Patent Application
20140298507
Sexual reproduction in flowering plants involves two fertilization events: fusion of a sperm cell with the egg cell to give a zygote; and fusion of a second sperm nucleus with the central cell nucleus which initiates development of endosperm, the embryo nourishing tissue. Apomixis in nature occurs by a range of alterations to the regular sexual developmental pathway (
Apomixis, asexual reproduction through seeds, results in progeny that are genetic clones of the maternal parent [Bicknell, R. A. & Koltunow, A. M. (2004), Koltunow, A. M. & Grossniklaus, (2003)]. Cloning through seeds has potential revolutionary applications in agriculture because its introduction into sexual crops would allow perpetuation of any elite heterozygous genotype [Spillane, C. et al (2004), Spillane, C. et al. (2001)]. However, despite the natural occurrence of apomixis in hundreds of plant species, very few crop species reproduce via apomixis and attempts to introduce this trait by conventional breeding have failed [Spillane, C. et al. (2001), Savidan, Y. (2001)].
An alternative approach is to de novo engineer the production of clonal seeds [Spillane, C. et al (2004)]. A major component of apomixis, the initiation and formation of functional apomeiotic female gametes that are also genetically identical to the parent plant (apomeiosis), can be induced in a sexual plant using Arabidopsis thaliana mutants that affect meiosis (MiMe-1 or MiMe-2) [d'Erfurth, I. et al. (2009), or d'Erfurth, I. et al. (2010), respectively]. Apomeiotic gametes in these MiMe lines participate in sexual reproduction, giving rise to an increase in ploidy. In order to produce a clonal seed, apomeiotic female gametes must initiate embryo development without fertilization.
The controls governing the other steps of apomixis, initiation of egg cell and central cell division to begin seed development, are poorly understood. Mutations that mimic embryo development without fertilization (parthenogenesis) or those that initiate autonomous endosperm have been reported in Arabidopsis, but these genetic manipulations do not lead to the formation of viable seed [Guitton, A. E. & Berger, F. (2005), Rodrigues, J. C. et al. (2010)].
Here, the inventors demonstrate an alternative to seed development without fertilization, the conversion of apomeiotic gametes into clonal seeds by fertilizing them with a strain whose chromosomes are engineered to be eliminated from the resultant progeny.
Directional genome elimination occurs in certain wide crosses (both interspecific and intergeneric), and leads to the formation of haploid plants [Dunwell, J. M. (2010), Bains, G. S. & Howard, H. W. (1950), Barclay, I. R. (1975), Burk, L. G. et al. (1979), Clausen, R. E. & Mann, M. C. (1924), Hougas, H. W. & Peloquin, S. J. (1957), Kasha, K. J. & Kao, K. N. (1970).]. The molecular basis for genome elimination is not understood, but one theory posits that centromeres from the two parent species interact unequally with the mitotic spindle, causing selective chromosome loss [Bennett, M. D., et al. (1976); Finch, R. A. (1983), Laurie, D. A. & Bennett, M. D. (1989)].
Haploid inducer plants which induce genome elimination have been reported, particularly in maize [U.S. Pat. Nos. 5,749,169 and 5,639,95; published International applications WO 2005/004586 and WO 2008/097791, Barret, P. et al. (2008); Röber, F. K. et al. (2005), Lashermes, P. & Beckert, M. (1988)]. Many haploid inducers exhibit low rates of haploid induction. It has recently been shown that haploid plants can be generated through seed by altering the centromeric-specific histone variant CENH3 in Arabidopsis. Mutants expressing certain altered CENH3 proteins when crossed to wild-type exhibit function as haploid inducers in which progeny preferential eliminate chromosomes originating from the cenh3 mutant parent [Ravi, M. & Chan, S. W. (2010), Ravi, M., et al. Jul. 13, 2010]. The genome elimination strain GFP-tailswap was reported as having a very high frequency of generation of haploid plants (25-45%) in crosses to wild-type as the pollen donor. However, GFP-tailswap plants were reported to be mostly male sterile making crosses with female mutants difficult. In addition, GFP-tailswap plants were reported to give an extremely low frequency of viable seeds when crossed as the female to a tetraploid male that produces diploid gametes.
The present invention relates to the production of clonal embryos or seeds by conversion of apomeiotic gametes into clonal embryos or seeds. More specifically, clonal embryos or seeds are produced by crossing a MiMe plant, as either a female or male, with an appropriate plant which induces genome elimination (genome eliminator, GE). MiMe plants are those in which meiosis is totally replaced by mitosis. In specific embodiments MiMe plants are MiMe-1 plants. In specific embodiments MiMe plants are MiMe-2 plants. In specific embodiments MiMe plants are mutant plants. In a more specific embodiment, the genome eliminator is a haploid inducer exhibiting directed genome elimination of its own genome. More specifically, the genome eliminator exhibits a haploid production rate of 1% or higher viable haploids and more preferably exhibits 10% or higher viable haploids when crossed with its corresponding wild-type. In another specific embodiment, the genome eliminator is a plant that expresses one or more altered CENH3 proteins, for example GFP-tailswap or GFP-CENH3. In a specific embodiment, the genome eliminator is a mutant plant or progeny thereof. In a specific embodiment, the genome eliminator is a transformed plant or progeny thereof.
In one aspect, the present invention relates to use of efficient genome elimination strains having altered CENH3 proteins with improved fertility and seed viability (compared to GFP-tailswap) for production of clonal embryos or seeds. In specific embodiments, the genome eliminator is a plant that expresses one or more altered CENH3 proteins. In specific embodiments, the genome eliminator is a plant that expresses two or more altered CENH3 proteins. In specific embodiments, the genome eliminator is a plant that expresses two altered CENH3 proteins, one of which proteins is GFP-CENH3. In another specific embodiment, the genome eliminator is a plant that expresses two altered CENH3 proteins, one of which proteins is GFP-tailswap. In another specific embodiment, the genome eliminator is a plant that expresses at least two altered CENH3 proteins, one of which proteins is GFP-tailswap and another of which is GFP-CENH3.
The invention also relates to clonal progeny produced by crossing a MiMe plant with a genome eliminator plant and to plant cells and tissue of such progeny. In specific embodiments the progeny are produced by crossing a MiMe plant with a genome eliminator which is a plant that expresses one or more altered CENH3 proteins.
In specific embodiments, MiMe plants form asexual diploid gametophytes which are then pollinated with pollen of the genome eliminator, the chromosome of the genome eliminator is selectively eliminated and an embryo develops solely from the diploid egg cell genome (gynogenesis). In other specific embodiments, genome eliminator plants form haploid gametophytes which are double fertilized by diploid pollen of a MiMe plant, the maternal genome of the genome eliminator is selectively eliminated and a diploid embryo develops from the sperm cell (androgenesis).
In specific embodiments, the MiMe plants and genome eliminator plants are Arabidopsis, particularly Arabidopsis thaliana. In specific embodiments, the MiMe plants and Arabidopsis plants are Oryza sativa. In specific embodiments, the MiMe plants and genome eliminator plants are Zea mays.
The invention relates to a method for generating clonal embryos or clonal seed which comprises the steps of crossing a MiMe plant as a male or female with a genome eliminator plant and selecting viable clonal embryos or seeds.
The invention also relates to methods of cultivating a clonal plant that is obtained by the methods of this invention and recovering gametes, particularly viable gametes, produced by that plant.
Plants produced by the methods of this invention are for example useful in plant breeding.
Other aspects of the invention will be apparent to one of ordinary skill in the art on consideration of the following detailed description, examples and figures. It is to be understood, however, that this detailed description, as well as any examples and figures are exemplary only and do not limit the invention.
×GEM
(A) and GEM
×osd1
(B) offspring as discussed in the Examples.
×GEM
(A), cloned MiMe
×GEM
(B) and GEM
×
(C) offspring as discussed in the Examples. Color coding is provided in
As illustrated in
1) Megasporogenesis: The formation of a megaspore from the archesporial cell of the ovule by meiosis.
2) Megagametogenesis: The formation of an embryo sac (female gametophyte) by the mitotic division of the haploid megaspore.
3) Double fertilization. One sperm cell fuses with the egg cell to form the zygote (2n) and the other sperm cell fertilizes the central cell to form the triploid (3n) embryo nourishing tissue, the endosperm.
As illustrated in
As illustrated in
Clonal reproduction though seeds is of great interest for agriculture because it allows the propagation of a chosen genotype to the infinite. Endless propagation requires that clonal reproduction can be achieved from generation to generation. As discussed below, the present invention demonstrates that clonal reproduction can be achieved from generation to generation and in principle indefinitely, by crossed a maternal MiMe clone to the exemplary genome eliminator strain GEM for a second generation with the result that the progeny of this cross, produce a large proportion (24%, n=79) of plants genetically identical to their mother and grandmother.
The strategies described herein reflect a de novo synthetic approach to creating apomixis in sexual plants. Given that apomixis in nature occurs by a range of developmental mechanisms it is not unexpected that there would be more than one way of achieving synthetic apomixis. The molecular mechanisms underlying apomixis have resisted elucidation and the genomic regions to which apomixis loci have been mapped are large and show reduced levels of recombination [Ozias-Akins and van Dijk (2007)], making it difficult to identify specific genetic elements that control the trait. It is not unlikely that apomixis as it occurs in nature may be highly context dependent and not readily amenable to transfer to other plant species. The de novo synthesis approach provided herein overcomes this limitation as the genes involved have clear homologues across plant species.
MiMe Plants
A plant having the MiMe (mitosis instead of meiosis) genotype is a plant in which a deregulation of meiosis results in a mitotic-like division and in which meiosis is replaced by mitosis. MiMe plants are exemplified by MiMe-1 plants as described by d'Erfurth, I. et al. (2009) and International patent application WO2001/079432, published Jul. 15, 2010) and MiMe-2 plants as described by d'Erfurth, I. et al. (2010). Each of these three references is incorporated by reference herein in its entirety to provide details of plants having the MiMe genotype and the OSD1 gene and the TAM gene (also designated CYCLIN-A CYCA1;2/TAM, which encodes the Cyclin A CycA1;2 protein) and to provide methods for making MiMe plants. Additional detailed methods provided in these references include sources of plant material, plant growth conditions, genotyping employing PCR and primers useful for such genotyping, and methods of cytology and flow cytometry. These references also provide details of specific mutants employed to produce MiMe plants.
Mercier R. & Grelon M. (2008) provide a recent review of plant meiotic genes which have been functionally characterized, particularly in Arabidopsis, rice and maize. This reference provides an overview of methods employed for such characterization.
Plants having the MiMe genotype produce functional diploid gametes that are genetically identical to their parent. Exemplary MiMe plants combine phenotypes of (1) no second meiotic division, (2) no recombination and (3) modified chromatid segregation.
Exemplary MiMe-1 plants combine inactivation of the OSD1 gene, with the inactivation of two or more other genes, one which encodes a protein necessary for efficient meiotic recombination in plants (e.g., SPO11-1, SPO11-2, PRD1, PRD2, or PAIR1), and whose inhibition eliminates recombination and pairing [Grelon et al., (2001)], and another which encodes a protein necessary for the monopolar orientation of the kinetochores during meiosis, e.g., REC8, and whose inhibition modifies chromatid segregation [Chelysheva et al (2005)]. Exemplary MiMe-2 plants combine inactivation of the TAM gene [d'Erfurth, I. et al. (2010)] with the inactivation of two or more other genes, one which encodes a protein necessary for efficient meiotic recombination in plants (e.g., SPO11-1, SPO11-2, PRD1, PRD2, or PAIR1), and whose inhibition eliminates recombination and pairing [Grelon et al., (2001)], and another which encodes a protein necessary for the monopolar orientation of the kinetochores during meiosis, e.g., REC8, and whose inhibition modifies chromatid segregation [Chelysheva et al (2005)]. MiMe-1 plants are distinguished from MiMe-2 in that MiMe-1 plants are generally more efficient for production of 2N female gametes. For example, in Arabidopsis thaliana specific MiMe-2 mutants generate ˜30% of 2N female gametes, compared to 80% in comparable MiMe-1 mutants [d'Erfurth, I. et al. (2009) and d'Erfurth, I. et al. (2010)].
The replacement of meiosis by mitosis results in apomeiotic gametes, retaining all of the parent's genetic information. The apomeiotic gametes produced by the MiMe mutant can be used, in the same way as SDR (Second Division Restitution) 2n gametes, for producing polyploids plants, or for crossing plants of different ploidy level. They are, however of particularly interest for the production of apomictic plants.
Inactivation of the OSD1 gene (omission of second division) in plants results in the skipping of the second meiotic division. This generates diploid male and female spores, giving rise to viable diploid male and female gametes, which are SDR gametes. The sequence of the OSD1 gene of Arabidopsis thaliana is available in the TAIR database under the accession number At3g57860, or in the GenBank database under the accession number NM—115648. This gene encodes a protein of 243 amino acids (GenBank NP—191345), whose sequence is also represented in the enclosed sequence listing as SEQ ID No. 1, Table 1. The OSD1 gene of Arabidopsis thaliana had previously been designated “UVI4-Like” gene (UVI4-L), which describes its paralogue UVI4 as a suppressor of endo-reduplication and necessary for maintaining the mitotic state (Hase et al. Plant J, 46, 317-26, 2006). However, OSD1 (UVI4-L) does not appear to be required for this process, but is necessary for allowing the transition from meiosis I to meiosis II. An ortholog of the OSD1 gene of Arabidopsis thaliana has been identified in rice (Oryza sativa). The sequence of this gene is available as accession number Os02g37850 in the TAIR database and the gene encodes a protein of 234 amino acid (sequence provided as SEQ ID No. 2, Table 2). The OSD1 proteins of Arabidopsis thaliana and Oryza sativa have 23.6% sequence identity and 35% sequence similarity over the whole length of their sequences. A plant producing Second Division Restitution 2N gametes can, for example, be obtained by inhibition in the plant of an OSD1 protein. Table 13 (SEQ ID Nos. 24-46) provides additional exemplary OSD1/UV14 protein sequences.
Inactivation of the TAM gene in plants can result in skipping of the second meiotic division giving a phenotype similar to that of osd1 mutants leading to the production of dyads of spores and diploid gametes that have undergone recombination. More specifically, Arabidopsis mutants including tam-2, tam-3, tam-4, tam-5, tam-6 and tam-7 as described in d'Erfurth, I. et al. (2010) express the dyad phenotype at normal growing temperatures and systematically produce mostly dyads. Plant mutants exhibiting inactivation of the TAM gene as in such mutants are useful in preparation of MiMe-2 plants. In contrast, Arabidopsis mutants such as tam-1 [Magnard, J. L. et al. (2001)] which exhibit a delay in the progression of meiosis and progress beyond the dyad stage are not useful in preparation of MiMe-2 plants. The TAM gene encodes a protein exhibiting cyclin-dependent protein kinase activity. The sequence of the TAM gene of Arabidopsis thaliana is available in the TAIR database under the accession number At1 G77390 (Table 9, SEQ ID No. 9). This gene encodes a protein of 442 amino acids (GenBank NP 177863). Cyclin-dependent kinases are reported to be highly conserved among plants and a CycA1;2 gene has been identified in rice (La, H. et al. (2006)]. A Cyclin-A1-2 protein of rice (Accession Q0JPA4-1 in UniProtKB/Swiss-Prot. Database) is identified as having 477 amino acid (Table 10, SEQ ID No. 10). A plant producing Second Division Restitution 2N gametes can, for example, be obtained by inhibition in the plant of an TAM (CycA1;2) protein. Table 12 provides the protein sequence of CYCA1; 2 of A. lyrata (SEQ ID No. 23).
Published International application WO 2010/07943 provides a schematic comparison (reproduced as
SPO11-1 and SPO11-2 proteins are related orthologs, both of which are required for meiotic recombination. [Grelon et al. (2001); Stacey et al. (2006); Hartung et al. (2007)]. Inhibition of one or both of SPO11-1 or SPO11-2 is useful in a MiMe plant of this invention. Examples of SPO11-1 and SPO11-2 proteins are provided in Table 3 (SEQ ID No. 3) and Table 4 (SEQ ID No. 4).
PRD1 protein is required for meiotic double stand break (DSB) formation and is exemplified by AtPRD1, a protein of 1330 amino acids (Table 5, SEQ ID No. 5) exhibiting significant sequence similarity with OsPRD1 (NCB1 Accession number CAE02100) SEQ ID No. 47 (Table 14). PRD1 homologs have also been identified in Physcomitrella patens (PpPRD1) from ASYA488561.b1; Medicago truncatula (MtPRD1) from sequences AC147484 (start 93451-end 101276) and Populus trichocarpa (PtPRD1) from LG_II:20125180-20129370 (http://genome.jgi-psf.org/Poptr1—1/Poptr1—1.home.html), see De Muyt et al. 2007,
PRD2 protein is a DSB-forming protein exemplified by AtPRD2, a protein of 378 amino acids (Table 6, SEQ ID No: 6) amino acids (identified as a protein of 385 amino acids in De Muyt et al. (2009) see Sequence Accession NP 568869 (Table 11, SEQ ID No. 18), with homologues identified in the monocot Oryza sativa, Populous trichocarpa, Vitis vinifera and Physcomitrella patens [De Muyt et al. (2009)] and see (Table 11, SEQ ID Nos. 19-22). PAIR1 (also called PRD3) is a DSB-forming protein exemplified by AtPAIR1, a protein a 449 amino acid protein (Table 7, SEQ ID No. 7) and its presumed ortholog OsPAIR1 [Nonomura et al. (2004)] a 492-amino acid protein, see Table 15, SEQ ID No. 50.
REC8 protein is a subunit of the cohesion complex. In plants, exemplified by Arabidopsis, REC8 protein (Table 8, SEQ ID No. 8) is necessary for monopolar orientation of the kinetochores [Chelysheva et al. (2005)].
In specific embodiments, plants producing apomeiotic gametes are produced by inhibition in the plant of the following proteins (a) a TAM (Cylin A CYCA1;2) protein (as described herein); (b) a protein involved in initiation of meiotic recombination in plants exemplified herein as SPO11-1; SPO11-2; PRD; PRD2; or PAIR1 (also called PRD3); and (c) a protein necessary for the monopolar orientation of the kinetochores during meiosis exemplified herein as REC8 protein.
In specific embodiments, plants producing apomeiotic gametes are produced by inhibition in the plant of the following proteins (a) an OSD 1 protein (as described herein); (b) a protein involved in initiation of meiotic recombination in plants exemplified herein as SPO11-1; SPO11-2; PRD; PRD2; or PAIR1 (also called PRD3); and (c) a protein necessary for the monopolar orientation of the kinetochores during meiosis exemplified herein as REC8 protein.
The OSD1 protein is exemplified by the AtOSD1 protein (SEQ ID No. 1) or the Os OSD1 protein (SEQ ID No. 2) and includes OSD1 protein wherein said protein has at least 20%, and by order of increasing preference, at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 29%, and by order of increasing preference, at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the AtOSD1 protein of SEQ ID No. 1 or with the OsOSD1 protein of SEQ ID No. 2.
The Cyclin-A CYCA1;2 (TAM) protein is exemplified by the CYCA1; 2 protein of Arabidopsis (SEQ ID No. 9) or the CYCA1; 2 protein of rice (SEQ ID No. 10) protein wherein said protein has at least 20%, and by order of increasing preference, at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 29%, and by order of increasing preference, at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98%
The protein involved in initiation of meiotic recombination in plants is exemplified by an SPO11-1 or SPO11-2 protein and particularly the AtSPO11-1 protein (SEQ ID No. 3), the AtSPO11-2 protein (SEQ ID No. 4) and includes SPO11-1 and SPO11-2 proteins having at least 40%, and by order of increasing preference, at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 60%, and by order of increasing preference, at least, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the SPO11-1 protein of SEQ ID No. 3 or the SPO11-2 protein of SEQ ID No. 4.
The protein involved in initiation of meiotic recombination in plants is also exemplified by a PRD1 or PRD2 protein and particularly the AtPRD1 protein (SEQ ID No. 5), and the AtPRD2 protein (SEQ ID No. 6) and includes PRD1 or PRD2 proteins having at least 25%, and by order of increasing preference, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 35%, and by order of increasing preference, at least, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the PRD1 protein of SEQ ID No. 5) or PRD2 protein of SEQ ID No. 6).
The protein involved in initiation of meiotic recombination in plants is also exemplified by a PAIR1 protein (also known as a PRD3 protein) and particularly the AtPAIR1 protein (SEQ ID No. 7), and includes PAIR1 proteins having at least 30%, and by order of increasing preference, at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 40%, and by order of increasing preference, at least, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the PAIR1 protein of SEQ ID No. 7.
The protein necessary for the monopolar orientation of the kinetochores during meiosis is exemplified herein as a REC8 protein (also designated DIF1/SYN1) a member of the cohesion complex in plants, particularly Arabidopsis. REC8 protein includes AtREC8 protein (SEQ ID No. 8) and includes REC8 protein having at least 40%, and by order of increasing preference, at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 45%, and by order of increasing preference at least, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the REC8 protein of SEQ ID No. 8.
The SPO11-1, SPO11-2, PRD1, PRD2, PAIR1, and REC8 proteins are conserved in higher plants, monocotyledons as well as dicotyledons. By way of non-limitative examples of orthologs of SPO11-1, SPO11-2, PRD1, PRD2, PAIR1 and REC8 proteins of Arabidopsis thaliana in monocotyledonous plants, one can cite the Oryza sativa SPO11-1, SPO11-2, PRD1, PRD2, PAIR1, and REC8 proteins. The sequence of the Oryza sativa SPO11-1 protein is available in GenBank under the accession number AAP68363 see Table 15 SEQ ID No. 48; the sequence of the Oryza sativa SPO11-2 protein is available in GenBank under the accession number NP—001061027 see Table 15 SEQ ID No. 49; the sequence of the Oryza sativa PRD1 protein is provided as SEQ ID No. 47 (Table 14); the sequence of the Oryza sativa PRD2 protein is provided (SEQ ID No. 21); the sequence of the Oryza sativa PAIR1 protein is available in SwissProt under the accession number Q75RY2, see Table 15 SEQ ID No. 50; the sequence of the Oryza sativa REC8 protein (also designated RAD21-4) is available in GenBank under the accession number AAQ75095., see Table 15, SEQ ID No. 51. Additional non-limiting examples of orthologs of PRD2 include Vitis vinifera VvPRD2 (accession number CAO66652) see Table 11, SEQ ID No. 20; Populous trichocarpa PtPRD2 (obtained from JCI (fgenesh4_pm.C_LG_VI000547) see Table 11 SEQ ID NO. 20 and Physcomitrella patens PpPRD2 obtained from JGI (jgi|Phypa1—1|73600|fgenesh1_pg.scaffold—42000158).
The inhibition of the above mentioned OSD1, Cyclin-A CYCA1;2 (TAM), SPO11-1, SPO11-2, PRD1, PRD2, PAIR1, or REC8 proteins can be obtained either by abolishing, blocking, or decreasing their function, or advantageously, by preventing or down-regulating the expression of the corresponding genes. By way of example, inhibition of said protein can be obtained by mutagenesis of the corresponding gene or of its promoter, and selection of the mutants having partially or totally lost the activity of said protein. For instance, a mutation within the coding sequence can induce, depending on the nature of the mutation, the expression of an inactive protein, or of a protein with impaired activity; in the same way, a mutation within the promoter sequence can induce a lack of expression of said protein, or decrease thereof.
Mutagenesis can be performed for instance by targeted deletion of the coding sequence or of the promoter of the gene encoding said protein or of a portion thereof, or by targeted insertion of an exogenous sequence within said coding sequence or said promoter. It can also be performed by inducing random mutations, for instance through EMS mutagenesis or random insertional mutagenesis, followed by screening of the mutants within the desired gene. Methods for high throughput mutagenesis and screening are available in the art. By way of example, one can mention TILLING (Targeting Induced Local Lesions In Genomes) described by McCallum et al., 2000).
Among the mutations within the OSD1 gene or TAM gene, those resulting in the ability to produce SDR 2n gametes can be identified on the basis of the phenotypic characteristics of the plants which are homozygous for this mutation: these plants can form at least 5%, preferably at least 10%, more preferably at least 20%, yet more preferably 30% or more, still more preferably at least 50%, and up to 100% of dyads as a product of meiosis.
Among the mutations within a gene encoding a protein involved in initiation of meiotic recombination in plants, such as the SPO11-1 gene or the SPO11-2, PRD1, PRD2 or PAIR1 gene, those useful for obtaining a plant producing apomeiotic gametes can be identified on the basis of the phenotypic characteristics of the plants which are homozygous for this mutation, in particular the presence of univalents instead of bivalents at meiosis I, and the sterility of the plant. Among the mutants having a mutation within the REC8 gene, those useful for obtaining a plant producing apomeiotic gametes can be identified on the basis of the phenotypic characteristics of the plants which are homozygous for this mutation, in particular chromosome fragmentation at meiosis, and sterility of the plant.
Alternatively, the inhibition of the target protein is obtained by silencing of the corresponding gene. [See, for example, the review Baulcombe, D. (2004)]. Methods for gene silencing in plants are known in the art. For instance, antisense inhibition or co-suppression, as described by way of example in U.S. Pat. Nos. 5,190,065 and 5,283,323 can be used. It is also possible to use ribozymes targeting the mRNA of said protein. Preferred methods are those wherein gene silencing is induced by means of RNA interference (RNAi), using a silencing RNA targeting the gene to be silenced. Various methods and DNA constructs for delivery of silencing RNAs are available in the art.
A “silencing RNA” is herein defined as a small RNA that can silence a target gene in a sequence-specific manner by base pairing to complementary mRNA molecules. Silencing RNAs include in particular small interfering RNAs (siRNAs) and microRNAs (miRNAs).
Initially, DNA constructs for delivering a silencing RNA in a plant included a fragment of 300 bp or more (generally 300-800 bp, although shorter sequences may sometime induce efficient silencing) of the cDNA of the target gene, under transcriptional control of a promoter active in said plant. Currently, the more widely used silencing RNA constructs are those that can produce hairpin RNA (hpRNA) transcripts. In these constructs, the fragment of the target gene is inversely repeated, with generally a spacer region between the repeats [for a review, see Watson et al., (2005)]. One can also use artificial microRNAs (amiRNAs) directed against the gene to be silenced (for review about the design and applications of silencing RNAs, including in particular amiRNAs, in plants see for instance [Ossowski et al., (2008)].
Tools for silencing one or more target gene(s) selected among OSD1, TAM, SPO11-1 SPO11-2, PRD1, PAIR1, PRD2, and REC8, including expression cassettes for hpRNA or amiRNA targeting said gene (s) are described and provided in PCT application WO 2010/079432. Useful expression cassettes comprise a promoter functional in a plant cell; one or more DNA construct(s) of 200 to 1000 bp, preferably of 300 to 900 bp, each comprising a fragment of a cDNA of a target gene selected among OSD1, TAM, SPO11-1, SPO11-2, PRD1, PRD2, PAIR1, and REC8, or of its complement, or having at least 95% identity, and by order of increasing preference, at least 96%, 97%, 98%, or 99% identity with said fragment, where the DNA construct(s) is placed under transcriptional control of the promoter. Additional useful expression cassettes for hpRNA comprise a promoter functional in a plant cell, one or more hairpin DNA construct(s) capable, when transcribed, of forming a hairpin RNA targeting a gene selected among OSD1, TAM, SPO11-1, SPO11-2, PRD1, PRD2, PAIR1, and REC8; where the DNA construct(s) is placed under transcriptional control of the promoter.
Generally, useful hairpin DNA constructs comprise: i) a first DNA sequence of 200 to 1000 bp, preferably of 300 to 900 bp, such as a fragment of a cDNA of the target gene, or having at least 95% identity, and by order of increasing preference, at least 96%, 97%, 98%, or 99% identity with the fragment; ii) a second DNA sequence that is the complement of the first DNA, said first and second sequences being in opposite orientations and ii) a spacer sequence separating the first and second sequence, such that these first and second DNA sequences are capable, when transcribed, of forming a single double-stranded RNA molecule. The spacer can be a random fragment of DNA. However, preferably, one will use an intron which is spliceable by the target plant cell. Its size is generally 400 to 2000 nucleotides in length. A useful expression cassette for an amiRNA comprises: a promoter functional in a plant cell, one or more DNA construct(s) capable, when transcribed, of forming an amiRNA targeting a gene selected among OSD1, TAM, SPI11-1, SPO11-2, PRD1, PRD2, PAIR1, and REC8; where the DNA construct(s) is placed under transcriptional control of the promoter. Useful expression cassettes comprise a DNA construct targeting the OSD1 gene or comprise a DNA construct targeting the OSD1 gene, and a DNA construct targeting a gene selected from one or more of SPO11-1, SPO11-2, PRD1, PRD2, or PAIR1, and a DNA construct targeting REC8. Useful expression cassettes comprise a DNA construct targeting the TAM gene or comprise a DNA construct targeting the TAM gene, and a DNA construct targeting a gene selected from one or more of SPO11-1, SPO11-2, PRD1, PRD2, or PAIR1, and a DNA construct targeting REC8. Additional useful expression cassettes comprise a DNA construct targeting the OSD1 gene and/or the TAM gene and/or comprise a DNA construct targeting the OSD1 gene and or the TAM gene, and/or a DNA construct targeting a gene selected from one or more of SPO11-1, SPO11-2, PRD1, PRD2, or PAIR1.
It will be appreciated by one of ordinary skill in the art that a large choice of promoters suitable for expression of heterologous genes in plants is available in the art. Useful promoters include those obtained from plants, plant viruses, or bacteria, such as Agrobacterium. Promoters include constitutive promoters, i.e. promoters which are active in most tissues and cells and under most environmental conditions, as well as tissue-specific or cell-specific promoters which are active only or mainly in certain tissues or certain cell types, and inducible promoters that are activated by physical or chemical stimuli, such as those resulting from nematode infection. Non-limiting examples of constitutive promoters that are commonly used in plant cells are the cauliflower mosaic virus (CaMV) 35S promoter, the Nos promoter, the rubisco promoter, or the Cassava vein Mosaic Virus (CsVMV) promoter. Organ or tissue specific promoters that can be used in such expression cassettes include in particular promoters able to confer meiosis-associated expression, such as the DMC1 promoter [Klimyuk & Jones (1997)]; one can also use any of the endogenous promoters of the genes OSD1, TAM, SPO11-1, SPO11-2, PRD1, PRD2, PAIR1, or REC8. Useful DNA constructs of the invention generally also include a transcriptional terminator (for instance the 35S transcriptional terminator, or the nopaline synthase (Nos) transcriptional terminator).
Recombinant vectors, host cells comprising recombinant DNA constructs, transgenic plants, transgenic plant cells and methods of transforming plants with a vector targeting the OSD1 gene and/or the TAM gene and/or a vector targeting one or more of the SPO11-1, SPO11-2, PRD1, PRD2, or PAIR1 genes and/or a vector targeting the REC8 gene and regenerating such transgenic plants are described and provided in PCT application WO 2010/079432 and are useful in preparation of MiMe plants useful in this invention. The expression of a chimeric DNA construct targeting the OSD1 gene, and which results in a down regulation of the OSD1 protein, provides to a transgenic plant the ability to produce 2n SDR gametes. The expression of a chimeric DNA construct targeting the TAM gene, and which results in a down regulation of the Cyclin A CycA1;2 protein, provides to a transgenic plant the ability to produce 2n SDR gametes. The co-expression of a chimeric DNA construct targeting the OSD1 gene and/or the TAM gene, a chimeric DNA construct targeting a gene selected among one or more of SPO11-1, SPO11-2, PRD1, PRD2 and PAIR1, and a chimeric DNA construct targeting the REC8 gene and which results in down regulation of the proteins encoded by these genes provides to a transgenic plant the ability to produce apomeiotic gametes. MiMe plants include those which produce at least 10%, more preferably at least 20%, and by order of increasing preference, at least 30%, 40%, 50%, or 60%, 70%, 80%, or 90% of viable apomeiotic gametes. MiMe plants also include those that are heterozygous for the MiMe.
The genes discussed above which confer the MiMe genotype are strongly conserved among plants, including monocots and dicots, thus, the MiMe genotype can be engineered, for example, as described herein in any plant species, including crop species. In specific embodiments, the MiMe genotype can be engineered as described herein in various species of Arabidopsis, in various crop plants including without limitation, rice, soybean, corn or maize, rye, cotton, oats, barley, wheat, alfalfa, sorghum, sunflower, various legumes, various Brassica, potato, peanuts, clover, sweet potato, cassava (manioc), rye-grass, banana, melon, watermelon, sugar beets, sugar cane, lettuce, carrots, spinach, endive, leeks, celery, artichokes, beets, radishes, turnips or tomato or ornamental plants such as roses, lilies, tulips or narcissus.
MiMe plants of this invention can be further engineered employing techniques that are well known to one of ordinary skill in the art to contain one or more non-endogenous genes or mutated endogenous genes the expression of which provides: (1) one or more gene products useful for screening or selection of such plants; or (2) one or more agriculturally useful traits. Methods of the present invention allow generation of clonal embryos or seeds which will retain such one or more non-endogenous genes or mutated genes.
Genome Eliminator Strains
Haploid inducer plants with directed genome elimination have been identified, generated or engineered in various plants and in particular in maize and Arabidopsis. Plants which induce genome elimination as described herein function for genome elimination in crossings with any MiMe plant.
U.S. Pat. No. 5,749,169 describes certain haploid inducer maize plants which induce genome elimination (ig plants-indeterminate gametophyte), including homozygous (igig) plants which can be used to generate androgenetic haploids. Female ig plants are pollinated with pollen from a selected maize plant, e.g., one carrying a mutation associated with a desirable phenotype. Progeny from such crosses include a significantly enhanced percentage of androgenetic haploids containing chromosomes derived only from the male parent. Maize ig plants exhibiting approximately 1 to 3% androgenetic haploids of total progeny are reported. Maize ig plants induce haploids of both male and female origin. The ig trait was initially reported as arising in the inbred Wisconsin-23 (W23) strain (Kermicle, J. L., 1969). U.S. Pat. No. 5,749,169 is incorporated by reference herein for its description of haploid inducers, particularly in maize and for methods of making and identifying such haploid inducers.
U.S. Pat. No. 5,639,951 describes maize haploid inducers, particularly those exhibiting the ig genotype and having a least one dominant gene which may be a conditional lethal gene, a screenable marker gene or a selectable marker gene. The presence of the dominant gene is useful in screening and selection methods. U.S. Pat. No. 5,639,951 is incorporated by reference herein for its description of haploid inducers with dominant genes as described, particularly in maize, and for methods of making an identifying such haploid inducers.
Maize genotypes which induce gynogenesis producing maternal haploids with chromosomes derived from the female parent have been described. Such inducer lines for maize include, but are not limited to, Stock 6 and Stock 6 derivatives [Coe, (1959); Sarkar & Coe, (1966); Sarkar et al. (1972), Lashermes & Beckert (1988), Chalyk, S. T. (1994), Bordes, J. R. et al. (1997), Eder J. & Chalyk, S. (2002) RWS [Röber et al. (2005)], KEMS [Deimling, et al. (1997)], or KMS and ZMS [Chalyk, S. T. et al. (1994), Chalyk & Chebotar (2000)]. The Stock 6 derivative WS14 [Lashermes & Beckert (1988)] is reported to exhibit haploid induction rate that is 1.2 to 5.5 times higher than that of Stock 6. A WS14 derivative designated FIGH 1 [Bordes et al. (1997)] is also of interest. Crosses between two haploid-inducing lines can be used generate progeny haploid inducers exhibiting higher rates of haploid induction compared to their parents, for examples crosses between KMS and ZMS lines are reported to be capable of inducing 7 to 9% of haploids [Chalyk et al. (1994)]. The disclosure of each of the foregoing references is incorporated by reference herein in its entirety for its description of haploid inducer lines, methods for identifying and/or making such lines, and sources of material for making such lines.
International patent application WO 2005/004586 describes certain gynogenetic haploids in maize which are designated as in the PK6 line of maize or derivative lines thereof. Haploid inducers of this maize line are reported to exhibit rates of gynogenetic haploid induction much superior to those observed with prior art haploid inducers. WO 2005/004586 is incorporated by reference herein in its entirety for descriptions of PK6 plants and derivatives thereof as well as for methods of making such plants by breeding and/or transformation methods.
Geiger H. H. & Gordillo (2009) provide a description of measurement of haploid induction rates and provide examples of maize haploid inducer lines (e.g., RWS, RWK-76 and the cross RWS×RWK-76) having higher haploid inducer rates (e.g., greater than 1%). This reference is incorporated by reference herein for details of the measurement of haploid induction rate and for sources of haploid inducers having higher haploid inducer rates.
Genome eliminator strains of this invention include all such haploid inducers and derivatives thereof. Haploid inducers include derivatives of the specifically mentioned haploid inducers which are generated by conventional plant breeding methods.
Mutants Having Altered CENH3 Protein
Mutants having altered CENH3 protein are exemplified by those described in Ravi, M & Chan, S. W-L. 2010 and Ravi, M. et al. Jul. 13, 2010. Each of which references is incorporated by reference in its entirety herein for description of such mutants and methods for making such mutants. Published patent application US 2011/0083202 A1 (Chan and Maruthachalam, Apr. 7, 2011) provides description of altered CENH3 protein and is incorporated by reference herein in its entirety for that description.
It will be appreciated however that CENH3 variants other than those specifically described in Ravi, M & Chan, S. W-L. 2010 and Ravi, M. et al. Jul. 13, 2010 are useful for making genome eliminator plants of this invention. It will be appreciated for example that useful CENH3 variants for a given plant can be obtained by replacing the N-terminal tail domain of the CENH3 endogenous in that plant with the N-terminal tail domain of a centromere specific histone of the same species of plant or that of a different species of plant or that of another organism.
It will be appreciated that any GFP-tag in an altered variant of CENH3 can be replaced with various other known tags (e.g., β-galactosidase, cyan fluorescent protein (CYP), or yellow fluorescent protein (YFP)) by methods that are well known in the art. Thus, tagged-CENH3 variants are useful in the methods of this invention.
Additional altered CENH3 useful in this invention preferably exhibits overall % identity of amino acid sequence to the endogenous CENH3 that is at least 25% and by order of increasing preference, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98%, or at least 35%, and by order of increasing preference at least, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% overall sequence similarity to the endogenous CENH3.
In specific embodiments, altered CENH3 having a GFP tag or functionally equivalent other tag (e.g., β-galactosidase, cyan fluorescent protein (CYP), yellow fluorescent protein (YFP, e.g., PhiYFP (Trademark, Evrogen)) can exhibit overall % identity of amino acid sequence to the endogenous CENH3 that is at least 50% and by order of increasing preference, at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, or 98%, or at least 60%, and by order of increasing preference at least, 65, 70, 75, 80, 85, 90, 95, 96 or 98% overall sequence similarity to the endogenous CENH3.
Further additional altered CENH3 useful in this invention preferably exhibit % identity of amino acid sequence to the histone fold region of the endogenous CENH3 that is at least 50% and by order of increasing preference, at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96 or 98%, or at least 60%, and by order of increasing preference at least, 65, 70, 75, 80, 85, 90, 95, 96 or 98% sequence similarity to the histone fold region of the endogenous CENH3.
In specific embodiments, altered CENH3 having a GFP tag or functionally equivalent other tag, can exhibit overall % identity of amino acid sequence to the histone fold region of endogenous CENH3 that is at least 50% and by order of increasing preference, at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, or 98%, or at least 60%, and by order of increasing preference at least, 65, 70, 75, 80, 85, 90, 95, 96 or 98% overall sequence similarity to the histone fold region of endogenous CENH3.
Plants expressing one, two or more altered CENH3 proteins which are haploid inducers preferably exhibit haploid induction rates of 1% or more and by order of increasing preference, 3% or more, 5% or more, 10% or more, 20% or more or 30% or more.
It will be appreciated that transformant plants expressing altered CENH3 may exhibit differences in expression level caused by position effects. One of ordinary skill in the art knows how to detect such position effects which may affect expression levels of altered CENH3 protein and select transformants with expression levels which exhibit levels of expression of one, two or more altered CENH3 protein that provide for haploid induction.
Useful CENH3 variants can be prepared by methods as described in Ravi, M & Chan, S. W-L. 2010 and Ravi, M. et al. Jul. 13, 2010 employing expression cassettes and plant transformation methods as described therein or by any means know in the art which would be appreciated by one of ordinary skill in the art to provide for expression of such variants in plants.
It will be appreciated that plants expressing CENH3 variants useful as haploid inducers can be prepared in various plants including without limitation in both monocots or dicots. Plants expressing such altered CENH3 genotypes can be engineered, for example, as described herein in any plant species, including crop species. In specific embodiments, the altered CENH3 genotype can be engineered as described herein in various species of Arabidopsis, in various crop plants including without limitation, rice, soybean, corn or maize, rye, cotton, oats, barley, wheat, alfalfa, sorghum, sunflower, various legumes, various Brassica, potato, peanuts, clover, sweet potato, cassava (manioc), rye-grass, banana, melon, watermelon, sugar beets, sugar cane, lettuce, carrots, spinach, endive, leeks, celery, artichokes, beets, radishes, turnips or tomato or ornamental plants such as roses, lilies, tulips or narcissus.
Unless otherwise specified, the protein sequence identity and similarity values provided herein are calculated over the whole length of the sequences, using the BLASTP program under default parameters, or the Needleman-Wunsch global alignment algorithm (EMBOSS pairwise alignment Needle tool under default parameters). Similarity calculations are performed using the scoring matrix BLOSUM62.
As used herein, the term “plant” includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. “Plant cell”, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
MiMe plants or any of the various haploid inducer plants useful in this invention can include, or be bred or engineered to include and express a selectable or screenable marker gene. Selectable markers generally include genes encoding antibiotic resistance or resistance to herbicide, which are known in the art. Screenable markers include β-galactosidase, green fluorescent protein (GFP), cyan fluorescent protein (CYP), yellow fluorescent protein (YFP, e.g., PhiYFP (Trademark, Evrogen)). MiMe plants or any of the various haploid inducer plants useful in this invention can include, or be bred or engineered to include and express a gene or combination of genes conveying a phenotype or trait of interest, such a phenotype or trait of agricultural interest. Conventional plant breeding methods or plant transformation methods may be used to generate such derivatives of MiMe plants and/or haploid inducer plants.
A portion of the subject matter of this application is reported in Marimuthu M. P et al. 2011, which is incorporated by reference herein in its entirety.
When a grouping is used herein, all individual members of the group and all possible combinations and subcombinations of the members of the groups therein are intended to be individually included in the disclosure. Every plant mutant, line or strain, or combination thereof described or exemplified herein can be used to practice the invention, unless otherwise stated.
One of ordinary skill in the art will appreciate that methods, procedures and materials, such as methods for detecting the presence or absence of genes or proteins, hybridization methods, PCR methods, culturing methods and media, other than those specifically exemplified herein can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such methods, materials and conditions are intended to be included in this invention.
Whenever a range is given in the specification, for example, a range of numbers, a range of any integer, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure.
As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the broad term “comprising”, particularly in a description of components of a composition, the recitation of steps in a method or in a description of elements of a device, is intended to encompass and describe the terms “consisting essentially of” or “consisting of”.
Although the description herein contains many specific details, these should not be construed as limiting the scope of the invention, but as merely providing illustrations of some of the embodiments of the invention. Each patent document and publication referenced in this specification is incorporated by reference herein to the same extent as if each individual document or publication was specifically and individually indicated to be incorporated by reference. In the case of any inconsistency between the content of a cited reference and the disclosure herein, the disclosure of this specification is to be given priority. Some references cited herein are incorporated by reference herein to provide details of haploid inducers and methods of making such haploid inducers, methods for making and mutants useful for making MiMe plants, methods for crossing specified plants, hybridization methods for the detection of genes, other methods for the detection of expression of certain genes in plants, PCR methods for the detection of expression of certain genes, methods for generating CENH3 variants, assay conditions, particularly hybridization assay conditions and PCR assay conditions, additional methods of analysis and additional uses of the invention.
Arabidopsis thaliana At
Arabidopsis thaliana SPO11-1 (SEQ ID No. 3):
Arabidopsis thaliana SPO11-2 9SEQ ID No. 4):
Arabidopsis thaliana PRD1 sequence (SEQ ID No. 5):
Arabidopsis thaliana gi|260590345|emb|CAX83745.1|
Arabidopsis thaliana PAIR1 (SEQ ID No. 7):
Arabidopsis thaliana REC8 (SEQ ID No. 8):
Arabidopsis thaliana ACCESSION NP_177863 442 aa
Arabidopsis thaliana ACCESSION (NP_568869) (385 aa) [DeMuyt et al.
Populus trichocarpa gi|224091813|ref|XP_002309357.1 (SEQ ID NO. 19):
Vitis vinifera gi|225445826|ref|XP_002275398.1| (SEQ ID NO. 20):
Oryza sativa Japonica Group gi|297608983|ref|NP_001062471.2|
Zea mays gi|212275736|ref|NP_001130070.1| LOC100191163 (SEQ ID No. 22):
Arabidopsis lyrata subsp. lyrata ACCESSION
Arabidopsis lyrata Al JGI907257 XP_002876442
Brassica rapa Br ESTs3 (SEQ ID No. 25):
Arabidopsis thanliana UVI4 NP_181755 (SEQ ID No.
Arabidopsis lyrata Al JGI903574 (SEQ ID No. 27):
Brassica rapa EX107108 (SEQ ID No. 28):
Populus Pt JGI576299 XP_002323297 (SEQ ID No. 29):
Populus Pt ABK93885 XP_002330993 (SEQ ID No. 30):
Vitis Vv CAO23523 gi|225441692|ref|XP_002277253
Glycine max Gm|JGI_Gm0077x00122 (SEQ ID No. 32):
Glycine max Gm|JGI_Gm0128x00128 (SEQ ID No. 33):
Oriza Os|CAH67433 Os04g39670 (SEQ ID No. 34):
Sorghum Sb|JGI5057365 (SEQ ID No. 35):
Sorghum Sb|JGI4979131 (SEQ ID No. 36):
Sorghum Sb|JGI5055355 (SEQ ID No. 37):
Zea mays Zm|ESTs (SEQ ID No. 38):
Zea mays Zm|ESTs2 (SEQ ID No. 39):
Zea mays Zm|ESTs3 (SEQ ID No. 40):
Medicago Mt|AC141114_13.2 (SEQ ID No. 41):
Mallus md|ESTs (SEQ ID No. 42):
Mallus md|ESTs2 (SEQ ID No. 43):
Ricinus communis gi|255583278|ref|XP_002532403.1
Oryza sativa Japonica Group OsPRD1 (NCBI
Oryra sativa Protein Sequences:
Oryza sativa SPO11-1 protein sequence GenBank AAP68363
Oryza sativa SPO11-2 protein sequence GenBank NP_001061027
Oryza sativa PAIR1 protein SwissProt Q75RY2 (SEQ ID NO. 50):
Oryza sativa REC8 Gen bank AAQ75095 (SEQ ID No. 51):
The GFP-tailswap plant (cenh3-1 mutant plants rescued by a GFP-tailswap transgene) is a very efficient haploid inducer, but is difficult to cross as the pollen donor, because it is mostly male sterile. Further, GFP-tailswap plants give an extremely low frequency of viable seeds (2%) when crossed as female to a tetraploid male that produces diploid gametes. In comparison, GFP-CENH3 (cenh3-1 mutant plants rescued by a GFP-tailswap transgene) is a weaker haploid inducer, but is much more fertile than GFP-tailswap (Ravi and Chan 2010).
In order to develop an efficient genome elimination strain with improved fertility and seed viability, cenh3-1 plants expressing combinations of CENH3 variants were screened. A cenh3-1 line that co-expresses two altered versions of the CENH3 protein, specifically GFP-CENH3 and GFP-tailswap, was found to produce more viable pollen and give better seed set than GFP-tailswap, yet still induces genome elimination when crossed to wild-type tetraploid plants and induced genome elimination in either direction of a cross. GEM is produced by crossing a GFP-tailswap plant with a GFP-CENH3 plant and selecting progeny which express both altered CENH3 proteins.
Indeed, cenh3-1 plants carrying both GFP-CENH3 and GFP-tailswap transgenes (GEM; Genome Elimination caused by a Mix of cenh3 variants) produced ample pollen for crosses, although pollen viability was still lower than wild-type (
Detailed description of plants expressing certain altered CENH3 proteins are provided in Ravi, M. & Chan, S. W-L. (2010) and Ravi, M. et al. (Jul. 13, 2010), each of which is incorporated by reference herein in its entirety for such description. In particular these references provide detail description of the null mutant cenh3-1, GFP-tagged variants of CENH3, of GFP-CENH3, GFP-tailswap (in which endogenous CENH3 is replaced with a variant CENH3 in which the N-terminal tail domain of CENH3 is replaced with the N-terminal tail domain of H3 (centromere-specific histone H3). Heterologous CENH3 variants were expressed from the CENH3 promoter in some cases with an N-terminal GFP tagged.
Crosses Between osd1 and GEM Lead to Diploid Uniparental, but Recombined Progeny
Diploid mutants of osd1 produce diploid male and female gametes because of an absence of second division of meiosis (d'Erfurth, Jolivet et al. 2009). We have found that crossing osd1 to GEM gave rise to diploid progeny originated only from the diploid osd1 parent because of elimination of the GEM parent genome. This was demonstrated by taking advantage of the three different genetic backgrounds of the osd1-1 (No-0) and osd1-2 mutants (Ler) and GEM (Col-0). We crossed osd1-1/osd1-2 plants that were heterozygous for polymorphism between No-0 and Ler, to GEM and followed parental origin in the progeny using trimorphic markers.
Among the progeny issued from crosses between osd1 and GEM 13% were parthenogenetic and 20% were androgenetic, depending on the direction of the cross.
Crossing osd1-1/osd1-2 as female with GEM as male resulted in 29 viable seeds per fruit, 26% of them being diploid (Table 16). Among these diploid progeny, half (24/50) were from sexual origin, carrying alleles of both parents (
The possibility of androgenesis was tested by crossing GEM as female with osd1-1/osd1-2 as male. This resulted in 3-4 viable seeds per fruit (Table X), 20% of them being diploid suggestive of androgenesis, because osd1 produces only 2n pollen grains (d'Erfurth, Jolivet et al. 2009). All of these 2n plants carried exclusively paternal alleles (
siliqua
1Deduced from FIGS. 6A-C. Tetraploid wild-type was in the C24 accession.
Crosses Between MiMe and GEM Lead to Diploid Uniparental Progeny
In this example we test the combination of apomeiosis with uniparental genome elimination. We crossed MiMe plants as female to the GEM line and looked for genome elimination events in the progeny. The MiMe parent had been previously genotyped and found to be either heterozygous or homozygous for a set of microsatellite markers across the genome (
MiMe×GEM gave an average of 14 viable seeds per fruit (˜1/3 of wild type), 35% of them being diploid (Table 18). Among these 2n plants, 98% (51/52) were entirely of maternal origin, lacking paternal contribution for eight loci tested at which the parents were homozygous for distinct alleles (
MiMe also produces male apomeiotic gametes. We tested if MiMe plants could be cloned as male. The GEM line was crossed as a female to MiMe plants and the elimination events were characterized Although seed viability was much lower in this cross, likely due to the fact that the Col-0 strain is very sensitive to paternal genome excess [Dilkes, B. P. et al. (2008)], 42% of progeny were diploid (Table XII). They all lacked maternal contribution and systemically kept heterozygosity of the male parent for all tested loci (
×
)
siliqua
1Deduced from FIGS. 7A-C data.
Genotype Analysis of GEM×MiMe Progeny
(female)×GEM
(male). Diploid plants were identified by flow cytometry, confirmed by mitotic chromosome spreads and genotyped. 51/52 had the same genotype as their mother (clonal progeny) and one had a hybrid genotype. (B) GEM
(female)×MiMe
(male). All diploid progeny had the same genotype as their mother. (E) Cloned MiMe
(female)×GEM
(male). One of the cloned plants shown in A was crossed to GEM
(male) and in the progeny 19/20 diploid plants had the same genotype as their mother and grandmother and one had a hybrid genotype.
Genotype Analysis of osd1×GEM and GEM×osd1 Offspring
As illustrated in ×GEM
. Among the diploid plants, half had a genotype of maternal origin (recombined), lacking paternal contribution and the other half had a hybrid genotype. (B) GEM
×osd1
. Among the diploid plants, all had a genotype of paternal origin (recombined), lacking maternal contribution.
Genotyping and Microsatellite Marker Analysis
Primers sequences and genotyping of plants for cenh3, GFP-tailswap, and GFP-CENH3 are listed below. Primers for osd1-1, Atspo11-1 and Atrec8-3 (MiMe) genotyping are described in [d'Erfurth, I. et al. (2009)]. Microsatellite markers (Table 17, above) were analyzed as described therein. [See also d'Erfurth, I. et al. (2008). and Dolezel, J et al. (2007)]. The cyclin-A CYCA1;2/TAM is required for the meiosis I to meiosis II transition and cooperates with OSD1 for the prophase to first meiotic division transition. Primer sequences were obtained from TAIR (www.arabidopsis.org) or from the MSAT database (INRA).
Identification of Diploid Plants from GEM×C24 Wild Type Tetraploid and its Reciprocal Cross
1. Putative diploid plants were first screened by their phenotype. Aneuploid plants can be morphologically distinguished from diploid and triploid plants. Triploid plants are hybrids containing Col-0 and C24 chromosomes. They are thus very late flowering, partially because of the combination of Col-0 FRIGIDA and C24 FLOWERING LOCUS C alleles [Sanda S. L. & Amasino R. M. (1995)]
2. All putative diploid plants along with randomly chosen sexual aneuploids and triploids were genotyped for at least one marker per chromosome. Pure diploids had only C24 alleles. Triploids had both C24 and Col-0 alleles. Aneuploids had all C24 alleles and lacked certainCol-0 alleles depending on the absence of a particular chromosome.
3. True diploid plants formed by genome elimination show a lack of GFP fluorescence because of the absence of GFP-tailswap whereas sexual aneuploids and triploids show GFP fluorescence at centromeres.
4. Random diploid plants were further confirmed by karyotyping in mitotic or meiotic spreads.
Diploid plants were genotyped to confirm their 4n C24 parental origin using the markers listed in Table 19
Genotyping the cenh3-1 Mutation and the GFP-Tailswap Transgene.
cenh3-1 is a point mutation in the CENH3 gene (also known as HTR12). The mutation is G161A relative to ATG=+1. cenh3-1 is genotyped with the following dCAPS primers:
GFP-tailswap is on chromosome 1 (identified by TAIL PCR). We genotype GFP-tailswap with the following primers:
The presence of GFP-CENH3 can be detected using the following primers:
Plant Material and Growth Conditions
Plants were grown in artificial soil mix at 20° C. under fluorescent lighting. Wild type and mutant strains of Arabidopsis were obtained from ABRC, Ohio or NASC, UK. MiMe plants were by construction a mixture of Col-0 from Atspo11-1/Atrec8 and No-0 from osd1-1 [d'Erfurth, I. et al. (2009)].
Ploidy Analysis
MiMe and osd1 offspring ploidy analyses were performed by flow cytometry and chromosome spreads as described [d'Erfurth, I. et al. (2009) and d'Erfurth, I. et al. (2010)].
This application claims the benefit of U.S. provisional application 61/418,792, filed Dec. 1, 2010. This application is incorporated by reference herein in its entirety.
This invention was made with government support under Grant No. 1026094 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/62718 | 11/30/2011 | WO | 00 | 4/28/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61418792 | Dec 2010 | US |
