珍稀濒危植物太行菊遗传多样性的ISSR分析

利用ISSR分子标记技术对太行山特有濒危物种太行菊11个自然居群的遗传多样性进行研究。用10个引物对11个居群的122个样品进行扩增,共得到150个扩增位点,其中多态性位点149个,多态位点百分率(PPL)为99.33%。POPGENE分析显示,太行菊具有较高的遗传多样性(H=0.2149,I=0.3455)。沁阳市大西天居群的遗传多样性水平最高(H=0.1910,I=0.2969),山西陵川县大双村居群的遗传多样性水平最低(H=0.1356,I=0.2155)。Nei’s遗传多样性分析表明,11个自然居群间出现了较高的遗传分化(基因分化系数Gst=0.2566,基因流Nm=1.4488)。生境的的片段化和基因流障碍可能是导致太行菊居群间遗传分化显著的主要原因。通过对太行菊居群遗传多样性和遗传结构的分析,该文提出了一些保护策略。     

白木香遗传多样性研究

用ISSR分子标记技术对白木香(Aquilaria sinensis(Lour.)Gilg)的遗传多样性进行分析.结果表明,白木香物种水平的遗传多样性较高,而居群水平的遗传多样性相对较低,其中广东茂名居群的遗传多样性最高.白木香居群间存在较大的遗传分化,遗传分化系数G_(ST)=0.4425,表明居群内遗传分化大于居群间的分化.UPGMA聚类分析表明白木香分化为两个谱系,其中谱系Ⅰ由广东、福建、海南的5居群组成,谱系Ⅱ由广西、云南的3个居群组成.居群间的基因交流受到阻碍(Nm=0.6633<1),阻碍主要产生于两个谱系间,而谱系内部的居群间在较近的历史时期基因交流频繁(Nm分别为1.4382和1.2333),谱系分化的原因主要是地理因素,两谱系交界处有云开山脉等形成的天然屏障,阻碍物种的扩展和基因交流.     

濒危物种珊瑚菜遗传多样性的ISSR分析

该研究采用ISSR分子标记对中国10个居群的241个珊瑚菜样本进行了遗传多样性分析。结果显示:8条引物共检测到76条清晰谱带,其中多样性条带64条;POPGENE分析显示,其物种水平多样性条带百分率(PPB)为84.21%,有效等位基因数(Ne)为1.562 8,Shannon多样性指数(I*)为0.866 3,Nei’s遗传多样性指数(h*)为0.342 5,居群间的遗传分化系数(GST)为0.205,基因流(Nm)为1.939 1,表明野生珊瑚菜具有较高的遗传多样性,且大部分遗传多样性存在于居群内;AMOVA分析显示,珊瑚菜居群间遗传分化水平(FST)为0.259 1,也表明珊瑚菜居群内变异大于居群间变异。研究认为,珊瑚菜的濒危原因主要来源于野生生态环境的破坏,应当加强种质资源的保护。     

濒危植物山白树遗传多样性及亲缘关系的ISSR分析

利用ISSR分子标记,对我国特有种金缕梅科(Hamamelidaceae)濒危植物山白树(Sinowilsonia fienryi Hemsl)6个野生居群和2个栽培居群75份试验材料的遗传多样性进行研究,以期为该物种种质资源的保护和合理利用提供科学依据。试验结果显示:(1)10条引物共检测到89条清晰的谱带,其中多态性条带85条,多态位点百分率(PPB)为95.5%,说明山白树在物种水平上遗传多样性较高;(2)Nei's基因多样度指数(H)为0.283 3,Shannon's多样性信息指数(I)为0.4034;Nei's遗传分化系数(G_(st))为0.3507,居群间与居群内变异百分率分别是25.49%,74.51%,说明山白树居群内与居群间都存在遗传分化,且主要遗传分化来自居群内部;(3)聚类分析(UPGMA法)得到,在阈值0.87处,山白树8个调查居群分为三类,分类结果与其地域分布存在一定规律。     

极小种群野生植物海南风吹楠的遗传多样性研究

为探讨海南风吹楠(Horsfieldiahainanensis)的濒危原因,利用限制性酶切位点相关的DNA测序技术(RAD-seq)开发单核苷酸多态性(SNPs),评估居群的遗传多样性和遗传结构。结果表明,海南风吹楠的遗传多样性较低(Ho=0.167),其中BWL居群表现出最高的遗传多样性;居群间存在中等程度的遗传分化(Fst=0.120)。Structure分析表明居群的最佳聚类值为2,但个别居群的遗传结构混杂,Mantel检测结果也表明遗传距离和地理距离没有相关性(r=0.733,P0.075)。自身更新能力低以及过度的人为活动干扰,可能是导致其濒危的主要原因。建议加强对遗传多样性高的居群(BWL和YGL)进行就地保护;对生境破坏严重的居群(EXL和DLS)进行近地或迁地保护,以增加居群间的基因交流,同时构建核心种质,防止遗传资源丢失加剧。     

基于保护基因组学揭示荷叶铁线蕨的濒危机制

理解物种的濒危机制对生物多样性的科学保护至关重要。荷叶铁线蕨(Adiantum nelumboides)作为国家一级重点保护野生植物, 其遗传多样性状况和濒危机制一直存在较大争议。本文利用简化基因组测序技术(genotyping by sequencing, GBS)对来自6个居群的28个荷叶铁线蕨样本测序, 共获得29.6 Gb的数据, 并筛选得到9,423个高质量单核苷酸变异位点(SNP), 通过遗传多样性和居群遗传结构分析, 并结合不同气候情景下物种潜在分布区差异, 探讨了荷叶铁线蕨的濒危原因和科学保护策略。结果表明: (1)荷叶铁线蕨具有较低的遗传多样性(H_o = 0.138、H_e = 0.232、P_i = 0.373), 同时种群间具有较低的遗传分化(F_st = 0.0202)和基因流(N_m = 1.9613); (2)所有样本均来自2个遗传分组, 基因组大小为 5.01‒5.83 Gb, 且均为四倍体, GC含量约为 39%‒41%; (3)生态位模拟表明, 与现代气候相比, 在未来气候变化下荷叶铁线蕨的潜在分布区面积略有增加, 但高适生区面积减小。其主要适生区向北迁移, 影响其分布的主导因子为昼夜温差月均值和最冷季降水量。正是由于荷叶铁线蕨遗传多样性低, 不同种群间遗传分化较低, 再加上气候条件的变化, 其适生区狭窄, 导致其遗传多样性和种群数量急剧下降。因此, 自身更新能力低以及过度的人为活动干扰可能是导致其濒危的主要原因。建议加强对荷叶铁线蕨的就地保护; 通过生境恢复及自然回归等措施, 增加居群间的基因交流, 防止遗传资源丢失加剧。     

濒危植物版纳青梅保护遗传学研究初报

运用20个10碱基随机引物,对中国龙脑香科(Dipterocarpaceae)特有的珍稀濒危植物版纳青梅(Vatica guangxiensis X. L. Mo)进行了RAPD多态性分析.3个自然居群和1 个迁地保护居群(分布于云南和广西)共扩增出231个位点,多态位点所占比例(PPB)为53.68%;观察等位基因数na=1.536*!8,有效等位基因数ne=1.287*!8,Nei基因多样性指数h为0.168*!6,居群内的遗传多样性水平较低.基于AMOVA和POPGENE的结果均表明居群内的遗传变异大于居群间的遗传变异.居群内的遗传变异为55.09%,居群间的变异为44.91% (AMOVA);基因分化系数Gst为0.374*!6 (POPGENE),表明居群间存在高水平的遗传分化.研究结果对该濒危植物的保护有重要意义.考虑到低水平的遗传多样性和高水平的居群分化,通过居群间种子和幼苗的交换来促进基因流是可行的保护方案.迁地保护居群(ML)不具最高的遗传多样性,表明为了保护此濒危物种的全部遗传变异,需要进一步采集更多个体补充到迁地保护居群中.     

武陵山区蛇足石杉遗传多样性的AFLP分析

采用AFLP分子标记对武陵山区蛇足石杉(Huperzia serrata)4个居群进行遗传多样性的研究,结果表明:(1)7对引物组合共扩增出条带615条,其中549条为多态性条带;在物种水平上,多态性条带百分率PPB=89.27%,有效等位基因数Ne=1.257,Nei’s基因多样度指数H=0.178,Shannon多样性信息指数Isp=0.298;在居群水平上,PPB=71.42%,Ne=1.235,H=0.154,Shannon多样性信息指数Ipop=0.251;遗传多样性在居群间有明显的差别,其中坪坝营(PBY)居群最高(PPB=81.95%),而铁峰山(TFS)居群最低(PPB=64.55%)。(2)居群间的遗传分化较低,基于Nei’s基因多样性分析结果显示,居群间遗传分化系数GST=0.159;Shannon’s居群分化系数[(Isp-Ipop)/Isp]为0.16;WINAMOVA分析显示,武陵山区蛇足石杉的遗传变异主要存在于居群内,居群内的遗传变异分量为65.057,占总变异的75.77%,而居群间的遗传变异分量为20.804,占总变异的24.23%;居群内存在极显著的遗传分化(ΦST=0.242,P0.001)。(3)由遗传分化系数(GST)估计,武陵山区蛇足石杉居群间的基因流Nm=2.647,表明蛇足石杉属于异交种。(4)两两居群间的Nei’s遗传一致度(IN)范围为0.031 0~0.969 4;Mantel检测结果显示,居群间的遗传距离与地理距离之间不存在显著的正相关关系(r=0.269,P=0.887)。研究认为,遗传多样性与遗传结构主要决定于居群历史,较少干扰而稳定的居群偏向克隆生殖,遗传多样性较低,而新建居群的遗传多样性则较高;克隆生长、生态位选择、异交,以及有效的孢子风媒传播等可能是其维持较高遗传多样性水平的因素,而过度采挖等人类活动和生境片断化是导致蛇足石杉濒危的主要因素。     

湖北野生春兰资源遗传多样性的ISSR分析

近年来,由于过度采挖和生境片断化,湖北野生春兰(Cymbidiumgoeringii)资源正面临着灭绝的危险。本文采用ISSR分子标记技术对湖北省内的11个春兰野生居群共325个个体的遗传多样性水平及居群遗传结构进行了研究。11个引物共检测到127个位点,其中112个为多态位点,占88.19%。POPGENE分析结果表明:与其他兰科植物相比,春兰具有丰富的遗传变异(在物种水平上,He=0.2628,Ho=0.4037;在居群水平上,PPL=63.06%,He=0.1945,Ho=0.2958)。Nei's遗传多样性分析和AMOVA分析表明,各居群间产生了一定程度的遗传分化(GST=0.2440,FST=0.2207)。居群间一定程度的遗传分化可能是由生境破坏和基因流障碍(Nm=0.8828)引起。UPGMA聚类分析可知,与其他居群相比,恩施地区的5个居群,即巴东(BD)、福宝山(FBS)、宣恩(XE)、毛坝(MB)、来凤(LF)优先聚成一支,而大悟(DW)居群单独聚为一支。同时本研究也表明,虽然春兰自交亲和,但在自然界中其繁育系统还是以异交为主。鉴于春兰资源的遗传多样性现状和其相应的居群遗传结构,我们建议在遗传多样性较高的来凤(LF)、京山(JS)、大悟(DW)居群设立保护点进行就地保护;而对资源破坏最为严重的毛坝(MB)和宣恩(XE)居群要实行迁地保护。     

云南南部野生大叶千斤拔资源遗传多样性的ISSR分析

应用ISSR分子标记技术,对云南南部7个地区的野生大叶千斤拔( Flemingia macrophylla)居群进行了遗传多样性分析。结果表明：云南野生大叶千斤拔具有较高的遗传多样性。在物种水平上,平均每个位点的多态位点百分率(PPL)为94.85％,有效等位基因数(Ne)为1.4627,Nei’s基因多样性指数(He)为0.2815, Shannon’s多样性信息指数(Ho)为0.4337;在居群水平上,PPL ＝43.44％,Ne ＝1.2981,He ＝0.1704,Ho ＝0.2499。基于Nei’ s遗传多样性分析可得出,居群间的遗传分化系数( Gst)为0.3975,表明居群内的遗传变异为60.25％,居群间的遗传变异为39.75％,这说明居群间的遗传分化要低于居群内的遗传分化。根据遗传多样性分析和聚类结果,应在大叶千金拔遗传多样性较高的勐腊易武( MY)、丘北( QB)和宁洱( NE)地区,设立保护点对其进行就地保护。     

Genetic diversity in endangered Notopterygium forbesii Boissieu based on intraspecies sequence variation of chloroplast DNA and implications for conservation

A thorough understanding of the levels and partitioning of genetic variation across populations and geographical regions of endangered species is a prerequisite to ensure effective conservation and/or restoration activities. Here, we examined chloroplast DNA (cpDNA) trnH-psbA intergenic spacer sequences variation within Notopterygium forbesii, an endangered and endemic perennial herb in China. Sequence data obtained from 141 individuals in 14 populations revealed twenty-two haplotypes. A high level of haplotype diversity (Hd = 0.81) and low level of nucleotide diversity (Pi = 0.0047) were detected. Low genetic differentiation among populations and also among regions was consistently indicated by both hierarchical analyses of molecular variance (AMOVA) and the structure of a neighbor-joining tree. Low level of population differentiation between populations or between regions in cpDNA sequences may be due to effects of the abundance of ancestral haplotype sharing and the high number of private haplotypes fixed for each population. Based on our results, we proposed some conservation strategies.     

基于ITS和trnL-trnF序列的草木樨种群遗传多样性研究

以白花草木樨(Melilotus alba)和黄花草木樨(Melilotus officinalis)18个地理种群植物为材料,用ITS序列和trnL-trnF序列研究了2种草木樨不同种群间的遗传多样性。结果表明:(1)trnL-trnF序列对位后长度为459bp,其中包括6个变异位点,6个简约信息位点,G+C含量为33.1%;ITS序列对位后长度为714bp,其中包括5个变异位点,3个简约信息位点,G+C含量为48.9%。(2)在基于trnL-trnF序列构建的系统发育树中,2种草木樨能够形成单系分支,说明trnL-trnF序列在草木樨中的鉴别能力较强。(3)单倍型多样性以及核苷酸多样性分析表明,黄花草木樨的遗传多样性高于白花草木樨。     

Strong Genetic Differentiation of Primula sikkimensis in the East Himalaya–Hengduan Mountains

The East Himalaya-Hengduan Mountains region is the center of diversity of the genus Primula, and P. sikkimensis is one of the most common members of the genus in the region. In this study, the genetic diversity and structure of P. sikkimensis populations in China were assessed using inter-simple sequence repeat (ISSR) and chloroplast microsatellite markers. The 254 individuals analyzed represented 13 populations. High levels of genetic diversity were revealed by ISSR markers. At the species level, the expected heterozygosity and Shannon’s index were 0.4032 and 0.5576, respectively. AMOVA analysis showed that 50.3% of the total genetic diversity was partitioned among populations. Three pairs of chloroplast microsatellite primers tested yielded a total of 12 size variants and 15 chloroplast haplotypes. Strong cpDNA genetic differentiation (G _ST = 0.697) and evidence for phylogeographic structure were detected (N _ST = 0.788, significantly higher than G _ST). Estimated rates of pollen-mediated gene flow are approximately 27% greater than estimated rates of seed-mediated gene flow in P. sikkimensis. Both seed and pollen dispersal, however, are limited, and gene flow among populations appears to be hindered by the patchiness of the species’ habitats and their geographic isolation. These features may have played important roles in shaping the genetic structure of P. sikkimensis. A minimum-spanning tree of chloroplast DNA haplotypes was constructed, and possible glacial refugia of P. sikkimensis were identified.     

基于叶绿体DNA非编码序列的蒙古扁桃谱系地理学研究

该研究选取中国西北干旱区第三纪孑遗植物蒙古扁桃（Amygdalus mongolica）,基于叶绿体DNA非编码trnH psbA序列对蒙古扁桃17个居群324个个体进行了谱系地理学研究。结果表明：（1）蒙古扁桃trnH psbA序列长度350 bp,变异位点63个,共有9种单倍型,居群间总遗传多样性为（H_t）为0.758,居群内平均遗传多样性为（H_s）为0.203,贺兰山东麓及阴山南麓边缘的居群具有较高的单倍型多样性及核苷酸多样性并固定较多特有单倍型,推测这2个地区是蒙古扁桃在第四纪冰期时的重要避难所。（2）AMOVA分析表明,居群间的遗传变异为83.84%,居群内的遗传变异为16.16%,居群间遗传分化系数N_st>G_st（N_st=0.733, G_st=0.655, P>0.05）,表明蒙古扁桃不存在明显的谱系地理结构;根据单倍型地理分布及网络关系图,把蒙古扁桃自然地理居群分为东、西两大地理组群,而且东、西地理组群没有共享单倍型;居群遗传结构分析表明,两大地理组群遗传分化较大。（3）蒙古扁桃居群在间冰期或冰期后经历了近期的居群扩张,由于奠基者效应使得多数居群只固定了单一的单倍型。     

中国东部沿海水仙归化群体的遗传多样性

为揭示我国东部归化水仙(Narcissus tazetta var. chinensis)的群体遗传多样性,利用2个叶绿体基因mat K和trn H-psb A片段对采自沪、浙、闽的5个代表群体的49株水仙进行了评估。结果表明,双基因联合序列的总长为1443bp,共定义6个单倍型,各归化群体的遗传多样性水平为DLSYPTDNJD=ZZCMD。AMOVA分析表明,群体内变异为遗传变异的主要来源(91.98%),群体间的遗传分化较低(Fst=0.080 22)。群体物种水平上的谱系结构不显著(Nst=0.020Gst=0.031;P0.05)。Mantel检验表明水仙群体间的遗传距离与地理距离呈显著的线性相关(r=0.929,P=0.02 0.05)。中性检验和错配分布分析结果均暗示水仙群体背离了快速扩张模型的假设。单倍型分布的中介网络图结合系统发育NJ树均将所有群体划分为2大分支。因此,我国东部沿海水仙归化群体整体遗传多样性水平较低,各群体间遗传分化较弱,遗传变异主要来自群体内,物种近期未经历扩张事件,可能是基因流受海岛隔离、自身生物学特征、生境异质性与及人为干扰的综合作用影响。     

Genetic diversity of wild populations of Rheum tanguticum endemic to China as revealed by ISSR analysis

Rheum tanguticum is an important but endangered traditional Chinese medicine endemic to China. The wild resources have been declining. Establishing the genetic diversity of the species would assist in its conservation and breeding program. Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population genetic structure in 13 wild populations of R. tanguticum from Qinghai Province. Thirteen selected primers produced 329 discernible bands, with 326 (92.94%) being polymorphic, indicating high genetic diversity at the species level. The Nei's gene diversity (He) was estimated to be 0.1724 within populations (range 0.1026–0.2104), and 0.2689 at the species level. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly within populations (71.02%), but variance among populations was only 28.98%. In addition, Nei's differentiation coefficients (G_st) was found to be high (0.3585), confirming the relatively high level of genetic differentiation among populations. Mantel test revealed a significant correlation between genetic and geographic distances (r = 0.573, P = 0.002), and the unweighted pair-group method using arithmetic average (UPGMA) clustering and Principal coordinates analysis (PCoA) demonstrated similar results. Meanwhile, the genetic diversity of R. tanguticum positively correlated with altitude and annual mean precipitation, but negatively correlated with latitude and annual mean temperature. This result might be an explanation that the natural distribution of R. tanguticum is limited to alpine cold areas. We propose conservation strategy and breeding program for this plant.     

Phylogeography of <Emphasis Type="Italic">Ceriops tagal</Emphasis> (Rhizophoraceae) in Southeast Asia: the land barrier of the Malay Peninsula has caused population differentiation between the Indian Ocean and South China Sea

The genetic structure of mangrove species is greatly affected by their geographic history. Nine natural populations of Ceriops tagal were collected from Borneo, the Malay Peninsula, and India for this phylogeographic study. Completely different haplotype compositions on the east versus west coasts of the Malay Peninsula were revealed using the atpB-rbcL and trnL-trnF spacers of chloroplast DNA. The average haplotype diversity (Hd) of the total population was 0.549, nucleotide diversity (θ) was 0.030, and nucleotide difference (π) was 0.0074. The cladogram constructed by the index of population differentiation (G _ST) clearly separated the South China Sea populations from the Indian Ocean populations. In the analysis of the minimum spanning network, the Indian Ocean haplotypes were all derived from South China Sea haplotypes, suggesting a dispersal route of C. tagal from Southeast Asia to South Asia. The Sunda Land river system and surface currents might be accountable for the gene flow directions in the South China Sea and Bay of Bengal, respectively. The historical geography not only affected the present genotype distribution but also the evolution of C. tagal. These processes result in the genetic differentiation and the differentiated populations that should be considered as Management Units (MUs) for conservation measurements instead of random forestation, which might lead to gene mixing and reduction of genetic variability of mangrove species. According to this phylogeographic study, populations in Borneo, and east and west Malay Peninsula that have unique genotypes should be considered as distinct MUs, and any activities resulting in gene mixing with each other ought to be prevented.     

Genetic diversity in Artemisia halodendron (Asteraceae) based on chloroplast DNA psbA–trnH region from different hydrothermal conditions in Horqin sandy land, northern China

The genetic diversity of Artemisia halodendron (Asteraceae), a constructive and dominant species in the Horqin sandy land, was investigated to examine the genetic relationships with different hydrothermal regions in the Horqin sandy land. We sequenced chloroplast DNA (cpDNA) fragments psbA–trnH of 243 plants from ten populations across the Horqin sandy land. The analyses of cpDNA variation identified seven haplotypes. A high level of haplotype diversity (H _d = 0.831) and low level of nucleotide diversity (π = 0.0018) were detected. Haplotypes clustered into three tentative clades. Low genetic differentiation among regions was consistently indicated by hierarchical analyses of molecular variance (AMOVA).     

Clonal diversity and geographic structure in <Emphasis Type="Italic">Pleurochaete squarrosa</Emphasis> (Pottiaceae): different sampling scale approach

This paper describes our investigation of genetic variation and clonal structure of the Mediterranean moss Pleurochaete squarrosa (Brid.) Lindb. (Pottiaceae), using inter-simple sequence repeat (ISSR) molecular markers and trnL_UAA (intron of plastid gene for Leu tRNA) sequence, choosing different sampling strategies and scales on 16 European populations. Moreover, the intercontinental distribution of two trnL haplotypes, previously found over a large area and including 24 populations in three continents, was also investigated. Despite the prevalent asexual reproduction, P. squarrosa shows a high level of genetic diversity. Some site features seem to affect the clonal structure at the local scale, influencing the relocation of detached fragments and the level of intermingling, but they do not substantially affect genetic diversity. The peculiar vegetative reproduction coupled with somatic mutation could partly account for the genetic variation detected. Genetic distances highlight geographic isolation and limited gene flow among populations. We found only two trnL haplotypes in Europe due to length polymorphism, but, over an intercontinental scale, only non-delete trnL was found in Africa and the USA. ISSR analysis within each population detected a higher genetic distance between the samples with different trnL haplotypes, suggesting the presence of two different genetic lineages within this species, geographically overlapping in the Mediterranean Basin.     

Phylogeography of Chrysanthemum indicum L. (Compositae) in China based on trnL-F sequences

Using chloroplast DNA (cpDNA) trnL-F sequences, we studied the phylogeographic pattern and demographic history of Chrysanthemum indicum with 81 individuals from 27 populations representing the partial geographic range in China. Fifteen haplotypes were identified based on nucleotide variation. Relatively high levels of haplotype diversity (Hd = 0.783) and low levels of nucleotide diversity (Pi = 0.00521) were detected in C. indicum. An analysis of molecular variance (AMOVA) indicated that most of the genetic variation (59.9%, P < 0.001) was partitioned within populations. Genetic differentiation among populations was high (F_ST = 0.35), suggesting that there is little gene flow among populations. Nested clade analysis showed that 15 haplotypes formed twelve 1-step clades, five 2-step clades and two 3-step clades, with four clades showing significant geographical association. We concluded that contiguous range expansion and restricted gene flow/dispersal but with some long distance dispersal seemed to have shaped the present-day distribution of the haplotypes of C. indicum in China.