小型猪微卫星标记多重PCR体系的建立与应用

目的建立实验用小型猪微卫星标记的多重PCR体系和进行实验猪群的遗传监测。方法利用3种不同荧光标记的微卫星引物结合ABI3700遗传分析仪测序的方法,通过筛选和优化反应条件,建立可用于实验用小型猪遗传质量控制的稳定的多重PCR反应体系。在此基础上进一步检测实验用小型猪近交群体的遗传变异以验证建立体系的效率。结果筛选出了2组理想的组合：组合1包括SW742、S0228和S0218座位,复性温度58℃和56℃;组合2包括S0155、SW902和S0227三个座位,复性温度为60℃和58℃。组合内不同座位标记不同的荧光染料。还以此检测了实验用小型猪群体中的遗传变异。结论初步建立了中国三种实验用小型猪微卫星标记检测的多重PCR体系,为快速、大通量、准确的小型猪遗传监测提供了初步的技术基础。     

8个亚洲水牛群体的遗传结构分析

应用13个微卫星标记结合荧光–多重PCR技术, 对德昌水牛、兴隆水牛、富钟水牛、温州水牛、东流水牛、福安水牛和两个引进品种摩拉水牛、尼里－拉菲水牛进行遗传结构分析。结果表明: 8个水牛群体在13个微卫星座位中共检测到157个等位基因, 其中7个群体具有各自的特有等位基因, 其和为23; 8个群体的有效等位基因数在2.2908～4.2308之间, 杂合度在0.4951～0.7194之间, 多态信息含量在0.4495～0.6776之间; 有11个座位为高度多态座位, 是适合分析水牛遗传多样性的多态标记; 聚类分析表明富钟水牛和东流水牛先聚在一起, 再与兴隆水牛聚在一起, 然后与温州水牛和福安水牛聚在一起, 德昌水牛独自聚为一类; 两个引进品种聚为一类。     

德宏水牛微卫星标记分析的群体遗传变异

德宏水牛是云南省地方水牛的优良品种之一,为了进一步阐明其群体遗传变异和遗传结构,筛选了分别位于水牛14条染色体上的15对微卫星引物,对德宏水牛81个个体进行了检测分析.共检测到62个等位基因,每个座位等位基因数目从2到6个不等,平均等位基因数为4.13,该水牛群体期望杂合度和多态信息含量分别为0.6520±0.1526和0.5863±0.1789,各座位的遗传分化系数在0～0.0919之间,平均值为0.0202.每个座位的基因流较大,平均12.1502.研究结果表明德宏水牛群体遗传多样性较丰富,亚群间的遗传分化程度低,基因流较大,且很少发生近交.     

德宏水牛微卫星标记分析的群体遗传变异

德宏水牛是云南省地方水牛的优良品种之一，为了进一步阐明其群体遗传变异和遗传结构，筛选了分别位于水牛14条染色体上的15对微卫星引物，对德宏水牛81个个体进行了检测分析。共检测到62个等位基因，每个座位等位基因数目从2到6个不等，平均等位基因数为4.13，该水牛群体期望杂合度和多态信息含量分别为0.6520±0.1526和0.5863±0.1789，各座位的遗传分化系数在0～0.0919之间，平均值为0.0202。每个座位的基因流较大，平均12.1502。研究结果表明德宏水牛群体遗传多样性较丰富，亚群间的遗传分化程度低，基因流较大，且很少发生近交。     

利用微卫星标记评估富钟水牛群体遗传多样性和保种效果

利用微卫星分子标记对富钟水牛群体遗传结构和保种效果进行评估,旨在掌握保种区富钟水牛目前的保种现状,为下一步制定有效的保种措施提供参考依据。结果表明:15个微卫星座位中,共检测到86个等位基因,平均等位基因NA为5.733 3;平均有效等位基因NE为2.902 9;期望杂合度、观察杂合度、多态信息含量PIC分别为0.596 4、0.487 2、0.551 9;1个座位为低度多态,3个座位为中度多态,9个座位为高度多态,这些位点可用于遗传多样性分析。     

雪峰乌骨鸡自然群体遗传多样性的微卫星分析

雪峰乌骨鸡是具有独特遗传特性的优质肉药兼用地方家禽品种.为了评价雪峰乌骨鸡现有品种资源的遗传背景、群体遗传结构等,提出合理的保种方法和措施,本研究筛选了家鸡基因组23条染色体上的23个微卫星标记,对采自湖南怀化的50只纯种乌骨鸡个体进行了多态性检测.结果表明:23个微卫星座位共检测到79个等位基因,所有座位都呈现出多态性,每个座位的等位基因数在2-6个之间,平均为3.435个.群体表现出较高的平均杂合度和平均多态信息含量,分别为0.6285和0.5496,说明雪峰乌骨鸡自然群体具有较丰富的遗传多样性.研究结果将为雪峰乌骨鸡种的保护和利用提供重要的理论依据.     

基于微卫星标记的鼋亲子鉴定技术

为研究鼋(Pelochelys cantorii)保种群体的遗传信息,利用鼋转录组文库, 筛选出了10对微卫星引物, 以此建立了基于 2 组各含5个微卫星位点多重 PCR 体系的鼋亲子鉴定技术, 并应用 2 组微卫星多重 PCR 体系, 通过 ABI3130 遗传分析仪及 PopGene3.2和CERVUS软件对56只子一代鼋进行个体基因型检测和群体遗传多样性分析, 结果显示, 鼋子一代群体的平均等位基因数为2.3, 微卫星位点的平均多态信息含量为0.3829, 平均观测杂合度和期望杂合度分别为0.6305和0.4767, 处中高度水平。对子代进行亲子分析, 10个微卫星位点的累积排除概率分别为73.14%(NE-1P)、90.98%(NE-2P)和98%(NE-3P), 能够满足亲子鉴定的需要。且4只亲本在生殖选择中表现出差异。鼋多重 PCR 亲子鉴定技术的建立为群体遗传多样性分析、家系鉴定管理和选择育种提供了有效的技术手段。     

布氏罗非鱼微卫星位点筛选和群体遗传多样性分析

为阐明布氏罗非(Tilapia buttikoferi)群体遗传变异和遗传结构状况,采用50个尼罗罗非鱼特异性的微卫星分子标记对45个布氏罗非鱼个体进行遗传检测.结果有27对引物能获得稳定的特异性条带,占总数的54%,其中16个多态性微卫星座位共检测出52个等位基因.每个座位的等位基因数为2～6之间,平均每个座位为3.24;平均观测杂合度(Ho)为0.5266,平均期望杂合度(He)为0.5237,平均多态信息含量为0.4652,表明布氏罗非鱼群体遗传多样性较丰富,种群结构处于合理状态.     

黄瓜SRAP遗传连锁图的构建及侧枝基因定位

在由黄瓜的两个自交系S06与S52杂交产生的F₂群体中, 应用SRAP(sequence-related amplified polymorphism)标记构建遗传连锁图谱, 检测控制黄瓜侧枝数(lbn)和侧枝平均长度(lbl)的数量性状座位(QTL). 使用筛选出的64个多态性引物组合对F2群体进行分析, 得到108个多态性位点. 经MAPMAKER/EXP3.0分析(LOD≥3.0), 获得由92个标记座位组成、覆盖7个连锁群的遗传图谱, 总长1164.2 cM, 标记平均间距12.6 cM. 应用QTLMapper1.6, 各检测到4个控制lbn和lbl的QTL, 其中, 对lbn贡献率最大的QTL位于第二连锁群的ME11SA4B-ME5EM5区间, 其S06基因型具有增效作用; 对lbl贡献率最大的QTL位于第二连锁群的DC1OD3-DC1EM14区间, 其S06基因型具有增效作用.     

基于微卫星多重PCR技术的黄喉拟水龟亲子鉴定

利用黄喉拟水龟(Mauremys mutica)微卫星标记,筛选出16对微卫星引物,通过优化各引物比例、荧光接头浓度、退火温度和循环次数,建立了基于2组各含8个微卫星位点多重PCR体系的黄喉拟水龟亲子鉴定技术。应用2组微卫星多重PCR体系,通过ABI3130遗传分析仪以及cervus3.0软件对428只黄喉拟水龟进行了个体基因型检测和遗传多样性分析,结果显示,群体的平均等位基因数为14.190,平均多态信息含量为0.748,平均观测杂合度和期望杂合度分别为0.687、0.771。对89只候选母本及296只子代进行亲子鉴定分析,结果显示:在父本信息未知时,母本鉴定率为87%;母本获得的子代个数范围为1-12,个体间表现出巨大的差异,这为选择育种提供了物质基础。黄喉拟水龟多重PCR亲子鉴定技术的建立为群体遗传多样性分析、家系鉴定管理和选择育种提供了有效的技术手段。       

Genetic diversity and differentiation of Chinese domestic buffalo based on 30 microsatellite markers

To determine genetic diversity and evolutionary relationships among Chinese buffalo populations, 18 indigenous swamp buffalo populations and two introduced river buffalo breeds were genotyped for 30 microsatellite loci. The mean number of alleles across the 30 loci was 8.13, and the expected heterozygosity ranged from 0.517 (Yanjin) to 0.609 (Dehong). Although there was abundant genetic variation, genetic differentiation between Chinese buffalo populations was low, with only 2.8% of the total genetic variance among populations. The genetic differentiation pattern and genetic relationships among Chinese buffalo populations were consistent with their geographic distribution. The Dehong population was discerned as a distinct indigenous population, but suffered genetic admixture with river buffalo. The remaining populations were divided into four major clusters, i.e. the Upper and Middle Reaches of Yangtze Valley cluster (Guizhou, Guizhoubai, Yanjin, Fuling, Enshi and Jianghan), the Lower Reaches of Yangtze Valley cluster (Haizi, Shanqu and Dongliu), the South China cluster (Fuan and Xinfeng) and the Southwest China cluster (Xinglong, Xilin, Diandongnan and Dechang).     

Genetic diversity of Asian water buffalo (Bubalus bubalis): microsatellite variation and a comparison with protein-coding loci

Twenty-one microsatellite loci in 11 populations of Asian water buffalo (eight swamp, three river type) were analysed and, within and among populations, genetic variability was compared with results from 25 polymorphic protein-coding loci. Within-population mean heterozygosity ranged from 0·380–0·615, approximately twice that estimated from the protein-coding loci (0·184– 0·346). Only eight significant departures from Hardy–Weinberg equilibrium (involving four loci) were detected; global tests showed significant heterozygote deficiencies for these four loci. Non-amplifying alleles are likely to be segregating in some or all populations for one of these loci, and probably for the other three. There was significant differentiation between the swamp and river types of water buffalo, and among populations within each buffalo type. Estimates of θ (measure of population differentiation) for each locus for the eight swamp populations were all highly significant (mean θ = 0·168 ± 0·018). Mean θ for protein-coding loci was not significantly different (0·182 ± 0·041). The variance among protein-coding loci was significantly higher than among microsatellite loci, suggesting balancing selection affecting allele frequencies at some protein-coding loci. Genetic distances show clear separation of the swamp and river types, which were estimated to have diverged at least 10 000–15 000 years ago. The topology of the swamp populations’ microsatellite tree is consistent with their geographical distribution and their presumed spread through south-east Asia. By contrast, the tree based on the protein-coding loci distances is quite different, being clearly distorted by a bottleneck effect in one population, and possibly in at least two others. As many domestic livestock breeds are possibly descended from small numbers of founders, microsatellite-based trees are to be preferred in assessing breed genetic relationships.     

Cross-species amplification of Bovidae microsatellites and low diversity of the endangered Korean goral

The Korean goral (Nemorhaedus caudatus) is an endangered species of wild goat. The conservation and management of this species could benefit from a better understanding of its genetic diversity and structure. Cross-species amplification of 34 Bovidae microsatellite loci was tested on a panel of 6 Korean gorals and 10 domestic goats. After polymerase chain reaction (PCR) optimization, 29 (85.3%) microsatellite loci amplified successfully for the Korean gorals and 27 (79.4%) for the domestic goats. Of the amplified products, 16 (55.2%) were polymorphic in the Korean goral and 22 (81.5%) in domestic goats. Nei's unbiased mean heterozygosity and mean allele number per locus were, respectively, 0.356 and 2.6 in the Korean goral and 0.636 and 4.8 in domestic goats. Low genetic diversity in the Korean gorals observed in this preliminary microsatellite survey suggests an urgent need for further detailed study of genetic diversity in Korean goral populations and a population management strategy based on these studies. Current results of cross-species amplification of domestic Bovidae microsatellites could be employed for the necessary population genetic studies on the Korean goral and other endangered Caprinae species.     

Genetic diversity and structure in a natural Elymus caninus population from Denmark based on microsatellite and isozyme analyses

 Genetic diversity in a natural Elymus caninus population from Denmark was assessed using isozyme and microsatellite markers. A total of 119 individuals from 46 maternal plants were assayed. Microsatellite loci are shown to display higher levels of variation than isozyme loci. The mean number of alleles per locus was 1.04 for isozymes and 1.38 for microsatellites. The percentage of polymorphic loci for isozymes and microsatellites was 4.7% and 23.6% across the maternal plant, respectively. The genetic diversity at population level was 0.1 for isozymes, and 0.63 for microsatellites. The mean genetic diversity at maternal plant level was 0.027 for isozyme loci and 0.117 for microsatellite loci. The average of total allozyme diversity (H_T) was 0.22. The average of total microsatellite diversity was 0.56. Isozyme and microsatellite variation showed the same pattern of differentiation between maternal plants. More than 75% total genetic diversity was found among maternal plants. About 25% total genetic diversity was detected within maternal plants. Ten (22.7%) maternal plants produced heterozygous offspring at allozyme loci, and 30 (68.2%) maternal plants gave heterozygous offspring at microsatellite loci. Both types of markers revealed a relatively high genetic diversity in this population. Received November 7, 2000 Accepted February 15, 2001     

沼泽型水牛Y染色体微卫星标记的筛选与多态性检测

为了研究水牛Y染色体的遗传多样性, 文章以滇东南水牛3个地方群体- 红河(HH)、西双版纳(BN)和普洱(PR)共31头公牛为研究对象, 选取14个家牛Y染色体特异性微卫星标记, 以检测这些标记在水牛Y染色体遗传多样性研究中的可行性。结果表明, 3个标记(INRA008、UMN0103和UMN0504)只有1个等位基因, 表现为单态; 3个标记(UMN1113、UMN0304和BC1.2)均为3个等位基因, 但呈单态; 3个标记(UMN0920、UMN0307和UMN3008)呈现无规律的梯状条带, 所以这9个标记都不适用于水牛的Y染色体遗传多样性研究; 只有5个标记(INRA124、INRA189、BM861、PBR1F1和UMN2001)具有多态性, 表明适用于水牛的Y染色体遗传多样性研究。这5个多态性Y染色体特异微卫星标记在滇东南水牛群体中的平均等位基因数(NA)为2.8000, 平均期望杂合度(He)为0.3998, 基因多样性(GD)为0.4144, 多态信息含量(PIC)为0.3245, Shannon信息熵(SI)为0.5849, 表明滇东南水牛群体的Y染色体具有中等遗传多态性。     

Analysis of conserved microsatellite sequences suggests closer relationship between water buffalo Bubalus bubalis and sheep Ovis aries.

The distribution and evolutionary pattern of the conserved microsatellite repeat sequences (CA)n, (TGG)6, and (GGAT)4 were studied to determine the divergence time and phylogenetic position of the water buffalo, Bubalus bubalis. The mean allelic frequencies of these repeat loci showed a high level of heterozygosity among the euartiodactyls (buffalo, cattle, sheep, and goat). Genetic distances calculated from the allelic frequencies of these microsatellites were used to position Bubalus bubalis in the phylogenetic tree. The tree topology revealed a closer proximity of the Bubalus bubalis to the Ovis aries (sheep) genome than to other domestic species. The estimated time of divergence of the water buffalo genome relative to cattle, goat, sheep, pig, rabbit, and horse was found to be 21, 0.5, 0.7, 94, 20.3, and 408 million years (Myr), respectively. Although water buffaloes share morphological and biochemical similarities with cattle, our study using the microsatellite sequences places the bubaline species in an entirely new phylogenetic position. Our results also suggest that with respect to these repeat loci, the water buffalo genome shares a common ancestry with sheep and goat after the divergence of subfamily Bovinae (Bos taurus) from the family Bovidae.     

Development of a genome-wide anchored microsatellite map for common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

A total of 150 microsatellite markers developed for common bean (Phaseolus vulgaris L.) were tested for parental polymorphism and used to determine the positions of 100 genetic loci on an integrated genetic map of the species. The value of these single-copy markers was evident in their ability to link two existing RFLP-based genetic maps with a base map developed for the Mesoamerican × Andean population, DOR364 × G19833. Two types of microsatellites were mapped, based respectively on gene-coding and anonymous genomic-sequences. Gene-based microsatellites proved to be less polymorphic (46.3%) than anonymous genomic microsatellites (64.3%) between the parents of two inter-genepool crosses. The majority of the microsatellites produced single bands and detected single loci, however four of the gene-based and three of the genomic microsatellites produced consistent double or multiple banding patterns and detected more than one locus. Microsatellite loci were found on each of the 11 chromosomes of common bean, the number per chromosome ranging from 5 to 17 with an average of ten microsatellites each. Total map length for the base map was 1,720 cM and the average chromosome length was 156.4 cM, with an average distance between microsatellite loci of 19.5 cM. The development of new microsatellites from sequences in the Genbank database and the implication of these results for genetic mapping, quantitative trait locus analysis and marker-assisted selection in common bean are described.Communicated by H.F. Linskens     

A set of polymorphic DNA microsatellites useful in swamp and river buffalo (Bubalus bubalis)

DNA microsatellites have found widespread application in gene mapping, pedigree determination and population genetics. In closely related species such as bovids, heterologous polymerase chain reaction (PCR) primers may in some cases be used, bypassing the need to isolate and characterize microsatellite-containing sequences and design PCR primers. We report on the ability of a set of eighty bovine derived DNA microsatellite primers to amplify sequences in the two types (swamp and river) of water buffalo ( Bubalus bubalis ). Number of alleles and per cent heterozygosities in a large number of animals were determined on a subset of microsatellite loci selected on the robustness of the primers. These loci will form the basis of a set of polymorphic DNA markers for use in water buffalo.     

Cloning and characterization of highly polymorphic porcine microsatellites.

A size-fractionated (200-400 bp) porcine genomic library was screened with the dinucleotide motifs (TG)n and (TC)n. The number of TG- and TC-positive clones was 83 and four, respectively, implying that the former motif is more frequent in the porcine genome, as previously reported in other species. Twenty-six TG-clones were sequenced, and the number of repeats varied between 16 and 42 with different compositions of the repetitive sequences; 17 clones had a perfect stretch of TG-repeats, four had imperfect stretches, and five had a compound structure with TG-repeats followed by TC-repeats. Primers for DNA amplification using the polymerase chain reaction (PCR) were synthesized for six loci. Ten unrelated individuals (two wild boars and eight domestic pigs of the Swedish Yorkshire breed) were screened for microsatellite polymorphism. All six microsatellite loci were polymorphic with two to seven alleles and observed heterozygosities in the range of 0.42-0.84; the inheritance of the observed polymorphism was confirmed by family studies. The characteristics of microsatellites make them highly suitable as genetic markers, and these microsatellites were isolated as a part of a pig gene mapping project.     

Abundance, polymorphism and genetic mapping of microsatellites in rice

Dinucleotide microsatellites have been characterized and used as genetic markers in rice. Screening of a rice genomic library with poly(dG-dA)·(dC-dT) and poly(dG-dT)·(dC-dA) probes indicated that (GA)_n repeats occurred, on average, once every 225 kb and (GT)_n repeats once every 480 kb. DNA sequencing of ten randomly selected microsatellites indicated that the numbers of repeats ranged from 12 to 34 and that the patterns of microsatellites in rice were similar to those of humans and other mammals. Primers to these microsatellite loci as well as to four published microsatellite-containing sequences have been designed and degrees of polymorphism has been examined with 20 rice accessions. Multiple alleles, ranging from 5 to 11, have been observed at all the microsatellite loci in 20 rice accessions. Alleles specific to two cultivated subspecies, indica and japonica, were found in some microsatellite loci. Heterozygosity values of all the microsatellite markers were significantly higher than those of RFLP markers, based upon a parallel comparison. Ten microsatellite loci have been genetically mapped to four rice chromosomes. The genomic distribution of microsatellites appears to be random in rice.