一个玉米CMS-C恢复基因的精细定位

Fine Mapping of a CMS-C Restorer Gene in Maize

为挖掘玉米C型细胞质雄性不育(CMS-C,C-type cytoplasmic male sterility)的育性恢复基因并解析基因功能,用恢复系K932R与2个CMS-C同质异核不育系K169S和K932S分别构建2个F2定位群体,结合SSR分子标记和分离群体分析法全基因组重测序(BSA-reseq)技术定位育性恢复基因,通过PCR技术克隆目的基因并进行基因表达分析。结果表明,用BSA-SSR分子标记法将育性恢复基因Rf 932定位于8号染色体短臂,位于SSR标记8-21与8-30之间,遗传距离分别为2.3 cM和7.7 cM;通过BSA-reseq技术,将Rf 932定位于8号染色体0.38Mb的区域,包含7个候选基因;对候选基因分段克隆和测序比对,获得Rf 932序列全长2549 bp,由4个外显子和3个内含子组成,编码385个氨基酸;其中控制育性恢复的关键位点发生了与Rf4相同的氨基酸变异F187Y,是1个新的Rf4等位基因;该基因编码bHLH转录因子,调控绒毡层发育;据qRT-PCR分析,Rf 932在不育系与恢复系花药中3个发育时期的表达量均无显著差异。研究结果丰富了玉米CMS-C恢复基因库,并为进一步解析育性恢复机理奠定了基础。

To explore the fertility restorer genes of CMS-C(C-type cytoplasmic male sterility)in maize,and analyze the gene functions,two F2 mapping populations were developed with restorer line K932R and two CMS-C allogeneic heterogeneous sterile lines K169S and K932S,respectively.Combined with SSR molecular markers and BSA-reseq technology,the fertility restorer gene in K932R was mapped.The target gene was cloned by PCR technology and gene expression analysis was performed.The results showed that the fertility restorer gene Rf 932 was mapped on the short arm of chromosome 8 by BSA-SSR molecular markers,between SSR markers 8-21 and 8-30,with the genetic distance of 2.3 cM and 7.7 cM,respectively.Rf 932 was located in a 0.38 Mb region on chromosome 8 by BSA-reseq technology,which contained 7 candidate genes.After the candidate genes being cloned in segments and aligned in sequences,Rf 932 was obtained with a full length of 2549 bp,consisting of 4 exons and 3 introns,encoding 385 amino acids.One of the key sites controlling fertility restoration,F187Y,had the same amino acid variation as Rf4,which was a new Rf4 allele.Rf 932 encoded a bHLH transcription factor that regulated the tapetum development.qRT-PCR analysis showed that there was no significant difference in the relative expression levels of Rf 932 in anthers between the sterile line and the restorer line at 3 developmental stages.The results enriched the restore gene bank of maize CMS-C,and laid a foundation for further analyzing the mechanism of fertility restoration.