Molecular Mapping Of Fertility Restorer Gene For WA Cytoplasmic Male Sterility In Nipponbare

Three-line indica hybrids have been extensively used in commercial rice hybrids,and they have been developed mainly on wild abortive(WA)-type cytoplasmic male sterility(CMS)because of stable sterility and well recoverability of WA-type CMS lines.However,some WA-type CMS lines used in rice production exhibited,under the influence of the external environment,the selfed seeds,resulting in poor purity of hybrid seeds,which has influence on the utilization of such hybrids.Meanwhile,studies on the genetic analysis of the fertility restoration of WA-type male sterility line had been development lag,which could not attach with the utilization of three-hybrids.Therefore,a proposal to analyze the fertility restoring genes(Rf)in WA-type male sterility line and maintainer had been put forward based on the former study by our group,which would provide more information of the genetic mechanism of fertility restoration and be helpful for breeding of three-line rice hybrids.In the present study,a wide population consisting of 135 chromosome segment substitution lines(CSSLs)(designated as C1-C135)derived from the cross between two sequenced rice cultivars:9311,as the recipient and Nipponbare,as the donor,were used,and one Rf for WA-type indica CMS lines from Nipponbare was identified.Furthermore,the target Rf was fine mapped by constructing chromosome single substitution segment lines.The main results are as followed:1.Six CSSLs,carrying the substituted chromosome segment covering Rf5 locus,and 93-11 were as the male parent to cross with WA-TianfengA,a typical indica CMS line used in rice production,and seven test-crossing Fi were produced.The natural spikelet fertility levels of WA-TianfengA/C119 Fi plants were 80%,indicating these plants were fertile.But,The natural spikelet fertility levels of other six test-crossing Fi plants were 0-12.7%,and such plants were considered to be sterile.This result indicated there were more Rfs,from Nipponbare,in C119,and these Rfs were located on substituted chromosome segments.2.WA-TianfengA,WA-WufengA and WA-Guang8A,three typical indica CMS lines,were crossed with 93-11 and C119,respectively.The test-crossing F1 plants from C119 were fertile,and F1 plants from 93-11 were sterile,indicating that the interaction of Rfs from Nipponbare and 93-11 were responded for the fertility restoration of WA-type CMS lines.3.According to high-throughput sequencing output,six substituted chromosome segments were found in C119,and these substituted segments were confirmed by detection with molecular markers.From the CSSL population,another 18 CSSLs,carrying the target substituted segments,were selected to cross with WA-TianfengA and WA-WufengA,respectively.By the fertility levels of the testcrossing F1 plants and sequencing output of the CSSLs,a new gene,named as RJ21(t)temporarily,was preliminary mapped to a candidate region from 43.6?44.9Mb on chromosome 1,with a physical distance of 1.40Mb.4.The F2 population was constructed from the cross between 9311 and C119,of which all plants were detected with four target markers.49 plants,harboring the different donor chromosome segments,were selected as males to cross with WA-TianfengA and WA-WufengA,respectively.The natural spikelet fertility levels of plants in the testcrossing F1 populations were observed,by which Rf21(t)was identified to be flanked by RM5310 and ID01M29151 on chromosome 1.RM5310 located at 41198296bp-41198316bp,and ID01M29151 located at 41894866bp-41894884bp.The physical distance between these markers was about 697kb.5.A total of 14 CSSLs,with the substituted chromosome segment covering the target region on chromosome 1,were selected to cross with WA-TianfengA and WA-WufengA,respectively,and these CSSLs were molecular detected with more dense markers located in the target region.Thus,Rf21(t)was delimited to a region between the markers RM5310 and STS 1-170.4 on chromosome 1,with a physical distance of 900kb.Furthermore,chromosome single substitution segment lines were constructed by using 9311/C119 F3 populations,and the target plants in the 9311/C119 F3 populations were selected to cross with WA-TianfengA and WA-WufengA,respectively.According to the natural spikelet fertility levels of plants in testcrossing Fi populations,Rf21(t)was delimited to a region between the markers RM5310 and RM12182 with a physical distances of 187kb.