岛屿植物舟山新木姜子居群遗传多样性的RAPD分析

基于随机扩增多态 DNA(RAPD)方法分析了舟山群岛濒危植物舟山新木姜子 (N eolitsea sericea) 6个居群的遗传多样性及分化程度。 10条随机引物扩增出 84个可分析位点 ,多态位点百分比 (PPL)为 3 8.10 %。经 POPOGENE分析发现 ,舟山新木姜子居群平均水平的多态位点百分比 (PPL )为 2 3 .18% ,Nei' s基因多样度 (HE)为 0 .0 793 ,Shannon信息指数 (H )为 0 .12 0 1,与其它岛屿植物比较具有中等偏低水平的遗传多样性 ;岛屿各居群间遗传分化程度较高 (Gst=0 .3 646) ;地理距离与遗传距离之间具有显著相关性 (r=0 .7697,P=96.62 % ) ,岛屿隔离效应是导致居群间遗传分化的重要因素。结合居群遗传多样性及UPGMA聚类分析 ,推测普陀山岛舟山新木姜子部分个体可能为大猫岛迁入的后裔 ,而朱家尖岛舟山新木姜子则由人为移植自普陀山岛。基于舟山新木姜子的物种保护及资源利用 ,建议加强现有自然居群的就地保护 ,促进居群自然更新 ;建立种质资源库 ,收集不同岛屿的种源进行混合繁殖 ,促进基因交流 ;选育优良品系用于海岛植被恢复及园林观赏     

Isolation of compound microsatellite markers for the endangered plant Neolitsea sericea (Lauraceae)

? Premise of the study: The development of compound microsatellite markers was conducted in Neolitsea sericea to investigate genetic diversity and population genetic structure of this endangered insular species. ? Methods and Results: Using the compound microsatellite marker technique, 10 compound microsatellite markers that were successfully amplified showed polymorphism when assessed in 55 individuals from two populations in East China and Japan. Overall, the number of alleles ranged from 3 to 17, with an average of 7.9 alleles per locus. In addition, these primers could be easily amplified in Neolitsea aurata var. paraciculata and N. aurata var. chekiangensis. ? Conclusions: The highly polymorphic markers developed and characterized in this study will be useful for population genetic studies of N. sericea.     

Development and characterization of polymorphic microsatellite markers for Neolitsea sericea using Illumina paired‐end draft sequencing data

Simple sequence repeat (SSR) markers were developed and characterized for Neolitsea sericea (Bl.) Koidz. (Lauraceae). Out of 196 designed primer pairs, a total of 144 pairs showed amplification, of which 44 had clear and stable chromatograms. Polymorphism of these 44 loci was tested using 32 individuals sampled from a single population of N. sericea. The number of alleles and the polymorphism information content varied from 3 to 12 and 0.271 to 0.853, respectively. A significant departure from the Hardy‐Weinberg equilibrium was observed in one of the 44 loci. These SSR markers are useful for population genetic studies and parentage analysis in N. sericea, which is one of the most common evergreen species in coastal Pinus thunbergii forests in central‐western Japan.     

Late Pleistocene lineage divergence among populations of Neolitsea sericea (Lauraceae) across a deep sea‐barrier in the Ryukyu Islands

Aim Our goals were: (1) to assess the population genetic structure and demographic divergence history of a bird‐dispersed tree, Neolitsea sericea, endemic to East Asian land‐bridge islands; and (2) to interpret the results in the light of controversies over the dating and configurations of land bridges through the Japanese Ryukyu Island Arc. Location Japan–Ryukyu–Taiwan Island Arc and Chinese/Korean offshore islands. Methods We applied 10 nuclear microsatellites (nSSRs) and one chloroplast (cp) DNA sequence marker (psbA–trnH intergenic spacer) to 31 populations (397 and 326 individuals, respectively) from throughout the species’ range to infer current patterns of genetic diversity and structure, and pollen‐to‐seed migration ratios (r). A coalescent‐based isolation‐with‐migration (IM) model was applied to the combined nSSR/cpDNA data set to estimate lineage divergence times and population demographic parameters. Results The geographic structure of nSSRs and the distribution of most cpDNA haplotypes revealed two distinct lineages located in areas north and south of the ‘Tokara Gap’, a narrow (c. 37 km wide) but deep (> 1000 m) sea‐strait between the northern and central Ryukyus. Based on the IM analyses, we (1) dated the divergence of these northern and southern lineages to c. 0.07 Ma (90% highest posterior density interval: 0.02–0.38 Ma); (2) estimated a slightly smaller effective population size for the northern compared to the southern lineage; and (3) recovered only trivial signals of post‐divergence gene flow between them. Main conclusions The estimated divergence time for northern and southern lineages is consistent with geological evidence for the existence of land connections in the Tokara region during cold stages of the latest Pleistocene; it is thus incompatible with an ‘ancient sea‐barrier hypothesis’ for the Ryukyu Arc, where we would have expected much older divergences related to the initial formation of the Tokara and Kerama tectonic straits during the Pliocene. Multiple factors are likely to have had a role in the divergence of N. sericea, including not only land‐bridge submergence, but also island configuration, and/or constraints on adaptation along a latitudinal temperature gradient.     

Random amplified polymorphic DNA (RAPD) markers reveal less genetic variation in the endangered plant Cerastiumfischerianum var. molle than in the widespread conspecific C. fischerianum var. fischerianum (Caryophyllaceae)

Random amplified polymorphic DNA (RAPD) analysis was used to assess the level of genetic diversity in an endangered plant Cerastiumfischerianum var. molle and the widespread conspecific C. fischerianum var. fischerianum. At both the variety and the population level, C. fischerianum var. molle had a lower level of RAPD variation than var. fischerianum. Analysis of molecular variance ( AMOVA ) showed that most of the genetic diversity exists among populations for both varieties. Genetic differentiation among populations probably resulted from the highly selfing habits of both varieties. To conserve C. fischerianum var. molle genetically, preservation of only a part of the population is insufficient.     

浙江沿海地区舟山新木姜子群落及种群结构特征分析

采用立木级结构代替年龄结构的方法,对浙江沿海地区(包括嵊泗县大戢山岛、定海区大猫岛、普陀区朱家尖岛、鄞州区瞻岐镇、北仑区春晓镇和宁海市力洋镇)的舟山新木姜子也Neolitsea sericea ( Bl.) Koidz.页群落的物种组成和垂直结构及其种群的年龄结构、静态生命表和分布格局进行了分析。结果表明：这6个舟山新木姜子天然群落共有维管束植物52科76属92种(含6变种),以木本植物为主,其中常绿木本植物34种,落叶木本植物31种。各群落的物种多样性指数差异较大,其中位于普陀区朱家尖岛的群落乔木层Shannon-Wiener指数( H忆)最高,位于定海区大猫岛的群落灌木层和草本层的H忆值均最高;且存在灌木层的H忆值大于乔木层和草本层的规律。种群个体年龄结构存在差异,但6个种群均为增长型种群,幼苗储备丰富,其他龄级的个体数量总体上随径级增大而减少。6个种群的分布格局均为显著的聚集分布,其中位于北仑区春晓镇的种群聚集强度最高,位于定海区大猫岛的种群聚集强度最低。从静态生命表看,舟山新木姜子岛屿类型种群的个体死亡率在径级玉( DBH<5 cm)和径级Ⅳ(15 cm≤DBH<20 cm)较高,大陆类型种群的个体死亡率在径级玉较高。研究结果显示：浙江沿海地区舟山新木姜子群落主要为常绿阔叶林,群落间的物种多样性差异明显源于人类活动的干扰;总体上看舟山新木姜子种群具有很强的更新能力。根据研究结果,对浙江沿海地区舟山新木姜子种群的保护提出了一些建议。     

Lack of genetic variability in the rare and endangered Limonium cavanillesii (Plumbaginaceae) using RAPD markers

Limonium cavanillesii is an extremely endangered plant species endemic to the east Mediterranean region of Spain. Regarded as extinct for several years, the recent discovery of a small population (only 29 individuals) has prompted the adoption of measures for its conservation by official agencies. As part of this effort, we have analysed genetic variation in this population by means of random amplified polymorphic DNA (RAPDs). The analysis of 29 individuals with 11 different primers produced 131 monomorphic bands. To our knowledge, this is the lowest level of genetic variation detected in plants using RAPD markers. This result could be explained both by the apomictic reproductive system of this species and by the passage through a severe bottleneck in recent times, after which there has been no chance for mutation to restore detectable genetic variation.     

用RAPD检测华山新麦草自然居群的遗传结构和居群分化

华山新麦草特产于我国陕西华山,为国家一级重点保护植物。应用RAPD技术,用2 0条随机引物对黄埔峪、仙峪、华山峪3个居群不同海拔的13个亚居群共2 6 6个华山新麦草取样个体进行扩增,共得12 2个扩增片段。平均每个引物得到的扩增片段数为6 .1(2～10 )。实验数据的统计分析表明:黄埔峪居群的多态性位点比率为6 0 .6 6 % ,仙峪居群的多态性位点比率为90 .98% ,华山峪居群的多态性位点比率为95 .0 8% ,总的多态性位点比率为95 .0 8% ,说明华山新麦草具有较高的遗传变异性。Shannon多样性指数(0 .330 6 )和基因分化系数(GST=0 .32 6 3)揭示了华山新麦草居群遗传变异多存在于亚居群内。华山新麦草亚居群间的基因流N m=1.0 32 2 ,低于一般风媒传粉植物(N m=5 .2 4 )的基因流水平。亚居群间平均的遗传距离为0 .15 71(变化范围:0 .0 0 2 2～0 .2 90 1) ,最大的遗传距离值出现在黄埔峪亚居群(hp1)和华山峪高海拔亚居群(h8)之间,仙峪亚居群与华山峪高海拔亚居群之间以及华山峪高海拔亚居群与低海拔亚居群之间的遗传距离值也较大。遗传距离与海拔垂直距离之间的相关性分析表明二者有显著的相关性(p<0 .0 1)。聚类分析和主成分分析也显示华山新麦草自然居群已发生明显分化,主要表现为黄埔峪居群、仙峪居群与华山峪居群     

濒危植物三棱栎遗传多样性的RAPD分析

用随机扩增多态DNA (RAPD)标记对 5个三棱栎 (Trigonobalanusdoichangensis)居群共 99个个体进行遗传多样性和居群遗传结构分析。 16个引物共检测到 15 7个位点 ,其中多态位点 83个 ,占 5 2 87%。物种水平Shannon多样性指数I =0 2 4 31,Nei基因多样度h =0 15 95 ,种内总遗传变异量Ht=0 16 0 0 ,居群内遗传变异量Hs =0 0 74 9,居群间变异量大于居群内变异量 ,表明三棱栎的遗传变异主要存在于居群之间。与同科植物相比 ,三棱栎遗传多样性较低 ,遗传分化系数Gst =0 5 32 0 ,说明居群间的遗传变异占 5 3 2 0 % ,居群间已出现强烈的遗传分化。当地人的强烈活动造成的生境破碎化和居群隔离 ,以及三棱栎演化过程中的地史变化对其种群发展的影响等 ,可能是造成其居群间强烈的遗传分化和较低遗传多样性的原因。基于本研究结果 ,提出了三棱栎遗传多样性的保护策略。     

Population genetic structure in the dioecious pioneer plant species Hippophae rhamnoides investigated by random amplified polymorphic DNA (RAPD) markers

Hippophae rhamnoides is an outcrossing pioneer plant species with a severely fragmented distribution. Random amplified polymorphic DNA (RAPD) marker variation was analysed in 10 populations of ssp. rhamnoides and in one population of ssp. mongolica to estimate the amount and distribution of genetic variability. No less than 89.7% of the scorable markers were polymorphic, but few of these were fixed and populations consequently differed mainly by frequency variation of individual markers. Within-population gene diversity was somewhat low for an outcrossing plant species: 0.192 or 0.159 for ssp. rhamnoides , depending on whether it was based on all 156 polymorphic RAPDs or on only those 63 RAPDs that fulfilled the 3/ N criterion. Analysis of molecular variance applied to the ssp. rhamnoides showed only 15% between-population variability, indicating a relatively restricted population differentiation as expected in outcrossing species and shown in several other AMOVA studies. The tendency for island populations to be somewhat more differentiated, and to have less within-population diversity than mainland populations, may indicate an effect of population fragmentation. Genetic distance estimates among populations, obtained with and without pruning of RAPD loci on the basis of the 3/ N criterion, were generally in very good agreement. Cluster analyses and principal coordinate analyses showed populations of ssp. rhamnoides to be rather close, but quite isolated from the single ssp. mongolica population. Genetic and geographical distances between the ssp. rhamnoides populations were not associated, indicating that large-scale geographical and ecotypic differentiation was not reflected in the RAPD profiles.     

Genetic diversity and population structure of endangered Isoetes coreana in South Korea based on RAPD analysis

Isoetes coreana Chung and Choi is on the list of critically endangered species in South Korea. Using randomly amplified polymorphic DNA (RAPD) markers, we investigated the genetic diversity within and between seven local populations of I. coreana. Ten RAPD primers produced a total of 94 bands, of which 59 (62.8%) were polymorphic. A low level of genetic diversity was recognized within the populations of I. coreana: polymorphic loci (P), with values ranging from 3.4% to 33.9%, and a mean value of 15.5% being observed. Analysis of molecular variance (AMOVA) showed that genetic diversity was greater among populations (81.6%) than within populations (19.7%) or among the three regions included in this study (Han River, Youngsan River, and Nakdong River). In addition, a high degree of genetic differentiation (θ^B = 0.742) was detected among the populations. These results indicate that genetic differentiation has occurred very rapidly. However, the rate of gene flow between populations was found to be as low as 0.059, irrespective of the genetic and geographical distances between the populations, which indicates that genetic drift must have played an important role in forming the present populations of I. coreana. Because a reduction of genetic diversity as a result of genetic drift is undesirable, increasing the gene flow between populations of Korean quillwort I. coreana should be considered as a conservation strategy.     

Genetic diversity and structure of the endangered lady's slipper orchid Cypripedium japonicum Thunb. (Orchidaceae) in Japan

Cypripedium japonicum Thunb. (Orchidaceae), once a common perennial herb, is now designated as endangered throughout most of its distribution due to habitat destruction and fragmentation, and the impacts of horticultural collection. We investigated the genetic characteristics of this species for conservation purposes, using microsatellite markers to examine the genetic diversity and structure of 15 native and 5 ex situ populations in Japan. The results imply that although allelic variation is low in Japanese C. japonicum, sexual reproduction by seed, as well as clonal propagation, may occur in some populations. Both native and ex situ populations were found to be genetically differentiated, indicating that some populations may have experienced recent population declines, genetic fragmentation, or bottlenecks. The degree of genetic drift from the putative ancestral population, inferred through STRUCTURE analysis, was more pronounced in northern populations than in southern populations. Some of the ex situ conserved populations exhibited a low degree of differentiation from ancestral native populations. Our results imply that conservation of C. japonicum in Japan is best supported by maintaining individual populations and their unique genetic characteristics.     

On the verge of extinction: genetics of the critically endangered Iberian plant species, Borderea chouardii (Dioscoreaceae) and implications for conservation management

Borderea chouardii is a relictual and dioecious, strictly sexually reproducing, long-living geophyte of the Dioscoreaceae family. Previous biological and demographic studies have indicated the existence of a uniformly distributed panmictic population of this taxon at the southernmost Spanish pre-Pyrenean mountain ranges where it occurs in rather inaccessible crevices of a single limestone cliff. However, individuals of B. chouardii are spatially subdivided into two subpopulations located, respectively, on the upper and lower parts of the cliff, and vertically separated 150 m. Because of its extreme rarity, B. chouardii was the first Iberian taxon to have a specific conservation plan and has been included in several red lists under the category of critically endangered (CR). However, no previous attempts have been conducted to analyse the fine scale evolutionary mechanisms involved in its present microspatial distribution. Genetic diversity and population structure have been investigated through the analysis of neutral hypervariable markers such as simple sequence repeats (SSRs) and randomly amplified polymorphic DNAs (RAPDs) to unravel the impact of life history traits in the differentiation of the two subpopulations. Both types of molecular markers were unequivocal in distinguishing two genetically distinct groups of individuals corresponding to their spatial separation. However, SSRs detected a higher level of subpopulation differentiation (F(ST) = 0.35, R(ST) = 0.32) than RAPDs (F(ST) = 0.21). SSR data indicated significant deviation from random dispersal of genes and genotypes between the two groups, suggesting that mating occurs mainly among individuals within subpopulations, thus, favouring the divergence between the two groups. This microevolutionary differentiation scenario might have been caused by a coupled effect of past genetic drift and reproductive isolation, as a result of strong glacial age bottlenecks and inefficient dispersal system of pollen and seeds, respectively. The identification of such genetic structure in this narrow endemic prompts a modification of the management strategies of its single extant population.     

Genetic diversity and population structure of the endangered medicinal plant Phellodendron amurense in China revealed by SSR markers

Phellodendron amurense is an endangered tree with important medicinal and economic value in China. In this study, eight nuclear SSR primer pairs were employed to assess the genetic diversity and structure of 22 natural populations, including 516 individuals. A total of 66 alleles were detected with an average of 8.3 alleles per locus ranging from 3 to 17. The expected heterozygosity (He) of each SSR locus varied from 0.347 to 0.877 (average 0.627). Analysis of molecular variance (AMOVA) revealed that the main variation component existed within populations (95.11%) rather than among populations (4.89%). The Wilcoxon's sign-rank tests did not show any recent bottleneck effect in any population. A Mantel test displayed a significant correlation between the geographic distances and genetic distances for all populations (r = 0.566, P = 0.0001), indicating conformity to the isolation by distance model. Bayesian clustering and UPGMA supported grouping the populations into two groups. The present genetic structure of P. amurense may be explained by geographical isolation. The lack of genetic structure and genetic diversity decreased with increasing latitude within the Northeast China group may be due to postglacial northward expansion from a single refugium. Proper conservation measures are proposed for this species.     

舟山群岛四个养殖獐种群遗传多样性和遗传结构

基于ISSR分子标记技术,对来自舟山群岛4个獐(Hydropotes inermis)养殖种群的遗传多样性和遗传结构进行了分析.26条ISSR引物共扩增出286个可分析位点,多态位点百分比(PPL)为64.34％.獐物种水平上的Nei's遗传多样性指数(H)为0.210,Shannon's多态信息指数(Ⅰ)为0.318,各种群的H介于0.157-0.190之间,I在0.228-0.278之间,与已报道的ISSR标记在其它动物物种中的应用结果相比,其遗传多样性较为丰富.Structure软件分析结果显示,所有个体根据遗传信息的不同可以被分为4个组群,每个养殖场内的个体基本上都在各自独立的组群中,这与地理区域的划分相似.另外发现各养殖场獐种群间已表现出了较大的遗传分化(Gst=0.163).同时,为了解各种群间的遗传关系,计算了各种群间的Nei's遗传距离,结果显示舟山种群(ZS)和秀山种群(XS)之间的遗传距离最近,为0.045,而岱山种群(DS)和朱家尖种群(ZJJ)间的遗传距离最大,为0.066.基于以上结果,建议加强不同岛屿种群间的个体交流,特别是朱家尖种群(ZJJ)与其它种群间的交流.     

Genetic differentiation in Thai populations of the rare species Afgekia sericea Craib (Leguminosae) revealed by RAPDPCR assays

Genetic diversity within and among populations was investigated using RAPD-PCR assays in a rare species, Afgekia sericea Craib. Two hundred and sixty-nine individuals were sampled from nine geographically isolated populations from northeastern Thailand. This study includes 73 RAPD markers. Within population polymorphism as measured by percentage of polymorphic RAPDs, varied between 38.4% and 60.3%. Genetic variability was measured using Shannon's information index and partitioned into between- and within-population components. Overall, genetic variation among A. sericea populations was high (H_sp-H_pop)/H_sp=48.2%. The genetic diversity for the species (H_sp) was 43.2%. Mean of within-population values (H_pop) for all populations was 0.224. The total genetic diversity was explained by high variation among populations (mean Gst=0.426), which is consistent with low gene flow among populations (Nm=0.35). High between-population genetic variation observed in this study could be explained by limited migration through seed and/or pollen dispersal among populations. Conservation strategies of A. sericea are discussed in the context of these results. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic structure of the rare and endangered Hieracium wiesbaurianum group (Asteraceae) in Bavaria

The rare and endangered Hieracium wiesbaurianum species group shows a scattered relictual distribution in Bavaria. Recently, a couple of populations were discovered which clearly differ from all other populations. If these must be considered as taxonomically independent units, they would be of crucial conservation interest, because of the sole responsibility that Bavaria has for these worldwide endemics. We therefore analysed the genetic structure of H. wiesbaurianum in a comparative approach. Our analysis comprised 37 populations of 13 taxa of H. wiesbaurianum, H. bifidum and H. laevigatum, including three potentially new taxa. We applied amplified fragment length polymorphism (AFLP) analysis and observed only limited genetic variation within populations and taxa. Nevertheless, each studied individual exhibited a unique genotype. An analysis of molecular variance revealed high levels of genetic variation between taxa, but populations were genetically less different. The clear genetic differentiation between the studied taxa was supported by neighbor‐joining cluster analyses and principal coordinate analyses in which every individual was clearly assigned to its respective taxon. The three potentially new taxa were genetically as well differentiated as the other taxa included in our study. This supports the assumption that they should be treated as taxonomically independent units of high conservation interest. Therefore, the genetic analysis confirmed the morphologically based classification of the studied Hieracium taxa. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 112–123.     

Use of RAPD analysis in devising conservation strategies for the rare and endangered Grevillea scapigera (Proteaceae)

Extensive human impact in south western Australia has resulted in a high incidence of rarity throughout the highly endemic flora of the region. Grevillea scapigera (Proteaceae) is a typical example, with 27 plants (represented by four extant populations) remaining in the wild. In order to devise an appropriate strategy for the conservation of this species, its population genetics were studied using RAPD analysis, which enabled the discrimination of individual plants and the detection of a relatively high amount of variability (V = 0.32) within G. scapigera. This variability was found to be evenly distributed within the plants analysed despite the clear distinction between most populations (87% of the variability being attributable to single plant difference and 13% to population difference). Finally, RAPD analysis was used to select a small group of plants that captured maximum genetic variability to be used in the recovery program of the species. Because of the low genetic difference between populations, the mixing of these selected plants during the recovery process should not create genetic imbalances. The methods used in this study provide a useful model for future projects involving the recovery of rare flora.     

濒危植物资源冷杉遗传多样性研究

资源冷杉(Abies ziyuanensis)属松科(Pinaceae)冷杉属(Abies),是国家一级保护的濒危植物。利用RAPD分子标记对分布于广西资源县银竹老山和湖南省炎陵县大院的2个资源冷杉种群共54株进行了遗传多样性分析。用20个随机引物共扩增到126个位点,其中66个位点是多态性的,总的多态位点百分率为52.4％,在两个种群分别为33.6％和31。2％。Nei's基因多样性为0.312,Shannon信息指数为0.475,两个种群的遗传分化系数Gst为0.455。基于Nei's遗传距离进行了UPGMA聚类分析,结果两种群的样品彼此明显区分开来。分析结果表明资源冷杉种群内的遗传多样性较低,但两种群间的遗传分化明显。需要同时重视两分布地的资源冷杉的保护工作。     

Highly efficient in vitro proliferation and genetic stability analysis of micropropagated Ceropegiaevansii by RAPD and ISSR markers: A critically endangered plant of Western Ghats

Ceropegiaevansii McCann (family: Asclepiadaceae), a critically endangered plant of Western Ghats has acquired significant importance due to its medicinal implications, edible tubers, and ornamental flowers. This study deals with the optimization of axillary bud proliferation using nodal explants followed by genetic stability analysis of regenerants. Maximum number of shoots (11.6 ± 1.1) was observed on the Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (4.0 mg/l) and indole-3-acetic acid (0.3 mg/l) with 85% shoot multiplication frequency. In vitro-grown shoots were rooted best in 1/2 MS medium supplemented with indole-3-butyric acid (1.0 mg/l) with an average of 10.3 ± 0.9 roots per shoot and 92% rooting frequency. Plantlets were acclimatized best (90%) in a mixture of sterile soil, sand, and coco peat (1:2:1). Micropropagated plants were subjected to random amplified polymorphic DNA and inter simple sequence repeat markers analyses. Collectively, 759 bands were generated which were monomorphic and similar to the mother plant. Findings of this study are the first report on micropropagation and assessment of genetic stability of micropropagated plantlets in C. evansii which suggests that axillary shoot proliferation can safely be used as an effective tool for propagation and conservation of C. evansii.