Fine Mapping And Cloning Of Recessive Genic Male Sterile Gene Bnms1 In Brassica Napus

Utilization of heterosis in rapeseed is an efficient path to enhance yield,improve quality,and to strengthen resistance.The genie male sterile(GMS) system has been applied to this crop as an effective and economical pollination control system in China for its stable and complete sterility,rich sources of cytoplasm and widely spread of restorers.S45AB,a recessive GMS two-type line in Brassica napus,was derived from male sterile mutant of the Brassica napus canola variety Oro,genetic analysis indicated that two duplicate recessive genes controlled the male sterility in S45 populations(Pan et al.1988).In this study,S45AB was used to fine map and clone the recessive GMS gene,Bnms1.Main results are as follows:1.The NIL population displayed a ratio of ms to mf plants that did not differ significantly from 1:1.The segregation of fertile to sterile plants in the selfing generation from the male fertile plant of NIL did not differ significantly from 3:1. This result indicated that the recessive allele at the first locus(designed as Bnms1) was heterozygous,while the recessive allele at the second locus(designed as Bnms2) was homozygous.2.The observation on anther microscopical structure showed that it was no difference between S45A and S45B before the stage of meiosis.The tapetum of S45A was a little thicker than that of S45B at the stage of tetrad,but the tetrad in S45A was normal.Soon after the microspore released from tetrad,there was distinct difference between the microspore of S45A and S45B,the surface of microspore was smooth in S45A without the exine formation,the mierospore stopped developping and disaggregated;The tapetum of male sterile anther enlarged radial with a number of large vacuoles.The key stage taking place male sterility was from microsporocyte to mierospores.Extine development defective caused male sterlity.3.AFLP technology combined with bulked segregant analysis(BSA) was used to identify the genetic markers for Bnms1 gene in the NIL population.2560 pairs of AFLP primers were screened,and seven genetic markers AF1-AF7 linked to the Bnms1 gene were obtained.An NIL population including 310 individuals was used to map the Bnms1 gene,two nearest markers AF3 and AF7 bracketed Bnms1 at distances of 1.6cM and 0.3cM,respectively,AF1 and AF2 were co-segregated with Bnms1 gene.4.The fragments were cloned and sequenced,which indicated that AF1-AF7 were 203bp,241bp,211bp,186bp,187bp,284bp and 362bp in length,respectively. The four AFLP markers(AF1,AF3,AF6 and AF7) were successfully extended by PCR-walking.The four extended sequences were 7,499bp,997bp,1,203bp and 656bp, respectively,and they were converted into SCAR markers(designated as SC1,SC3, SC6 and SC7).Another population in the same NIL including 1,974 individuals was used for fine mapping of the Bnms1 gene.As a result,the Bnms1 gene was flanked by two SCAR markers,SC1 and SC7,with genetic distance of 0.1cM and 0.3cM, respectively.5.Using a DH population derived from the cross Zhongyou821×Bao604,a genetic linkage map spanning 1,625.6cM was constructed,which including 2 RFLP markers,65 RAPD markers,86 SSR markers and 84 SRAP markers.Based on two linakge map constructed using DH populations(Tapidor×Ningyou7 and Zhongyou821×Bao604),the Bnms1 gene was mapped on linkage group N7.One co-dominant SSR marker located on linkage group N7,Na12A02,was confirmed to link to the Bnms1 gene.The distance between the marker and Bnms1 gene was 2.6cM.6.Three probes(SC7,SC1 and SC6) screened the Tapidor BAC library,and 41 positive BAC clone were identified.One BAC clone BAC1 which might contain the Bnms1 gene was identified by two blacketing SCAR markers,SC1 and SC7.The BAC clone was sequenced using the shotgun sequencing strategy.Two and six recombinants with the Bnms1 gene were detected by two SCAR markers(SC8 and SC11) based on the sequence of BAC1,respectively.The physical distance between SC8 and SC11 was 21.2-kb.That meaned the Bnms1 gene was narrowed into a 21.2-kb DNA fragment.7.Searches for putative candidate gene were performed using TBLASTN searches in the plant EST database at http://www.ncbi.nlm.nih.gov/and AGI Genes database at http://www.arabidopsis.org/using 21.2-kb sequence as the query sequence. The result indicated that the 21.2-kb region included four candidate genes,and the gene order was the same between Brassica napus and Arabidopsis,it indicated that the Bnms1 gene located in a collinearity region of Brassica napus and Arabidopsis. The functions of the first and the fourth candidate were unknow,the second candidate gene was a NAP like type,the third candidated gene was a member of CPY450 family, and both the second and the third candidate gene were related with pollen development,which were designed as G14 and G15,respectively.8.Northern blot analysis showed that the expressions of G14 and G15 were neither distinctly different between S45A and S45B.The expression of G14 was not different among four stages tested and expression was low in bud,while G15 specially expressed from the microsporocyte stage to microsorpe stage.9.Comparative sequence analysis identified 2 missense mutations between S45A and S45B at both candidate genes,and those caused two amino acid conversion,V to A and A to S in G14,G to R and V to A in G15,respectively. 10.Plant expression vectors pBnGA15 and RNA interference expression vectors, pAtG15RNAi-1 and pAtG15RNAi-2,were constructed.Five transgenic Brassica napus plants and two male sterile Arabidopsis plants were obtained,respectively.