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基于ITS2序列及其二级结构对矩镰荚苜蓿和近缘种的分子鉴定
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引用本文:王 霞,刘 艳,贾秀秀,方强恩.基于ITS2序列及其二级结构对矩镰荚苜蓿和近缘种的分子鉴定[J].西北农业学报,2024,(3):397~405
DOI:10.7606/j.issn.1004-1389.2024.03.003
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作者单位
王 霞,刘 艳,贾秀秀,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兰州 730070) 
基金项目:国家自然科学基金(31760703);甘肃省高校教师创新基金(2023A-49);草业生态系统教育部重点实验室开放课题(KLGE202212)。
中文摘要:为准确鉴定矩镰荚苜蓿(Medicago archiducis-nicolai)及其近缘种花苜蓿(M.ruthenica )、阔荚苜蓿(M.platycarpos )和毛荚苜蓿(M.edgeworthii ),利用DNA条形码技术对4个物种的ITS2序列进行注释、比对、检验,计算种内种间遗传距离,邻接法(neighbor-joining,NJ)构建系统发育树,通过RNAfold web server预测4个近缘种的ITS2二级结构。结果显示,矩镰荚苜蓿及其近缘种的ITS2序列长度为220~221 bp,稳定性好;种间遗传距离(0.004 55~0.022 73)明显大于种内遗传距离(均为0),存在明显的“Barcoding Gap”区域;NJ系统发育树显示,毛荚苜蓿聚为一支,矩镰荚苜蓿、花苜蓿和阔荚苜蓿聚为另一支,然后矩镰荚苜蓿与阔荚苜蓿又各自形成单系;在二级结构中,矩镰荚苜蓿与阔荚苜蓿、毛荚苜蓿存在明显差异,但与花苜蓿极为相似,难以区分。分析表明:除花苜蓿外,以ITS2序列作为条形码并辅以二级结构,可以达到对矩镰荚苜蓿、阔荚苜蓿和毛荚苜蓿准确、快速鉴定的目的。
中文关键词:矩镰荚苜蓿  ITS2序列  二级结构  分子鉴定
 
Molecular Identification of Medicago archiducis-nicolai and Its Related Species Based on ITS2 Sequence and Its Secondary Structure
Abstract:In order to accurately identify Medicago archiducis-nicolai and its relative species,M.ruthenica,M.platycarpos and M.edgeworthii,the ITS2 sequences of four species were annotated,compared and examined by DNA barcoding,and the genetic distances of intra-and inter-species were calculated,the neighbor-joining phylogenetic tree was constructed,the ITS2 secondary structures of four related species were predicted by RNA fold web server. The results showed that the length of ITS2 sequence of four species was between 220-221 bp and showed good stability. The genetic distance of inter-species (0.004 55-0.022 73) was significantly larger than that of the intra-species (0),and there was an obvious barcoding gap region. The NJ phylogenetic tree showed that M.edgeworthii clustered into one branch,and Medicago archiducis-nicolai,M.ruthenica and M.platycarpos clustered into another branch,then Medicago archiducis-nicolai and M.platycarpos formed monophyletic,respectively. The ITS2 secondary structure of Medicago archiducis-nicolai was obviously different from that of M.platycarpos and M.edgeworthii,but it was very similar to that of M.ruthenica,and difficult to distinguish. The analysis showed that ITS2 sequence as a barcode and supplemented with secondary structure can be accurately and rapidly used for the identification of M.archiducis-nicolai,M.platycarpos and M.edgeworthii exception for M.ruthenica.
keywords:Medicago archiducis-nicolai  ITS2 sequence  Secondary structure  Molecular identification
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