METHOD FOR THE PRODUCTION OF HAPLOID AND SUBSEQUENT DOUBLED HAPLOID PLANTS

Abstract

The present disclosure provides a modified CenH3 protein that, when present in a plant, allows the plant to be used as a haploid inducer line for plant breeding purposes. Polynucleotides encoding such modified CenH3 proteins, chimeric genes and vectors comprising such polynucleotides, host cells, and plants comprising such polynucleotides, chimeric genes or vectors are also provided. Additionally, methods for making such plants as well as methods for producing haploid or doubled haploid plants using such plants are disclosed.

Claims

1-60. (canceled)

61. A CenH3 protein of plant origin comprising one or more active mutations in the CenH3 motif block 1 of the N-terminal tail domain having an amino acid sequence of SEQ ID NO: 4.

62. The CenH3 protein according to claim 61, wherein the active mutation is at position 9 or 10 of SEQ ID NO: 4.

63. The CenH3 protein according to claim 61, which is derived from an endogenous CenH3 protein having at least 70% sequence identity to any one of SEQ ID NO: 1, 2, 3, 11 and 12, or is encoded by a polynucleotide having at least 70% sequence identity to any one of SEQ ID NO: 6, 9, 16, or 20.

64. The CenH3 protein according to claim 63, wherein the derivation is by introducing mutations in the polynucleotide encoding the endogenous CenH3 protein using targeted nucleotide exchange or by applying an endonuclease.

65. The CenH3 protein according to claim 61, wherein the active mutation is a point mutation.

66. The CenH3 protein according to claim 61, wherein the CenH3 protein of plant origin comprises the amino acid sequence of SEQ ID NO: 8 or 13, or is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 7, 10, 17 or 21.

67. A polynucleotide encoding the CenH3 protein according to claim 61.

68. The polynucleotide according to claim 67, comprising the nucleic acid sequence of SEQ ID NO: 7, 10, 17 or 21.

69. A chimeric gene comprising the polynucleotide according to claim 67.

70. A vector comprising the polynucleotide according to claim 67.

71. A vector comprising the chimeric gene according to claim 68.

72. A host cell comprising the polynucleotide according to claim 67.

73. The host cell according to claim 72, wherein the host cell is a plant cell.

74. The host cell according to claim 73, wherein the plant cell is a tomato or rice plant cell or a tomato or rice protoplast.

75. A plant expressing the CenH3 protein according to claim 61.

76. The plant according to claim 75, wherein endogenous CenH3 protein is not expressed.

77. The plant according to claim 76, wherein the plant is a Solanum plant or an Oryza plant.

78. The plant according to claim 77, wherein the plant is a Solanum lycopersicum plant or an Oryza sativa plant.

79. A method for making a plant according to claim 75, comprising the steps of: (a) modifying a polynucleotide encoding an endogenous CenH3 protein within a plant cell to obtain a mutated polynucleotide encoding a CenH3 protein of plant origin comprising one or more active mutations in the CenH3 motif block 1 of the N-terminal tail domain having an amino acid sequence of SEQ ID NO: 4; (b) selecting a plant cell comprising the mutated polynucleotide; and (c) optionally, regenerating a plant from the plant cell.

80. A method of generating a haploid plant, a plant with aberrant ploidy or a doubled haploid plant, the method comprising the steps of: (a) crossing a plant expressing an endogenous CenH3 protein to the plant of claim 75, wherein the plant according to claim 75 does not express an endogenous CenH3 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 the 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.

81. A method of generating a doubled haploid plant, the method comprising the step of: (a) crossing a plant expressing an endogenous CenH3 protein to the plant of claim 75, wherein the plant according to claim 75 does not express an endogenous CenH3 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 the seed, (d) selecting a haploid seedling, plantlet or plant; a seedling, plantlet or plant with aberrant ploidy; or a doubled haploid seedling, plantlet or plant; and (e) converting the haploid seedling, plantlet or plant into a doubled haploid plant.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0321] FIG. 1. depicts a micronucleus (arrow) in pollen tetrad of CenH3_K9E, DAPI staining.

SEQUENCE LISTING

[0322] SEQ ID NO: 1: Plant CenH3 consensus protein sequence

[0323] SEQ ID NO: 2: Consensus Solanaceae CenH3 protein sequence

[0324] SEQ ID NO: 3: Solanum lycopersicum CenH3 protein sequence (Solyc01g095650.2.1)

[0325] SEQ ID NO: 4: Plant consensus CenH3 motif 1 domain protein sequence

[0326] SEQ ID NO: 5: Consensus Solanaceae CenH3 motif 1 domain protein sequence

[0327] SEQ ID NO: 6: Solanum lycopersicum CenH3 coding sequence (Solyc01g095650.2.1)

[0328] SEQ ID NO: 7: Solanum lycopersicum CenH3_K9E coding sequence

[0329] SEQ ID NO: 8: Solanum lycopersicum CenH3_K9E protein sequence

[0330] SEQ ID NO: 9: Solanum lycopersicum CenH3 genomic DNA sequence (Solyc01g095650.2.1)

[0331] SEQ ID NO: 10: Solanum lycopersicum CenH3_K9E genomic DNA sequence

[0332] SEQ ID NO: 11: Monocotyledon consensus CenH3 protein sequence

[0333] SEQ ID NO: 12: Oryza sativa L. ssp. japonica CenH3 protein sequence (LOC_Os05g41080)

[0334] SEQ ID NO: 13: Oryza sativa L. ssp. japonica CenH3_V9M protein sequence

[0335] SEQ ID NO: 14: Oryza sativa L. ssp. japonica CenH3_P16S protein sequence

[0336] SEQ ID NO: 15: Oryza sativa L. ssp. japonica CenH3_P26L protein sequence

[0337] SEQ ID NO: 16: Oryza sativa L. ssp. japonica CenH3 coding sequence (LOC_Os05g41080)

[0338] SEQ ID NO: 17: Oryza sativa L. ssp. japonica CenH3_V9M coding sequence

[0339] SEQ ID NO: 18: Oryza sativa L. ssp. japonica CenH3_P16S coding sequence

[0340] SEQ ID NO: 19: Oryza sativa L. ssp. japonica CenH3_P26L coding sequence

[0341] SEQ ID NO: 20: Oryza sativa L. ssp. japonica CenH3 genomic DNA sequence (LOC_Os05g41080)

[0342] SEQ ID NO: 21: Oryza sativa L. ssp. japonica CenH3_V9M genomic DNA sequence

[0343] SEQ ID NO: 22: Oryza sativa L. ssp. japonica CenH3_P16S genomic DNA sequence

[0344] SEQ ID NO: 23: Oryza sativa L. ssp. japonica CenH3_P26L genomic DNA sequence

EXAMPLES

Example 1: Generation of a Haploid Plant

[0345] Plant Material

[0346] 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, a somatic non-synonymous mutant in the gene CenH3 was selected, namely CenH3_K9E, which is mutated at amino acid position 9. 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.

[0347] Method

[0348] Uniparental 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 uniparental 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 uniparental genome elimination (Sanei et al. PNAS 108.33 (2011): E498-E505).

[0349] Results

[0350] The non-synonymous mutation of A to G in the CenH3_K9E mutant resulted in an amino acid modification of a lysine to a glutamate (SEQ ID NO: 8). The synonymous mutation of C to T in the Msi2_D337D mutant did not result in an amino acid modification. Both mutant plants homozygous for the CenH3_K9E 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.

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. Experiments with MicroTom as female are shown from two subsequent years. Number Number of Year of plants with cross Plant used as Plant used as Background plants MicroTom was female male mutant parent tested phenotype made MicroTom CenH3_K9E RZ52201 516 6 2014 CenH3_K9E MicroTom RZ52201 564 1 2015 MicroTom CenH3_K9E RZ52201 297 13 2015 RZ52201 MicroTom 188 0 2015 MicroTom RZ52201 188 0 2015 MoneyBergTMV+ MicroTom 188 0 2015 MicroTom MoneyBergTMV+ 188 0 2015 Msi2_D337D MicroTom MoneyBergTMV+ 160 0 2015 MicroTom Msi2_D337D MoneyBergTMV+ 36 0 2015

[0351] 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+. A single exception was found; for the cross in 2014 of MicroTom (female)CenH3_K9E (male), one offspring plant was found to be aneuploid (i.e., having an aberrant ploidy) based on flow cytometry analysis.

[0352] 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. 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. Using the CenH3_K9E mutant as male or female parent, in total 20 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 20 offspring plants are of haploid MicroTom origin. The ploidy of all plants of the latter 20 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 62December 2012).

[0353] In order to determine whether and to what extent uniparental genome elimination had occurred, a single nucleotide polymorphism (SNP) assay was run for in total 24 positions, 2 SNPs on each of the 12 tomato chromosomes for the 2015 crosses. For the 2014 crosses SNP assays were run on in total 8 positions, one on chromosomes 2, 7, 9, and 12 and two on chromosomes 3 and 6. The single 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.

[0354] 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 20 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 CenH3_K9E mutant was no longer present in the offspring.

[0355] Therefore, it can be concluded that the CenH3_K9E mutant functions as a highly efficient haploid inducer line. In the crosses in which the CenH3_K9E 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 in two subsequent years of making crosses (only 6 seeds out of 516 and 13 out of 297), and the fact that only homozygous base pairs were scored.

[0356] Pollen tetrads of the CenH3_K9E mutant and of RZ52201 control plants were checked for occurrence of aberrancies.

[0357] From two flowers, the anthers were squashed in order to look at pollen tetrads. For the CenH3_K9E mutant, scoring 2 flowers an average 2.600.25 percent of micronuclei were observed in all tetrads. FIG. 1 shows an example of such a micronucleus. For the RZ52201 control, rarely an anther was observed containing pollen tetrads with micronuclei. Scoring 5 flowers an average 0.580.36 percent of micronuclei were observed in all tetrads. It is concluded that the separation of chromosomes during meiosis is considerably more frequently disturbed as a result on the CenH3_K9E 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 CenH_K9E 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 CenH_K9E zygotes takes place and that this results in the observed induction of haploids.

Example 2: Uniparental Genome Elimination in Rice

[0358] Plant Material

[0359] Oryza sativa L. ssp. japonica cv. Volano are used to generate a mutant population by means of chemical mutagenesis. From this mutant population, following methods described in WO2007/037678 and WO2009/041810, three somatic non-synonymous mutants in the gene CenH3 (LOC_Os05g41080; SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15) are selected, namely CenH3_V9M, CenH3_P16S and CenH3_P26L. The selected mutant plants are self-pollinated and in the offspring, plants are selected that are homozygous for the mutated locus. A non-mutated Oryza sativa L. ssp. japonica (encoding SEQ ID NO: 12) cv. Volano plant is used as well.

[0360] Method

[0361] 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 non-mutated Oryza sativa L. ssp. japonica cv. Volano plant. The ploidy of the offspring is measured to determine whether they are diploid or haploid. To include the possibility that a haploid offspring plant is spontaneously doubled to a diploid state, the total absence of either of the three listed CenH3 mutant SNPs is tested as well. In a spontaneously doubled provoked haploid plant none of three separate CenH3 mutant SNPs (SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15), not even as heterozygous allele, will be present.

[0362] Results

[0363] The non-synonymous mutation of G to A in the CenH3_V9M mutant resulted in an amino acid modification of a valine to a methionine (SEQ ID NO: 13). The non-synonymous mutation of C to T in the CenH3_P16S mutant resulted in an amino acid modification of a proline to a serine (SEQ ID NO: 14). The non-synonymous mutation of C to T in the CenH3_P26L mutant resulted in an amino acid modification of a proline a leucine (SEQ ID NO: 15). Each of the three mutant plants homozygous for the CenH3_V9M, the CenH3_P16S or the CenH3_P26L mutation were used as pollen donor using non-mutated wild type Oryza sativa L. ssp. japonica cv. Volano as female. Table 2 lists an overview of all crosses and the seeds that are sown which are evaluated for ploidy levels. A reciprocal cross may yield similar results.

TABLE-US-00002 TABLE 2 Example list of crosses which can be made; genetic background of all plants is Oryza sativa L. ssp. japonica cv. Nipponbare, number of offspring plants which are tested and number of haploid offspring plants based on flow cytometry. Plant used as Plant used Number of plants Number of haploid female as male tested plants Wild type CenH3_V9M 300 3 Wild type CenH3_P16S 300 1 Wild type CenH3_P26L 300 2 Wild type Wild type 300 0

[0364] Seeds derived from the crosses listed in table 1 are sown and the plants are evaluated for their DNA content by means of flow cytometry. Presence of CenH3_V9M, the CenH3_P16S or CenH3_P26L mutant SNP is tested in plants determined to be haploid by flow cytometry analysis. Absence of the mutant SNP indicates that the mutant parent genetic material is not part of the resulting offspring and that these offspring plants are of haploid wild type parent origin, i.e. that each of the CenH3_V9M, the CenH3_P16S or the CenH3_P26L mutants function as a highly efficient haploid inducer line.