The Fine Mapping And Candidate Identification Of The Recessive Genic Male Sterile Restorer Gene BnMs2 In Brassica Napus L.

Rapeseed is one of the major oil crops in China and the utilization of heterosis in rapeseed is an efficient way to increase yield, to mitigate the contradiction between yield and quality, and to improve resistance/tolerance. In China the recessive genic male sterility system (GMS) has become one of the most important approaches in virtue of the characteristics of complete and stable sterility, rich sources of cytoplasm and affluent restorer resources. However, the production of F1 hybrid seed requires the removal of 50% of the fertile plants in the female line, which limits its wide application. S45AB is a recessive GMS two-type line in Brassica napus and genetic analysis indicated that two duplicate recessive genes controlled the male sterility in S45A (Pan et al.,1988). For S45B, the BnMsl locus is heterozygous and Bnms2 locus is homozygous. In this study, for the fine mapping and cloning of BnMs2, a near isogenic line S4516AB, derived from S45A×IL362-2-4, was constructed by crossing, selfing, allelism testing and repeated full-sib mating. The objectives of this research are:(1) to develop a set of markers tightly linked to BnMs2 for marker assisted selection (MAS) in breeding of RGMS lines; (2) to isolate the BnMs2 gene and analyse its promoter to better our understanding of the mechanism of male sterility. The main results are as follows:1. The allelism analysis between S4516B and S45B indicated that the heterozygous locus of S4516B was not allelic to that of S45B. After assaying the segregation ratio of sterile plants and fertile plants in the NIL population of 262 individuals, S4516B was proved to be a single locus segregating line. Combining with the result of allelism study, the genotype of S4516A was Bnms1 Bnms1 Bnms2Bnms2 and that of S4516B was Bnms1Bnms1BnMs2Bnms2.2. From survey of 1024 AFLP primer combinations,12 AFLP markers tightly linked to the BnMs2 gene were idendified by the method of AFLP and BSA. In the NIL population of 262 plants, BnMs2 was mapped in the interval of 3.5cM and 8 AFLP markers co-segregated with BnMs2.3. Twelve AFLP markers were cloned and sequenced. By PCR walking we extended all the sequences of the eight co-segregation AFLP markers, the size range of which was 815-2661bp. Five SCAR markers were successfully obtained and two of them were co-dominant markers. Fine mapping was conducted in 2650 sterile plants with the methodology of pooled sampling strategy and flanking marker analysis. The eight AFLP markers and five SCAR markers were all mapped in a 1.0cM region around BnMs2 gene. Among these flanking markers of BnMs2 gene, AFLP markers Lm10 and Lm11 were the most closely linked ones, which were 0.038cM and 0.075cM from BnMs2 gene, respectively.4. We integrated four BnMs2 linked AFLP or SCAR markers to two doubled-haploid (DH) populations (Tapidor×Ningyou7 and Quantum×No.2127-17), and BnMs2 was mapped on N16. On the N16 linkage map one co-dominant SSR marker Na12A05 was identified by analyzing the selfing progeny of S4516B and the NIL population of 262 plants.5. BLAST searches were carried out against the Arabidopsis genome with the extended marker sequences and a collinear region comprising 121 Arabidopsis genes was identified. On the basis of Arabidopsis genome information and many Brassica EST, GSS and BAC sequences, one ACGM marker B14 was developed and Lm10 was mapped between two Arabidopsis genes i.e., Atlg69510 and Atlg69520. Thus marker B14 and Lm10 delimited a region only containing 12 Arabidopsis genes in which the homolog of BnMs2 might exist. As both of BnMs2 and BnMs1 can restore the sterility of S45A and S4516A, and the 2 candidate genes of BnMs1 was included in the 12 candidate genes of BnMs2, therefore, we conclude that BnMs2 candidates should be the homologs of the two candidates of BnMs1. The two candidates of BnMs2 were then designated as BnG1 and BnG2, respectively.6. For the 2 candidate genes of BnG1 and BnG2, plant expression vectors p2301G1 and p2301G2 were constructed, respectively. Using S4516AB as explants, transgenic positive plants were obtained by Agrobacterium-mediated transformation. In the 7 BnG1 transgenic plants, no recovery of fertility was observed. In the 3 BnG2 transgenic plants, no plants showed recovery of fertility, as well.7. Antisense expression vector p2301AntiG2 was constructed according to the 5' reserved cDNA sequence of BnMsl and BnG2. Using Westar(Brassica napus) as explants,20 positive transgenic plants were recovered,12 of which were assayed as fertile at flowering time. No expected phenotype was found.8. Gus fusion gene drived by the putative BnG2 promoter was introduced into Westar and 11 positive plants were assayed for GUS activity at different developmental stages. Results indicated that the expression of GUS was restricted specifically to the developing anthers (flower buds of 2-3mm). No GUS expression was observed in vegetative organs of plants.