羊草叶绿体非编码区多态性标记筛选及群体遗传多样性
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摘要
为筛选多态性丰富的羊草叶绿体非编码区片段,本研究以9个不同地理位置的羊草居群为材料,通过对12个叶绿体非编码区DNA片段测序及其序列间变异分析试图从中找出有遗传差异的叶绿体DNA(cpDNA)片段,并利用有多态性的cpDNA片段进行遗传多样性分析。结果表明,序列ndhF-rpl32、trnL-trnF、trnC-ycf6、aptI-aptH具有比较丰富的变异,可作为下一步研究野生羊草群体遗传学和谱系地理学较为理想的分子标记。在37个羊草个体4条非编码区片段的合并序列中,共检测到15种单倍型,单倍型多态性(Hd)为0.928,核苷酸多态性(Pi)为0.00101;遗传分化指数(Fst)为0.58884,基因流(Nm)为0.17,基因流较小;中性检验Tajima’s D(-1.08542)和Fu’s Fs(-5.301)均为负值,且差异不显著(P>0.10),推测羊草遵循中性进化理论,可能经历过种群扩张;AMOVA结果显示,65%的分子变异出现在居群间,35%出现在居群内;Mantel检验得到,遗传距离与地理距离具有显著相关性(r=0.449,P<0.05);居群分化值Nst 0.386(0.1865)大于Gst 0.234(0.1506),差异显著(P<0.05),表明羊草居群存在分子谱系地理结构。通过系统发育树分析得到,羊草15个单倍型分为两大分支,H2和H10聚为一支,它们与别的居群个体亲缘关系较远,其余单倍型聚为另一支;单倍型网络图显示,H2和H12、H10和H12亲缘关系较远,与系统发育树分析结果基本一致。本研究为羊草的种质资源保护、谱系地理学研究等工作奠定了基础。
The aim of this research was to screen for and classify polymorphic loci of chloroplast non-coding fragments in Leymus chinensis.The study evaluated 9 L.chinensis populations from different geographical locations to identify polymorphic loci from 12 chloroplast DNA(cpDNA)non-coding fragments,and then genetic diversity of the polymorphic cpDNA fragments was analyzed.The results showed that the polymorphic loci of ndhF-rpl32,trnL-trnF,trnC-ycf6 and aptI-aptH were rich,and they had a relatively fast evolution rate.Hence,they could be regarded as ideal molecular markers for future research on population genetics and phylogeography.Based on combined sequences there were 15 haplotypes identified.The haplotype diversity(Hd)and nucleotide diversity(Pi)were 0.928 and 0.00101 respectively.The genetic differentiation coefficient Fst was 0.58884 with the low gene flow(Nm=0.17).Tajima's Dand Fu's Fs values of neutrality tests were negative(Tajima's D=-1.0854;Fu's Fs=-5.301),indicating that L.chinensis had experienced historical population expansion.Analysis of molecular variance showed that most of genetic variation was distributed among populations.The Mantel test showed a significantly positive correlation between genetic and geographical distance(r=0.449,P<0.05).The genetic differentiation coefficients Nst(0.386)>Gst(0.234)implied a significant phylogeographic structure for the populations.The neighbor-joining tree based on combined sequences showed that all haplotypes divided into two major clades,H2 and H10 clustered into a category.The network for cpDNA haplotypes based on combined sequence data showed that the relationships among H2 and H12,and H10 and H12 were further than others,which was similar to the pattern identified by the neighbor-joining tree.These results will be useful for future research on germplasm resources and phylogeography.
The aim of this research was to screen for and classify polymorphic loci of chloroplast non-coding fragments in Leymus chinensis.The study evaluated 9 L.chinensis populations from different geographical locations to identify polymorphic loci from 12 chloroplast DNA(cpDNA)non-coding fragments,and then genetic diversity of the polymorphic cpDNA fragments was analyzed.The results showed that the polymorphic loci of ndhF-rpl32,trnL-trnF,trnC-ycf6 and aptI-aptH were rich,and they had a relatively fast evolution rate.Hence,they could be regarded as ideal molecular markers for future research on population genetics and phylogeography.Based on combined sequences there were 15 haplotypes identified.The haplotype diversity(Hd)and nucleotide diversity(Pi)were 0.928 and 0.00101 respectively.The genetic differentiation coefficient Fst was 0.58884 with the low gene flow(Nm=0.17).Tajima's Dand Fu's Fs values of neutrality tests were negative(Tajima's D=-1.0854;Fu's Fs=-5.301),indicating that L.chinensis had experienced historical population expansion.Analysis of molecular variance showed that most of genetic variation was distributed among populations.The Mantel test showed a significantly positive correlation between genetic and geographical distance(r=0.449,P<0.05).The genetic differentiation coefficients Nst(0.386)>Gst(0.234)implied a significant phylogeographic structure for the populations.The neighbor-joining tree based on combined sequences showed that all haplotypes divided into two major clades,H2 and H10 clustered into a category.The network for cpDNA haplotypes based on combined sequence data showed that the relationships among H2 and H12,and H10 and H12 were further than others,which was similar to the pattern identified by the neighbor-joining tree.These results will be useful for future research on germplasm resources and phylogeography.
引文
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[20]Silva C,Snak C,Schnadelbach A S,et al.Phylogenetic relationships of Echinolaena and Ichnanthus within Panicoideae(Poaceae)reveal two new genera of tropical grasses.Molecular Phylogenetics and Evolution,2015,93:212-233.
[21]Dong Z Z,Fan X,Sha L N,et al.Phylogeny and differentiation of the St genome in Elymus L.sensu lato(Triticeae;Poaceae)based on one nuclear DNA and two chloroplast genes.BMC Plant Biology,2015,15(1):179-192.
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[29]Wright S.Evolution and the genetics of populations.London:Chicago University of Chicago Press,1968.
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[31]Chen L L,Peng G Z,Zhang W L,et al.The significance of mutations in genomic evolution.Hereditas,2006,28(5):631-638.陈玲玲,彭贵子,张伟丽,等.突变在基因组进化中的意义.遗传,2006,28(5):631-638.
[32]Timme R E,Kuehl J V,Boore J L,et al.A comparative analysis of the Lactuca and Helianthus(Asteraceae)plastid genomes:Identification of divergent regions and categorization of shared repeats.American Journal of Botany,2007,94(3):302-312.
[33]Liu J,Zhang H Q,Fan X,et al.Phylogenetic relationships and maternal donor of Hystrixand Leymus species as revealed by chloroplast atpB-rbcLsequence.Acta Prataculturae Sinica,2012,21(5):77-85.刘静,张海琴,凡星,等.基于叶绿体atpB-rbcL序列探讨猬草属和赖草属植物的系统发育和母系起源.草业学报,2012,21(5):77-85.
[34]Xu M P.Genetic diversity of Scutellaria tsinyunensis C.Y.Wu et S.Chow and its related species based on ITS and cp DNAsequence.Chongqing:Southwest University,2016:29.徐梦平.缙云黄芩(Scutellaria tsinyunensis C.Y.Wu et S.Chow)及近缘种基于ITS和cpDNA序列的遗传多样性研究.重庆:西南大学,2016:29.
[35]Liu H F,Gao Y B,Wang D,et al.Genetic differentiation in eight populations of Leymus chinensis in Inner Mongolia Steppe.Acta Ecologica Sinica,2004,24(3):423-431.刘惠芬,高玉葆,王丹,等.内蒙古典型草原羊草种群遗传分化的RAPD分析.生态学报,2004,24(3):423-431.
[36]Ren W W,Qian J.A comparative study on genetic differentiation of Leymus chinensis in different geographic populations.Acta Ecologica Sinica,1999,19(5):689-696.任文伟,钱吉.不同地理种群羊草的遗传分化研究.生态学报,1999,19(5):689-696.
[37]Qian J,Ma Y H,Ren W W,et al.Comparative study on ecotype differentiation of Leymus chinensis in different geographic populations at moleculat level.Acta Ecologica Sinica,2000,20(3):440-443.钱吉,马玉虹,任文伟,等.不同地理种群羊草分子水平上生态型分化的研究.生态学报,2000,20(3):440-443.
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[41]Fu C L,Dai X K,Li X Y,et al.Screening cpDNA markers and preliminary study on the genetic diversity of Hydrilla verticillatafrom China.Plant Science Journal,2017,35(1):87-92.付春霖,戴小康,李小燕,等.黑藻cpDNA分子标记的筛选及其遗传多样性的初步研究.植物科学学报,2017,35(1):87-92.
[2]Liu X,Cui J Z,Zhang J,et al.The genetic diversity and differentiation of Leymus chinensis.Northern Horticulture,2015,(8):182-185.刘欣,崔继哲,张隽,等.羊草的遗传多样性及其分化.北方园艺,2015,(8):182-185.
[3]Hu B Z,Liu D,Hu G F,et al.Morphological variation and genetic diversity in Aneurolepidium chinense.Chinese Journal of Plant Ecology,2001,25(1):83-89.胡宝忠,刘娣,胡国富,等.羊草遗传多样性的研究.植物生态学报,2001,25(1):83-89.
[4]Yasuyuki H,Tomoyuki K,Seiro K,et al.Comparative phylogeography of two bitterlings,Tanakia lanceolata and T.limbata(Teleostei,Cyprinidae),in Kyushu and adjacent districts of Western Japan,based on mitochondrial DNA analysis.Zoological Science,2015,23(4):309-322.
[5]Zhang H,Yan J,Zhang G Q,et al.Phylogeography and demographic history of Chinese black-spotted frog populations(Pelophylax nigromaculata):Evidence for independent refugia expansion and secondary contact.BMC Evolutionary Biology,2008,8(1):1-16.
[6]Badenes M L,Parfitt D E.Phylogenetic relationships of cultivated Prunus species from an analysis of chloroplast DNA variation.Theoretical and Applied Genetics,1995,90(7/8):1035-1041.
[7]Pierre T,Ludovic G,Guy P,et al.Universal primers for amplification of three non-coding regions of chloroplast DNA.Plant Molecular Biology,1991,17(5):1105-1109.
[8]Douglas E S,Pamela S S.Choosing an approach and an appropriate gene for phylogenetic analysis//Molecular Systematics of Plants II.New York:Springer US,1998:1-42.
[9]Albert V A,Backlund A,Bremer K,et al.Functional constraints and rbcLevidence for land plant phylogeny.Annals of the Missouri Botanical Garden,1994,81(3):534-567.
[10]Wu J Z,Wang Q,Zhong X X,et al.Analysis of the genetic structure of Spartina alterniflora populations in China based on cpDNAtrnT-trnFsequences.Acta Prataculturae Sinica,2012,21(4):134-140.吴娟子,王强,钟小仙,等.根据cpDNAtrnT-trnF序列变异分析中国互花米草种群的遗传结构.草业学报,2012,21(4):134-140.
[11]Zhao Y N.Molecular phylogeographic study of Stipa breviflora.Hohhot:Inner Mongolia University,2015:22-24.赵艳宁.基于cpDNA序列的短花针茅的分子谱系地理学研究.呼和浩特:内蒙古大学,2015:22-24
[12]Yang Q S,Xiong Y,Hu L,et al.Cp DNA psbA-trnH sequence characteristics and genetic structure of Sambucus adnata Wall.at different altitudes populations.Biotechnology,2015,25(8):337-345.杨青松,熊勇,胡琳,等.不同海拔血满草居群cpDNApsbA-trnH序列特征及其遗传结构.生物技术,2015,25(8):337-345.
[13]Sun G.Genetic diversity of rbcL gene in Elymus trachycaulus complex and their phylogenetic relationships to several Triticeae species.Genetic Resources&Crop Evolution,2007,54(8):1737-1746.
[14]Liu Z P,Chen Z Y,Pan J,et al.Phylogenetic relationships in Leymus(Poaceae:Triticeae)revealed by the nuclear ribosomal internal transcribed spacer and chloroplast trnL-F sequences.Molecular Phylogenetics and Evolution,2008,46(1):278-289.
[15]Guo G Y,Yang R W,Ding C B,et al.Phylogenetic relationships among Leymus and related diploid genera(Triticeae:Poaceae)based on chloroplast trnQ-rps16sequences.Nordic Journal of Botany,2014,32(5):658-666.
[16]Zhang J,Huang X,Huang D J,et al.Studies on genetic diversity and phylogenetic relationships of limpograss(Hemarthria altissima)and related species based on combined chloroplast DNA intergenic spacer data.Biochemical Systematics and Ecology,2016,69:91-100.
[17]Yamane K,Kawahara T.Intra-and interspecific phylogenetic relationships among diploid Triticum-Aegilops species(Poaceae)based on base-pair substitutions,indels,and microsatellites in chloroplast noncoding sequences.American Journal of Botany,2005,92(11):1887-1898.
[18]Shaw J,Lickey E B,Beck J T,et al.The tortoise and the hare II:Relative utility of 21noncoding chloroplast DNA sequences for phylogenetic analysis.American Journal of Botany,2005,92(1):142-166.
[19]Nomura Y,Shimono Y,Tominaga T.Development of chloroplast DNA markers in Japanese Imperata cylindrica.Weed Research,2015,55(4):329-333.
[20]Silva C,Snak C,Schnadelbach A S,et al.Phylogenetic relationships of Echinolaena and Ichnanthus within Panicoideae(Poaceae)reveal two new genera of tropical grasses.Molecular Phylogenetics and Evolution,2015,93:212-233.
[21]Dong Z Z,Fan X,Sha L N,et al.Phylogeny and differentiation of the St genome in Elymus L.sensu lato(Triticeae;Poaceae)based on one nuclear DNA and two chloroplast genes.BMC Plant Biology,2015,15(1):179-192.
[22]Thomas D C.Phylogenetics and historical biogeography of Southeast Asian Begonia L.(Begoniaceae).Glasgow:University of Glasgow,2010.
[23]Shaw J,Lickey E B,Schilling E E,et al.Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms:The tortoise and the hare III.American Journal of Botany,2007,94(3):275-288.
[24]Xu D H,Ban T.Phylogenetic and evolutionary relationships between Elymus humidus and other Elymus species based on sequencing of non-coding regions of cpDNA and AFLP of nuclear DNA.Theoretical and Applied Genetics,2004,108(8):1443-1448.
[25]DvoǐákováH,Fér T,Marhold K.Phylogeographic pattern of the European forest grass species Hordelymus europaeus:cpDNA evidence.Flora-Morphology,Distribution,Functional Ecology of Plants,2010,205(6):418-423.
[26]Meimberg H,Rice K J,Milan N F,et al.Multiple origins promote the ecological amplitude of allopolyploid Aegilops(Poaceae).American Journal of Botany,2009,96(7):1262-1273.
[27]Qi X S.Biogeographic studies on the genus Platycrater(Hydrangeaceae)endemic to East China and South Japan.Hangzhou:Zhejiang University,2013.祁新帅.东亚特有间断分布植物蛛网萼属的生物地理学研究.杭州:浙江大学,2013.
[28]Tao X L,Wang Y R,Liu Z P.Progress in chloroplast genome analysis of herbage.Pratacultural Science,2015,32(6):978-987.陶晓丽,王彦荣,刘志鹏.牧草叶绿体基因组研究进展.草业科学,2015,32(6):978-987.
[29]Wright S.Evolution and the genetics of populations.London:Chicago University of Chicago Press,1968.
[30]Small R L,Ryburn J A,Cronn R C,et al.The tortoise and the hare:Choosing between noncoding plastome and nuclear Adh sequences for phylogeny reconstruction in a recently diverged plant group.American Journal of Botany,1998,85(9):1301-1315.
[31]Chen L L,Peng G Z,Zhang W L,et al.The significance of mutations in genomic evolution.Hereditas,2006,28(5):631-638.陈玲玲,彭贵子,张伟丽,等.突变在基因组进化中的意义.遗传,2006,28(5):631-638.
[32]Timme R E,Kuehl J V,Boore J L,et al.A comparative analysis of the Lactuca and Helianthus(Asteraceae)plastid genomes:Identification of divergent regions and categorization of shared repeats.American Journal of Botany,2007,94(3):302-312.
[33]Liu J,Zhang H Q,Fan X,et al.Phylogenetic relationships and maternal donor of Hystrixand Leymus species as revealed by chloroplast atpB-rbcLsequence.Acta Prataculturae Sinica,2012,21(5):77-85.刘静,张海琴,凡星,等.基于叶绿体atpB-rbcL序列探讨猬草属和赖草属植物的系统发育和母系起源.草业学报,2012,21(5):77-85.
[34]Xu M P.Genetic diversity of Scutellaria tsinyunensis C.Y.Wu et S.Chow and its related species based on ITS and cp DNAsequence.Chongqing:Southwest University,2016:29.徐梦平.缙云黄芩(Scutellaria tsinyunensis C.Y.Wu et S.Chow)及近缘种基于ITS和cpDNA序列的遗传多样性研究.重庆:西南大学,2016:29.
[35]Liu H F,Gao Y B,Wang D,et al.Genetic differentiation in eight populations of Leymus chinensis in Inner Mongolia Steppe.Acta Ecologica Sinica,2004,24(3):423-431.刘惠芬,高玉葆,王丹,等.内蒙古典型草原羊草种群遗传分化的RAPD分析.生态学报,2004,24(3):423-431.
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