覆盆子与其相似品种鉴别的分子标记筛选与评价研究

Study on molecular marker screening and evaluation of Rubi Fructus and its analogues

目的利用ITS2和matK分子标记对覆盆子及其相似品种进行鉴别,规范覆盆子的基源。方法收集覆盆子及其相似品山莓、蓬蘽、茅莓、寒莓、高粱泡的ITS和matK序列,经隐马尔可夫模型去除两端的5.8S和28S序列,共得到ITS2序列25条,经Clustal校对共获得matK序列22条。运用MEGA软件分析各分类群的ITS2和matK序列,计算种内和种间遗传距离,构建邻接系统进化树(NJ);通过ITS2 database预测各分类群的ITS2二级结构,采用4Sale软件比对二级结构,通过ProfDistS软件构建基于联合ITS2一级序列及其二级结构的剖面邻接(PNJ)系统发育树。结果分别基于ITS2和matK标记的不同物种种间具有显著的遗传间隔。NJ树和PNJ树拓扑关系一致,各分类群表现出单系性。覆盆子ITS2二级结构与其相似品种均具有显著差异。结论建议ITS2和matK序列均可作为鉴别覆盆子与其相似品种的DNA条形码,ITS2二级结构信息可丰富鉴定结果,可为覆盆子及悬钩子属植物的资源研究及品种选育提供依据。

Objective To standardize the origin of Rubi Fructus by using ITS2 and matK molecular markers to identify Rubi Fructus and its analogues.Methods The ITS and matK sequences of Rubus chingii Hu,Rubus corchorifolius L.f.,Rubus hirsutus Thunb.,Rubus parvifolius L.,Rubus buergeri Miq.and Rubus lambertianus Ser.were amplified and sequenced.The hidden Markov model was used to remove 5.8S and 28S from ITS sequences.A total of 25 ITS2 sequences were obtained.And a total of 22 matK sequences were obtained by proofreading by Glustal software.MEGA software was used for matK and ITS2 sequences analysis,intraspecific and interspecific genetic distances caculation,and neighbor joining(NJ)phylogenetic tree construction.ITS2 secondary structure was predicted by ITS2 database and aligned by 4Sale software.Profile neighbor-joining(PNJ)phylogenetic tree was constructed based on combined ITS2 sequence and its secondary structure by ProfDistS software.Results An obvious barcoding gap between Rubi Fructus and its analogues was showed.The topological relationship between NJ tree and PNJ tree was consistent,and each taxon exhibits monophyly.The ITS2 secondary structure of Rubi Fructus was significant different from its analogues.Conclusions It is recommended that both ITS2 and matK markers can serve as DNA barcodes for identifying Rubi Fructus and its analogues.The addition of ITS2 secondary structure information can enrich the identification results and provide theoretical support for resource research and variety selection of Rubi Fructus.