基于SLAF-seq技术的沙带鱼(Lepturacanthus savala)微卫星标记开发以及在近缘种中的通用性

沙带鱼是带鱼科中一种重要的海洋经济鱼类,广泛分布于印度-西太平洋海域。近年来,过度捕捞和渔业生态环境变化使沙带鱼的自然资源和种群结构受到了严重影响。因此,为了制定出科学合理的资源保护和管理措施,急需开发出大量多态的沙带鱼微卫星位点以促进其种群遗传学研究。本研究采用SLAF-seq技术,首次在沙带鱼基因组DNA中筛选出28个多态性微卫星位点。每个位点的等位基因数为2~18,平均值9.11;观测杂合度和期望杂合度分别为0.077~0.871(平均值0.634)和0.151~0.942(平均值0.711);各位点多态信息含量为0.137~0.919,平均值0.670。经过Bonferroni校正后,有3个位点显著偏离哈迪-温伯格平衡(HWE),并显示出较高的近交系数和无效等位基因频率,其余25个位点均符合HWE。这些多态性微卫星DNA标记可为沙带鱼的种群遗传资源研究提供有效的分子标记和技术支撑。此外,跨物种扩增结果表明,有19个微卫星标记在4种带鱼属鱼类中具有较好的通用性,这些标记可为阐明带鱼科属间或种间的系统进化关系提供新的研究手段和视角。     

梨小食心虫微卫星标记的扩增稳定性及多态性分析

【目的】筛选适合我国梨小食心虫Grapholita molesta种群遗传学研究的微卫星位点,并依据所筛选的微卫星位点进行梨小食心虫地理种群的遗传多样性分析。【方法】利用欧洲梨小食心虫和苹果蠹蛾Cydia pomonella种群中已报道的11个微卫星位点, 分析各位点在我国12个种群257头梨小食心虫样本中的扩增稳定性,再进行其多态性分析,筛选适合的位点,然后进行种群遗传多态性分析。【结果】在分析的11个微卫星位点中, 位点Gm01, Gm03, Gm04和Cyd15无法稳定扩增; 位点Gm05扩增成功率较低, 位点Gm07遗传多态性较低; 而位点Gm02, Gm06, Gm08, Gm09和Gm10等扩增效果稳定且遗传多态性丰富。这5个稳定扩增的微卫星位点平均等位基因数量（N_A）为7.417~12.500, 平均观察杂合度（H_o）为0.366~0.655, 平均期望杂合度（H_e）为0.642~0.846, 多态信息含量（PIC）为0.800~0.935。【结论】本研究成功筛选出位点Gm02, Gm06, Gm08, Gm09和Gm10等5个微卫星位点。基于这5个微卫星位点标记的结果显示, 山东和陕西不同梨小食心虫地理种群均具有丰富的遗传多样性。 这5个位点可以适用于我国梨小食心虫种群的进一步遗传分析研究。     

基于粪便DNA的岩羊微卫星引物的筛选及个体识别

贺兰山保护区岩羊Pseudois nayaur种群数量近年来增长很快,为了解贺兰山保护区的岩羊种群的遗传健康状况,从而更好地保护和管理岩羊种群,需要对岩羊种群的遗传多样性进行研究。在其近缘物种中选出36对微卫星引物对收集到的岩羊粪便DNA样品进行PCR扩增。结果发现,有9对引物在岩羊粪便DNA中扩增出了多态性位点。各位点基因杂合度介于0.26~0.95之间,平均杂合度为0.48,各位点多态信息含量介于0.23~0.68之间,平均多态性为0.48。     

金黄地鼠封闭群SPF化后的微卫星遗传学检测

目的检测和评估金黄地鼠封闭群SPF化后的遗传学变化,为SPF金黄地鼠遗传质量的控制提供技术资料。方法应用小鼠和大鼠的微卫星标记筛选适于金黄地鼠遗传检测的微卫星标记,并结合微卫星荧光标记-半自动基因分型技术,对成都生物制品研究所的SPF级金黄地鼠及其来源的普通级金黄地鼠进行遗传检测,计算其群体遗传学参数。结果对18个小鼠和6个大鼠微卫星标记进行了筛选,分别有2个小鼠和2个大鼠微卫星标记在金黄地鼠种群中具PCR扩增多态性。4个检测的微卫星位点在普通级金黄地鼠和SPF金黄地鼠种群分别发现25和20个等位基因,两群体的期望杂合度分别为0.4979和0.5048,其群体遗传多样性无显著差异;群体间的不同微卫星位点FST范围从0.0095到0.0367,平均为0.0315,表明两群体间的遗传分化很弱,其遗传多样性主要存在于群体内;Nei(1972)遗传距离和Nei(1978)无偏遗传距离分别为0.0678和0.0570,表明了2群体之间很高的遗传相似度和非常近的亲缘关系;Hardy-Weinberg平衡检验表明普通级和SPF金黄地鼠分别有2个和3个位点偏离遗传平衡,且偏离位点均表现为杂合子缺陷。结论该SPF金黄地鼠基本保持了其来源普通级黄地鼠的遗传多样性,两群体间遗传分化程度和遗传差异很小,但应进一步加强其封闭群的繁育控制,保持其遗传稳定性。     

秦川母牛群体遗传特性的微卫星标记研究

为了从DNA分子水平揭示秦川牛群体遗传多态性和群体遗传结构,寻找可用于秦川牛的微卫星标记,本研究选择了12个普通牛（Bos taurus）微卫星标记检测了90头秦川母牛各微卫星位点的遗传变异及多态性。结果表明,在秦川母牛群体中,12个微卫星位点共检测到了247个等位基因,各位点的等位基因数在13（INRA005）～33个（HEL13）之间,平均每个微卫星位点的等位基因数为21个;总有效等位基因数和平均每个位点平均有效等位基因数（Ne）分别分为142．6229和11．8852。各位点平均基因频率取样方差（V（pij））为2．6036×10^-4。12个微卫星位点平均观察杂合度（Ho）和平均期望杂合度（He）在0．7842（INRA005）～0．9775（BM315）和0．7952（BM315）～0．9446（HEL13）之间。12个位点平均多态信息含量（PIC）在0．7653（INRA005）～0．9420（HEL13）之间,平均为0．8965．12个微卫星位点均属于高度多态位点,这表明秦川母牛群体中所检测各微卫星位点具有丰富的遗传多态性,具备较大的选择潜力。12个微卫星位点的平均固定指数（F）为-0．0076,即各位点杂合子的缺陷度不高,即偏离Hardy—Weinberg平衡的程度不大。     

我国粘虫种群的微卫星位点筛选及遗传多样性分析

【目的】筛选适用于我国粘虫Mythimna separata种群遗传学研究的微卫星位点,从分子水平揭示粘虫种群的遗传多样性。【方法】利用已报道的微卫星标记及本实验室粘虫转录组测序的SSR序列,采用PCR产物荧光标记与自动扫描分型方法,分析各位点在我国河南、陕西、山西3个省份的7个粘虫地理种群200头试虫中的扩增稳定性和多态性。【结果】7个粘虫地理种群有7个位点能稳定扩增且具有较高的多态性。这7个位点等位基因丰富度(Ar)为4.167~12.402,观测杂合度(Ho)平均为0.640,期望杂合度(He)平均为0.752,多态信息含量(PIC)为0.547~0.884;各位点均存在无效等位基因且偏离哈迪-温伯格平衡,所有成对位点不存在显著连锁不平衡情况。【结论】从来自河南、陕西、山西的7个不同粘虫地理种群中成功筛选了7个能稳定扩增的SSR位点,且在这7个不同的粘虫地理种群中均具有较高的多态性,可用于我国粘虫种群遗传结构研究。粘虫不同地理种群间基因交流频繁,基因交流阻止了由遗传漂变引起的群体间分化,不同地理种群间遗传分化很低甚至不存在遗传分化。     

基于转录组平台的蛤仔微卫星标记筛选

以菲律宾蛤仔转录组测序所得拼接序列为基础,采用MISA软件进行微卫星分析,对其中的145个微卫星位点进行引物设计,得到具有清晰扩增条带的微卫星位点58个。对大连庄河野生蛤仔群体的扩增结果表明,18个位点显示单态性,40个位点表现为多态性。该群体40个多态性微卫星位点得到的等位基因数在2—6之间,平均等位基因数为3.4250±0.9718,观测杂合度和期望杂合度分别在0.0000—1.0000和0.0615—0.7996之间,平均值分别为0.2727±0.2272和0.4739±0.1902,群体平均Nei指数为0.4664±0.1872。多态信息含量(PIC)在0.0586—0.7529之间,平均值为0.4148±0.1707,其中16个微卫星位点的PIC值大于0.5,为高度多态性,15个位点0.25PIC0.5,为中度多态性,其余9个为低度多态性。经Sequential Bonferroni校正的Hardy-Weinberg平衡检验,有10个位点尚未偏离平衡。基于转录组平台筛选微卫星标记的方法,在很大程度上推动了DNA分子标记的开发。研究开发的微卫星标记可用于蛤仔群体遗传学、遗传连锁图谱构建及其他相关研究,为蛤仔分子标记辅助育种及群体种质保护等工作提供技术支持。     

大鸨东方亚种遗传多样性的微卫星分析

为了探索东方亚种数量正在减少的原因和制定科学、有效的保护措施,利用微卫星DNA对大鸨东方亚种(Otistardadybowskii)的遗传多样性进行了研究。应用3个大鸨指名亚种的微卫星位点和13个波斑鸨的微卫星位点扩增了47个个体的基因组DNA,筛选出8个具有多态性的微卫星。其中有3个微卫星的多态性较低,其余5个微卫星的多态性较高。各位点的观察杂合度为0.0435-1.0000,平均杂合度(h)为0.6595;各位点多态信息含量(PIC)为0.0416-0.8520,平均为0.5497;有效等位基因数(E)为1.04-7.46,平均为3.61。4个位点符合HardyWeinberg平衡,4个位点偏离了HardyWeinberg平衡。多方面比较发现,大鸨东方亚种遗传多样性很低,且低于指名亚种,这可能由于其种群较小、历史遗传瓶颈作用、生境破碎化、分布地域紧缩等原因造成的。     

白条草蜥多态性微卫星位点的分离与鉴定

白条草蜥(Takydromus wolteri)是一种年产多窝卵的蜥蜴。为了对其婚配制度、同一雌性个体所产卵的窝内和窝间的父权状况、种群的遗传结构和物种的进化历史等研究内容进行探讨,本研究筛选出白条草蜥的9个具有高度多态性的微卫星位点。微卫星位点筛选自包含(AC)n和(ATAG)n重复片度的微卫星富集文库。在白条草蜥安徽滁州种群的16～32个个体中对上述位点进行检测后发现,上述座位的等位基因数目范围为12～20个,期望杂合度范围为0.894～0.955,观测杂合度范围0.565～0.938,表明这些微卫星标记具有良好的遗传多样性,它们将在白条草蜥的种群遗传结构、基因流水平、种群分化和婚配制度的研究中发挥重要作用。     

宽口光唇鱼微卫星位点的筛选与特征分析

通过FIASCO法(Fast Isolation by AFLP Sequences Containing Repeats)筛选宽口光唇鱼Acrossocheilus monticola(Günter)基因组微卫星位点,利用生物素标记的寡核苷酸探针(AC)8、(CT)8、(GGT)8、(GATA)8和(GATT)7首次成功构建宽口光唇鱼基因组微卫星富集文库。从文库中共筛选495个克隆测序,成功设计163对微卫星引物,经PCR扩增检测,获得了18个多态性微卫星标记。利用这18对多态性引物,分析了赤水河赤水市宽口光唇鱼种群的遗传多样性,数据显示:18对多态性微卫星引物的等位基因数为8～31个,平均等位基因数为19.6个,平均期望杂合度为0.8701,平均观测杂合度为0.8312,其中引物Am07、Am35、Am47、Am69、Am78和Am127显著偏离哈迪温伯格平衡(P<0.05),以上数据表明赤水河赤水市宽口光唇鱼种群的遗传多样性水平较高。筛选的微卫星标记对于宽口光唇鱼的遗传背景分析和生物种质资源的保护有重要作用。     

Development and characterization of 70 novel microsatellite markers for the sea cucumber (Apostichopus japonicus)

The sea cucumber (Apostichopus japonicus) is an important item in Asian cuisine. It is currently produced through aquaculture, especially in China, after being overexploited in the wild in the 1990s. We isolated 70 novel polymorphic microsatellite loci using an enrichment-colony hybridization protocol. All loci were characterized in 48 individuals from a natural population in Rongcheng (Shandong, China) using genomic DNA isolated from muscle tissue. The number of alleles ranged from 2 to 17 (mean 7.0), and the observed and expected heterozygosities varied from 0.0010 to 1.0000 and from 0.2125 to 0.9477, respectively. Thirty-one of the 70 loci exhibited departure from Hardy-Weinberg equilibrium. These microsatellite markers should be useful resources for population genetic studies and for molecular marker-assisted breeding of A. japonicus.     

Seventeen polymorphic microsatellite markers developed for the Javelin goby, Synechogobius hasta (Gobiidae)

Synechogobius hasta is an important commercial marine fish with distinctive features of rapid growth and short lifespan. We isolated and characterized 17 microsatellite markers for S. hasta using a (GT)(13)-enriched genomic library. Polymorphism was assessed in 48 individuals from a single population collected from the northern coastal waters of the Yellow Sea. The number of alleles per locus ranged from 2 to 23, with a mean of 11.3. The observed and expected heterozygosities ranged from 0.130 to 1.000 and from 0.123 to 0.939, with means of 0.758 and 0.774, respectively. Fourteen of 17 loci conformed to Hardy-Weinberg equilibrium and no significant linkage disequilibrium between locus pairs was detected. These microsatellite markers will be useful for population genetic structure analyses.     

Isolation and characterization of 19 highly polymorphic microsatellite markers in the devil stinger, Inimicus japonicus (Synanceiidae)

Inimicus japonicus, the devil stinger, has an extensive distribution along the coast of China, Japan and the Korean Peninsula. Nineteen highly polymorphic microsatellite markers were isolated and characterized in I. japonicus. Twenty-eight individuals from a wild population were tested for polymorphism using this set of polymorphic microsatellite markers. The number of alleles per locus ranged from 4 to 14. The ranges of observed and expected heterozygosity were 0.500-0.892 and 0.521-0.910, respectively. Significant deviations from Hardy-Weinberg equilibrium were detected at two loci. To the best of our knowledge, these were the first microsatellite loci characterized from the Synanceiidae; they can be used for estimating genetic diversity, population structure studies, parentage analysis, genetic linkage map construction, germplasm classification and identification, gene identification, quantitative trait loci mapping, and marker-assisted selection in breeding of I. japonicus and other species of this family.     

Development of 21 polymorphic microsatellite markers for the black-banded sea krait, <Emphasis Type="Italic">Laticauda semifasciata</Emphasis> (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species

The genus Laticauda (Reptilia: Elapidae), commonly known as sea kraits, is venomous marine amphibious snakes distributed throughout the south and southeast Asian islands and mostly found in coastal waters. To facilitate genetic studies, we have developed microsatellite loci for L. semifasciata using the 454 GS-FLX pyrosequencing technique. A total of 65,680 sequences containing a minimum of five repeat motifs were identified from 451,659 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5%) produced strong PCR products, of which 21 were polymorphic among 36 samples of L. semifasciata. All loci exhibited high genetic variability, with an average of 7.38 alleles per locus, and the mean observed and expected heterozygosities were 0.73 and 0.76, respectively. The cross-species amplification of these loci in two laticaudine species, L. colubrina and L. laticaudata, revealed a high transferability (78.6%) and polymorphism (59.5%) of the loci. Our work demonstrated the utility of next-generation 454 sequencing as the rapid and cost-effective method for development of microsatellite markers. The high level of polymorphism in these microsatellite loci will be useful for the detection of population subdivision and the study of migration, gene flow, relatedness and philopatry of L. semifasciata and other laticaudine species.     

Development of microsatellite markers in the marine phytoplankton Karenia mikimotoi (Dinophyceae)

The marine phytoplankton, Karenia mikimotoi, causes severe red tides which are associated with mass mortality of marine fish, and have expanded their distributions in the coastal waters of western Japan. To assess the dispersal mechanism, a population genetic study using highly polymorphic genetic markers is one of the crucial approaches. Here we developed 12 polymorphic microsatellite markers from K. mikimotoi. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from 5 to 23, and the estimate of gene diversity was from 0.551 to 0.933 across the 12 microsatellites. We consider these loci potentially useful for detailing the genetic structure and gene flow among K. mikimotoi populations.     

Polymorphic microsatellite loci for the Japanese anchovy Engraulis japonicus (Engraulidae)

We isolated and characterized 14 polymorphic microsatellite loci in the Japanese anchovy (Engraulis japonicus) using a (GT)(13)-enriched genomic library. The numbers of alleles per locus ranged from 6 to 31, with a mean of 17.8. The observed and expected heterozygosities ranged from 0.180 to 0.949 and from 0.172 to 0.966, with means of 0.731 and 0.825, respectively. All 14 loci were in Hardy-Weinberg equilibrium and no significant linkage disequilibrium between loci pairs was detected. These microsatellite markers will be useful for analyzing the population genetic structure and gene flow of E. japonicus.     

Isolation and characterization of nine polymorphic microsatellite markers of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae)

Sebastiscus marmoratus is a typical sedentary fish and usually lives at the bottom of the coastal zone from Japan to the East China Sea. Next-generation sequencing platform was used to obtain microsatellite loci sequences of S. marmoratus, among which forty-eight perfect microsatellite repeats were suitable for primer design. Nine polymorphic microsatellite markers were exploited and characterized for it. The allele number of all polymorphic microsatellite markers ranged from 4 to 12. Expected and observed heterozygosity varied from 0.302 to 0.909 and from 0.190 to 0.625, respectively. In further studies, the genetic variation and population structure of this species would be investigated using these microsatellite loci.     

Development and characterization of novel microsatellite markers from the olive ridley sea turtle (Lepidochelys olivacea)

Olive ridley turtles, although widely distributed globally and in Indian coastal waters, have undergone declines in recent years due to anthropogenic factors, particularly fishery‐related mortality. Assessment of genetic variability in existing populations is critical to the development of effective conservation strategies. Here we describe the development of six highly polymorphic microsatellite loci from a simple sequence repeat‐enriched genomic DNA library of olive ridley turtle. Characterization of five of these loci using 83 individual olive ridley turtles revealed eight to 24 alleles per locus, high observed and expected heterozygosity values and broad cross‐species amplifications. The sixth microsatellite was found to be monomorphic in the olive ridley samples but was polymorphic in two related marine turtle species. These microsatellites thus provide efficient genetic markers to understand the population structure, phylogeography and species relationships of olive ridley and other marine turtle species.     

Microsatellite development for the endangered Yangtze sturgeon (Acipenser dabryanus Duméril, 1869) using 454 sequencing

The Yangtze sturgeon (Acipenser dabryanus) is an endemic species in China. Using 454 sequencing, eight polymorphic tri‐, tetra‐, penta‐, and hexanucleotide microsatellite loci were isolated in this study. The raw sequence data from a one‐eighth run of 454 sequencings were 38.0 Mbp containing 94 222 reads/sequences. Of 80 microsatellite loci, only eight loci were polymorphic in a population of 30 individuals. The number of alleles per locus ranged from 4 to 14 (mean 7.62), and the observed heterozygosities varied between 0.46 and 0.88 (mean 0.74). Cross amplification was tested in congeneric species Acipenser sturio and Acipenser sinensis. These new microsatellite markers will be useful for further studies on genetic variation, parentage analysis, and conservation management for this critically endangered species.     

Restricted dispersal in a continuously distributed marine species: common bottlenose dolphins Tursiops truncatus in coastal waters of the western North Atlantic

The marine environment provides an opportunity to examine population structure in species with high dispersal capabilities and often no obvious barriers to genetic exchange. In coastal waters of the western North Atlantic, common bottlenose dolphins, Tursiops truncatus, are a highly mobile species with a continuous distribution from New York to Florida. We examine if the highly mobile nature coupled with no obvious geographic barriers to movement in this region result in a large panmictic population. Mitochondrial control region sequences and 18 microsatellite loci indicate dolphins are partitioning the habitat both latitudinally and longitudinally. A minimum of five genetically differentiated populations were identified among 404 samples collected in the range of New Jersey to northern Florida using both genetic marker types, some inhabiting nearshore coastal waters and others utilizing inshore estuarine waters. The genetic results reject the hypothesis of a single stock of coastal bottlenose dolphins put forth after the 1987–1988 epizootic that caused a large‐scale die‐off of dolphins and suggest instead the disease vector was transferred from one population to the next as a result of seasonal migratory movements of some populations. These coastal Atlantic populations also differ significantly from bottlenose dolphin samples collected in coastal waters of the northern Gulf of Mexico, implying a long‐term barrier to movement between the two basins.