SELF-INCOMPATIBLE DIHAPLOID POTATO LINE AND PRODUCTION METHOD THEREFOR, AND HYBRID POTATO AND SEED PRODUCTION METHOD THEREFOR

Abstract

Disclosed is a self-incompatible dihaploid potato and a production method therefor, and a hybrid potato and a seed production method therefor. The production method includes the following steps: haploid-inducing a diploid potato carrying one or more heterozygous self-compatibility genes by a haploid induction line, and screening haploid mutants; selecting a haploid mutant which does not contain any self-compatible gene; and performing chromosome doubling on the haploid mutant which does not contain any self-compatibility gene to obtain a self-incompatible dihaploid potato line. The self-incompatible dihaploid potato is used as a female parent for seed production, such that emasculation may be avoided, hybrids do not bear fruits, and the yield of the potato is increased.

Claims

1. A method for the production of a self-incompatible dihaploid potato line, comprising the following steps: (1) haploid-inducing a diploid potato carrying one or more heterozygous self-compatibility genes by a haploid induction line, and screening haploid mutants; (2) selecting a haploid mutant which does not comprise any self-compatible gene; and (3) performing chromosome doubling on the haploid mutant which does not comprise any self-compatibility gene to obtain a self-incompatible dihaploid potato line.

2. The method of claim 1, wherein the self-incompatible dihaploid potato line is fertile.

3. The method of claim 1, wherein one or more self-compatibility genes comprises S.sub.S11 gene and/or Sli gene.

4. The method of claim 1, wherein the method for screening haploid mutants comprises one or more of a seed appearance characteristic marker, a molecular marker, a fluorescent marker, and flow cytometry.

5. The method of claim 4, wherein a seed appearance characteristic marker comprises the presence or absence of embryonic spots.

6. The method of claim 1, wherein the haploid mutants are one or more of seeds, plants, and tissue cells.

7. The method of claim 1, wherein the method for chromosome doubling comprises one or more of: a chemical induction method, a low temperature induction method, and a callus regeneration method.

8. The method of claim 7, wherein the method for chromosome doubling is a chemical induction method; the chemical induction method comprises treating the haploid mutant with one or more of colchicine, cytochalasin B, and/or phenylmercury-p-toluene sulfonilide.

9. A self-incompatible dihaploid potato line, wherein the self-incompatible dihaploid potato line is obtained by the method of claim 1.

10. A self-incompatible dihaploid potato line, wherein a plant of the dihaploid potato line does not comprise any self-compatibility gene, and the plant is fertile.

11. The self-incompatible dihaploid potato line of claim 9, wherein the plant of the dihaploid potato line further comprises one or more beneficial alleles; and/or, the plant of the dihaploid potato line further does not comprise one or more harmful alleles.

12. The self-incompatible dihaploid potato line of claim 11, wherein the beneficial alleles comprise Floral Bud Abortion 1 (FBA1) allele, and/or yellow (Y) allele, and/or, the harmful alleles comprise floral bud abortion 1 (fbal) allele, and/or large-effect deleterious mutation 1 (led1) allele.

13. A plant or part thereof of the self-incompatible dihaploid potato line of claim 9, wherein the part thereof is a tuber or tuber part, plant cell, pollen, or seed.

14. A seed production method for obtaining a hybrid potato, wherein comprising using a plant of the self-incompatible dihaploid potato line obtained by the method of claim 1 as a female parent for hybrid seed production; or using a plant of the self-incompatible dihaploid potato line as a female parent for hybrid seed production, the plant does not comprise any self-compatibility gene and is fertile.

15. The seed production method of claim 14, wherein a male parent for hybrid seed production is selected from: a plant of the self-incompatible dihaploid potato line obtained by the method for the production of a self-incompatible dihaploid potato line; or a plant of the self-incompatible dihaploid potato line that does not comprise any self-compatibility gene, and the plant is fertile; or a plant of other self-incompatible potato lines.

16. The seed production method of claim 14, wherein a male parent of the hybrid seed production is a plant of a self-compatible potato line.

17. A hybrid potato seed obtained by the seed production method of claim 14.

18. A plant or part thereof grown from the seed of claim 17, wherein the part thereof is a tuber or tuber part, plant cell, pollen, or seed.

19. A feed or food product comprising materials from the plant or part thereof of claim 18.

20. The feed or food product of claim 19, wherein the feed comprises: liquid feed, solid feed, semi-solid feed or feed raw materials; and the food comprises: fresh potatoes, dried potatoes, frozen potatoes, French fries, potato chips, potato flour or potato starch.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 is a schematic diagram of the production of a self-incompatible dihaploid potato.

[0054] FIG. 2 shows a comparison between seeds without embryo spots and seeds with embryo spots.

[0055] FIG. 3 shows self-incompatible haploid plants and diploid plants.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0056] The present disclosure discloses a self-incompatible dihaploid potato and a production method therefor, and a hybrid potato and a seed production method therefor. Persons skilled in the art may learn from the contents of this disclosure, and appropriately improve the process parameters for implementation. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be encompassed in the present disclosure. The methods and applications of the present disclosure have been described through preferred embodiments. Relevant persons may make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the contents, principle, and scope of the present disclosure to achieve and apply the technology of this disclosure.

[0057] The disclosure provides a method for avoiding emasculation during seed production and inhibiting fruit-bearing of the above-ground parts of a hybrid potato, the method mainly includes the following steps.

[0058] (1) Haploid-inducing a diploid potato carrying one or more heterozygous self-compatibility genes by a haploid induction line, and screening haploid mutants.

[0059] Diploid potatoes carrying one or more heterozygous self-compatibility genes were selected for haploid induction. Generally, diploid potatoes are directly induced into haploids, and the haploids obtained are difficult to survive mainly due to carrying harmful mutations with large effects. As for diploid materials carrying self-compatibility genes, some harmful mutations with large effects may have been eliminated through selfing. These diploid materials that have removed harmful mutations with large effects are selected for performing haploid induction to obtain haploid mutants with a higher survival probability.

[0060] (2) Selecting a single plant which does not contain any self-compatible gene from the induced haploid mutants, and performing chromosome doubling to obtain a dihaploid potato plant which is self-incompatible.

[0061] A single plant which does not contain any self-compatible gene is selected from the induced haploid mutants. A diploid material carrying one or more heterozygous self-compatibility gene is used for haploid induction, and there is a separation of self-compatible and self-incompatible mutants. A self-incompatible single plant screened by molecular marker is chromosome-doubled to obtain a dihaploid material having a homozygous genome but being self-incompatible, i.e., a dihaploid line.

[0062] (3) Using the above self-incompatible dihaploid material as a parent for hybridization, emasculation may be avoided, and the resulting hybrids are also self-incompatible.

[0063] Using the dihaploid plant obtained from steps (1)-(2) as a female parent to hybridize with other parents, the problem of emasculation may be avoided. This step is only limited to the fact that the female parent is self-incompatible, and emasculation is not needed.

[0064] The above steps (1)-(2) are used to create dihaploid materials with different genetic backgrounds. Crossbreeding between these materials for hybrid seed production not only avoids emasculation but also produces the resulting hybrid offspring that are also self-incompatible, thereby avoiding the problem of the fruit-bearing of the above-ground parts affecting the underground yield. In this step, both the female and male parents are self-incompatible, thereby simplifying the seed production process and increasing the yield.

Terminology Explanation

[0065] Allele: refers to a gene located at the same position on a pair of homologous chromosomes that controls a relative trait. An individual developing from a zygote formed by the union of gametes of the same gene is called a homozygote (such as AA and aa), while an individual developing from a zygote formed by the union of gametes of different genes is called a heterozygote (such as Aa).

[0066] Self-incompatibility: refers to the self-sterility of a plant that has complete flowers and may form normal male and female gametes, but lacks the ability to self-pollinate and set fruit.

[0067] Fertile: refers to a plant or seed that, when grown into a plant, has complete flowers and may form normal female and male gametes, and may be used as a male or female parent to produce offspring through fertile pollen or fertile ovules (i.e. male flowers and female flowers are all viable).

[0068] Chromosome ploidy: refers to the number of chromosome complements or genomes contained in a cell. The number of chromosomes contained in normal gametocytes or half of the number of chromosomes in somatic cells is called a set of haploid chromosomes, represented by the symbol n. The complete set of haploid chromosomes of an organism is called the genome or chromosome complement of that organism. Cells with one chromosome complement and individuals composed of such cells are called haploidy (n). Cells or individuals with two chromosome complements are called diploidy (2n). Cells or individuals with more than two chromosome complements are called polyploidy, including triploidy (3n), tetraploidy (4n), etc. Polyploidy formed from chromosome complements from the same source is called autopolyploidy, and polyploidy formed from different chromosome complements from different sources is called allopolyploidy.

[0069] Dihaploid (double haploid): refers to cells or individuals obtained by conducting chromosome doubling on haploid, a dihaploid usually has a high genomic homozygosity rate (99.99%).

[0070] Hybridization: refers to the crossing of two individuals with different genotypes, and also refers to mating between different varieties, for example, cross-pollination between different plant varieties for plants.

[0071] Selfing: refers to the crossing of two individuals with the same genotype, for example, self-pollinating of plants.

[0072] Male parent: refers to one of the parents participating in the hybridization. In animals and plants, it is a male individual or an individual that produces male germ cells.

[0073] Female parent: herein refers to a plant that receives pollen when flowers of different plants undergo cross-pollination. During plant reproduction, it refers to the female plant of the previous generation.

[0074] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0075] The biological materials, sequences, reagents, or instruments used in the examples of the present disclosure may all be purchased from the market.

[0076] The present disclosure will be further described below in conjunction with the following examples.

Example 1: Creation of Self-Incompatible Dihaploid Materials

[0077] The male parent and female parent of hybrid potato Upotato 1 (Cell, 184:3873-3883, 2021,hybrid H1 of A6-26 and E4-63) were selected for hybridization to produce F.sub.1 hybrids. The female parent and male parent contain different self-compatibility genes respectively. The female parent contains the S.sub.S11 gene located on chromosome 1, and the male parent contains the Sli gene located on chromosome 12. In the F.sub.1 hybrid, the two genes are in a heterozygous state (see FIG. 1).

[0078] The haploid induction line PL4 introduced from the United States Department of Agriculture was used (other haploid induction lines may also be used) to haploid induce (pollinate into) the obtained hybrids, then screening haploid mutants. The screening of the haploid mutants is mainly divided into three steps.

[0079] (1) Eliminating seeds with purple embryonic spots. Since male parent PL4 is homozygous for purple embryonic spots (see FIG. 2), and the seeds of the female parent do not have embryonic spots, theoretically the seeds produced after hybridization should have purple embryonic spots. If not, it means that it may be a haploid mutant developing from a female gamete of the female parent.

[0080] (2) Using molecular markers to detect whether seeds without embryonic spots are obtained by selfing. Since the female parent is self-compatible, there may be a certain probability of selfing seeds due to untimely or incomplete emasculation. These seeds also do not have embryonic spots, and they may be eliminated through molecular markers. The primer sequences of the molecular markers include:

TABLE-US-00004 1)chr01_623388: Forwardprimer: (SEQIDNO:1) 5-TACAAATATTCAGCGAAGGG-3; Reverseprimer: (SEQIDNO:2) 5-GCTCAATTGTCTTCTTCAATCC-3. 2)chr02_112634: Forwardprimer: (SEQIDNO:3) 5-TCCTCCATCTCAGATTCACT-3; Reverseprimer: (SEQIDNO:4) 5-GGGTACGATTGTGAACATCT-3. 3)chr03_265493: Forwardprimer: (SEQIDNO:5) 5-CTATAATTTGGGCACGTGAT-3; Reverseprimer: (SEQIDNO:6) 5-CTAGTTGCATTCAGCCTTCT-3. 4)chr06_537151: Forwardprimer: (SEQIDNO:7) 5-CAGAGACAGGAACAGAAAATG-3; Reverseprimer: (SEQIDNO:8) 5-TGAAGGGACTAATTTGGCTA-3. 5)chr07_377300: Forwardprimer: (SEQIDNO:9) 5-GGGATCATTTCGATCTTTCT-3; Reverseprimer: (SEQIDNO:10) 5-GTAAATTGGAAATCCCTCCT-3. 6)chr08_844961: Forwardprimer: (SEQIDNO:11) 5-TGACCCTTTGGATGAAATAC-3; Reverseprimer: (SEQIDNO:12) 5-CACCTTATCAAACGAAGCTC-3.

[0081] (3) Using flow cytometry to detect the chromosome ploidy of suspected haploid plants and ultimately determine which plants are haploid.

[0082] The above method was used to screen out 17 haploid plants from 36,801 hybrid seeds. Subsequently, two primers respectively specific to self-compatibility genes Ssu and Sli were used to detect the presence of the two genes, and it was found that 4 individual plants do not contain these two genes, and they are respectively numbered YS1-187, YS1-192, YS1-311, and YS1-366.

[0083] S.sub.S11 gene-specific primers:

TABLE-US-00005 Forwardprimer: (SEQIDNO:13) 5-GAAGAAAGGAAATGAAGTGAGTTGTTC-3; Reverseprimer: (SEQIDNO:14) 5-GAATAATAACTTATTTCTTGATGGGATTG-3.

[0084] Sli gene-specific primers:

TABLE-US-00006 Forwardprimer: (SEQIDNO:15) 5-CGTCGGATTCAGCAGCAGAGTT-3; Reverseprimer: (SEQIDNO:16) 5-AAGCGAATTACAAGCCTGTTTAGATTGAC-3.

[0085] Then the top shoots of the haploid plants were taken to conduct chromosome doubling treatment through 0.35-0.55% colchicine for at least 2 days, thereby obtaining dihaploid plants (see FIG. 3). These dihaploid plants were transplanted to the greenhouse, and self-pollinated during flowering. It was found that they did not bear fruits finally, proving that they were indeed self-incompatible. Through the above process, self-incompatible dihaploid plants were finally obtained, and they may be used for hybrid potato production.

Example 2: Using Self-incompatible Parents for Seed Production to Increase the Rate of Hybrids

[0086] Hybridization was conducted by using the self-incompatible dihaploid material obtained in Example 1 as the female parent, and the self-compatible homozygous diploid material D180 [D180 was obtained from the hybridization of CIP 703280 and E172 (Cell, 184:3873-3883, 2021)] as the male parent. The female parent will not be emasculated during pollination. In the control group, the female parent A6-26 (A6-26 was obtained from the selfing of CIP 705468) and the male parent E4-63 [E4-63 was obtained from the hybridization of CIP 703767 and E172 (Cell, 184:3873-3883, 2021)] were both self-compatible homozygous diploid materials, and the female parent is artificially emasculated during the hybridization process.

[0087] Enough hybrids were obtained by using the above two methods, but the first method is more time-saving and labor-saving. For each method, 500 seeds were selected for purity testing of the hybrids through molecular markers. The results prove that the rate of hybrids in the first method is 100% and that in the second method is 97.5%. Therefore, using self-incompatible dihaploid materials as female parents for hybrid seed production may not only avoid emasculation during the hybridization process, and save manpower, but also increase the rate of the hybrids.

[0088] The above contents are only preferred embodiments of the present disclosure. It should be noted that those skilled in the art may make several improvements and modifications without departing from the principles of the present disclosure. These improvements and modifications should also be regarded as falling in the protection scope of the present disclosure.