山葡萄种质资源DNA条形码通用序列的筛选

Screening of Universal DNA Barcodes for Vitis amurensis

对山葡萄(Vitis amurensis)种质资源样品的ITS、ITS2、psb A-trn H、rbc L和mat K序列进行PCR扩增及测序,优化PCR反应的退火温度,比较各序列的扩增效率、测序成功率、品种间和品种内的差异及barcoding gap图,使用BLAST和NJ树法比较不同序列的鉴定能力,最终从5条DNA片段中筛选出可用于山葡萄种质资源鉴定的DNA条形码通用序列。结果表明,在采集的11份33个山葡萄样品中,psb A-trn H和ITS2序列的扩增与测序成功率较高,其品种间、品种内差异及barcoding gap较ITS、rbc L和mat K序列具有明显的优势,且ITS2序列能够鉴别psb A-trn H序列无法鉴别的品种。实验证明,ITS2和psb A-trn H序列是较适合鉴别山葡萄资源的DNA条形码序列组合。DNA条形码弥补了形态学鉴定的不足,可为山葡萄种质资源的准确鉴定提供科学依据。

To determine the best candidate sequence that can be used as a universal DNA barcode to identify Vitis amurensis varieties, we used PCR amplification with the ideal annealing temperature and sequenced the DNA fragments of five different regions（ITS, ITS2, psb A-trn H, rbc L and mat K） of Amur grape samples. The effective sequence ratios obtained by PCR amplification and sequencing, divergence of intra-and inter-varieties, DNA barcoding gap and identification by using BLAST and NJ tree methods were used to evaluate the discrimination ability of these candidate sequences. ITS2 and psb A-trn H had fairly high amplification efficiency and sequencing efficiency in 33 samples belonging to 11 V. amurensis resources; they had larger divergences of intra-and inter-varieties and better DNA barcoding gaps than the other regions tested（ITS, rbc L and mat K）. In addition, ITS2 could discriminate some varieties that could not be successfully discriminated by the psb A-trn H region. ITS2 and psb A-trn H may be promising DNA barcode region combinations for identifying V. amurensis resources. DNA barcoding can compensate for morphological identification and help in the exact identification of V. amurensis resources.