Detection of occurrence of a recent genetic bottleneck event in Indian hill cattle breed Bargur using microsatellite markers

The effective number of breedable individuals is a crucial determinant for maintaining genetic variability within a population. The population of Bargur, the hill cattle of South India, has gone down drastically by more than 93 % in the past three decades, and only a few thousand animals are available at present. The present study was undertaken to evaluate Bargur cattle for mutation drift equilibrium and to detect the occurrence of recent genetic bottleneck event, if any, in this population. About 50 unrelated animals, true to the type, were sampled and genotyped at 25 microsatellite loci. The mean observed heterozygosity (0.808?±?0.023) was higher than the mean expected heterozygosity (0.762?±?0.008) with 15 out of 25 microsatellite loci exhibiting heterozygosity excess when assumed under Hardy–Weinberg equilibrium. To evaluate Bargur cattle for mutation drift equilibrium, three tests were performed under three different mutation models, viz., infinite allele model (IAM), stepwise mutation model (SMM) and two-phase model (TPM). The observed gene diversity (H _e) and expected equilibrium gene diversity (H _eq) were estimated under different models of microsatellite evolution. All the 25 loci were found to exhibit gene diversity excess under IAM and TPM, while 22 loci were having gene diversity excess under SMM. All the three statistical tests, viz., sign test, standardized differences test, and Wilcoxon sign rank test, revealed significant (P?<?0.01) deviation of Bargur cattle population from mutation-drift equilibrium under all the three models of mutation. Furthermore, the qualitative test of allele frequency distribution in Bargur cattle population revealed a strong mode shift from the normal L-shaped form suggesting that the population had experienced genetic bottleneck in the recent past. The occurrence of genetic bottleneck might have led to the loss of several rare alleles in the population, which point towards the need for efforts to conserve this important cattle germplasm. The present study is the first report in demonstrating the genetic basis of demographic bottleneck in an Indian cattle population.     

Evaluation of genetic variability and mutation drift equilibrium of Banni buffalo using multi locus microsatellite markers

The present study was conducted to evaluate genetic diversity of Banni buffalo and its relationship/differentiation with Murrah using genotypic data on 24 heterologus bovine specific microsatellite marker loci. A total of 138 alleles were observed with a mean of 5.75 alleles/locus across two populations. The mean observed and expected heterozygosities were found to be 0.441 and 0.572 respectively in Banni buffaloes while it was 0.464 and 0.610 respectively in Murrah buffaloes. The average heterozygosity deficit was significantly positive with substantially higher values observed in Banni (22.3%) and Murrah (24%) buffalo populations. Banni buffalo population, when evaluated for mutation drift equilibrium revealed significant heterozygosity excess under IAM while no such excess was observed under SMM and TPM. The qualitative graphical test revealed a normal L-shaped distribution of allele frequencies indicating the absence of genetic bottleneck in Banni buffaloes. The mean estimates of F-statistics over all the loci were 0.376 for F_IT, 0.187 for F_ST and 0.232 for F_IS respectively. Analysis of molecular variance (AMOVA) revealed 18.95% of the total variation being explained by between breed differences while 14.36% of the variation explained differences between individuals within each breed. Genotype assignment test revealed distinct clustering of Banni and Murrah buffaloes. Genetic distance was estimated using three different methods, the results of which revealed considerable genetic differentiation between these two buffalo populations. The divergence time between Banni and Murrah buffaloes was estimated to be around 7286 years. The results of the present study may be helpful in decision making for conservation programs as Banni buffalo population is on decline.     

Microsatellite-based genetic monitoring to detect cryptic demographic bottleneck in Indian riverine buffaloes (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The present study was undertaken to evaluate different Indian riverine buffalo breeds (Bubalus bubalis) for mutation drift equilibrium and occurrence of any recent genetic bottleneck. A total of 330 animals from seven different breeds were analyzed with a set of 24 heterologous microsatellite markers. Three different tests revealed significant heterozygosity excess in all the seven buffalo breeds studied when assumed under infinite alleles model of microsatellite evolution, while it was the reverse with no significant heterozygosity excess when assumed under conservative stepwise mutation model. Under the two-phase model, all the buffalo breeds except Mehsana were found to be in mutation drift equilibrium when evaluated by all the three statistical methods. Standardized differences test and Wilcoxon signed-rank test revealed significant heterozygosity excess suggesting possible cryptic demographic bottleneck in Mehsana buffaloes of Western India.     

中国5个矮马品种的瓶颈效应分析

利用13个微卫星基因座对中国5个矮马品种(德保、贵州、宁强、四川、云南)群体进行了遗传平衡检测。根据微卫星等位基因频率,应用无限等位基因模型(infinite allele model,IAM)、双相突变模型(two-phased model of mutation,TPM)和逐步突变模型(step-wise mutation model,SMM)3种模型,符号和Wilcoxon 符号—秩2种检验方法,对5个矮马群体的突变－漂移平衡进行分析。结果表明,在IAM下,Wilcoxon 符号－秩检验中,德保矮马和云南矮马达到了显著性水平(P<0.05);在SMM下,宁强矮马在符号检验中达到了显著水平(P<0.05);在TPM下,5个群体均未达到显著水平(P>0.05)。因此,德保、云南和宁强矮马近期可能经历了瓶颈效应。本研究结果对中国矮马的保护和利用具有重大意义。     

Riverine status and genetic structure of Chilika buffalo of eastern India as inferred from cytogenetic and molecular marker-based analysis

The present study aimed at assessing the status of the Chilika buffalo population of eastern India employing cytogenetic and molecular markers. The Chilika buffaloes investigated cytogenetically possess a somatic chromosome count of 50, identical to that of typical riverine buffaloes. Various diversity estimates, viz. observed number of alleles (4.68), effective number of alleles (2.79), and observed (0.487) and expected (0.602) heterozygosity across 25 heterologous microsatellite markers indicated the presence of a moderate level of genetic diversity in Chilika buffaloes, comparable with three other prominent Indian riverine buffalo breeds (Murrah, Nagpuri and Toda) included in this study. Across the four buffalo populations, mean estimates of F -statistics from Jackknifing over loci were significantly different from zero (p < 0.05), with F _IT (total inbreeding estimate) = 0.315 ± 0.038, F _IS (within-population inbreeding estimate) = 0.178 ± 0.038, and F _ST (population differentiation) = 0.166 ± 0.025. Inter-breed analysis reflected Chilika buffaloes to be genetically close to Nagpuri followed by Murrah and Toda buffaloes. Factorial correspondence analysis (FCA) revealed low breed-specific clustering of Chilika and Nagpuri buffaloes. Additionally, the neighbour-joining tree structure of mitochondrial DNA D-loop haplotypes indicated clear grouping of the Chilika haplotypes with the riverine buffalo. Thus the cytogenetic, microsatellite and mitochondrial data analysed in the present study classify Chilika buffalo of eastern India to be of the riverine type and not swamp-type buffalo.     

塔里木马鹿微卫星遗传多样性与产茸量的相关性研究

利用7个微卫星遗传标记,采用PCR扩增,12%非变性聚丙烯酰胺凝胶电泳、Sanguinetti银染法显色,对新疆塔里木马鹿亚种遗传多样性进行检测,统计各群体的等位基因组成、平均有效等位基因数(E)和平均基因纯合率(Rh),根据等位基因频率计算出各群体的平均遗传杂合度(h)、多态信息含量(PIC)和群体间的遗传距离,利用分子进化遗传分析软件(MEGA),采用邻结法(NJ)重建系统发生树。根据等位基因频率,利用PHYLIP(3.6)分析软件,采用最大似然法(ML)构建系统发生树,应用自举检验(bootstrap test)估计系统树中节点的自引导值(bootstrap value),并进行了系统发生分析。同时对部分马鹿群体个体基因型与产茸量之间进行了相关分析。结果表明:7个微卫星位点在3个塔里木马鹿中的多态信息含量除BMS2508和Celjp0023没有多态性,以及BM5004为中度多态外,其他4个微卫星均为高度多态,可作为有效的遗传标记用于3个塔里木马鹿群体遗传多样性和系统发生关系的分析。所有马鹿群体的平均PIC为(0.5196)、h(0.5552)和E(2.45),其基因多态性和遗传多样性相对丰富。塔里木马鹿35团群体与沙雅群体的血缘关系及遗传距离近于阿拉尔群体,3个塔里木马鹿群体的系统发育关系基本符合其地理分布和育成史。微卫星位点BM4208的166bp/185bp基因型和微卫星BM888的208bp/208bp基因型可以对塔里木马鹿35团群体高产茸量进行分子遗传标记。     

Biodiversity in Mediterranean buffalo using two microsatellite multiplexes

The present study was carried out to investigate genetic diversity in Nile-Delta and Southern-Egypt buffalo populations in comparison with the Italian buffalo utilizing two microsatellite multiplexes. A total of 104 animals classified into three groups were used, 28, 38 and 38 representing the Nile-Delta, Southern-Egypt and Italian buffalo, respectively. The 15 studied microsatellites were CSSM38, CSSM70, CYP21, CSSM42, CSSM60, MAF65, BM0922, CSSM19, INRA006, ETH02, BM1706, BMC1013, CSSM47, INRA026 and CA004. All studied microsatellites showed allelic polymorphism. Number of polymorphic alleles ranged between 4 alleles (CSSM38, CSSM70 and CYP21) and 11 alleles (CA004). Pairwise Chi-square test for Nile Delta and Southern Egypt showed significant differences in allelic distribution at five loci, CSSM70, CSSM38, BM0922, ETH02 and BM1706. Italian buffalo showed the lowest percentage of observed heterozygotes (65%), while the Southern Egypt showed the highest (71%). Both the Italian and the Delta populations deviated significantly (P < 0.05) from HW equilibrium. Italian buffalo is relatively the most inbred population while the Southern-Egypt buffalo is the only outbred population. High level of genetic differentiation (F_ST estimates) between the Italian group and each of the Delta and Southern-Egypt group (0.083 and 0.076, respectively) was observed while Southern-Egypt group showed a lower level of genetic differentiation with the Delta group (0.014). Italian buffalo had the greatest genetic distance values with the two Egyptian groups (0.25 and 0.23) while much lower values between the Southern-Egypt and the Delta groups (0.06) was observed. Genetic variation between the Italian buffalo and the Egyptian buffalo was detected in 14 (out of 15) microsatellite loci. While a lower level (than that between Egyptian and Italian) of genetic variation between Southern-Egypt and Delta buffalo populations was expressed by 5 loci. It was concluded that the Southern-Egypt buffalo could be considered as distinct population from the Delta buffalo. In addition, Southern-Egypt group, being the only group with non-significant global deviation from HW equilibrium, the most heterozygous, and the only outbred population as well, is expected to respond more favorably to selection.     

Genetic variation of ten microsatellite loci in Makui sheep of Iran

The native breeds, because of their natural selection against harsh environment and adaptation to regional conditions are important to resource-poor farmers and pastoralists. The molecular characterization of genetic variation is a fundamental step to manage and conserve indigenous breeds. The aim of this study was to determine the genetic variation of Makui sheep. Totally, 100 sheep (60 ewes, 10 ram and 30 lambs) were used in this study. Polymerase Chain Reaction (PCR) was performed using microsatellite primers BM1329, OarAE101, OarFCB11, OarFCB128, OarFCB129, OarFCB20, OarFCB304, OarFCB5, OarHH35 and OarHH55 in a standard 25 μl reaction. All microsatellite loci were amplified and produced minimum 2 and maximum 14 alleles ranging from 90 to 185 bp in size. The mean number of alleles for each locus was 6.8. Loci OarFCB128 and OarAE101 produced the highest (8.5288) and the lowest (1.0304) effective number of alleles, respectively. The mean expected heterozygosity for all loci was 0.6893 (range 0.0295–0.8837). The highest (2.12) and the lowest (0.07) Shanon Index was observed in OarFCB11 and OarAE101, respectively. Only two (OarFCB5 and OarAE101) out of ten loci were in Hardy-Weinberg equilibrium. The present study was able to demonstrate a reasonable genetic variation and polymorphism across all microsatellite loci studied in the Makui sheep.     

Analysis of genetic diversity and phylogenetic relationship of red deer subspecies in XinJiang,China

Polymorphisms for seven microsatellite loci in three red deer subspecies (9 populations) found in XinJiang were detected by polymerase chain reaction (PCR), 12% nondenaturation polyacrylamide gel electrophoresis and the Sanguinetti silver staining method. Numbers of alleles, average effective numbers of alleles (E) and the average rate of homozygosity, allelic frequencies of seven microsatellite loci, polymorphism information content (PIC), mean heterozygosity (H) and genetic distances among the populations were calculated for each population. Dendrograms were constructed based on genetic distances by the neighbor‐joining method (NJ), utilizing molecular evolutionary genetics analysis software PHYLIP (3.6). The phylogenetic tree was constructed based on allelic frequencies using maximum likelihood (ML); the bootstrap value was estimated by bootstrap test in the tree. Lastly, phylogenesis was analyzed. The results showed that four of the seven microsatellite loci were highly polymorphic, but BMS2508 and Celjp0023 showed no polymorphism and BM5004 was a neutral polymorphism. It is our conclusion that the four microsatellite loci are effective DNA markers for the analysis of genetic diversity and phylogenetic relationships among the three red deer subspecies. The mean PIC, H and E‐values across the microsatellite loci were 0.5393, 0.5736 and 2.64, which showed that these microsatellite loci are effective DNA markers for the genetic analysis of red deer. C.e. songaricus populations from Regiment 104, 151 and Hami are clustered together. C.e. yarkandensis populations from Regiment 35, Xaya and Alaer are clustered together. These two clusters also cluster together. Lastly, C.e. sibiricus populations from Burqin, Regiment 188 and the first two clusters were clustered together. The phylogenetic relationship among different red deer populations is consistent with the known origin, history of breeding and geographic distributions of populations.     

青海高原牦牛遗传多样性研究

以中心产区简单随机抽样在青海省西宁市共抽取青海高原牦牛76头,用垂直平板聚丙烯酰胺凝胶电泳（PAGE）和水平淀粉凝胶电泳技术检测21个编码血液蛋白（酶）结构基因座及12个微卫星位点的多态性,并进行中性检验及连锁不平衡分析。结果表明：在所检测的21个血液蛋白基因座中,有6个存在多型,多态位点百分比为28．57％,12个微卫星位点全部为多态,共检测出63个等位基因;两层次标记所反映的群体内遗传变异水平均较低,21个血液蛋白基因座平均有效等位基因数、观察杂合度、期望杂合度和多态信息含量分别为1．118、0．0665、0．0729和0．0603,12个微卫星位点相应指标分别为2．9005、0．4138、0．5325和0．4903;除Alp外,其余5个血液蛋白多态基因座和12个微卫星位点均为中性基因。除Alp-CA、LDH1-ADH两对血液蛋白基因座和8对微卫星显著（P〈0．05）或极显著（P〈0．01）处于连锁不平衡状态外,其余13对血液蛋白基因座和58对微卫星均处于连锁平衡状态（P〉0．05）;另外,6个血液蛋白多态基因座、12个微卫星位点或是两者的合并数据从群体水平上均处于连锁平衡状态（P〉0．05）。     

The Genetic Polymorphism Analysis in Tree Shrew(T.belangeri chinensis) based on Fluorescent Microsatellite

A total of 10 fluorescent labeling microsatellite markers detected by capillary electrophoresis were selected to analyze the genetic diversity of tree shrew(T.belangeri chinensis).Microsatellite Toolkit and PopGene softwares were used to calculate the genetic correlation indexes to assess the genetic diversity of the population.64 alleles with 6.4 alleles per locus were identified among 10 microsatellite loci.The average effective number of alleles, observed heterozygosity, expected heterozygosity and polymorphic information content were 3.7892, 0.6156, 0.6963, and 0.6367, respectively.Seven of all loci were high polymorphic, and three were moderate polymorphic.Five loci extremely significantly deviated from Hardy-Weinberg equilibrium(P<0.01).These novel polymorphic microsatellite markers indicated high genetic diversity, which will be used to evaluate genetic diversity in tree shrew.     

荧光标记微卫星分析广西树鼩群体的遗传多态性

选取10个微卫星位点,结合荧光标记(FAM) PCR扩增技术,运用毛细管电泳检测扩增产物,利用Microsatellite Toolkit、PopGene软件计算遗传相关指标,探讨广西树鼩群体的遗传多态性。结果发现,在10个微卫星基因座中,共检测到64个等位基因,平均等位基因数为6.4个,平均有效等位基因数为3.7892,平均观察杂合度和平均期望杂合度为0.6156和0.6963,平均多态信息含量为0.6367,其中7个是高度多态位点,3个属中度多态位点。此外,在10个微卫星基因座中,有5个位点偏离Hardy-Weinberg平衡(P<0.01)。结果表明广西树鼩遗传多态性较丰富,所选微卫星位点基本可用于评估广西树鼩群体遗传多样性。     

Genetic relationship and diversity analysis of Indian water buffalo (Bubalus bubalis)

The water buffalo (Bubalus bubalis) is an important dairy animal on the Indian subcontinent and in Southeast Asian countries. The diversity and differentiation among 12 populations or breeds of buffalo were studied. Data were generated and analyzed from 527 animals belonging to 10 recognized breeds and 2 additional populations of Indian buffalo by using 22 microsatellite loci. Relationships among buffalo breeds and populations were estimated based on genetic distances. The Bayesian analysis grouped 12 populations into 8 distinctive clusters. Geographically close breeds clustered together, except for the Jaffarabadi and Murrah, which were not in geographic contiguity. The Mantel test revealed nonsignificant correlations between genetic and geographic distances. This supports the hypothesis that buffaloes have been domesticated at different places for specific purposes. The phylogenetic relationship based on microsatellite loci supported the breed classification based on body size. The Toda breed, which is considered to be endangered, had genotypes similar to those of the surrounding buffalo populations.     

Genetic Characterization of Mangalarga Marchador Breed Horses Based on Microsatellite Molecular Markers

The Mangalarga Marchador (MM) horse breed has expressive importance in the Brazilian economy. Thus, the aim of this study was to investigate diversity in the MM breed. A database with a total of 3,193 genotyped horses was used (MM, n = 2,829; Andalusian – AND, n = 67; Pure Blood Lusitano – LUS, n = 43; English Thoroughbred – THO, n = 54; Arabian – ARA, n = 99; Campolina – CAM, n = 61; and Mangalarga – MAN, n = 40) for 13 microsatellite. Diversity parameters were estimates, such as mean number of alleles (Nm_a) and the number of rare alleles (AR), expected heterozygosity (He), F statistics, genetic distances, Hardy-Weinberg equilibrium test (HWE), population structure, and others. The Nm_a was 10.85, the AR was prevalent in the MM, and the He was 0.7402. In MM, the values of Fis (−0.0195), Fit (0.0566), Fst (0.0748), and deviations of HWE were observed. The genetic distances of the ARA and THO breeds with the other breeds were greater than the distances between the Brazilian breeds and between these and the breeds in the Iberian Peninsula. The population structure indicated that MM was substructured, yet there were some more genetically defined breeding farms. The genetic diversity is satisfactory for MM conservation, but the population is substructure, and parameters indicate moderate gene flow and the existence, though few, of crosses with other horse breeds. Immediate implementation of a genetic breeding program is required, especially seeking to conserve the structure of the MM breed as a well-defined genetic entity.     

3个品种兔微卫星标记的遗传多样性研究

为分析福建黄兔、新西兰兔、日本大耳白兔群体内的遗传变异情况和群体间的遗传相关性,本试验选取3个品种兔各25只,分别耳静脉采血并采用试剂盒抽提全血基因组,运用15个微卫星标记,结合荧光PCR技术和毛细管电泳方法获得目的片段,通过计算等位基因数、有效等位基因数、杂合度、多态信息含量和遗传距离,分析3个品种兔的遗传多样性。结果显示,3个品种兔在15个座位共检测到110个等位基因,平均等位基因数为7.3个,其中福建黄兔、新西兰兔和日本大耳白兔分别检测到95、95和88个等位基因,且分别在11、12和11个微卫星座位表现为高度多态,表明3个品种兔在筛选的15个微卫星座位均呈现较丰富的遗传多态信息。Hardy-Weinberg遗传平衡检验结果表明,福建黄兔、新西兰兔和日本大耳白兔分别有8、8和6个座位处于Hardy-Weinberg不平衡状态。Nei氏遗传距离分析显示,福建黄兔与新西兰兔、福建黄兔与日本大耳白兔、新西兰兔与日本大耳白兔的Nei氏遗传距离分别为0.1761、0.2347和0.0432。遗传距离越小时,相似度越高,亲缘关系越近,因此,新西兰兔与日本大耳白兔间的亲缘关系最近,福建黄兔与日本大耳白兔间的亲缘关系最远。     

Genetic diversity analysis of two buffalo populations of northern India using microsatellite markers

The genetic diversity in two buffalo populations of northern India, the Bhadawari and the Tarai was assessed using a set of 22 heterologous (bovine) microsatellite markers. The average number of alleles across all loci in both populations was found to be 4.7, indicating that this set of 22 bovine microsatellite markers could be used to study genetic variation in buffalo species also. The overall polymorphic information content (PIC) value for these markers was 0.54. The average observed and expected heterozygosities for both populations were 0.59 and 0.64, respectively. Common alleles with varying allele frequencies in both populations also represented the genetic variability existing between Bhadawari and Tarai buffaloes. However the θ estimates for population differentiation indicated low levels of differentiation between the two populations. This was further supported by the low genetic distance (0.155) between Bhadawari and Tarai, which was calculated using Nei's standard genetic distance method. The present study on Bhadawari and Tarai populations represents a much‐needed preliminary effort that could be extended to other local buffalo populations of India as well.     

Estimating allelic richness and its diversity

Assessing allelic richness in a set of populations requires that variations of sample size be taken into account. One way of doing this is to estimate the number of alleles expected in samples of specified size, using the rarefaction method applied in ecology. An alternative method, based on extrapolation, consists of adding to the number of alleles actually seen in a population the expected number of alleles missing, given the number of genes examined in the population and the allelic frequencies observed over the whole set of populations. Heterogeneity of allelic richness across populations and across loci can also be tested in this framework by numerical re-sampling. Both methods provide a measure of “private” allelic richness, a useful criterion in genetic diversity preservation, by allowing evaluation of the uniqueness of each population in terms of allele numbers. The two methods are compared on isozyme loci in the argan tree of Morocco and on microsatellite genotypes in the European pig. In both species, allelic richness and gene diversity behave quasi-independently over the populations compared and a higher differentiation is observed in allelic richness compared to gene diversity. In general, the rarefaction technique is sensitive to the sample size of reference and may lack sensitivity to rare alleles when the sample size of reference is small. Extrapolation may thus be recommended especially when the sample sizes of the populations are either low on average or highly unbalanced among populations.     

Genetic variation within and relationships among populations of Asian goats (Capra hircus)

Genetic variation at 59 protein coding loci (16 polymorphic) and 25 microsatellite loci was analysed for 11 indigenous south-east Asian goat populations, and the Australian feral population, to determine the magnitude of genetic differentiation and the genetic relationships among the populations. Significant deviations from Hardy–Weinberg equilibrium were detected in one or more populations for eight of the nine protein loci with codominant alleles, and for microsatellites for all except the two Sri Lankan populations and for all but four loci. For both marker types, average inbreeding coefficients ( F _IS) were exceptionally high. Heterogeneity of deviations from Hardy–Weinberg equilibrium for the microsatellites showed no differences for among loci within populations as compared with among populations within loci. For protein loci, however, the former was higher, indicating selection affecting allele frequencies at some loci. The variance among protein loci was significantly higher than among microsatellite loci, further indicating selection at some protein loci. There was significant differentiation among populations for both protein and microsatellite loci, most likely reflecting the geography of south-east Asia, and the presumed spread of goats throughout the region. Phylogenies derived from pair-wise genetic distance estimates show some similar clustering for the microsatellite and protein based trees, but bootstrap support was generally low for both. A phylogeny based on the combined set of 38 protein and microsatellite loci showed better consistency with geography and higher bootstrap values. The genetic distance phylogeny and the Weitzman diversity tree derived from microsatellite data showed some identical clusters, and both identified the Ujung Pandang and Australia populations as contributing most to overall genetic diversity.     

High genetic diversity in the endangered and narrowly distributed amphibian species Leptobrachium leishanense

Threatened species typically have a small or declining population size, which make them highly susceptible to loss of genetic diversity through genetic drift and inbreeding. Genetic diversity determines the evolutionary potential of a species; therefore, maintaining the genetic diversity of threatened species is essential for their conservation. In this study, we assessed the genetic diversity of the adaptive major histocompatibility complex (MHC) genes in an endangered and narrowly distributed amphibian species, Leptobrachium leishanense in Southwest China. We compared the genetic variation of MHC class I genes with that observed in neutral markers (5 microsatellite loci and cytochrome b gene) to elucidate the relative roles of genetic drift and natural selection in shaping the current MHC polymorphism in this species. We found a high level of genetic diversity in this population at both MHC and neutral markers compared with other threatened amphibian species. Historical positive selection was evident in the MHC class I genes. The higher allelic richness in MHC markers compared with that of microsatellite loci suggests that selection rather than genetic drift plays a prominent role in shaping the MHC variation pattern, as drift can affect all the genome in a similar way but selection directly targets MHC genes. Although demographic analysis revealed no recent bottleneck events in L. leishanense, additional population decline will accelerate the dangerous status for this species. We suggest that the conservation management of L. leishanense should concentrate on maximizing the retention of genetic diversity through preventing their continuous population decline. Protecting their living habitats and forbidding illegal hunting are the most important measures for conservation of L. leishanense.     

Screening the Polymorphic Tri-nucleotide Repeat Microsatellites from the Genome of Maiwa Yak and Their Genetic Diversity Analysis

In order to study the genetic diversity and differentiation characteristics of the existing population of Maiwa yak and provide a theoretical basis for utilization in the future, a total of 9 466 tri-nucleotide repeats microsatellites loci were screened based on the release of genome of yaks by bioinformatics method. The tri-nucleotide repeats microsatellites were amplified by PCR using the genomic DNA of 191 Maiwa yaks as templates,and the polymorphic microsatellite loci were genotyped through polyacrylamide electrophoresis experiment. After the Hardy-Weinberg equilibrium test, 5 tri-nucleotide microsatellite loci were deviated from the Hardy-Weinberg equilibrium (P <0.05). 15 microsatellite loci were highly polymorphic (PIC >0.5), the mean observed heterozygosity (Ho),mean expected heterozygosity(He) and mean polymorphic information content(PIC) were 0.5041,0.8152 and 0.7831,respectively. Maiwa yak population exhibited no characteristics of differentiation by Structure 3.0 software analysis. Therefore, Maiwa yak had plentiful genetic polymorphism while no signs of genetic differentiation were observed within the population.