番椒雄不稔性系統相關分子標誌之鑑識

Abstract

雄不稔性應用於生產番椒一代雜交種子有許多優點，若能搜尋到雄不稔性相關分子標誌則能增進育種效率。TSIPS(行政院種苗改良繁殖場)所提供之穩定雄不稔性番椒材料經性狀調查及分析顯示：同質顯性結合則為雄不稔株，其稔性分離符合孟德爾遺傳律，並為一對對偶基因所控制。以RAPD-BSA方式針對材料之稔性分類後進行分析，共篩選508個隨機引子，但無法獲得具潛力之雄不稔性分子標誌，前人所述具潛力之OPP13及OPW19引子亦未能增幅出預期條帶。 在特殊引子設計的試驗結果方面，以CRF-S引子對TSIPS及AVRDC(世界-亞洲蔬菜研究中心)試驗材料進行單株檢定，除AVRDC的5032號品系之外，其餘材料皆能分別在850bp及950bp處增幅出稔性恢復基因Rf及rf之專一性條帶，証明此分子標誌具共顯性、專一性及穩定性，具潛力成為篩選雄不稔性番椒的工具。由於CRF-S引子的增幅產物為稔性恢復基因的專一性條帶，故可推測TSIPS試驗材料屬核質互作型雄不稔(G-CMS)。而由不稔細胞質atp6-2及orf456基因為基礎所設計的引子CMS P1、CMS P2、Atp1、Atp2、Atp3則能在TSIPS及AVRDC參試材料中分別增幅出400bp、350bp、650bp、550bp、420bp的不稔細胞質專一性條帶，試驗結果除顯示此五引子具高專一性及穩定性之外，亦證明TSIPS參試材料具不稔細胞質，屬於G-CMS型雄不稔。綜合試本研究中的六組特殊引子，可廣泛用於多種來源的材料，並正確的早期辨認各種雄不稔性基因型，具應用於番椒雄不稔性育種之潛力。

Abstract The identification molecular marker relevant to male sterility is very important in breeding program. Precise prediction of male sterility at seedling stage can certainly shorten the breeding protocol and schedule. According to the segregation ratio, the male sterility of the peppers form TSIPS (Taiwan Seed Improvement and Propagation Station) was controlled by a single gene. RAPD-BSA (random amplified polymorphic DNA - bulked segregant analysis) was used to identify potential markers; however, none of the 508 tested primers was able to identify either male sterile or fertile bulk including OPP13 and OPW19. Primer sets that designed according to the mitochondria apt6-2 gene and male sterility related protein ORF456 were able to identified the male sterile lines indicated that the tested TSIPS pepper lines contain male-sterile cytoplasm. The CRF-S primer set was reported to be specific to the nuclear fertility restorer gene, Rf, in hot pepper; such primers set was able to identified the male fertile progenies of the male sterile lines indicating that the material contained Rf gene. Therefore, the male sterility of the material belongs to genic-cytoplasmic male sterility system. In this study, other male sterile lines were collected from TSIPS and AVRDC (the World Vegetable Center) to test for the discriminate ability of the markers to male-sterility. The result of our study indicated that the primer sets for the male sterile cytoplasm were able to identify all the lines and the CRF-S primer could discriminate the Rf_ and rfrf genotype from all the collected lines except AVRDC-5032. Therefore, we concluded that the primer sets we used could be potentially applied in pepper breeding program.