開發適用於鑑別不同地理來源水稻品種之SSR DNA 分子標誌

Abstract

由於DNA分子標誌技術蓬勃發展，在品種鑑別具有高度潛力，國際種子檢查協會欲建立分別適用於全世界的玉米、小麥、水稻和大豆品種之鑑別技術。以往水稻 (Oryza sativa L.) 品種鑑別分子技術的研究，多是針對一個特定區域或國家中的品種所建立。本研究利用192個不同地理來源的水稻品系進行分析，篩選出在品種鑑別具有潛力的簡單重複序列分子標誌。本研究中使用54組SSR分子標誌，使用螢光標定多重PCR組合方式，分成11個組合，進行毛細管電泳。初步篩選排除增幅效果弱，或是由於嚴重的加一個鹼基效應使資料不易判讀之6組分子標誌後，剩下的48組SSR分子標誌，依據選擇無連鎖、資料易於判讀、沒有無對偶基因現象和PIC值較高者的原則下，共選出12組分子標誌。選出的12組分子標誌最少具有5個對偶基因，最多有26個對偶基因，共產生153個對偶基因，平均每個基因座具有12.8個對偶基因；PIC值範圍由0.64至0.90，平均PIC值為0.79。以此12組分子標誌對於192個水稻品系進行主成分分析與群集分析。主成分分析的結果，前三個主成分的解釋量分別為：17.7%、6.44% 和5.41%。主成分分析與群集分析結果相似，分為兩大群集。此次分析的品系分為兩群，台灣的稉稻 (japonica) 與4個義大利稉稻品種皆屬同一群，總共包含56個品系。台灣的3個秈稻 (indica) 品種則屬另一較大的群集，共有136個品系。較大的群集中，除了在第一及第二主成分上的差異較小群集大之外，在第三主成分上也有很大的差異，表示此群集中各品系的差異較大。無法由此12組SSR標誌分開的品系，共有27對，包含39個品系。而最佳組合分析結果顯示，只要選對分子標誌，即使將分子標誌數目減至9個標誌，無法解開的品系對數與使用全部12個誌時相同，有27對，對於解析力沒有影響。

Molecular markers are potentially useful for crop variety identification. International Seed Testing Association is currently developing DNA-based markers system of variety identification for four major crops including Zea mays (maize), Oryza sativa (rice), Triticum durum and Triticum aestivum (wheat), and Glycine max L Mer. (soybean). Previous researches conducted on rice variety identification focused on particular regions or countries. The object of this study was to select simple sequence repeat (SSR) markers from 54 fluorescently-labeled SSR markers in 11 multiplex panels for rice, using 192 lines originated from geographically diverse regions. After excluding 6 markers which were poorly amplified or difficult to score due to strong stutter with incomplete effect of adding terminal adenine, a group of 12 unlinked makers were selected based on their easiness to score, absent of null alleles, and polymorphic information content (PIC) value. Numbers of alleles per locus of the selected markers ranged from 5 to 26, with an average of 12.8. PIC values ranged from 0.64 to 0.9, with an average of 0.79. Principal coordinate analysis (PCoA) result of the 192 rice lines showed that there were two groups. All Taiwan japonica varieties and four Italian varieties were assigned to the smaller group, which consist of 56 lines. The larger group contained the other 136 lines, including three Taiwan indica varieties. The first three dimensions account for 17.7%, 6.44% and 5.41% of total variability respectively. The larger group was more diverse on the third dimension, indicating its higher genetic diversity. Cluster analysis result was similar to PCoA result. There were 27 unresolved pairs, including 39 lines, by using these 12 selected markers. The best combination analysis indicated that if choose properly, the number of markers may be further reduced to 9 with no effect on the resolution