采用高通量技术开发花鲈二碱基重复微卫星标记

花鲈是中国重要的捕捞和养殖对象之一。本研究利用高通量测序法开发花鲈微卫星标记,共获得60632个二至六碱基重复微卫星序列,从52747个二碱基重复序列中随机挑选150个位点进行引物合成及多态性检测,最终开发出27个具有多态性的微卫星标记。群体遗传学检测结果显示,27个微卫星标记的等位基因数为6~22(均值12.667),观测杂合度(Ho)为0.323~1.000(均值0.614),期望杂合度(He)为0.723~0.934(均值0.861),多态信息含量(PIC)为0.665~0.915(均值0.830),各位点PIC值均大于0.500,表明所开发的二碱基重复微卫星标记均具有较高的多态性。哈迪-温伯格平衡(HWE)检测结果显示,14个标记符合HWE,其中LM2-11与LM2-2、LM2-16之间存在连锁不平衡(p<0.050),其他符合HWE的标记间不存在连锁不平衡。Wilcoxon检验结果显示,花鲈群体并未偏离L-shaped分布,表明其经历过近期的遗传瓶颈效应。本研究开发的27个微卫星位点可为花鲈的分子标记辅助育种、增殖放流遗传效应评估、群体遗传学等研究提供有效的分子标记。     

中国特有种爆杖花的微卫星分子标记开发与评价

爆杖花（Rhododendron spinuliferum）是中国西南地区特有的观赏和药用植物。为了研究爆杖花和碎米花之间的杂交物种形成过程,本研究利用FIASCO方法对爆杖花进行微卫星引物开发,从100对引物中筛选出28个微卫星标记,其中22个为多态。利用爆杖花两个居群共24个个体对22个多态性位点进行分析,结果显示：每个位点具有2～5个等位基因,平均为3．4个,其观测杂合度和期望杂合度分别为0．083～0．792和0．153～0．744。对筛出的28个微卫星标记在碎米花的两个自然居群中也做了检测,结果显示：有22个微卫星标记成功扩增,其中20个有多态性;每个多态位点有2。6个等位基因,平均为3．2个,其观测杂合度和期望杂合度分别为0．000～0．833和0．117～0．736。开发的微卫星标记可用于爆杖花及其近缘物种的居群遗传学分析和杂交物种形成研究。     

中国特有种爆杖花的微卫星分子标记开发与评价

爆杖花（Rhododendron spinuliferum）是中国西南地区特有的观赏和药用植物。为了研究爆杖花和碎米花之间的杂交物种形成过程,本研究利用FIASCO方法对爆杖花进行微卫星引物开发,从100对引物中筛选出28个微卫星标记,其中22个为多态。利用爆杖花两个居群共24个个体对22个多态性位点进行分析,结果显示：每个位点具有2~5个等位基因,平均为34个,其观测杂合度和期望杂合度分别为0083~0792和0153~0744。对筛出的28个微卫星标记在碎米花的两个自然居群中也做了检测,结果显示：有22个微卫星标记成功扩增,其中20个有多态性;每个多态位点有2~6个等位基因,平均为32个,其观测杂合度和期望杂合度分别为0000~0833和0117~0736。开发的微卫星标记可用于爆杖花及其近缘物种的居群遗传学分析和杂交物种形成研究。     

微卫星标记的制备策略

微卫星标记作为一类重要的、成熟的分子遗传标记,凭借自身的高变异性、孟德尔遗传模 式、共显性遗传方式等优点,在遗传及其它相关领域发挥了十分重要的作用,而其主要的缺点是 必须知道所研究生物的微卫星序列及其旁侧序列。随着人类及模式生物基因组的深入研究,微 卫星标记的制备经历了从费时、费力、低效的传统法到周期短、效率高的富集法(选择性杂交法和 FIASCO法);从基因组构建文库筛选到公共数据库ESTs发掘;从实验室自制到花钱购买。总之, 随着方法与技术的不断改进,从目标生物中获得微卫星标记变得越来越简单、经济。     

微卫星标记对中国引进加州鲈养殖群体遗传多样性的分析

为了解我国引进加州鲈养殖群体的种群遗传结构和种质资源现状,本文发展了加州鲈的微卫星标记。用磁珠富集法获得加州鲈微卫星标记267个,设计并合成了85对微卫星引物对加州鲈的基因组进行遗传多样性分析,从中筛选出18对并从网上查询获得3对共21对引物对加州鲈的基因组DNA进行扩增。PCR扩增产物在8%的聚丙烯酰胺凝胶中分离,硝酸银染色,经紫外成像后对电泳条带进行统计分析。计算得到三个群体的平均等位基因数分别为2.3、2.3和2.1;多态信息含量（PIC）为0.0906—0.7480;平均杂合度观测值分别为0.384、0.396和0.356;杂合度期望值为0.375、0.403和0.368。统计结果初步表明3个不同群体平均杂合度处于中等偏低水平,遗传多样性不高。     

伊犁鲈微卫星位点的筛选及近缘物种通用性

为开发伊犁鲈(Perca schrenkii)分子标记用于鲈属鱼类种质资源保护,以伊犁鲈为材料,应用磁珠富集法进行了微卫星标记的筛选.从伊犁鲈尾鳍提取总DNA,进行酶切、接头连接、PCR扩增,再采用生物素标记(CA)15探针及生物素标记(TG)15探针对扩增产物进行杂交富集,经再次PCR扩增及T-A克隆,成功构建了伊犁鲈基因组微卫星富集文库.采用重复序列引物筛选获得阳性克隆,随机选取48个阳性克隆进行测序,测得序列46个,其中38个克隆含有微卫星序列,41个位点的微卫星重复数在8次以上.根据测得序列设计17对微卫星引物,均能在伊犁鲈群体中扩增获得目的条带.采用该17对引物对河鲈(P.fluviatilis)及黄金鲈(P.flavescens)群体样本进行扩增,10对引物具有通用性,其中6对在河鲈中具有高度多态性(PIC>0.5),5对在黄金鲈中具有高度多态性.     

异型花多态性微卫星标记的开发（英文）

异型花是青藏高原特有的一年生草本植物。为了更好地利用和保护这一重要的植物资源,本研究开发了异型花的多态性微卫星标记。首先,基于限制性位点相关DNA测序(RAD-seq)技术,开发了异型花14个多态性微卫星位点和2个单态性微卫星位点的引物;随后,对异型花的3个自然居群的57个个体的14个位点进行了分析。结果显示:14个位点观察到的等位基因数为4～7个,平均为5.071个;期望杂合度范围为0.003～0.312 (平均值为0.194),而观测杂合度范围为0～0.281 (平均值为0.047)。其中,仅有3对引物在龙胆科的椭圆叶花锚中具有通用性。本研究开发的多态标记对研究异型花种群结构和遗传多样性具有重要意义。     

花鲈水霉病及其病原的初步研究

山东省高青县水产试验场于1997年11月底开始发生花邮水霉病,此后不断有鱼因此病而死亡,12月中旬达到高峰,最严重时日死亡率达到1%左右。发病期间水温在5℃以下,有时水面还有薄冰。虽然水霉病是鱼类越冬期间的一种常见病,但由于该县地处氯化物水型的盐碱地区,在这种水体中发生的水霉病国内还未报道。为此,对该病进行了初步研究。       

长江江豚微卫星DNA分离的初步研究

为了开发物种特异性微卫星标记,本文采用一种改良的快速微卫星分离法(FIASCO)从长江江豚(Neophocaena phocaenoides asiaeorientalis)的基因组中筛选得到72条微卫星DNA序列。根据重复单元的排列特点,完美型、非完美型及复合型序列所占的比例分别为58.3%、22.2%和19.5%。选择其中30条序列设计PCR扩增引物,并用12个随机选择的长江江豚样品进行多态性筛选。初步结果表明其中14对引物的扩增产物稳定并且具有多态性;在每个座位上获得2-13个等位基因,平均等位基因数为5.87个;14个微卫星座位的平均观察杂合度(Ho)和期望杂合度(He)分别为0.560和0.709。本研究获得了长江江豚的第一批物种特异性微卫星座位及其扩增引物,这些微卫星标记将在后续的保护遗传学研究中发挥重要作用。       

花鲈群体的遗传变异

采用水平淀粉凝胶电泳技术对花鲈群体的遗传结构进行了研究 ,共检测了中国沿海花鲈 14 6尾和 40尾日本东京湾花鲈。其群体的多态位点比例为 0 2 667— 0 53 3 3 ,观测杂合度和预期杂合度分别为 0 0 2 11— 0 0 515和0 0 3 98— 0 0 797。中日花鲈在LDH ,GPI 1 ,GPI 2 基因位点上的等位基因接近完全置换。中国花鲈各群体之间的根井遗传距离为 0 0 0 0 4— 0 0 0 11,平均值约为 0 0 0 80 ;而中日花鲈间的根井遗传距离为 0 1870— 0 1954,平均值为 0 192 6。以上结果表明中国花鲈群体间遗传变异很小 ,中日花鲈间遗传变异远大于中国花鲈群体间的遗传距离。     

Isolation and characterization of polymorphic microsatellite markers for Alternaria alternata

Alternaria alternata is of major significance as a food and feed contaminant and is able to produce a range of mycotoxins that may elicit adverse effects in both animals and humans. We describe the isolation and characterization of five microsatellite markers for studying A. alternata. Marker polymorphism was screened in 64 isolates of A. alternata. The number of alleles per locus ranged from eight to 24, and allelic diversity ranged from 0.425 to 0.882. These markers will be useful in the study of relationships and population genetics amongst isolates of A. alternata.     

Isolation and characterization of polymorphic microsatellite markers in the red snow crab (Chionoecetes japonicus)

A total of 12 polymorphic microsatellite DNA loci were isolated from the red snow crab, Chionoecetes japonicus (Brachyura: Majidae), one of important fisheries resources in the Far East. The number of alleles observed at each locus ranged from two to 19, with the observed and expected heterozygosities of 0.125-0.875 and 0.156-0.949, respectively, suggesting these loci to be a useful molecular marker for population analysis in this species. Of the 12 loci, seven also were available for genotyping of the snow crab, Chionoecetes opilio, implying these loci as a useful molecular marker in the genus Chionoecetes.     

Isolation and characterization of 22 polymorphic microsatellite DNA markers of Japanese sea bass (Laterolabrax japonicus)

Japanese sea bass (Laterolabrax japonicus) inhabits the coast of East Asia and is cage‐cultured currently in China as well. Twenty‐two microsatellite DNA markers were developed and used to type 35 individuals collected along the Chinese coast. The number of alleles per locus ranged from three to 23. The expected heterozygosity ranged from 0.111 to 0.938, while the observed heterozygosity ranged from 0.114 to 0.914. All 22 loci are neutral and independent of each other; two deviated significantly from Hardy–Weinberg equilibrium. These microsatellite DNA markers are moderately informative, which will certainly facilitate the management and exploitation of the genetic resource of L. japonicus.     

Isolation of polymorphic microsatellite markers for British Euphrasia L.

Euphrasia species in Britain attract a large amount of conservation attention due to the recognition of numerous endemic taxa in what is essentially a species‐poor flora. To develop a set of research tools to investigate the evolutionary processes underlying this diversification, a membrane enrichment procedure has been used to isolate five polymorphic microsatellite loci from Euphrasia nemorosa (Pers.) Wallr. These loci amplify polymorphic products in several other British Euphrasia species.     

Isolation and characterization of polymorphic microsatellite markers in Anthyllis vulneraria

To study the reproductive system of the putative autogamous species Anthyllis vulneraria in Belgian calcareous grasslands, eight polymorphic microsatellite loci were developed using the sequence‐tagged microsatellite profiling technique and 35 plants were typed for all loci. No linkage disequilibrium was found between any pair of loci. Over the loci, the number of alleles ranged from two to five. Observed and expected heterozygosities ranged from 0.053 to 0.688 and from 0.053 to 0.631, respectively. The estimated F_IS values (F_IS = 0.031 and 0.122) are not in accordance with a predominantly selfing reproductive system.     

Isolation of polymorphic tetranucleotide microsatellite markers for the skink Mabuya affinis

We isolated 10 polymorphic microsatellite markers for the skink Mabuya affinis from genomic libraries enriched for (AAGG)_n and (AAAG)_n repetitive elements. The number of alleles ranged from eight to 13 per locus with the observed heterozygosity ranging from 0.60 to 0.92. These markers will be useful for analysis of questions concerning population genetic structure and models of speciation.     

Isolation and characterization of polymorphic microsatellite markers for the black rockfishSebastes inermis

Black rockfish (Sebastes inermis, Scorpaenidae) is a commercially important fisheries resource in Korea. However, no genetic diversity information is available for wild or released hatchery populations. To study the effects of stock enhancement programs and conservation biology, we developed microsatellite DNA markers fromS. inermis. We report the isolation and characterization of seven microsatellite loci, designated KSi221B, KSi239A, KSi25A, KSi252B, KSi193A, KSi259B, and KSi234, isolated using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. To characterize each locus, 30 individuals from a naturalS. inermis population in Yeosu, Korea, were genotyped. All loci except one, KSi193A, were polymorphic with an average of 9.5 alleles per locus (range 2–20). The mean observed and expected heterozygosities were 0.71 (range 0.43–1.00) and 0.76 (range 0.41–0.90), respectively. Significant deviation from Hardy-Weinberg equilibrium was observed at one locus, KSi221B. Such high variability indicates that these microsatellites should provide useful markers for studies of kinship and population genetics.     

Isolation and characterization of 10 polymorphic dinucleotide microsatellite markers for llama and guanaco

We isolated and characterized five polymorphic dinucleotide microsatellite markers from llama (Lama glama) and five from guanaco (Lama guanicoe). All loci were assayed on wild llamas and guanacos from Argentina, as all of the primers were able to amplify in both species.     

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.     

Isolation of polymorphic microsatellite markers for Begonia sutherlandii Hook. f.

Seven polymorphic microsatellite loci have been characterized for investigating population structure in the patchily distributed herb Begonia sutherlandii. Two loci (BSU3 and BSU4) exhibited population specific null alleles; primer redesign and allele sequencing for one of these loci showed two transition mutations in the original primer site. Two loci exhibited imperfect repeat polymorphisms due to single base pair indels in the flanking region (locus BSU6) and in the microsatellite region itself (BSU7). Transversion mutations were also found in the microsatellite region of locus BSU7. The remaining three loci amplified in all individuals tested and appeared to conform to a simple stepwise mutation pattern.