青鱼微卫星标记的开发与特性分析

青鱼(Mylopharyngodon piceus)是中国最为重要的淡水养殖鱼类。开发青鱼的微卫星标记能为青鱼的遗传多样性分析提供更多工具。本研究使用磁珠富集法,利用生物素探针(CA)10和(GACA)6,富集得到青鱼基因组微卫星片段,进一步通过设计微卫星引物检验其在青鱼原种群体中的有效性和多态性水平。结果显示,所构建文库中849个克隆含有微卫星序列,通过利用PCR技术在吴江原种青鱼36个个体中进行多态性筛选,获得了25个多态性微卫星位点。其平均等位基因数(Na)和有效等位基因数(Ne)分别为7.08和3.526,平均观测杂合度(Ho)和期望杂合度(He)分别为0.602和0.619,平均多态信息含量(PIC)为0.568。其中,Mp23、Mp27和Mp35这3个位点极显著偏离哈迪-温伯格平衡(P 0.01)。本研究开发的微卫星标记能为青鱼种质资源的评价和保护等研究提供工具。     

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。     

大黄鱼连续两代雌核发育群体的微卫星标记分析

通过对大黄鱼(Pseudosciaena crocea)异质雌核发育一代群体(meio-G1)与二代群体(meio-G2)微卫星位点的纯合度进行分析,研究异质雌核发育对大黄鱼基因纯化的效率。结果显示:meio-G1和meio-G215个微卫星座位的平均纯合度分别为0.661和0.803,纯合位点比例最高个体分别为0.867(13/15)和0.933(14/15),两个群体内个体间的平均相似系数分别为0.5903和0.8672,最高分别达0.9286和1.0(遗传距离为0.0741和0),远高于两性交配繁殖群体(平均纯合度0.376,平均相似系数0.4687,个体间最小遗传距离0.2288);其中meio-G2群体有7个位点(46.7%)已经完全纯合固定,并与普通养殖群体产生较明显的遗传分化;表明人工诱导异质雌核发育可大大加速大黄鱼大多数基因位点的纯合,是快速建立高纯品系的有效手段。但不同位点的纯合度差异很大,部分位点在异质雌核发育后代中迅速纯合,在meio-G1中就达到很高的纯合度,而有些位点则在meio-G1和meio-G2中仍保持很高的杂合度;meio-G1和meio-G2群体中不同个体纯合位点比例差异也很大。研究培育的雌核发育群体为大黄鱼进一步选育提供了良好的遗传材料。       

利用微卫星遗传标记探讨达氏鳇的多倍体倍性

鲟形目(Acipenseriformes)鱼类是一类多倍体起源的鱼类,种问易于杂交,且由于存在大量微型染色体,其染色体数目和倍性也难于确定.到目前为止包括达氏鳇(Huso dauricus)在内的一些鲟鱼物种基因组大小和染色体倍性仍旧存在争议.本实验采用微卫星遗传标记技术,通过观察9个微卫星位点的等位基因数目,发现达氏鳇在大部分位点中显示的倍性大于二倍性,同时利用多倍体分析软件POLYSAT推断了26尾达氏鳇的个体倍性.结果表明,26尾达氏鳇中有20尾显示为六倍体,占总数的76.92％;4尾显示为八倍体,占总数的15.38％;2尾显示为四倍体,占总数的7.79％.因此,我们认为达氏鳇应该为八倍体物种,结果中出现的四倍体和六倍体个体,是由于这些个体在我们选取的微卫星位点中存在部分纯合等位基因,导致了多数个体显示为六倍体.这一结论与近年来支持达氏鳇为进化性八倍体物种的研究结果一致.     

吉富罗非鱼第二十一代选育群体微卫星标记研究

采用10对微卫星引物对广东省化州光辉养殖场有限公司新选育的吉富罗非鱼第21代群体进行遗传多样性研究。结果显示:10对引物在48尾吉富罗非鱼中共检测到57个等位基因(Na),平均值为5.700;有效等位基因(Ne)平均值为3.726;观测杂合度(Ho)在0.750~1之间,平均值为0.896;多态信息含量(PIC)在0.390~0.797之间,平均值为0.640。表明该吉富罗非鱼选育群体具有较高的遗传多样性水平,可作为选育和养殖基础群体。     

基于高通量测序的厚唇裸重唇鱼微卫星分子标记筛选

本研究对厚唇裸重唇鱼(Gymnodiptychus pachycheilus)基因组进行Illumina HiSeq高通量测序，共获得85 905条序列，包含567 200个微卫星位点，从中筛选出15个微卫星位点，采用雅砻江新龙种群和黄河渭河种群对其多态性进行了验证。新龙种群中，15个位点的平均等位基因数为6.9(3 ~ 13)，观测杂合度(Ho)为0.712 4，多态信息含量(PIC)为0.630 3，有8个位点显著偏离哈迪-温伯格平衡，1对位点表现出连锁。渭河种群中，15个位点的平均等位基因数为8.4(4 ~ 16)，观测杂合度(Ho)为0.719 5，多态信息含量(PIC)为0.680 7，有7个位点显著偏离了哈迪-温伯格平衡，4对位点表现出连锁。筛选获得的15个微卫星位点多态性较高，适合用于厚唇裸重唇鱼种群遗传学研究。     

中国特有单种属血水草的微卫星分子标记开发与评价

血水草(Eomecon chionantha Hance)是中国亚热带地区特有的单种属植物。本研究采用(AG)15、(AC)15两种生物素探针及磁珠富集法从血水草基因组中开发并筛选出13个微卫星位点,且在该物种中能进行稳定的PCR扩增并表现出多态性。利用筛选出的13个SSR多态性标记对血水草4个野生居群24个个体进行PCR扩增,结果显示每个微卫星位点的等位基因数为2~7个,观测杂合度(HO)和期望杂合度(HE)分别为0.125~0.792和0.299~0.691。说明这些SSR标记可用于血水草遗传多样性的后续研究,并揭示该物种地理分布格局形成的原因,有助于保护和利用中国亚热带地区的血水草野生资源。     

甘肃金鳟遗传资源评价及其与日本金鳟和道氏虹鳟遗传分化研究

为分析甘肃金鳟遗传资源状况, 研究利用18 个微卫星DNA 标记, 对甘肃金鳟、日本金鳟和道氏虹鳟3 群体各50 尾个体进行了遗传分析, 以评价甘肃金鳟遗传资源状况并研究其与日本金鳟和道氏虹鳟遗传分化情况。结果表明甘肃金鳟具有较丰富的遗传变异, 15 个微卫星DNA 座位共获得了51 个等位基因, 有效等位基因数(Ne)为1.13, 多态信息含量(PIC)为0.440, 平均观察杂合度(Ho)和期望杂合度(He)分别为0.430 和0.498。在Hardy-Weinberg 平衡条件下, 进行了P 检验, 发现3 个群体均有位点发生了显著偏离; 对3 个群体进行了Fst 值计算, 表明群体间存在着一定程度的遗传分化, 但遗传变异绝大部分存在于品种内。综合分析,认为甘肃金鳟遗传性状基本稳定, 遗传变异度较高, 遗传多样性相对丰富, 具有较大的遗传潜力, 与道氏虹鳟和日本金鳟群体间分化明显, 人工选育对群体的遗传变异产生了一定的影响。研究结果对甘肃金鳟的保种、选育及其合理的推广应用具有一定的指导作用。       

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。     

虾夷扇贝遗传结构及微卫星标记与经济性状相关分析

选用30个多态性微卫星分子标记对大连大长山(96个)、大连獐子岛(96个)及日本青森县陆奥湾(96个)3个地区虾夷扇贝养殖群体(Patinopecten yessoensis)进行遗传多样性分析。结果显示,30个基因座共检测到198个等位基因(Na),平均等位基因数为6.63个,每个座位检测到的等位基因数为3-12,有效等位基因数(Ne)每个位点平均为4.70个,观测杂合度(Ho)平均值0.58,期望杂合度(He)的平均值为0.75,30个位点的平均多态信息含量为0.703,说明3个地区虾夷扇贝群体遗传多样性水平较高。运用统计软件SPSS11.5对30个微卫星标记与大长山群体生长性状相关性进行了分析。结果表明,位点DQ221714和FJ262378与壳长、壳高、壳宽和鲜重显著相关(P0.05)位点;位点FJ262372与壳宽、软体部重和闭壳肌重显著相关;位点FJ262369与软体部重及闭壳肌重显著相关。对差异显著的位点进行不同基因型间的多重比较,找到了与虾夷扇贝生长性状相关的基因型,进一步将所得显著性标记对大长山虾夷扇贝个体最大的17个和个体最小的13个基因组DNA进行检测,验证其准确性。结果表明,位点DQ221714在两组虾夷扇贝中存在特异性条带,可作为QTL定位候选标记。     

Characterization of 35 novel microsatellite DNA markers from the duck (Anas platyrhynchos) genome and cross-amplification in other birds

In order to study duck microsatellites, we constructed a library enriched for (CA)n, (CAG)n, (GCC)n and (TTTC)n. A total of 35 pairs of primers from these microsatellites were developed and used to detect polymorphisms in 31 unrelated Peking ducks. Twenty-eight loci were polymorphic and seven loci were monomorphic. A total of 117 alleles were observed from these polymorphic microsatellite markers, which ranged from 2 to 14 with an average of 4.18 per locus. The frequencies of the 117 alleles ranged from 0.02 to 0.98. The highest heterozygosity (0.97) was observed at the CAUD019 microsatellite locus and the lowest heterozygosity (0.04) at the CAUD008 locus, and 11 loci had heterozygosities greater than 0.50 (46.43%). The polymorphism information content (PIC) of 28 loci ranged from 0.04 to 0.88 with an average of 0.42. All the above markers were used to screen the polymorphism in other bird species. Two markers produced specific monomorphic products with the chicken DNA. Fourteen markers generated specific fragments with the goose DNA: 5 were polymorphic and 9 were monomorphic. But no specific product was detected with the peacock DNA. Based on sequence comparisons of the flanking sequence and repeat, we conclude that 2 chicken loci and 14 goose loci were true homologous loci of the duck loci. The microsatellite markers identified and characterized in the present study will contribute to the genetic map, quantitative traits mapping, and phylogenetic analysis in the duck and goose.     

Isolation and characterization of microsatellite loci from forest musk deer (Moschus berezovskii)

This study reported the isolation and characterization of eight polymorphic microsatellite loci in endangered forest musk deer Moschus berezovskii. An improved enrichment protocol was used to isolate microsatellites, and polymorphism was explored with samples from wild musk deer population collected in Miyalo of Sichuan Province in China. Approximately 70% of clones from the genomic library constructed in current study contained dinucleotide (AC) repeats. Eight microsatellite loci amplified were highly polymorphic within forest musk deer population. The number of alleles per locus ranged from 6 to 14, and the observed and expected heterozygosities ranged from 0.41 approximately 1.0 and from 0.8 approximately 0.9, respectively. The average polymorphic information content (PIC) value for these markers was 0.82. This demonstrated that the eight microsatellite loci developed here are highly polymorphic, and can be used as genetic markers for further investigation of musk deer. Also, the results showed that the musk deer distributed in Miyalo had a relatively higher level of genetic variation.     

Development of microsatellite markers in Rhizophora stylosa using a dual‐suppression‐polymerase chain reaction technique

Rhizophora stylosa is an ecologically important mangrove tree species that is found in tropical and subtropical regions. We isolated five polymorphic microsatellite loci from R. stylosa using a dual‐suppression‐polymerase chain reaction technique. These loci provided microsatellite markers with polymorphism of four alleles in each locus for overall population. The mean expected heterozygosities ranged from 0.113 to 0.473.     

Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

Development of microsatellite markers for the wood cricket, Nemobius sylvestris (Orthoptera: Gryllidae)

Thirty-four novel microsatellite markers developed for wood cricket (Nemobius sylvestris) were tested and optimized. Twenty-five microsatellite loci were polymorphic, exhibiting between two and nine alleles. Observed heterozygosities ranged from 0.038 to 0.925. The microsatellites were also tested in a species belonging to another genus of the Gryllidae family (Gryllus bimaculatus). Two markers produced clear banding patterns with the expected product size. These markers will be used to study the effects of forest fragmentation on genetic connectivity using wood cricket as a model species.     

Isolation and characterization of polymorphic microsatellite loci in the field vole,Microtus agrestis,and their cross‐utility in the common vole,Microtus arvalis

We developed nine polymorphic microsatellite loci for the field vole, Microtus agrestis. The number of alleles ranged from five to 15 and observed heterozygosities ranged from 0.40 to 1.00. We also tested the microsatellite loci for amplification and polymorphism in the congeneric species Microtus arvalis. Five of the nine loci were successfully analysed in this species. The microsatellite markers will be employed in studies of reproductive success and fine‐scale spatial genetic structure.     

Isolation and characterization of 12 microsatellite loci for Rhamnus alaternus (Rhamnaceae)

Twelve polymorphic microsatellite loci were developed for Rhamnus alaternus using an enriched-library approach. We detected 69 alleles in 49 individuals genotyped (mean number of alleles per locus was 4.79) in two different populations. The values of observed and expected heterozygosities ranged from 0.045 to 0.963 and 0.089 to 0.873 respectively. Levels of polymorphism and the exclusionary power of the developed markers render them readily applicable for studies of contemporary pollen and seed gene flow through parentage analyses.     

Isolation and characterization of microsatellite markers from the great hornbill, Buceros bicornis

Thirteen polymorphic microsatellite markers were isolated and characterized from the great hornbill, Buceros bicornis. In analyses of 20 individuals, the numbers of alleles per locus varied from two to 11. The expected and observed heterozygosities ranged from 0.22 to 0.88 and from 0.20 to 1.00, respectively. The mean polymorphic information content was 0.62. Among these, three loci deviated from the Hardy-Weinberg equilibrium. However, no significant genotypic disequilibrium was detected between any pair of loci. These microsatellite markers are useful for the population genetic study of the great hornbill.     

Identification and characterization of ten polymorphic microsatellite loci in the red panda Ailurus fulgens

Ten polymorphic microsatellite loci were characterized from two genomic DNA-enriched libraries of the red panda (Ailurus fulgens). The number of observed alleles among 35 samples of red pandas ranged from five to 12. Observed and expected heterozygosities were 0.286–0.971 and 0.443–0.894, and the mean polymorphic information content was 0.712. All loci followed Hardy–Weinberg expectations except Aifu-14 and Aifu-16, which may due to the presence of inbreeding or null alleles. Three pairs of loci exhibited significant linkage disequilibrium after Bonferroni correction for multiple comparisons. These microsatellites would be useful to strengthen population management, genetic diversity exploration, and demographic history speculation of this species.