摘要
为了筛选适于鉴定易混淆种矩镰荚苜蓿(Medicago archiducis-nicolai)和花苜蓿(M.ruthenica)的DNA条形码,本研究采集了两近缘种的113个样本,对6个候选条形码序列(通用序列rbcL,psbA-trnH,trnL-trnF,trnK-matK,ITS 2和新序列GA3ox1)进行PCR扩增、测序和序列比对,经过barcoding gap分析、wilcoxn检验以及构建NJ系统发育树评价各序列的鉴定能力。结果显示:6条候选序列的扩增和测序成功率在85%以上;rbcL序列在两近缘种间不存在变异位点,其余5条候选序列各有不同的种内变异和种间变异;5条候选序列的种间最小遗传距离均大于种内最大遗传距离,GA3ox1,ITS2和psbA-trnH序列在种内种间存在明显的“Barcoding Gap”区域;在候选序列构建的NJ系统进化树中,利用GA3ox1和psbA-trnH,矩镰荚苜蓿和花苜蓿均能各自形成单系,但trnL-trnF,t rnK-matK和ITS2不能将两个近缘种区分开。通过分析,我们推荐使用核编码基因GA3ox1作为鉴定矩镰荚苜蓿和花苜蓿的首选序列,叶绿体基因psbA-trnH作为辅助条形码。
In order to screen DNA barcodes suitable for identification of closely related species Medicago archiducis-nicolai and M.ruthenica,in this study,PCR amplification,sequencing and sequence alignment were performed out for six candidate barcode sequences(commonly used sequences rbcL,psbA-trnH,trnL-trnF,trnK-matK,ITS2 and a novel sequence GA3ox1)in 113 samples of both species.The identification ability of different sequences was evaluated by barcoding gap analysis,wilcoxn test and NJ phylogenetic tree.The results showed that the amplification rate of six candidate sequences and the success rate of sequencing were more than 85%.There was no variation site in rbcL sequence between the two related species,but the other five candidate sequences had different intraspecific variation and interspecific variation.The minimum interspecific genetic distance of the five candidate sequences was greater than the maximum interspecific genetic distance for both species.There was a significant“Barcoding Gap”between the intraspecific and interspecific for GA3ox1,ITS2 and psbA-trnH sequences.In the NJ phylogenetic tree constructed with candidate sequences,both M.archiducis-nicolai and M.ruthenica could form individual monophyletic groups respectively by using GA3ox1 and psbA-trnH,but trnL-trnF,trnK-matK and ITS2 could not distinguish the two related species from each other.In this analysis,we recommend the nuclear coding gene GA3ox1 as a preferred sequence for identification between M.archiducis-nicolai and M.ruthenica,and the chloroplast gene psbA-trnH as the auxiliary barcode.
作者
王霞
刘艳
贾秀秀
李永强
方强恩
WANG Xia;LIU Yan;JIA Xiu-xiu;LI Yong-qiang;FANG Qiang-en(College of Pratacultural Sciences,Gansu Agricultural University,Key Laboratory of Pratacultural Ecosystem,Ministry of Education,Sino-US Research Center for Sustainable Development of Grassland Animal Husbandry,Lanzhou,Gansu Province 730070,China)
出处
《草地学报》
CAS
CSCD
北大核心
2023年第8期2323-2333,共11页
Acta Agrestia Sinica
基金
国家自然科学基金项目“花苜蓿越冬芽适应青藏高原寒冷胁迫的分子机制”(31760703),甘肃省高校教师创新基金项目“野生特异种质矩镰荚苜蓿根茎芽抗寒基因筛选与功能分析”(2023A-49)、草业生态系统教育部重点实验室开放课题“青藏高原特有种矩镰荚苜蓿的遗传多样性与分子谱系地理研究”(KLGE202212)资助。
