以多重單一核苷酸多型性基因定型技術建立臺灣稻米品種DNA指紋資料庫及其用於品種純度檢測之可行性研究

Abstract

為提昇國產稻米之競爭力及區隔性並保持稻種純度，除了以外觀、生理或生化特性進行稻種鑑定外，以不同DNA分子標記產生之DNA指紋進行稻米品種鑑定已成為目前常用之分析技術。本研究以過去建立之國內外稻米品種單一核苷酸多型性 (Single nucleotide polymorphisms, SNPs)資料庫，篩選解析度最大之核心單一核苷酸多型性組(core SNP marker set)，發展多重單一核苷酸多型性之基因定型（multiplex SNP genotyping）分析平台以利國、內外稻米品種鑑定。本研究以多重PCR的方式，設計10或15個專一SNP位點之引子對於單一反應中進行多個PCR產物的增幅，再以毛細管電泳或電泳膠片分析產物大小，以確認該些SNP位點之多型性，並與己建立之不同稻米SNP資料庫互相比對進行稻種鑑識。本試驗嘗試以此技術應用於國內外水稻品種DNA資料庫之建立，市售稻米生產過程中容易產生品種混雜階段之檢出，並檢測不同生產階段的稻米混雜比率，作為稻米生產過程中合理混雜可能上限值之探討。台灣現有水稻品種DNA資料庫分析50個品種，結果發現其中21個品種具有一致的DNA指紋表現，19個品種有一主要表現型，而10個品種則具有2-3種主要表現型，顯示同一稻種內之DNA指紋表現均一度不佳，不易建立標準DNA指紋資料庫。另一方面，稻米生產過程中不可避免的混雜比率，若以分析結果最主要之DNA指紋為標準，顯示不論碾米廠規模大小、有無契作，異表現型的比率大致為10%左右。多重單一核苷酸多型性之基因定型技術雖有諸多限制性性，但若應用於鑑定稻米品種大量的刻意摻雜與混充，實為一有效的工具，亦可進一步推廣為國、內外米之檢測，促進稻米產業之健全發展。

In order to increase the competitiveness and marketing segment of Taiwan’s domestic rice, and maintain its genetic purity, the DNA fingerprinting technique which is based on DNA molecular markers for rice varieties identification has become a commonly used platform. In this study, we chose a core SNP marker set from previously established rice SNP database to develop a multiplex-SNP genotyping analysis platform for domestic and foreign rice varieties characterization. Using 10 or 15 sets of allelic-specific SNP primer pairs in the same PCR reaction and analysis PCR amplicons by capillary or regular gel electrophoresis, we were able to distinguish specific SNP loci among various rice cultivars. The DNA fingerprinting generated by multiplex-SNP genotyping analysis from rice grain sample can be further compared with available database to confirm specific rice identity. In this research, first, we attempted to establish an up-dated DNA fingerprinting database of domestic and foreign rice. From the genotyping analysis of 50 domestic rice varieties, we found that 21 varieties showed similar DNA fingerprinting pattern and 19 varieties showed one major type of DNA fingerprinting. However, 10 varieties had 2-3 major types of DNA fingerprinting profile, indicating that the results were too complicated to build up a thorough DNA database. Second, we tried to address the issue related to the assessment of admixture ratio along production processes and set up a standard for tolerance of mixed-rice ratio in rice market. The results showed that the acceptable admixture ratio can be as less as 10%. Multiplex SNP genotyping technique has some limitations, but it can provide a reliable and effective method for characterization and purity test of rice varieties.