Isolation of microsatellite loci and reliable genotyping using noninvasive samples of a critically endangered primate,Trachypithecus leucocephalus

Genetic information can be critical in identifying conservation priorities and developing conservation strategies. There is an urgent need for noninvasive genetic tools to study the wild populations of Asian colobine monkeys. The majority of these species are threatened with habitat destruction, population reduction and even extinction, but generally lack information on their genetic diversity and population structure. Genetic sampling and tissue collection have been scarce in these species owing to strict regulations on manipulation of endangered species, and the difficulties and risks associated with capturing these arboreal and fast‐moving monkeys in the challenging environments that they inhabit. These difficulties have hindered the development of molecular genetic markers, which are usually derived from tissues or blood. In this study, we present a method for de novo microsatellite isolation and genotyping using DNA from noninvasive origins of a critically endangered Asian colobine, the white‐headed langur (Trachypithecus leucocephalus). Genomic DNA isolated from hair was shown to be sufficient for microsatellite enrichment and isolation, with similar isolation efficiencies as from tissue DNA. We identified and characterized 20 polymorphic microsatellite loci, and evaluated their amplification success and genotyping reliability with 86 field‐collected fecal samples. These results show that this panel of loci can produce reliable genotypes from fecal samples, and represent a useful tool for noninvasive investigation of genetic structure, individual identification and kinship assessment in this highly endangered species. Our approach can be applied to conservation genetic studies of other wild species that lack sequence information and tissue samples.     

An evaluation of genetic diversity indices of the Red Bororo and White Fulani cattle breeds with different molecular markers and their implications for current and future improvement options

The genetic diversity of the Red Bororo and White Fulani cattle breeds of Cameroon and Nigeria was assessed with a panel of 32 markers. Estimates for the various indices of genetic diversity, total number of alleles (TNA), mean observed number of alleles (MNA), mean effective number of alleles (MNE), observed heterozygosity (H _ob) and expected heterozygosity (H _ex), were higher at microsatellite loci than at protein loci. Mean H _ex values were above 71% at microsatellite loci in all the breeds and ranged from 37% to 41.6% at milk protein loci and from 40.9% to 45.6% at blood protein loci. The highest TNA and MNA of microsatellites were recorded for the Nigerian White Fulani. MNE of milk protein loci was highest in the Cameroonian Red Bororo, while TNA of blood protein loci was highest in the Cameroonian White Fulani. The high genetic diversity levels indicate the presence of the necessary ingredients for improvement breeding and conservation. Multi-locus estimates of within-population inbreeding (f), total inbreeding (F) and population differentiation (θ) of the breeds were significantly different from zero, except for θ of blood proteins. A high level of gene flow was found between the breeds (5.829). The phylogenetic relationship existing among the four breeds is greatly influenced by location. The high gene flow between the breeds may lead to a loss of genetic diversity through genetic uniformity and a reduction in opportunities for future breed development. We propose an improvement scheme with aims to prevent loss of genetic diversity, improve productivity and reduce uncontrolled genetic exchanges between breeds.     

Characterization of 18 polymorphic microsatellite loci in the red‐crowned crane (Grus japonensis), an endangered bird

Red‐crowned cranes (Grus japonensis) were classified as an endangered species by the International Union for Conservation of Nature, but recently, their population has decreased dramatically. For the purpose of conserving this endangered species, 18 microsatellite markers were developed, including 12 newly isolated ones from a genomic library and 6 modified from another crane species. The markers were characterized in 26 red‐crowned cranes. As a result, these markers displayed 3–13 alleles, and the observed and expected heterozygosities ranged from 0.462 to 1.000 and from 0.483 to 0.884, respectively. The marker suite averaged 6.390 alleles per locus with an average polymorphic information content of 0.631. The combined exclusion probability (PE‐1) was 0.9985, and the combined exclusion probability (PE‐2) was 0.9999. Three of the 18 microsatellite loci presented significant deviations from Hardy–Weinberg Equilibrium (P < 0.05), likely due to sampling bias and unknown founder relatedness in a semi‐free population. Our results show that microsatellite loci can provide a standard protocol for genetic information in red‐crowned crane populations upon which strategies for effective conservation and management may be based.     

Evaluation of genetic architecture and mutation drift equilibrium of Marathwada buffalo population in Central India

In the present study, microsatellite data on 24 loci were generated and utilized to evaluate the genetic architecture and mutation drift equilibrium of Marathwada buffaloes, a Central Indian population maintained under low input system. Sufficient allelic diversity was observed with a total of 109 alleles across different loci. The genetic diversity analysis of Marathwada buffaloes displayed moderate level of within breed variability in terms of mean number of alleles per locus (4.48) and heterozygosity values (Ho = 0.532, He = 0.624). The studied Indian buffalo population showed considerable heterozygote deficiency (F_IS = 0.138) and deviation from HWE at many investigated loci. Three quantitative tests viz. sign test, standardized difference test and Wilcoxon sign rank test and a qualitative test for mode shift distortion of allelic frequencies were employed to evaluate mutation drift equilibrium under three different models of microsatellite evolution. The population was found to deviate significantly under IAM and TPM, while it was reverse under SMM. The qualitative test for mode shift supported the results under SMM indicating the absence of genetic bottleneck in the recent past in Marathwada buffaloes.     

东方白鹳微卫星遗传标记的筛选及其在种群遗传分析中的应用

东方白鹳(Ciconia boyciana)是分布于东亚的大型濒危涉禽,亟待深入开展其种群的保护遗传学研究.本研究采用微卫星标记技术筛选东方白鹤的微卫星位点,并进行种群的遗传多样性分析.所用的36对微卫星引物7对来自白鹳(Ciconia ciconia),12对来自朱鹮(Nipponia nippon),17对来自大蓝...     

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.     

太湖鸡资源群体遗传变异的检测

应用微卫星标记对江苏省无锡市祖代鸡场有限公司太湖鸡的保种群体进行了遗传变异的分析,计算了群体的杂和度(H)和多态信息含量(PIC)。研究结果表明:30个微卫星标记在太湖鸡保种群体的杂和度为0.504～0.858之间,平均杂和度为0.688,群体的遗传多样性丰富;其中24个微卫星标记在太湖鸡中多态信息含量为高度多态,可作为有效遗传标记用于太湖鸡保种群的遗传分析,而且各座位杂和度的高低与PIC值的大小体现了较高的一致性。本研究结果为太湖鸡开发利用研究提供了基础,也为太湖鸡的合理保护提供了科学根据。     

Study on Simplified Monitoring Method of Genetic Diversity for Shenxian Pig Conservation Population

In order to establish a simple detecting index system for the genetic diversity of Shen-xian pig population, 179 samples in 4 generations of Shenxian pigs were collected,and the population genetic diversity were detected using 27 microsatellite markers which were jointly recommended by the Food and Agriculture Organization of the United Nations (FAO) and the International Society for Animal Genetics (ISAG). 2-stage and multi-gradient sampling were conducted to the 27 microsatellite markers. The suitable number of microsatellite loci was determined according to the comparation of the polymorphic information content (PIC) of the sampled groups and total population,then validated with 4 generations of samples. The results showed that the relative deviation value PIC of eight microsatellite markers was no more than 5% comparing with all markers, and the relative deviation was about 10% in the different generations. 8 microsatellite markers including S0155, S0228, SW632, S0090, CGA, S0101, S0227 and SW122 constituted the final simplified index system, which could represent the changes of genetic diversity in Shenxian pig conservation population. Therefore, the genetic diversity monitoring of the population of Shenxian pig could be simplified to detect the eight microsatellite markers. At the same time, the method used in this study could also provide a reference for the simplified treatment of genetic diversity monitoring methods.     

深县猪保种群遗传多样性简化监测方法研究

为建立深县猪种群多样性监测的简化指标体系,试验采集河北省深县猪4个世代179头猪样本,采用联合国粮农组织和国际动物遗传协会联合推荐的27个微卫星标记进行群体遗传多样性分析,对27个微卫星标记进行两阶段、多梯度抽样,并根据抽样群体和总群体的多态信息含量（PIC）值的变化确定适宜抽样微卫星位点数,再以4个世代样本进行验证。结果表明,选取8个微卫星位点时与全部位点的比较相对偏差可控制在5%以内,且以各分世代的验证比较相对偏差在10%左右,通过比较确定以S0155、S0228、SW632、S0090、CGA、S0101、S0227、SW122这8个微卫星位点构成的简化指标体系可较好地反映深县猪种群遗传多样性的变化。因此,深县猪的保种群群体遗传多样性监测可以简化为检测这8个微卫星位点,同时本研究采用的方法也可为其他保种群的遗传多样性监测方法的简化处理提供参考。     

Past climate change and recent anthropogenic activities affect genetic structure and population demography of the greater long‐tailed hamster in northern China

The genetic diversity and the spatial structure of a species are likely consequences of both past and recent evolutionary processes, but relevant studies are still rare in East Asia where the Pleistocene climate has unique influences. In this study, we examined the impact of past climate change and recent anthropogenic activities on the genetic structure and population size of the greater long‐tailed hamster (Tscherskia triton), an agricultural rodent pest species in northern China. DNA sequence data of 2 mitochondrial genes and genotypic data of 11 microsatellite DNA loci from 41 populations (545 individuals) were gathered. Phylogenetic and population genetic analyses, as well as species distribution modeling and coalescent simulations, were conducted to infer its historical and demographic patterns and processes. Two deeply diverged mitochondrial clades were recovered. A small one was restricted to the Shandong Peninsula while the main clade was further divided into 3 geographic clusters by their microsatellite DNA genotypes: Northwest, North‐center and Northeast. Divergence dating indicated a Middle‐to‐Late Pleistocene divergence between the 2 clades. Demographic analysis indicated that all 3 and pooled populations showed consistent long‐period expansions during last glacial period; but not during the Holocene, probably due to the impact of climate warming and human disturbances. Conflicting patterns between mtDNA and microsatellite markers imply an anthropogenic impact on North‐center populations due to intensified agricultural cultivation in this region. Our study demonstrated that the impact of past glaciation on organisms in East Asia significantly differs from that of Europe and North America, and human activity is an important factor in determining the genetic diversity of a species, as well as its spatial structure.     

Application of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis)

The aim of the present study was to assess the applicability of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis). A total of 127 microsatellite markers were tested on a male and a young female Saola. An efficient amplification was observed for 123 markers (96.8%), 73 markers (59.3%) were polymorphic. Four loci (BM2304, BMS1928, BMS779 and ILSTS006) on cattle chromosomes 1, 4, 7 and 8, respectively, failed to amplify in Saola. Two cattle Y‐chromosome‐specific microsatellite markers (INRA126 and BM861) were successfully amplified from both sexes in Saola. However, two additional markers (INRA124 and INRA189) on Y‐chromosome failed to amplify in the female animal. These results show that most of the bovine microsatellite markers are applicable in Saola and therefore they can be used to study the phylogenetic relationships and the genetic diversity of the Saola population.     

Genetic monitoring of ex situ African Penguin (Spheniscus demersus) populations in South Africa

The African Penguin (Spheniscus demersus) has suffered population declines and is listed in the IUCN Red List as Endangered. The species is endemic to the coast of southern Africa, and breeding colonies are distributed on the south-western coast of Africa. Currently, African Penguins are being kept in zoo and aquarium facilities throughout South Africa. In this study, molecular genetic data based on 12 microsatellite markers from 1 119 African Penguin samples from four facilities were generated in order to determine the level of genetic variation, population structure and differentiation, and effective population size to assist in the development of an effective captive management plan. Expected heterozygosity ranged from 0.57 to 0.62, and allelic richness from 4.2 to 5.1. However, based on differences between first- and second-generation captive birds, we conclude that the ex situ population is at risk of losing genetic variability in the future and management programmes should include exchange of birds between captive facilities in order to induce gene flow and increase effective population size. Adding individuals from in situ populations should also be considered in the future in cases where these birds cannot be rehabilitated. Molecular genetic analyses of wild penguin populations should be carried out for comparison, and to ascertain to what degree ‘in situ genetic diversity’ is represented among ex situ populations. With regular resampling and analyses, the extent of the effect of processes such as genetic drift on diversity in the ex situ penguin populations will become evident.     

Effect of selection for scrapie resistance on genetic diversity in a rare and locally adapted sheep breed: The case of Sambucana

In Italy, since 2005 a breeding plan to increase scrapie resistance has been adopted. The impact of this selection on genetic diversity was assessed on Sambucana, an autochtonous sheep breed reared in southern Piedmont, by analysing the evolution of allele frequencies at different levels: PRNP (prion protein) gene, microsatellite loci on OAR13 (where PRNR maps), and microsatellite loci on other chromosomes, not subjected to selection for scrapie resistance. A total of 147 young rams, 80 born in 2004 and 67 in 2008–2009 were analysed. Evidence of diversity loss was observed for PRNP gene as a consequence of the directional selection. Diversity was affected in the immediate vicinity of PRNP but the effect on more distant loci on the same chromosome was trivial. With regard to neutral markers, lack of heterozygosis with no changeover of allele frequencies was observed suggesting an increase of inbreeding. Mating policies would be sufficient to solve these problems. A selection scheme based on genotyping rams and eliminating carriers of both susceptible and high susceptible alleles is the best way to improve natural resistance to scrapie with low costs and minimal problems in the current conservation programmes targeting rare breeds.     

Combining spawn egg counts,individual photo-ID and genetic fingerprinting to estimate the population size and sex ratio of an endangered amphibian

According to the International Union for Conservation of Nature Red List, 41% of the world's amphibian species are threatened with extinction, making them more threatened than any other vertebrate group nowadays. Given the global amphibian crisis, comprehensive understanding of demographics and population trends of declining and threatened species is essential for effective management and conservation strategies. Counting egg spawns is widely used to assess population abundance in pond breeding anurans. However, it is unknown how such counts translate into robust population size estimations. We monitored the breeding activity of the Natterjack toad (Epidalea calamita), combining egg string counts and individual photo-identification with Capture-Mark-Recapture population size and operational sex ratio estimation. Male Natterjack toads were identified by the pattern of natural markings with repeated ID of the same individual confirmed for 10% of the samples using genetic fingerprinting. We identified 647 unique individuals within a closed study population at Caherdaniel, Co Kerry. Population estimates derived from egg string counts estimated a breeding population of 368 females (95% CI 353–384) and Capture-Mark-Recapture estimated a breeding population of 1698 males (95% CI 1000–2397). The female:male sex ratio was conservatively estimated at 1:5 (95% CI 1:3–1:6) where 62% ± 6% of females were assumed to spawn. These substantially departed from any priori assumption of 1:1 which could have underestimated the breeding population by up to 72%. Where amphibian absolute population size estimation is necessary, methods should include empirical survey data on operational sex ratios and not rely on assumptions or those derived from the literature which may be highly population and/or context-dependent.     

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.     

Genetic structure and recent population demographic history of Taihangshan macaque (Macaca mulatta tcheliensis), North China

Massive actions have been and are being taken into protecting the world's primates from extinction, while the study of the properties of genetic diversity, demographic history, and ecological relationships will benefit the understanding of the long-term survival of a species. The Taihangshan macaque (Macaca mulatta tcheliensis), a subspecies of rhesus macaque (Macaca mulatta), is endemic to China and currently restricted to southern Mt. Taihangshan area. Herein, we evaluated the genetic diversity, population structure, and demographic history of this subspecies using mitochondrial (Cytb and high variable region I: HVR I) and nuclear markers (microsatellite loci) of 131 individuals collected from 9 localities covering the distribution range of this subspecies. Both phylogenetic analyses and genetic assignment revealed that the wild populations of Taihangshan macaques could be divided into 2 major highly divergent clades, THS-east and THS-west. Low genetic diversity (π: 0.00266 ± 0.00016) but high haplotype diversity (H_d: 0.80352 ± 0.015) were detected in the Taihangshan macaques, particularly in THS-east. Analyses of demographic history suggested that the Taihangshan macaques experienced first a stable historical population size from Holocene to early 19th century but a subtle decline and then slight growth in the recent 200 years. We suggest that bridging the neighbor populations (i.e. setting corridors) would facilitate the male-mediated gene flow and subsequently increase the genetic diversity of the Taihangshan macaque populations.     

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。     

新疆塔里木马鹿遗传多样性的微卫星分析

利用5个微卫星标记对新疆塔里木马鹿遗传多样性进行了检测。统计了塔里木马鹿3个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),利用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)。结果表明5个微卫星位点在库尔勒、阿拉尔和沙雅县塔里木马鹿3个群体的平均多态信息含量分别为0.5884、0.5754、0.5344,除微卫星位点BM5004外均为高度多态,可作为有效的遗传标记用于塔里木马鹿遗传多样性分析;塔里木马鹿三个群体总平均PIC、h、Rh和E分别为:0.5661、0.5995、0.4474和2.7。分析认为塔里木马鹿遗传变异度较高,遗传多样性相对丰富,具有较大的遗传潜力。     

Aggregate diversity: New approach combining within- and between-breed genetic diversity

Between-breed genetic diversity is classically considered as a major criterion to be taken into account when setting priorities for conservation of domestic animal breeds. However, it has been argued that methods based on the between-breed component of genetic diversity may not be optimal because they ignore the within-breed component of variation. The paper considers the most common methods used to evaluate those two components when genetic diversity is evaluated on the basis of genetic markers, and proposes to define an aggregate diversity combining linearly the two components. This implies defining for each breed (or population) its contributions to the between-breed and to the within-breed diversity. When defining an aggregate diversity, one can weight these contributions by F_ST and 1−F_ST, respectively, since the fixation index F_ST of Wright represents the proportion of the total genetic variation which is due to differences in allelic frequencies between populations. Such an approach is valid when the objective is genetic improvement by selection within a so-called “meta-population”. However, in a more general context of animal breeding, when heterosis and complementarities between breeds have to be considered, as well as adaptation to specific environments, more weight should be given to the between-breed variation. The proper weight to apply may require solutions adapted to each particular situation. In a long-term conservation perspective, priorities should also take into account the degree of endangerment of each breed. By combining diversity contributions and probability of extinction, a cryopreservation potential (or priority) may be estimated for each breed. The problem is illustrated on a sample of 11 European pig breeds typed for 18 microsatellite loci.     

Patterns of geographic variation between mitochondrial and nuclear markers in Heaviside's (Benguela) dolphins (Cephalorhynchus heavisidii)

The Heaviside's (or Benguela) dolphin (Cephalorhynchus heavisidii) is endemic to the west coast of southern Africa. The present study investigated the population genetic structure across a large portion of the species distribution using mitochondrial control region and nuclear (microsatellite) markers. A total of 395 biopsy skin samples were analyzed; they were collected from free‐ranging Heaviside's dolphins in 7 locations along 1650 km of coast between Table Bay, South Africa and Walvis Bay, Namibia. Both genetic markers rejected the hypothesis of 1 homogenous population but revealed contrasting results in the genetic structuring of putative populations. Mitochondrial DNA suggested either 2 populations or a fine‐scale division with 6 (sub) populations, while microsatellite markers were indicative of 2 widespread populations with measurable gene flow between them. Neutrality tests and mismatch distribution of the mitochondrial sequences indicated a departure from mutation–drift equilibrium due to a population expansion at the 2 extremes of the geographic range, but not towards the middle of the distribution. These results highlight the importance of evaluating multiple genetic markers to gain reliable insights into population processes and structure.