Patent Grant
8445752
This application is the national phase under 35 U.S.C. §371 of PCT International Application No. PCT/NL2008/050232 which has an International filing date of 22 Apr. 2008, which designated the United States of America and which claims priority on Netherlands Application No. 2000622, the entire contents of each of which are hereby incorporated herein by reference.
The material in the ASCII text file entitled 1470765—1.txt is hereby incorporated herein by reference in its entirety. The ASCII text file entitled 1470765—1.txt was created on 16 May 2012 and the size is 222 KB.
The present invention relates to Brassica oleracea plants which are resistant to Albugo candida, the cause of white blister. The invention also relates to the seeds, fruits and/or other plant parts from these resistant plants. The present invention further relates to a method for providing B. oleracea plants which are resistant to A. candida. The invention also relates to the use of specific DNA markers which are linked to the A. candida resistance gene for the purpose of identifying resistant B. oleracea plants.
White blister (A. candida; synonyms: A. cruciferum, A. cruciferatum, white rust, staghead) is a plant disease which causes many problems in vegetables crops of cabbage, but also in related species such as rape, mustard and radish. The disease can in principle occur on all cruciferae, so also on wild species such as shepherd's purse (Capsella bursa-pastoris) and wild mustard (charlock mustard, Sinapis arvensis). Contrary to what the name suggests, this is not a rust fungus but an oomycete closely related to downy mildew (Peronospora parasitica) and Phytophtora. Oomycetes are not fungi and, although they also grow in threads, they are more related to algae.
The oomycete causes blisters with spores (sori, pustules) on the leaves, stems and ovaries (siliques) of Brassica plants. Distortions in the form of spots/are also often present. Systemic infection of plants results in abnormal growth, deformations and sometimes sterility of the flowers or inflorescence. The oomycete thrives best at temperatures between and 20° C. and in moist conditions. A leaf wetness period of 2.5 hours is sufficient to result in infection, at which there is an incubation period of 10 to 14 days. Moist weather conditions with moderate temperatures are therefore ideal for infection and spreading of the oomycete.
When spores of A. candida land on a cabbage leaf, they form a germ tube with which they penetrate the leaf. In here, the mycelium grows intercellularly and absorbs nutrients via haustoria. The vegetative spore formation takes place in the zoosporangium which develops under the epidermis. Created herein are the asexual zoospores which, when there is sufficient moisture, are released from the zoosporangia and can then cause new infections. The spores have two whiplash tails (flagellae), one for forward movement and one for the swimming direction.
A. candida can overwinter in the ground in sexual form with thick-walled oospores, which may or may not be on infected plant remnants, or in asexual form (mycelium) on winter-hardened host plants. During mild winters the oomycete does not really go to rest but remains active at a lower level. New plants can be infected in the spring. Plant material can also be already infected on the plant bed without symptoms becoming visible. Spread of the oomycete takes place through sporangia being carried away by air movements, hard rainfall, watering, machines, farm workers and insects, whereby other plants are infected.
Host specialization in A. candida is known and different physiological species and formae specialis are distinguished on the basis of the species or the line which is infected and the aggressiveness of the isolate on the line.
Brassica is a plant genus in the family Brassicaceae (formerly referred to as Cruciferae). The members of this genus are referred to as cabbage or mustard. The genus Brassica comprises a number of important agricultural and horticultural crops, including rape, cauliflower, red cabbage, savoy cabbage, white cabbage, oxheart cabbage, curly cale cabbage, broccoli, Brussels sprouts, Chinese cabbage, turnip cabbage and Portuguese cabbage (tronchuda). Almost all parts of the plants are used as food, such as the roots (turnip), stalks (turnip cabbage), leaves (white cabbage), axillary buds (sprouts), flowers (cauliflower, broccoli) and seeds (rape). Rape and rape seed are also used for oil, both for consumption and for fuel. Some species with white or purple flowers or distinct colour or shape of the leaves are cultivated for ornamental purposes. The Brassica family occurs worldwide and consists of annuals, biennials and perennials. The family also comprises a large number of wild species.
At the moment few agents are known which can be used to control white blister in Brassica. An increasing number of countries in Europe moreover have a policy aimed at reducing the use of crop protection agents. If the use of control agents is no longer allowed at all, this can result in major problems in the cultivation of Brassica species. In crops such as for instance Brassica rapa (syn. campestris) (turnip rape), Brassica juncea (mustard) and Brassica napus (rapeseed) white blister can cause huge losses in yield (Bernier, Can. Plant Dis. Surv. 52: 108, 1972; Fan et al., Can. J. Genet. Cytol. 25: 420-424, 1983); Harper and Pittman, Phytopathology 64: 408-410, 1974; Varshney et al., Theoretical and Applied Genetics 109: 153-159, 2004). In vegetable crops the quality aspect is particularly important. Vegetables such as sprouts, headed cabbage and curly cale cabbage infected by white blister are no longer sellable because of the cosmetic damage. There is therefore a great need for Brassica vegetable crops which are resistant to white blister.
Resistance to white blister is described in diverse Brassica species such as B. rapa, B. napus and B. juncea (Ebrahimi et al., Proc. Am. Phytopathol. Soc. 3: 273, 1976; Delwiche and Williams, Proc. Am. Phytopathol. Soc. 1: 66, 1974; Tiwari et al., Can. J. Of Plant Science 68: 297-300, 1988; Kole et al., Genome 45: 22-27, 2002; Varshney et al., Theoretical and Applied Genetics 109: 153-159, 2004; Tanhuanpad, Theoretical and Applied Genetics 108: 1039-1046, 2004). In addition, partial resistance has been demonstrated in B. oleracea lines (Santos and Dias, Genetic Resources and Crop Evolution 51: 713-722, 2004). Full resistance to white blister in B. oleracea vegetable crops has however not as yet been described.
An object of the present invention is to provide a B. oleracea plant with a resistance to A. candida, the cause of white blister.
The invention provides to this end a B. oleracea plant comprising a resistance gene to A. candida.
The resistance gene according to the invention provides a monogenic and dominant resistance to A. candida. The resistance gene is preferably present in heterozygous form, and more preferably the resistance gene is present in homozygous form.
According to the invention the resistance gene to A. candida preferably comes from the B. oleracea plant, the seeds of which were deposited in the American Type Culture Collection (ATCC, Patent Depository, 10801 University Boulevard, Manassas, Va. 20110, United States of America) on 1 Mar. 2006 under number PTA 74-12. Surprisingly, it has been found that with the resistance gene according to the invention a dominant resistance is provided to A. candida.
In order to obtain a full resistance to A. candida in B. oleracea the transmission is described in this invention of a dominant, monogenic resistance to A. candida from a first B. oleracea source to different other B. oleracea types such as white cabbage, Brussels sprouts, cauliflower and turnip cabbage.
Using a disease test for white blister resistance B. oleracea lines were screened and a white blister resistance source was identified. The resistance was then transmitted from the source to existing quality lines by means of repeated backcrossing, in some cases as many as four to six times, followed by multiple generations of self-pollination. A disease test was performed here each time in order to select the resistant plants for the continuation of the backcrossing program. In the evaluation of these disease tests, plants were grouped into the classes resistant (no visible reaction or necrotic spots), susceptible (many sporulating blisters) and intermediate (necrotic spots and several sporulating blisters). It was found from the segregation ratios found during the backcrossing program that the resistance was a monogenic dominant trait. A lack of resistant plants was however found in many genetic backgrounds and, in addition, a great variation in numbers in the intermediate class (from several plants to half the population). The penetration of this gene was thus very incomplete in these genetic backgrounds and the breeding program was greatly hampered as a result.
In a further preferred embodiment of the invention the resistance gene is linked to one or more specific DNA markers. So as to be better able to monitor the resistance and transmit it more quickly, DNA markers have been developed according to the present invention which are closely linked to the introgression having thereon the disease-resistance gene against white blister. These markers have been developed by means of a BSA (Bulked Segregant Analysis). For this purpose individuals from a correct (1:1) segregating BC population were divided on the basis of the disease test into a resistant and a susceptible class. DNA was then isolated from all plants, and the resistant plants were bulked to form a resistant pool, and the susceptible plants to form a susceptible pool. Marker analyses were then performed on these pools by means of the RAMP technique and markers were identified which were closely linked to the resistance. By means of an analysis with the closely linked markers the plants were selected with certainty which contained the resistance gene in populations where the disease test does not give an unambiguous picture (many intermediary reactions, not a good segregation ratio). In addition, the homozygous resistant plants are directly differentiated from the heterozygous resistant plants during inbreeding. This results in an accelerated breeding program.
In a preferred embodiment of the invention the presence of the introgression with the resistance gene to A. candida can be demonstrated using at least two, preferably at least three, more preferably at least four, more preferably at least five, six, seven or eight, most preferably nine DNA markers linked to the resistance gene, wherein the DNA markers enclose the resistance gene. Enclose in the present application is understood to mean that the DNA markers are located on the genome on both sides of the resistance gene, i.e. “upstream” as well as “downstream” of the resistance gene. Demonstrating the presence of a plurality of DNA markers, which are linked to the resistance gene, and moreover enclosing the resistance gene ensure that the introgression with the resistance gene is actually present.
The DNA markers according to the invention are preferably chosen from table 1, wherein the presence of the DNA markers in the genome of the plant is demonstrated using the primer sequences chosen from the group consisting of SEQ ID NO: 1 up to and including SEQ ID NO: 10 (table 2).
In the research which has led to the present invention it has been demonstrated that the relevant DNA markers are characteristic for the introgression of the resistance to A. candida. The DNA markers according to the invention are DNA fragments which are linked to the relevant resistance gene, have a determined size (bp) as indicated in table 1, and can be demonstrated by using specific primer combinations.
The plant according to the invention is preferably chosen from the group consisting of B. oleracea convar. botrytis var. botrytis (cauliflower, romanesco), B. oleracea convar. botrytis var. cymosa (broccoli), B. oleracea convar. botrytis var. asparagoides (sprouting broccoli), B. oleracea convar. oleracea var. gemnifera (Brussels sprouts), B. oleracea convar. capitata var. alba (white cabbage, oxheart cabbage), B. oleracea convar. capitata var. rubra (red cabbage), B. oleracea convar. capitata var. sabauda (savoy cabbage), B. oleracea convar. acephela var. sabellica (curly cale cabbage), B. oleracea convar. acephela var. gongyloides (turnip cabbage) and B. oleracea var. tronchuda syn. costata (Portuguese cabbage).
The invention also relates to the seeds, fruits and/or other plant parts from the above described plants. Plant parts are here understood to mean, among others, the edible parts of the plant, such as for instance axillary buds (sprouts).
The invention also relates to a method for obtaining a B. oleracea plant with a resistance to A. candida, which method comprises at least the following steps of:
(a) providing a first B. oleracea plant, which plant comprises a resistance gene to A. candida;
(b) crossing the resistant plant with a susceptible second B. oleracea plant;
(c) isolating genomic DNA from the progeny for detecting the presence of an introgression with the resistance gene using one or more specific DNA markers linked to the resistance gene; and
(d) selecting from the progeny a B. oleracea plant in which the presence of the introgression with the resistance gene has been demonstrated in step (c).
With the method according to the invention resistant B. oleracea plants can be provided in rapid and simple manner by making use of DNA markers which are specific to the introgression with the resistance gene according to the invention.
Using the method according to the present invention and the use of the specific DNA markers linked to a resistance gene it is possible to determine in simple manner wether a plant contains the resistance gene. Performing the disease test is a very time-consuming procedure. Selection of resistant plants by utilizing the specific DNA markers linked to a resistance gene is much more efficient. Larger numbers of plants can hereby be tested more easily. The introgression with the resistance gene can also be more readily mapped, whereby plants with the smallest possible introgression can be selected. Furthermore, distinction can be made between homozygous and heterozygous resistant plants.
The plants selected in step (d) of the method according to the invention can optionally be subjected to additional steps, such as back-crossing or self-pollination of the plant obtained in step (d) one or more times with a susceptible B. oleracea plant and subsequently selecting once again from the progeny a resistant B. oleracea plant using the specific DNA markers. The plants obtained in step (d) can for instance also be made homozygous by means of techniques known to the skilled person such as anther and/or microspore culture.
In a preferred embodiment of the method the presence of the introgression with the resistance gene in the selected plants is confirmed by means of a disease test. The presence and effect of the resistance gene can be definitively confirmed by performing a disease test.
The first B. oleracea plant preferably comprises a resistance gene which gives a monogenic and dominant resistance to A. candida. In a preferred embodiment of the invention the resistance gene is present in heterozygous form, preferably in a homozygous form.
In a preferred embodiment the first B. oleracea plant comprises a resistance gene from the B. oleracea plant, the seeds of which were deposited in the American Type Culture Collection (ATCC, Patent Depository, 10801 University Boulevard, Manassas, Va. 20110, United States of America) on 1 Mar. 2006 under number PTA 74-12.
In a further preferred embodiment of the method according to the invention the selection of the resistant B. oleracea plant in step (d) comprises of selecting a B. oleracea plant which comprises at least two, preferably at least three, more preferably at least four, more preferably at least five, six, seven or eight, and most preferably nine DNA markers linked to the resistance gene, wherein the DNA markers enclose the resistance gene. It is hereby possible to determine with certainty that the plant actually possesses the introgression with the resistance gene.
The DNA markers according to the invention are preferably chosen from table 1, wherein the presence of the DNA markers in the genome of the plant is demonstrated using the primer sequences chosen from the group consisting of SEQ ID NO: 1 up to and including SEQ ID NO: 10 (table 2).
In a particular embodiment according to the invention the first B. oleracea plant comprises a resistance gene to A. candida originating from the B. oleracea plant, the seeds of which were deposited in the American Type Culture Collection (ATCC, Patent Depository, 10801 University Boulevard, Manassas, Va. 20110, United States of America) on 1 Mar. 2006 under number PTA 74-12.)
The susceptible B. oleracea plant into which the resistance gene is inserted is preferably chosen from the group consisting of B. oleracea convar. botrytis var. botrytis (cauliflower, romanesco), B. oleracea convar. botrytis var. cymosa (broccoli), B. oleracea convar. botrytis var. asparagoides (sprouting broccoli), B. oleracea convar. oleracea var. gemnifera (Brussels sprouts), B. oleracea convar. capitata var. alba (white cabbage, oxheart cabbage), B. oleracea convar. capitata var. rubra (red cabbage), B. oleracea convar. capitata var. sabauda (savoy cabbage) B. oleracea convar. acephela var. sabellica (curly cale cabbage), B. oleracea convar. acephela var. gongyloides (turnip cabbage) and B. oleracea var. tronchuda syn. costata (Portuguese cabbage).
The invention further relates to the B. oleracea plants obtainable with the above described method, and to the seeds and/or plant parts thereof.
The invention also relates to the use of at least one DNA marker linked to a resistance gene to A. candida for identifying a B. oleracea plant which is resistant to A. candida, wherein the DNA marker is chosen from the DNA markers of table 1 and wherein the DNA marker is demonstrated with the primer sequences chosen from the group consisting of SEQ ID NO: 1 up to and including SEQ ID NO: 10 (table 2).
The resistance gene preferably originates from the B. oleracea plant of which the seeds were deposited in the American Type Culture Collection (ATCC, Patent Depository, 10801 University Boulevard, Manassas, Va. 20110, United States of America) on 1 Mar. 2006 under number PTA 74-12.
The invention is further elucidated on the basis of the following examples.
The white blister resistance source originates from the parent line 9002757 of Bejo Zaden BV, seeds of which were deposited at the (ATCC, Patent Depository, 10801 University Boulevard, Manassas, Va. 20110, United States of America) on 1 Mar. 2006 under number PTA 74-12. Using this source crossings were made with different B. oleracea species (curly cale cabbage, turnip cabbage, broccoli, sprouting broccoli, white cabbage, oxheart cabbage, red cabbage, savoy cabbage, tronchuda, Brussels sprouts and cauliflower). BC1 populations were obtained after backcrossing with susceptible parent lines. Use was made of a disease test in order to select the resistant plants from these populations.
In order to preserve A. candida isolates which are used for the disease test, zoosporangia from susceptible B. oleracea plants from the field were isolated. After germination in water the spores were used to inoculate susceptible plants. After development of the blisters, these zoosporangia were harvested and stored in liquid nitrogen until use. The eventual disease test took place in the glasshouse on seedlings of the BC1 population, the seed leaves of which had developed 24 to 48 hours before. The plants were inoculated with a fresh zoospore suspension (5×104 zoospores per ml) which was prepared by washing zoosporangia from susceptible plants and allowing them to germinate in water. Several drops of zoospore suspension were pipetted onto the seed leaves. After the pipetting the plants were further grown under a plastic tunnel in order to ensure optimum conditions for infection. Two weeks after inoculation the plants were assessed, wherein they were grouped into the classes resistant, susceptible or intermediate (Williams, Screening crucifers for multiple disease resistance. Workshop, Sep. 2-3, 1981, J.F. Friedrick Center, University of Wisconsin, Madison, USA).
After performing of the disease test on the seedlings, the resistant plants were retained for the following step in the backcrossing program. The results of the disease test showed that the resistance was in principle a monogenic dominant trait. Plants with intermediate reactions were however also often found in addition to plants with susceptible and resistant reactions. This was found to be highly dependent upon the genetic background in which work was being done. Different populations were selected from the programme in which there was no, or hardly any, intermediate reaction and in which the expected segregation ratio (1:1 for a BC and 3:1 for a self-pollination) was also found.
For the development of linked DNA markers, four populations of about 200 individuals were used (cauliflower, curly cale cabbage, tronchuda, white cabbage). DNA of all individuals was isolated from leaf punches (˜0.3 cm2/leaf punch). A BSA method was used to generate closely linked DNA markers, with the aid of the RAMP technique (Random Amplified Microsatellite Polymorphisms) (Matsumoto et al., Mammalian Genome 9: 531-535, 1998; Reiter, PCR-based marker systems, in: R. L. Philips & I. K. Vasil (eds.), DNA-based markers in plants, Kluwer Academic Publishers, 2001; Weising et al., DNA fingerprinting in plants, principles, methods and application, CRC Press, 2nd ed., 21-73, 2005).
The RAMP technique, wherein an iSSR and a RAPD-primer were combined, produced band patterns having fragments therein which specifically co-segregated with the resistance, and wherein a distinction could be made between plants with and without the resistance gene. By mapping the RAMP-fragments, closely linked RAMP-markers were identified which enclose the resistance gene.
The PCR conditions used for the RAMP reactions are as follows:
PCR mix
75 mM Tris-HCL (pH 8.8)
20 mM NH4SO4
0.01% (v/v) Tween20
2.8 mM MgCl2
0.25 mM dNTPs
0.15 μM forward primer
0.2 μM reverse primer
0.04 units/μl Red Hot® DNA Polymerase (ABgene, Epsom, UK)
˜0.2 ng/μl genomic plant DNA
PCR Program:
step 1: 2 min. 93° C.
step 2: 30 sec. 93° C.
step 3: 30 sec. 35° C.
step 4: heating by 0.3°/sec to 72° C.
step 5: 1 min. 30 sec 72° C.
steps 2-5: repeat 40×
step 6: 5 min 72° C.
Polyacryl Gel Electrophoresis
For analysis of the RAMP patterns use was made of “Gene ReadIR 4200 DNA analyzers” (Licor Inc.). On the basis of an optimal concentration of 6.5% acryl amide, fragments can be separated which have a difference in size of a single base.
In order to make the fragments visible on this system it is necessary to use labelled (IRDye labels) primers. For this purpose a third of the quantity of the forward primer was replaced by a labelled primer with the same sequence.
Given in tables 1 and 2 for the different RAMP markers are the sequences of the primers, the size of the informative fragment and the estimated distance from the resistance in cM based on the number of crossing-overs in the population. Analysis of the number of crossing-overs between the different markers shows that the markers enclose the resistance gene.
BSA—Bulked Segregant Analysis—Selection strategy wherein, in large segregating populations, individuals with the same trait (phenotype) or DNA of these individuals are bulked into “pools”. After screening of these pools with DNA techniques, markers are identified which are linked to the relevant phenotype.
cM—centimorgan—Unit for the genetic distance between markers, based on the number of crossing-overs per hundred individuals.
DNA marker—A DNA fragment which is linked to a gene or another piece of DNA with a known location on the genome, which is used to monitor heritability of this gene or this location.
Dominant—Allel which masks the phenotypical expression of another allel when both are present.
Gel-electrophoresis—Method for separating molecules (DNA, RNA, protein among others), on the basis of their size, shape or charge, in a matrix (agarose or polyacrylamide) under the influence of an electric field.
Gene—The basic unit of heredity, whereby hereditary traits are transmitted from parents to progeny.
Introgression—A chromosome fragment of a line which can for instance be inserted into another line by crossing.
IRDye labels—Labels which are used for Licor imaging systems, the detection of which takes place at 700 nm or 800 nm.
iSSR-primer (inter Simple Sequence Repeat primer)—A primer designed on the 5′ end of an SSR (Single Sequence Repeat), a piece of DNA consisting of a repetition of 2 or 3 nucleotides.
Monogenic—Determined by a single gene.
PCR (Polymerase Chain Reaction)—An in vitro amplification method for multiplying a specific DNA fragment. This synthesis reaction makes use of a minimum of one oligonucleotide primer which hybridizes with a piece of DNA, after which a DNA polymerase amplifies the flanking region via successive temperature cycles.
Primer—A short oligonucleotide (−20-50 bp) complementary to the sequence of a single-strand DNA molecule, which serves as starting point of a polymerase.
RAPD-primer (Random Amplified Polymorphic DNA primer)—A 10-mer with a “random” sequence, wherein the GC-content lies between 60% and 70% and wherein the primer ends are not self-complementary.
RAMPs (Random Amplified Microsatellite Polymorphisms)-DNA fingerprinting technique based on RAPD and iSSR primers with which polymorphisms between different DNA monsters are detected.
Resistance—The ability of a plant to wholly or partially prevent the effects and/or growth of a pathogen.
BC (Backcrossing)—Crossing of an individual with one of the original parents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2000622 | May 2007 | NL | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL2008/050232 | 4/22/2008 | WO | 00 | 10/9/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/133503 | 11/6/2008 | WO | A |
| Number | Date | Country |
|---|---|---|
| WO 0008189 | Feb 2000 | WO |
| Entry |
|---|
| Bernier, Can. Plant Dis. Surv. 52: 108, 1972. |
| Fan et al., Can. J. Genet. Cytol. 25: 420-424, 1983. |
| Harper and Pittman, Phytopathology 64: 408-410. 1974. |
| Varshney et al., Theoretical and Applied Genetics 109: 153-159, 2004. |
| Ebrahimi et al., Proc. Am. Phytopathol. Soc. 3: 273, 1976. |
| Delwiche and Williams, Proc. Am. Phytopathol. Soc. 1: 66, 1974. |
| Tiwari et al., Can. J. of Plant Science 68: 297-300, 1988. |
| Kole et al., Genome 45: 22-27, 2002. |
| Tanhuanpää, Theoretical and Applied Genetics 108: 1039-1046, 2004. |
| International Preliminary Report on Patentability; 2008. |
| M.R. Santos et al., “Evaluation of a core collection of Brassica oleracea accessions for resistance to white rust of crucifiers (Albugo candida) at the cotyledon stage”, Genetic Resources and Crop Evolution, Kluwer Academic Publishers,, vol. 51, No. 7, Nov. 1, 2004, pp. 713-722. |
| A.W. May et al., “Linkage Studies in Brassica-Oleracea of Morphologic and Isozymic Markers and Disease Resistance” Phytopathology, vol. 77, No. 12, 1987, p. 1772. |
| W.Y. Cheung et al., Identification of RFLP markers linked to the white rust resistance gene (Acr) in mustard (Brassica juncea (L.) Czern. and Coss.), Genome, vol. 41, No. 4, Aug. 1998, pp. 626-628. |
| Mohammad H. Borhan et al., “White rust (Albugo candida) resistance loci on three Arabidopsis chromosomes are closely linked to downy mildew (Peronospora parasitica) resistance loci” Molecular Plant Pathology, vol. 2, No. 2, Mar. 2001, pp. 87-95. |
| International Search Report, 2008. |
| Number | Date | Country | |
|---|---|---|---|
| 20110030085 A1 | Feb 2011 | US |
