Method for the production of haploid and subsequent doubled haploid plants

Abstract

It was found that plants with loss of functional Msi2 protein due to a nucleotide polymorphism resulting in the introduction of a premature stop codon in the Msi2 protein, are able to induce haploid offspring after a cross to or with a wild type plant comprising a functional Msi2 protein. The invention relates to generation of haploid and doubled haploid plants.

Claims

1. A Msi2 gene comprising a loss-of-expression mutation, wherein the Msi2 gene is derived from a gene encoding an Msi2 protein having at least 70% sequence identity with at least one of SEQ ID NO. 1, 2, 3 and 10, wherein the loss-of-expression mutation results in reduced expression of the Msi2 protein, wherein the gene is not an Arabidopsis thaliana-derived Msi2 gene, and wherein the Msi2 gene, when present in a plant in the absence of the endogenous Msi2 gene, allows generation of haploid progeny, or progeny with aberrant ploidy, at a more than normal frequency when the plant is crossed with a wild-type plant.

2. The gene according to claim 1, wherein the sequence encoding the Msi2 protein has at least 90% sequence identity with at least one of SEQ ID NO: 5 and 7.

3. The gene according to claim 1, wherein the expression of the Msi2 protein is knocked out.

4. The gene according to claim 1, wherein the loss-of-expression mutation is in a transcription regulation sequence.

5. The gene according to claim 1, wherein the mutation is at least one of a frame-shift mutation, deletion, substitution, rearrangement or insertion of nucleotides in the Msi2-coding region.

6. The gene according to claim 1, herein at least 10% of the Msi2-coding region is removed.

7. A plant, seed, or plant cell comprising the Msi2 gene of claim 1.

8. The plant, seed or plant cell according to claim 7, wherein the expression of an endogenous Msi2 protein is reduced or knocked out.

9. The plant, seed or plant cell according to claim 7, which is a Solanum plant, seed or plant cell.

10. The plant, seed or plant cell according to claim 8, wherein the Solanum plant, seed or plant cell is a Solanum lycopersicum.

11. A method for producing a plant, seed, or plant cell, the method comprising: (a) modifying an endogenous Msi2 gene within a plant cell to obtain the mutated Msi2 gene, wherein the gene comprises a loss-of-expression mutation resulting in a reduced expression of the Msi2 protein, and wherein the gene is not an Arabidopsis thaliana-derived Msi2 gene; (b) selecting a plant cell comprising the mutated Msi2 gene; and (c) optionally, regenerating a plant from said plant cell.

12. A method of generating a haploid plant, a plant with aberrant ploidy, or a doubled haploid plant, comprising: (a) crossing a plant expressing an endogenous Msi2 protein with a plant of claim 7 wherein the plant lacks expression of endogenous Msi2 protein at least in its reproductive parts and/or during embryonic development; (b) harvesting seed; (c) growing at least one seedling, plantlet or plant from said seed; and (d) selecting a haploid seedling, plantlet or plant, a seedling, plantlet or plant with aberrant ploidy, or a doubled haploid seedling, plantlet or plant.

13. A method of generating a doubled haploid plant, comprising: (a) crossing a plant expressing an endogenous Msi2 protein to the plant of claim 7, wherein the plant lacks expression of endogenous Msi2 protein at least in its reproductive parts and/or during embryonic development; (b) selecting a haploid plant; and (c) converting said haploid plant into a doubled haploid plant.

14. The method according to claim 13, wherein the conversion in (c) is performed by treatment with colchicine.

Description

BRIEF DESCRIPTION OF THE FIGURE

(1) FIG. 1 shows a tetrad of a Msi2-K126-stop mutant. The arrow shows a micronucleus.

SEQUENCE LISTING

(2) SEQ ID NO:1: plant Msi2 consensus protein sequence

(3) SEQ ID NO:2: Solanum consensus Msi2 protein sequence

(4) SEQ ID NO:3: Solanum lycopersicum Msi2 protein sequence

(5) SEQ ID NO:4: Solanum lycopersicum Msi2_K126* coding sequence

(6) SEQ ID NO:5: Solanum lycopersicum Msi2 coding sequence

(7) SEQ ID NO:6: Solanum lycopersicum Msi2_K126* truncated protein sequence

(8) SEQ ID NO:7: Solanum consensus Msi2 coding sequence

(9) SEQ ID NO:8—Solanum lycopersicum Msi2 genomic DNA sequence

(10) SEQ ID NO:9—Solanum lycopersicum Msi2_K126* genomic DNA sequence

(11) SEQ ID NO:10—Solanaceae consensus Msi2 protein sequence

EXAMPLES

Example 1: Uniparental Genome Elimination in Tomato

Material and Methods

Plant Material

(12) Three tomato cultivars were used namely “MoneyBerg TMV+”, “MicroTom” and “RZ52201”. From a tomato RZ52201 mutant population, following methods described in WO 2007/037678 and WO2009/041810, two somatic non-synonymous mutants in the gene Msi2 were selected, namely Msi2_K126-STOP and Msi2_K126M, which are both mutated at amino acid position 126. The selected mutant plant was self-pollinated and in the offspring, plants were selected that were homozygous for the mutated locus. From a tomato MoneyBerg TMV+ mutant population a somatic synonymous mutant was selected, following methods described in WO 2007/037678 and WO2009/041810, in the gene Msi2, namely Msi2_D337D, which is mutated at amino acid position 337 (C to T). The selected mutant plant was self-pollinated and in the offspring, plants were selected that were homozygous for the mutated locus.

Method

(13) Uni-parental genome elimination and the resulting production of a haploid plant was provoked by making a cross between a so called haploid inducer line and another non-haploid inducer line, for example a breeding line. Crosses of tomato lines for uni-parental genome elimination were performed at relatively high temperatures (26-28° C.), since it is known that an elevated temperature can, but only in some cases, have a positive effect on the occurrence of uni-parental genome elimination (Sanei et al. PNAS 108.33 (2011): E498-E505).

Results

(14) The non-synonymous mutation of A to Tin the Msi2_K126-STOP mutant resulted in the introduction of a premature stop codon and thereby the production of a truncated protein (SEQ ID NO:6). The non-synonymous mutation of A to T in the Msi2_K126M mutant resulted in an amino acid modification of a lysine to a methionine. Furthermore a SIFT analysis was run for the Msi2 protein and the mutation of a lysine to a methionine at position 126 was rated by this analysis to be neutral (Kumar et al. Nat Protoc. 2009; 4(7):1073-81). The synonymous mutation of C to T in the Msi2_D337D mutant did not result in an amino acid modification. Each of the three mutant plants homozygous for the Msi2_K126-STOP, the Msi2_K126M or the Msi2_D337D mutation were used as pollen donor and as female in crosses at relatively high temperatures (26-28° C.) using non-mutated wild type MicroTom plants as female or pollen donor, respectively. Table 1 lists an overview of all crosses made and the sown seeds which were evaluated for the MicroTom phenotype.

(15) TABLE-US-00001 TABLE 1 List of crosses made; genetic background of the parents used, number of offspring plants tested and number of offspring plants which showed MicroTom dwarf phenotype. Number of Number plants with Plant used Plant used Background of plants MicroTom as female as male mutant parent tested phenotype Year Msi2_K126- MicroTom RZ52201 98 3 2014 STOP MicroTom Msi2_K126- RZ52201 89 2 2014 STOP Msi2_K126- MicroTom RZ52201 564 4 2015 STOP MicroTom Msi2_K126- RZ52201 325 1 2015 STOP Msi2_K126M MicroTom RZ52201 19 0 2014 MicroTom Msi2_K126M RZ52201 205 0 2014 Msi2_D337D MicroTom MoneyBergTMV+ 160 0 2014 MicroTom Msi2_D337D MoneyBergTMV+ 36 0 2014 RZ52201 MicroTom — 188 0 2015 MicroTom RZ52201 — 188 0 2015

(16) Seeds derived from the crosses listed in table 1 were sown and the plants were evaluated for their DNA content by means of flow cytometry. The flow cytometry analysis resulted in a determination of only normal diploid ploidy levels for all plants tested, similar to wild type tomato cultivars such as MoneyBergTMV+. The cultivar MicroTom has a dwarf phenotype, which is known to be recessive (Marti et al, J Exp Bot, Vol. 57, No. 9, pp. 2037-2047, 2006). After a cross of MicroTom to or with, for instance a MoneyBerg TMV+ or RZ52201 wild type cultivar, one only finds offspring with the indeterminate non-dwarf phenotype of the MoneyBerg TMV+ or RZ52201 wild type cultivar, respectively. The same was found for crosses with the Msi2_D337D synonymous mutant and MicroTom; all offspring of a MicroTom and Msi2_D337D mutant crosses showed the indeterminate non-dwarf phenotype of the MoneyBerg TMV+ parent. Reciprocal crosses of MicroTom and the Msi2_K126M mutant did not result in offspring with the MicroTom phenotype. Using the Msi2_K126-STOP mutant as male or female parent, in total 10 plants were found which showed a MicroTom phenotype. This indicates that the RZ52201 parent genetic material is not part of the resulting offspring and this indicates that these 10 offspring plants are of haploid MicroTom origin. The ploidy of all plants of the latter 10 plants was found to be diploid, indicating that spontaneous doubling had occurred, a phenomena which has been described to have an exceptional high frequency of appearance for tomato (Report of the Tomato Genetics Cooperative Number 62-December 2012).

(17) In order to determine whether and to what extent uni-parental genome elimination had occurred, a single nucleotide polymorphism (SNP) assay was run for in total 44 positions for the 2014 offspring, spread across each of the 12 tomato chromosomes (4 SNPs on chromosome 1, 2, 3, 4, 5, 6, 11 and 12; 3 SNPs on chromosome 8 and 10; 2 SNPs on chromosome 9). The same analysis was performed for the 2015 offspring, now on 22 positions (2 SNPs on chromosome 1, 2, 3, 4, 5, 6, 7, 8, 10 and 12; 1 SNP on chromosome 9 and 11). The single 5 nucleotide polymorphisms selected were homozygous for one base pair for the MicroTom parent and homozygous for all but not the MicroTom base pair in the RZ52201 parent. A regular cross between a wild type MicroTom cultivar and the RZ52201 cultivar would result in a heterozygous single nucleotide polymorphism score. However, when the process of uniparental genome elimination has occurred, one expects the loss of the haploid inducer line genome. The single nucleotide polymorphism test resulted in calling of only homozygous base pair scores from the MicroTom parent for each of the 5 offspring plants which also showed the MicroTom phenotype and none of the RZ52201 parent were called. Based on the single nucleotide polymorphism scores it was concluded that the complete genome of the Msi2_K126-STOP mutant was no longer present in the offspring. Therefore, it can be concluded that the Msi2_K126-STOP mutant functions as a highly efficient haploid inducer line. In the crosses in which the Msi2_K126-STOP mutant was used as female parent, a selfing of MicroTom can be ruled out. It is highly unlikely that in the experiment using MicroTom as female parent selfing took place, given the very low number of offspring showing the MicroTom phenotype (only 2 seeds out of 89 and 1 seed out of 325), and the fact that only homozygous base pairs were scored.

(18) Pollen tetrads of the Msi2-K126-stop mutant and of RZ52201 control plants were checked for occurrence of aberrancies. From four different flower trusses at least two flowers were taken and anthers were squashed in order to look at pollen tetrads. For the Msi2-K126-stop mutant, in all 10 observed anthers from 5 individual flowers, micronuclei were observed (see FIG. 1). In each observed anther several examples of micronuclei were found, however not more than 1% of the pollen tetrads in an anther showed the aberrancies. For the RZ52201 control, rarely an anther was observed containing pollen tetrads with micronuclei. Two anthers were found in which in total only two or three examples of micronuclei could be observed. In a second round of experiments, the micronuclei were counted; For the Msi2-K126-stop mutant, micronuclei were observed with a frequency of 1.94% (n=1). For the RZ52201 control plant flowers, micronuclei were observed with an average frequency of 0.58±0.36% (n=5). It is concluded that the separation of chromosomes during meiosis is considerably more frequently disturbed as a result on the Msi2-K126-stop mutation compared to the control. Aberrant mitosis, for instance observations of micronuclei, are often used as direct evidences of chromosome elimination and haploid production in inter-, intra-specific hybridizations in crops. For example, aberrant mitosis as well as aberrant meiosis, for instance micronuclei, were found in a study of a maize DH-inducer line (Qiu, Fazhan, et al. Current Plant Biology 1 (2014): 83-90). The observations of meiosis micronuclei in the Msi2-K126-stop mutant, suggest that during mitosis similar processes occur. It is likely that the process of uniparental genome elimination during the first mitotic divisions after fusion of wild type and Msi2-K126-stop zygotes takes place and that this results in the observed induction of haploids.

Example 2: Uniparental Genome Elimination in Arabidopsis

Materials and Methods

Plant Material

(19) The following Arabidopsis NASC stock centre accessions were used; Columbia (background line, Col-0, N1092), Col-5 (N1644), Arabidopsis Msi2 gene (At2g16780) T-DNA insertion lines (N720344 and N501214, in Col-0 background) and, since it is not known whether the Msi2 and Msi3 gene are functionally redundant genes, Arabidopsis Msi3 gene (At4g35050) T-DNA insertion mutants (N309860, N309863 and N564092 in Col-0 background). The T-DNA insertion lines were evaluated by means of PCR amplification and subsequent sequencing of the putative T-DNA insertion locus in order to determine the exact insertion in the Arabidopsis Msi2 and Msi3 genes. Based on the finding that the insertions were located in exons of either Msi2 or Msi3 genes it was concluded that these T-DNA insertion lines are true knock-outs for either the Msi2 or Msi3 gene. The exact positions as counted in number of bases downstream of the start codon were; N720344 (position 429, exon 2), N501214 (position 111, exon 1), N309863/N309860 (both in position 559, exon 2) and N564092 (position 1237, exon 6). By making crosses between two insertion lines and selecting for homozygous T-DNA insertions in both the Msi2 and the Msi3 gene, two novel double T-DNA insertion lines were produced; N720344+N309860 and N309863+N501214.

Method

(20) Uni-parental genome elimination and the resulting production of a haploid plant is provoked by making a cross between a so called haploid inducer line and another non-haploid inducer line, for example a Columbia background (Col-0) control line.

Results

(21) Either a single T-DNA insertion line for Msi2 (N720344 and N501214), a Msi3 T-DNA insertion line (N309863 and N564092), the two newly generated Msi2/Msi3 double T-DNA insertion lines (N720344+N309860 and N309863+N501214) or Col-0 background plants are used as pollen donor and as female in crosses using Col-5 as female or pollen donor, respectively. Table 2 lists an example of typical crosses which can be made and an example of the evaluation of the offspring for the Col-5 phenotype. Col-5 has a clear distinct recessive phenotype compared to the T-DNA insertion lines and Col-0, namely trichomeless leaves.

(22) TABLE-US-00002 TABLE 2 List of crosses which can be made; genetic background of all insertion lines was Col-0, and number of offspring plants which are tested. Plant used as Plant used as Number of female male plants tested Msi2 (N720344) Col-5 300 Col-5 Msi2 (N720344) 300 Msi2 (N501214) Col-5 300 Col-5 Msi2 (N501214) 300 Msi3 (N309863) Col-5 300 Col-5 Msi3 (N309863) 300 Msi3 (N564092) Col-5 300 Col-5 Msi3 (N564092) 300 Msi2/Msi3 Col-5 300 (N720344 + N309860) Col-5 Msi2/Msi3 300 (N720344 + N309860) Msi2/Msi3 Col-5 300 (N309863 + N501214) Col-5 Msi2/Msi3 300 (N309863 + N501214) Col-0 Col-5 300 Col-5 Col-0 300

(23) The Col-5 accession harbours the gl1-1/gl1-1 locus giving it a trichomeless phenotype, which is known to be recessive (Kuppu et al. PLoS Genet 11.9 2015 e1005494). After a cross of Col-5 to or with, for instance a Col-0 wild type cultivar, one only finds offspring with trichomes coming from the dominant Col-0 allele. Using Msi2 single, Msi3 single or Msi2/Msi3 double T-DNA insertion lines as male or female parent, in total several plants are found which show a trichomeless phenotype. This indicates that the Col-0 parent genetic material is not part of the resulting offspring and this indicates that these offspring plants are of haploid Col-5 origin.

(24) Based on the single Col-5 phenotype individuals among the offspring of the crosses performed, it is concluded that the complete genome of the respective T-DNA insertion line in the Col-0 background is no longer present in the offspring. Therefore, it is concluded that the T-DNA insertion lines “N720344, N501214, N309863, N564092, N720344+N309860 (double insertion line) and N309863+N501214 (double insertion line) function as highly efficient (doubled) haploid inducer lines.