Phylogeography and conservation genetics of Eld's deer (Cervus eldi)

Eld's deer (Cervus eldi) is a highly endangered cervid, distributed historically throughout much of South Asia and Indochina. We analysed variation in the mitochondrial DNA (mtDNA) control region for representatives of all three Eld's deer subspecies to gain a better understanding of the genetic population structure and evolutionary history of this species. A phylogeny of mtDNA haplotypes indicates that the critically endangered and ecologically divergent C. eldi eldi is related more closely to C. e. thamin than to C. e. siamensis, a result that is consistent with biogeographic considerations. The results also suggest a strong degree of phylogeographic structure both between subspecies and among populations within subspecies, suggesting that dispersal of individuals between populations has been very limited historically. Haplotype diversity was relatively high for two of the three subspecies (thamin and siamensis), indicating that recent population declines have not yet substantially eroded genetic diversity. In contrast, we found no haplotype variation within C. eldi eldi or the Hainan Island population of C. eldi siamensis, two populations which are known to have suffered severe population bottlenecks. We also compared levels of haplotype and nucleotide diversity in an unmanaged captive population, a managed captive population and a relatively healthy wild population. Diversity indices were higher in the latter two, suggesting the efficacy of well-designed breeding programmes for maintaining genetic diversity in captivity. Based on significant genetic differentiation among Eld's deer subspecies, we recommend the continued management of this species in three distinct evolutionarily significant units (ESUs). Where possible, it may be advisable to translocate individuals between isolated populations within a subspecies to maintain levels of genetic variation in remaining Eld's deer populations.     

海南坡鹿的起源、进化及保护

坡鹿是世界濒危物种,三个亚种分布在东南亚大陆,仅海南坡鹿种群分布在中国海南岛.2003年,国际社会的专家和学者提出了将海南坡鹿引入泰国亚种原分布区,重建已经绝灭野生种群的建议.在此种情况下,明确海南坡鹿的起源、与其它亚种间的系统发生关系、以及遗传多样性水平对有效保护坡鹿具有重要意义.本研究以线粒体DNA D-loop区490 bp基因片段为分子标记,比较分析了海南坡鹿、泰国亚种和缅甸亚种共35个样本的序列差异.我们所测的样本中,总共发现4种单倍型.所有21个海南坡鹿样品共享1种单倍型.利用最大似然法(ML)、最大简约法(MP)、邻接法(NJ)和贝叶斯法(Bayesian)构建的系统进化树表明海南坡鹿种群与泰国亚种的关系较近.但是,二者也发生一定程度的遗传分化.海南坡鹿与泰国亚种的遗传距离均值为0.026.我们推测海南坡鹿可能是在更新世冰期(69万年前)通过陆桥由东南亚大陆迁入中国海南岛.我们的结论说明海南坡鹿的遗传多样性很低,并且已独立进化很长时间.因此,我们不支持将海南坡鹿引入泰国亚种的原分布区,重建已经绝灭的野生种群的设想和建议.我们建议将海南坡鹿与泰国亚种分别作为两个独立的进化显著单元(ESUs)进行管理.     

梅花鹿的微卫星多态性及种群的遗传结构

梅花鹿是我国极度濒危的鹿科动物,其野生种群已濒临灭绝。为了探讨我国野生梅花鹿的保护和管理对策,我们选用了16 个微卫星位点检测来自东北、四川、江西和浙江种群的122 份样品,以此分析我国野生梅花鹿种群的遗传多样性和遗传结构。费希尔确切性检验表明,四个种群中均存在偏离哈迪- 温伯格平衡的现象。与其它的濒危动物相比,中国梅花鹿有着相对较高的遗传多样性:每个位点的平均等位基因数为4 个,平均期望杂合度为0. 559。四个种群中,东北种群拥有最高的平均等位基因数(n = 3. 688),东北种群、四川种群、江西种群以及浙江种群的平均期望杂合度分别为0. 584,0. 477,0. 585 和0.589,它们之间不存在显著的差异。同时,利用逐步突变模型、双相突变模型和无限等位基因突变模型检测了种群的瓶颈效应,结果表明:除四川种群外,其他种群在近期内都经历过遗传瓶颈。费希尔确切性检验及配对样品F ST 的结果均表明:四个梅花鹿种群间存在显著的遗传分化(P < 0. 001)。因此,我们建议将我国梅花鹿的野生种群划分为4 个管理单元进行保护和
管理。     

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

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

Development and characterization of microsatellite markers in sawih tree (Duabanga moluccana Blume) using ISSR-suppression PCR techniques

Duabanga moluccana (or locally known as sawih) is an indigenous fast growing tropical tree species that confers various advantages for the timber industry and for planted forests development. In this paper, we isolated and characterized 8 polymorphic microsatellite markers from the D. moluccana genome using ISSR-suppression PCR techniques. The number of alleles and PIC values ranged from 3 to 8 alleles per locus and from 0.488 to 0.792, respectively. Three microsatellite loci were deviated from Hardy-Weinberg equilibrium (P < 0.05). The transferability rate ranged from 24 to 100 % among the three indigenous tree species tested. This indicates that the newly developed microsatellite markers would be useful tools for population genetic studies on D. moluccana and other indigenous tree species.     

Application of bovine microsatellite markers for genetic diversity analysis of Swiss yak (Poephagus grunniens)

In order to assess the applicability of bovine microsatellite markers for population genetic studies in Swiss yak, 131 bovine microsatellite markers were tested on a panel of 10 animals. Efficient amplification was observed for 124 markers (94.6%) with a total of 476 alleles, of which 117 markers (94.3%) were polymorphic. The number of alleles per locus among the polymorphic markers ranged from two to nine. Seven loci (ILSTS005, BMS424B, BMS1825, BMS672, BM1314, ETH123 and BM6017) failed to amplify yak genomic DNA. Two cattle Y-chromosome specific microsatellite markers (INRA126 and BM861) amplified genomic DNA from both male and female yaks. However, two additional markers on cattle Y-chromosome (INRA124 and INRA189) amplified DNA from only males. Of the polymorphic markers, 24 microsatellites proposed by CaDBase for within- and cross-species comparisons and two additional highly polymorphic markers (MHCII and TGLA73) were used to investigate the genetic variability and the population structure of a Swiss yak herd that included 51 additional animals. The polymorphic information content ranged from 0.355 to 0.752, while observed heterozygosity (HO) ranged from 0.348 to 0.823. Furthermore, a set of 13 markers, organized into three multiplex polymerase chain reactions, was evaluated for routine parentage testing. This set provided an exclusion probability in a family of four yaks (both parents and two offspring) of 0.995. These microsatellites serve as useful tools for genetic characterization of the yak, which continues to be an important domestic livestock species.     

Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo‐Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co‐amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent–offspring relationships in natural populations, with over 99.6% accuracy in parent–offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28–96% of loci. The multiplex PCRs developed here provide a reliable and cost‐effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species.     

Isolation and characterization of microsatellite loci in the finless porpoise (Neophocaena phocaenoides)

Eight dinucleotide microsatellite loci were isolated from the finless porpoise (Neophocaena phocaenoides). Analysis of 30 individuals showed the number of alleles ranged from four to 21 with observed heterozygosity ranging from 0.300 to 0.833, and expected heterozygosity ranging from 0.437 to 0.932. Cross‐species amplification was tested in four other cetacean species. These microsatellite markers would be valuable tools for population genetic studies of finless porpoises and other cetacean species.     

棕果蝠微卫星位点的筛选及其对近缘种的通用性

棕果蝠在我国热带和亚热带地区分布广、种群数量大,对龙眼、荔枝等水果种植业造成巨大危害。近年来,棕果蝠在国内被大范围捕杀,造成其种群数量急剧下降。为了获得用于研究棕果蝠的种群结构和遗传多样性的分子标记,我们利用探针富集法构建了棕果蝠部分微卫星基因组文库,筛选出棕果蝠的4 个高度多态性位点;并结合前期本研究组已经发表的棕果蝠微卫星位点,共检测了10 个棕果蝠特异的微卫星标记在其它4 种果蝠(短耳犬蝠、犬蝠、大长舌果蝠、小长舌果蝠)内的通用性,为不同果蝠种群遗传学研究提供了更多的微卫星分子标记。     

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.)

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref‐sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit‐SMsa1 and kit‐SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.     

Plant microsatellite genotyping with 4-color fluorescent detection using multiple-tailed primers

We extended the concept of fluorescent microsatellite genotyping with a single-universal tailed primer to the simultaneous use of three different tailed primers to allow multiplexed 4-color detection for medium throughput genotyping of plant species. The method was tested on Eucalyptus DNA samples using three forward primer sequences of human microsatellite markers labeled with different fluorescent dyes. The robustness of the method was tested for the simultaneous detection and genetic analysis of microsatellites in a genetic mapping experiment. This method allows reliable and cost-effective genotyping with the same level of multiplexing attained in regular microsatellite fluorescent detection assays. Besides the enhanced quality of the genotypic data provided by the fluorescent detection method when compared to colorimetric ones, the economy brought about by this method becomes greater with an increasing number of microsatellite markers. This method has been particularly useful for genotyping populations of several tropical tree species addressing community-wide population genetics and conservation questions.     

Pedigree Analysis of the Sika Deer (Cervus nippon) using Microsatellite Markers

The usefulness of microsatellite markers in pedigree analysis of the sika deer (Cervus nippon) was tested in a herd in which the maternal lineages were recorded. Eighteen sets of microsatellite primers originally designed for bovine, ovine, and cervine loci successfully amplified polymorphic DNA in the deer. The numbers of alleles per locus ranged from two to seven, and the observed heterozygosity ranged from 0.350 to 0.900. The resolution power of the markers in paternity testing was then determined by calculating exclusion probabilities and paternity indices. Parentages of the study population were efficiently discriminated by genotyping 17 microsatellite loci. The microsatellite data were also used to calculate the genetic relatedness between individuals, which significantly correlated with coancestry coefficients for the pairs. Our results demonstrate that the microsatellite markers are efficient tools in studying the social structure and behavior of the sika deer, as well as in monitoring the inbreeding status.     

Determination of the minimum number of microsatellite markers for individual genotyping in wild boar (Sus scrofa) using a test with close relatives

In the context of developing a noninvasive, practicable method for population size estimation in wild boar, we present a stepwise procedure to reduce the number of required microsatellite markers for individual genotyping. Step1: an initial marker set of 12 microsatellite loci was tested for species specificity with nontarget DNA and resulted in an exclusion of two markers. Step 2: a variability test regarding heterozygosity and deviations from Hardy–Weinberg equilibrium led to the rejection of two further markers. Step 3: the remaining eight markers were tested for transferability across populations with three separate wild boar sample sets. Step 4: on the basis of probability of identity values, a reduction from eight to five markers was possible. Step 5: a novel test using tissue samples from female wild boars and their embryos provided evidence that four variable microsatellite markers and one sex marker are sufficient for individual identification of close relatives. Step 6: feces samples were finally used to estimate PCR (PS) and genotyping success (GS). In conclusion, we recommend a specific four-marker combination with both PS and GS >50% for a reliable individual identification in noninvasive population size estimation of wild boar.     

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.     

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.     

Development and characterization of microsatellite loci in the marsh deer (Blastocerus dichotomus Cervidae)

Marsh deer (Blastocerus dichotomus) is one of the most exposed large mammals in South America. To aid in the conservation management of the species, nine polymorphic microsatellite loci were isolated and tested on up to 50 animals, showing 3–12 alleles and expected heterozygosity values varying from 0.69 to 0.89. These markers should be of considerable utility in future population and ecological genetics studies of this species. The marsh deer (Blastocerus dichotomus) is the biggest South American species of deer. Originally distributed across a large part of South America, stretching from the south bank of the Amazon river to northern Argentina, significant wild populations are now restricted to the Pantanal, swamp-lands that cover about 40% of southwest Brazil. The marsh deer is listed as Vulnerable on the Red List of the IUCN. Three populations of the species from three areas in the Paraná River basin (between the states of São Paulo and Mato Grosso do Sul) were recently studied by observing protein polymorphism at 17 loci (Oliveira et al. 2005). Now we are presenting data about isolation of microsatellite markers to improve the results regarding population structure.     

Genetic diversity of Metrodorea nigra (Rutaceae) from a small forest remnant in Brazil assessed with microsatellite markers

Metrodorea nigra (Rutaceae) is an endemic Brazilian tree of great ecological importance, frequently found in the submontane regions of ombrophilous dense and semideciduous forests. This tree is useful for reforesting degraded areas and the wood can be employed in construction. We developed 12 microsatellite markers from a genomic library enriched for GA/CA repeats, for this species. Polymorphisms were assessed in 40 trees of a highly fragmented population found in Cravinhos, State of S?o Paulo, in southeastern Brazil. Among the 12 loci, 8 were polymorphic and only one had fixed alleles in this population. The number of alleles per locus and expected heterozygosity ranged from 2 to 11 and from 0.190 to 0.889, respectively. These results revealed moderate levels of genetic variation in M. nigra population when compared to other tropical species. Additionally, transferability of the 12 primers was tested in seven other Brazilian Rutaceae tree species (endemics: M. stipularis, Galipea jasminiflora, Esenbeckia leiocarpa and non-endemics: E. febrifuga, E. grandiflora, Balfourodendron riedelianum, Zanthoxylum riedelianum). Transferability ranged among species, but at least 8 loci (~67%) amplified in M. stipularis, demonstrating a high potential for transferring microsatellite markers between species of the same genus in the Rutaceae family.     

Microsatellite loci for Paspalum atratum (Poaceae) and cross-amplification in other species

? Premise of the study: Paspalum atratum is a perennial, cespitose, tropical grass native to Central and South America. This species belongs to a polyploid complex (Plicatula group) little known at the genetic level. The characterized microsatellite markers provide new informative tools for further studies of the hybridization, mating systems, and structure of the population. ? Methods and Results: Using the microsatellite-enriched library method, we isolated and characterized 19 microsatellite markers from P. atratum. Eleven of them were polymorphic, showing a variable degree of variation, while eight were monomorphic in the samples analyzed. Additionally, the transferability of these microsatellite markers was tested in other species. ? Conclusions: These results suggest that the characterized markers have enough discriminatory potential to be used in genetic characterizations of Paspalum taxa, which are based on an understanding of their mating systems and genetic structure, as well as in understanding the evolutionary processes involved in the evolution of groups of Paspalum.     

New polymorphic microsatellite loci,cross‐species amplification and PCR multiplexing in the black aphid,Aphis fabae Scopoli

Aphis fabae includes four morphological cryptic subspecies, which are mostly identified by their partially distinct secondary host range. To determine the extent of gene flow and isolation between these four taxa, we isolated and characterized 12 microsatellite loci from Aphis fabae fabae and tested cross‐species amplification of eight loci from the closely related species Aphis gossypii. Using eight previously described microsatellite loci, we have developed the polymerase chain reaction (PCR) multiplexing of 24 loci, which were separated in tree sets and five PCRs. These sets of microsatellite loci provide high throughput capacity for large‐scale population genetic studies at a minimum cost.     

Development and characterization of 10 novel microsatellite markers from Chital deer (Cervus axis) and their cross–amplification in other related species

Ten polymorphic microsatellite loci were isolated and characterized from Chital deer (Cervus axis). These loci show high levels of allelic diversity with four to eight alleles per locus in the 22 individuals of the free‐ranging population of Chital deer in Nehru Zoological Park, Hyderabad. In addition, we found that all the loci show cross–amplification in closely related as well as distantly related deer species. The amplification of these markers in different genera further indicates that these can be applied to a wide range of endangered deer species for their population genetics studies and conservation management.