以簡單重複序列開發水稻品種鑑別系統

Abstract

水稻 (Oryza sativa L.) 為臺灣地區重要的糧食作物，本研究針對水稻提供一套有效的品種純化與品種鑑別工具，以提升國內種苗純度。本研究篩選重複單位為4鹼基對的SSR分子標誌，所建立的套件包含12個SSR分子標誌，分散於12對染色體上。以4種螢光分別標定由3組分子量不同標誌所組成的次群，全部12組SSR分子標誌可於單次的多重PCR反應 (Multiplex PCR) 同步增幅，並於單次的多重毛細管電泳 (Multiplex capillary electrophoresis) 完成基因型分析。分析315個水稻種原得到12組SSR分子標誌的PIC值範圍為0.49至0.84，期望相符率 (Probability of identity) 為1.91x10-7。與以3鹼基為重複單位的SSR分子標誌相較，此套件的痕跡條帶高度明顯降低，整體螢光訊號強弱相當對平均，並未隨標誌分子量增高而有漸次遞減的情形。雖然在水稻種原具有高度的鑑別率，但由於臺灣地區稉稻品種間的高度親緣關係，此套件有18組無法區別的兩兩臺灣品種組合。本套件以12個臺灣水稻品種建立標準品種，可涵蓋53個臺灣品種96.2%的對偶基因型。

Rice (Oryza sativa L.) is the most important food crop in Taiwan. The goal of this study is to develop a set of simple sequence repeat (SSR) markers for rice variety identification. By screening SSRs with four-nucleotide repeat units from the published rice whole genome sequence, we have successfully developed a set of 12 SSR markers that are distributed over the 12 rice chromosomes. The complete set of 12 SSR markers are arranged into 4 non-overlapping groups that are labeled with 4 different fluorophores and may be combined into a multiplex-PCR reaction and analyzed concurrently with multiplex capillary electrophoresis. A total of 315 varieties from a wide geographical area were evaluated with these 12 SSR markers, polymorphism information contents (PICs) of the markers ranged from 0.49 to 0.84. The probability of identity (PID) value for this set is 1.91x10-7. Compared with SSR markers with three-nucleotide repeat units, the set of markers developed in the study have significantly lower stutter ratio and more uniform signal strength. These 12 selected markers having high discriminatory power allow to discriminate all of Taiwan rice varieties except 18 unresolved pairs of Taiwan rice varieties with high kinship. We have also selected 12 Taiwan rice varieties as the reference varieties, which effectively cover 96.2% of the alleles of 53 Taiwan rice varieties.