【摘要】 【目的】利用核糖体DNA(nrDNA)ITS序列和叶绿体DNA(cpDNA)psbA-trnH序列对云南不同地理区域的黄毛草莓进行分子鉴定,并分析不同地理居群间的分子进化及地理分布特征,为黄毛草莓种质鉴定、保护及开发利用提供理论依据。【方法】以云南省12个不同地理区域的黄毛草莓居群样品为材料,PCR扩增其ITS和psbA-trnH序列,并进行双向测序及序列合并,分别基于ITS和psbA-trnH序列及二者合并序列构建系统发育进化树。最后利用DnaSP5.10对ITS和psbA-trnH序列进行核苷酸多样性(π)及单倍型数目、类型和多样性(Hd)分析,并对黄毛草莓居群进行中性检验及分子进化特征分析。【结果】基于ITS和psbA-trnH序列的聚类分析结果均显示,12个不同地理区域的黄毛草莓居群样品均与GenBank数据库中下载的黄毛草莓聚在一个分支上,分支自展值均大于阈值(75%),表明这两种序列均可用于黄毛草莓种质的分子鉴定。基于二者合并序列的聚类分析结果与上述结果基本一致,但分支自展值达99%,表明该聚类分析结果更可靠。不同地理区域的黄毛草莓ITS序列间的遗传距离为0～0.014,排序为迪庆州>昆明市>文山州,psbA-trnH序列间的遗传距离为0～0.025,排序为昆明市>迪庆州>文山州,推测ITS和psbA-trnH序列均能显示黄毛草莓居群遗传分化与地理分布格局的相关性。ITS序列长度为668 bp,变异位点百分率为1.8%,共有8种单倍型,Hd和π分别为0.894±0.078和0.006,以昆明市黄毛草莓样品的单倍型最多,为4种;psbA-trnH序列有203 bp,变异位点百分率为2.5%,psbA-trnH序列共有5种单倍型,Hd和π分别为0.788±0.090和0.009,以昆明市黄毛草莓样品单倍型最多,为3种,表明两种序列的单倍型均呈现地理分布格局。黄毛草莓ITS和psbA-trnH序列的中性检验Tajima’s D值分别为-0.673和0.227(P>0.1),表明云南省12个不同地理区域的黄毛草莓居群保持稳定状态,在截至目前的历史时间内不存在扩张。【结论】从云南不同地理区域采集的样品均为黄毛草莓。ITS序列和psbA-trnH序列均可作为黄毛草莓的DNA条形码,二者的合并序列更能准确鉴定黄毛草莓种,适用于黄毛草莓分子谱系地理学研究。更多还原

【Abstract】 【Objective】The molecular identification of Fragaria nilgerrensis samples in different geographical regions of Yunnan was performed by using ribosomal DNA(nrDNA)ITS sequences and chloroplast DNA(cpDNA)psbA-trnH sequences. At the same time,the characteristics of molecular evolution and geographical distribution in different geographical populations were analyzed,which provided with a theoretical basis on molecular identification,protection,and exploitation and utilization of F. nilgerrensis germplasm resources.【Method】Taking samples of F. nilgerrensis populations from12 different geographical regions in Yunnan as the research object. Subsequently,PCR was used to amplify the ITS and psbA-trnH sequences from genomic DNAs of these samples,and performed bidirectional sequencing and sequence combination. Then phylogenetic trees were constructed based on ITS and psbA-trnH sequences and their combined sequences,respectively. Finally,the DnaSP ver5.10 software was used to analyze the nucleotide diversity(π)and the number/type/diversity(Hd)of haplotype for ITS and psbA-trnH sequences. At the same time,the neutrality test and the molecular evolution characteristics of F. nilgerrensis populations were analyzed.【Result】The clustering analysis results based on ITS and psbA-trnH sequences showed that all samples of the F. nilgerrensis populations from 12 different geographical regions were clustered on one branch with the samples from GenBank database,and bootstrap value in this branch was higher than the threshold of 75%,indicating that both sequences could be used for molecular identification of this species germplasm(resources). The results of the cluster analysis based on the combination sequence of above DNA markers were consistent with the above results,the bootstrap value reached 99%,indicating that the cluster analysis results were more scientific and accurate. The genetic distance between ITS sequences of the F. nilgerrensis populations from different geographical regions was 0-0.014,the order of genetic distance in different regions was Diqing>Kunming>Wenshan. The genetic distance between psbA-trnH sequences was 0-0.025,the order of genetic distance in different regions was Kunming>Diqing>Wenshan. Therefore,it was speculated that both the ITS and psbA-trnH sequences could show the correlation between the genetic differentiation and geographical distribution pattern of F. nilgerrensis populations. The length of the ITS sequence was 668 bp,the mutation rate was 1.8%,the number of haplotypes was eight. Furthermore,the values of Hdand π were0.894±0.078 and 0.006,respectively. Kunming had the most haplotypes from ITS sequences,with four haplotypes;while the psbA-trnH sequence was 203 bp,the mutation rate was 2.5%,these sequences have five haplotypes,the values of Hd and π were 0.788±0.090 and 0.009,respectively. Kunming had the most haplotypes from psbA-trnH sequences,with three haplotypes. Therefore,the results showed that the haplotypes of both ITS and psbA-trnH sequences presented a specific correlation between evolutionary characteristics and geographical distribution of F. nilgerrensis. The Tajima’s D value(neutral test)of the ITS and psbA-trnH sequences were-0.673 and 0.227(P>0.1),respectively. It showed that the populations of F. nilgerrensis from 12 different geographical regions in Yunnan Province were stable and did not experience a recent population expansion event.【Conclusion】All samples collected from different geographical regions of Yunnan are the species of F. nilgerrensis. The ITS sequence and the psbA-trnH sequence can be used as the DNA barcode,their combined sequence of these DNA barcodes can more accurately identify this species, and both of them are suitable for the molecular phylogeographic study of F. nilgerrensis.更多还原