Molecular breeding method for improving stigma exsertion rate of sterile line of three-line rice

[0024] Examples, see figure 1 with 2 ,

[0025] 1. Fine mapping of rice head exposure rate gene qES7

[0026] (1) Huhan 1B/II-32B BC3F2 population construction

[0027] Yue Gaohong et al. (2009) used Huhan 1B/Ⅱ-32B to construct F 2 The population has conducted QTL analysis on the traits of rice stigma exposure rate, and initially located a QTL-qES7 that affects both unilateral stigma exposure rate and total stigma exposure rate. It is located between the molecular markers RM3859 and RM5436 on chromosome 7 with additive effect. From parent II-32B with high stigma exposure rate. Therefore, using Huhan 1B as the recurrent parent, select individual plants containing qES7 locus in the F2 population to backcross with Huhan 1B to obtain BC1F1, and then use molecular markers RM3859 and RM5436 to detect the backcrossed progeny. The single plants of the zygotic genotype continue to be backcrossed with Huhan 1B to obtain BC2F1, and the backcross progeny whose markers on both sides are heterozygous genotypes are also selected to be backcrossed with Huhan 1B to obtain BC3F1, and then 6720 BC3F2 are obtained from the self-bred species Individual plant in the population.

[0028] (2) Molecular marker analysis of the constructed BC3F2 population

[0029] Refer to and compare the sequenced rice line 9311 and Nipponbare's corresponding genome sequences between RM3859 and RM5436, use the microsatellite DNA locus search software SSRHunter to find the SSR sequence, and use the molecular biology application software DNAMAN to find the Indel difference sequence. The above sequence was designed by the primer design software PRIME PRIMER 5.

[0030] According to the conventional CTAB method, the DNA of individual plants of the parent populations of Huhan 1B, II-32B and BC3F2 was extracted, and the parents were screened for polymorphism with the designed Indel marker primers. The PCR amplification reaction system was: 20ng rice genomic DNA template, 10uL Taq PCR Mastermix (Tiangen Biochemical Technology (Beijing) Co., Ltd.), 10uM front and rear primers 1uL each, supplemented with ddH2O to 20uL. The PCR amplification procedure is: pre-denaturation at 95°C for 5 minutes, then 30 cycles at 95°C for 30s, 55°C for 30s, 72°C for 45s, and finally 72°C to extend for 5 minutes. The PCR amplification products were electrophoresed on a 6% non-denaturing polyacrylamide gel, and finally 6 labeled primers were obtained from 23 designed and synthesized Indel markers in the target range. There are polymorphisms among the parents (Table 1) .

[0031] Table 1 Molecular marker information

[0032]

[0033]

[0034] The genetic distance between the markers RM3859 and RM5436 is 1.2cM, and the physical distance is about 200kb. Using RM3859 and RM5436 to screen out 18 heterozygous recombinant single plants from 6720 BC3F2 population single plants, using new design and development screening to obtain 6 polymorphic molecular markers of Indel4373, Indel4380, Indel4385, Indel4419, Indel4459, Indel4477 to detect recombination The genomic DNA of a single plant was found to be divided into 6 types of recombination (Table 2). The 18 recombined single plants were harvested and planted into BC3F3 strains. 48 strains were planted in each strain, and each strain in each strain was investigated. The stigma exposure rate phenotype of the plant, the phenotype investigation method refers to Yue Gaohong et al. (2009), according to the separation of the phenotypic value, the genotype of the BC3F2 single plant is inferred, and the seeds of 6 pure and recombinant single plants are collected and planted. The BC3F4 strain was formed, and each strain was planted with 24 strains. Theoretically, the pure and recombined types will not be separated again. Repeated investigation of the phenotype of stigma exposure, combined with the phenotype and genotype, the qES7 gene was finally finely mapped to the molecular markers RM3859 and 28.5k interval between Indel4373 (Table 3).

[0035] Table 2 Genotype of BC3F2 recombinant single plant

[0036]

[0037]

[0038] Table 3 Genotype and phenotype of BC3F4 recombinant single plant

[0039]

[0040]

[0041] 2. Molecular breeding methods to increase the exposure rate of stigma of three-line sterile rice lines

[0042] Among the pure and recombinant single plants of BC3F3 generation obtained in Example 1, the high stigma exsertion rate was selected, the agronomic traits were similar to the recurrent parent Huhan 1B, and the markers RM3859 and Indel4373 were genotype II-32B, and the remaining markers were Huhan 1B The genotype of a single plant was crossed with the wild abortive sterile line Huhan 1A, and a single plant was bagged and self-crossed to obtain BC3F4; three consecutive backcrossings, each generation selected a single with high stigma exudation rate and agronomic traits similar to Huhan 1A Plants are backcrossed in pairs, that is, improved sterile lines with increased stigma exposure rate and paired maintainer lines.

[0043] The flowering habit, stigma vitality, stigma exposure rate, outcrossing seed setting rate and seed production yield of the newly improved sterile line were investigated. The results showed that the agronomic traits of the improved sterile line were similar to those of Huhan 1A. Good, the flowering period is concentrated, the proportion of flowers in the early afternoon is high, the stigma is strong, the total stigma exposure rate is 69.7%, and the outcrossing seed setting rate is as high as 68.0%. The seed production yield of Hanhui 3 was 3.72t/hm2, which was 2.33t/hm2 higher than the seed production (1.39t/hm2) of the control Huhan 1A and Hanhui 3.