Exploration Of Differential Genomic Fragments In Near-isogenic Lines Of Male-sterile And Fertile Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge is a kind of perennial herb in the mint family. It distributes inmost areas of China, and it is one of the most famous traditional Chinese medicines, whichhas more than one-thousand-year long clinical history. There are more than100sorts ofactive chemical components isolated and purified from Salvia. The predominant activecomponents are salvianolic acid, tanshinone, and tanshiquinone. Our lab had obtained anAFLP fragment tightly associated with sterile gene in salvia. But the mechanism of malesterile in salvia is still unclear. Thus, successfully cloning full-length relative genes onfertility, as well as localizing them seems to be extremely urgent, which can help us toexplore the machnism of sterility in depth and restore the fertility.In this study, EST-derived microsatellite (EST-SSR) and genome-walking are employedto identify the differences between fertile and sterile strains of salvia. The main results andconclusions are listed as follows:1. Optimal EST sequences of salvia from11747EST sequences in NCBI GENEBANKwere sifted out. Employing optimal EST sequences and Primer5.0, we designed andsynthesized76primer pairs to screen the differences between the sterile and fertile strains.10primer pairs out of them showed stable differences between two strains. Then, two primerpairs were subjected to population test including40individuals, respectively. The resultindicated that there were only two primers pairs（SmSSR56， S019） showed a stabledifference between two strains.2. Taking the differential fragment obtained by AFLP in the previous study as thetemplate, three sense and three anti-sense primers were designed by Primer5.0. Genome-walking method was applied and conducted nest-PCR by using universal primer (AP1, AP2,AP3and AP4), respectively. Then a2028bp gene fragment on sterile phynotype wasidentified. Then this fragment was employed as the template to design a specific pair ofprimer. A2027bp-length sequence was obtained from fertile strains by PCR.3. On-line software NCBI ORF finder and DNAMAN were applied to blast twosequences. The result showed that there were four-base mutations between the sterile andfertile sequences. And all of mutations were located on the intron area.