番椒細胞質雄不稔品系之DNA鑑定與相關基因之研究

Study on Identifying Cytoplasmic Male Sterility (CMS) by DNA Marker and Associated CMS Genes in Pepper

利用雄不稔性狀DNA鑑定標誌，於苗期輔助雄不稔育種篩選，可縮短育種時程。本試驗利用番椒細胞質雄不稔相關蛋白基因序列（DQ116040, ORF456）設計引子組CMSP1、CMSP2，以番椒第三級花蕾cDNA為模版，CMSP1為引子，可於種苗改良繁殖場培育之核質共作雄不稔品系擴增出預期的402 bp雄不稔專一性條帶。此外，利用CMSP1也可鑑定其他蒐集自亞蔬－－世界蔬菜中心、農業試驗所以及種苗改良繁殖場的其他番椒品系的雄不稔細胞質。以CMSP2為引子時，除350bp之預期片段外，又擴增出約1200 bp及1500 bp兩明顯雄不稔專一性片段，經選殖及定序分析，結果顯示這兩片段由粒線體coxIⅡ基因（DQ126683.1；Cap. coxⅡ）後段及番椒細胞質雄不稔相關蛋白基因前段序列所組成；雖然引子不在序列兩端，但此二雄不稔專一性條帶可穩定的被擴增出來。此外，根據番椒atp6-2基因所設計之引子組ATP-1、ATP-2及ATP3，也都可於番椒細胞質雄不稔品系正確擴增出預期大小的雄不稔專一性條帶。雖然，引子組的標的基因為粒線體基因，由於PCR僅需微量模版DNA，檢定上苗期葉片DNA萃取時的殘餘粒線體DNA即足以穩定擴增出雄不稔專一性條帶，因此既可在苗期早期鑑定，也可省去複雜的粒線體DNA純化步驟，提高了此檢測技術之應用性。 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. In this study, CMSP1 and CMSP2 primer sets were designed according to Capsicum annuum cytoplasmic male sterilityassociated protein gene (DQ116040, ORF456). A 402bp DNA fragment specific to pepper male-sterile cytoplasm could be successfully amplified from cDNA of flower buds(3-5mm in length)of the TSIPS male-sterile pepper lines by CMSP1 primers. Moreover, such specific PCR product could be amplified by CMSP1 from other male-sterile pepper lines, including lines developed from AVRDC-TWVC, ARI, and TSIPS. Using CMSP2 primer set, two additional reproductive PCR products(1200bp, 1500bp)besides the expected 350bp fragment were amplified and cloned from the male-sterile lines. The result of sequence alignment indicated that such clones were composed of sequences of coxⅡ gene (DQ126683.1；Cap. coxⅡ ) and cytoplasmic male sterilityassociated protein gene. Interestingly, although their sequences contained no primer sequences, these fragments were stably produced. Besides CMSP1 and CMSP2, ATP-1, ATP-2 and ATP-3 primer sets designed according to pepper atp6-2 gene sequence could also produce reliable marker specific to pepper cytoplasmic male sterility lines. Moreover, the residual mitochondrial DNA in DNA extracts of seedling leaves was adequate for PCR amplification by these primer sets, which means the laborious mitochondria DNA extraction may not be necessary.