矩镰荚苜蓿与近缘种花苜蓿的DNA条形码筛选

doi:10.11733/j.issn.1007-0435.2023.08.008

草地学报 ›› 2023, Vol. 31 ›› Issue (8): 2323-2333.DOI: 10.11733/j.issn.1007-0435.2023.08.008

矩镰荚苜蓿与近缘种花苜蓿的DNA条形码筛选

王霞, 刘艳, 贾秀秀, 李永强, 方强恩

甘肃农业大学草业学院, 草业生态系统教育部重点实验室, 中-美草地畜牧业可持续发展研究中心, 甘肃兰州 730070

收稿日期:2023-02-20 修回日期:2023-04-07 出版日期:2023-08-15 发布日期:2023-09-01
通讯作者: 方强恩,E-mail:fangqen@163.com
作者简介:王霞(1999-),女,汉族,甘肃会宁人,硕士研究生,主要从事草地生物多样性研究,E-mail:18394151945@163.com
基金资助:
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

Screening of DNA Barcodes for Medicago archiducis-nicolai and Its Relative Medicago ruthenica

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

Received:2023-02-20 Revised:2023-04-07 Online:2023-08-15 Published:2023-09-01

摘要/Abstract

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，ITS₂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，trnK-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。

关键词: 矩镰荚苜蓿, 物种界定, psbA-trnH, GA3ox1, DNA条形码

Abstract: In order to screen DNA barcodes suitable for identification of closely related speciesMedicago 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,ITS2and 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.

Key words: Medicago archiducis-nicolai, Species identification, psbA-trnH, GA3ox1, DNA barcoding

中图分类号:

S326

王霞, 刘艳, 贾秀秀, 李永强, 方强恩. 矩镰荚苜蓿与近缘种花苜蓿的DNA条形码筛选[J]. 草地学报, 2023, 31(8): 2323-2333.

WANG Xia, LIU Yan, JIA Xiu-xiu, LI Yong-qiang, FANG Qiang-en. Screening of DNA Barcodes for Medicago archiducis-nicolai and Its Relative Medicago ruthenica[J]. Acta Agrestia Sinica, 2023, 31(8): 2323-2333.

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矩镰荚苜蓿与近缘种花苜蓿的DNA条形码筛选

Screening of DNA Barcodes for Medicago archiducis-nicolai and Its Relative Medicago ruthenica

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