEF DESCRIPTION OF FIGURES AND TABLES

FIG. 1: RNAi effect on Meloidogyne incognita in ex vitro tomato composite plants. Egg counts (average number of eggs per μl from three samplings) from tomato composite roots expressing hairpins of different targets. The control lines were transformed with a binary vector without hairpin (empty vector control). Error bars are 95% confidence intervals.

FIG. 2: RNAi effect on Meloidogyne incognita in transgenic tomato hairy roots. Egg counts per gram of root mass (average number of eggs from three samplings) as % to egg counts per gram of control roots (100%) from tomato hairy roots expressing hairpins of different targets. The control lines were transformed with a binary vector containing a gus hairpin. Error bars are standard error bars.

FIG. 3: RNAi effect on Meloidogyne incognita in transgenic Arabidopsis thaliana plants expressing a MiCC2 hairpin. Number of galls (determined 7 days post inoculation) (average of 10 replicates) and number of egg laying females ELF (determined 8 weeks post inoculation)(average of 10 replicates) of three independent transgenic lines (MiCC2 HP expressing Ara) are presented. The control plants are wild type Arabidopsis thaliana. Error bars are standard deviations.

FIG. 4: RNAi effect on Meloidogyne incognita in transgenic Arabidopsis thaliana plants expressing a Mi40 hairpin. Number of galls (determined 7 days post inoculation) (average of 10 replicates) and number of egg laying females (ELF) (determined 8 weeks post inoculation) (average of 10 replicates) of two independent transgenic lines (Mi40 HP expressing Ara plants) are presented. The control plants are wild type Arabidopsis thaliana. Error bars are standard deviations.

FIG. 5: RNAi effect on Meloidogyne incognita in transgenic Arabidopsis thaliana plants expressing a Mi116 hairpin. Number of galls (determined 7 days post inoculation) (average of 10 replicates) and number of egg laying females (ELF) (determined 8 weeks post inoculation) (average of 10 replicates) of two independent transgenic lines (Mi116 HP expressing Ara plants) are presented. The control plants are wild type Arabidopsis thaliana. Error bars are standard deviations.

Table 1: Examples of novel identified nematode target genes. Gene function assigned is based on the Wormbase orthologue.

Table 2: Overview of cloning details of cDNA's of Meloidogyne incognita target genes including primer sequences and cDNA sequences obtained.

Table 3: Amino acid sequence of Meloidogyne incognita cDNA clones.

Table 4: Nucleotide sequences of fragments of the Meloidogyne incognita cDNA clones.

Table 5: Hairpin sequences.

Table 6: Selected sequences* of target genes. Fragments of at least 17 by of the sequences* are present in any of the orthologues sequences in nematode species (represented by GI number in the right column; several database entry numbers were found for each species but only one is given by way of example).

Table 7: Selected sequences* of target genes. Fragments of at least 17 by of the sequences* are present in any of the orthologues sequences in insect species (represented by GI number in the right column; several database entry numbers were found for each species but only one is given by way of example).

Table 8: Selected sequences* of target genes. Fragments of at least 17 by of the sequences* are present in any of the orthologues sequences in fungi species (represented by GI number in the right column; several database entry numbers were found for each species but only one is given by way of example).

The invention will be further understood with reference to the following non-limiting examples.

EXAMPLES
Example 1
The Effect of Silencing the C. elegans Target Genes Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi127, Mi128 and Mi129 in C. elegans

The C. elegans orthologs of the target genes Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi127, Mi128 and Mi129 were isolated via PCR from genomic DNA of N2-staged wild-type worms. A fragment of these orthologs was chosen to maximize the number of exons in overlap, and this fragment was used in further dsRNA mediated silencing experiments.

Each fragment was cloned in the pGN49A vector (WO01/88121) between two identical T7-promoters and terminators, driving its expression in the sense and antisense direction upon expression of the T7 polymerase, which was induced by IPTG.

This vector was transformed into the bacterial strain AB301-105 (DE3). Subsequently these bacterial cells were fed to a nuclease deficient C. elegans strain.

Feeding the dsRNA produced in bacterial strain AB301-105 (DE3), to C. elegans nuc-1 (e1392) worms, was performed in a 96 well plate as follows nuc-1 eggs were transferred to a separate plate and allowed to hatch simultaneously at 20° C. for synchronization of the L1 generation. A 96 well plate was filled with 100 μL liquid growth medium comprising IPTG and with 10 μL bacterial cell culture of OD₆₀₀1 AB301-105 (DE3) carrying the vector with the fragment for expression of the dsRNA. To each well, 4 of the synchronized L1 worms were added and were incubated at 25° C. for at least 4 to 5 days. These experiments were performed in quadruplicate. As a negative control, C. elegans was fed with bacteria carrying a vector without the 1216 by fragment.

After 5 days the phenotype of the C. elegans nuc-1 (e1392) worms fed with the bacteria producing dsRNA was compared to the phenotype of worms fed with the empty vector.

The worms that were fed with dsRNA targeting Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi127, Mi128 and Mi129 showed acute lethality when L1 were put in the experiment or lethal when later larval stages were tested.

The worms that were fed with dsRNA targeting Mi11 showed embryonic lethality or were sterile (based on public RNAi data (www.wormbase.org)).

Based on these experiments, it was concluded that silencing the above mentioned C. elegans target genes had a fatal effect on the growth and viability of the worm and that the target gene is essential to the viability of nematodes. Therefore these genes are good target genes to control (kill or prevent from growing) nematodes via dsRNA mediated gene silencing. Accordingly, the present invention encompasses the use of a nematode ortholog of the above C. elegans target genes, to control nematode infestation, such as nematode infestation of plants.

Example 2
Cloning, Identification or Assembly of M. incognita Target Genes Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi127, Mi128 and Mi129
Example 2.1
Cloning of a Partial Sequence of the M. incognita Mi05 Gene Via Family PCR

To isolate cDNA sequences from M. incognita comprising a portion of the Mi05 gene, a series of PCR reactions were performed on cDNA (prepared from M. incognita total RNA), using Amplitaq Gold (Cat. NO. N8080240; Applied Biosystems) as prescribed by the manufacturer.

First, the degenerate primers oGAUH009 and oGAUH018 (represented herein as SEQ ID NO 3 and SEQ ID NO 4 respectively) were used in a PCR reaction with the following conditions: 10 minutes at 95° C., followed by 40 cycles of 30 seconds at 95° C., 40 seconds at 45° C. and 1 minute 10 seconds at 72° C., followed by 5 minutes at 72° C. The resulting PCR product was analyzed on agarose gel, isolated, cloned into the pCR4-TOPO vector (Cat. NO. K4575-40; Invitrogen) and sequenced.

Subsequently, specific primers were designed in order to perform 3′ RACE PCR in combination with SMART primers (Clontech). This resulted in the identification of more 3′ coding sequence and the 3′ UTR of Mi05. The assembled cDNA contig is herein represented by SEQ ID NO 1 and is referred to as the partial sequence of the M. incognita Mi05 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 2.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 1 or comprising any fragment thereof.

Based on the partial sequence of the Mi05 target gene, represented herein as SEQ ID NO 1, the full length sequence of Mi05 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 1.

Example 2.2
Identification of a Sequence of the M. incognita Mi11 Gene

To identify a cDNA sequence from M. incognita Mi11 gene, one EST was found in the public database Genbank under accession number BM882119. Based on this EST sequence, specific primers oGAUL001 and oGAUL008 (represented herein as SEQ ID NO 10 and SEQ ID NO 11 respectively) were designed and used in a PCR reaction on cDNA (prepared from M. incognita total RNA), using Amplitaq Gold (Cat. NO. 8080240; Applied Biosystems) as prescribed by the manufacturer. Two independent PCR reactions were set up with the following conditions: 10 minutes at 95° C., followed by 35 cycles of 30 seconds at 95° C., 30 seconds at 53° C. and 30 seconds at 72° C., followed by 7 minutes at 72° C. The resulting PCR products were analyzed on agarose gel, isolated, cloned into the pCR4-TOPO vector (Cat. NO. K4575-40; Invitrogen) and sequenced. The sequences of 3 clones from each independent PCR reactions were used to assemble the contig of Mi11. The assembled cDNA contig is herein represented by SEQ ID NO 8 and is referred to as the partial sequence of the M. incognita Mi11 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 9.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 8 or comprising any fragment thereof.

Based on the partial sequence of the Mi11 target gene, represented herein as SEQ ID NO 8, the full length sequence of Mi11 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 8.

Example 2.3
Identification of a Sequence of the M. incognita Mi38 Gene

To identify a cDNA sequence from M. incognita Mi38 gene, one EST was found in the public database Genbank under accession number AW829338. Based on this EST sequence, specific primers oGAU072 and oGAU073 (represented herein as SEQ ID NO 16 and SEQ ID NO 17 respectively) were designed and used in a PCR reaction on cDNA (prepared from M. incognita total RNA), using PerfectShot Ex Taq (Cat. NO. RR005A , Takara) as prescribed by the manufacturer. Two independent PCR reactions were set up with the following conditions: 5 minutes at 94° C., followed by 30 cycles of 30 seconds at 94° C., 30 seconds at 45° C. and 2 minutes at 72° C., followed by 7 minutes at 72° C. The resulting PCR products were analyzed on agarose gel, isolated, cloned into the pCR4-TOPO vector (Cat. NO. K4575-40; Invitrogen) and sequenced. The sequences of 3 clones from each independent PCR reactions were used to assemble the contig of Mi38.

The assembled cDNA contig is herein represented by SEQ ID NO 14 and is referred to as the partial sequence of the M. incognita Mi38 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 15.

Based on the partial sequence of the Mi38 target gene, represented herein as SEQ ID NO 14, the full length sequence of Mi38 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 14.

Example 2.4
Identification of a Sequence of the M. incognita Mi40 Gene

To identify a cDNA sequence from M. incognita Mi40 gene, one EST was found in the public database Genbank under accession number BQ519760. Based on this EST sequence, specific primers oGAU048 and oGAU049 (represented herein as SEQ ID NO 22 and SEQ ID NO 23 respectively) were designed and used in a PCR reaction on cDNA (prepared from M. incognita total RNA), using PerfectShot Ex Taq (Cat. NO. RR005A , Takara) as prescribed by the manufacturer. Two independent PCR reactions were set up with the following conditions: 5 minutes at 94° C., followed by 30 cycles of 30 seconds at 94° C., 30 seconds at 45° C. and 2 minutes at 72° C., followed by 7 minutes at 72° C. The resulting PCR products were analyzed on agarose gel, isolated, cloned into the pCR4-TOPO vector (Cat. NO. K4575-40; Invitrogen) and sequenced. The sequences of 3 clones from each independent PCR reactions were used to assemble the contig of Mi40.

The assembled cDNA contig is herein represented by SEQ ID NO 20 and is referred to as the partial sequence of the M. incognita Mi40 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 21.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 20 or comprising any fragment thereof.

Based on the partial sequence of the Mi40 target gene, represented herein as SEQ ID NO 20, the full length sequence of Mi40 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 20.

Example 2.5
Identification of a Sequence of the M. incognita Mi101 Gene

To identify a cDNA sequence from M. incognita Mi101 gene, one EST was found in the public database Genbank.

The EST CF803049 is herein represented by SEQ ID NO 26 and is referred to as the partial sequence of the M. incognita Mi101 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 27.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 26 or comprising any fragment thereof.

Based on the partial sequence of the Mi101 target gene, represented herein as SEQ ID NO 26, the full length sequence of Mi101 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 26.

Example 2.6
Identification of a Sequence of the M. incognita Mi109 Gene

To identify a cDNA sequence from M. incognita Mi109 gene, one EST was found in the public database Genbank.

The EST CK233410 is herein represented by SEQ ID NO 31 and is referred to as the partial sequence of the M. incognita Mi109 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 32.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 31 or comprising any fragment thereof.

Based on the partial sequence of the Mi109 target gene, represented herein as SEQ ID NO 31, the full length sequence of Mi109 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 31.

Example 2.7
Assembly of a Sequence of the M. incognita Mi111 Gene Via Contig Building

To assemble a cDNA sequence from M. incognita Mi111 gene, several ESTs were used in the contig builder program TGICL (TIGR Gene Indices clustering tools). The ESTs used were CD749362 and CK233691, originating from the public database Genbank.

The assembled cDNA contig is herein represented by SEQ ID NO 35 and is referred to as the partial sequence of the M. incognita Mi111 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 36.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 35 or comprising any fragment thereof.

Based on the partial sequence of the Mi111 target gene, represented herein as SEQ ID NO 35, the full length sequence of Mi111 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 35.

Example 2.8
Assembly of a Sequence of the M. incognita Mi116 Gene Via Contig Building

To assemble a cDNA sequence from M. incognita Mi116 gene, several ESTs were used in the contig builder program TGICL (TIGR Gene Indices clustering tools). The ESTs used were BE239183, CF099482 and BM880799, originating from the public database Genbank.

The assembled cDNA contig is herein represented by SEQ ID NO 39 and is referred to as the partial sequence of the M. incognita Mi116 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 40.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 39 or comprising any fragment thereof. Based on the partial sequence of the Mi116 target gene, represented herein as SEQ ID NO 39, the full length sequence of Mi116 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 39.

Example 2.9
Assembly of a Sequence of the M. incognita Mi125 Gene Via Contig Building

To assemble a cDNA sequence from M. incognita Mi125 gene, several ESTs were used in the contig builder program TGICL (TIGR Gene Indices clustering tools). The ESTs used were BQ548495, CD749619, BQ625388, CK983617, CF802860, BQ519621, CN578439, CK984202, CK985275, CK985196, CN578210, CF980909, CN443-475, CK984712, CK984808, CN443437, CN443424, CN443495, CN443459 and CN578277, originating from the public database Genbank.

The assembled cDNA contig is herein represented by SEQ ID NO 43 and is referred to as the partial sequence of the M. incognita Mi125 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 44.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 43 or comprising any fragment thereof. Based on the partial sequence of the Mi125 target gene, represented herein as SEQ ID NO 43, the full length sequence of Mi125 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 43.

Example 2.10
Assembly of a Sequence of the M. incognita Mi127 Gene Via Contig Building

To assemble a cDNA sequence from M. incognita Mi127 gene, several ESTs were used in the contig builder program TGICL (TIGR Gene Indices clustering tools). The ESTs used were CD749453, CF980531, CF803087, CF980430, CF803126, CN578375, CN443332 and CK984842, originating from the public database Genbank.

The assembled cDNA contig is herein represented by SEQ ID NO 49 and is referred to as the partial sequence of the M. incognita Mi127 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 50.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 49 or comprising any fragment thereof. Based on the partial sequence of the Mi127 target gene, represented herein as SEQ ID NO 49, the full length sequence of Mi127 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 49.

Example 2.11
Assembly of a Sequence of the M. incognita Mi128 Gene Via Contig Building

To assemble a cDNA sequence from M. incognita Mi128 gene, several ESTs were used in the contig builder program TGICL (TIGR Gene Indices clustering tools). The ESTs used were CK233383, BQ519732, CN578199, CN578113, CF803167, CK233325 and CK233386, originating from the public database Genbank.

The assembled cDNA contig is herein represented by SEQ ID NO 52 and is referred to as the partial sequence of the M. incognita Mi128 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 53.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 52 or comprising any fragment thereof. Based on the partial sequence of the Mi128 target gene, represented herein as SEQ ID NO 52, the full length sequence of Mi128 target gene is isolated from cDNA prepared from M. incognita total RNA. The “full length gene” is meant to encompass the coding region with or without the 5′ UTR and/or 3′ UTR. This is achieved via a 5′ RACE PCR where necessary, and/or a 3′ RACE PCR where necessary and the specific primers used herein are designed based on the sequence of SEQ ID NO 52.

Example 2.12
Assembly of a Sequence of the M. incognita Mi129 Gene Via Contig Building

To assemble a cDNA sequence from M. incognita Mi129 gene, several ESTs were used in the contig builder program TGICL (TIGR Gene Indices clustering tools). The ESTs used were BM774428, CK233774, CK983824, CK984252, CK985154, CK983765, CK984305, CN443466, CK984908, CN443767 and CN443135 originating from the public database Genbank. Before running the contig builder program, the full insert of the ESTs CK984305 and CN443135 were sequenced. These full insert sequences together with BM774428, CK233774, CK983824, CK984252, CK985154, CK983765, CN443466, CK984908 and CN443767were used to assemble the contig of Mi129.

The assembled cDNA contig is herein represented by SEQ ID NO 55 and is referred to as the partial sequence of the M. incognita Mi129 gene. The corresponding partial amino acid sequence is herein represented as SEQ ID NO 56.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 55 or comprising any fragment thereof.

Example 3
Identification of a Fragment of the M. incognita Target Genes Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi125, Mi116, Mi127, Mi128 and Mi129 with No Substantial Homology to Non-Target Organisms

The sequences of the Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi125, Mi116, Mi127, Mi128 and Mi129 genes were analyzed for regions that show no substantial homology to non-target organisms at the siRNA level. Such regions free of non-target organism sequences were named “freefrags” herein.

For this analysis, non-target organisms used were tomato (Lycopersicum esculentum), cotton (Gossypium hirsutum), potato (Solanum tuberosum), Arabidopsis thaliana, tobacco (Nicotiana tabacum) and human (Homo sapiens). Within the freefrags not any fragment of 21 contiguous nucleotides occur in the non-target organisms.

Other examples may be fragments of 22 contiguous nucleotides allowing one mismatch, or fragments of 24 contiguous nucleotides allowing three mismatches.

A sequence of Mi05 free of non-target organism sequences was selected and was used partially in further RNA interference experiments. Similarly, a sequence of Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi125, Mi116, Mi127, Mi128 and Mi129 free of non-target organism sequences was selected and was each used partially in further RNA interference experiments.

The Mi05 (SEQ ID NOs 5 and 6), Mi11 (SEQ ID NO 12), Mi38 (SEQ ID NO 18), Mi40 (SEQ ID NO 24), Mi101 (SEQ ID NO 28), Mi101 used for MiCC2 (SEQ ID NO 29), Mi109 (SEQ ID NO 33), Mi111 (SEQ ID NO 37), Mi116 (SEQ ID NO 41), Mi125a (SEQ ID NO 45), Mi125b (SEQ ID NO 46), Mi127 (SEQ ID NO 51), Mi128 (SEQ ID NO 54), Mi129 (SEQ ID NO 57), freefrags represented by their respective SEQ ID NO (in brackets) and their nucleotide sequence is given in Table 4. A person skilled in the art will recognize that more freefrags, of various lengths, may be identified in the Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi125, Mi116, Mi127, Mi128 and Mi129 cDNA sequences, and that the present invention also extends to these freefrags. The sequences of Mi125a (SEQ ID NO 45) and Mi125b (SEQ ID NO 46) are examples of freefrags of different length in the Mi125 target gene. These sequences are suitable for nematode control by RNA interference when expressed in a plant and taken up by a nematode feeding from the plant.

Example 4
Assembly of the M. incognita MiCC2 Sequence Via Concatenating

MiCC2 is a concatemer that comprises sequences of targets Mi127-Mi128-Mi101. To assemble a sequence for the M. incognita MiCC2, the following freefrag sequences as described in Example 3 were concatenated: Mi127freefrag (SEQ ID NO 51), Mi128freefrag (SEQ ID NO 54) and Mi101 freefrag for MiCC2 (SEQ ID NO 29).

The assembled MiCC2 sequence is herein represented by SEQ ID NO 59 and is referred to as the sense MiCC2freefrag. The sense MiCC2freefrag sequence was generated by chemical synthesis of DNA.

Accordingly, a further embodiment of the present invention includes an isolated polynucleotide comprising a sequence substantially identical to SEQ ID NO 59 or comprising any fragment thereof.

Example 5
Recombination of the M. incognita Target Genes Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125a, Mi125b, Mi129 and MiCC2 into a Destination Vector

Since the mechanism of RNA interference operates through dsRNA fragments, the Mi05 freefrag polynucleotide (see Example 3) was cloned in sense and antisense orientation, separated by an Arabidopsis-intron sequence, to form a dsRNA hairpin construct. Similarly the Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125a, Mi125b, Mi129 and MiCC2 freefrag polynucleotides (see Example 3) were each cloned in sense and antisense orientation, separated by the Arabidopsis-intron to form a dsRNA hairpin construct.

The sense sequences were isolated as a PCR product (with flanking AttB recombination sites) and ligated via BP clonase into a Gateway™ Entry clone (pDONR221) or were cloned according to Collier et al., (2005) The Plant Journal 43: 449-457. This Entry clone provides attL recombination sites flanking the cloned sense sequences.

A destination vector was made based on the binary pBIN19 plasmid, which can replicate in A. tumefaciens as well as in A. rhizogenes (RK2 broad host range origin of replication) and which comprises the RB and LB borders sequences of a Ti plasmid.

The nucleotide sequences from the Entry clone were recombined into this destination vector in sense and antisense orientation in an LR recombination reaction. The reaction products were analyzed and the correct hairpin cassettes of the format “sense-Arabidopsis:intron-antisense” were selected.

The hairpin cassette comprising the Mi05 freefrag is herein represented by SEQ ID NO 7. The hairpin cassette comprising the Mill freefrag (i.e. SEQ ID NO 12) is represented by SEQ ID NO 13. The hairpin cassette comprising the Mi38 freefrag (i.e. SEQ ID NO 18) is represented by SEQ ID NO 19. The hairpin cassette comprising the Mi40 freefrag (i.e. SEQ ID NO 24) is represented by SEQ ID NO 25. The hairpin cassette comprising the Mi101 freefrag (i.e. SEQ ID NO 28) is represented by SEQ ID NO 30. The hairpin cassette comprising the Mi109 freefrag (i.e. SEQ ID NO 33) is represented by SEQ ID NO 34. The hairpin cassette comprising the Mi111 freefrag (i.e. SEQ ID NO 37) is represented by SEQ ID NO 38. The hairpin cassette comprising the Mi116 freefrag (i.e. SEQ ID NO 41) is represented by SEQ ID NO 42. The hairpin cassette comprising the Mi125a freefrag (i.e. SEQ ID NO 45) is represented by SEQ ID NO 47; the Mi125b freefrag (i.e. SEQ ID NO 46) is represented by SEQ ID NO 48. The hairpin cassette comprising the Mi129 freefrag (i.e. SEQ ID NO 57) is represented by SEQ ID NO 58. The hairpin cassette comprising the MiCC2 freefrag is (i.e. SEQ ID NO 59) represented by SEQ ID NO 60. The sequences of the hairpin cassettes are given in Table 5.

Example 6
Recombinant Constructs to Produce Dsrna Derived from the M. incognita Ortholog Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi129 and MiCC2 in Transformed Tomato, Cotton, Tobacco, Potato, Rice and Arabidopsis Plants

In the example below the following abbreviations for nucleic acid fragments comprising the different components are used: “RB” and “LB” correspond to right and left borders of the T-DNA, P35S, pCvMv and pSBV2 are constitutive promoters originating from viruses for use in plants, pUbi U4 is a constitutive promoter originating from tobacco, pTobRB7, pPGM is a nematode feeding site specific promoter originating from tobacco and Arabidopsis, respectively, pLaccase and pAct7 are constitutive promoters originating from Arabidopsis, pSU is a constitutive promoter for use in plants and is described in U.S. Publication 2003/0101478, pNOS corresponds to the nopaline synthase promoter, tNOS corresponds to the nopaline synthase terminator, t35S corresponds to the 35S terminator.

The hairpin cassettes as described in Example 5 were embedded in a binary vector, suitable for transformation into A. tumefaciens as well as in A. rhizogenes which can be used for transformation of the hairpin into any plant species such as cotton, potato, tobacco, Arabidopsis, tomato or rice. For driving the expression of the hairpin in cotton, potato, tobacco, Arabidopsis and tomato plants, different promoters were cloned preceding the hairpin cassette. Such promoters include p35S, pCvMv, pTobRB7, pLaccase, pUbi U4, pAct7, pPGM and pSBV2. Within the RB and LB borders also a selectable marker cassette of the format pNOS-selectable marker (e.g. nptII)-tNOS and a scorable marker cassette of the format promoter pSU—scorable marker (e.g. gfp)—tNOS, were cloned.

The plant expression vectors comprising the hairpin of Mi05, Mi11, Mi38, Mi40, Mi101, Mi125, Mi129, Mi109, Mi111, Mi116 and MiCC2 fragments were transformed either into Agrobacterium rhizogenes for generation of ex vitro composite plants (see example 7) or hairy roots (example 8) or into Agrobacterium tumefaciens for the regeneration of whole transgenic plants (see example 9).

Example 7
Bioassay of M. incognita Infection on Ex Vitro Composite Tomato Plants

Generation of Ex Vitro Composite Plants

The resulting binary constructs as described in example 6 were introduced into A. rhizogenes strain NCPPB 2659 (Combard et al., (1987) Plasmid 18: 70-75) via electroporation. A. rhizogenes strain NCPPB 2659 harboring the described binary vector was grown in Luria Bertani broth in a flask at 28° C. on a rotary shaker at 200 rpm overnight. Cells were collected by centrifugation at 3000 g for 10 min, and resuspended in ¼×Murashige and Skoog basal medium (pH 5.8) (Sigma-Aldrich, St Louis, Mo., USA) to an OD₆₀₀=0.3. Sterilized Fibrgro® cubes (Hummed International, Earth City, Mo., USA) were saturated with resuspended cells via pipetting, typically between 4 and 7 ml. Apical stem sections, excised from greenhouse-grown Lycopersicon esculentum cv. Marmande plants, were inserted into inoculated Fibrgro® cubes, placed in open Petri dishes within plant growth trays and covered with clear plastic domes and incubated overnight at room temperature. The trays were subsequently opened and the cubes were allowed to dry until the plant material was fully wilted. Cubes were saturated with deionized water and domes were returned to closed position. Cubes were checked periodically and watered when necessary for the remainder of the induction period. Nascent composite plants were incubated at room temperature in normal light conditions until roots emerged from the teratoma. Roots were analyzed for the presence of GFP using a microscope outfitted with a FITC filter (Collier et al., (2005) The Plant Journal 43: 449-457).

M. incognita Bioassay

Composite tomato plants were transferred to sand 36 days after induction; subsequently, 5 days after planting, each composite plant was infested with 1,500 M. incognita eggs (Collier et al., (2005) The Plant Journal 43: 449-457). Between 5 and 16 plant root systems, for both empty vector control as well as the RNAi Mi constructs, were assayed for M. incognita infection level with a modified egg collection protocol (Hussey and Barker (1973) Plant Disease Reports 57: 1025-1028). Eight weeks upon inoculation sand was removed from the entire root ball by a 1-min wash in Milli-Q water, followed by a 5-min wash in 5% bleach. Three samples of 100 μl were delivered to a Petri dish and counted under a dissecting microscope and number of nematode eggs (FIG. 1) was reported as an average.

Example 8
Bioassay of M. incognita Infection on Transformed Hairy Roots of Cotton and Tomato

Generation of Transformed Hairy Roots

Cotton (Gossypium hirsutum) or tomato (e.g. Lycopersicum esculentum cv. Marmande) cotyledons were transformed with the Agrobacterium rhizogenes strain comprising the Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi129 and MiCC2 hairpin (see Example 6), on an agar plate according to the protocols described in Plovie et al. (Nematology 2003, vol. 5(6): 831-841). The transformed hairy roots were subsequently tested for nematode resistance.

M. incognita Bioassay

The necessary number of independent transformed lines (e.g. 15) and replicates per line (e.g. 5) were inoculated with M. incognita J2 larvae. Approximately 1,000 eggs of axenically grown root-knot nematode M. incognita species were added to the plate. The plates were incubated in the dark at a constant temperature of 20° C. Tomato roots were harvested and weighed at eight weeks post infection. Eggs were harvested from the roots using a 5% bleach solution. Eggs were separated from root material using a 50% sucrose gradient and centrifugation (130g; 750 rpm). Eggs were removed from the gradient interface and counted. All nematode egg counts were normalized to appropriate control plants being lines transformed with a binary vector containing a gus hairpin (FIG. 2).

Example 9
Bioassay of M. incognita Infection on Transgenic Cotton, Potato, Tobacco, Rice, Arabidopsis and Tomato Plants Expressing a Fragment of the Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi129 and MiCC2 Genes

Generation of Transgenic Plants

Cotton, potato, tobacco, rice or tomato plant tissues are transformed with the Agrobacterium tumefaciens strain (e.g. C58) comprising the Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi129 and MiCC2 hairpin (see Example 5), and regenerated into whole plants via protocols described for example in “Transgenic plants, Methods and Protocols. (2005) Methods in Molecular Biology, Volume 286, edited by Leandro Peña, Humana Press, Totowa N.J.”

Arabidopsis thaliana plants were transformed using the floral dip method (Clough and Bent (1998) Plant Journal 16:735-743). Aerial parts of the plants were incubated for a few seconds in a solution containing 5% sucrose, resuspended A. tumefaciens cells from an overnight culture and 0.03% of the surfactant Silwet L-77. After inoculation plants were covered for 16 hours with a transparent plastic to maintain humidity. To increase the transformation efficiency, the procedure was repeated after one week. Watering was stopped as seeds become mature and dry seeds are harvested and cold treated for two days. After sterilization seeds were plated on an antibiotic containing growth medium for selection of transformed plants. The selected plants are transferred to soil for optimal seed production.

M. incognita Bioassay

Transgenic plants were germinated on growth medium supplemented with the appropriate selectable agent and one week old plants are transferred to 10×10 cm²Petri dishes, containing Gamborg's B5 medium. For each transgenic line 10 replicate plates were made and on each plate ten plants are lined up. The Petri dishes were placed slightly tilted to promote unidirectional root growth. One week later, the roots of each plant were inoculated with 100 freshly hatched M. incognita second stage juveniles. The whole procedure was done in sterile conditions under a binocular microscope. Seven days after inoculation the number of root galls was determined and six weeks after inoculation adult females were counted and compared with the appropriate control lines (Examples are given in FIGS. 3, 4 & 5). Mature females were collected and put to hatch to check the progeny.

Example 10
Control of Nematodes in Transgenic Cotton, Potato, Tobacco, Arabidopsis, Rice or Tomato, Based on the Silencing of Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi129 and MiCC2

Small plastic pots (e.g. 3 inch) or plastic seedling trays with wells of 60 mL capacity are filled with about 40 ml of soil per well, the soil originating from a plot heavily infested with M. incognita. Part of this soil is sterilized by autoclaving and is used as negative control. 5 ml of water per well is used for irrigation.

Tomato, potato, tobacco, Arabidopsis, rice or cotton seeds of the transgenic plants generated as described in Example 9 are sown in the wells, at about 2 cm deep. A light cover of vermiculite on the surface is used to avoid dehydration. Plants are irrigated by micro-nebulization 3-4 times per week at a rate of 3 L/m², weeded by hand and treated with fertilizers, fungicide and insecticides when needed.

The phenotype of the plants is monitored and the efficacy of the dsRNA complementary to Mi05, Mi11, Mi38, Mi40, Mi101, Mi109, Mi111, Mi116, Mi125, Mi129 and MiCC2 gene or gene-fragment is measured as follows. Plants are grown to reach good development of the roots, which corresponds to plant grown to about 10 cm high at about one month after emergence. After removal of the soil from the roots by rinsing with water, the roots are visually inspected and root galls are counted. The number of galls and the corresponding degree of damage is scored on the 0 to 5 infestation scale described by Lambertini (1971), Tobacco, 738: 5-10). The infestation scale is as follows:

0 is “NO ATTACK: No galls on root system (healthy plant), free from galls”;

1 is “LOW ATTACK: 1-5 small galls located in a region of root system”;

2 is “MODERATE ATTACK: 6-20 small galls, located on entire root system”;

3 is “HIGH ATTACK: More than 20 galls, located on entire root system;”

4 is “VERY HIGH ATTACK: Root system reduced and severely deformed by few and big galls, with some galls grown together”; and

5 is “EXTREMELY HEAVY ATTACK: Root system completely reduced and deformed by big gall (plant and roots dead, rotten root).”

TABLE 1

Function

(identified by

Target

applicant)

ID
Identifier
Function (based on Wormbase)
(NA = not available)

Mi05
LiN-26
encodes a putative C2H2 zinc finger-containing protein required
Cell fate

for proper differentiation of ectodermal and mesodermal

epithelial cells

Mi11
cdl-1
histone stem-loop binding protein
Apoptosis

Mi38
rpt-1
26S proteasome regulatory complex, ATPase RPT1
NA

Mi40
phi-7
mRNA splicing protein CDC5 (Myb superfamily)
NA

Mi101
SKP-1
mRNA splicing factor/probable chromatin binding snw family
NA

nuclear protein

Mi109
rpl-19
60s ribosomal protein L19
NA

Mi111
RPT-2
26S proteasome regulatory complex, ATPase RPT2
NA

Mi116
PAS-4
20S proteasome, regulatory subunit alpha type PSMA7/PRE6
NA

Mi125
rps-9
Ribosomal protein S4
NA

Mi127
hsp-1
Molecular chaperones HSP70/HSC70, HSP70 superfamily
NA

Mi128
rpl-36
60S ribosomal protein L36
NA

Mi129
tbb-2
Beta tubulin
NA

TABLE 2

Target
Primer
Primer
cDNA Sequence (Contig)

ID
Forward
Reverse
sense strand 5′ → 3′

Mi05
SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 1

CCAACAACAC
TCGCAGATTC
CTCCGTCACGTTATTATGATCCATTCGGACAGCAAGCCCTACCAGTGCTTCGACTGTGAT

TGAATTCTAC
CATTCAGCTC
TTTATCGGGATCAAGAGCAATGTGGTCAGTCATGCGAGACAGAGCGGACACCGGAATGAC

AGGAWSNMGN
KCATYTCRTC
GATGCCCTGGACATTACCACTGATGACATGAGGGCCGAATGGAATACCATGCTCCATGCT

TGGAA

TGTTTCCCTGACTATGTTAAGGCAAAGAGCCGTGGATGGCAGCCCGAAACCGAGGTCAAA

GACGAGCCTCTGGAATCCAACAACGAACAGGACACTACCGTCAAAATTGAAGTCTAATCA

TCTTACTCAAAAGAACATATCTCAAGAGTTGCTTTCCTTTAGTAGATTTTCTATATAACT

GGGTCTCTGTCTCTCTCCCGCCTTGCCCCAACTGTGATCCCCCTACCGTCTTGCCTTTTT

TCTTCCAACGAGCCCCGGGTGTATCTTCTCAACCAGCCACAAAAGTATTTCCACTTTGAT

ATCGCATATTAGTGCTATACACCGAGCATACAAACAACCTATACATAATAAAAGTTATTA

TTTCAAAAAAAAAAAAAAAAAAAAAAAA

Mi11
SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO:8

CCATTGCTCG
GAACGAGTAA
CCATTGCTCGTCGTAAAAAGGAAATCAATAAAATGAAGGATCGTCCAACTTATGAGCGTT

TCGTAAAAAG
CAGGGCCATT
ACATAGAACAAGTTCCAAAGGATGACAGGAAGAAAGGTGTTCATCCTAAAACACCTAACA

G
AGG
AATTTCTCAATTGGAGTAGACGATCATGGGACAAACAGATCAAATTATGGAAACTTGCAA

TATATGAATGGGCAGGGGAATCGCCATCTTCAAGTGTTVACGGCTCTTATTGTTCATCGG

AATGTAGTGACGAGGATCGAGCGCAGTCTTTGGAACGGCCGCATTCAGCGGAGCCTTCAA

CATCAAAAAAGATTAAAATTGAGAGGGACGTTGAACCGAAGAATAGGATTGTTAATATTC

CACCATCAGCCGACACTATGGCTTCATTGTTAGGTCACTTTGATTTGGACACGCTTCGTG

AAGCACCAGCACCTATTATGAACACAACTGGAGATGAATCAACATTGAAAGATATTCCTA

AACCTAATGGCCCTGTTACTCGTTC

Mi38
SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 14

GTGTCATATA
CGAGATGGTT
CGAGATGGTTTCGTAAATTATTCAGATGGCAAAAACAAAGAAGGCTTGTATTATTTTCTT

ACTTGGAGTT
TCGTAAATTA
TGATGAAGTTGATGCTATTGGCGGAGCTCGTTTTGATGATGGAATGGGGGGAGATAATGA

GAACTAAACT
TTCAGATGG
AGTTCAACGGACAATGCTCGAATTGATCAATCAGCTGGATGGATTTGACTCGCGTGGTGC

TTGC

AATCAAAGTTTTAATGGCCACAAATCGTCCTGATACTTTGGATCCTGCACTTGTACGGCC

AGGACGTATTGATCGTCGTATCGAATTCGCCGTGCCTGATTTGGAAGCTCGTGCAAACAT

TCTAAAGATTCATACAAAGCGGATGAGCGTCGATCGGAATATVCGTTATGAATTGATATC

GCGTCTTTGCCCTAATACAACAGGAGCTGATATTCGCTCTGTCTGTACTGAAGCCGGAAT

GTTTGCATTACGTGCTCGTCGTAAAGCAATAACCGAAAAGGATTTTATTGACGCTGTTCA

GAAAGTGGTGAAAGGATATGCAAAGTTTAGTTCAACTCCAAGTTATATGACAC

Mi40
SEQ ID NO: 22
SEQ ID NO: 23
SEQ ID NO: 20

CTGGAAGAAT
GCTGGAACTT
CTGGAAGAATACAGATTGCGAAATTCTTAAAGCTGCTATTATGAAATATGGAAAAAATCA

ACAGATTGCG
GCTTTTCAAA
ATGGAGCCGAATTGCCTCATTACTTCATCGAAAATCTGCGAAACAATGTAAAGCCCGTTG

AAATTCTTAA
AGGTATTC
GTATGAATGGCTTGATCCTCGAATTAAAAAGACGGAATGGTCCAGAGAAGAGGACGAAAA

AG

ACTTCTCCATTTGGCCAAAATTATGCCTACTCAATGGCGTACAATTGCTCCTATTGTTGG

AAGAACTGCTGCTCAGTGTTTGGAAAGATATGAATATCTTCTTGACGAAGCTCAACGTCG

TGCTGAAGGTATTGAAGAGGATGATTCATTAAAAGAGGCTAAGAAGTTAAAGCCAGGCGA

TGTTGATCCAACGCCAGAAACTAAACCGGCTAGGCCTGATCCTATTGATATGGATGAAGA

TGAATTGGAAATGTTATCAGAAGCCCGAGCTCGTTTGGCCAATACACAAGGAAAGAAAGC

AAAACGCAAAGCTCGTGAAAAGCAATTAGGCGAGGCTCGAAGACTTGCTTCTCTACAAAA

ACGAAGAGAACTTCGGGCTGCTGGAATAATTATTGGATCTGGTTGGGCTTATAAAATGAA

GAAGCATCGTCTAATGGATTATAATAATGGAATACCTTTTGAAAAGCAAGTTCCAGC

Mi101

SEQ ID NO: 26

GGTTATTACCCATGCTTGAGACAAAAGCACAATATTCTGAGAAATTTTAAACAACAACAA

TGCATTTGTCTTTAGCTAAAGTTGTTGCTATTAAAGAGCCGCCTCTATATGATCGTCGAC

AAGGATTTGTGCCCCGAACGCAAGATGATTTTGGTGATGGTGGGGCTTTTCCAGAAATAC

ATATTGCTCAGTTTCCAATCGGTATGGGCGCCGATAAGCCCGGCACTGGGGCAAAGAACA

CAGTTGCGCTTCAGTTTGACTCTGAGGGCAAACTTCGATTTGATGAGCTTACACGAATTG

GGCATGGAAAAGACAAGATTGTTCATTCCCGCCTTTCTGATATGAAATCCAACCATATTG

ATGATGAAGACGAAAGTTTCAAAAAACCAACAGATGAAGAAATACATGAAACAGCCGAAG

CAACTCGTGTTTCCTTAGAGAAAATTACAGCTGTTAAGATCGCCGCATCATTACCAGTCC

AGCATGCAAAGAAAACAGCGCCTGCACAATATATTCGTTACACTCCTAGCCAGCAGGCTG

GTGGTTTTCATACAAGTGGAGCACAACAAAGAATTATTCGTTTGGTTGAAGAGCAGAAGG

ATCCAATGGAGCCGCCACGTTTTCAAATAAACCAGAAA

Mi109

SEQ ID NO: 31

GCGTTTGGCGGCCTCGCTTCAAAAGTGCGGCAAAGGCAAAATTTGGTTGGATCCTAATGA

GGTCAACGTGATTTCCAACGCAAATTCTCGTCAGAACGTTCGTCGTTTGGTCAAAGATGG

TCTTATAATTCGTAAGCCTGGCAAAGTTCATTCGCGTTTCCGAGCTCGTGAACGTTTGGA

AGCACGCAGAAAGGGTCGTCATATGGGTTTTGGAAAACGACGCGGAACTCGTAACGCTCG

AATGCCGGAGAAGGTCTTATGGATGCGTTTTTTTGTTGTACTCCGTCATCTTTTAAAGCG

CTATCGAGCTGCGAAGAAGATTGACAAGCATCTTTACCACGAGCTTTACTTGAAGGCTAA

AGGAAACGCATTTAAGAATAAGAGAAATTTGATGGAATATATCTTCAAAAAGAAGACTGA

GAACCAGCGTTCTAAACAACTTGCTGAACAGGCTGAGGCTCGCCGAAACAAGAACAAGGA

AACGAGAAAGAGGCGTGAGGAGCGTTTGATTTTGAAACGTAAGGAATTGCTCCACAAACT

TTCTGAGAGCGAAAAGCAGAAGACTCCACAGCTTCAAGAAGAGGAAGCGAAGGAAGCTTC

GCCTTCTCCACCACC

Mi111

SEQ ID NO: 35

GGGAAATCAGCAATTCCAACAATTTACCCCAGGTTCAGGAGATAAGCAAGGAGAAGGAGA

CAAGAAGCGTCGTTTTGGGCCTCCAATCCCTACACGCTTTGGGAAGCGAAAGAAGGGAAG

TAAAGGACCTGAAGCTAGCAATAAAATGCCTAATGTGACACCAGTAACTCGTTGCAAATT

GCGTCTTCTTAAATATGAACGGATTAAAGATTATTTGTTAATGGAAGAAGAATTTATTCG

AAATCAGGAAAGACATAAACCTCAAGAGGAAAGGCAGGAAGAAGAACGGACAAAAGTAGA

TGAAATGAGAGGGTCACCAATGGCTGTGGGTACATTGGAGGAGATTATTGACGATCAACA

CGCAGTCGTTTCTACGAATGTTGGCAGCGAACATTATGTTAACATTCTTTCATTTGTGGA

CAAGGAACAATTGGAGCCAGGCTGTGCAGTTCTTCTAAATCACAAAACACACGCAGTTGT

TGGTGTTTTGGCTGATGACACGGATCCAATGGTTTCTGTAATGAAATTGGAAAAGGCTCC

AACTGAAACGTACGCTGATGTTGGCGGTTTGGAACAACAAATTCAAGAAATTAAGGAGGC

AGTGGAATTGCCACTTACACATCCAGAGTACTACGAAGAAATTGGAATCAAGCCTC

M1116

SEQ ID NO: 39

CAGCCAGTGGTCTTTCCCCTTCAATCACACCAAATTCCTTGATATTTTCTTTAACATTTT

TTTCAAACTGTGGGTCTCCAAGCAATAAGTTTTCAGGGTATAGGTCAGGTGGTGACTCAT

TTTTCAATATTTGATAGTGGTTTGTATTTTTGACTTTACTTAACTTGTTTACTGAAATCT

GTTCCTGTTCTTCAAGATGACCGCACAATTCGAAAGATACACAAATTGGATGATCACGTA

ATGGCTGCTTTTGCTGGTTTGAGTGCAGATGCTCGTGTTTTGATTGACCGTGCCAGAGTT

GCTTGTGAAAATTACAAATTGACTTTGGAGGATCCCGTCACACTTGAGCACATCTCCCGC

ACTATTGCGGACTTGAAACATGAATTTACGCAAACTACAGGTCGTCGTCCTTTTGGTGTC

TCTTTACTTGTTGGCGGTTTTGATCCCGATGGAACTCCTCATCTTTTTATTACTGAACCA

TCTGGTGTGTATTACGAATTACTTGCTGGCTCAATCGGCCGGAATGAGAAGGTTGTCAAA

GAATATTTGGAGGAGAATTATAATGATGGCAATGTTTCAAACGAAAACTTGGTCTTGAAG

TTGGTGGTCAAAGCTCTTGTCCCAGTTGTGCAAACTGGAGCGCGTAATATGGAGATATCG

GTGTTGAAGCGTGATAATGAGGGGAATATTGTTCAACGTGTTCTACTCACTGAGGAGTTG

GAGGAAATTTTACGTCAAATTGAAGCTGAAACTATGGAGACTGTTTAGACTTTCTTTGAT

TTTTTTAAAAATTCAGTCGAACCTCATTTTTAGATACTCTTGGGGCTGAAAAATCGTATC

TTATTTGAGGTCTTATAATGAGGTCCAACTATACATACATTTGCTATTTTTGTTAATAAA

TGTTTCGCTATTCTG

M1125

SEQ ID NO: 43

GGTTTAATTACTCCAGTTTGAGCTTAAAAAGTAAAACAAGATGCCTCGCATAAAGCATGT

ATTTTCTAAAGTTTATGACGTGCCTCGTCGTCCATTCGAGAAGGAGCGTTTAGATCAGGA

ATTAAAATTAATTGGCGAGTTTGGTTTACGAAATAAACGTGAAGTTTGGCGTGTCAAATT

AACTTTGGCAAAGATTCGTAAAGCTGCTCGTGAACTTTTAACATTGGAAGACAAGGATCC

TCGACGCTTATTTGAAGGAAATGCTCTTCTTCGTCGTTTGGTTCGTATTGGAGTTCTTTT

GGAGGATAAGATGAAATTGGACTATGTTCTTGGTTTGCGTGTTGAAGACTTTTTGGAGCG

TCGTTTGCAGACGCAAGTTTTCAAATTGGGATTGGCTAAATCAATTCACCATGCTCGTGT

TCTTATTCGTCAAAAGCATATTCGAATTCGTCGACAAGTAGTCAATATTCCATCATTCAT

TGTTCGTCTCGACTCTCAAAAGCACATTGACTTCTCCACAAAATCTTATCTTGCTGGTGG

ACCTCCTGGACGTGTCAAGAGAAAGAATTTGAAGAAGGCTGCTTCGGGAGGTGGAGGAGG

AGGCGGTGACGATGAAGAGGATTGAATAATATCTAAAGGTTTATAAAACGTTGAAAGTTA

ATTCTGTTAAGTTGTTAAAAGATTTTGGATTTGAAAAAATGTTTTTGTTGA

Mi127

SEQ ID NO: 49

GGTTTAATTACCCAAGTTTGAGCTATCATTTTAAAAAAATCTTTTAAAAATGTCTAAAGC

TAACGCTGTCGGAATTGATCTGGGAACGACTTATTTATGTGTCGGTGTATTCCAGCATGG

AAAAGTCGAGATCATTGCCAATGATCAAGGAAATCGCACGACTCCATCTTATGTGGCTTT

TACAGACACGGAACGTCTGATTGGTGATGCTGCGAAAAATCAAGTGGCAATGAACCCGTC

GAATACTGTTTTTGACGCAAAGCGCTTGATTGGACGCAAATTTGACGATCCAGCTGTTCA

ATCTGACATGAAGCATTGGCCGTTCAAGGTTATTCAAGGAGAAGGTGCTCGTCCAAAAAT

CCAAGTGGAGGTTAAGGGTGAAATGAAGGCGTTCTTCCCTGAGGAAGTCTCCGCCATGGT

TTTGACTAAAATGAAGGAAACGGCTGAGGCATTTTTGGGCCAAACCGTTAAGGATGCTGT

CATCACTGTGCCTGCGTACTTTAATGACAGTCAACGCCAAGCCACAAAGGATGCTGGAAC

GATTTCTGGACTTAATGTTTTGCGTATCATCAACGAGCCAACTGCTGCGGCTATTGCTTA

TGGTTTGGATAAGAAAGGCCAAGGCGAGCGCAACGTCTTGATTTTTGACTTGGGCGGTGG

AACTTTTGATGTGTCTATTTTGACTATTGAAGATGGTATTTTTGAGGT

Mi128

SEQ ID NO: 52

GGTTTAATTACCTACGTTTGAGAAATTTAAAGAATGACAAAGTTACAACATCCAGCTGTT

GAGGGTCTTGCTGTTGGTTTGGATCGTGGCCATCCTGTGACTAAAAATGTTCGCAAGCCA

CGTCAATCTCGTAAAAAAGGCCGCATTACGAAGAAGACTCGAATTGTTCGTGAAGTTGTT

CGCGAGGTTGTTGGTTTTTCACCTTATGAGCGTCGAACTATGGAATTGCTTCGTATCAGT

AAGGACAAGAAGGCTTTGAAGTTTTTGAAGAAGCGAATTGGACAACATGTCCGTGCAAAG

AGAAAGCGTGACGAAATCCAACGAATACTCGTTGAGATGAGAAAGCATCATAAGTGAGAA

AATATAAGGGAAGGAAAACATTCAATCACAGTTTTAATTGTTAAAAATAATGTTTTATGC

GATTGAATAAATTTGTTAGAATTCAAACAAAA

Mi129

SEQ ID NO: 55

AGGTTTAATTACCCAAGTTTGAGTTTTACTATTVAATTTATCTAACAAAAGATCTCAAAA

AATGCGTGAAATCGTCCATATCCAGGCTGGACAATGTGGTAACCAAATTGGTTCCAAGTT

CTGGGAAGTCATTTCGGATGAACACGGAATTCAACCTGATGGATCATACAAGGGAGAATC

TGACTTGCAATTGGAACGAATCAATGTTTACTACAATGAGGCTCATGGTGGAAAGTATGT

CCCAAGAGCTGTTCTTGTTGACTTGGAGCCGGGAACCATGGACTCGATTAGAGCTGGGCC

ATACGGAGAACTCTTCCGTCCTGACAACTTTGTCTTTGGCCAGAGCGGTGCAGGAAACAA

TTGGGCCAAAGGACATTACACTGAGGGAGCGGAGTTGGTCGACAATGTTTTGGATGTTGT

TCGAAAAGAGGCTGAGGGTTGTGATTGTCTTCAAGGCTTTCAATTGACTCACTCGCTTGG

TGGTGGAACTGGCTCTGGAATGGGAACTTTGCTCATTTCAAAGATTCGTGAGGAGTATCC

GGACCGCATTATGTCTTCCTTCTCTGTTGTTCCGTCGCCTAAGGTCTCTGACACGGTCGT

TGAGCCCTACAACGCAACTCTTTCTGTTCATCAACTTGTTGAGAACACCGACGAGACTTA

CTGCATCGACAACGAGGCTTTGTACGACATCTGCTTCCGAACTCTGAAGCTTCAAAATCC

AACATATGGAGATTTGAATCATCTCGTATCCGTAACCATGTCTGGAGTGACTACTTGTCT

TCGCTTCCCAGGCCAATTGAACGCTGATCTTCGCAAATTGGCTGTCAACATGGTTCCATT

CCCACGTCTTCACTTTTTCATGCCTGGTTTCGCTCCACTTTCGGCTAAGGGCGCAGCTGC

CTACCAGGCCCAAAATGTTGCTGAATTGACCAAGCAAATGTTTGATGCCAAGAATATGAT

GGCTGCTTGTGATCCACGCCATGGACGCTATTTAACTGTTGCCGCTATCTTCCGTGGACG

TATGTCAATGCGTGAAGTAGACGATCAAATGATGTCGGTTCAAAACAAGAATTCGTCATA

CTTTGTTGAATGGATTCCAAACAACGTTAAGACTGCGGTTTGTGACATTCCTCCGCGTGG

TCTCAAGATGTCTGCGACCTTCATTGGCAACTCCACGGCCATTCAAGAACTGTTCAAACG

CATCTCTGAGCAATTCACGGCCATGTTCCGTCGCAAGGCTTTCCTTCACTGGTACACTGG

CGAAGGTATGGACGAGATGGAATTCACTGAAGCCGAGTCCAACATGAACGACTTGATTTC

TACCAATCAAGCAACTGTTGAACAAGAATAAAAAAGAGAGAAGTTTTTGTTGAATACCAG

CAATATCAAGATGCGACTGTTGAAGACGAAGGGGAATTTGAAGGTGAAGATTTGAATTAC

CACGTATTTTTTTAATTTTATTTTGCTTTTAACCGCATATTTCCGAAATAATTCCTCGCTT

AACTTTATTTATGCCAAAAAA

TABLE 3

Target
Amino acid sequence of cDNA sequence

ID
(Translation of Contig) sense strand 5′ → 3′

Mi05
SEQ ID NO: 2

LRHVIMIHSDSKPYQCFDCDFIGIKSNWSHARQSGHRNDDALDI

TTDDMRAEWNTMLHACFPDYVKAKSRGWQPETEVKDEPLESNNE

QDTTVKIEV

Mi11
SEQ ID NO: 9

IARRKKEINKMKDRPTYERYIEQVPKDDRKKGVHPKTPNKFLNW

SRRSWDKQIKLWKLAIYEWAGESPSSSVYGSYCSSECSDEDRAQ

SLERPHSAEPSTSKKIKIERDVEPKNRIVNIPPSADTMASLLGH

FDLDTLREAPAPIMNTTGDESTLKDIPKPNGPVTR

Mi38
SEQ ID NO: 15 (partial translation)

IIQMAKTKKACIIFFDEVDAIGGARFDDGMGGDNEVQRTMLELI

NQLDGFDSRGAIKVLMATNRPDTLDPALVRPGRIDRRIEFAVPD

LEARANILKIHTKRMSVDRNIRYELISRLCPNTTGADIRSVCTE

AGMFALRARRKAITEKDFIDAVQKVVKGYAKFSSTPSYMT

Mi40
SEQ ID NO: 21

WKNTDCEILKAAIMKYGKNQWSRIASLLHRKSAKQCKARWYEWL

DPRIKKTEWSREEDEKLLHLAKIMPTQWRTIAPIVGRTAAQCLE

RYEYLLDEAQRRAEGIEEDDSLKEAKKLKPGDVDPTPETKPARP

DPIDMDEDELEMLSEARARLANTQGKKAKRKAREKQLGEARRLA

SLQKRRELRAAGIIIGSGWAYKMKKHRLMDYNNGIPFEKQVP

Mi101
SEQ ID NO: 27 (partial translation)

TTTMHLSLAKVVAIKEPPLYDRRQGFVPRTQDDFGDGGAFPEIH

IAQFPIGMGADKPGTGAKNTVALQFDSEGKLRFDELTRIGHGKD

KIVHSRLSDMKSNHIDDEDESFKKPTDEEIHETAEATRVSLEKI

TAVKIAASLPVQHAKKTAPAQYIRYTPSQQAGGFHTSGAQQRII

RLVEEQKDPMEPPRFQINQK

Mi109
SEQ ID NO: 32

RLAASLQKCGKGKIWLDPNEVNVISNANSRQNVRRLVKDGLIIR

KPGKVHSRFRARERLEARRKGRHMGFGKRRGTRNARMPEKVLWM

RFFVVLRHLLKRYRAAKKIDKHLYHELYLKAKGNAFKNKRNLME

YIFKKKTENQRSKQLAEQAEARRNKNKETRKRREERLILKRKEL

LHKLSESEKQKTPQLQEEEAKEASPSPP

Mi111
SEQ ID NO: 36

GNQQFQQFTPGSGDKQGEGDKKRRFGPPIPTRFGKRKKGSKGPE

ASNKMPNVTPVTRCKLRLLKYERIKDYLLMEEEFIRNQERHKPQ

EERQEEERTKVDEMRGSPMAVGTLEEIIDDQHAWSTNVGSEHYV

NILSFVDKEQLEPGCAVLLNHKTHAVVGVLADDTDPMVSVMKLE

KAPTETYADVGGLEQQIQEIKEAVELPLTHPEYYEEIGIKP

Mi116
SEQ ID NO: 40 (partial translation)

KSVPVLQDDRTIRKIHKLDDHVMAAFAGLSADARVLIDRARVAC

ENYKLTLEDPVTLEHISRTIADLKHEFTQTTGRRPFGVSLLVGG

FDPDGTPHLFITEPSGVYYELLAGSIGRNEKWKEYLEENYNDGN

VSNENLVLKLVVKALVPVVQTGARNMEISVLKRDNEGNIVQRVL

LTEELEEILRQIEAE

Mi125
SEQ ID NO: 44

MPRIKHVFSKVYDVPRRPFEKERLDQELKLIGEFGLRNKREVWR

VKLTLAKIRKAARELLTLEDKDPRRLFEGNALLRRLVRIGVLLE

DKMKLDYVLGLRVEDFLERRLQTQVFKLGLAKSIHHARVLIRQK

HIRIRRQVVNIPSFIVRLDSQKHIDFSTKSYLAGGPPGRVKRKN

LKKAASGGGGGGGDDEED

Mi127
SEQ ID NO: 50

MSKANAVGIDLGTTYLCVGVFQHGKVEIIANDQGNRTTPSYVAF

TDTERLIGDAAKNQVAMNPSNTVFDAKRLIGRKFDDPAVQSDMK

HSPFKVIQGEGARPKIQVEVKGEMKAFFPEEVSAMVLTKMKETA

EAFLGQTVKDAVITVPAYFNDSQRQATKDAGTISGLNVLRIINE

PTAAAIAYGLDKKGQGERNVLIFDLGGGTFDVSILTIEDGIFE

Mi128
SEQ ID NO: 53

MTKLQHPAVEGLAVGLDRGHPVTKNVRKPRQSRKKGRITKKTRI

VREVVREWGFSPYERRTMELLRISKDKKALKFLKKRIGQHVRAK

RKRDEIQRILVEMRKHHK

Mi129
SEQ ID NO: 56

MREIVHIQAGQCGNQIGSKFWEVISDEHGIQPDGSYKGESDLQL

ERINVYYNEAHGGKYVPRAVLVDLEPGTMDSIRAGPYGELFRPD

NFVFGQSGAGNNWAKGHYTEGAELVDNVLDWRKEAEGCDCLQGF

QLTHSLGGGTGSGMGTLLISKIREEYPDRIMSSFSWSPKVSDTV

VEPYNATLSVHQLVENTDETYCIDNEALYDICFRTLKLQNPTYG

DLNHLVSVTMSGVTTCLRFPGQLNADLRKLAVNMVPFPRLHFFM

PGFAPLSAKGAAAYQAQNVAELTKQMFDAKNMMAACDPRHGRYL

TVAAIFRGRMSMREVDDQMMSVQNKNSSYFVEWIPNNVKTAVCD

IPPRGLKMSATFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYT

GEGMDEMEFTEAESNMNDLISEYQQYQDATVEDEGEFEGEDTNQ

ATVEQE

TABLE 4

Target
DNA Sequence of fragments (sense

ID
freefrag) and concatemer constructs

Mi05
SEQ ID NO: 5

AGGCAAAGAGCCGTGGATGGCAGCCCGAAACCGAGGTCAAAGAC

GAGCCTCTGGAATCCAACAACGAACAGGACACTACCGTCAAAAT

TGAAGTCTAATCATCTTACTCAAAAGAACATATCTCAAGAGTTG

CTTTCCTTTAGTAGATTTTCTATATAACTGGGTCTCTGTCTCTC

TCCCGCCTTGCCCCAACTGTGATCCCCCTACCGTCTTGCCTTTT

TTCTTCCAACGAGCCCCGGGTGTATCTTCTCAACCAGCCACAAA

AGTATTTCCACTTTGATATCGCATATTAGTGCTATACACCGAGC

ATACAAACAACCTATACATAATAAAA

Mi05
SEQ ID NO: 6

TACCGGCTCAAGGCAAAGAGCCGTGGATGGCAGCCCGAAACCGA

GGTCAAAGACGAGCCTCTGGAATCCAACAACGAACAGGACACTA

CCGTCAAAATTA

Mi11
SEQ ID NO: 12

TTTGATCTGTTTGTCCCATGATCGTCTACTCCAATTGAGAAATT

TGTTAGGTGTTTTAGGATGAACACCTTTCTTCCTGTCATCCTTT

GGAACTTGTTCTATGTAACGCTCATAAGTTGGACGATCCTTCAT

TTTATTGATTTCCTTTTTACGACGAGCAATGG

Mi38
SEQ ID NO: 18

CGATCGACGCTCATCCGCTTTGTATGAATCTTTAGAATGTTTGC

ACGAGCTTCCAAATCAGGCACGGCGAATTCGATACGACGATCAA

TACGTCCTGGCCGTACAAGTGCAGGATCCAAAGTATCAGGACGA

TTTGTGGCCAT

Mi40
SEQ ID NO: 24

AAGAAGATATTCATATCTTTCCAAACACTGAGCAGCAGTTCTTC

CAACAATAGGAGCAATTGTACGCCATTGAGTAGGCATAATTTTG

GCCAAATGGAGAAGTTTTTCGTCCTCTTCTCTGGACCATTCCGT

CTTTTTAATTCGAGGATCAAGCCATTCATACCAACGGGCTTTAC

A

Mi101
SEQ ID NO: 28

GAGCCGCCTCTATATGATCGTCGACAAGGATTTGTGCCCCGAAC

GCAAGATGATTTTGGTGATGGTGGGGCTTTTCCAGAAATACATA

TTGCTCAGTTTCCAATCGGTATGGGCGCCGATAAGCCCGGCACT

GGGGCAAAGAACACAGTTGCGCTTCAGTTTGACTCTGAGGGCAA

ACTTCGATTTGATGAGCTTACACGAATTGGGCATGGAAAAGACA

AGATTGTTCATTCCCGCCTTTCTGATATGAAATCCAACCATATT

GATGATGAAGACGAAAGTTTCAAAAAACCAACAGATGAAGAAAT

ACATGAAACAGCCGAAGCAACTCGTGTTTCCTTAGAGAAAATTA

CAGCTGTTAAGATCGCCGCATCATTACCAGTCCAGCATGCAAAG

AAAACAGCGCCTGCACAATATATTCG

Mi101
SEQ ID NO: 29

for
ACTCGTGTTTCCTTAGAGAAAATTACAGCTGTTAAGATCGCCGC

MiCC2
ATCATTACCAGTCCAGCATGCAAAGAAAACAGCGCCTGCACAAT

ATATTCGTTACACTCCTAGCCAGCAGGCTGGTGGTTTTCATACA

AGTGGAGCACAACAAAGA

Mi109
SEQ ID NO: 33

GTTGGATCCTAATGAGGTCAACGTGATTTCCAACGCAAATTCTC

GTCAGAACGTTCGTCGTTTGGTCAAAGATGGTCTTATAATTCGT

AAGCCTGGCAAAGTTCATTCGCGTTTCCGAGCTCGTGAACGTTT

GGAAGCACGCAGAAAGGGTCGTCATATGGGTTTTGGAAAACGAC

GCGGAACTCGTAACGCTCGAATGCCGGAGAAGGTCTTATGGATG

CGTTTTTTTGTTGTACTCCGTCATCTTTTAAAGCGCTATCGAGC

TGCGAAGAAGATTGACAAGCATCTTTACCACGAGCTTTACTTGA

AGGCTAAAGGAAACGCATTTAAGAATAAGAGAAATTTGATGGAA

TATATCTTCAAAAAGAAGACTGAGAACCAGCGTTCTAAACAACT

TGCTGAACAGGCTGAGGCTCG

Mi111
SEQ ID NO: 37

GGCCTCCAATCCCTACACGCTTTGGGAAGCGAAAGAAGGGAAGT

AAAGGACCTGAAGCTAGCAATAAAATGCCTAATGTGACACCAGT

AACTCGTTGCAAATTGCGTCTTCTTAAATATGAACGGATTAAAG

ATTATTTGTTAATGGAAGAAGAATTTATTCGAAATCAGGAAAGA

CATAAACCTCAAGAGGAAAGGCAGGAAGAAGAACGGACAAAAGT

AGATGAAATGAGAGGGTCACCAATGGCTGTGGGTACATTGGAGG

AGATTATTGACGATCAACACGCAGTCGTTTCTACGAATGTTGGC

AGCGAACATTATGTTAACATTCTTTCATTTGTGGACAAGGAACA

ATTGGAGCCAGGCTGTGCAGTTCTTCTAAATCACAAAACACACG

CAGTTGTTGG

Mi116
SEQ ID NO: 41

TCACACCAAATTCCTTGATATTTTCTTTTAACATTTTTTTCAAA

CTGTGGGTCTCCAAGCAATAAGTTTTCAGGGTATAGGTCAGGTG

GTGACTCATTTTTCAATATTTGATAGTGGTTTGTATTTTTGACT

TTACTTAACTTGTTTACTGAAATCTGTTCCTGTTCTTCAAGATG

ACCGCACAATTCGAAAGATACACAAATTGGATGATCACGTAATG

GCTGCTTTTGCTGGTTTGAGTGCAGATGCTCGTGTTTTGATTGA

CCGTGCCAGAGTTGCTTGTGAAAATTACAAATTGACTTTGGAGG

ATCCCGTCACACTTGAGCACATCTCCCGCACTATTGCGGACTTG

AAACATGAATTTACGCAAACTACAGGTCGTCGTCCCTTTGGTGT

CTCTTTACTTGTTGGCGGTTTTGATCCCGATGGAACTCCTCATC

TTTTTATTAC

Mi125a
SEQ ID NO: 45

ATCCTCTTCATCGTCACCGCCTCCTCCTCCACCTCCCGAAGCAG

CCTTCTTCAAATTCTTTCTCTTGACACGTCCAGGAGGTCCACCA

GCAAGATAAGATTTTGTGGAGAAGTCAATGTGCTTTTGAGAGTC

GAGACGAACAATGAATGATGGAATATTGACTACTTGTCGACGAA

TTCGAATATGCTTTTGACGAATAAGAACACGAGCATGGTGAATT

GATTTAGCCAATCCCAATTTGAAAACTTGCGTCTGCAAACGACG

CTCCAAAAAGTCTTCAACACGCAAACCAAGAACATAGTCCAATT

TCATCTTATCCTCCAAAAGAACTCCAATACGAACCAAACGACGA

AGAAGAGCATTTCCTTCAAATAAGCGTCGAGGATCCTTGTCTTC

CAATGTTAAAAGTTCACGAGCAGCTTTACGAATCTTTGCCAAAG

TTAATTTGACACGCCAAACTTCACGTTTATTTCGTAAACCAAAC

TCGCCAATTAATTTTAATTCCTGATCTAAACGCTCCTTCTCGAA

TGGACGACGAGGCAC

Mi125b
SEQ ID NO: 46

TTCATCTTATCCTCCAAAAGAACTCCAATACGAACCAAACGACG

AAGAAGAGCATTTCCTTCAAATAAGCGTCGAGGATCCTTGTCTT

CCAATGTTAAAAGTTCACGAGCAGCTTTACGAATCTTTGCCAAA

GTTAATTTGACACGCCAAACTTCACGTTTATTTCGTAAACCAAA

CTCGCCAATTAATTTTAATTCCTG

Mi127
SEQ ID NO: 51

ATTTTTCGCAGCATCACCAATCAGACGTTCCGTGTCTGTAAAAG

CCACATAAGATGGAGTCGTGCGATTTCCTTGATCATTGGCAATG

ATCTCGACTTTTCCATGCTGGAATACACCGACACATAAATAAGT

CGTTCCCAGATCAATTCC

Mi128
SEQ ID NO: 54

TGAGAAATTTAAAGAATGACAAAGTTACAACATCCAGCTGTTGA

GGGTCTTGCTGTTGGTTTGGATCGTGGCCATCCTGTGACTAAAA

ATGTTCGCAAGCCACGTCAATCTCGTAAAAAAGGCCGCATTACG

AAGAAGACTCGAATTGTT

Mi129
SEQ ID NO: 57

CCGACGAGACTTACTGCATCGACAACGAGGCTTTGTACGACATC

TGCTTCCGAACTCTGAAGCTTCAAAATCCAACATATGGAGATTT

GAATCATCTCGTATCCGTAACCATGTCTGGAGTGACTACTTGTC

TTCGCTTCCCAGGCCAATTGAACGCTGATCTTCGCAAATTGGCT

GTCAACATGGTTCCATTCCCACGTCTTCACTTTTTCATGCCTGG

TTTCGCTCCACTTTCGGCTAAGGGCGCAGCTGCCTACCAGGCCC

AAAATGTTGCTGAATTGACCAAGCAAATGTTTGATGCCAAGAAT

ATGATGGCTGCTTGTGATCCACGCCATGGACGCTATTTAACTGT

TGCCGCTATCTTCCGTGGACGTATGTCAATGCGTGAAGTAGAC

MiCC2
SEQ ID NO: 59

ATTTTTCGCAGCATCACCAATCAGACGTTCCGTGTCTGTAAAAG

CCACATAAGATGGAGTCGTGCGATTTCCTTGATCATTGGCAATG

ATCTCGACTTTTCCATGCTGGAATACACCGACACATAAATAAGT

CGTTCCCAGATCAATTCCTGAGAAATTTAAAGAATGACAAAGTT

ACAACATCCAGCTGTTGAGGGTCTTGCTGTTGGTTTGGATCGTG

GCCATCCTGTGACTAAAAATGTTCGCAAGCCACGTCAATCTCGT

AAAAAAGGCCGCATTACGAAGAAGACTCGAATTGTTACTCGTGT

TTCCTTAGAGAAAATTACAGCTGTTAAGATCGCCGCATCATTAC

CAGTCCAGCATGCAAAGAAAACAGCGCCTGCACAATATATTCGT

TACACTCCTAGCCAGCAGGCTGGTGGTTTTCATACAAGTGGAGC

ACAACAAAGA

TABLE 5

Target

ID
Hairpin Sequence

M105
SEQ ID NO: 7

AGGCAAAGAGCCGTGGATGGCAGCCCGAAACCGAGGTCAAAGAC

GAGCCTCTGGAATCCAACAACGAACAGGACACTACCGTCAAAAT

TGAAGTCTAATCATCTTACTCAAAAGAACATATCTCAAGAGTTG

CTTTCCTTTAGTAGATTTTCTATATAACTGGGTCTCTGTCTCTC

TCCCGCCTTGCCCCAACTGTGATCCCCCTACCGTCTTGCCTTTT

TTCTTCCAACGAGCCCCGGGTGTATCTTCTCAACCAGCCACAAA

AGTATTTCCACTTTGATATCGCATATTAGTGCTATACACCGAGC

ATACAAACAACCTATACATAATAAAAATCGTCTCTAGACCCAGC

TTTCTTGTACAAAGTGGTTGATCCTGCAGGGTCCGTCGCTTCTC

TTCCATTTCTTCTCATTTTCGATTTTGATTCTTATTTCTTTCCA

GTAGCTCCTGCTCTGTGAATTTCTCCGCTCACGATAGATCTGCT

TATACTCCTTACATTCAACCTTAGATCTGGTCTCGATTCTCTGT

TTCTCTGTTTTTTTCTTTTGGTCGAGAATCTGATGTTTGTTTAT

GTTCTGTCACCATTAATAATAATGAACTCTCTCATTCATACAAT

GATTAGTTTCTCTCGTCTACAAAACGATATGTTGCATTTTCACT

TTTCTTCTTTTTTTCTAAGATGATTTGCTTTGACCAATTTGTTT

AGATCTTTATTCTATTTTATTTTCTGGTGGGTTGGTGGAAATTG

AAAAAAAAAAAACAGCATAAATTGTTATTTGTTAATGTATTCAT

TTTTTGGCTATTTGTTCTGGGTAAAAATCTGCTTCTACTATTGA

ATCTTTCCTGGATTTTTTACTCCTATTGGGTTTTTATAGTAAAA

ATACATAATAAAAGGAAAACAAAAGTTTTATAGATTCTCTTAAA

CCCCTTACGATAAAAGTTGGAATCAAAATAATTCAGGATCAGAT

GCTCTTTGATTGATTCAGATGCGATTACAGTTGCATGGCAAATT

TTCTAGATCCGTCGTCACATTTTATTTTCTGTTTAAATATCTAA

ATCTGATATATGATGTCGACAAATTCTGGTGGCTTATACATCAC

TTCAACTGTTTTCTTTTGGCTTTGTTTGTCAACTTGGTTTTCAA

TACGATTTGTGATTTCGATCGCTGAATTTTTAATACAAGCAAAC

TGATGTTAACCACAAGCAAGAGATGTGACCTGCCTTATTAACAT

CGTATTACTTACTACTAGTCGTATTCTCAACGCAATCGTTTTTG

TATTTCTCACATTATGCCGCTTCTCTACTCTTTATTCCTTTTGG

TCCACGCATTTTCTATTTGTGGCAATCCCTTTCACAACCTGATT

TCCCACTTTGGATCATTTGTCTGAAGACTCTCTTGAATCGTTAC

CACTTGTTTCTTGTGCATGCTCTGTTTTTTAGAATTAATGATAA

AACTATTCCATAGTCTTGAGTTTTCAGCTTGTTGATTCTTTTGC

TTTTGGTTTTCTGCAGGTTTAAACATCAACCACTTTGTACAAGA

AAGCTGGGTCTAGAGACGATTTTTATTATGTATAGGTTGTTTGT

ATGCTCGGTGTATAGCACTAATATGCGATATCAAAGTGGAAATA

CTTTTGTGGCTGGTTGAGAAGATACACCCGGGGCTCGTTGGAAG

AAAAAAGGCAAGACGGTAGGGGGATCACAGTTGGGGCAAGGCGG

GAGAGAGACAGAGACCCAGTTATATAGAAAATCTACTAAAGGAA

AGCAACTCTTGAGATATGTTCTTTTGAGTAAGATGATTAGACTT

CAATTTTGACGGTAGTGTCCTGTTCGTTGTTGGATTCCAGAGGC

TCGTCAAGACCTCGGTTTCGGGCTGCCATCCACGGCTCTTTGCC

T

Mi11
SEQ ID NO: 13

TTTGATCTGTTTGTCCCATGATCGTCTACTCCAATTGAGAAATT

TGTTAGGTGTTTTAGGATGAACACCTTTCTTCCTGTCATCCTTT

GGAACTTGTTCTATGTAACGCTCATAAGTTGGACGATCCTTCAT

TTTATTGATTTCCTTTTTACGACGAGCAATGGATCGTCTCTAGA

CCCAGCTTTCTTGTACAAAGTGGTTGATCCTGCAGGGTCCGTCG

CTTCTCTTCCATTTCTTCTCATTTTCGATTTTGATTCTTATTTC

TTTCCAGTAGCTCCTGCTCTGTGAATTTCTCCGCTCACGATAGA

TCTGCTTATACTCCTTACATTCAACCTTAGATCTGGTCTCGATT

CTCTGTTTCTCTGTTTTTTTCTTTTGGTCGAGAATCTGATGTTT

GTTTATGTTCTGTCACCATTAATAATAATGAACTCTCTCATTCA

TACAATGATTAGTTTCTCTCGTCTACAAAACGATATGTTGCATT

TTCACTTTTCTTCTTTTTTTCTAAGATGATTTGCTTTGACCAAT

TTGTTTAGATCTTTATTCTATTTTATTTTCTGGTGGGTTGGTGG

AAATTGAAAAAAAAAAAACAGCATAAATTGTTATTTGTTAATGT

ATTCATTTTTTGGCTATTTGTTCTGGGTAAAAATCTGCTTCTAC

TATTGAATCTTTCCTGGATTTTTTACTCCTATTGGGTTTTTATA

GTAAAAATACATAATAAAAGGAAAACAAAAGTTTTATAGATTCT

CTTAAACCCCTTACGATAAAAGTTGGAATCAAAATAATTCAGGA

TCAGATGCTCTTTGATTGATTCAGATGCGATTACAGTTGCATGG

CAAATTTTCTAGATCCGTCGTCACATTTTATTTTCTGTTTAAAT

ATCTAAATCTGATATATGATGTCGACAAATTCTGGTGGCTTATA

CATCACTTCAACTGTTTTCTTTTGGCTTTGTTTGTCAACTTGGT

TTTCAATACGATTTGTGATTTCGATCGCTGAATTTTTAATACAA

GCAAACTGATGTTAACCACAAGCAAGAGATGTGACCTGCCTTAT

TAACATCGTATTACTTACTACTAGTCGTATTCTCAACGCAATCG

TTTTTGTATTTCTCACATTATGCCGCTTCTCTACTCTTTATTCC

TTTTGGTCCACGCATTTTCTATTTGTGGCAATCCCTTTCACAAC

CTGATTTCCCACTTTGGATCATTTGTCTGAAGACTCTCTTGAAT

CGTTACCACTTGTTTCTTGTGCATGCTCTGTTTTTTAGAATTAA

TGATAAAACTATTCCATAGTCTTGAGTTTTCAGCTTGTTGATTC

TTTTGCTTTTGGTTTTCTGCAGGTTTAAACATCAACCACTTTGT

ACAAGAAAGCTGGGTCTAGAGACGATCCATTGCTCGTCGTAAAA

AGGAAATCAATAAAATGAAGGATCGTCCAACTTATGAGCGTTAC

ATAGAACAAGTTCCAAAGGATGACAGGAAGAAAGGTGTTCATCC

TAAAACACCTAACAAATTTCTCAATTGGAGTAGACGATCATGGG

ACAAACAGATCAAA

Mi38
SEQ ID NO: 19

CGATCGACGCTCATCCGCTTTGTATGAATCTTTAGAATGTTTGC

ACGAGCTTCCAAATCAGGCACGGCGAATTCGATACGACGATCAA

TACGTCCTGGCCGTACAAGTGCAGGATCCAAAGTATCAGGACGA

TTTGTGGCCATATCGTCTCTAGACCCAGCTTTCTTGTACAAAGT

GGTTGATCCTGCAGGGTCCGTCGCTTCTCTTCCATTTCTTCTCA

TTTTCGATTTTGATTCTTATTTCTTTCCAGTAGCTCCTGCTCTG

TGAATTTCTCCGCTCACGATAGATCTGCTTATACTCCTTACATT

CAACCTTAGATCTGGTCTCGATTCTCTGTTTCTCTGTTTTTTTC

TTTTGGTCGAGAATCTGATGTTTGTTTATGTTCTGTCACCATTA

ATAATAATGAACTCTCTCATTCATACAATGATTAGTTTCTCTCG

TCTACAAAACGATATGTTGCATTTTCACTTTTCTTCTTTTTTTC

TAAGATGATTTGCTTTGACCAATTTGTTTAGATCTTTATTCTAT

TTTATTTTCTGGTGGGTTGGTGGAAATTGAAAAAAAAAAAACAG

CATAAATTGTTATTTGTTAATGTATTCATTTTTTGGCTATTTGT

TCTGGGTAAAAATCTGCTTCTACTATTGAATCTTTCCTGGATTT

TTTACTCCTATTGGGTTTTTATAGTAAAAATACATAATAAAAGG

AAAACAAAAGTTTTATAGATTCTCTTAAACCCCTTACGATAAAA

GTTGGAATCAAAATAATTCAGGATCAGATGCTCTTTGATTGATT

CAGATGCGATTACAGTTGCATGGCAAATTTTCTAGATCCGTCGT

CACATTTTATTTTCTGTTTAAATATCTAAATCTGATATATGATG

TCGACAAATTCTGGTGGCTTATACATCACTTCAACTGTTTTCTT

TTGGCTTTGTTTGTCAACTTGGTTTTCAATACGATTTGTGATTT

CGATCGCTGAATTTTTAATACAAGCAAACTGATGTTAACCACAA

GCAAGAGATGTGACCTGCCTTATTAACATCGTATTACTTACTAC

TAGTCGTATTCTCAACGCAATCGTTTTTGTATTTCTCACATTAT

GCCGCTTCTCTACTCTTTATTCCTTTTGGTCCACGCATTTTCTA

TTTGTGGCAATCCCTTTCACAACCTGATTVCCCACTTTGGATCA

TTTGTCTGAAGACTCTCTTGAATCGTTACCACTTGTTTCTTGTG

CATGCTCTGTTTTTTAGAATTAATGATAAAACTATTCCATAGTC

TTGAGTTTTCAGCTTGTTGATTCTTTTGCTTTTGGTTTTCTGCA

GGTTTAAACATCAACCACTTTGTACAAGAAAGCTGGGTCTAGAG

ACGATATGGCCACAAATCGTCCTGATACTTTGGATCCTGCACTT

GTACGGCCAGGACGTATTGATCGTCGTATCGAATTCGCCGTGCC

TGATTTGGAAGCTCGTGCAAACATTCTAAAGATTCATACAAAGC

GGATGAGCGTCGATCG

Mi40
SEQ ID NO: 25

AAGAAGATATTCATATCTTTCCAAACACTGAGCAGCAGTTCTTC

CAACAATAGGAGCAATTGTACGCCATTGAGTAGGCATAATTTTG

GCCAAATGGAGAAGTTTTTCGTCCTCTTCTCTGGACCATTCCGT

CTTTTTAATTCGAGGATCAAGCCATTCATACCAACGGGCTTTAC

AATCGTCTCTAGACCCAGCTTTCTTGTACAAAGTGGTTGATCCT

GCAGGGTCCGTCGCTTCTCTTCCATTTCTTCTCATTTTCGATTT

TGATTCTTATTTCTTTCCAGTAGCTCCTGCTCTGTGAATTTCTC

CGCTCACGATAGATCTGCTTATACTCCTTACATTCAACCTTAGA

TCTGGTCTCGATTCTCTGTTTCTCTGTTTTTTTCTTTTGGTCGA

GAATCTGATGTTTGTTTATGTTCTGTCACCATTAATAATAATGA

ACTCTCTCATTCATACAATGATTAGTTTCTCTCGTCTACAAAAC

GATATGTTGCATTTTCACTTTTCTTCTTTTTTTCTAAGATGATT

TGCTTTGACCAATTTGTTTAGATCTTTATTCTATTTTATTTTCT

GGTGGGTTGGTGGAAATTGAAAAAAAAAAAACAGCATAAATTGT

TATTTGTTAATGTATTCATTTTTTGGCTATTTGTTCTGGGTAAA

AATCTGCTTCTACTATTGAATCTTTCCTGGATTTTTTACTCCTA

TTGGGTTTTTATAGTAAAAATACATAATAAAAGGAAAACAAAAG

TTTTATAGATTCTCTTAAACCCCTTACGATAAAAGTTGGAATCA

AAATAATTCAGGATCAGATGCTCTTTGATTGATTCAGATGCGAT

TACAGTTGCATGGCAAATTTTCTAGATCCGTCGTCACATTTTAT

TTTCTGTTTAAATATCTAAATCTGATATATGATGTCGACAAATT

CTGGTGGCTTATACATCACTTCAACTGTTTTCTTTTGGCTTTGT

TTGTCAACTTGGTTTTCAATACGATTTGTGATTTCGATCGCTGA

ATTTTTAATACAAGCAAACTGATGTTAACCACAAGCAAGAGATG

TGACCTGCCTTATTAACATCGTATTACTTACTACTAGTCGTATT

CTCAACGCAATCGTTTTTGTATTTCTCACATTATGCCGCTTCTC

TACTCTTTATTCCTTTTGGTCCACGCATTTTCTATTTGTGGCAA

TCCCTTTCACAACCTGATTTCCCACTTTGGATCATTTGTCTGAA

GACTCTCTTGAATCGTTACCACTTGTTTCTTGTGCATGCTCTGT

TTTTTAGAATTAATGATAAAACTATTCCATAGTCTTGAGTTTTC

AGCTTGTTGATTCTTTTGCTTTTGGTTTTCTGCAGGTTTAAACA

TCAACCACTTTGTACAAGAAAGCTGGGTCTAGAGACGATTGTAA

AGCCCGTTGGTATGAATGGCTTGATCCTCGAATTAAAAAGACGG

AATGGTCCAGAGAAGAGGACGAAAAACTTCTCCATTTGGCCAAA

ATTATGCCTACTCAATGGCGTACAATTGCTCCTATTGTTGGAAG

AACTGCTGCTCAGTGTTTGGAAAGATATGAATATCTTCTT

Mi101
SEQ ID NO: 30

GAGCCGCCTCTATATGATCGTCGACAAGGATTTGTGCCCCGAAC

GCAAGATGATTTTGGTGATGGTGGGGCTTTTCCAGAAATACATA

TTGCTCAGTTTCCAATCGGTATGGGCGCCGATAAGCCCGGCACT

GGGGCAAAGAACACAGTTGCGCTTCAGTTTGACTCTGAGGGCAA

ACTTCGATTTGATGAGCTTACACGAATTGGGCATGGAAAAGACA

AGATTGTTCATTCCCGCCTTTCTGATATGAAATCCAACCATATT

GATGATGAAGACGAAAGTTTCAAAAAACCAACAGATGAAGAAAT

ACATGAAACAGCCGAAGCAACTCGTGTTTCCTTAGAGAAAATTA

CAGCTGTTAAGATCGCCGCATCATTACCAGTCCAGCATGCAAAG

AAAACAGCGCCTGCACAATATATTCGATCGTCTCTAGACCCAGC

TTTCTTGTACAAAGTGGTTGATCCTGCAGGGTCCGTCGCTTCTC

TTCCATTTCTTCTCATTTTCGATTTTGATTCTTATTTCTTTCCA

GTAGCTCCTGCTCTGTGAATTTCTCCGCTCACGATAGATCTGCT

TATACTCCTTACATTCAACCTTAGATCTGGTCTCGATTCTCTGT

TTCTCTGTTTITTTCTTTTGGTCGAGAATCTGATGTTTGTTTAT

GTTCTGTCACCATTAATAATAATGAACTCTCTCATTCATACAAT

GATTAGTTTCTCTCGTCTACAAAACGATATGTTGCATTTTCACT

TTTCTTCTTTTTTTCTAAGATGATTTGCTTTGACCAATTTGTTT

AGATCTTTATTCTATTTTATTTTCTGGTGGGTTGGTGGAAATTG

AAAAAAAAAAAACAGCATAAATTGTTATTTGTTAATGTATTCAT

TTTTTGGCTATTTGTTCTGGGTAAAAATCTGCTTCTACTATTGA

ATCTTTCCTGGATTTTTTACTCCTATTGGGTTTTTATAGTAAAA

ATACATAATAAAAGGAAAACAAAAGTTTTATAGATTCTCTTAAA

CCCCTTACGATAAAAGTTGGAATCAAAATAATTCAGGATCAGAT

GCTCTTTGATTGATTCAGATGCGATTACAGTTGCATGGCAAATT

TTCTAGATCCGTCGTCACATTTTATTTTCTGTTTAAATATCTAA

ATCTGATATATGATGTCGACAAATTCTGGTGGCTTATACATCAC

TTCAACTGTTTTCTTTTGGCTTTGTTTGTCAACTTGGTTTTCAA

TACGATTTGTGATTTCGATCGCTGAATTTTTAATACAAGCAAAC

TGATGTTAACCACAAGCAAGAGATGTGACCTGCCTTATTAACAT

CGTATTACTTACTACTAGTCGTATTCTCAACGCAATCGTTTTTG

TATTTCTCACATTATGCCGCTTCTCTACTCTTTATTCCTTTTGG

TCCACGCATTTTCTATTTGTGGCAATCCCTTTCACAACCTGATT

TCCCACTTTGGATCATTTGTCTGAAGACTCTCTTGAATCGTTAC

CACTTGTTTCTTGTGCATGCTCTGTTTTTTAGAATTAATGATAA

AACTATTCCATAGTCTTGAGTTTTCAGCTTGTTGATTCTTTTGC

TTTTGGTTTTCTGCAGGTTTAAACATCAACCACTTTGTACAAGA

AAGCTGGGTCTAGAGACGATCGAATATATTGTGCAGGCGCTGTT

TTCTTTGCATGCTGGACTGGTAATGATGCGGCGATCTTAACAGC

TGTAATTTTCTCTAAGGAAACACGAGTTGCTTCGGCTGTTTCAT

GTATTTCTTCATCTGTTGGTTTTTTGAAACTTTCGTCTTCATCA

TCAATATGGTTGGATTTCATATCAGAAAGGCGGGAATGAACAAT

CTTGTCTTTTCCATGCCCAATTCGTTGTAAGCTCATCAAATCGA

AGTTTGCCCTCAGAGTCAAACTGAAGCGCAACTGTGTTCTTTGC

CCCAGTGCCGGGCTTATCGGCGCCCATACCGATTGGAAACTGAG

CAATATGTATTTCTGGAAAAGCCCCACCATCACCAAAATCATCT

TGCGTTCGGGGCACAAATCCTTGTCGACGATCATATAGAGGCGG

CTC

Mi109
SEQ ID NO: 34

GTTGGATCCTAATGAGGTCAACGTGATTTCCAACGCAAATTCTC

GTCAGAACGTTCGTCGTTTGGTCAAAGATGGTCTTATAATTCGT

AAGCCTGGCAAAGTTCATTCGCGTTTCCGAGCTCGTGAACGTTT

GGAAGCACGCAGAAAGGGTCGTCATATGGGTTTTGGAAAACGAC

GCGGAACTCGTAACGCTCGAATGCCGGAGAAGGTCTTATGGATG

CGTTTTTTTGTTGTACTCCGTcATCTTTTAAAGCGCTATCGAGC

TGCGAAGAAGATTGACAAGCATCTTTACCACGAGCTTTACTTGA

AGGCTAAAGGAAACGCATTTAAGAATAAGAGAAATTTGATGGAA

TATATCTTCAAAAAGAAGACTGAGAACCAGCGTTCTAAACAACT

TGCTGAACAGGCTGAGGCTCGATCGTCTCTAGACCCAGCTTTCT

TGTACAAAGTGGTTGATCCTGCAGGGTCCGTCGCTTCTCTTCCA

TTTCTTCTCATTTTCGATTTTGATTCTTATTTCTTTCGAGTAGC

TCCTGCTCTGTGAATTTCTCCGCTCACGATAGATCTGCTTATAC

TCCTTACATTCAACCTTAGATCTGGTCTCGATTCTCTGTTTCTC

TGTTTTTTTCTTTTGGTCGAGAATCTGATGTTTGTTTATGTTCT

GTCACCATTAATAATAATGAACTCTCTCATTCATACAATGATTA

GTTTCTCTCGTCTACAAAACGATATGTTGCATTTTCACTTTTCT

TCTTTTTTTCTAAGATGATTTGCTTTGACCAATTTGTTTAGATC

TTTATTCTATTTTATTTTCTGGTGGGTTGGTGGAAATTGAAAAA

AAAAAAACAGCATAAATTGTTATTTGTTAATGTATTCATTTTTT

GGCTATTTGTTCTGGGTAAAAATCTGCTTCTACTATTGAATCTT

TCCTGGATTTTTTACTCCTATTGGGTTTTTATAGTAAAAATACA

TAATAAAAGGAAAACAAAAGTTTTATAGATTCTCTTAAACCCCT

TACGATAAAAGTTGGAATCAAAATAATTCAGGATCAGATGCTCT

TTGATTGATTCAGATGCGATTACAGTTGCATGGCAAATTTTCTA

GATCCGTCGTCACATTTTATTTTCTGTTTAAATATCTAAATCTG

ATATATGATGTCGACAAATTCTGGTGGCTTATACATCACTTCAA

CTGTTTTCTTTTGGCTTTGTTTGTCAACTTGGTTTTCAATACGA

TTTGTGATTTCGATCGCTGAATTTTTAATACAAGCAAACTGATG

TTAACCACAAGCAAGAGATGTGACCTGCCTTATTAACATCGTAT

TACTTACTACTAGTCGTATTCTCAACGCAATCGTTTTTGTATTT

CTCACATTATGCCGCTTCTCTACTCTTTATTCCTTTTGGTCCAC

GCATTTTCTATTTGTGGCAATCCCTTTCACAACCTGATTTCCCA

CTTTGGATCATTTGTCTGAAGACTCTCTTGAATCGTTACCACTT

GTTTCTTGTGCATGCTCTGTTTTTTAGAATTAATGATAAAACTA

TTCCATAGTCTTGAGTTTTCAGCTTGTTGATTCTTTTGCTTTTG

GTTTTCTGCAGGTTTAAACATCAACCACTTTGTACAAGAAAGCT

GGGTCTAGAGACGATCGAGCCTCAGCCTGTTCAGCAAGTTGTTT

AGAACGCTGGTTCTCAGTCTTCTTTTTGAAGATATATTCCATCA

AATTTCTCTTATTCTTAAATGCGTTTCCTTTAGCCTTCAAGTAA

AGCTCGTGGTAAAGATGCTTGTCAATCTTCTTCGCAGCTCGATA

GCGCTTTAAAAGATGACGGAGTACAACAAAAAAACGCATCCATA

AGACCTTCTCCGGCATTCGAGCGTTACGAGTTCCGCGTCGTTTT

CCAAAACCCATATGACGACCCTTTCTGCGTGCTTCCAAACGTTC

ACGAGCTCGGAAACGCGAATGAACTTTGCCAGGCTTACGAATTA

TAAGACCATCTTTGACCAAACGACGAACGTTCTGACGAGAATTT

GCGTTGGAAATCACGTTGACCTCATTAGGATCCAAC

Mi111
SEQ ID NO: 38

GGCCTCCAATCCCTACACGCTTTGGGAAGCGAAAGAAGGGAAGT

AAAGGACCTGAAGCTAGCAATAAAATGCCTAATGTGACACCAGT

AACTCGTTGCAAATTGCGTCTTCTTAAATATGAACGGATTAAAG

ATTATTTGTTAATGGAAGAAGAATTTATTCGAAATCAGGAAAGA

CATAAACCTCAAGAGGAAAGGCAGGAAGAAGAACGGACAAAAGT

AGATGAAATGAGAGGGTCACCAATGGCTGTGGGTACATTGGAGG

AGATTATTGACGATCAACACGCAGTCGTTTCTACGAATGTTGGC

AGCGAACATTATGTTAACATTCTTTCATTTGTGGACAAGGAACA

ATTGGAGCCAGGCTGTGCAGTTCTTCTAAATCACAAAACACACG

CAGTTGTTGGATCGTCTCTAGACCCAGCTTTCTTGTACAAAGTG

GTTGATCCTGCAGGGTCCGTCGCTTCTCTTCCATTTCTTCTCAT

TTTCGATTTTGATTCTTATTTCTTTCCAGTAGCTCCTGCTCTGT

GAATTTCTCCGCTCACGATAGATCTGCTTATACTCCTTACATTC

AACCTTAGATCTGGTCTCGATTCTCTGTTTCTCTGTTTTTTTCT

TTTGGTCGAGAATCTGATGTTTGTTTATGTTCTGTCACCATTAA

TAATAATGAACTCTCTCATTCATACAATGATTAGTTTCTCTCGT

CTACAAAACGATATGTTGCATTTTCACTTTTCTTCTTTTTTTCT

AAGATGATTTGCTTTGACCAATTTGTTTAGATCTTTATTCTATT

TTATTTTCTGGTGGGTTGGTGGAAATTGAAAAAAAAAAAACAGC

ATAAATTGTTATTTGTTAATGTATTCATTTTTTGGCTATTTGTT

CTGGGTAAAAATCTGCTTCTACTATTGAATCTTTCCTGGATTTT

TTACTCCTATTGGGTTTTTATAGTAAAAATACATAATAAAAGGA

AAACAAAAGTTTTATAGATTCTCTTAAACCCCTTACGATAAAAG

TTGGAATCAAAATAATTCAGGATCAGATGCTCTTTGATTGATTC

AGATGCGATTACAGTTGCATGGCAAATTTTCTAGATCCGTCGTC

ACATTTTATTTTCTGTTTAAATATCTAAATCTGATATATGATGT

CGACAAATTCTGGTGGCTTATACATCACTTCAACTGTTTTCTTT

TGGCTTTGTTTGTCAACTTGGTTTTCAATACGATTTGTGATTTC

GATCGCTGAATTTTTAATACAAGCAAACTGATGTTAACCACAAG

CAAGAGATGTGACCTGCCTTATTAACATCGTATTACTTACTACT

AGTCGTATTCTCAACGCAATCGTTTTTGTATTTCTCACATTATG

CCGCTTCTCTACTCTTTATTCCTTTTGGTCCACGCATTTTCTAT

TTGTGGCAATCCCTTTCACAACCTGATTTCCCACTTTGGATCAT

TTGTCTGAAGACTCTCTTGAATCGTTACCACTTGTTTCTTGTGC

ATGCTCTGTTTTTTAGAATTAATGATAAAACTATTCCATAGTCT

TGAGTTTTCAGCTTGTTGATTCTTTTGCTTTTGGTTTTCTGCAG

GTTTAAACATCAACCACTTTGTACAAGAAAGCTGGGTCTAGAGA

CGATCCAACAACTGCGTGTGTTTTGTGATTTAGAAGAACTGCAC

AGCCTGGCTCCAATTGTTCCTTGTCCACAAATGAAAGAATGTTA

ACATAATGTTCGCTGCCAACATTCGTAGAAACGACTGCGTGTTG

ATCGTCAATAATCTCCTCCAATGTACCCACAGCCATTGGTGACC

CTCTCATTTCATCTACTTTTGTCCGTTCTTCTTCCTGCCTTTCC

TCTTGAGGTTTATGTCTTTCCTGATTTCGAATAAATTCTTCTTC

CATTAACAAATAATCTTTAATCCGTTCATATTTAAGAAGACGCA

ATTTGCAACGAGTTACTGGTGTCACATTAGGCATTTTATTGCTA

GCTTCAGGTCCTTTACTTCCCTTCTTTCGCTTCCCAAAGCGTGT

AGGGATTGGAGGCC

Mi116
SEQ ID NO: 42

TCACACCAAATTCCTTGATATTTTCTTTTAACATTTTTTTCAAA

CTGTGGGTCTCCAAGCAATAAGTTTTCAGGGTATAGGTCAGGTG

GTGACTCATTTTTCAATATTTGATAGTGGTTTGTATTTTTGACT

TTACTTAACTTGTTTACTGAAATCTGTTCCTGTTCTTCAAGATG

ACCGCACAATTCGAAAGATACACAAATTGGATGATCACGTAATG

GCTGCTTTTGCTGGTTTGAGTGCAGATGCTCGTGTTTTGATTGA

CCGTGCCAGAGTTGCTTGTGAAAATTACAAATTGACTTTGGAGG

ATCCCGTCACACTTGAGCACATCTCCCGCACTATTGCGGACTTG

AAACATGAATTTACGCAAACTACAGGTCGTCGTCCCTTTGGTGT

CTCTTTACTTGTTGGCGGTTTTGATCCCGATGGAACTCCTCATC

TTTTTATTACACCCAGCTTTCTTGTACAAAGTGGTGATATCCCG

CGGGATCAGAAGCAACCTCATGGAAATGATGAGGTAAGGTTTCA

TACTCTTGCCTCTTCTTACGGCTTTCTGTGTCTTCACTGTAAGT

TTCTATGATTTGAGCCACCAATATATATGCTCTGGTGTGCTGAG

TTATGTTTATCTGGTCACGCTTAGTGGGTAAAATTATGCTTATT

TTAGCATAAACTTTAATGAGATTAGGTTTTGTATCACACCGATC

TTTAGTTGTTTAGTAAGATGACAGAAATTCTTGGTAAAACACTC

TAAATCGTCTTCTTTAGTGAAGTTTTCCTTAGAGTAGCATAAAT

TTTGGCTTTTTTCTTGATGGTTGAATAAGGTGGCACTTGTTGGT

ATGAGACTTTATTGAGAGTCATATTAAGCTGATCCACGCGTTTA

CGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAA

GCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACC

TGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATA

TTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGG

CCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCT

GAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGC

CAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTA

GAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAA

AACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAAC

ACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGA

ATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAG

GCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAA

GGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAG

CAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGG

GATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTT

AGCTTCCTTAGCTCCTGAAAATCTCGCCGGATCAGCTTAGCGTT

CATTGAATTTGATGGCCATAGGGGTTTAGATGCAACTGTTTCTT

TGAACATTGTAGAAATATATAAAGATTTTACATTAGCCTACTCT

TGAAAGTCAAATTGTCGAATTTGATTATATTATACTCTAGAGGT

GATATTAGTTAATGAGTTTATACTCGGTTATTTACAGCTTATTC

ATATACCAGTTAACGTGTCTCATATATTCTAACTTCTTAGCATT

TAACGTGTTTGCAGGTCAGCTTGACACTGAACATAACAGCATCA

CTAGTGCGGCCGCCTGCAGGTCGACCATATGGTCGACCTGCAGG

CGGCCGCACTAGTGATATCACCACTTTGTACAAGAAAGCTGGGT

GTAATAAAAAGATGAGGAGTTCCATCGGGATCAAAACCGCCAAC

AAGTAAAGAGACACCAAAGGGACGACGACCTGTAGTTTGCGTAA

ATTCATGTTTCAAGTCCGCAATAGTGCGGGAGATGTGCTCAAGT

GTGACGGGATCCTCCAAGTCAATTTGTAATTTTCACAAGCAACT

CTGGCACGGTCAATCAAAACACGAGCATCTGCACTCAAACCAGC

AAAAGCAGCCATTACGTGATCATCCAATTTGTGTATCTTTCGAA

TTGTGCGGTCATCTTGAAGAACAGGAACAGATTTCAGTAAACAA

GTTAAGTAAAGTCAAAAATACAAACCACTATCAAATATTGAAAA

ATGAGTCACCACCTGACCTATACCCTGAAAACTTATTGCTTGGA

GACCCACAGTTTGAAAAAAATGTTAAAAGAAAATATCAAGGAAT

TTGGTGTGA

Mi125a
SEQ ID NO: 47

ATCCTCTTCATCGTCACCGCCTCCTCCTCCACCTCCCGAAGCAG

CCTTCTTCAAATTCTTTCTCTTGACACGTCCAGGAGGTCCACCA

GCAAGATAAGATTTTGTGGAGAAGTCAATGTGCTTTTGAGAGTC

GAGACGAACAATGAATGATGGAATATTGACTACTTGTCGACGAA

TTCGAATATGCTTTTGACGAATAAGAACACGAGCATGGTGAATT

GATTTAGCCAATCCCAATTTGAAAACTTGCGTCTGCAAACGACG

CTCCAAAAAGTCTTCAACACGCAAACCAAGAACATAGTCCAATT

TCATCTTATCCTCCAAAAGAACTCCAATACGAACCAAACGACGA

AGAAGAGCATTTCCTTCAAATAAGCGTCGAGGATCCTTGTCTTC

CAATGTTAAAAGTTCACGAGCAGCTTTACGAATCTTTGCCAAAG

TTAATTTGACACGCCAAACTTCACGTTTATTTCGTAAACCAAAC

TCGCCAATTAATTTTAATTCCTGATCTAAACGCTCCTTCTCGAA

TGGACGACGAGGCACATCGTCTCTAGACCCAGCTTTCTTGTACA

AAGTGGTTGATCCTGCAGGGTCCGTCGCTTCTCTTCCATTTCTT

CTCATTTTCGATTTTGATTCTTATTTCTTTCCAGTAGCTCCTGC

TCTGTGAATTTCTCCGCTCACGATAGATCTGCTTATACTCCTTA

CATTCAACCTTAGATCTGGTCTCGATTCTCTGTTTCTCTGTTTT

TTTCTTTTGGTCGAGAATCTGATGTTTGTTTATGTTCTGTCACC

ATTAATAATAATGAACTCTCTCATTCATACAATGATTAGTTTCT

CTCGTCTACAAAACGATATGTTGCATTTTCACTTTTCTTCTTTT

TTTCTAAGATGATTTGCTTTGACCAATTTGTTTAGATCTTTATT

CTATTTTATTTTCTGGTGGGTTGGTGGAAATTGAAAAAAAAAAA

ACAGCATAAATTGTTATTTGTTAATGTATTCATTTTTTGGCTAT

TTGTTCTGGGTAAAAATCTGCTTCTACTATTGAATCTTTCCTGG

ATTTTTTACTCCTATTGGGTTTTTATAGTAAAAATACATAATAA

AAGGAAAACAAAAGTTTTATAGATTCTCTTAAACCCCTTACGAT

AAAAGTTGGAATCAAAATAATTCAGGATCAGATGCTCTTTGATT

GATTCAGATGCGATTACAGTTGCATGGCAAATTTTCTAGATCCG

TCGTCACATTTTATTTTCTGTTTAAATATCTAAATCTGATATAT

GATGTCGACAAATTCTGGTGGCTTATACATCACTTCAACTGTTT

TCTTTTGGCTTTGTTTGTCAACTTGGTTTTCAATACGATTTGTG

ATTTCGATCGCTGAATTTTTAATACAAGCAAACTGATGTTAACC

ACAAGCAAGAGATGTGACCTGCCTTATTAACATCGTATTACTTA

CTACTAGTCGTATTCTCAACGCAATCGTTTTTGTATTTCTCACA

TTATGCCGCTTCTCTACTCTTTATTCCTTTTGGTCCACGCATTT

TCTATTTGTGGCAATCCCTTTCACAACCTGATTTCCCACTTTGG

ATCATTTGTCTGAAGACTCTCTTGAATCGTTACCACTTGTTTCT

TGTGCATGCTCTGTTTTTTAGAATTAATGATAAAACTATTCCAT

AGTCTTGAGTTTTCAGCTTGTTGATTCTTTTGCTTTTGGTTTTC

TGCAGGTTTAAACATCAACCACTTTGTACAAGAAAGCTGGGTCT

AGAGACGATGTGCCTCGTCGTCCATTCGAGAAGGAGCGTTTAGA

TCAGGAATTAAAATTAATTGGCGAGTTTGGTTTACGAAATAAAC

GTGAAGTTTGGCGTGTCAAATTAATTTTGGCAAAGATTCGTAAA

GCTGCTCGTGAACTTTTAACATTGGAAGACAAGGATCCTCGACG

CTTATTTGAAGGAAATGCTCTTCTTCGTCGTTTGGTTCGTATTG

GAGTTCTTTTGGAGGATAAGATGAAATTGGACTATGTTCTTGGT

TTGCGTGTTGAAGACTTTTTGGAGCGTCGTTTGCAGACGCAAGT

TTTCAAATTGGGATTGGCTAAATCAATTCACCATGCTCGTGTTC

TTATTCGTCAAAAGCATATTCGAATTCGTCGACAAGTAGTCAAT

ATTCCATCATTCATTGTTCGTCTCGACTCTCAAAAGCACATTGA

CTTCTCCACAAAATCTTATCTTGCTGGTGGACCTCCTGGACGTG

TCAAGAGAAAGAATTTGAAGAAGGCTGCTTCGGGAGGTGGAGGA

GGAGGCGGTGACGATGAAGAGGAT

Mi125b
SEQ ID NO: 48

TTCATCTTATCCTCCAAAAGAACTCCAATACGAACCAAACGACG

AAGAAGAGCATTTCCTTCAAATAAGCGTCGAGGATCCTTGTCTT

CCAATGTTAAAAGTTCACGAGCAGCTTTACGAATCTTTGCCAAA

GTTAATTTGACACGCCAAACTTCACGTTTATTTCGTAAACCAAA

CTCGCCAATTAATTTTAATTCCTGATCGTCTCTAGACCCAGCTT

TCTTGTACAAAGTGGTTGATCCTGCAGGGTCCGTCGCTTCTCTT

CCATTTCTTCTCATTTTCGATTTTGATTCTTATTTCTTTCCAGT

AGCTCCTGCTCTGTGAATTTCTCCGCTCACGATAGATCTGCTTA

TACTCCTTACATTCAACCTTAGATCTGGTCTCGATTCTCTGTTT

CTCTGTTTTTTTCTTTTGGTCGAGAATCTGATGTTTGTTTATGT

TCTGTCACCATTAATAATAATGAACTCTCTCATTCATACAATGA

TTAGTTTCTCTCGTCTACAAAACGATATGTTGCATTTTCACTTT

TCTTCTTTTTTTCTAAGATGATTTGCTTTGACCAATTTGTTTAG

ATCTTTATTCTATTTTATTTTCTGGTGGGTTGGTGGAAATTGAA

AAAAAAAAAACAGCATAAATTGTTATTTGTTAATGTATTCATTT

TTTGGCTATTTGTTCTGGGTAAAAATCTGCTTCTACTATTGAAT

CTTTCCTGGATTTTTTACTCCTATTGGGTTTTTATAGTAAAAAT

ACATAATAAAAGGAAAACAAAAGTTTTATAGATTCTCTTAAACC

CCTTACGATAAAAGTTGGAATCAAAATAATTCAGGATCAGATGC

TCTTTGATTGATTCAGATGCGATTACAGTTGCATGGCAAATTTT

CTAGATCCGTCGTCACATTTTATTTTCTGTTTAAATATCTAAAT

CTGATATATGATGTCGACAAATTCTGGTGGCTTATACATCACTT

CAACTGTTTTCTTTTGGCTTTGTTTGTCAACTTGGTTTTCAATA

CGATTTGTGATTTCGATCGCTGAATTTTTAATACAAGCAAACTG

ATGTTAACCACAAGCAAGAGATGTGACCTGCCTTATTAACATCG

TATTACTTACTACTAGTCGTATTCTCAACGCAATCGTTTTTGTA

TTTCTCACATTATGCCGCTTCTCTACTCTTTATTCCTTTTGGTC

CACGCATTTTCTATTTGTGGCAATCCCTTTCACAACCTGATTTC

CCACTTTGGATCATTTGTCTGAAGACTCTCTTGAATCGTTACCA

CTTGTTTCTTGTGCATGCTCTGTTTTTTAGAATTAATGATAAAA

CTATTCCATAGTCTTGAGTTTTCAGCTTGTTGATTCT1TTGCTT

TTGGTTTTCTGCAGGTTTAAACATCAACCACTTTGTACAAGAAA

GCTGGGTCTAGAGACGATCAGGAATTAAAATTAATTGGCGAGTT

TGGTTTACGAAATAAACGTGAAGTTTGGCGTGTCAAATTAACTT

TGGCAAAGATTCGTAAAGCTGCTCGTGAACTTTTAACATTGGAA

GACAAGGATCCTCGACGCTTATTTGAAGGAAATGCTCTTCTTCG

TCGTTTGGTTCGTATTGGAGTTCTTTTGGAGGATAAGATGAA

Mi129
SEQ ID NO: 58

CCGACGAGACTTACTGCATCGACAACGAGGCTTTGTACGACATC

TGCTTCCGAACTCTGAAGCTTCAAAATCCAACATATGGAGATTT

GAATCATCTCGTATCCGTAACCATGTCTGGAGTGACTACTTGTC

TTCGCTTCCCAGGCCAATTGAACGCTGATCTTCGCAAATTGGCT

GTCAACATGGTTCCATTCCCACGTCTTCACTTTTTCATGCCTGG

TTTCGCTCCACTTTCGGCTAAGGGCGCAGCTGCCTACCAGGCCC

AAAATGTTGCTGAATTGACCAAGCAAATGTTTGATGCCAAGAAT

ATGATGGCTGCTTGTGATCCACGCCATGGACGCTATTTAACTGT

TGCCGCTATCTTCCGTGGACGTATGTCAATGCGTGAAGTAGACA

TCGTCTCTAGACCCAGCTTTCTTGTACAAAGTGGTTGATCCTGC

AGGGTCCGTCGCTTCTCTTCCATTTCTTCTCATTTTCGATTTTG

ATTCTTATTTCTTTCCAGTAGCTCCTGCTCTGTGAATTTCTCCG

CTCACGATAGATCTGCTTATACTCCTTACATTCAACCTTAGATC

TGGTCTCGATTCTCTGTTTCTCTGTTTTTTTCTTTTGGTCGAGA

ATCTGATGTTTGTTTATGTTCTGTCACCATTAATAATAATGAAC

TCTCTCATTCATACAATGATTAGTTTCTCTCGTCTACAAAACGA

TATGTTGCATTTTCACTTTTCTTCTTTTTTTCTAAGATGATTTG

CTTTGACCAATTTGTTTAGATCTTTATTCTATTTTATTTTCTGG

TGGGTTGGTGGAAATTGAAAAAAAAAAAACAGCATAAATTGTTA

TTTGTTAATGTATTCATTTTTTGGCTATTTGTTCTGGGTAAAAA

TCTGCTTCTACTATTGAATCTTTCCTGGATTTTTTACTCCTATT

GGGTTTTTATAGTAAAAATACATAATAAAAGGAAAACAAAAGTT

TTATAGATTCTCTTAAACCCCTTACGATAAAAGTTGGAATCAAA

ATAATTCAGGATCAGATGCTCTTTGATTGATTCAGATGCGATTA

CAGTTGCATGGCAAATTTTCTAGATCCGTCGTCACATTTTATTT

TCTGTTTAAATATCTAAATCTGATATATGATGTCGACAAATTCT

GGTGGCTTATACATCACTTCAACTG1TTTCTTTTGGCTTTGTTT

GTCAACTTGGTTTTCAATACGATTTGTGATTTCGATCGCTGAAT

TTTTAATACAAGCAAACTGATGTTAACCACAAGCAAGAGATGTG

ACCTGCCTTATTAACATCGTATTACTTACTACTAGTCGTATTCT

CAACGCAATCGTTTTTGTATTTCTCACATTATGCCGCTTCTCTA

CTCTTTATTCCTTTTGGTCCACGCATTTTCTATTTGTGGCAATC

CCTTTCACAACCTGATTTCCCACTTTGGATCATTTGTCTGAAGA

CTCTCTTGAATCGTTACCACTTGTTTCTTGTGCATGCTCTGTTT

TTTAGAATTAATGATAAAACTATTCCATAGTCTTGAGTTTTCAG

CTTGTTGATTCTTTTGCTTTTGGTTTTCTGCAGGTTTAAACATC

AACCACTTTGTACAAGAAAGCTGGGTCTAGAGACGATGTCTACT

TCACGCATTGACATACGTCCACGGAAGATAGCGGCAACAGTTAA

ATAGCGTCCATGGCGTGGATCACAAGCAGCCATCATTTTCTTGG

CATCAAACATTTGCTTGGTCAATTCAGCAACATTTTGGGCCTGG

TAGGCAGCTGCGCCCTTAGCCGAAAGTGGAGCGAAACCAGGCAT

GAAAAAGTGAAGACGTGGGAATGGAACCATGTTGACAGCCAATT

TGCGAAGATCAGCGTTCAATTGGCCTGGGAAGCGAAGACAAGTA

GTCACTCCAGACATGGTTACGGATACGAGATGATTCAAATCTCC

ATATGTTGGATTTTGAAGCTTCAGAGTTCGGAAGCAGATGTCGT

ACAAAGCCTCGTTGTCGATGCAGTAAGTCTCGTCGG

MiCC2
SEQ ID NO: 60

ATTTTTCGCAGCATCACCAATCAGACGTTCCGTGTCTGTAAAAG

CCACATAAGATGGAGTCGTGCGATTTCCTTGATCATTGGCAATG

ATCTCGACTTTTCCATGCTGGAATACACCGACACATAAATAAGT

CGTTCCCAGATCAATTCCTGAGAAATTTAAAGAATGACAAAGTT

ACAACATCCAGCTGTTGAGGGTCTTGCTGTTGGTTTGGATCGTG

GCCATCCTGTGACTAAAATGTTCGCAAGCCACGTCAATCTCGTA

AAAAAGGCCGCATTACGAAGAAGACTCGAATTGTTACTCGTGTT

TCCTTAGAGAAAATTACAGCTGTTAAGATCGCCGCATCATTACC

AGTCCAGCATGCAAAGAAAACAGCGCCTGCACAATATATTCGTT

ACACTCCTAGCCAGCAGGCTGGTGGTTTTCATACAAGTGGAGCA

CAACAAAGAACCCAGCTTTCTTGTACAAAGTGGTGATATCCCGC

GGGATCAGAAGCAACCTCATGGAAATGATGAGGTAAGGTTTCAT

ACTCTTGCCTCTTCTTACGGCTTTCTGTGTCTTCACTGTAAGTT

TCTATGATTTGAGCCACCAATATATATGCTCTGGTGTGCTGAGT

TATGTTTATCTGGTCACGCTTAGTGGGTAAAATTATGCTTATTT

TAGCATAACTTTAATGAGATTAGGTTTTGTATCACACCGATCTT

TAGTTGTTTAGTAAGATGACAGAAATTCTTGGTAAAACACTCTA

AATCGTCTTCTTTAGTGAAGTTTTCCTTAGAGTAGCATAAATTT

TGGCTTTTTTCTTGATGGTTGAATAAGGTGGCACTTGTTGGTAT

GAGACTTTATTGAGAGTCATATTAAGCTGATCCACGCGTTTACG

CCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGC

ATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTG

AATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATT

TGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCC

ACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGA

GACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCA

GGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGA

AACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAA

CGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACAC

TATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAAT

TCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGC

CGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGG

CCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCA

ACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGA

TATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAG

CTTCCTTAGCTCCTGAAAATCTCGCCGGATCAGCTTAGCGTTCA

TTGAATTTGATGGCCATAGGGGTTTAGATGCAACTGTTTCTTTG

AACATTGTAGAAATATATAAAGATTTTACATTAGCCTACTCTTG

AAAGTCAAATTGTCGAATTTGATTATATTATACTCTAGAGGTGA

TATTAGTTAATGAGTTTATACTCGGTTATTTACAGCTTATTCAT

ATACCAGTTAACGTGTCTCATATATTCTAACTTCTTAGCATTTA

ACGTGTTTGCAGGTCAGCTTGACACTGAACATAACAGCATCACT

AGTGCGGCCGCCTGCAGGTCGACCATATGGTCGACCTGCAGGCG

GCCGCACTAGTGATATCACCACTTTGTACAAGAAAGCTGGGTTC

TTTGTTGTGCTCCACTTGTATGAAAACCACCAGCCTGCTGGCTA

GGAGTGTAACGAATATATTGTGCAGGCGCTGTTTTCTTTGCATG

CTGGACTGGTAATGATGCGGCGATCTTAACAGCTGTAATTTTCT

CTAAGGAAACACGAGTAACAATTCGAGTCTTCTTCGTAATGCGG

CCTTTTTTACGAGATTGACGTGGCTTGCGAACATTTTTAGTCAC

AGGATGGCCACGATCCAAACCAACAGCAAGACCCTCAACAGCTG

GATGTTGTAACTTTGTCATTCTTTAAATTTCTCAGGAATTGATC

TGGGAACGACTTATTTATGTGTCGGTGTATTCCAGCATGGAAAA

GTCGAGATCATTGCCAATGATCAAGGAAATCGCACGACTCCATC

TTATGTGGCTTTTACAGACACGGAACGTCTGATTGGTGATGCTG

CGAAAAAT

TABLE 6

Target
SEQ

ID
ID NO
Sequence*
Example Gi-number and species

Mi11
61
AAAAGGAAATCAATAAAATGAA
15767577 (Meloidogyne arenaria)

Mi11
62
ATGAACACAACTGGAGATGAATCAACATTGAAAGATATTCC
15767577 (Meloidogyne arenaria)

Mi11
63
GCGCAGTCTTTGGAACGGCCGCATTC
15767577 (Meloidogyne arenaria)

Mi11
64
GGGGAATCGCCATCTTCAAGTGTTTACGGCTCTTATTGTTCATC
15767577 (Meloidogyne arenaria)

Mi11
65
TAGAACAAGTTCCAAAGGATGACAGGAAGAAAGG
15767577 (Meloidogyne arenaria)

Mi11
66
TGGGACAAACAGATCAAAUATGGAAACTTGCAATATATGAATGGGC
15767577 (Meloidogyne arenaria)

Mi38
67
AACTTGGAGTTGAACTAAACTTTGCATATCCTTTCACCACTTTCTGAAC
46983636 (Meloidogyne hapla)

AGCGTCAATAAAATC

Mi38
68
ACTTCATCAAAGAAAATAATACAAGCCTTCTT
19268096 (Meloidogyne hapla)

Mi38
69
ATACGACGATCAATACGTCCTGGCCGTACAAGTGC
46987842 (Meloidogyne hapla)

Mi38
70
CCCATTCCATCATCAAAACGAGCTCC
46987842 (Meloidogyne hapla)

Mi38
71
GCACGTAATGCAAACATTCCGGCTTC
46987842 (Meloidogyne hapla)

Mi38
72
GGACGATTTGTGGCCATTAAAACTTT
19268096 (Meloidogyne hapla)

Mi38
73
GGGCAAAGACGCGATATCAATTC
46987842 (Meloidogyne hapla)

Mi38
74
GTACAGACAGAGCGAATATCAGC
46987842 (Meloidogyne hapla)

Mi38
75
GTGTCATATAACTTGGAGTTGAACTAAACTTTGCATATCCTTTCACC
46219310 (Meloidogyne hapla)

Mi38
76
GTGTCATATAACTTGGAGTTGAACTAAACTTTGCATATCCTTTCACCAC
46987842 (Meioidogyne hapla)

TTTCTGAACAGCGTCAATAAAATC

Mi38
77
ATACAAGCCTTCTTTGTTTTTGCCATCT
15766735 (Meloidogyne javanica)

Mi38
78
CCGTACAAGTGCAGGATCCAAAGTATCAGGACGATTTGTGGCCATTAAA
15766735 (Meloidogyne javanica)

ACTTTGATTGCACCACGCGAGTCAAATCCATCCAGCTGATTGATCAATT

CGAGCATTGTCCGTTGAACTTCATTATCTCCCCCCATTCCATCATCAAA

ACGAGCTCCGCCAATAGCATC

Mi40
79
GATCCTATTGATATGGATGAAGATGAATT
31546893 (Strongyloides ratti)

Mi101
80
CAGTTTGACTCTGAGGGCAAACTTCGATTTGATGA
19383725 (Meloidogyne hapla)

Mi101
81
CATTCCCGCCTTTCTGATATGAAATC
19383725 (Meloidogyne hapla)

Mi101
82
GAACGCAAGATGATTTTGGTGATGGTGGGGCTTT
19383725 (Meloidogyne hapla)

Mi109
83
CGCAGAAAGGGTCGTCATATGGGT
944801 (Brugia malayi)

Mi109
84
GACAAGCATCTTTACCACGAGCTTTAC
752661 (Caenorhabditis briggsae)

Mi109
85
GACAAGCATCTTTACCACGAGCT
68275989 (Caenorhabditis remanei)

Mi109
86
AAGATTGACAAGCATCTTTACC
18382417 (Globodera pallida)

21819186 (Trichinella spiralis)

Mi109
87
ACAAGCATCTTTACCACGAGCT
18382417 (Globodera pallida)

Mi109
88
AAGATTGACAAGCATCTTTACCACGAGCT
111166140 (Globodera rostochiensis)

Mi109
89
AAGATTGACAAGCATCTTTACCACGAGCTTTAC
111165991 (Globodera rostochiensis)

Mi109
90
AGATTGACAAGCATCTTTACCA
18382417 (Globodera pallida)

Mi109
91
ATTGACAAGCATCTTTACCACG
18382417 (Globodera pallida)

Mi109
92
CAAGCATCTTTACCACGAGCT
18382417 (Globodera pallida)

Mi109
93
GACAAGCATCTTTACCACGAGC
18382417 (Globodera pallida)

Mi109
94
GATTGACAAGCATCTTTACCAC
18382417 (Globodera pallida)

Mi109
95
TGACAAGCATCTTTACCACGAG
18382417 (Globodera pallida)

Mi109
96
AAGATTGACAAGCATCTTTACCACGAGCT
7143692 (Globodera rostochiensis)

Mi109
97
AGATTGACAAGCATCTTTACCACGAGCT
7143685 (Globodera rostochiensis)

Mi109
98
GCTCGTGAACGTTTGGAAGCA
28567874 (Heterodera glycines)

Mi109
99
GCTCGTGAACGTTTGGAAGCACGCAGAAAGGG
28566936 (Heterodera glycines)

33139882 (Heterodera schachtii)

Mi109
100
GCTCGTGAACGTTTGGAAGCACGCAGA
33139171 (Heterodera schachtii)

Mi109
101
TGAACAGGCTGAGGCTCGCCGAAACAAGAACAAGGAAACGAGAAAGAGG
15769093 (Meloidogyne arenaria)

CGTGAGGAGCGTTTGATTTTGAAACGTAAGGAATTGCTCCACAAACTTT

CTGAGAGCGAAAAGCAGAAGACTCCACAGCTTCAAGAAGAGGAAGCGAA

GGAAGCTTCGCCTTCTCCACCAC

Mi109
102
AGAAAGGGTCGTCATATGGGTTTTGG
30029158 (Meloidogyne chitwoodi)

Mi109
103
AAAGTTCATTCGCGTTTCCGAGCTCGTGAACGTTTGGAAGCACGCAGAA
39746570 (Meloidogyne paranaensis)

AGGGTCGTCATATGGGTTTTGGAAAACG

Mi109
104
AAGAGAAATTTGATGGAATATATCTTCAAAAAGAAGACTGAGAACCAGC
39746570 (Meloidogyne paranaensis)

GTTCTAAACAACTTGCTGAACA

Mi109
105
AAGCAGAAGACTCCACAGCTTCAAGAAGAGGAAGCGAAGGAAGCTTCGC
39746570 (Meloidogyne paranaensis)

CTTCTCCACC

Mi109
106
AAGGAATTGCTCCACAAACTTTCTGAGAGCGA
39746570 (Meloidogyne paranaensis)

Mi109
107
ATGCCGGAGAAGGTCTTATGGATGCGT
39746570 (Meloidogyne paranaensis)

Mi109
108
ATTCGTAAGCCTGGCAAAGTTCATTCGCGTTTCCGAGCTCGTGAACGTT
39746726 (Meloidogyne paranaensis)

TGGAAGCACGCAGAAAGGGTCGTCATATGGGTTTTGGAAAACGACGCGG

AACTCGTAACGCTCGAATGCCGGAGAAGGTCTTATGGATGCGT

Mi109
109
CATCTTTTAAAGCGCTATCGAGCTGCGAAGAAGATTGACAAGCATCTTT
39746570 (Meloidogyne paranaensis)

ACCACGAGCTTTAC

Mi109
110
GATTTCCAACGCAAATTCTCGTCAGAACGTTCGTCGTTT
39746570 (Meloidogyne paranaensis)

Mi109
111
GATTTCCAACGCAAATTCTCGTCAGAACGTTCGTCGTTTG
39746726 (Meloidogyne paranaensis)

Mi109
112
GATTTCCAACGCAAATTCTCGTCAGAACGTTCGTCGTTTGGTCAAAGAT
39746574 (Meloidogyne paranaensis)

GGTCTTATAATTCGTAAGCCTGGCAAAGTTCATTCGCGTTTCCGAGCTC

GTGAACGTTTGGAAGCACGCAGAAAGGGTCGTCATATGGGTTTTGGAAA

ACGACGCGGAACTCGTAACGCTCGAATGCCGGAGAAGGTCTTATGGATG

CGT

Mi109
123
GCTGAGGCTCGCCGAAACAAGAACAAGGA
39746570 (Meloidogyne paranaensis)

Mi109
114
GTCAAAGATGGTCTTATAATTCGTAAGCCTGG
39746570 (Meloidogyne paranaensis)

Mi109
115
TGGATCCTAATGAGGTCAACG
39746574 (Meloidogyne paranaensis)

Mi109
116
TGTACTCCGTCATCTTTTAAAGCGCTATCGAGCTGCGAAGAAGATTGAC
39746726 (Meloidogyne paranaensis)

AAGCATCTTTACCACGAGCTTTACTTGAAGGCTAAAGGAAACGCATTTA

AGAATAAGAGAAATTTGATGGAATATATCTTCAAAAAGAAGACTGAGAA

CCAGCGTTCTAAACAACTTGCTGAA

Mi109
117
TGTACTCCGTCATCTTTTAAAGCGCTATCGAGCTGCGAAGAAGATTGAC
39746574 (Meloidogyne paranaensis)

AAGCATCTTTACCACGAGCTTTACTTGAAGGCTAAAGGAAACGCATTTA

AGAATAAGAGAAATTTGATGGAATATATCTTCAAAAAGAAGACTGAGAA

CCAGCGTTCTAAACAACTTGCTGAACAGGCTGAGGCTCGCCGAAACAAG

AACAAGGAAACGAGAAAGAGGCGTGAGGAGCGTTTGATTTTGAAACGTA

AGGAATTGCTCCACAAACTTTCTGAGAGCGAAAAGCAGAAGACTCCACA

GCTTCAAGAAGAGGAAGCGAAGGAAGCTTCGCCTTCTCCACCAC

Mi109
118
CGCAGAAAGGGTCGTCATATGGGT
4136397 (Onchocerca volvulus),

2898199 (Toxocara canis)

M1109
119
CGCAGAAAGGGTCGTCATATGGGT
45214539 (Wuchereria bancrofti)

Mi109
120
AATTCTCGTCAGAACGTTCGTCG
51863033 (Xiphinema index)

Mi111
121
CTTACACATCCAGAGTACTACGAAGAAATTGGAATCAAGCCT
15767736 (Meloidogyne arenaria)

Mi111
122
GGAAAAGGCTCCAACTGAAACGTACGCTGATGTTGGCGGTTTGGAACAA
15767736 (Meloidogyne arenaria)

CAAATTCAAGAAATTAAGGAGGCAGTGGAATTGCC

Mi111
123
AAGCGAAAGAAGGGAAGTAAAGGACCTGAAGCTAGCAATAAAATGCC
19384087 (Meloidogyne hapla)

Mi111
124
ATGGAAGAAGAATTTATTCGAAATCAGGAAAGAC
19384087 (Meloidogyne hapla)

Mi111
125
GAACATTATGTTAACATTCTTTCA
19384692 (Meloidogyne hapla)

Mi111
126
GAACATTATGTTAACATTCTTTCATTTGT
19384750 (Meloidogyne hapla)

Mi111
127
GAATTGCCACTTACACATCCAGA
19435681 (Meloidogyne hapia)

Mi111
128
GATGAAATGAGAGGGTCACCAATGGCTGTGGGTACATT
19384087 (Meloidogyne hapia)

Mi111
129
GGTTTGGAACAACAAATTCAAGAAATTAA
19435681 (Meloidogyne hapla)

Mi111
130
TGTGCAGTTCTTCTAAATCACAA
19435681 (Meloidogyne hapla)

Mi111
131
AACATTCTTTCATTTGTGGACAAGGAACAATTGGAGCCAGGCTGTGCAG
15767091 (Meloidogyne javanica)

TTCTTCTAAATCACAAAACACACGCAGTTGTTGGTGTTTTGGCTGATGA

CACGGATCCAATGGT

Mi111
132
AAGAAGGGAAGTAAAGGACCTGAAGCTAGCAATAAAATGCCTAATGTGA
40670817 (Meloidogyne javanica)

CACCAGTAACTCGTTGCAAATTGCG

Mi111
133
AAGGACCTGAAGCTAGCAATAAAATGCCTAATGTGACACCAGTAACTCG
15767091 (Meloidogyne javanica)

TTGCAAATTGCG

Mi111
134
CAGGAAGAAGAACGGACAAAAGTAGATGAAATGAGAGGGTCACCAATGG
15767091 (Meloidogyne javanica)

CTGTGGGTACATTGGAGGAGATTATTGACGATCAACACGC

Mi111
135
CAGGAAGAAGAACGGACAAAAGTAGATGAAATGAGAGGGTCACCAATGG
40670817 (Meloidogyne javanica)

CTGTGGGTACATTGGAGGAGATTATTGACGATCAACACGCAGTCGTTTC

Mi111
136
CCAGGTTCAGGAGATAAGCAAGGAGAAGGAGACAAGAAGCGTCGTTTTG
40670817 (Meloidogyne javanica)

GGCCTCCAATCCC

Mi111
137
CTTCTTAAATATGAACGGATTAAAGATTATTTGTTAATGGAAGAAGAAT
15767091 (Meloidogyne javanica)

TTATTCGAAATCAGGAAAGACATAAACCTCAAGA

Mi111
138
GTTGGCAGCGAACATTATGTTAACATTCTTTCATTTGTGGACAAGGAAC
40670817 (Meloidogyne javanica)

AATTGGAGCCAGGCTGTGCAGTTCTTCTAAATCACAAAACACACGCAGT

TGTTGGTGTTTTTGGCTGATGACACGGATCCAATGGTTTCTGTAATGAA

ATTGGAAAAGGCTCCAACTGAAACGTACGCTGATGTTGGCGGTTTGGAA

CAACAAATTCAAGAA

Mi111
139
TCTGTAATGAAATTGGAAAAGGCTCCAACTGAAACGTACGCTGATGTTG
15767091 (Meloidogyne javanica)

GCGGTTTGGAACAACAAATTCAAGAAATTAAGGAGGCAGTGGAATTGCC

ACTTACACATCCAGAGTACTA

Mi116
140
ATTGTGCGGTCATCTTGAAGA
1120680 (Caenorhabditis elegans)

Mi116
141
ACGGGATCCTCCAAAGTCAATTTGTAATTTTCACA
16004901 (Meloidogyne arenaria)

Mi16
142
GCAACTCTGGCACGGTCAATCAAAACACGAGCATCTGCACTCAAACCAG
16004901 (Meloidogyne arenaria)

CAAAAGCAGCCATTACGTGATCATCCAATTTGTGTATCTTTCGAATTGT

GCGGTCATCTTGAAGAACAGGAACAGATTTC

Mi116
143
GGTTCAGTAATAAAAAGATGAGGAGTTCCATCGGGATCAAAACCGCCAA
16004901 (Meloidogyne arenaria)

CAAGTAAAGAGACACCAAAAGGACGACGACCTGTAGTTTGCGTAAATTC

ATGTTTCAAGTCCGCAATAG

Mi116
144
TTCTCATTCCGGCCGATTGAGCCAGCAAGTAATTCGTAATACACACCAG
15767794 (Meloidogyne arenaria)

ATGGTTCAGTAATAAAAAGATGAGGAGTTCCATCGGGATCAAAACCGCC

AACAAGTAAAGAGACACCAAAAGGACGACGACCTGTAGTTTGCGTAAAT

TCATGTTTCAAGTCCGCAATAGTGCGGGAGATGTGCTCAAGTGTGACGG

GATCCTCCAAAGTCAATTTGTAATTTTCACAAGCAACTCTGGCACGGTC

AATCAAAACACGAGCATCTGCACTCAAACCAGCAAAAGCAGCCATTACG

TGATCATCCAATTTGTGTATCTTTCGAATTGTGCGGTCATCTTGAAGAA

CAGGAACAGATTTC

Mi116
145
AAAAAAATGTTAAAAGAAAATAT
30049385 (Meloidogyne chitwoodi)

Mi116
146
AAAAAATGTTAAAAGAAAATAT
30050083 (Meloidogyne chitwoodi)

Mi116
147
AAAAATGTTAAAAGAAAATAT
30166852 (Meloidogyne chitwoodi)

Mi116
148
GAAAAAAATGTTAAAAGAAAA
31325468 (Meloidogyne chitwoodi)

Mi116
149
GAAAAAAATGTTAAAAGAAAAT
31325428 (Meloidogyne chitwoodi)

Mi116
150
GAAAAAAATGTTAAAAGAAAATA
31325427 (Meloidogyne chitwoodi)

Mi116
151
GAAAAAAATGTTAAAAGAAAATAT
30049285 (Meloidogyne chitwoodi)

Mi116
152
GACAAGCATCTTTACCACGAGCT
88681932 (Caenorhabditis remanei)

Mi125
153
ATAAGAACACGAGCATGGTGAATTGATTTAGCCAATCCC
16004848 (Meloidogyne arenaria)

Mi125
154
GGAGGTCCACCAGCAAGATAAGATTTTGTGGAGAAGTCAAT
16004848 (Meloidogyne arenaria)

Mi125
155
TCCAAAAGAACTCCAATACGAACCAAACGACGAAGAAGAGCATTT
16004848 (Meloidogyne arenaria)

Mi125
156
TCCTCCACCTCCCGAAGCAGCCTTCTTCAAATTCTTTCTCTTGACACGT
16004848 (Meloidogyne arenaria)

CC

Mi125
157
TGCTTTTGAGAGTCGAGACGAACAATGAATGATGGAATATTGACTACTT
16004848 (Meloidogyne arenaria)

GTCGACGAATTCGAAT

Mi125
158
TTTTATAAACCTTTAGATATTATTCAATCCTCTTCAT
16004848 (Meloidogyne arenaria)

Mi125
159
AAAAGTTCACGAGCAGCTTTACG
31325000 (Meloidogyne chitwoodi)

Mi125
160
AAGAACTCCAATACGAACCAAACGACGAAGAAGAGCATTTCCTTC
32185135 (Meloidogyne chitwoodi)

Mi125
161
AATGATGGAATATTGACTACTTGTC
32185135 (Meloidogyne chitwoodi)

Mi125
162
AATTTTAATTCCTGATCTAAACGCTC
32185135 (Meloidogyne chitwoodi)

Mi125
163
ACACGTCCAGGAGGTCCACCAGCAAGATAAGATTTTGT
32185135 (Meloidogyne chitwoodi)

Mi125
164
CCAAGAACATAGTCCAATTTCAT
32185135 (Meloidogyne chitwoodi)

Mi125
165
CTCAAACTGGAGTAATTAAAG
37819228 (Meloidogyne chitwoodi)

Mi125
166
GCTCAAACTGGAGTAATTAAAC
37819133 (Meloidogyne chitwoodi)

Mi125
167
GTCTGCAAACGACGCTCCAAAAAGTCTT
32183696 (Meloidogyne chitwoodi)

Mi125
168
GTCTGCAAACGACGCTCCAAAAAGTCTTCAACACGCAA
32185135 (Meloidogyne chitwoodi)

Mi125
169
AGCTCAAACTGGAGTAATTAAAC
38513039 (Meloidogyne hapla)

Mi125
170
CTCAAACTGGAGTAATTAAAC
37970857 (Meloidogyne hapla)

Mi125
171
GCTCAAACTGGAGTAATTAAAC
37971092 (Meloidogyne hapla)

Mi125
172
TAAGCTCAAACTGGAGTAATTAAAC
37970990 (Meloidogyne hapla)

Mi125
173
AATAAGCGTCGAGGATCCTTGTCTTCCAATGTTAAAAGTTCACGAGC
9829373 (Meloidogyne javanica)

Mi125
174
AATTTTAATTCCTGATCTAAACGCTCCTTCTCGAATGGACG
9829373 (Meloidogyne javanica)

Mi125
175
ATAAGAACACGAGCATGGTGAATTGATTTAGCCAATCCC
15766790 (Meloidogyne javanica)

Mi125
176
ATAAGAACACGAGCATGGTGAATTGATTTAGCCAATCCCAATTTGAAAA
9829373 (Meloidogyne javanica)

CTTG

Mi125
177
CGAGGCACGTCATAAACTTTAGAAAATACATGCTTTAT
9829373 (Meloidogyne javanica)

Mi125
178
CTCCTCCACCTCCCGAAGCAGCCTTCTTCAAATTCTTTCTCTTGACACG
9829373 (Meloidogyne javanica)

TCC

Mi125
179
GAATCTTTGCCAAAGTTAATTTGAC
9829639 (Meloidogyne javanica)

Mi125
180
GACGCTCCAAAAAGTCTTCAACACGCAAACCAAGAAC
9829564 (Meloidogyne javanica)

Mi125
181
GCTTTACGAATCTTTGCCAAAGTTAATTTGACACGCCAAACTTCACG
9829373 (Meloidogyne javanica)

Mi125
182
GGAGGTCCACCAGCAAGATAAGATTTTGTGGAGAAGTCAAT
15766790 (Meloidogyne javanica)

Mi125
183
TCAACAAAAACATTTTTTCAAATCCAAAATCTTTTAACAACTTAACAG
15766790 (Meloidogyne javan Ca)

Mi125
184
TCCAAAAGAACTCCAATACGAACCAAACGACGAAGAAGAGCATTTCCTT
15766790 (Meloidogyne javan ica)

C

Mi125
185
TGCAAACGACGCTCCAAAAAGTCTTCAACACGCAAACCAAGAAC
9829373 (Meloidogyne javanica)

Mi125
186
TGCTTTTGAGAGTCGAGACGAACAATGAATGATGGAATATTGACTACTT
15766790 (Meloidogyne javanica)

GTCGACGAATTCGAAT

Mi125
187
TTTTATAAACCTTTAGATATTATTCAATCCTCTTCATCGTCACCGCCTC
15766790 (Meloidogyne javanica)

CTCCTCCACCTCCCGAAGCAGCCTTCTTCAAATTCTTTCTCTTGACACG

TCC

Mi125
188
AAGCTCAAACTGGAGTAATTA
39746721 (Meloidogyne paranaensis)

Mi125
189
ACGCCAAACTTCACGTTTATTTCGTAA
39747139 (Meloidogyne paranaensis)

Mi125
190
AGCTCAAACTGGAGTAATTAAAC
39746611 (Meloidogyne paranaensis)

Mi125
191
CAGCTTTACGAATCTTTGCCAAAGTTAATTTGA
39747139 (Meloidogyne paranaensis)

Mi125
192
CAGCTTTACGAATCTTTGCCAAAGTTAATTTGACACGCCAAACTTCACG
46498505 (Meloidogyne paranaensis)

TTTATTTCGTAA

Mi125
193
CAGCTTTACGAATCTTTGCCAAAGTTAATTTGACACGCCAAACTTCACG
39747346 (Meloidogyne paranaensis)

TTTATTTCGTAAACC

Mi125
194
CCAAACTCGCCAATTAATTTTAATTCCTGATCTAAACGCTCCTTCTCGA
46498505 (Meloidogyne paranaensis)

ATGGACGA

Mi125
195
CCAAACTCGCCAATTAATTTTAATTCCTGATCTAAACGCTCCTTCTCGA
39747139 (Meloidogyne paranaensis)

ATGGACGACGAGGCACGTCATA

Mi125
196
CCAAACTCGCCAATTAATTTTAATTCCTGATCTAAACGCTCCTTCTCGA
46498634 (Meloidogyne paranaensis)

ATGGACGACGAGGCACGTCATAAACTTTAGAAAATACATGC1TTATGCG

AGGCATCTTGTTTTACTTTTTAAGCTCAAACT

Mi125
197
CTCAAACTGGAGTAATTAAAC
39746528 (Meloidogyne paranaensis)

Mi125
198
CTTTCAACGTTTTATAAACCTTTAGATATTATTCAATCCTCTTCATCGT
39747032 (Meloidogyne paranaensis)

CACCGCCTCCTCCTCCACCTCCCGAAGCAGCCTTCTTCAAATTCTTTCT

CTTGACACGTCCAGGAGGTCCACCAGCAAGATAAGA

Mi125
199
GCTCAAACTGGAGTAATTAAAC
39746628 (Meloidogyne paranaensis)

Mi125
200
GTCTGCAAACGACGCTCCAAAAAGTCTTCAACACGCAAACCAAGAAC
39747032 (Meloidogyne paranaensis)

Mi125
201
TCCAAAAGAACTCCAATACGAACCAAACGACGAAGAAGAGCATTTCCTT
39747032 (Meloidogyne paranaensis)

C

Mi125
202
TCGAATGGACGACGAGGCACGTCATAAACTTTAGAAAATACATGCTTTA
39747346 (Meloidogyne paranaensis)

T

Mi125
203
TCGCCAATTAATTTTAATTCCTGATCTAAACGCTCCTT
39747346 (Meloidogyne paranaensis)

Mi125
204
TGCTTTTGACGAATAAGAACACGAGCATGGTGAATTGATTTAGCCAATC
39747032 (Meloidogyne paranaensis)

CCAATTTGAAAACTTG

Mi125
205
TGCTTTTGAGAGTCGAGACGAACAATGAATGATGGAATATTGACTACTT
39747032 (Meloidogyne paranaensis)

GTCGACGAATTCGAAT

Mi125
206
TTTTACTTTTTAAGCTCAAAC
39747346 (Meloidogyne paranaensis)

Mi125
207
TCTCGAATGGACGACGAGGCACGTCATA
51237570 (Radopholus similis)

Mi125
208
TTCTCGAATGGACGACGAGGCACGTCATA
51237458 (Radopholus similis)

Mi125
209
AAGAAGAGCATTTCCTTCAAA
9830677 (Strongyloides stercoralis)

Mi125
210
AAGTTCACGAGCAGCTTTACG
9832721 (Strongyloides stercoralis)

Mi125
211
AATAAACGTGAAGTTTGGCGTGT
115342194 (Heterorhabditis bacteriophora)

Mi125
212
TTCGTAAAGCTGCTCGTGAACTT
115342194 (Heterorhabditis bacteriophora)

Mi125
213
GAAGGAAATGCTCTTCTTCGTCGTTTGGT
115342194 (Heterorhabditis bacteriophora)

Mi127
214
CCTTGATCATTGGCAATGATCTC
19556133 (Ascaris suum)

Mi127
215
GTTGGCTCGTTGATGATACGCAAAAC
14019989 (Ascaris suum)

Mi127
216
AGCTCAAACTTGGGTAATTAAA
1850670 (Brugia malayi)

Mi127
217
AGCTCAAACTTGGGTAATTAAAC
1893269 (Brugia malayi)

Mi127
218
CTCAAACTTGGGTAATTAAAC
1017391 (Brugia malayi)

M1127
219
GCTCAAACTTGGGTAATTAAA
1193277 (Brugia malayi)

Mi127
220
GCTCAAACTTGGGTAATTAAAC
1871110 (Brugia malayi)

Mi127
221
TAGCTCAAACTTGGGTAATTA
1850558 (Brugia malayi)

Mi127
222
TAGCTCAAACTTGGGTAATTAAAC
1917062 (Brugia malayi)

Mi127
223
AGCTCAAACTTGGGTAATTAAAC
6578024 (Brugia pahangi)

Mi127
224
CTCAAACTTGGGTAATTAAAC
6578007 (Brugia pahangi)

Mi127
225
GCTCAAACTTGGGTAATTAAAC
6578012 (Brugia pahangi)

Mi127
226
TAGCTCAAACTTGGGTAATTAAAC
6578013 (Brugia pahangi)

Mi127
227
AGCTCAAACTTGGGTAATTAA
14833886 (Caenorhabditis elegans)

Mi127
228
AGCTCAAACTTGGGTAATTAAA
10925161 (Caenorhabditis elegans)

Mi127
229
AGCTCAAACTTGGGTAATTAAAC
10924962 (Caenorhabditis elegans)

Mi127
230
ATAGCTCAAACTTGGGTAATTA
31233223 (Caenorhabditis elegans)

Mi127
231
ATAGCTCAAACTTGGGTAATTAA
14835549 (Caenorhabditis elegans)

Mi127
232
ATAGCTCAAACTTGGGTAATTAAA
10925347 (Caenorhabditis elegans)

Mi127
233
CTCAAACTTGGGTAATTAAAC
2423515 (Caenorhabditis elegans)

Mi127
234
GATAGCTCAAACTTGGGTAATTA
33943631 (Caenorhabditis elegans)

Mi127
235
GATAGCTCAAACTTGGGTAATTAA
18287564 (Caenorhabditis elegans)

Mi127
236
GCAGCAGTTGGCTCGTTGATGA
71986023 (Caenorhabditis elegans)

Mi127
237
GCTCAAACTTGGGTAATTAAA
10925190 (Caenorhabditis elegans)

Mi127
238
GCTCAAACTTGGGTAATTAAAC
10923462 (Caenorhabditis elegans)

Mi127
239
TAGCTCAAACTTGGGTAATTA
684987 (Caenorhabditis elegans)

Mi127
240
TAGCTCAAACTTGGGTAATTAA
14838520 (Caenorhabditis elegans)

Mi127
241
TAGCTCAAACTTGGGTAATTAAA
14825166 (Caenorhabditis elegans)

Mi127
242
GCAGCAGTTGGCTCGTTGATGATACG
68276618 (Caenorhabditis remanei)

Mi127
243
AGCTCAAACTTGGGTAATTAA
11068960 (Haemonchus contortus)

Mi127
244
AGCTCAAACTTGGGTAATTAAA
11068981 (Haemonchus contortus)

Mi127
245
GCTCAAACTTGGGTAATTAAA
11068764 (Haemonchus contortus)

Mi127
246
TAGCTCAAACTTGGGTAATTA
61116185 (Litomosoides sigmodontis)

Mi127
247
CGATTTCCTTGATCATTGGCAATGAT
32184674 (Meloidogyne chitwoodi)

Mi127
248
AATAAGTCGTTCCCAGATCAATTCCGACAGCGTTAGCTTTAGACATTTT
39747367 (Meloidogyne paranaensis)

T

Mi127
249
AATAAGTCGTTCCCAGATCAATTCCGACAGCGTTAGCTTTAGACATTTT
39747357 (Meloidogyne paranaensis)

TAAAAG

Mi127
250
ACGTTGCGCTCGCCTTGGCCTTTCTTATCCAAACCATAAGCAATAGCCG
39747367 (Meloidogyne paranaensis)

CAGCAGT

Mi127
251
GGCTCGTTGATGATACGCAAAACATTAAGTCCAGAAATCGTTCCAGCAT
39747367 (Meloidogyne paranaensis)

CCTTTGTGGCTTGGCGTTGACTGTCATTAAAGTACGCAGGCACAGTGAT

GACAGCATCCTTAACGGTTTGGCCCAAAAATGCCTCAGCCGTTTCCTTC

ATTTTAGTCAAAACCATGGCGGAGACTTCCTCAGGGAAGAACGCCTTCA

TTTCACCCTTAACCTCCACTTGGATTTTTGGACGAGCACCTTCTCCTTG

AATAACCTTGAACGGCCAATGCTTCATGTCAGATTGAACAGCTGGATCG

TCAAATTTGCGTCCAATCAAGCGCTTTGCGTCAAAAACAGTATTCGACG

GGTTCATTGCCACTTGATTTTTCGCAGCATCACCAATCAGACGTTCCGT

GTCTGTAAAAGCCACATAAGATGGAGTCGTGCGATTTCCTTGATCATTG

GCAATGATCTCGACTTTTCCATGCTGGAATACACC

Mi127
252
TAGCTCAAACTTGGGTAATTA
46498358 (Meloidogyne paranaensis)

Mi127
253
TCAAAAGTTCCACCGCCCAAGTCAAAAATCA
39747367 (Meloidogyne paranaensis)

Mi127
254
CTCAAACTTGGGTAATTAAAC
10840960 (Onchocerca volvulus)

Mi127
255
TCAGCCGTTTCCTTCATTTTA
4191634 (Onchocerca volvulus)

Mi127
256
ACCTTGAACGGCCAATGCTTCAT
20062709 (Toxocara canis)

Mi127
257
CCTTGATCATTGGCAATGATCTC
20499328 (Toxocara canis)

Mi127
258
CTCAAACTTGGGTAATTAAAC
1498474 (Toxocara canis)

Mi127
259
GTCAAAATAGACACATCAAAAGT
2104671 (Trichinella britovi)

Mi127
260
TCGTTGATGATACGCAAAACATT
2104671 (Trichinella britovi)

Mi127
261
TCGTTGATGATACGCAAAACATT
13199118 (Trichinella spiralis)

Mi127
262
ACCTTGAACGGCCAATGCTTCATGTC
17353707 (Trichuris muris)

Mi127
263
ACCTTGAACGGCCAATGCTTCAT
28561608 (Trichuris vulpis)

Mi127
264
TAGCTCAAACTTGGGTAATTA
2789663 (Wuchereria bancrofti)

Mi127
265
AAAGCTAACGCTGTCGGAATTGATCT
115342160 (Heterorhabditis bacteriophora)

Mi127
266
CGTATCATCAACGAGCCAACTGCTGC
88679042 (Caenorhabditis remanei)

Mi127
267
GGTTTAATTACCCAAGTTTGA
9112994Nippostrongylus brasiliensis)

Mi127
268
GGTTTAATTACCCAAGTTTGAG
83656004Heterorhabditis bacteriophora)

Mi127
269
GGTTTAATTACCCAAGTTTGAG
91103082 (Nippostrongylus brasiliensis)

Mi127
270
GGTTTAATTACCCAAGTTTGAGCT
91102738 (Nippostrongylus brasiliensis)

Mi127
271
GGTTTAATTACCCAAGTTTGAGCTA
91102815 (Nippostrongylus brasiliensis)

Mi127
272
GTTTAATTACCCAAGTTTGAG
91102668 (Nippostrongylus brasiliensis)

Mi127
273
GTTTAATTACCCAAGTTTGAGC
91103120 (Nippostrongylus brasiliensis)

Mi127
274
TCATCAACGAGCCAACTGCTGC
86563795 (Caenorhabditis elegans)

Mi127
275
TTAATTACCCAAGTTTGAGCT
3096950Brugia pahangi)

Mi127
276
TTAATTACCCAAGTTTGAGCT
14832559 (Caenorhabditis elegans)

Mi127
277
TTAATTACCCAAGTTTGAGCTA
14829900 (Caenorhabditis elegans)

Mi127
278
TTAATTACCCAAGTTTGAGCTAT
14842179 (Caenorhabditis elegans)

Mi127
279
TTTAATTACCCAAGTTTGAGC
31237454 (Caenorhabditis elegans)

Mi128
280
CGTGCAAAGAGAAAGCGTGACGA
808508 (Brugia malayi)

Mi128
281
TTGTTCGCGAGGTTGTTGGTTT
10713865 (Heterodera glycines)

Mi128
282
TTGTTCGCGAGGTTGTTGGTTT
35504991 (Heterodera schachtii)

Mi128
283
AAGAAGGCTTTGAAGTTTTTGAAGAA
32183499 (Meloidogyne chitwoodi)

Mi128
284
GGTTTAATTACCTACGTTTGA
37820376 (Meloidogyne chitwoodi)

Mi128
285
GGTTTAATTACCTACGTTTGAG
37819977 (Meloidogyne chitwoodi)

Mi128
286
GGTTTAATTACCTACGTTTGAGA
37819540 (Meloidogyne chitwoodi)

Mi128
287
GGTTTAATTACCTACGTTTGAGAA
37820112 (Meloidogyne chitwoodi)

Mi128
288
GGTTTAATTACCTACGTTTGAGAAATTT
37819939 (Meloidogyne chitwoodi)

Mi128
289
AGAAGGCTTTGAAGTTTTTGAAGAA
46988391 (Meloidogyne hapla)

Mi128
290
AGAATGACAAAGTTACAACAT
37972035 (Meloidogyne hapla)

Mi128
291
ATGAGAAAGCATCATAAGTGA
37972035 (Meloidogyne hapla)

Mi128
292
ATGGAATTGCTTCGTATCAGTAAGG
27540048 (Meloidogyne hapla)

Mi128
293
ATGGAATTGCTTCGTATCAGTAAGGACAAGAAGG
46987443 (Meloidogyne hapla)

Mi128
294
ATGGAATTGCTTCGTATCAGTAAGGACAAGAAGGCTTTGAAGTTTTTGA
37972035 (Meloidogyne hapla)

AGAA

Mi128
295
GGACAAGAAGGCTTTGAAGTTTTTGAAGAA
27540048 (Meloidogyne hapla)

Mi128
296
GGTTTAATTACCTACGTTTGAG
37970947 (Meloidogyne hapla)

Mi128
297
GGTTTAATTACCTACGTTTGAGA
37970869 (Meloidogyne hapla)

Mi128
298
GGTTTAATTACCTACGTTTGAGAA
38513179 (Meloidogyne hapla)

Mi128
299
GGTTTAATTACCTACGTTTGAGAAA
38513227 (Meloidogyne hapla)

Mi128
300
AACATCCAGCTGTTGAGGGTCTTGCTGTTGGTTTGGATCGTGGCCATCC
9829663 (Meloidogyne javanica)

Mi128
301
AACATCCAGCTGTTGAGGGTCTTGCTGTTGGTTTGGATCGTGGCCATCC
9828974 (Meloidogyne javanica)

TGTGACTAAAAATGTTCGCAAGCCACG

Mi128
302
AAGGAAAACATTCAATCACAGTTTTAATTGTTAAAAA
15003757 (Meloidogyne javanica)

Mi128
303
ACATCCAGCTGTTGAGGGTCTT
9829249 (Meloidogyne javanica)

Mi128
304
ACTATGGAATTGCTTCGTATCAGTA
9829867 (Meloidogyne javanica)

Mi128
305
ACTATGGAATTGCTTCGTATCAGTAAGGACAAGAAGGCTTTGAAGTTTT
9828974 (Meloidogyne javanica)

TGAAGAAGCGAATTGGACA

Mi128
306
AGAAGCGAATTGGACAACATGTCCGTGCAAAGAGAAAGCGTGACGA
15003757 (Meloidogyne javanica)

Mi128
307
CAGCTGTTGAGGGTCTTGCTGTTGGTTTGGATCGTGGCCATCCTGTGAC
9829867 (Meloidogyne javanica)

TAAAAATGTTCGCAAGCCACG

Mi128
308
CATCCAGCTGTTGAGGGTCTTGCTGTTGGTTTGGATCGTGGCCATCCTG
9829640 (Meloidogyne javanica)

TGACTAAAAATGTTCGCAAGCCACG

Mi128
309
CATCCTGTGACTAAAAATGTTCGCAAGCCACGTCAATCTCGTAAAAAAG
9829249 (Meloidogyne javanica)

G

Mi128
310
CATGTCCGTGCAAAGAGAAAGCGTGACGAAATCCAACGAATACTCGTTG
9829663 (Meloidogyne javanica)

AGATGAGAAAGCATCATAAGTGAGAAAATATAAGGGAAGGAAAACATTC

AATCACAGTTTTAATTGTTAAAAA

Mi128
311
CATGTCCGTGCAAAGAGAAAGCGTGACGAAATCCAACGAATACTCGTTG
9828974 (Meloidogyne javanica)

AGATGAGAAAGCATCATAAGTGAGAAAATATAAGGGAAGGAAAACATTC

AATCACAGTTTTAATTGTTAAAAATAATGTT

Mi128
312
CGAATTGTTCGTGAAGTTGTTCGCGAGGTTGTTGGTTTTTC
9828974 (Meloidogyne javanica)

Mi128
313
CGAATTGTTCGTGAAGTTGTTCGCGAGGTTGTTGGTTTTTCACCTTATG
9829249 (Meloidogyne javanica)

AGCGTCGAACTATGGAATTGCTTCGTATCAGTAAGGACAAGAAGGCTTT

GAAGTTTTTGAAGAAGCGAATTGGACAACATGTCCGTGCAAAGAGAAAG

CGTGACGA

Mi128
314
GAATTGTTCGTGAAGTTGTTCGCGAGGTTGTTGGTTTTTCACCTTATGA
15003757 (Meloidogyne javanica)

GCGTCGAACTATGGAATTG

Mi128
315
GTGACTAAAAATGTTCGCAAGCCACG
9829663 (Meloidogyne javanica)

Mi128
316
GTTGAGATGAGAAAGCATCATAAGT
9829249 (Meloidogyne javanica)

Mi128
317
TATGCGATTGAATAAATTTGTTAGAATTCA
9829640 (Meloidogyne javanica)

Mi128
318
TGAGATGAGAAAGCATCATAAGT
15003757 (Meloidogyne javanica)

Mi28
319
TTCGTATCAGTAAGGACAAGAAGGCTTTGAAGTTTTTG
15003757 (Meloidogyne javanica)

Mi128
320
CGTGCAAAGAGAAAGCGTGACGA
31229640 (Wuchereria bancrofti)

Mi128
321
AACATGGTTCCATTCCCACGTCTTCA
14851165 (Caenorhabditis elegans)

Mi128
322
GTTGAATGGATTCCAAACAACGT
14851165 (Caenorhabditis elegans)

Mi129
323
AACATGGTTCCATTCCCACGTCTTCACTT
21809191 (Ancylostoma caninum),

24559473 (Ancylostoma ceylanicum),

24559473 (Ancylostoma ceylanicum),

68303302 (Ancylostoma caninum),

103002658 (Caenorhabditis Elegans),

72003699 (Caenorhabditis elegans)

Mi129
324
ATGGACGAGATGGAATTCACTGA
103002658 (Caenorhabditis Elegans)

Mi129
325
AACATGGTTCCATTCCCACGTCTTCACTT
24559473 (Ancylostoma ceylanicum)

Mi129
326
AACATGGTTCCATTCCCACGTCTTCACTT
66734013 (Ancylostoma duodenale)

Mi129
327
AAGATTCGTGAGGAGTATCCGGACCG
17972056 (Ascaris suum)

Mi129
328
AGGTTTAATTACCCAAGTTTG
16746749 (Ascaris suum);

14854924Caenorhabditis elegans

Mi129
329
GAGGCTTTGTACGACATCTGCT
16747657 (Ascaris suum)

Mi129
330
GAGGCTTTGTACGACATCTGCTTC
11140987 (Ascaris suum)

Mi129
331
GCTTTGTACGACATCTGCTTC
15784432 (Ascaris suum)

Mi129
332
GGTTTAATTACCCAAGTTTGA
1871111 (Brugia malayi),

91102994 (Nippostrongylus brasiliensis),

10925140 (Caenorhabditis elegans),

6578011 (Brugia pahangi)

Mi129
333
GGTTTAATTACCCAAGTTTGAG
1917158 (Brugia malayi),

83655795 (Heterorhabditis bacteriophora),

91103082 (Nippostrongylus brasiliensis),

10923350 (Caenorhabditis elegans),

1498474 (Toxocara canis),

6578007 (Brugia pahangi)

M1129
334
GGTTTAATTACCCAAGTTTGAGT
1017391 (Brugia malayi),

91102995 Nippostrongylus brasiliensis,

10923353 (Caenorhabditis elegans),

6578016 (Brugia pahangi)

Mi129
335
GGTTTAATTACCCAAGTTTGAGTT
1345290 (Brugia malayi),

83656004 (Heterorhabditis bacteriophora),

91102904 (Nippostrongylus brasiliensis),

10924806 (Caenorhabditis elegans),

1498477 (Toxocara canis)

Mi129
336
GGTTTAATTACCCAAGTTTGAGTTT
1870969 (Brugia malayi),

10923537 (Caenorhabditis elegans)

Mi129
337
GTTTAATTACCCAAGTTTGAG
1017394 (Brugia malayi),

91102668 (Nippostrongylus brasiliensis),

2423515 (Caenorhabditis elegans)

Mi129
338
GTTTAATTACCCAAGTTTGAGT
1870808 (Brugia malayi)

Mi129
339
GTTTAATTACCCAAGTTTGAGTT
1871103 (Brugia malayi)

Mi129
340
GTTTAATTACCCAAGTTTGAGTTT
1893261 (Brugia malayi)

Mi129
341
TTAATTACCCAAGTTTGAGTT
1345262 (Brugia malayi)

Mi129
342
AACATGGTTCCATTCCCACGTCTTCA
14851165 (Caenorhabditis elegans)

Mi129
343
AACATGGTTCCATTCCCACGTCTTCACTTT
47671577 (Caenorhabditis elegans)

Mi129
344
AATTACCCAAGTTTGAGTTTT
40643118 (Caenorhabditis elegans)

Mi129
345
AATTACCCAAGTTTGAGTTTTA
47594382 (Caenorhabditis elegans)

Mi129
346
AATTACCCAAGTTTGAGTTTTAC
47628305 (Caenorhabditis elegans)

Mi129
347
ACATGGTTCCATTCCCACGTCTTCACTT
14854924 (Caenorhabditis elegans)

Mi129
348
AGGTTTAATTACCCAAGTTTG
14824308 (Caenorhabditis elegans)

Mi129
349
AGGTTTAATTACCCAAGTTTGA
10925161 (Caenorhabditis elegans)

Mi129
350
AGGTTTAATTACCCAAGTTTGAG
10924911 (Caenorhabditis elegans)

Mi129
351
ATGGACGAGATGGAATTCACTGA
277183 (Caenorhabditis elegans)

Mi129
352
ATGGACGAGATGGAATTCACTGAAGC
14641526 (Caenorhabditis elegans)

Mi129
353
CAACATGGTTCCATTCCCACGTCTTCACTT
1121008 (Caenorhabditis elegans)

Mi129
354
CTTGTTGAGAACACCGACGAGACTTACTG
2386822 (Caenorhabditis elegans)

Mi129
355
GGAAACAATTGGGCCAAAGGACA
18246795 (Caenorhabditis elegans)

Mi129
356
GGTTCCATTCCCACGTCTTCA
14855460 (Caenorhabditis elegans)

Mi129
357
GTCAACATGGTTCCATTCCCACGT
47651088 (Caenorhabditis elegans)

Mi129
358
GTCAACATGGTTCCATTCCCACGTCTT
18321206 (Caenorhabditis elegans)

Mi129
359
GTCAACATGGTTCCATTCCCACGTCTTCACTT
1120739 (Caenorhabditis elegans)

Mi129
360
GTCAACATGGTTCCATTCCCACGTCTTCACTTT
47656473 (Caenorhabditis elegans)

Mi129
361
TAATTACCCAAGTTTGAGTTT
18248382 (Caenorhabditis elegans)

Mi129
362
TAATTACCCAAGTTTGAGTTTT
18268138 (Caenorhabditis elegans)

Mi129
363
TAATTACCCAAGTTTGAGTTTTA
18269837 (Caenorhabditis elegans)

Mi129
364
TACAACGCAACTCTTTCTGTTCATCAACTTGT
47641436 (Caenorhabditis elegans)

Mi129
365
TACAACGCAACTCTTTCTGTTCATCAACTTGTTGA
1120154 (Caenorhabditis elegans)

Mi129
366
TACTTTGTTGAATGGATTCCAAACAACGT
30744925 (Caenorhabditis elegans)

Mi129
367
TGAATGGATTCCAAACAACGT
1121447 (Caenorhabditis elegans)

Mi129
368
TGGTTCCATTCCCACGTCTTCACTT
18323141 (Caenorhabditis elegans)

Mi129
369
TGTCAACATGGTTCCATTCCCACGTCTTCA
56145926 (Caenorhabditis elegans)

Mi129
370
TTAATTACCCAAGTTTGAGTT
14833204 (Caenorhabd itis elegans)

Mi129
371
TTAATTACCCAAGTTTGAGTTT
14831587 (Caenorhabditis elegans)

Mi129
372
TTAATTACCCAAGTTTGAGTTT
14833268 (Caenorhabditis elegans)

Mi129
373
TTAATTACCCAAGTTTGAGTTTTA
14837998 (Caenorhabditis elegans),

15767282 (Meloidogyne javanica)

Mi129
374
TTCCGTGGACGTATGTCAATG
14641526 (Caenorhabditis elegans)

Mi129
375
TTGGCTGTCAACATGGTTCCATTCCC
47725169 (Caenorhabditis elegans)

Mi129
376
TTGGCTGTCAACATGGTTCCATTCCCACGTC
18324147 (Caenorhabditis elegans)

Mi129
377
TTGGCTGTCAACATGGTTCCATTCCCACGTCTTCA
71987928 (Caenorhabditis elegans)

Mi129
378
TTTAATTACCCAAGTTTGAGT
10925180 (Caenorhabditis elegans),

91103983 (Nippostrongylus brasiliensis),

11068715 (Haemonchus contortus)

Mi129
379
TTTAATTACCCAAGTTTGAGTT
14824445 (Caenorhabditis elegans),

91103246 (Nippostrongylus brasiliensis)

Mi129
380
TTTAATTACCCAAGTTTGAGTTT
14832338 (Caenorhabditis elegans),

91104236 (Nippostrongylus brasiliensis)

Mi129
381
TTTAATTACCCAAGTTTGAGTTTT
14825571 (Caenorhabditis elegans)

Mi129
382
TTTAATTACCCAAGTTTGAGTTTTA
14824404 (Caenorhabditis elegans)

Mi129
383
TTTAATTACCCAAGTTTGAGTTTTAC
14829782 (Caenorhabditis elegans)

Mi129
384
GGTTCCAAGTTCTGGGAAGTCAT
68277783 (Caenorhabditis remanel)

Mi129
385
GAATCAATGTTTACTACAATGA
37594691 (Cooperia oncophora)

Mi129
386
GTTGAATGGATTCCAAACAACGT
37594691 (Cooperia oncophora),

14855460 (Caenorhabditis elegans),

Mi129
387
AACCAAATTGGTTCCAAGTTCTGGGAAGT
53748584 (Cyathostomum catinatum)

Mi129
388
GAAGGTATGGACGAGATGGAATTCACTGAAGCCGAGTC
50402529 (Cyathostomum catinatum)

Mi129
389
GATGCCAAGAATATGATGGCTGCTTG
50402529 (Cyathostomum catinatum)

Mi129
390
TTCACTGAAGCCGAGTCCAACATGAA
22297092 (Cyathostomum catinatum),

22297112 (Cyathostomum pateratum)

Mi129
391
AACCAAATTGGTTCCAAGTTCTGGGAAGT
22297084 (Cyathostomum coronatum)

Mi129
392
GAAGGTATGGACGAGATGGAATTCACTGAAGCCGAGTC
22297134 (Cyathostomum coronatum)

Mi129
393
AACCAAATTGGTTCCAAGTTCTGGGAAGT
22297080 (Cyathostomum pateratum)

Mi129
394
GAAGGTATGGACGAGATGGAATTCACTGAAGCCGAGTC
53748578 (Cyathostomum pateratum)

Mi129
395
GATGCCAAGAATATGATGGCTGCTTG
53748578 (Cyathostomum pateratum)

Mi129
396
AACCAAATTGGTTCCAAGTTCTGGGAAGT
22297100 (Cylicocyclus insigne)

Mi129
397
ATGTTTGATGCCAAGAATATGATGGCTGCTTG
22297100 (Cylicocyclus insigne)

Mi129
398
TTGGTTCCAAGTTCTGGGAAGT
22297102 (Cylicocyclus insigne)

Mi129
399
AACCAAATTGGTTCCAAGTTCTGGGAAGT
10179955 (Cylicocyclus nassatus)

Mi129
400
TTCACTGAAGCCGAGTCCAACATGAA
10179955 (Cylicocyclus nassatus)

Mi129
401
AACCAAATTGGTTCCAAGTTCTGGGAAGT
22297108 (Cylicocyclus radiatus)

Mi129
402
ATGTTTGATGCCAAGAATATGATGGCTGCTTG
22297108 (Cylicocyclus radiatus)

Mi129
403
ACTGAAGCCGAGTCCAACATGAA
53748590 (Cylicostephanus goldi)

Mi129
404
GATGCCAAGAATATGATGGCTGCTTG
53748590 (Cylicostephanus goldi)

Mi129
405
AACCAAATTGGTTCCAAGTTCTGGGAAGT
53748588 (Cylicostephanus longibursatus)

Mi129
406
AAGAATATGATGGCTGCTTGTGATCC
53748588 (Cylicostephanus longibursatus)

Mi129
407
TTCACTGAAGCCGAGTCCAACATGAA
53748588 (Cylicostephanus longibursatus)

Mi129
408
AATTACCCAAGTTTGAGTTTT
452448 (Dirofilaria immitis)

Mi129
409
AGGTTTAATTACCCAAGTTTG
15499065 (Haemonchus contortus);

91103285Nippostrongylus brasiliensis

Mi129
410
GAACGAATCAATGTTTACTACAATGAGGCT
159156 (Haemonchus contortus)

Mi129
411
GAATGGATTCCAAACAACGTTAAGACTGC
159160 (Haemonchus contortus)

Mi129
412
TAATTACCCAAGTTTGAGTTT
27539282 (Haemonchus contortus)

Mi129
413
TGAACACGGAATTCAACCTGATGG
10122229 (Haemonchus contortus)

Mi129
414
TGAACACGGAATTCAACCTGATGGA
27590544 (Haemonchus contortus)

Mi129
415
TGGTACACTGGCGAAGGTATGGACGA
27925410 (Haemonchus contortus)

Mi129
416
TTAATTACCCAAGTTTGAGTT
11068983 (Haemonchus contortus)

Mi129
417
TTAATTACCCAAGTTTGAGTT
11068983 (Haemonchus contortus)

Mi129
418
TTTAATTACCCAAGTTTGAGTT
11068785 (Haemonchus contortus)

Mi129
419
ATGTTCCGTCGCAAGGCTTTCCT
27428224 (Heterodera glycines)

Mi129
420
GAAGCCGAGTCCAACATGAACGA
27428224 (Heterodera glycines)

M1129
421
TAATTACCCAAGTTTGAGTTTT
40304404 (Heterodera glycines)

Mi129
422
TTCACGGCCATGTTCCGTCGCAAGGCTTTC
28706250 (Heterodera glycines)

Mi129
423
AATTACCCAAGTTTGAGTTTT
61116089 (Litomosoides sigmodontis)

Mi129
424
CAATGTGGTAACCAAATTGGT
61115778 (Litomosoides sigmodontis)

Mi129
425
AAAAGAGAGAAGTTTTTGTTTT
34024847 (Meloidogyne arenaria)

Mi129
426
AAACGCATCTCTGAGCAATTCACGGCCATGTTCCGTCGCAA
34024847 (Meloidogyne arenaria)

Mi129
427
AATATGATGGCTGCTTGTGATCC
15352144 (Meloidogyne arenaria)

Mi129
428
ACTCTGAAGCTTCAAAATCCAACATA
15767987 (Meloidogyne arenaria)

Mi129
429
ATGATGGCTGCTTGTGATCCACG
15767987 (Meloidogyne arenaria)

Mi129
430
ATGCGTGAAGTAGACGATCAAATGATGTCGGTTCAAAACAAGAATTCGT
15767987 (Meloidogyne arenaria)

CATACTTTGTTGAATGGATTCCAAACAACGTTAAGACTGC

Mi129
431
ATGTCTGCGACCTTCATTGGCAACTCCAC
34024847 (Meloidogyne arenaria)

Mi129
432
ATTCAACCTGATGGATCATACAAGGGAGAATCTGACTTGCAATTGGA
15769029 (Meloidogyne arenaria)

Mi129
433
CAGGCCCAAAATGTTGCTGAATTGACCAA
15767987 (Meloidogyne arenaria)

Mi129
434
CGAATCAATGTTTACTACAATGAGGCTCATGGTGGAAAG
15769029 (Meloidogyne arenaria)

Mi129
435
CTGGTTTCGCTCCACTTTCGGCTAAGGGCGCAGCTGCCTA
34027531 (Meloidogyne arenaria)

Mi129
436
CTTTGTTGAATGGATTCCAAACAACGTTAAGACTGC
34024847 (Meloidogyne arenaria)

Mi129
437
GCTATCTTCCGTGGACGTATGTC
34027531 (Meloidogyne arenaria)

Mi129
438
GTATCCGTAACCATGTCTGGAGTGACTACTTGTCTTCGCTTCCCAGGCC
15767987 (Meloidogyne arenaria)

AATTGAACGC

Mi129
439
TACCAGCAATATCAAGATGCGACTGTTGAAGACGAAGGGGAATTTGAAG
34027531 (Meloidogyne arenaria)

GTGAAGATACCAATCAAGCAACTGTTGAACAAGAATAAAA

Mi129
440
TCCAAGTTCTGGGAAGTCATTTCGGATGAACACGG
15769029 (Meloidogyne arenaria)

Mi129
441
TGTCGGTTCAAAACAAGAATTCGTCAT
34024847 (Meloidogyne arenaria)

Mi129
442
TTATTTTGCTTTTAACCGCATATTTCCGAAATAATTCCTCGCTTAACTT
34024847 (Meloidogyne arenaria)

TATTTATGCCA

Mi129
443
TTCCTTCACTGGTACACTGGCGAAGGTATGGACGAGATGGAATTCAC
34024847 (Meloidogyne arenaria)

Mi129
444
TTGGCTGTCAACATGGTTCCATTCCCACGTCTTCACTTTTTCATGCCTG
15767987 (Meloidogyne arenaria)

GTTTCGCTCCACTTTCGGCTAAGGGCGCAGCTGCCTA

Mi129
445
TTTATCTAACAAAAGATCTCAAAAAATGCGTGAAATCGTCCATATCCAG
15769029 (Meloidogyne arenaria)

GCTGGACAATGTGG

Mi129
446
TAATTACCCAAGTTTGAGTTTT
37820079 (Meloidogyne chitwoodi)

Mi129
447
TAATTACCCAAGTTTGAGTTTTA
30169860 (Meloidogyne chitwoodi)

Mi129
448
AACATGGTTCCATTCCCACGTCTTCACTTTTT
19267174 (Meloidogyne hapla)

Mi129
449
ACTACTTGTCTTCGCTTCCCAGG
19267247 (Meloidogyne hapla)

Mi129
450
AATTACCCAAGTTTGAGTTTT
4587962 (Meloidogyne javanica)

Mi129
451
ATGGACTCGATTAGAGCTGGGCCATA
14086909 (Meloidogyne javanica)

Mi129
452
GACAATGTTTTGGATGTTGTTCGAAAAGAGGCTGAGGGTTGTGATTGTC
14086909 (Meloidogyne javanica)

TTCAAGGCTTTCAATTGACTCACTCGCTTGGTGGTGGAACTGGCTCTGG

AATGGGAACTTTGCTCATTTCAAAGATTCGTGAGGAGTATCCGGACCG

Mi129
453
GGAGAACTCTTCCGTCCTGACAACTTTGTCTTTGGCCAGAGCGGTGCAG
14086909 (Meloidogyne javanica)

GAAACAATTGGGCCAAAGGACATTACACTGAGGGAGC

Mi129
454
GGTGGAAAGTATGTCCCAAGAGC
14086909 (Meloidogyne javanica)

Mi129
455
TAATTACCCAAGTTTGAGTTT
40671282 (Meloidogyne javanica)

Mi129
456
AAAAAAGAGAGAAGTTTTTGTTTT
40670115 (Meloidogyne paranaensis)

Mi129
457
AAAAAGAGAGAAGTTTTTGTTTT
40669765 (Meloidogyne paranaensis)

Mi129
458
AAACGCATCTCTGAGCAATTCACGGCCATGTTCCGTCGCAA
40670115 (Meloidogyne paranaensis)

Mi129
459
ACCTTCATTGGCAACTCCACGGCCAT
40670115 (Meloidogyne paranaensis)

Mi129
460
ATAATTCCTCGCTTAACTTTATTTATGCCA
40670115 (Meloidogyne paranaensis)

Mi129
461
ATTACCCAAG1TTGAGTTTTA
46498709 (Meloidogyne paranaensis)

Mi129
462
ATTTGAAGGTGAAGATACCAATCAAGCAAC
46497880 (Meloidogyne paranaensis)

Mi129
463
CAAAGGACATTACACTGAGGGAGC
39746828 (Meloidogyne paranaensis)

Mi129
464
CGCATATTTCCGAAATAATTCCTCGCTTAACTTTATTTATGCCAAAA
40669765 (Meloidogyne paranaensis)

Mi129
465
CGTCCTGACAACTTTGTCTTTGGCCAGAGCGGTGC
39746808 (Meloidogyne paranaensis)

Mi129
466
GAACTGTTCAAACGCATCTCTGAGCAATTCACGGCCATGTTCCGTCGCA
40669765 (Meloidogyne paranaensis)

AGGC

Mi129
467
GAAGACGAAGGGGAATTTGAAGG
40669765 (Meloidogyne paranaensis)

Mi129
468
GAAGCCGAGTCCAACATGAACGACTTGATTTCTGAATACCAGCAATATC
40669765 (Meloidogyne paraneensis)

AAGATGCGACTGT

Mi129
469
GACGATCAAATGATGTCGGTTCAAAACAAGAATTCGTCATACTTTGTTG
40669436 (Meloidogyne paranaensis)

AATGGATTCCAAACAACGTTAAGACTGCGGTTTGTGACATTCCTCC

Mi129
470
GAGATGGAATTCACTGAAGCCGAGTCCAACATGAACGACTTGATTTCTG
40670115 (Meloidogyne paranaensis)

AATACCAGCAATATCAAGATGCGACTGTTGAAGACGAAGGGGAATTTGA

AGGTGAAGATACCAATCAAGCAACTGTTGAACAAGAATAAAAAA

Mi129
471
GAGTTGGTCGACAATGTTTTGGATGTT
39746828 (Meloidogyne paranaensis)

Mi129
472
GCCAAGAATATGATGGCTGCTTGTGATCCACGCCATGGACGCTATTT
40669436 (Meloidogyne paranaensis)

Mi129
473
GCGACCTTCATTGGCAACTCCAC
40669765 (Meloidogyne paranaensis)

Mi129
474
GCTGTTCTTGTTGACTTGGAGCCGGG
39746779 (Meloidogyne paranaensis)

Mi129
475
GTTCCGTCGCCTAAGGTCTCTGACACGGT
40669436 (Meloidogyne paranaensis)

Mi129
476
GTTGAACAAGAATAAAAAAGAGAGAAGTTTTTGTTTT
46497880 (Meloidogyne paranaensis)

Mi129
477
TAATTACCCAAGTTTGAGTTT
39747478 (Meloidogyne paranaensis)

Mi129
478
TAATTACCCAAGTTTGAGTTTT
46498746 (Meloidogyne paranaensis)

Mi129
479
TAATTACCCAAGTTTGAGTTTTA
46498594 (Meloidogyne paranaensis)

Mi129
480
TCTGTTCATCAACTTGTTGAGAACAC
40669436 (Meloidogyne paranaensis)

Mi129
481
TGGAACTGGCTCTGGAATGGGAACTTTGCTCATTTCAAAGATTCGTGAG
40669436 (Meloidogyne paranaensis)

GAGTATCCGGACCGCATTATGTCTTCCTTCTCTGT

Mi129
482
TTATTTTGCTTTTAACCGCATATTTCCGA
40670147 (Meloidogyne paranaensis)

Mi129
483
TTATTTTGCTTTTAACCGCATATTTCCGAAATA
46497880 (Meloidogyne paranaensis)

Mi129
484
TTCCTTCACTGGTACACTGGCGAAGGTATGGA
40669765 (Meloidogyne paranaensis)

Mi129
485
GAATGGATTCCAAACAACGTTAAGAC
22527837 (Necator americanus)

Mi129
486
TTCATGCCTGGTTTCGCTCCACT
22528338 (Necator americanus)

Mi129
487
TTGGCTGTCAACATGGTTCCATTCCCACGTCTTCACTT
33242934 (Necator americanus)

Mi129
488
GTTGTTCGAAAAGAGGCTGAGGG
21393141 (Nippostrongylus brasiliensis)

Mi129
489
AGGTTTAATTACCCAAGTTTGAG
10840960 (Onchocerca volvulus)

Mi129
490
TACTTTGTTGAATGGATTCCAAA
3046904 (Onchocerca volvulus)

Mi129
491
ACTCTTTCTGTTCATCAACTTGTTGA
15765704 (Parastrongyloides trichosuri)

Mi129
492
CATACTTTGTTGAATGGATTCCAAACAA
15340288 (Parastrongyloides trichosuri)

Mi129
493
CAAAGATTCGTGAGGAGTATCC
31250007 (Parelaphostrongylus tenuis)

Mi129
494
AATATGATGGCTGCTTGTGATCCA
14494074 (Strongyloides ratti)

Mi129
495
CATACTTTGTTGAATGGATTCCAAA
19252051 (Strongyloides ratti)

Mi129
496
TGGTACACTGGCGAAGGTATGGA
15002387 (Strongyloides ratti)

Mi129
497
AATATGATGGCTGCTTGTGATCCA
12713662 (Strongyloides stercoralis)

Mi129
498
CATACTTTGTTGAATGGATTCCAAA
12713662 (Strongyloides stercoralis)

Mi129
499
TACTTTGTTGAATGGATTCCAA
9830375 (Strongyloides stercoralis)

Mi129
500
AGCAAATGTTTGATGCCAAGAA
27758042 (Teladorsagia circumcincta)

Mi129
501
CCAAGTTCTGGGAAGTCATTTC
27756411 (Teladorsagia circumcincta)

Mi129
502
GAACGAATCAATGTTTACTACAATGAGGCT
27756411 (Teladorsagia circumoincta)

Mi129
503
GGTATGGACGAGATGGAATTCAC
19559988 (Toxocara canis)

Mi129
504
ATGGACGAGATGGAATTCACTGA
2372083 (Caenorhabditis elegans)

Mi129
505
ATGGACGAGATGGAATTCACTGAAGC
1116997 (Caenorhabditis elegans)

Mi129
506
CTTGGTGGTGGAACTGGCTCTGGAATGGGAACT
108965893 (Bursaphelenchus xylophilus)

Mi129
507
GATGCCAAGAATATGATGGCTGC
108983689 (Bursaphelenchus xyiophilus)

Mi129
508
GCAACTCTTTCTGTTCATCAACT
115342288 (Heterorhabditis bacteriophora)

Mi129
509
GGAACTGGCTCTGGAATGGGA
108976152 (Bursaphelenchus mucronatus)

Mi129
510
GGTTCCATTCCCACGTCTTCA
14855460 (Caenorhabditis elegans)

Mi129
511
GTCAACATGGTTCCATTCCCACGT
103038769 (Caenorhabditis elegans)

Mi129
512
ATGGACGAGATGGAATTCACTGAAGC
71983644 (Caenorhabditis elegans)

Mi129
513
TACAACGCAACTCTTTCTGTTCATCAACTTGTTGA
1120154 (Caenorhabditis elegans)

Mi129
514
TTAATTACCCAAGTTTGAGTTTTAC
14829782 (Caenorhabditis elegans)

TABLE 7

Target
SEQ

ID
ID NO
Sequence*
Example Gi-number and species

Mi05
515
TTATTATTTCAAAAAAAAAAA
50829840 (Apis mellifera)

M138
516
TACGGCCAGGACGTATTGATCGT
47532028 (Acyrthosiphon pisum)

Mi40
517
GATATGGATGAAGATGAATTGGAAATGTTATC
66530331 (Apis mellifera)

Mi40
518
CAGTGTTTGGAAAGATATGAATAT
84647988 (Myzus persicae)

Mi40
519
GATATGGATGAAGATGAATTGGA
124238642 (Solenopsis invicta)

Mi40
520
GATATGGATGAAGATGAATTGGAAATGTTATC
110764965 (Apis mellifera)

Mi111
521
ACAAATTCAAGAAATTAAGGA
66509031 (Apis mellifera)

Mi111
522
ACAAATTCAAGAAATTAAGGAG
77329902 (Chironomus tentans)

Mi111
523
TTTAAAAAAATCTTTTAAAAAT
82830959 (Boophilus microplus)

Mi111
524
CACGCAGTTGTTGGTGTTTTGG
94429207 (Bombyx mori)

Mi111
525
GTGGAATTGCCACTTACACATCCAGA
78539405 (Glossina morsitans)

Mi125
526
CAAAAGCACATTGACTTCTCC
37952462 (Ips pini)

Mi125
527
CACATTGACTTCTCCACAAAATC
56156455 (Rhynchosciara americana)

Mi125
528
CGTAAAGCTGCTCGTGAACTT
77332739 (Chironomus tentans)

M1125
529
CGTCGTTTGGTTCGTATTGGAGT
62240069 (Diabrotica virgifera),

94438358 (Bombyx mori)

Mi125
530
GACTTTTTGGAGCGTCGTTTGCAGACGCA
41405201 (Drosophila melanogaster)

Mi125
531
GTTTTCAAATTGGGATTGGCTAAATC
31364772 (Toxoptera citricida)

Mi125
532
TTCAAATTGGGATTGGCTAAATC
37622137 (Sarcophaga crassipalpis)

Mi125
533
GACTTTTTGGAGCGTCGTTTGCAGAC
91826622 (Bombyx mori)

Mi125
534
TTGGAGCGTCGTTTGCAGACGCA
114056346 (Drosophila melanogaster)

Mi127
535
ATCATTGCCAATGATCAAGGAAA
60315026 (Tricholepisma aurea)

Mi127
536
ATGAAGCATTGGCCGTTCAAGGT
61951993 (Tribolium castaneum)

Mi127
537
ATTGCTTATGGTTTGGATAAGAAAG
33298660 (Glossina morsitans)

Mi127
538
ATTTGACGATCCAGCTGTTCA
60303218 (Julodis onopordi)

Mi127
539
CAGCATGGAAAAGTCGAGATCAT
61955760 (Tribolium castaneum)

Mi127
540
CATGGAAAAGTCGAGATCATTGC
77326551 (Chironomus tentans)

Mi127
541
GACATGAAGCATTGGCCGTTCAA
60308798 (Silpha atrata)

Mi127
542
GGTGTATTCCAGCATGGAAAAGT
16285055 (Apis mellifera)

Mi127
543
ATTGCTTATGGTTTGGATAAG
78525195 (Glossina morsitans)

Mi127
544
GAGATCATTGCCAATGATCAAGGAAA
107248738 (Drosophila melanogaster)

Mi127
545
GAGATCATTGCCAATGATCAAGGAAA
99005779 (Leptinotarsa decemlineata)

Mi127
546
TCGGAATTGATCTGGGAACGAC
113208384 (Mamestra brassicae)

Mi127
547
TTTAAAAAAATCTTTTAAAAA
124012837 (Oncopeltus fasciatus)

Mi128
548
GAAATTTAAAGAATGACAAAG
77321879 (Chironomus tentans)

Mi128
549
AATTGGGCCAAAGGACATTACACTGA
84114197 (Blomia tropicalis)

Mi128
550
CGCAAGGCTTTCCTTCACTGGTA
82849727 (Boophilus microplus)

Mi128
551
CGCAAGGCTTTCCTTCACTGGTACACTGG
82831082 (Boophilus microplus)

Mi128
552
GCAAGGCTTTCCTTCACTGGTA
82835811 (Boophilus microplus)

Mi128
553
GCCATGTTCCGTCGCAAGGCTTTCCT
110889758 (Argas monolakensis)

Mi128
554
TTTGTTGAATGGATTCCAAACAA
84114847 (Blomia tropicalis)

Mi128
555
GTCAACATGGTTCCATTCCCACGT
84114197 (Blomia tropicalis)

Mi129
556
AAATTGGCTGTCAACATGGTTCC
62239796 (Diabrotica virgifera)

Mi129
557
AACAATTGGGCCAAAGGACATTACAC
37659666 (Bombyx mori)

Mi129
558
AACATGGTTCCATTCCCACGT
22038826 (Ctenocephalides felis);

67884361 (Drosophila pseudoobscura)

Mi129
559
AACATGGTTCCATTCCCACGTCT
67841791 (Drosophila pseudoobscura);

67895980 (Drosophila pseudoobscura)

Mi129
560
AAGGCTTTCCTTCACTGGTACACTGG
55894639 (Locusta migratonia)

Mi129
561
AGCAAATGTTTGATGCCAAGAA
40917832 (Bombyx mori);

158744 (Drosophila hydei)

Mi129
562
ATCAATGTTTACTACAATGAGGC
50560478 (Homalodisca coagulata)

Mi129
563
ATGATGGCTGCTTGTGATCCACG
56150501 (Rhynchosciara americana),

123264037 (Laupala kohalensis)

Mi129
564
ATGGAATTCACTGAAGCCGAG
16898208 (Ctenocephalides felis)

Mi129
565
ATGGTTCCATTCCCACGTCTTCACTT
77324743 (Chironomus tentans);

67880163 (Drosophila pseudoobscura)

Mi129
566
ATGTTCCGTCGCAAGGCTTTC
77642397 (Aedes aegypti),

92040999 (Drosophila willistoni),

67893984 (Drosophila pseudoobscura),

56151969 (Rhynchosciara americana),,

45244777 (Bombyx mori),

Mi129
567
ATGTTCCGTCGCAAGGCTTTC
67893984 (Drosophila pseudoobscura)

Mi129
568
ATGTTCCGTCGCAAGGCTTTC
56151969 (Rhynchosciara americana)

Mi129
569
CAATTGAACGCTGATCTTCGCAAATTGGC
56150501 (Rhynchosciara americana)

Mi129
570
CATGGTTCCATTCCCACGTCT
67894145 (Drosophila pseudoobscura)

Mi129
571
CGTATTTTTTTAATTTTATTTT
55798845 (Acyrthosiphon pisum)

Mi129
572
CTGCATCGACAACGAGGCTTTGTA
24650775 (Drosophila melanogaster)

Mi129
573
CTTACTGCATCGACAACGAGGC
48095546 (Apis mellifera)

Mi129
574
GAAGGTATGGACGAGATGGAATTCACTGAAGC
73612788 (Aphis gossypii),

99009385 (Leptinotarsa decemlineata)

Mi129
575
GAGATGGAATTCACTGAAGCCGA
77759798 (Aedes aegypti),

83663743 (Myzus persicae)

Mi129
576
GATGCCAAGAATATGATGGCTGCTTG
77753173 (Aedes aegypti)

Mi129
577
GCCATTCAAGAACTGTTCAAACG
33429239 (Glossina morsitans)

Mi129
578
GGCGAAGGTATGGACGAGATGGAATTCACTGAAGCCGA
75466135 (Tribolium castaneum)

Mi129
579
GGTATGGACGAGATGGAATTCAC
33427916 (Glossina morsitans),

83930827 (Lutzomyia longipalpis)

Mi129
580
GTTGTGATTGTCTTCAAGGCTT
62239916 (Diabrotica ving ifera)

Mi129
581
TACTGCATCGACAACGAGGCT
29535296 (Bombyx mori);

4463876 (Drosophila melanogaster);

38047746 (Drosophila yakuba)

Mi129
582
TACTGCATCGACAACGAGGCTT
3944276 (Drosophila melanogaster)

Mi129
583
TACTGCATCGACAACGAGGCTTT
3719603 (Manduca sexta)

Mi129
584
TACTTTGTTGAATGGATTCCAAA
55882682 (Locusta migratoria)

Mi129
585
TCCATTCCCACGTCTTCACTT
67840488 (Drosophila pseudoobscura)

Mi129
586
TCCATTCCCACGTCTTCACTTTTTCATGCCTGG
33429092 (Glossina morsitans)

Mi129
587
TGGCTGTCAACATGGTTCCATTCCCACGTCT
77706291 (Aedes aegypti);

67842756 (Drosophila pseudoobscura)

Mi129
588
TGGTACACTGGCGAAGGTATGGA
56151969 (Rhynchosciara americana)

Mi129
589
TGGTACACTGGCGAAGGTATGGACGA
47534792 (Acyrthosiphon pisum)

Mi129
590
TGTACGACATCTGCTTCCGAAC
40918504 (Bombyx mori)

Mi129
591
TTCCGTGGACGTATGTCAATG
77324743 (Chironomus tentans),

118158757 (Lysiphlebus testaceipes)

Mi129
592
TTTTTAATTTTATTTTGCTTT
24641385 (Drosophila melanogaster)

Mi129
593
AAGGCTTTCCTTCACTGGTACACTGGCGA
124242563 (Solenopsis invicta)

Mi129
594
AAGGTATGGACGAGATGGAATTCAC
113022997 (Bemisia tabaci)

Mi129
595
AATATGATGGCTGCTTGTGATCCA
118158757 (Lysiphlebus testaceipes)

Mi129
596
AATTTGAAGGTGAAGATACCAA
123262489 (Laupala kohalensis)

Mi129
597
ACCTTCATTGGCAACTCCACGGCCAT
92944161 (Drosophila ananassae)

Mi129
598
TCATACTTTGTTGAATGGATTCCAAACAACGT
118158757 (Lysiphlebus testaceipes)

Mi129
599
ATGGACGAGATGGAATTCACTGA
91086094 (Tribolium castaneum)

Mi129
600
ATGGTTCCATTCCCACGTCTTCACTTTTTCATGCCTGG
78543404 (Glossina morsitans)

Mi129
601
ATGTTCCGTCGCAAGGCTTTC
93002796 (Drosophila grimshawi)

Mi129
602
CAAATTGGTTCCAAGTTCTGGGAA
118158037 (Lysiphlebus testaceipes)

Mi129
603
CGCAAGGCTTTCCTTCACTGGTACAC
110557210 (Drosophila ananassae)

Mi129
604
CGGCCATGTTCCGTCGCAAGGC
92940755 (Drosophila virilise)

Mi129
605
CTTTGTTGAATGGATTCCAAA
118158492 (Lysiphlebus testaceipes)

Mi129
606
GACAACTTTGTCTTTGGCCAG
92479391 (Drosophila erecta)

Mi129
607
ATGTTCCGTCGCAAGGCTTTC
114949473 (Aedes aegypti)

Mi129
608
GCCATTCAAGAACTGTTCAAACG
78532419 (Glossina morsitans)

Mi129
609
GCCGAGTCCAACATGAACGAC
83930973 (Lutzomyia longipalpis)

Mi129
610
GGCGAAGGTATGGACGAGATGGA
90982659 (Aedes aegypti)

Mi129
611
GTCAACATGGTTCCATTCCCACGTCT
109290429 (Culex pipiens),

93001606 (Drosophila grimshawi)

Mi129
612
ATGTTCCGTCGCAAGGCTTTC
93001606 (Drosophila grimshawi

Mi129
613
TACTGCATCGACAACGAGGCT
92952298 (Drosophila ananassae)

Mi129
614
TACTGCATCGACAACGAGGCT
92473113 (Drosophila erecta)

Mi129
615
TACTGCATCGACAACGAGGCT
107330711 (Drosophila melanogaster)

Mi129
616
TCATACTTTGTTGAATGGATTCCAAACAA
119363347 (Rhodnius prolixus)

Mi129
617
TGGAGCCGGGAACCATGGACTC
92931833 (Drosophila virilise)

Mi129
618
TGGAGCCGGGAACCATGGACTC
123283502 (Laupala kohalensis)

Mi129
619
TGGCTGTCAACATGGTTCCATTCCC
92047961 (Drosophila willistoni)

Mi129
620
TGGGCCAAAGGACATTACACTGAGGG
103793359 (Heliconius erato)

Mi129
621
TGGGCCAAAGGACATTACACTGAGGG
103793359 (Heliconius erato)

Mi129
622
TTCCGTGGACGTATGTCAATG
118159629 (Lysiphlebus testaceipes)

Mi129
623
TTTTACTATTTAATTTATCTA
83935851 (Lutzomyia longipalpis)

Mi129
624
TCATACTTTGTTGAATGGATTCCAAACAACGT
118159629 (Lysiphlebus testaceipes)

TABLE 8

Target
SEQ

ID
ID NO
Sequence*
Example Gi-number and species

Mi40
625
ATTGATATGGATGAAGATGAA
90555342 (Lentinula edodes)

Mi111
626
AACAAATTCAAGAAATTAAGGA
110464534 (Rhizopus oryzae)

Mi125
627
TTCGTCGTTTGGTTCGTATTGG
110460324 (Rhizopus oryzae)

Mi127
628
ATTGCTTATGGTTTGGATAAGAAAGGC
119201164 (Saccharomyces cerevisiae)

Mi127
629
GGAACGTCTGATTGGTGATGC
90538259 (Gloeophyllum trabeum)

Mi127
630
GAGATCATTGCCAATGATCAAGG
58160145 (Phytophthora infestans)

Mi129
631
AACTCTTCCGTCCTGACAACTTT
110459609 (Rhizopus oryzae)

Mi129
632
AGCAAATGTTTGATGCCAAGAA
98997118 (Spizellomyces punctatus)

Mi129
633
GGTATGGACGAGATGGAATTCACTGA
98997118 (Spizellomyces punctatus)

Mi129
634
ATCGACAACGAGGCTTTGTACGACAT
119495801 (Neosartorya fischeri)

Mi129
635
ATCGACAACGAGGCTTTGTACGACATCTG
110118467 (Saitoella complicate)

Mi129
636
ATGGACGAGATGGAATTCACTGAAGCCGAGTC
114217954 (Ophiostoma clavigerum)

Mi129
637
CAATGTGGTAACCAAATTGGT
116079742 (Botryotinia fuckeliana),

90392956 (Cunninghamella elegans),

110465715 (Rhizopus oryzae),

117792906 (Trichophyton rubrum)

Mi129
638
AACTCTTCCGTCCTGACAACTTT
110465715 (Rhizopus oryzae)

Mi129
639
CATACTTTGTTGAATGGATTCC
119184208 (Coccidioides immitis)

Mi129
640
CCTGACAACTTTGTCTTTGGCCAG
116002727 (Trichoderma atroviride)

Mi129
641
GACACGGTCGTTGAGCCCTACAACGC
90624483 (Corynascus heterothallicus)

Mi129
642
GCAAATGTTTGATGCCAAGAA
116360500 (Puccinia striiformis)

Mi129
643
GCCATGTTCCGTCGCAAGGCTTTCCTTCA
90610729 (Ophiostoma piliferum)

Mi129
644
GCCGAGTCCAACATGAACGAC
90610729 (Ophiostoma piliferum),

117422543 (Blastocladiella emersonii)

Mi129
645
GGAAACAATTGGGCCAAAGGA
90644351 (Trametes versicolor)

Mi129
646
GGTATGGACGAGATGGAATTCA
50551818 (Yarrowia lipolytica)

Mi129
647
AGGTATGGACGAGATGGAATTCAC
70689303 (Gibberella monil iformis)

Mi129
648
ATGGACGAGATGGAATTCACTGA
46343419 (Paracoccidloides brasiliensis)

Mi129
649
ATGTTCCGTCGCAAGGCTTTC
70995399 (Aspergillus fumigatus);

49086565 (Aspergillus nidulans FGSCA4);

70825342 (Aspergillus niger),

119495801 (Neosartorya fischeri),

70995399 (Aspergillus fumigatus),

70825342 (Aspergillus niger),

84573702 (Aspergillus oryzae),

116202308 (Chaetomium globosum),

116002543 (Trichoderma atroviride),

119184208 (Coccidloides immitis)

Mi129
650
GGTATGGACGAGATGGAATTCACTGA
98996315 (Spizellomyces punctatus)

Mi129
651
TACCAGCAATATCAAGATGCGAC
45739462 (Hebeloma cylindrosporum)

Mi129
652
TGCATCGACAACGAGGCTTTGTACGA
50980793 (Paxillus involutus)

Mi129
653
TGGGCCAAAGGACATTACACTGAGGGAGC
83922458 (Phakopsora pachyrhizi)

Mi129
654
TTGGCTGTCAACATGGTTCCATTCCC
90385723 (Amorphotheca resinae),

45743270 (Hebeloma cylindrosporurn)

Mi129
655
TTGGTGGTGGAACTGGCTCTGG
90535726 (Gloeophyllum trabeum),

90546970 (Lentinula edodes)

Mi129
656
CAATGTGGTAACCAAATTGGT
54397728 (Trichophyton rubrum)

Mi129
657
GAGTCCAACATGAACGACTTG
66909865 (Phaeosphaeria nodorum),

90357303 (Aureobasidium pullulans)

Mi129
658
GCCATGTTCCGTCGCAAGGCTTT
70709731 (Gibberella moniliformis)

Mi129
659
GCCATGTTCCGTCGCAAGGCTTTC
70662385 (Gibberella moniliformis),

90624483 (Corynascus heterothallicus),

121701810 (Aspergillus clavatus),

115491490 (Aspergillus terreus),

90357303 (Aureobasidium pullulans),

90618069 (Corynascus heterothallicus),

89975356 (Hypocrea lixii),

Mi129
660
GCCGAGTCCAACATGAACGACTTG
60674404 (Alternaria brassicicola),

116002543 (Trichoderma atroviride),

89975356 (Hypocrea lixii)

Mi129
661
GGTATGGACGAGATGGAATTCAC
27735638 (Aspergillus flavus);

70662385 (Gibberella moniliformis);

22499956 (Gibberella zeae),

84573811 (Aspergillus oryzae),

46124466 (Gibberella zeae)

Mi129
662
TTCCGTCCTGACAACTTTGTCT
21649805 (Conidiobolus coronatus)

Nematode Control

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