Cotton whole genome assisted recurrent selection breeding method and application thereof

Abstract

The invention discloses a cotton whole genome assisted recurrent selection breeding method and application. According to the invention, through agronomic character phenotype identification and genotype identification based on whole genome re-sequencing, target character excellent germplasm is screened; a single dominant genic male sterile material is used as a female parent, excellent germplasm with outstanding agronomic traits is screened as a male parent, an F1 plant is obtained through artificial hybridization, a sterile plant is planted and separated to continue backcross with the corresponding male parent, backcross filial generation seeds are obtained, and the backcross filial generation seeds are equivalently mixed to serve as a basic population for population improvement. Carrying out recurrent selection on germplasm with new excellent character loci as male parents, hybridizing the male parents with sterile plants in the recurrent selection group, introducing new excellent character gene loci into the recurrent selection group, and carrying out reselection to obtain an improved group. According to the invention, recurrent selection is carried out based on single-gene dominant genic male sterility germplasm and whole genome assisted selection, so that the predictability of breeding and the polymerization efficiency of excellent gene loci are further improved, and new germplasm with excellent multi-character polymerization is created.

Description

Technical Field

[0001] The present invention relates to the technical field of plant biological breeding, and in particular to a cotton whole genome assisted recurrent selection breeding method and an application method thereof. Background Art

[0002] Cotton is the crop with the largest planting area in Xinjiang. In 2023, the cotton planting area in Xinjiang was 35.54 million mu, and the total cotton output was 5.112 million tons, accounting for 91% of the national total output. Xinjiang's cotton area, yield, total output and commodity transfer volume have ranked first in the country for 29 consecutive years. However, the cotton varieties currently planted in Xinjiang generally have problems such as low fiber quality, poor resistance to Verticillium wilt, serious variety homogeneity, and incompatible agricultural machinery and agronomy. As we all know, excellent varieties play an important role in agricultural production. Excellent varieties are not only conducive to increasing crop yield per unit area, improving product quality, maintaining stable yield, and improving stress resistance, but also conducive to promoting the development of agricultural mechanization and improving economic benefits. However, the selection and breeding of breakthrough excellent varieties is inseparable from the quantity and quality of germplasm resources and the improvement and innovation of breeding technology that makes full use of modern scientific and technological achievements.

[0003] Recurrent selection is a new breeding system developed and improved by American corn geneticists after the 1960s in response to the continuous new requirements for new varieties and the bottleneck factors in corn breeding. Recurrent selection is one of the important ways of population improvement and germplasm innovation. This breeding method is to improve the genetic structure of crop populations, expand the genetic basis of existing germplasm, increase the frequency of excellent genes in the population, break linkage drag, and increase the frequency of excellent gene aggregation in different germplasms through repeated alternating hybridization and selection.

[0004] With the rapid development of genomics and bioinformatics, plant breeding has entered a new stage of molecular breeding. Whole genome selection-assisted breeding uses the close relationship between molecular markers and target traits to accurately detect genotypes to quickly screen excellent plants, thereby accelerating the breeding process and improving breeding efficiency. Whole genome assisted recurrent selection breeding is an organic combination of whole genome selection biological breeding methods and recurrent selection conventional breeding methods. It can efficiently aggregate multiple excellent genes / traits, significantly improve breeding efficiency, and accelerate the breeding process. Summary of the invention

[0005] The purpose of the present invention is to innovate cotton breeding technology, increase the frequency of favorable genes in a population, create new cotton germplasm, and propose a cotton whole genome assisted recurrent selection breeding method for efficiently aggregating excellent genes.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a cotton molecular assisted recurrent selection breeding method and an application method thereof, comprising the following steps: Figure 1 ):

[0008] (1) Screening out germplasm with excellent traits through phenotypic identification of agronomic traits and genotypic identification based on whole genome resequencing or liquid phase chip technology; the traits may be one or more of yield, quality, stress resistance, and suitability for machine harvesting;

[0009] (2) Using sterile material as the female parent and excellent germplasm with outstanding agronomic traits selected through phenotype and genotype as the male parent, hybridization is carried out through artificial pollination to obtain F1 plants, in which the sterile and fertile plants of each combination of F1 generation show a 1:1 separation;

[0010] (3) Planting the F1 generation seeds of each hybrid combination separately, and then backcrossing the sterile plants separated after planting with the corresponding male parent once or twice to obtain backcross progeny seeds; and mixing the backcross progeny seeds in equal amounts as the C0 base population for population improvement;

[0011] (4) The phenotype and genotype of individual plants in the C0 basic population are accurately identified. Based on the complementarity of excellent gene loci between fertile and sterile plants, hybridization is carried out, hybrid bolls are harvested from sterile plants, and the hybrid bolls are mixed to form a rotation selection population;

[0012] (6) Furthermore, the phenotype and genotype of each plant in the rotation selection population are accurately identified, and poor fertile and sterile plants are removed in a timely manner. Based on the number of excellent gene loci in fertile and sterile plants and the complementarity of excellent gene loci, accurate guidance is provided for plant selection and hybridization to obtain an improved population.

[0013] Specifically, during the recurrent selection process in step (4), superior trait gene loci that are not present in the recurrent selection population are introduced, that is, germplasm with new superior trait loci is selected as the male parent, hybridized with the sterile plants in the recurrent selection population, and the new superior trait gene loci are introduced into the recurrent selection population.

[0014] The method further includes: (7) finally, the excellent individual plants in the improved population are self-pollinated and homozygous through rapid breeding methods, so as to efficiently create new cotton germplasms and new varieties with excellent comprehensive traits.

[0015] Specifically, in step (1), the male parent is a cotton germplasm or variety with excellent agronomic traits selected through precise phenotypic and genotypic identification.

[0016] Specifically, based on whole genome resequencing or liquid phase chip technology, the genotype identification of the father of the recurrent selection population and the individual plants of each generation of hybrid offspring are carried out;

[0017] More specifically, the specific operation of genotype identification based on whole genome resequencing or liquid phase chip technology is: in the seedling stage, the genomic DNA of each individual leaf is extracted respectively, and the genotype information of the detected individual plant at the known important agronomic trait functional gene loci is obtained by using whole genome resequencing technology or liquid phase chip technology; the genotype identification results are further sorted out to determine the type and quantity of important agronomic trait functional genes carried by each individual plant.

[0018] Preferably, the female parent used in the construction of the recurrent selection population is a single dominant nuclear sterile material, and the emasculation step is omitted during hybridization.

[0019] Specifically, the female parent used for the construction of the recurrent selection population was the "Shida 98-6A" single dominant nuclear sterile germplasm.

[0020] In a specific embodiment, the genotype identification of individual plants in the recurrent selection population is carried out based on a low-density liquid phase chip of upland cotton based on targeted capture sequencing.

[0021] The present invention also provides application of the method in new cotton germplasm innovation or rapid breeding of new cotton varieties.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The method of the present invention can be used to complete the breeding process, thereby eliminating the need for manual emasculation, greatly reducing the workload, and improving the pollination rate of cotton.

[0024] 2. The present invention utilizes molecular markers covering the whole genome level for assisted breeding, which reduces the blindness and randomness of plant selection, improves accuracy, and greatly shortens the breeding period.

[0025] 3. The present invention can achieve high efficiency of multi-parent gene aggregation, increase the probability of aggregation and recombination of excellent genes, and provide greater possibilities for breaking the narrow genetic basis of cotton, creating excellent germplasm and further breeding excellent varieties.

[0026] 4. The present invention can introduce paternal groups with different traits / genes according to different breeding objectives, such as paternal materials with excellent performance in traits such as disease resistance and plant type, and ultimately establish ideal materials for excellent gene aggregation with different breeding objectives.

[0027] 5. The present invention can be used for whole genome assisted recurrent selection of upland cotton or whole genome assisted recurrent selection of sea island cotton, depending on the cultivated species.

[0028] 6. The present invention organically combines the whole genome selection biological breeding method with the conventional recurrent selection breeding method through whole genome assisted recurrent selection breeding, and efficiently aggregates a variety of excellent traits / genes in a targeted manner, significantly improving breeding efficiency and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The main process of the cotton whole genome assisted recurrent selection breeding method of the present invention. Figure 2 Genotype identification results are displayed. Figure 3 Flowering appearance of fertile and sterile lines. Among them: a is the fertile line; b is the sterile line. Figure 4 The offspring obtained by the method of the present invention result in aggregation of superior sites. Figure 5 The excellent genotype frequency of the reincarnation population after one whole genome assisted reincarnation selection in the method of the present invention. DETAILED DESCRIPTION

[0029] The present invention is described below through specific implementation methods in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0030] The cotton whole genome assisted recurrent selection breeding method of the present invention has the following main processes: Figure 1 The specific steps include: 1. Recurrent selection of backbone parent screening

[0031] (1) A total of 343 upland cotton germplasm resources were identified by phenotypic and molecular genotypic characterization, and 12 micro-core germplasm materials were selected as examples of the paternal population of the recurrent selection population.

[0032] Furthermore, cotton liquid phase array (https: / / molbreeding.com / index.php?m=home&c=Lists&a=index&tid=82; https: / / www.sciencedirect.com / science / article / pii / S0926669022011128) was used to identify excellent loci in 12 micro-core germplasms.

[0033] The genotype identification results showed that ( Figure 2), the number of excellent loci of Lu 211, Jinfeng 219, Zhejiang University 2062, Xin 19075, Ta 2, Chuang D2, China Cotton Research Institute 2102, C240, 3497, 3220, 3-43, and 20S2-3 were 99, 89, 91, 100, 97, 103, 103, 102, 89, 125, 101, and 123, respectively. Lu 211 has more excellent loci for fiber quality and agronomic traits. Jinfeng 219 has an outstanding performance in yield trait loci. Although Zhejiang University 2062 has few excellent loci overall, it has better performance in agronomic and yield traits, and has better resistance loci. Xin 19075 has 4% and 53% excellent loci for resistance and fiber quality, and performs well. C240 ​​has more excellent loci for agronomic traits and yield, accounting for 10.78% and 40.2%, respectively. 3220 itself contains the most excellent sites, with 65 and 5 excellent sites in fiber quality and stress resistance, respectively, accounting for 52.1% and 4% of the excellent sites, respectively. 20S2-3 excellent sites ranked second, with four types of excellent sites accounting for 8.94%, 46.34%, 39.84% and 4.88% respectively. Chuang D2 has more sites in stress resistance and fewer sites for other traits. The sites of 2102 of the China Cotton Research Institute in terms of yield quality are relatively uniform, and the number of excellent sites of Tower 2 is relatively low, with only 3 excellent sites for stress resistance. 3-43 has the highest proportion of excellent sites in fiber quality, accounting for 50.9% of the excellent sites, and 3497 performs well in fiber quality and yield, accounting for 48.5% and 35% respectively.

[0034] Figure 2 The number of dominant alleles in the micro-core germplasm and their distribution in the four types of germplasm; a) The number of dominant alleles in the micro-core germplasm; b) The distribution of the micro-core germplasm in the four trait types; Note: a) FL: fiber length; FS: fiber strength; FU: uniformity; M: micronaire value; FE: elongation; FT: flowering time; FBP: fruiting branch first node; FBT: fruiting branch type; NFB: number of fruiting branches; HNFFB: height of fruiting branch first node; PH: plant height; LP: lint percentage; SI: seed index; BW: boll weight; BN: boll number; CVLV: cotton leaf curl virus; SR: salt resistance; HTR: high temperature resistance; VW: Verticillium wilt resistance. b) From left to back are C240, 3220, Ta 2, Chuang D2, Zhongmian 2102, Jinfeng 219, 3497, Xin 19075, 3-43, Lu 211, 20S2-3.

[0035] The phenotypic data of 12 micro-core germplasms showed (Table 1) that Xin 19075 performed well in fiber length, fiber strength, and elongation; the plant height of Lu 211 reached 93.04 cm. Chuang D2 had higher fiber length and fiber strength; 20S2-3 and C240 ​​had better fiber length and fiber uniformity, and Zhejiang University 2062 had higher lint percentage, and also had better performance in the number of fruit branches and the number of bolls per plant. 3220 had better uniformity, micronaire value, and lint yield, Zhongmian Institute 2102 had higher single boll weight, 3497 had more fruit branches, and Jinfeng 219 performed well in the number of fruit branches and the number of bolls per plant.

[0036] Table 1. Phenotypic values ​​of 12 mini core collections

[0037]

[0038]

[0039] In summary, 12 materials including C240, 3220, Jinfeng 219, Xin 19075, Lu 211, Zhejiang University 2062, 20S2-3, Ta 2, Chuang D2, China Cotton Research Institute 2102, 3497, and 3-43 were finally selected as the male parents for reincarnation selection.

[0040] 2. Fertility survey of recurrent selection population

[0041] The dominant nuclear sterility gene of the single dominant nuclear sterile material "Shida 98-6A" is Ms20, and the genotype is Ms20ms20. 12 excellent germplasms with outstanding one or more agronomic traits selected by phenotype and genotype were used as male parents, and the corresponding fertility genotype was ms20ms20. The single dominant nuclear sterile material "Shida 98-6A" was used as the sterile female parent, and the 12 excellent germplasms selected were used as the fertile male parent. Through artificial pollination and hybridization, F1 plants were obtained. The sterile and fertile plants of each combination of F1 generation showed a separation of 1 (Ms20ms20): 1 (ms20ms20). The F1 generation seeds of each hybrid combination are planted separately. The sterile plants separated after planting are backcrossed with the corresponding male parent once or twice to obtain backcross progeny seeds. The backcross progeny seeds are mixed in equal amounts and used as the C0 base population for population improvement. The sterile and fertile plants in the C0 base population will still show a separation of 1(Ms20ms20):1(ms20ms20).

[0042] The fertility of the offspring of the hybrid combination of sterile plants and fertile plants in each generation can be identified by molecular identification of fertility at the seedling stage using the linked molecular marker of the single dominant nuclear sterile gene Ms20 of "Shida 98-6A" (CN202410582970.0), or by observing the pollen fertility of individual plants at the flowering stage. Figure 3As shown in Figure 1, pollen fertility can be divided into two types: (1) fertile lines, with complete petal structures, stamens in the petals, and a large amount of pollen on the stamens ( Figure 3 (a); (2) sterile line, petal structure is complete, with stamens and stigma reproductive organs, but stamens are smooth and pollen-free ( Figure 3 (b)

[0043] 3. Hybridization of selected strains in recurrent selection populations

[0044] In order to efficiently aggregate the excellent loci of multiple agronomic traits, the phenotype and genotype of the C0 basic population and the individual plants of the recurrent selection population of each generation were accurately identified. According to the results of chip genotyping, combined with the comprehensive performance of fertility identification and field agronomic traits, poor fertile and sterile plants were timely eliminated in the contemporary recurrent selection hybridization. According to the number of excellent gene loci of fertile and sterile plants and the complementarity of excellent gene loci, accurate guidance was given to plant selection and hybridization. Finally, 14 excellent fertile plants P9, P10, P155, P217, P230, P319, P321, P350, P396, P421, P501, P606, P759, P801 and 10 excellent sterile plants P2, P21, P92, P155, P212, P265, P322, P340, P492, P516 were selected.

[0045] Based on the number of excellent gene loci in fertile and sterile plants and the complementarity of excellent gene loci, precise guidance is provided for plant selection and hybridization. Taking the aggregation of excellent gene loci of fertile plant P319 and sterile plant P2 as an example, P319 carries excellent gene loci for four traits: fiber length, fiber strength, boll weight, and lint percentage. P2 carries excellent gene loci for five traits: fiber elongation, fiber strength, lint percentage, and boll weight. These loci are complementary to the excellent gene loci of fertile plant P319. By hybridizing P319 and P2, the offspring can be aggregated to the excellent loci of the above traits, thus enabling targeted and efficient genetic improvement of multiple traits of cotton ( Figure 4 : Blue means that the individual plant contains the corresponding trait SNP site, and yellow means that it does not contain the corresponding trait SNP site).

[0046] Through accurate identification of phenotype and genotype of C0 basic population, after one round of whole genome assisted reincarnation selection, namely, according to the number of excellent gene loci of fertile and sterile plants and the complementarity of excellent gene loci, accurate guidance of plant selection and hybridization, the frequency of excellent genes in the round selection population increased significantly. After one round of whole genome assisted reincarnation selection, the excellent genotype frequency of the round selection population was 66.89% (204 / 305), and the average number of excellent loci (204) was significantly higher than that of the paternal germplasm (185) and the basic population (186) ( Figure 5 ).

Claims

1. A cotton whole genome assisted recurrent selection breeding method, characterized in that: The following steps are involved: (1) Screening out germplasm with excellent traits through phenotypic identification of agronomic traits and genotypic identification based on whole genome resequencing or liquid phase chip technology; the traits may be one or more of yield, quality, stress resistance, and suitability for machine harvesting; (2) Using sterile material as the female parent and excellent germplasm with outstanding agronomic traits selected through phenotype and genotype as the male parent, hybridization is carried out through artificial pollination to obtain F1 plants, in which the sterile and fertile plants of each combination of F1 generation show a 1:1 separation; (3) Planting the F1 generation seeds of each hybrid combination separately, and then backcrossing the sterile plants separated after planting with the corresponding male parent once or twice to obtain backcross progeny seeds; and mixing the backcross progeny seeds in equal amounts as the C0 base population for population improvement; (4) The phenotype and genotype of individual plants in the C0 basic population are accurately identified. Based on the complementarity of excellent gene loci between fertile and sterile plants, hybridization is carried out, hybrid bolls are harvested from sterile plants, and the hybrid bolls are mixed to form a rotation selection population; (6) Furthermore, the phenotype and genotype of each plant in the rotation selection population are accurately identified, and poor fertile and sterile plants are removed in a timely manner. Based on the number of excellent gene loci in fertile and sterile plants and the complementarity of excellent gene loci, accurate guidance is provided for plant selection and hybridization to obtain an improved population.

2. The method according to claim 1, characterized in that During the recurrent selection process in step (4), superior trait gene loci that are not present in the recurrent selection population are introduced, that is, germplasm with new superior trait loci is selected as the male parent, hybridized with the sterile plants in the recurrent selection population, and the new superior trait gene loci are introduced into the recurrent selection population.

3. The method according to claim 1, characterized in that Further including: (7) Finally, the excellent individual plants in the improved population are self-pollinated through rapid breeding methods to efficiently create new cotton germplasms and new varieties with excellent comprehensive traits.

4. The method according to claim 1, characterized in that: In the step (1), the male parent is a cotton germplasm or variety with excellent agronomic traits selected through accurate phenotypic and genotypic identification.

5. The method according to claim 4, characterized in that Based on the genotype identification of whole genome resequencing or liquid phase chip technology, the genotype of the father of the recurrent selection population and the individual plants of each generation of hybrid offspring are identified.

6. The method according to claim 5, characterized in that The specific operation of genotype identification based on whole genome resequencing or liquid phase chip technology is as follows: at the seedling stage, the genomic DNA of each individual leaf is extracted respectively, and the genotype information of the detected individual plant at the known important agronomic trait functional gene loci is obtained by using whole genome resequencing technology or liquid phase chip technology; the genotype identification results are further sorted out to determine the type and quantity of important agronomic trait functional genes carried by each individual plant.

7. The method according to claim 1, characterized in that The female parent used in the construction of the recurrent selection population is a single dominant nuclear sterile material, and the emasculation step is omitted during hybridization.

8. The method according to claim 7, characterized in that The female parent used in the construction of the recurrent selection population was the "Shida 98-6A" single dominant nuclear sterile germplasm.

9. The method according to claim 1, characterized in that: The genotype identification of individual plants in the recurrent selection population was carried out based on a low-density liquid phase array of upland cotton based on targeted capture sequencing.

10. Use of the method according to any one of claims 1 to 9 in the innovation of new cotton germplasm or in the rapid breeding of new cotton varieties.

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