Mapping Of Restorer Gene Rf₂ Of CMS-D8 And The Development Of Markers

Cotton is the world's most important raw material for the textile industry,the utilization of heterosis in cotton increased the yield of cotton,and CMS systems hybrid seeds production is an ideal tool to utilize heterosis.The CMS-D8 has matched maintainer line and restorer line,but CMS-D8system has not been applied in practical,and the restored gene Rf₂ has not been successfully cloned and functionally verified so far.In this study,we expected to map the restorer gene Rf₂ of CMS-D8 and develop InDel markers closely linked with Rf₂.The results not only help to shorten the cycle of improved restorer line,but also accelerate the application of the CMS-D8 system to the practical production and breeding of hybrids lay a foundation for the separation of Rf₂.The main results of this study are as follows:1.In this study,Rf₂ was mapped in a BC₁F₁??sterile line×restorer line?×maintainer line?segregation population of 1623 individual plants.The fertility investigation results showed that there were 850 fertile and 773 sterile strains in the population,and the separation ratio was consistent with 1:1,indicating that the fertility recovery of CMS-D8 was controlled by a single gene dominant.2.The parents were resequenced,and BSA resequencing technology combined with high-throughput SNP genotyping were used to locate Rf₂ at 54.11 MB-55.99 Mb of Chromosome D05,with a candidate interval of 1.88MB.Base on the InDel variation within the candidate interval,16polymorphic In Del markers were developed,and fine localization of Rf₂ was performed using In Del markers and 6 exchange individual plants,reducing the interval to 1.48 Mb.In Del markers co-isolated from Rf₂ genes were selected to trace Rf₂ during restorer line improvement.3.The InDel1892 marker was obtained that could distinguish the restoration gene Rf₁ or Rf₂ at the same time.The mutation site sequence analysis revealed 32 bp insertions in the CMS-D8 restorer line and 182 bp insertions in the CMS-D2 restorer line.InDel1892 marker combined with atpA SCAR marker related to sterile cytoplasm developed earlier by our team can be used to identify CMS system hybrids and distinguish restorer gene types contained in hybrids.4.The q RT-PCR analysis explored the expression pattern of PPR family genes in the interval in CMS-D8 system,and the relative expression level of Gh?D05G3391 in restorer lines was significantly lower than that in sterile lines and maintainers.Based on RNA-Seq data of anther exfoliated from 3 mm bud of CMS-D8 system,it was identified that the expression level of Gh?D05G3374 encoding NB-ARC domain-containing disease resistance protein in restorer lines was significantly higher than that in sterile lines and maintainers.