Isolation and characterization of 12 microsatellite loci from Korean water deer (Hydropotes inermis argyropus) for population structure analysis in South Korea

The main goals of this study were to isolate microsatellites markers of Korean water deer (Hydropotes inermis argyropus) and understand the genetic status of the species in South Korea. Twelve new microsatellite loci were isolated and characterized to establish basic population genetic parameters for 45 H. i. argyropus specimens in South Korea. There were no significant regional or genetic structure differences between the mid-eastern and southwestern populations in South Korea according to the population genetic analyses. The number of alleles per locus ranged from two to 13 with an average of 6.08. Mean expected (H _E) and observed heterozygosity (H _O) were 0.622 and 0.533, respectively. Microsatellite variability was also not significant between the two regions (F _ST=0.012). These new markers should facilitate the future population genetics studies of Korean water deer and other closely related species.     

Complete mitochondrial genome sequence of the Korean water deer Hydropotes inermis argyropus (Cervidae, Capreolinae)

The complete mitochondrial genome sequence of Hydropotes inermis argyropus consists of 13 protein-coding, 22 tRNA, and two rRNA genes, and 1 control region (CR). Three overlaps among the 13 protein-coding genes were found: ATP8/ATP6, ND4L/ND4, and ND5/ND6. The CR was located between the tRNA-Pro and tRNA-Phe genes and is 928 bp in length. The typical conserved domains, such as TAS and CSB, were identified in the CR.     

Distribution, density, and habitat use of the Korean water deer (Hydropotes inermis argyropus) in Korea

Water deer (Hydropotes inermis) belong to the genus Hydropotes, which is ecologically well adapted for environments ranging from desert to forest. Water deer tend to occupy the richest areas between forest and grasslands. There are two distinct subspecies in far East Asia: one in China (H. inermis inermis), and the other in Korea (H. inermis argyropus). Despite being listed as vulnerable on the International Union for Conservation of Nature (IUCN) Red List, little is known about the water deer. The species occurs in most areas of the Korean peninsula, except for Seoul and Jeju Island. Kyunggi Province near Seoul showed the lowest rate (56%) due to urbanization. There was a high difference in their presence between inland (81%) and coastal (60%) areas . In addition, large cities (67%) showed much lower rates than did rural areas (83%) where human population size is relatively low. Water deer are distributed differently based on habitat type, with differences in mean density observed among lowland (6.93 ind./km²), mountainous (1.91), and urban (1.27) areas. There was also a difference in mean density between low-elevation and high-elevation areas. Finally, whereas the deer preferred landscapes with 20°–25° of slope and broadleaf forest (P < 0.01), they did not avoid areas with other types of slopes and habitats.     

Developing and testing a habitat suitability index model for Korean water deer (Hydropotes inermis argyropus) and its potential for landscape management decisions in Korea

Geographic information system (GIS) and landscape-level data offer a new opportunity for modeling and evaluating the quality of wildlife habitats. Models of habitat quality have not been developed for some species, and existing models could be improved by incorporating updated information on wildlife–habitat relationships and habitat variables. We developed a GIS-based habitat suitability index (HSI) model for the Korean water deer (Hydropotes inermis argyropus), which often causes human–wildlife conflicts in the Chungnam Province of Korea because of industrialization and urbanization. The model is based on logistic regression analysis, which addresses the impact of multiple habitat variables, such as habitat components, topographic characteristics, and human disturbances. The model yielded a p-value of .289 (χ²?=?9.672) and 65.4% correct prediction level with the overall observation–prediction comparison data. The model demonstrated that a large portion of the province (61.6%) could be regarded as a poor habitat (mean HSI value of the province?=?0.22), while the current habitats of the province could be considered of moderate quality (mean HSI value?=?0.31). In addition, the chance of observation of the deer increases as the HSI level increases, which means that the model yields a good predictive power. Lastly, we used the model to produce a habitat suitability map. Our HSI model enabled us to quantify habitat preferences, which could be the basis for decision-making on habitat protection, mitigation, and enhancement of the Korean water deer. The proposed model is also applicable for improving and enhancing the existing management practices, as well as for establishing an effective wildlife protection policy.     

Genetic diversity of Chinese water deer (Hydropotes inermis inermis): implications for conservation

The Chinese water deer (Hydropotes inermis inermis) is endemic to China. Historically, the species was widely distributed, but now, habitat loss and poaching have reduced its range and number drastically. In order to provide useful information for its conservation, we have investigated the genetic diversity and population structure of the Chinese water deer by analyzing the 403 bp fragment of the mitochondrial DNA (mtDNA) control region (D-loop). Eighteen different haplotypes were detected in 40 samples. Overall, Chinese water deer have a relatively high-genetic diversity compared to other rare cervid species, with a haplotype diversity of 0.923+/-0.025 and nucleotide diversity of 1.318 +/- 0.146%. No obvious phylogenetic structure among haplotypes was found for samples of different origin. An analysis of molecular variance (AMOVA) showed significant differentiation between the Zhoushan and the mainland population (F(ST)= 0.088, P < 0.001; Phi( ST ) = 0.075, P = 0.043), which suggests that exchanges of individuals between Zhoushan and the mainland should be avoided. We also recommend that a breeding center be set up for the mainland population.     

Prevalence of Anaplasma and Bartonella spp. in Ticks Collected from Korean Water Deer (Hydropotes inermis argyropus)

Deer serve as reservoirs of tick-borne pathogens that impact on medical and veterinary health worldwide. In the Republic of Korea, the population of Korean water deer (KWD, Hydropotes inermis argyropus) has greatly increased from 1982 to 2011, in part, as a result of reforestation programs established following the Korean War when much of the land was barren of trees. Eighty seven Haemaphysalis flava, 228 Haemaphysalis longicornis, 8 Ixodes nipponensis, and 40 Ixodes persulcatus (21 larvae, 114 nymphs, and 228 adults) were collected from 27 out of 70 KWD. A total of 89/363 ticks (266 pools, 24.5% minimum infection rate) and 5 (1.4%) fed ticks were positive for Anaplasma phagocytophilum using nested PCR targeting the 16S rRNA and groEL genes, respectively. The 16S rRNA gene fragment sequences of 88/89 (98.9%) of positive samples for A. phagocytophilum corresponded to previously described gene sequences from KWD spleen tissues. The 16S rRNA gene fragment sequences of 20/363 (5.5%) of the ticks were positive for A. bovis and were identical to previously reported sequences. Using the ITS specific nested PCR, 11/363 (3.0%) of the ticks were positive for Bartonella spp. This is the first report of Anaplasma and Bartonella spp. detected in ticks collected from KWD, suggesting that ticks are vectors of Anaplasma and Bartonella spp. between reservoir hosts in natural surroundings.     

Development of chromosome-arm-specific microsatellite markers in Triticum aestivum (Poaceae) using NGS technology

? Premise of the study: The aim of this study was to assess the feasibility of developing chromosome-arm-specific microsatellite markers in wheat on a large scale based on chromosome survey sequences obtained with next-generation sequencing (NGS) technology. ? Methods and Results: The Illumina Hi Seq2000 sequencing platform was used to sequence DNA of isolated wheat chromosome-arm 7DL. The data were assembled and microsatellite loci were identified computationally. In total, 16315 microsatellites were identified from 161061 assembled contigs. Thirty-three markers were randomly selected for validation across 20 diverse wheat cultivars. Two nulli-tetrasomic stocks were also screened to validate the specificity of the newly developed markers. ? Conclusions: This is the first study on identification of chromosome-arm-specific microsatellite markers using NGS technology. These new chromosome-arm-specific markers will facilitate saturation of the 7DL genetic map, and their availability will support genetic mapping and positional cloning in wheat.     

Chromosomal distribution and organization of three cervid satellite DNAs in Chinese water deer (Hydropotes inermis)

The species-specific profile and centromeric heterochromatin localization of satellite DNA in mammalian genomes imply that satellite DNA may play an important role in mammalian karyotype evolution and speciation. A satellite III DNA family, CCsatIII was thought to be specific to roe deer (Capreolus capreolus). In this study, however, this satellite DNA family was found also to exist in Chinese water deer (Hydropotes inermis) by PCR-Southern screening. A satellite III DNA element of this species was then generated from PCR-cloning by amplifying this satellite element using primer sequences from the roe deer satellite III clone (CCsatIII). The newly generated satellite III DNA along with previously obtained satellite I and II DNA clones were used as probes for FISH studies to investigate the genomic distribution and organization of these three satellite DNA families in centromeric heterochromatin regions of Chinese water deer chromosomes. Satellite I and II DNA were observed in the pericentric/centric regions of all chromosomes, whereas satellite III was distributed on 38 out of 70 chromosomes. The distribution and orientation of satellite DNAs I, II and III in the centromeric heterochromatin regions of the genome were further classified into four different types. The existence of a Capreolus-like satellite III in Chinese water deer implies that satellite III is not specific to the genus Capreolus (Buntjer et al., 1998) and supports the molecular phylogeny classification of Randi et al. (1998) which suggests that Chinese water deer and roe deer are closely related.     

獐ISSR-PCR反应体系的建立与优化及引物筛选

利用正交试验L₁₆(4⁵)对影响獐(Hydropotes inermis)ISSR-PCR反应的Taq DNA聚合酶浓度、dNTP浓度、引物浓度、Mg²⁺浓度及模板DNA浓度5个因素在4个水平上进行优化,同时对退火温度进行梯度PCR反应,以建立适合于獐ISSR-PCR反应的最佳体系.最终确定獐25μL ISSR-PCR反应体系为:Taq酶1.25 U·25μL^-1、Mg²⁺浓度2.5 mmol·L^-1、引物浓度0.3μmol·L^-1、DNA模板量350 ng·25μL^-1、dNTP浓度0.15 mmol·L^-1.在此基础上,利用优化的反应体系成功筛选出10条用于獐相关研究的ISSR引物并确定了各自的最佳退火温度,为今后利用ISSR技术进行獐的物种鉴定与分类、亲缘关系、系统发育和生理病理学研究奠定了技术基础,也为开展其它大型资源动物如黑麂的保护遗传学研究提供理论基础.     

圈养獐春夏季昼间行为时间分配及活动节律

2008 年3 ～7 月,在上海市浦东新区华夏公园进行獐重引入试点,采用瞬时扫描取样法和全事件记录法,对圈养条件下24 只獐(12 雌,12 雄)春夏季昼间行为时间分配及活动节律进行了观察。结果表明:休息是春夏季最主要的行为方式,其次是摄食和运动。獐的昼间行为具有明显的节律性,以晨昏活动为主,主要活动时间集中在06:00 ～08:00 和16:00 ～18:00。单因素方差分析(One-way ANOVA analysis)和Mann-Whitney U 检验表明,在时间分配上,春、夏季休息、站立、排遗、梳理和运动行为差异极显著;雌雄间休息、站立、反刍、梳理和警戒行为差异极显著。雌性春、夏季站立、排遗、梳理、运动和社会行为差异极显著,摄食差异显著;雄性春、夏季休息、摄食、梳理、警戒和社会行为差异极显著,站立、反刍、排遗和运动行为差异显著。本研究结果表明重引入獐的行为时间分配和活动节律与野生种群相似,动物福利得到较好满足,对下一步在上海地区的种群复壮和迁地野放等具有重要意义。     

舟山群岛春秋季獐栖息地的生态特征

2008 年3 月和2008 年11 月,在舟山群岛以獐的足迹、粪便和卧迹等新鲜活动痕迹为依据,对獐春、秋季栖息地利用特征进行研究。共设置样方420 个,对样方内生境类型、乔木盖度、灌木盖度、草本盖度、坡位、坡度、坡向、海拔、人为干扰距离和水源距离等10 个生态因子进行测量评估。结果发现,獐春、秋两季的栖息地利用特征是(1)隐蔽和食物因子:春、秋季利用阔叶林、农田和山坡地,秋季对灌木林也有较高的利用率,而对农田的利用率下降;春、秋季乔木盖度、灌木盖度和草本盖度较低处(≤50%) 利用率较高,但春季对乔木盖度较高处(> 50% )也有较高的利用率; (2) 地形因子:春季对坡度较缓、中下坡位、海拔较低处(< 100 m)利用率较高;秋季对坡度较缓、中下坡位、海拔较低(<100 m)的南坡利用率较高; (3) 水源因子:春季利用水源距离较近(< 200 m) 的区域;秋季对水源距离较近(< 200 m)和较远(>600 m) 区域的利用率均较高; (4) 干扰因子:春季主要利用距离人为干扰近处(< 100 m),秋季主要利用距离人为干扰远(> 200 m)处。逐步判别分析显示,春、秋季獐栖息地利用特征存在显著差异,乔木盖度、坡度、坡向、海拔和人为干扰5 个因子是主要的区分因子。舟山群岛人为干扰剧烈、次生乔木和灌木较为发达以及草本植物不发达等一系列特点,造成了舟山群岛獐特殊的栖息地现状。本研究将对制订适合于海岛的动物栖息地保护对策提供理论依据,也为了解海岛生境下獐不同季节的生存状况提供科学依据。     

Population genetic structure of wild and hatchery black rockfish Sebastes inermis in Korea, assessed using cross-species microsatellite markers

The population structure of the black rockfish, Sebastes inermis (Sebastidae), was estimated using 10 microsatellite loci developed for S. schlegeli on samples of 174 individuals collected from three wild and three hatchery populations in Korea. Reduced genetic variation was detected in hatchery strains [overall number of alleles (N(A)) = 8.07; allelic richness (A(R)) = 7.37; observed heterozygosity (H(O)) = 0.641] compared with the wild samples (overall N(A) = 8.43; A(R) = 7.83; H(O) = 0.670), but the difference was not significant. Genetic differentiation among the populations was significant (overall F(ST) = 0.0237, P < 0.05). Pairwise F(ST) tests, neighbor-joining tree, and principal component analyses showed significant genetic heterogeneity among the hatchery strains and between wild and hatchery strains, but not among the wild populations, indicating high levels of gene flow along the southern coast of Korea, even though the black rockfish is a benthic, non-migratory marine species. Genetic differentiation among the hatchery strains could reflect genetic drift due to intensive breeding practices. Thus, in the interests of optimal resource management, genetic variation should be monitored and inbreeding controlled within stocks in commercial breeding programs. Information on genetic population structure based on cross-species microsatellite markers can aid in the proper management of S. inermis populations.     

Microsatellites for ecologists: a practical guide to using and evaluating microsatellite markers

Recent improvements in genetic analysis and genotyping methods have resulted in a rapid expansion of the power of molecular markers to address ecological questions. Microsatellites have emerged as the most popular and versatile marker type for ecological applications. The rise of commercial services that can isolate microsatellites for new study species and genotype samples at reasonable prices presents ecologists with the unprecedented ability to employ genetic approaches without heavy investment in specialized equipment. Nevertheless, the lack of accessible, synthesized information on the practicalities and pitfalls of using genetic tools impedes ecologists' ability to make informed decisions on using molecular approaches and creates the risk that some will use microsatellites without understanding the steps needed to evaluate the quality of a genetic data set. The first goal of this synthesis is to provide an overview of the strengths and limitations of microsatellite markers and the risks, cost and time requirements of isolating and using microsatellites with the aid of commercial services. The second goal is to encourage the use and consistent reporting of thorough marker screening to ensure high quality data. To that end, we present a multistep screening process to evaluate candidate loci for inclusion in a genetic study that is broadly targeted to both novice and experienced geneticists alike.     

Comparative genetic diversity of wild and hachery populations of Korean threadsail filefish Stephanolepis cirrhifer using cross-species microsatellite markers

The threadsail filefish Stephanolepis cirrhifer is a highly commercial fisheries resource in Korea that suffers intensive anthropogenic pressure across much of its range. For basic information about its current genetic status in relation to stock enhancement, the level and distribution of genetic variation between a wild and a hatchery-bred population were investigated using 10 microsatellite markers developed for Thamnaconus modestus. High levels of polymorphism were observed between the two populations. A total of 95 different alleles were found at all loci, with some alleles being unique. The allelic variability ranged from six to 13 in the wild population and from five to 13 in the hatchery one. The average observed and expected heterozygosities were estimated to be 0.72 and 0.80 in the wild sample and 0.70 and 0.79 in the hatchery one, respectively. These results showed similar genetic variability in the hatchery population, as compared with the wild population and significant genetic differentiation between the wild population and the hatchery samples (F _ST = 0.016, P < 0.05). Genetic drift in the intensive breeding practices for stock enhancement has probably promoted differentiation between populations. Significant deviations from Hardy-Weinberg equilibrium were detected in both populations. Our results indicate that further studies using species-specific microsatellite markers will be necessary for a more reliable assessment of genetic diversity of the species.     

Handicapped males and extrapair paternity in pied flycatchers: a study using microsatellite markers

We report an attempt to induce extrapair copulations and fertilizations in a species with a low intensity of sperm competition, the pied flycatcher Ficedula hypoleuca. Shortly after pair formation males were made less attractive to females by removing certain wing and tail feathers. Earlier research has shown that this manipulation reduces a male's pairing success. The idea was to test whether females mated to such males (N= 9) were more likely to obtain extrapair fertilizations than females mated to unmanipulated controls (N= 9). Paternity testing was carried out on all 98 young in the 18 broods, using a set of six microsatellite markers isolated from the species. Extrapair fertilizations were revealed in only three (17%) broods; two broods of handicapped males and one of a control male. A total of seven (7%) offspring were not genetically related to their putative father, a level which agrees well with results of other studies of this and other populations. We conclude that there was no evidence to suggest that the fertilization pattern was altered by the experimental manipulation. One reason for the lack of response could be that female mate choice in this species is based on male phenotypic, and not genotypic, quality.     

Pseudo-Sanger sequencing: massively parallel production of long and near error-free reads using NGS technology

Background

Usually, next generation sequencing (NGS) technology has the property of ultra-high throughput but the read length is remarkably short compared to conventional Sanger sequencing. Paired-end NGS could computationally extend the read length but with a lot of practical inconvenience because of the inherent gaps. Now that Illumina paired-end sequencing has the ability of read both ends from 600 bp or even 800 bp DNA fragments, how to fill in the gaps between paired ends to produce accurate long reads is intriguing but challenging.

Results

We have developed a new technology, referred to as pseudo-Sanger (PS) sequencing. It tries to fill in the gaps between paired ends and could generate near error-free sequences equivalent to the conventional Sanger reads in length but with the high throughput of the Next Generation Sequencing. The major novelty of PS method lies on that the gap filling is based on local assembly of paired-end reads which have overlaps with at either end. Thus, we are able to fill in the gaps in repetitive genomic region correctly. The PS sequencing starts with short reads from NGS platforms, using a series of paired-end libraries of stepwise decreasing insert sizes. A computational method is introduced to transform these special paired-end reads into long and near error-free PS sequences, which correspond in length to those with the largest insert sizes. The PS construction has 3 advantages over untransformed reads: gap filling, error correction and heterozygote tolerance. Among the many applications of the PS construction is de novo genome assembly, which we tested in this study. Assembly of PS reads from a non-isogenic strain of Drosophila melanogaster yields an N50 contig of 190 kb, a 5 fold improvement over the existing de novo assembly methods and a 3 fold advantage over the assembly of long reads from 454 sequencing.

Conclusions

Our method generated near error-free long reads from NGS paired-end sequencing. We demonstrated that de novo assembly could benefit a lot from these Sanger-like reads. Besides, the characteristic of the long reads could be applied to such applications as structural variations detection and metagenomics.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-711) contains supplementary material, which is available to authorized users.     

Genetic diversity over multiple generations of supplementation: an example from Chinook salmon using microsatellite and demographic data

We examined demographic data and microsatellite loci in a supplemented population of Chinook salmon (Oncorhynchus tshawytscha) seeking evidence of changes in genetic diversity or for reduction of the effective size (N _e ) arising from supplementation (i.e., the Ryman-Laikre effect). A supplementation program in the North Fork Stillaguamish River (Washington State, USA) was intended to increase abundance (N) and maintain genetic diversity in the depressed population. Since supplementation expanded in 1986, about 9% of the population has been randomly collected for broodstock. The resulting progeny are released into the wild and comprised 10–60% of all returning adults. Genotypic data were obtained at 14 microsatellite loci from adult samples collected in four years between 1985 and 2001; these data indicated that the allelic richness and expected heterozygosity did not significantly change during this period and that genetic diversity in the captive and wild progeny was similar. The inbreeding and variance N _e estimated from adult escapement between 1974 and 2004 were different for the same generation, but the ratios of effective size to census size were very similar and decreased following supplementation. The variance N _e by the temporal method increased over time, but it is difficult to draw conclusions because of necessary assumptions made during the calculations. Based on these results we conclude that: (1) genetic diversity has been maintained over multiple generations of supplementation; (2) supplementation has not contributed to a loss of genetic diversity; and (3) monitoring genetic effects of supplementation is not straightforward, but it can be useful to look at both demographic and genetic data simultaneously.     

Development and characterization of 29 microsatellite markers for Ligumia nasuta (Bivalvia: Unionidae) using an Illumina sequencing approach

Ligumia nasuta (Say, 1817; Eastern Pondmussel) is an imperiled freshwater mussel (Unionidae) in eastern North America. Population declines in the Laurentian Great Lakes resulting from the introduction of dreissenid mussels and habitat destruction in the 20th Century have greatly reduced and limited its distribution. To properly inform restoration and management efforts for L. nasuta, fine-scale genetic analyses must be performed on the remnant populations. This study used Illumina paired-end shotgun sequencing to identify potential microsatellite loci for L. nasuta, utilizing two samples to develop the Illumina paired-end shotgun library. Forty-eight primer pairs were tested on the remaining 24 samples. Twenty-nine of the 48 microsatellite primer sets screened were successfully amplified using 24 L. nasuta samples collected from the Great Lakes watershed. The estimated fragment size ranged from 167 to 445 base-pairs (bp) and the number of alleles per locus varied between 5 and 16 (mean = 9.7). Only five of the loci deviated significantly from Hardy–Weinberg expectations after Bonferroni corrections. The development of these new microsatellite loci will greatly facilitate future genetic studies on L. nasuta.     

Development and characterization of 24 microsatellite markers in Primula tosaensis,an endangered primrose,using MiSeq

Primula tosaensis (Primulaceae) is an endangered primrose endemic to Japan. In this study, 24 novel microsatellite markers were developed using Illumina MiSeq sequencing to facilitate conservation of this endangered species. The genetic diversity and polymorphisms of these novel markers were measured in 32 individuals from a wild P. tosaensis population. The number of alleles and expected heterozygosities ranged from 2 to 5 (mean = 2.8) and from 0.119 to 0.724 (mean = 0.395), respectively. All loci were in Hardy–Weinberg equilibrium. The markers developed in this study will provide a powerful and practical tool for investigating the population structure and genetic diversity of P. tosaensis.