Fine Mapping Of A Male Sterility Gene Ms-cd1 In Brassica Oleracea

Cabbage (Brassica oleracea var. capitata) is an important leafy vegetable crop with a widely growing area only second to Chinese cabbage (Brassica rapa) in China. Utilization of male sterile lines is becoming the trends for cabbage heterosis breeding because of the main heterosis. At present, main cultivated varieties of cabbage are hybrid cultivars.A dominant male-sterility gene Ms-cd1 was identified as a spontaneous mutation in a spring cabbage line 79-399-3.Molecular markers linked to this gene were developed, including a RAPD (randomly amplified polymorphic DNA) marker with a linkage distance of 7.8 cM, a SCAR (sequence characterized amplified region) marker converted by the previous RAPD marker and an ERPAR (extended random primer amplified region) marker. Wang et al indirectly mapped MS-cd1 onto linkage group O9, corresponding to chromosome 3 of B. oleracea, corresponding to the top of chromosome 5 in Arabidopsis thaliana.Because traditional breeding is relatively long procedure, cheap, fast, and reliable PCR based markers were to be made available for the Ms-cd1 locus, this would greatly improve the efficiency of breeding programs using the Ms-cd1 allele. However, the genetic distance between these markers and MS-cd1 was not close enough for practical utilization in marker assisted selection. Hence, for practical use in MAS (marker-assisted selection) or fine mapping the need remains for simple PCR based markers, such as SCAR markers. In this study, we screened a large scale BC4 population segregating in male fertility with SRAP and SSR (simple sequence repeat) markers to identify markers closely linked to MS-cd1. The identified markers were converted into donminant SCAR markers, together with a dominant SSR marker and making use of the collinearity between B. oleracea and B. rapa, which may be useful in marker-assisted selection of breeding programs and map-based cloning.Main conclusions are listed as follows: 1. Bulked segregation analysis (BSA) was performed for 226 BC4 individuals using SRAP technology regarding of male sterility and fertility. Using 800 SRAP primer and1425 combinations of SRAP primers and AFLP primers, a primary map surrounding Ms-cd1 was constructed. Eleven markers closely linked to the target gene were identified, among which the closest one on each side to Ms-cd1 was 2.0 cM and 4.0 cM, respectively.2. BSA was performed for 2,028 BC4 individuals using SRAPs and SSRs regarding of male sterility and fertility. Using 26,417 SRAP primer combinations and 1300 SSRs, a high-resolution map surrounding Ms-cd1 was constructed. Thirteen SRAPs closely linked to the target gene were identified, among which the closest one on each side to Ms-cd1 was 0.18 cM and 2.16 cM, respectively. We identified a SSR marker 8C0909 closely linked to MS-cd1 gene with a distance 2.06 cM and being a dominant marker. Markers linked closely to the Ms-cd1 gene will enrich resources of molecular marker of Ms-cd1 locus, also serve to lay the foundations for molecular-assisted selection in breeding program, as well as fine mapping and map-based cloning of Ms-cd1 gene.3. Three of these SRAPs were successfully converted to dominant SCAR markers. Two SCAR markers, ENA20R-rem2SC and OD3R-Ni2E12FSC, flanked the Ms-cd1 gene at distances of 0.39 and 4.23 cM, respectively. Based on the information of BLAST analysis with these SCAR marker sequences, the MS-cd1 gene was located on a collinear genomic region about 650kb in scaffold 000010 on linkage group A10 in Brassica rapa, which contains 54 genes in the collinear region of Arabidopsis thaliana. Finally, F8 gene was primarily determined to be a candidate gene with Ms-cd1 gene because of high-efficiency expression in Arabidopsis thaliana by using online software.4. In order to overcome the limitations of the standard SRAP primers and to make greater use of the supply of alternative primers, potentially reducing laboratory costs, we described in this paper how random primer combinations of SRAP, AFLP (amplified fragment length polymorphism ) and SSR were used as new primers in SRAP analyses. We defined this technique as AP-SRAP (arbitrarily primed SRAP), which combines simplicity and reliability.