Fine Mapping Of A Recessive Genic Male Sterility Gene (ms3) In Brassica Napus L

9012AB is one type of the recessive genic male sterility(RGMS) in Brassica napus L.It has some advantages:such as complete and stable male sterility,widely spread of restorers and no negative cytoplasmic effect on yield as CMS might do.Genetic analysis indicated that the male sterility of 9012AB is controlled by two paris of recessive duplicate sterile genes(ms3ms3 and ms4ms4) interacting with one pair of a recessive epistatic inhibitor gene(rfrf)(Chen et al.,1998).Homozygous recessive sterile gene at both loci can result in male sterility,but homozygousity at the rf locus(rfrf) inhibits the expression of the recessive male sterility trait and restores the male fertility of plants. Based on this genetic model,a three-line system for hybrid seed production can be developed in this RGMS system.The three-line system can resolve the difficulty that 50%male fertile plants must be removed from the female lines during hybrid seed production.So,many researchers have put their eyes on the system in recent years.A three-line hybrid production on this RGMS system has been well-documented and the study about molecular mechanism has been started.Previous study had mapped the ms3 gene of 9012AB to a region of 0.5cM(Ke et al.,2005).In this study,9012AB was used to further fine map the ms3 gene based on the result above.Main results of the present study are as follows:1.AFLP technology combined with bulked segregant analysis(BSA) of recombinants was used to identify the genetic markers more tightly linked to ms3 in the 9012AB NIL population.Additional 2048 primer combinations were screened and 21 AFLP markers were obtained.Among them,2 markers(AH15 and AH17) co-segregated with the target gene in the tested population,14 markers(AH1,AH2,AH3,AH4,AH6,AH8,AH9,AH10,AH11,AH13,AH18,AH19,AH20,AH21) located at one side of the target locus,and other 5 markers(AH5,AH7,AH12,AH14,AH16) located at the other side of the target locus.The ms3 gene was mapped to a region of 0.14cM by the nearest flanking markers in a NIL population of 1506 plants.2.Seven AFLP markers(AH2,AH6,AH15,AH9,AH16,AH2 and AH23) were converted into SCAR markers successfully(designated as SW0,SW1,SW2,SW3,SW4, SW5 and SW6).These SCAR markers were used to analysis in three populations derived from 9012AB NIL including 6,407;4,768 and 8,041 individuals,respectively. As a result,three SCAR markers(SW2,SW5 and SW6) still co-segregated with the target gene;the ms3 gene was mapped between the nearest SCAR markers SW1 and SW4 with the distance of 0.029 cM each.Then the ms3 gene was delimited in a region of 0.058cM. 3.BLASTn analysis of seven markers' sequences above and their flanking squences on NCBI website can identify many homologues,most of which distribute on Arabidopsis chromosome v.And the order of their homologous loci in Arabidopsis chromosome v is the same as the marker loci in linkage map in B.napus;the homologous region spanning a physical distance of about 576kb in Arabidopsis chromosome v.The result of alignment above indicates that there is a good collinearity between the region flanking ms3 and Arabidopsis homologues.4.Specific PCR primers developed from the published EST and GSS of B.napus,B. rapa and B.oleracea which are comparatively located in the Arabidopsis syntenic region and Arabidopsis genomic sequences.Six ACGM markers which more tightly linked to ms3 locus were obtained(designated as AR23,AR44,AR45,AR48,AR52 and AR53). Subsequently,these ACGM markers were used to analysis in a small size population and recombinants derived from 9012AB NIL.As a result,AR23 cosegregated with the marker SW4 and owned the same recombinants;the other five ACGM markers shared only one recombinants and located at the same side with the marker SW1.The target gene was further delimited in a region of 0.034cM.5.Four probes(SW2 and flanking squences of AH9,AH16 and AH17) screen the Tapidor BAC library,and 86 BAC clones were obtained.14 positive BAC clones were identified by primers from probes.However,the 14 positive BAC clones can not construct a whole contigs spanning ms3 gene region.6.Based on the result above,the probe(o BAC clone digested with Hindâ…¢) was used to further screen the Tapidor BAC library,and 48 positive BAC clones were obtained.7 positive BAC clones which could be overlap with o BAC were identified by PCR analysis.Then,an overlap contigs of ms3 genomic region were constucted by the 7 positive BACs and 14 positive BACs obtained above.7.The BAC contigs covering the ms3 region was further confirmed by Southern hybridization of 21 BACs digested with restrction enzymes.Combining the PCR analysis with Southern analysis,the physical map of the ms3 gene region including 6 BACs at least was identified.Subsequently,a sequenced library which could be contains the target gene was constucted by subclone method.Now,about 150kb sequences were obtained from the subclone library.