Development and characterization of 24 microsatellite markers in Primula tosaensis,an endangered primrose,using MiSeq

Primula tosaensis (Primulaceae) is an endangered primrose endemic to Japan. In this study, 24 novel microsatellite markers were developed using Illumina MiSeq sequencing to facilitate conservation of this endangered species. The genetic diversity and polymorphisms of these novel markers were measured in 32 individuals from a wild P. tosaensis population. The number of alleles and expected heterozygosities ranged from 2 to 5 (mean = 2.8) and from 0.119 to 0.724 (mean = 0.395), respectively. All loci were in Hardy–Weinberg equilibrium. The markers developed in this study will provide a powerful and practical tool for investigating the population structure and genetic diversity of P. tosaensis.     

鹅掌楸贵州烂木山居群的微卫星遗传多样性及空间遗传结构

濒危植物鹅掌楸(Liriodendron chinense)目前仅零散分布于我国亚热带及越南北部地区, 残存居群生境片断化较为严重。研究濒危植物片断化居群的遗传多样性及小尺度空间遗传结构(spatial genetic structure)有助于了解物种的生态进化过程以及制定相关的保育策略。本研究采用13对微卫星引物, 对鹅掌楸的1个片断化居群进行了遗传多样性及空间遗传结构的研究, 旨在揭示生境片断化条件下鹅掌楸的遗传多样性及基因流状况。研究结果表明: 鹅掌楸烂木山居群内不同生境斑块及不同年龄阶段植株的遗传多样性水平差异不显著(P>0.05), 居群内存在寨内和山林2个遗传分化明显的亚居群。烂木山居群个体在200 m以内呈现显著的空间遗传结构, 而2个亚居群内的个体仅在20 m的距离范围内存在微弱或不显著的空间遗传结构。鹅掌楸的空间遗传结构强度较低(Sp = 0.0090), 且寨内亚居群的空间遗传结构强度(Sp = 0.0067)要高于山林亚居群(Sp = 0.0053)。鹅掌楸以异交为主, 种子较轻且具翅, 借助风力传播, 在一定程度上降低了空间遗传结构的强度。此外, 居群内个体密度及生境特征也对鹅掌楸的空间遗传结构产生了一定影响。该居群出现显著的杂合子缺失, 近交系数(F_IS)为0.099 (P < 0.01), 表明生境片断化的遗传效应正逐渐显现。因此, 对鹅掌楸的就地保护应注意维护与强化生境的连续性, 促进基因交流。迁地保护时, 取样距离应不小于20 m, 以涵盖足够多的遗传变异。     

基于3种线粒体标记探讨中日沿海角木叶鲽遗传多样性差异

角木叶鲽(Pleuronichthys cornutus)是东亚沿海重要的鲽形目经济鱼类, 为更好地保护和开发利用其种质资源, 有必要全面了解其遗传背景。本研究测定了中国和日本沿海7个群体200尾角木叶鲽线粒体控制区(CR) 5'端、细胞色素b (Cytb)和NADH脱氢酶第二亚基(ND2)基因序列, 比较不同标记在解析遗传多样性和种群结构上的可行性与有效性, 阐明中日沿海角木叶鲽群体间出现遗传分化的分子机制。CR序列分析发现中日沿海7个角木叶鲽群体遗传多样性表现出较高的单倍型多样性(H_d = 0.9699)和较低的核苷酸多样性(π = 0.0061); 各群体间无显著的遗传分化(F_ST = -0.0197-0.0184, P > 0.05); 单倍型网络未显示出明显的地理聚群和谱系结构; 分子方差分析(AMOVA)表明变异主要发生在群体内部(> 99.17%)。进一步通过Cytb和ND2基因分别与CR序列对比分析, 结果表明群体遗传多样性均表现为高H_d (0.9683-0.9829)低π (0.0050-0.0063)模式, 仅有ND2基因分析F_ST值(F_ST = 0.0302, P < 0.05)显示了中国碣石(GDJS)和日本明石(JAP)群体间显著的低水平遗传分化现象。CR、Cytb和ND2的单倍型网络图均无明显的地理聚类和谱系结构, AMOVA分析也显示变异主要来源于群体内(> 98.39%)。种群历史动态分析结果显示, 角木叶鲽可能在第四纪中更新世晚期经历了群体扩张事件, 扩张时间分别为31.93-9.58万年前(CR)、27.53-22.02万年前(Cytb)和26.99-18.75万年前(ND2)。综上所述, 中日沿海的角木叶鲽具有较高遗传多样性, GDJS和JAP群体间存在低度分化; ND2基因比CR和Cytb序列更适于分析角木叶鲽种群遗传结构, 选择多个遗传标记可有效弥补单一标记分析遗传多样性的局限性; 推测冰期两大独立避难所的形成及GDJS和JAP群体距离相隔较远是其发生遗传分化的主要原因。研究结果为中日沿海角木叶鲽渔业资源的种质保护与可持续利用提供了理论依据。     

Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.     

基于高通量测序的达氏鲟微卫星标记筛选

达氏鲟(Acipenser dabryanus)是我国特有的珍稀鱼类,主要分布在长江上游干支流。由于人类活动的影响,其种群数量急剧下降,被列为国家Ⅰ级重点保护动物、IUCN红色名录极危(CR)物种。本研究针对当前达氏鲟群体的濒危现状,应用微卫星标记开展达氏鲟保护遗传学研究。通过高通量测序成功筛选出25个微卫星位点,采用18尾野生个体和30尾人工繁殖个体对其多样性进行了检验,结果表明,25个位点共检测出181个等位基因,每个位点的等位基因数(A)为4~11(平均值7.2),观测杂合度(HO)为0.160~1.000(平均值0.744),期望杂合度(HE)为0.346~0.875(平均值0.727)。所有位点均不偏离"Hardy-Weinberg"平衡(P0.05),各位点间也无连锁不平衡现象。除了其中一个位点以外,所有位点的多态信息含量(PIC)均大于0.5,其多态性较好。本次筛选的微卫星位点将有助于达氏鲟资源保护和群体遗传研究。     

Molecular and paleo-climatic data uncover the impact of an ancient bottleneck on the demographic history and contemporary genetic structure of endangered Pinus uliginosa

With the current rate of biodiversity loss, conservation management practices require a comprehensive understanding of eco-evolutionary relationships, history, and genetic structure of species. Assessments of genetic diversity are crucial, especially in rare, endemic, or threatened forest tree species with small and isolated populations, such as peat bog pine (Pinus uliginosa N.). Here, we used a novel approach, combining genetic diversity assessment, ecological niche modeling, and population demography inference to explore the complex history of a few remnant populations of this endangered pine. To asses the relative influence of isolation and fragmentation on genetic diversity in the taxonomic context, the patterns of genetic variation found in P. uliginosa were contrasted with those observed in its close relatives with much bigger distribution ranges and larger populations (Pinus sylvestris, Pinus mugo, and Pinus uncinata). We found a similar level of genetic diversity across the species at nuclear loci but contrasting patterns of variability distribution at chloroplast markers. We detected the signatures of an ancient genetic bottleneck dated at around 26 400 years ago, indicating a drastic reduction in the population size of P. uligionosa during the Last Glacial Maximum. In addition, we found substantial differentiation between current populations as a result of enhanced genetic drift during long-lasting isolation. The research suggests potential conservation management strategies for peat bog pine and emphasizes the importance of using complementary approaches for their successful development.     

Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers

China is rich in chicken genetic resources, and many indigenous breeds can be found throughout the country. Due to poor productive ability, some of them are threatened by the commercial varieties from domestic and foreign breeding companies. In a large-scale investigation into the current status of Chinese poultry genetic resources, 78 indigenous chicken breeds were surveyed and their blood samples collected. The genomes of these chickens were screened using microsatellite analysis. A total of 2740 individuals were genotyped for 27 microsatellite markers on 13 chromosomes. The number of alleles of the 27 markers ranged from 6 to 51 per locus with a mean of 18.74. Heterozygosity (H) values of the 78 chicken breeds were all more than 0.5. The average H value (0.622) and polymorphism information content (PIC, 0.573) of these breeds suggested that the Chinese indigenous chickens possessed more genetic diversity than that reported in many other countries. The fixation coefficients of subpopulations within the total population (F _ST) for the 27 loci varied from 0.065 (LEI0166) to 0.209 (MCW0078), with a mean of 0.106. For all detected microsatellite loci, only one (LEI0194) deviated from Hardy-Weinberg equilibrium (HWE) across all the populations. As genetic drift or non-random mating can occur in small populations, breeds kept on conservation farms such as Langshan chicken generally had lower H values, while those kept on large populations within conservation regions possessed higher polymorphisms. The high genetic diversity in Chinese indigenous breeds is in agreement with great phenotypic variation of these breeds. Using Nei’s genetic distance and the Neighbor-Joining method, the indigenous Chinese chickens were classified into six categories that were generally consistent with their geographic distributions. The molecular information of genetic diversity will play an important role in conservation, supervision, and utilization of the chicken resources.     

中国西北地区天然胡杨群体遗传多样性及核心保护单元的构建

胡杨(Populus euphratica)是极端干旱荒漠区的珍稀乔木树种。为了确定天然胡杨群体遗传多样性保护单元并挖掘优异的种质资源, 本研究以中国西北地区新疆、青海、甘肃、宁夏、内蒙古的58个天然胡杨群体为研究对象, 利用120个位点的SNPs标记对这些胡杨群体进行群体遗传结构和遗传多样性分析, 并根据不同群体间Nei’s遗传相似度, 采用逐步聚类优先取样法对初始群体、遗传多样性保护单元和剩余群体进行t检验。群体结构和主成分分析表明, 胡杨群体可分为新疆南疆(SX)、新疆北疆(NX)、青海(QH)和混合群(甘肃、宁夏和内蒙古混合群, GNM) 4个分支, 遗传多样性分析表明新疆南疆(SX)遗传多样性高于其他群体。分子方差分析(AMOVA)表明天然胡杨群体的遗传变异主要分布在各个群体内。构建了天然胡杨群体一级核心保护单元3个群体(CU3), 二级核心保护单元33个群体(CU33)。南疆存在较多优异抗逆的天然胡杨古树资源, 南疆分布区的平均遗传多样性水平最高。综上所述, 南疆地区胡杨古树遗传多样性整体高于北疆及疆外地区, 结合新疆地区干旱严重指数等生境信息, 建议加大对南疆胡杨古树群体的保护力度, 重视北疆胡杨林的更新换代。     

Development and characterization of 20 new polymorphic microsatellite markers from Mangifera indica (Anacardiaceae)

? Premise of the study: Twenty microsatellite loci for mango (Mangifera indica), an important commercial fruit tree in East Asia, were developed to evaluate the genetic diversity and identification of cultivars. ? Methods and Results: The 20 new microsatellite markers were isolated from mango using a magnetic bead enrichment method, and polymorphisms were identified in 22 mango cultivars. The number of alleles ranged from one to nine, with expected heterozygosity ranging from 0 to 0.826. The polymorphism information content ranged from 0 to 0.756 with a mean of 0.525. ? Conclusions: These new microsatellite loci should be useful and convenient for further studies of the genetic diversity and identification of cultivars in mango.     

Development of microsatellite markers in Parakmeria nitida (Magnoliaceae)

? Premise of the study: We developed microsatellite makers for Parakmeria nitida to investigate its population structure and conservation genetics. ? Methods and Results: A total of 25 microsatellite primer pairs were developed using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, and polymorphism was assessed in three natural populations of P. nitida. Among these markers, 11 were monomorphic and 14 showed polymorphism. ? Conclusions: These markers are potentially useful for future population genetic analyses of P. nitida and will serve as an important tool for conservation efforts.     

Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers

China is regarded as one of the domestication cen-ters for chickens and archaeological studies provided evidence of chicken domestication in northern Chinaas early as 6000 BC[1]. At present, China has the larg-est chicken population in the world, represen…     

两种濒危水韭植物迁地保护居群的基因流动态及回归重建保育遗传管理策略

采用微卫星分子标记对中华水韭（Isoetessinensis）安徽休宁、浙江建德和东方水韭（I.orientalis）浙江松阳三个孑遗居群的迁地保护居群开展了遗传多样性检测与遗传结构分析。7对多态性微卫星引物在36个迁地保护亚居群的720个样本中共检测到59个等位基因,每位点平均等位基因数（A）为8·43。迁地保护亚居群均维持很高的遗传多样性,多态信息含量（PIC）平均为0·707。迁地保护亚居群间遗传分化较低,遗传分化系数GST仅为0·070,居群间具有较大基因流（Nm=3·59）。单因素方差分析发现水韭孢子或孢子体在沿主要水流方向上的长距离传播能力要强于弱水流方向上的短距离传播能力,水流动态对水韭植物的基因流有重要影响。这与UPGMA聚类分析中迁地保护亚居群按邻近位置或水流相通程度优先聚类的结果相一致,水流所带动的强大基因流导致了不同孑遗居群来源的迁地保护亚居群间的遗传混杂。建议在开展水韭植物的迁地保护或回归自然重建时,对具有地方适应分化或者显著性进化的水韭植物居群应相互隔离而不宜配置在一起,以避免远交衰退的遗传风险。     

Identification of Bactrian camel cell lines using genetic markers

Iranian Bactrian camel population is less than 100 animals. Iranian biological resource center produced more than 50 Bactrian camel fibroblast cell lines as a somatic cell bank for conservation animal genetic resources. We compared two type markers performance, including 14 random amplified polymorphic DNA (RAPDs) (dominant) and eight microsatellite (co-dominant) for cell line identification, individual identification and investigation genetic structure of these samples. Based on clarity, polymorphism, and repeatability, four RAPD primers were selected for future analysis. Four RAPD primers and eight microsatellite markers have generated a total of 21 fragments and 45 alleles, respectively. RAPD primers revealed fragment size between 150 to 2000 bp and gene diversity since 0.27 (IBRD) to 0.46 (GC10), with an average of 0.37. Microsatellite markers generated number of alleles per locus ranged from 3 to 11, with an average of 5.62 alleles. The observed heterozygosity ranged from 0.359 (IBRC02) to 0.978 (YWLL08), and expected heterozygosity ranged from 0.449 (IBRC02) to 0.879 (YWLL08). Bottleneck analysis and curve showed that Bactrian camel population did not experience a low diversity. RAPD profiles were especially suitable for investigation population genetics. All primers generated novel and polymorphic fragments. Briefly, our results show that a multiplex PCR based on these markers can still be valuable and suitable for authentication of cell lines, investigating gene diversity and conservation genetic resources in Bactrian camel, while new technologies are continuously developed.     

Assessment of population genetic structure in common wild rice Oryza rufipogon Griff. using microsatellite and allozyme markers

The genetic structure of five natural populations of common wild rice Oryza rufipogon Griff. from China, was investigated with 21 microsatellite loci and compared to estimates of genetic diversity and genetic differentiation detected by 22 allozyme loci. Microsatellite loci, as expected, have much higher levels of genetic diversity (mean values of A = 3.1, P = 73.3%, Ho = 0.358 and He = 0.345) than allozyme loci (mean values of A = 1.2, P = 12.7%, Ho = 0.020 and He = 0.030). Genetic differentiation detected by microsatellite loci ( FST = 0.468, mean I = 0.472) was higher than that for allozyme loci ( FST =0.388, mean I = 0.976). However, microsatellite markers showed less deviation from Hardy-Weinberg expectation (Wright's inbreeding coefficient FIS = -0.069) than do allozymes ( FIS = 0.337). These results suggest that microsatellite markers are powerful high-resolution tools for the accurate assessment of important parameters in population biology and conservation genetics of O. rufipogon, and offer advantages over allozyme markers.     

滇杨种群遗传多样性与遗传结构

滇杨(Populus yunnanensis)是我国西南地区的特有树种, 具有速生、易无性繁殖、适应性强等优良特性, 是典型的南方型杨属树种。研究滇杨遗传多样性及种群结构对其种质资源的收集、保存和利用具有重要的意义。本研究从我国滇杨主要分布区云南和四川共采集了6个种群, 包括云南的昭通(ZT)、会泽(HZ)、嵩明(SM)、洱源(EY)、拉市海(LS)以及四川的美姑(MG), 共64个个体, 利用34对SSR分子标记和3对cpDNA叶绿体标记开展遗传多样性与遗传结构研究。SSR引物共检测到154个等位基因, 平均等位基因数为4.529, 观测杂合度(Ho)与期望杂合度(He)分别为0.552和0.472, 遗传分化系数(Fst)平均值为0.238, 多态性信息含量指数(PIC)平均值为0.421, 基因流(Nm)为0.806。滇杨的遗传结构分析(DAPC)与遗传距离的主坐标分析(PCoA)、UPGMA聚类分析均将6个种群划分为3个亚类: 第І亚类包括昭通种群、会泽种群和嵩明种群的4个个体, 第ІІ亚类包括嵩明种群的6个个体以及洱源种群和拉市海种群, 第III亚类为美姑种群; 嵩明种群包含第І和第ІІ两个亚类的混合遗传成分。3个cpDNA联合序列中共检测到35个变异位点, 分为13个单倍型, 其中单倍型H5在种群中分布最为广泛, 其余的单倍型均为种群特有的单倍型。分子方差分析(AMOVA)表明种群内的遗传变异大于种群间变异。研究表明滇杨不同种群的遗传分化具有地域性, 可选择就地保护; 昭通种群遗传多样性最高, 且包含7种叶绿体单倍型, 单倍型类型最多, 应优先保护。     

Isolation and characteristics of 10 microsatellite markers from the endangered coconut crab (Birgus latro)

The coconut crab (Birgus latro), an endangered marine-dispersed crustacean, is facing severe and probably accelerating population extinction worldwide, but biological information on its conservation remains deficient. In order to reveal the genetic structure of B. latro, 10 microsatellite loci were developed. A high degree of polymorphism was observed with a mean number of alleles per locus of 16.9. The mean expected heterozygosities were also high, ranging from 0.742 to 0.965. The observed heterozygosities ranged from 0.210 to 0.925. Departures from Hardy-Weinberg equilibrium were observed at five loci after the Bonferroni correction. These hypervariable markers will be utilized to study the genetic diversity and conservation of B. latro throughout its distribution range in the Pacific and Indian Oceans.     

利用微卫星标记分析贵州白水牛与普通水牛的遗传多样性与亲缘关系

利用11对微卫星引物对贵州白水牛和6个普通水牛群体的有效等位基因数、基因杂合度、多态信息含量和遗传距离进行了分析。结果表明,11个微卫星基因座在7个水牛群体中均存在多态性,可以用于水牛的遗传多样性评估。贵州白水牛和6个普通水牛群体的平均杂合度和平均多态信息含量分别为0·7450～0·7922和0·7021～0·7605。贵州白水牛和遵义普通水牛的亲缘关系最近,遗传距离为0·0910。由UPGMA聚类法得到的系统聚类图反映了贵州白水牛和6个普通水牛群体的亲缘关系远近程度,贵州白水牛没有单独聚为一类,而是与同分布区的遵义普通水牛首先聚类,然后依次与其余地区的普通水牛聚类。研究提出了贵州白水牛应是其产地的普通水牛群体中的一个突变群的见解,为开展贵州白水牛的遗传资源评估、保护与利用提供了分子水平的遗传学依据。     

Low genetic diversity and population connectivity fuel vulnerability to climate change for the Tertiary relict pine Pinus bungeana

Endemic species are important components of regional biodiversity and hold the key to understanding local adaptation and evolutionary processes that shape species distributions. This study investigated the biogeographic history of a relict conifer Pinus bungeana Zucc. ex Endl. confined to central China. We examined genetic diversity in P. bungeana using genotyping-by-sequencing and chloroplast and mitochondrial DNA markers. We performed spatial and temporal inference of recent genetic and demographic changes, and dissected the impacts of geography and environmental gradients on population differentiation. We then projected P. bungeana's risk of decline under future climates. We found extremely low nucleotide diversity (average π 0.0014), and strong population structure (global F_ST 0.234) even at regional scales, reflecting long-term isolation in small populations. The species experienced severe bottlenecks in the early Pliocene and continued to decline in the Pleistocene in the western distribution, whereas the east expanded recently. Local adaptation played a small (8%) but significant role in population diversity. Low genetic diversity in fragmented populations makes the species highly vulnerable to climate change, particularly in marginal and relict populations. We suggest that conservation efforts should focus on enhancing gene pool and population growth through assisted migration within each genetic cluster to reduce the risk of further genetic drift and extinction.     

基于全基因组SNP分子标记分析青海云杉遗传结构

以张掖龙渠青海云杉（Picea crassifolia）无性系种子园中的106个青海云杉亲本无性系为研究材料,采用改良CTAB法,提取的青海云杉基因组DNA,构建SLAF文库并进行高通量测序,之后分析SLAF测序数据和筛选SNP位点,基于邻接法分析得到样品的聚类情况。研究得出：将测序的水稻日本晴reads与其参考基因组进行比对,显示本试验双端比对效率为95.33%,说明SLAF建库成功。本研究中所测序列的Q30较高,碱基测 序错误率低,测序质量高;本研究共开发4 058 883个SLAF标签,标签的平均测序深度为21.21×。共开发 12 275 765个青海云杉SNP标记,各青海云杉样本的SNP数量为1 890 934~4 487 841。利用已开发的高质量青海云杉SNP标记,构建了106个青海云杉的系统发育树,发现来自不同种源的青海云杉在各组中分布比较均匀,不同种源的青海云杉多聚为一类。通过SNP标记和主成分分析,这些无性系来源于同一个祖先的可能性较大,表明无性系间亲缘关系相近。为今后遗传多样性的分析、遗传图谱的构建等提供了基础数据,也为青海云杉初级种子园去劣疏伐提供依据,为高世代种子园的营建奠定基础。     

基于简化基因组技术的云南蓝果树群体遗传分析

极小种群野生植物云南蓝果树是国家和云南省实施极小种群野生植物保护工程的代表性物种。为有效保护其遗传资源,本研究通过二代测序技术,对其进行简化基因组测序,开发一批特异性高的单核苷酸多态性标记,分析现存群体的遗传结构和遗传多样性。经过遗传变异检测,本次研究中共获得SNP位点98 498个,通过样品最低测序深度>2,样品缺失率<0.5,次要基因型频率（MAF）>0.05筛选以后,得到有效SNP位点6 309个。基于过滤后的SNP,运用生物信息学分析方法,对云南蓝果树完成了群体的遗传分析,其中：系统进化树分析将云南蓝果树划分为3大类,研究分析了云南蓝果树各分类的私人等位基因数目（Private）、平均观测杂合度（H_o）、平均期望杂合度（H_e）、核苷酸多样性（π）和平均近交系数（F_IS）5个遗传多样性参数;群体结构和主成分分析进一步证明了,云南蓝果树现存植株之间亲缘关系较远,遗传多样性差异较大,具有很高的遗传资源保存价值。本研究结果将为基于遗传管理的云南蓝果树就地保护、遗传资源保存和种群重建等保护工程提供科学依据。