Identification Of SSR Markers Linked To Restorer Gene RF6 And Of Genes Associated With Fertility Restoration Of T-Type Cytoplasmic Male Sterility Of Wheat

Rf6 is a restorer gene for T-type cytoplasmic male sterility of wheat derived from chromosome 6U of Aegilops umbellulata Zhuk. N2114 is a restorer line possessing Rf6 with T-type cytoplasm. This study was conducted to identify SSR markers linked to Rf6 and genes associated with fertility restoration.A F2 segregation population derived from N2114xSumai No.3 was produced. x2 test indicates that the fertile and sterile plants segregated in 3:1 ratio. Through linkage analysis, six co-segregating loci, including Xgwm88, Xgwm644, Xbarcl46, Xbarcl98, Xwmc397 and Xwmc398, were identified to be tightly linked to Rf6 with a distance of 2.0 cM. The polymorphic bands of these markers are identical to the corresponding bands in Aegilops umbellulata Zhuk. The Rf6 was mapped to 6BS near the centromere.SSH (suppression subtractive hybridization) was conducted using the cDNA mixture from spikelets of Sumai No.3 and CS at meiosis as the driver and that from N2114 as the tester. The PCR products amplified using the template from the subtractive hybridization were cloned and a SSH library containing 350 recombinants was constructed. Differential screening of the SSH library using the cDNA mixture from spikelets of Sumai No.3 and CS at meiosis and that from N2114 as probes, respectively, showed that 51 of them were up-regulated in spikelets of N2114, among which 25 had strong signals.Based on the Msp I -digested fingerprinting patterns of the insert, the 25 clones were classified into 15 groups. One from each group was sequenced. BLAST analysis indicated y6 and y221 were homologous to the putative senescence-associated protein ACC synthase and ACC oxidase of Pisum sativum and Pyrus communis, yl35 was homologous to the putative diacylglycerol kinase of the Oryza sativa and Lycopersicon esculentum, y382 was homologous to the calmodulin-binding protein of Arabidopsis thaliana, y582 was homologous to the copper-transporting ATPase of Arabidopsis thaliana, y546 washomologous to the ATP synthase of Arabidopsis thaliana and Oryza sativa, y309 was homologous to the putative reverse transcriptase of Cicer arietinum, y587 was homologous to the DHHC-type zinc finger domain-containing protein of Oryza sativa, and y92 was homologous to the ethylene-responsive transcriptional coactivator of Oryza sativa.A full-length cDNA encoding the ATP synthase was isolated through RT-PCR. This gene was mapped to the short arm of group 6 chromosomes of wheat. This is a housekeeping gene but does not express in the anthers of the sterile plants. Its encoding product contains a putative mitochondrial targeted signal peptide. The amino acid sequences of ATP synthases in plants are highly conservative.