Fine Mapping Of The Epistatic Suppressor Gene (Esp) Of A Recessive Genic Male Sterility In Rapeseed (Brassica Napus L.)

It is widely accepted that recessive genic male sterility (RGMS) systems have great potential in rapeseed heterosis utilization because of their stable and complete sterility, extensive distribution of restorers and diverse cytoplasmic sources. However, for most of them, their potential application for the commercial production of rapeseed hybrids is limited by the inability to maintain the male-sterile female parent line in an efficient and cost-effective way. About 50% male fertile plants must be removed during hybrid seed production, which increases the cost of hybrid seeds and limits its wide application. Fortunately, this difficulty can be overcome when a novel RGMS line,9012AB, is involved. Genetic analysis indicated that the sterility of 9012AB is controlled by two recessive genes (ms3 and ms4) interacting with one recessive epistatic suppressor gene (esp). Homozygous recessive sterile gene at both loci can result in male sterility, but the fertility can be restored when the esp gene is in recessive homozygosity or either of the sterile genes is not recessively homozygous. Based on this genetic model, a 100% male-sterile population could be produced by crossing a homozygous male-sterile line with a temporary maintainer line. This strategy is used successfully for the commercial production of canola hybrids (Brassica napus) in China. Care needs to be taken that the breeding of elite RGMS lines and their temporary maintainers by conventional methods is very laborious and time-consuming. It is therefore highly desirable to develop molecular marker tightly linked to the genes (ms3, ms4 and esp), which will greatly accelerate these breeding process. The purposes of this investigation are to develop molecular markers tightly linked to the esp gene, using two BC1 mapping populations derived from recessive epistatic genic male sterility line (9012A and GosA) and their temporary maintainer (T45), and then to construct a high-resolution map surrounding the esp gene. The main results were as follows:1. Development of molecular markers linked to the esp gene. Several strategies were used to develop molecular markers associated with the target gene. Firstly, AFLP technology combined with bulked segregant analysis (BSA). From the survey of 2,816 AFLP primer combinations, seventeen tightly linked AFLP markers were obtained. Among them, ten AFLP makers co-segregated with the target gene in the populationⅠof 143 individuals. Given that AFLP has limitations in large-scale population analysis, seven co-segregated AFLP markers were converted to dominant SCAR makers. Secondly, genetic map integration. Considering the esp gene was mapped on linkage group N7 of Brassica napus, PCR markers surrounding corresponding region were selected from linkage group A7 to analyze the populationⅡof 188 plants. Results indicated that 14 PCR markers including 1 SCAR marker,7 SSR markers,2 IP markers and 3 BAC sequence-based markers were assicated with the esp gene. Thirdly, comparative mapping. Sequences of the esp-linked PCR markers were submitted to NCBI for BLASTN analysis. Combined with previous reports on comparative analysis, we found that the esp region in Brassica napus linkage group N7 is constituted of blocks G-H-F-B. The Arabidopsis genes of block F encompassing the esp gene were employed for the development of Intron Polymorphism (IP) markers. IP primers were designed from exon sequences which showed strong nucleotide conservation between Arabidopsis and the corresponding EST or GSS sequences described for any Brassica species, and 122 primer pairs were designed. After the polymorphism survey in parents and mapping population,14 IP markers were found to be tightly linked to the esp gene in both populationⅠand populationⅡ.2. Fine mapping of the esp gene. The esp-linked PCR markers described above were used to tested on 3,878 plants from populationⅠand 3,484 plants from populationⅡ. We employed the flanking marker approach to improve the efficiency of large population analysis. In the populationⅠ, the esp gene was then genetically restricted to a region of 0.335 cM,0.206 cM from SCAR marker WSC6 and 0.129 cM from IP marker IP5-3. In the populationⅡ, the target gene was mapped between IP marker IP36 and IP marker IP02-3 with the distance of 0.057 cM and 0.057 cM, respectively. Integrating the mapping results from two larger populations, the esp was fine-mapped to the interval between IP markers IP36 and IP05-3. Despite of 7,362 plants from two mapping population were analyzed, six SCAR makers and three IP makers were still co-segregated with the esp gene.3. Microcollinearity between Brassica napus and Arabidopsis at the esp locus region. The presence of many Arabidopsis-derived IP markers in the local high-resolution map allowed us to study the collinearity between Brassica napus and Arabidopsis at the esp locus region. Microcollinearity was found between the region on N7 carrying the esp gene and Arabidopsis chromosome 3, interrupted by chromosome segment inversion and gene inserts. Due to the inversion directly flanking the map position of esp, extending the search for candidate genes to an area delimited by the flanking markers, IP36 and IP5-3, identifies two regions on Arabidopsis chromosome 3. The first is a collinear region between IP markers IP5-3 and IP9-4 covering an interval of 54kb and containing 12 annotated genes. The second candidate region which locate between IP markers At3G24315 and IP36 cover an interval of 40kb and include 8 annotated genes. Unfortunately, no obvious candidate gene for esp was identified among 20 annotated genes in two Arabidopsis collinear region.