【作者】 杨萍；

【导师】 刘玉萍；

【作者基本信息】 青海师范大学 ， 生物学， 2024， 硕士

【摘要】 黄缨菊（Xanthopappus subacaulis）是菊科（Asteraceae）、黄缨菊属（Xanthopappus）的一种多年生药用草本植物,它广泛分布于青藏高原及其邻近地区的高寒草甸和干旱坡地,具有耐寒、抗旱、耐盐碱等多种抗逆性,并且有凉血、止血等药用功效。目前,国内外对黄缨菊的研究多集中在生物学特性、化学结构、药理活性、系统发育和种群动态历史等领域,尚未见基于单拷贝核基因探究黄缨菊种群遗传结构的研究,因而利用多种分子标记对黄缨菊进行群体遗传结构和遗传多样性研究显得尤为重要。鉴于此,本研究先基于转录组数据筛选多套单拷贝核基因,在此基础上开展黄缨菊自然居群的遗传结构分析,同时结合最大熵模型推测黄缨菊的冰期避难所,以期更好地阐明黄缨菊的动态进化历史。主要研究结果如下:⑴基于全长转录组测序数据,本研究获得28,826,664条子序列（Subreads）,平均长度为2,479 bp,N50为2,878 bp,其中环形一致性序列（CCS）有638,103个,包含全长非嵌合体序列（FLNC）575,533个;FLNC序列校正和去冗余后得到46,100个unigenes,平均长度2,620 bp,N50长度2,976 bp;同源基因家族分析获得407条单拷贝核基因,从中挑选60条同源性高的基因通过引物设计和验证,得到10对扩增性好、测序成功率高的核基因引物,扩增产物介于600～1,000bp之间;多态性分析显示,筛选出的10对单拷贝核基因引物隶属于中度多态性位点。⑵黄缨菊存在强烈的遗传分化F_ST:0.83035（P<0.01）,居群间基因流范围为0.019～0.246;分子变异方差分析表明,黄缨菊遗传变异主要存在于居群间（83.04%）,居群内遗传变异很少（16.96%）,整体呈现为群体间遗传差异很大;Mantel检验结果显示,黄缨菊居群间的遗传距离与地理距离呈显著正相关（r=0.537;P<0.01）,地理隔离对黄缨菊群体的遗传分化有明显影响;STRUCTURE分析表明,K=2时黄缨菊被明显分成两个不同的支系,其中Group 1包含祁连山和青海湖的30个居群,Group 2主要由阿尼玛卿山和横断山区的19个居群组成,并且祁连山东部、青海湖以南和阿尼玛卿以北地区的居群存在明显的基因渐渗,这也得到PCo A结果的支持。⑶黄缨菊多态性位点（S）介于1～6之间,单倍型多样性指数（H_d）范围在0.558～0.883之间,平均值为0.719,核苷酸多样性指数（π）范围在0.00132～0.00603之间,平均值为0.00275,不同的单拷贝核基因对黄缨菊的遗传变异影响不同;青海北部和甘肃黄缨菊居群的遗传多样性大于青海南部、四川和西藏的居群,呈现北部的居群略高于南部的居群,说明祁连山和青海湖附近的居群比阿尼玛卿山附近的居群具有更强的适应性;单倍型谱系分析显示,Group 1居群的多个位点有更多的特异单倍型,单体型丰富度高。⑷错配分布分析显示,黄缨菊的观测值与期望值吻合度较低,呈双峰曲线;中性检验结果表明,Tajima’s D=1.45398、Fu and Li’s D^*=2.37467、Fu and Li’s F^*=2.33334均为不显著正值（P>0.1）,说明黄缨菊没有经历过近期种群扩张;ABC模拟分析显示,黄缨菊的Group 1与Group 2居群均从祖先种分化而来,两个集群的分化约在3.33百万年前,认为黄缨菊可能于上新世早期开始分化。⑸最暖季度平均温度与海拔是影响黄缨菊自然地理分布的主要因子;末次间冰期,黄缨菊在我国西北地区呈大面积连续分布;末次间冰期（38.177 Km²）至末次盛冰期（37.668 Km²）,黄缨菊的整个分布区向青海湖附近缩减;末次盛冰期至当前（中全新世39.826 Km²,现在46.497 Km²）,黄缨菊的适宜分布区面积扩张,但其高适宜区基本保持不变,自西向东主要位于柴达木盆地东部、祁连山脉南部、阿尼玛卿山北部,甘肃武威等地;当前至未来,黄缨菊的分布区在2050S-RCP4.5条件下扩张最明显;青海湖、阿尼玛卿山及横断山等地可能是黄缨菊的主要冰期避难所。更多还原

【Abstract】 Xanthopappus subacaulis was a perennial medicinal herb.belongs to Xanthopappus（Asteraceae）.It is distributed in the alpine meadows and arid slopes of Qinghai-Xizang Plateau and its neighboring areas.It has many resistance characteristics such as cold,drought,salt-alkali resistance,and has medicinal value such as cooling blood and hemostasis.At present,the studies on X.subacaulis mainly focus on its biological characteristics,pharmacological activity,chemical structure,phylogeny and population dynamic history,but there are no studies on population genetic structure based on single copy nuclear genes.Therefore,it is very important to study the population genetic structure and genetic diversity of X.subacaulis using multiple molecular markers.In view of this,this study screened multiple sets of single-copy nuclear genes based on transcriptome data to analyze the genetic structure of X.subacaulis in different populations,and combined with the maxent to predict the glacial refuge of X.subacaulis to elucidated the dynamic evolutionary history of X.subacaulis preferably.The key findings were as follows:⑴Based on full-length transcriptome sequencing,28,826,664 Subreads were obtained,with an average length of 2,479 bp and an N50 of 2,878 bp.Among them,638,103 circular consensus sequences（CCS）were obtained,including 575,533full-length non-chimeric reads（FLNC）.After sequence correction and redundancy removal of FLNC,46,100 unigenes were retained,with an average length of 2,620 bp and N50 length of 2,976 bp.407 single-copy nuclear genes were obtained based on homologous gene family analysis.Then,we totally selected 60 genes with high homology,and finally obtained ten pairs of single-copy nuclear gene primers with good amplification and high success rate of sequencing.The results of polymorphism analysis showed that the amplified products ranged from 671 bp to 1,000 bp.In addition,the results of polymorphism analysis showed that the selected 10 sets of single copy nuclear gene primers belonged to moderately polymorphic loci.⑵There was strong genetic differentiation(F_ST)of 0.83035（P<0.01）,and the gene flow size of the population ranged from 0.019 to 0.246.Molecular variance analysis showed that the genetic variation was mainly came from among populations（83.04%）,and the genetic variation within populations was small（16.96%）.In general,there was a large genetic differentiation and limited gene flow among populations.The mantel test showed that there was a significant correlation between genetic distance and geographic distance among different populations（r=0.537;P<0.01）,geographical isolation significantly affected the genetic differentiation of X.subacaulis.In addition,the STRUCTURE analysis shows that when K=2 is the best Group,the population can be clearly divided into two distinct lineage branches,in which Group 1 branch contains 30 populations in the Qilian Mountains and Qinghai Lake region,and Group 2 branch mainly consists of 19 populations distributed in the Aemye Ma-chhen Range and Hengduan Mountains.The populations distributed in the eastern Qilian Mountains,the southern Qinghai Lake and the northern Aemye Ma-chhen showed significant introgression of genes.PCo A analysis also supported the division of the population into two genetic lineages.⑶Based on 10 sets of single copy nuclear gene loci,the genetic diversity analysis of 49 populations of X.subacaulis showed that the polymorphic loci（S）ranged from 1 to 6,and the haplotype diversity（H_d）ranged from 0.558 to 0.883,with an average value of 0.719.Nucleotide diversity（π）ranged from 0.00132 to 0.00603,with an average value of 0.00275.It was found that there were differences in the expression of variation degree of different single copy nuclear genes,but in general,10 sets of single copy nuclear gene primers belonged to moderate polymorphism loci.The genetic diversity of the populations in the northern Qinghai and Gansu was larger than that in the southern Qinghai,Sichuan and Xizang,indicating that the northern populations in the Qilian Mountains and Qinghai Lake were more adaptive than the southern populations in the Aemye Ma-chhen.The results of haplotype analysis showed that Group 1 had several unique haplotypes at multiple loci and had high haplotype richness.⑷The mismatch distribution shows that the agreement between the observed value and the expected value is low,showing a bimodal curve.neutrality test results showed that Tajima’s D=1.45398、Fu and Li’s D^*=2.37467、Fu and Li’s F^*=2.33334 were not significant positive values（P>0.1）.There was no recent population expansion in the current distribution area.Approximate Bayesian Computation model simulation analysis showed that Group 1 and Group 2 diverged from the ancestral species,and the divergence time of the two clusters was estimated to be about 3.33million years ago,suggesting that X.subacaulis may have diverged in the early pliocene epoch.⑸The results of Maxent showed that the average temperature and altitude in the warmest quarter were the main factors affecting the distribution of X.subacaulis.In the last interglacial period,the X.subacaulis distributed continuously in the northwest of China;From the last interglacial period（38.177 Km²）to the last glacial maximum period（37.668 Km²）,the whole distribution area decreased to the vicinity of the Qinghai Lake.From the last glacial maximum to the present（39.826 Km²in the Middle Holocene,46.497 Km²in the present）,the suitable distribution area of X.subacaulis expanded.On the whole,the distribution expansion history of X.subacaulis was consistent with the first contraction and then expansion.Its high suitable areas remain roughly unchanged from west to east,mainly located in the east of the Qaidam Basin,the south of the Qilian Mountains,the north of the Aemye Ma-chhen,Wuwei of Gansu Province,etc.From the present to the future,the distribution area of X.subacaulis will expand most obviously under the conditions of2050S-RCP4.5 Qinghai Lake,the Aemye Ma-chhen and the Hengduan Mountain may be the main Ice shelter of X.subacaulis.更多还原