| As one of the effective ways to improve crop yield,heterosis utilization has been widely used in hybrid breeding of rice,corn,rapeseed and other crops.Soybean,the crop for both oil and grain,is a natural cleistogamic crop and has the character of flowering and podding in leaf axils.The emergence of cytoplasmic male sterility/restorer-of-fertility（CMS/Rf）system of soybean realized the application of heterosis technology in soybean.At present,the utilization of heterosis in soybean is mostly realized by"three-line method".In view of the large candidate interval and a large number genes contained in the interval of cytoplasmic male sterility（CMS）restorer-of-fertility gene Rf3,the gene cloning and preliminary identification of Rf3 was carried out.This study not only has the theoretical value for the study of the interaction mechanism of soybean CMS/Rf system,but also has important practical significance for the selection and breeding of restorer lines.In this study,soybean RN-type cytoplasmic male sterile line JLCMS5A and restorer line JLR2 were selected as parents and used to construct F3 fertility isolation population.Furthermore,the fertility restorer gene Rf3 was fine mapped,and genes in the candidate interval were cloned and preliminary identified by gene annotation.The main conclusions are as follow:（1）Our team preliminary mapped CMS restorer gene Rf3 between molecular markers d CAPS 09-2 and BARCSOYSSR＿09＿1178 on chromosome 9.On this basis,the linkage map of the fertility restorer gene which contained in the restorer line JLR2 was established by Join Map 4.0 through combining SSR（Simple Sequence Repeat）,In Del（Insertion-Deletion）and d CAPS（Derived Cleaved Amplified Polymorphic Sequence）markers.Then the corresponding candidate interval of the fertility restorer gene on chromosome 9 was determined.The Rf3 gene was fine mapped between molecular marker d CAPs 09-2 and BARCSOYSSR＿09＿1170.The physical map of fertility restorer gene Rf3 was constructed by Map Chart and the physical mapping range was 86.433 Kb.There are 10 genes in the candidate interval:from Glyma.09G171100 to Glyma.09G172000.Moreover,functional annotation was performed on these genes which in the candidate interval.The result showed that Glyma.09g171200 encodes pentatricopeptide repeat（PPR）and has the characteristics of CMS fertility restorer gene in plants.（2）The 10 genes in candidate interval were cloned and sequenced.Through the consistency analysis by using DNAMAN software,only gene Glyma.09G171200 and Glyma.09G171800 had multiple single nucleotide polymorphism（SNP）sites.There are 47SNPs in the coding sequence（CDS）of Glyma.09G171200,including 38 non-synonymous mutations and 9 synonymous mutations.Non-synonymous mutations causing the variation of coding amino acids between parents.As for Glyma.09G171800,there are 2 SNPs in the coding region including 1 non-synonymous mutation and 1 synonymous mutation.（3）q RT-PCR was used to identify the expression level of Glyma.09G171200 and Glyma.09G171800 in five different organs of soybean including root,stem,leaf,bud and flower.Moreover,the gene expression level was higher in all detective organs of restorer line JLR2,especially in the stem,leaf,and flower organs.It is speculated that the Glyma.09G171200specific coding orf contained in JLR2 is a candidate Rf3 gene.（4）A vector which used for plant expression fused with the Rf3 candidate gene and green fluorescent protein（GFP）was constructed.Then the subcellular localization of Glyma.09G171200 was performed.It could be found that the fluorescent signal of the recombinant vector p CAMBIA1302:Rf3-GFP was only appeared in mitochondria.The result indicated that the protein encoded by the Rf3 candidate gene was located in mitochondria and possessed the characteristics of restorer gene.（5）Cleaved Amplified Polymorphic Sequence（CAPS）molecular marker,CAPS1712 and CAPS1718,closely linked to Rf3 gene were developed by using SNPs of Glyma.09G171200and Glyma.09G171800.Combined with CAPS 1712 marker and restriction enzyme Sca I,the male parent could be cut into 897 bp and 1204 bp fragments,but the female parent could not be cut.Combined with CAPS 1718 maker and restriction enzyme Bsp EI,the male parent could be cut into 604 bp and 1330 bp fragments,but the female parent material could not be cut.The above markers can be used for molecular marker assisted breeding of materials containing Rf3. |
