Molecular Marker Analysis And Mapping Of The Genic Male Sterility Genes In Brassica Rapa L.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important vegetable crop in China. It originated in China and has many advantageous economic characteristics. It has quite strong heterosis. Self-incompatible lines and male sterile lines are widely used in F1 seed production. The application of self-incompatible lines is limited by the reduction of fitness and vigor, the inconvenience of selfing, and the susceptibility of plants to environmental conditions. Male sterile lines are ideal systems for hybrid production. Plant male sterility can be generally classified into cytoplasmic male sterility (CMS) and genic male sterility (GMS). In GMS lines, sterility is stable and complete, and GMS does not result in reduced vigor. The study of genic male sterility of Brassica rapa showed different genetic models. The methods of transferring male sterility genes depend on certain genetic models. The genetic models of the genic male sterility lines had to be identified before they can be used for breeding. Molecular markers analysis of those male sterility genes just started in recent years.This thesis deals with two recessive genic male sterility lines (452AB and 454AB) and one domiant genic male sterility line (451AB) to confirm their genetic models, to investigate distribution of different male sterile genes in male fertile inbred lines, and to find the molecular markers linked to the restoring genes and male sterility gene, and to map these genes. The main conclusions are as follows:1. The genetic analysis in the recessive genic male sterility lines (452AB and 454AB) and dominant genic male sterility line (451AB) was conducted. The sterile plants and fertile plants in 452AB line and 454AB line were crossed with the fertile plants in 451AB line. According to the results, BrMs1 gene in 451AB and BrMsf3 gene in 454AB line were at the same allelic locus. So the genetic model of 451AB line and 454AB line was conformed to be a multiple-allele model. 452AB line had no relationship with 451AB line and 454AB line. For transferring these genes to other lines, male sterile plants were backcrossed with 17 inbred lines to confirm their genotypes. Four inbred lines can maintain sterility gene in 452AB line and resulted in 100% male sterile populations. The further experiments confirmed the genetic model of 452AB line also to be multiple-allele, but the genespositions were different from those in line 451AB and line 454AB.2. Line 452AB was recessive genic male sterility (RGMS), which contained 164 individuals. Bulked segregant analysis (BSA) was used to screen markers linked to restoring gene BrMsf2. After SRAP (Sequence-related amplified polymorphism) markers system was optimized, 1 256 SRAP primer pairs and 187 simple sequence repeat (SSR) primer pairs were analyzed between fertile bulks. Two SRAP markers PM8K4 and Me2M49 were found, and the distance of PM8K4 and Me2M49 was 2.98 cM and 10.92 cM, respectively. PM8K4 was subsequently mapped on chromosome A08 using a doubled-haploid mapping population. Screened 23 Indel markers on A08 from Brassica rapa genome sequence, one marker (S20-1) in scaffold 20 showed polymorphism, and the genetic distance was 4.22 cM. According the positions of S20-1 and PM8K4 on chromosome A08, BrMsf2 was mapped around scaffold 20 in A08. 3. Line 451AB was dominant genic male sterility (DGMS), which produced 480 individuals. SRAP, SSR and sequence-characterized amplified region (SCAR) analyses were performed to screen markers linked to BrMs1. Five of the 1 378 SRAP primer pair combinations (CuMe7BEm12, BMe9E32, PM8E38, E35BEm10, and M51K2) and one of 238 SSRs (ENA6) revealed polymorphisms between bulks from male sterile and fertile progeny. PM8E38, the nearest marker linked to BrMs1 (0.37 cM), was converted to the SCAR marker PM8E38S. ENA6 was subsequently mapped on chromosome A07 using a doubled haploid mapping population.4. A fine mapping population which contained 2 357 individuals was constructed in 451AB line. In this larger population, the distance between PM8E38S and BrMs1 was changed to 0.59 cM. None of them was mapped on A07 according to the alignment between the sequences of markers found in Chapter four and the whole genome of Brassica rapa. Firstly, Screened 13 Intron Polymorphism (IP) markers designed from Brassica rapa genome and 28 IP markers from Brassica juncea genome, one marker (A0703-2) showed polymorphism, but the genetic distance was larger than other markers. Secondly, a new marker technique based on genome sequencing named Specific Length Amplified Fragment (SLAF) was constructed, and first used on gene fine mapping. After screened 76 SLAF markers, 10 showed polymorphism. Four of them were position-known (S17-52,S1727-5,S1727-6,S1729-5), and six of them were position-unknown (SNONE-2,SNONE-7,SNONE-9,SNONE-16,SNONE-27å’ŒSNONE-44). The nearest position-known marker was S1727-6 (0.76 cM), and the nearest position-unknown marker was SNONE-9 (0.51 cM). Finally, 26 Indel markers on A07 were screened, four markers (S17-1,S17-2,S69-1 and S69-2) linked to BrMs1 gene. According the position of SLAF markers and Indel markers, the genetic distances of markers and gene corresponded to physical distances, and the predicted area in physical map was confirmed. By the sequence analysis in the predicted area, the position of BrMs1 was predicted in centriolar region.5. Line 454AB was a RGMS line, which gave 320 individuals. SRAP and SSR techniques and bulked segregant analysis (BSA) were used to screen markers linked to the RGMS restoring gene (BrMsf3). Among the 1 128 SRAP primers, only BMe10/SA4 and M52/K2 showed polymorphism between bulks. The polymorphic bands were named as BMe10SA4 and M52K2. The distance of BMe10SA4 and M52K2 was 4.38 cM and 7.46 cM, respectively, and these two markers were on the same side of BrMsf3. SSR marker ENA6 was linked to BrMsf3, and the genetic distance was 11.57 cM. According to the position of ENA6 in genetic map, BrMsf3 was mapped on A07. Subsequently, 26 Indel markers and 65 SLAF markers from A07 were screened, and no one showed polymorphism. This result confirmed the multiple-allele genetic model for 451AB line and 454AB line.6. According to the genetic analysis and results of gene mapping, 451AB line and 454AB line were multiple-allele male sterility lines, and the male sterility genes were on A07. 452AB line was also multiple-allele male sterility line, and the male sterility genes were on A08. That verified there were at least two multiple-allele male sterility systems in Chinese cabbage.