基于DNA条形码psbA-trnH,matK,trnL对不同地理居群野菊和药用菊的鉴定研究
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摘要
利用DNA条形码技术通过psbA-trnH,mat K,trn L序列分析,建立一种快速鉴定野菊和药用菊的方法。提取21份样品的基因组DNA,对psbA-trnH,mat K,trn L片段进行PCR扩增,双向测序,获得其psbA-trnH,mat K,trn L序列信息;运用MEGA 7. 0对所有样品的psbA-trnH,mat K,trn L序列共63条进行比对、计算种内种间遗传距离,分析psbA-trnH+mat K+trn L组合序列SNPs分布,构建Neighbor-Joining(NJ)系统进化树。结果显示,野菊,野菊(菊花脑)和药用菊的种内种间遗传距离重合; psbA-trnH+mat K+trn L组合序列的SNPs分析显示其组合序列共有19个核苷酸多态性位点(SNPs),野菊较野菊(菊花脑)、药用菊呈现更为明显的序列多态性,psbA-trnH序列表现出较为明显的长度变异; NJ树显示药用菊与野菊、野菊(菊花脑)区别开来,其中编号为C2～C5的药用菊单独聚为一亚支,置信度为62%,编号为Z9,Z10的野菊均采自甘肃省,单独聚为一支,置信度为77%;同时也发现来自西北地域的野菊样品,其psbA-trnH和trn L序列信息变化与地域和环境之间有明显的相关,野菊与野菊(菊花脑)具有明显的分化,同时还是药用菊的进化来源。因此,将psbA-trnH+mat K+trn L组合序列作为DNA条形码能准确、快速的鉴别野菊和药用菊,为其种质资源鉴定及物种鉴别提供了重要依据。
DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum and Ch. morifolium based on psbA-trn H,mat K and trn L sequences. The total DNA was extracted from 21 samples collected,and the psbA-trn H,mat K,trn L sequences were amplified by PCR and sequenced. The information of these sequences were obtained. We aligned all 63 sequences,calculated the intraspecific and interspecific distances,analysed the SNPs distribution of psbA-trn H+mat K+trn L combination sequences and constructed the Neighbor-joining( NJ) Tree,using MEGA 7. 0. The results showed that the genetic distances of Ch. indicum,Ch. indicum( Juhuanao)and Ch. morifolium were overlapped. The SNPs analysis of psbA-trn H+mat K+trn L combination sequences showed that there were 19 nucleotide polymorphism loci( SNPs) and nine parsim-informative sites in the combination sequences. In addition,Ch. indicum showed more obvious sequence polymorphism than those of Ch. indicum( Juhuanao) and Ch. morifolium. The psbA-trn H sequences showed obvious length variation.The NJ Tree showed that Ch. morifolium numbered C2-C5 were clustered into a single subbranch with a bootstrap value of 62%,and Ch.morifolium could be distinguished from Ch. indicum and Ch. indicum( Juhuanao). Moreover,Ch. indicum numbered Z9 and Z10 collected from Gansu province were singly clustered into one branch with a bootstrap value of 77%. It was also found that the changes of psbA-trn H and trn L sequences information of Ch. indicum samples from the northwest were obviously related to the geography and environment. Moreover,Ch.indicum and Ch. indicum( Juhuanao) had obvious differentiation,were also regarded as the evolutionary sources of Ch. morifolium. Therefore,psbA-trn H+mat K+trn L combination sequences as DNA barcode can identify Ch. indicum and Ch. morifolium accurately and rapidly,which provides an important basis for germplasm resources identification and species identification.
DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum and Ch. morifolium based on psbA-trn H,mat K and trn L sequences. The total DNA was extracted from 21 samples collected,and the psbA-trn H,mat K,trn L sequences were amplified by PCR and sequenced. The information of these sequences were obtained. We aligned all 63 sequences,calculated the intraspecific and interspecific distances,analysed the SNPs distribution of psbA-trn H+mat K+trn L combination sequences and constructed the Neighbor-joining( NJ) Tree,using MEGA 7. 0. The results showed that the genetic distances of Ch. indicum,Ch. indicum( Juhuanao)and Ch. morifolium were overlapped. The SNPs analysis of psbA-trn H+mat K+trn L combination sequences showed that there were 19 nucleotide polymorphism loci( SNPs) and nine parsim-informative sites in the combination sequences. In addition,Ch. indicum showed more obvious sequence polymorphism than those of Ch. indicum( Juhuanao) and Ch. morifolium. The psbA-trn H sequences showed obvious length variation.The NJ Tree showed that Ch. morifolium numbered C2-C5 were clustered into a single subbranch with a bootstrap value of 62%,and Ch.morifolium could be distinguished from Ch. indicum and Ch. indicum( Juhuanao). Moreover,Ch. indicum numbered Z9 and Z10 collected from Gansu province were singly clustered into one branch with a bootstrap value of 77%. It was also found that the changes of psbA-trn H and trn L sequences information of Ch. indicum samples from the northwest were obviously related to the geography and environment. Moreover,Ch.indicum and Ch. indicum( Juhuanao) had obvious differentiation,were also regarded as the evolutionary sources of Ch. morifolium. Therefore,psbA-trn H+mat K+trn L combination sequences as DNA barcode can identify Ch. indicum and Ch. morifolium accurately and rapidly,which provides an important basis for germplasm resources identification and species identification.
引文
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[23]孙春青,陈发棣,房伟民,等.野菊与菊花杂交中花粉活力和柱头可授性及胚胎发育研究[J].西北植物学报,2009,29(7):1335.
[24]夏至,李家美,张红瑞,等.中药材菊花和野菊花的DNA条形码鉴定研究[J].时珍国医国药,2013,24(8):1902.
[25] Hollingsworth P M,Li D Z,Michelle V D B,et al. Telling plantspecies apart with DNA:from barcodes to genomes[J]. PhilosTrans R Soc Lond B Biol Sci,2016,371(1702):20150338.
[26] Fang H L,Guo Q S,Shen H J,et al. Phylogeography of Chrys-anthemum indicum L.(Compositae)in China based on trnL-Fsequences[J]. Biochem Syst Ecol,2010,38(6):1204.
[27]韩建萍,李美妮,罗焜,等.DNA条形码鉴定洋金花及其伪品[J].药学学报,2011(11):1408.
[28] Fang H L,Guo Q S,Shen H J,et al. Genetic diversity evalua-tion of Chrysanthemum indicum L. by medicinal compounds andmolecular biology tools[J]. Biochem Syst Ecol,2012,41:26.
[29]戴思兰,李文彬.菊花起源的RAPD分析[J].植物学报,1998,40(11):1053.
[2]中国药典.一部[S].2015.
[3]钟赣生.中药学[M].北京:中国中医药出版社,2012:114.
[4] Wang T,Guo Q S,Mao P F. Flavonoid accumulation during flo-rescence in three Chrysanthemum morifolium Ramat cv.'Hangju'genotypes[J]. Biochem Syst Ecol,2014,55:79.
[5] Wang T,Zhu Z B,Guo Q S,et al. Variation in major flavonoidsglycosides and caffeoylquinic acids during florescence of threeChrysanthemum morifolium Ramat cv.'Hangju'genotypes[J].Biochem Syst Ecol,2013,47:74.
[6]刘丽,郭巧生,徐文斌.药用菊花不同栽培类型植物学形态比较[J].中国中药杂志,2008,33(24):2891.
[7]徐文斌,郭巧生,王长林,等.药用菊花遗传多样性的RAPD分析[J].中国中药杂志,2006,31(1):8.
[8]吕琳,秦民坚,贺丹霞,等.不同种源药用菊花、野菊和菊花脑的ISSR分子标记及遗传关系分析[J].植物资源与环境学报,2008,17(1):7.
[9] Shao Q S,Guo Q S,Deng Y M,et al. A comparative analysis ofgenetic diversity in medicinal Chrysanthemum morifolium basedon morphology,ISSR and SRAP markers[J]. Biochem SystEcol,2010,38(6):1160.
[10] Cheng X,Chen S M,Chen F D,et al. Interspecific hybrids be-tween Dendranthema morifolium(Ramat.)Kitamura and D.nankingense(Nakai)Tzvel. achieved using ovary rescue andtheir cold tolerance characteristics[J]. Euphytica,2010,172(1):101.
[11]付涛,胡仲义,何月秋,等.基于cpDNA条形码鉴定铁皮石斛种质资源[J].核农学报,2017,31(2):255.
[12]丁鸽,张代臻,丁小余.石斛资源分子水平研究进展[J].中国实验方剂学杂志,2018,24(4):208.
[13] Nithaniyal S,Parani M. Evaluation of chloroplast and nuclearDNA barcodes for species identification in Terminalia L.[J].Biochem Syst Ecol,2016,68:223.
[14]张玉霄,许宇星,马朋飞,等.竹亚科叶绿体DNA条形码筛选[J].植物分类与资源学报,2013,35(6):743.
[15]谭秀芳,杜向红,牛善策,等.基于叶绿体序列分析紫茎泽兰在菊科的系统发育[J].西北农业学报,2011,20(4):138.
[16]何维颖,潘跃芝.菊科橐吾属植物的DNA条形码研究[J].植物分类与资源学报,2015,37(6):693.
[17] Stech M,Frey W. A morpho-molecular classification of the mos-ses(Bryophyta)[J]. Nova Hedwigia,2008,86(2):1.
[18] Kumar S,Stecher G,Tamura K. MEGA 7:Molecular Evolution-ary Genetics Analysis Version 7. 0 for Bigger Datasets[J]. MolBiol Evol,2016,33(7):msw054.
[19] Saitou N,Nei M. The neighbor-joining method:a new methodfor reconstructing phylogenetic trees[J]. Mol Biol Evol,1987,4(4):406.
[20] Felsenstein J. Confidence limits on phylogenies:an approachusing the bootstrap[J]. Evolution,1985,39(4):783.
[21]刘思余,张飞,陈素梅,等.四倍体菊花脑与栽培菊种间杂交及F_1杂种的遗传表现[J].中国农业科学,2010,43(12):2500.
[22] Li J,Wan Q,Yan P G,et al. Should I stay or should I go:bio-geographic and evolutionary history of a polyploid complex(Chrysanthemum indicum complex)in response to pleistoceneclimate change in China[J]. New Phytologist,2014,201(3):1031.
[23]孙春青,陈发棣,房伟民,等.野菊与菊花杂交中花粉活力和柱头可授性及胚胎发育研究[J].西北植物学报,2009,29(7):1335.
[24]夏至,李家美,张红瑞,等.中药材菊花和野菊花的DNA条形码鉴定研究[J].时珍国医国药,2013,24(8):1902.
[25] Hollingsworth P M,Li D Z,Michelle V D B,et al. Telling plantspecies apart with DNA:from barcodes to genomes[J]. PhilosTrans R Soc Lond B Biol Sci,2016,371(1702):20150338.
[26] Fang H L,Guo Q S,Shen H J,et al. Phylogeography of Chrys-anthemum indicum L.(Compositae)in China based on trnL-Fsequences[J]. Biochem Syst Ecol,2010,38(6):1204.
[27]韩建萍,李美妮,罗焜,等.DNA条形码鉴定洋金花及其伪品[J].药学学报,2011(11):1408.
[28] Fang H L,Guo Q S,Shen H J,et al. Genetic diversity evalua-tion of Chrysanthemum indicum L. by medicinal compounds andmolecular biology tools[J]. Biochem Syst Ecol,2012,41:26.
[29]戴思兰,李文彬.菊花起源的RAPD分析[J].植物学报,1998,40(11):1053.
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