Genetic Variations Revealed by Microsatellite Markers ina Small Population of the Sika Deer (Cervus nippon)on Kinkazan Island, Northern Japan

Genetic variations within a population of the Japanese sika deer, Cervus nippon, on Kinkazan Island were studied by microsatellite analysis. Seventeen pairs of polymerase chain reaction primers designed for several species of ungulates successfully amplified polymorphic microsatellite DNA in sika deer. About 20% of the Kinkazan population was sampled and genotyped for nine diagnostic microsatellite loci. Alleles at those loci in the Kinkazan population were found to be under the Hardy-Weinberg equilibrium. To determine whether the Kinkazan deer have a reduced level of genetic variability, an average heterozygosity in the population was calculated and compared with the values determined for other populations from Hyogo, Yamaguchi, Shimane, Tsushima, and Nagasaki. Neither the observed nor the expected heterozygosity in the Kinkazan deer significantly differed from that in the other populations. Our result indicated that, despite its small population size, the Kinkazan deer preserve extensive microsatellite variations.     

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.     

Eighteen novel polymorphic microsatellite loci developed from the Père David’s deer (<Emphasis Type="Italic">Elaphurus davidianus</Emphasis>)

Père David’s deer is a severely bottlenecked species but without showing inbreeding depression, making it essential to develop molecular markers to explore her genetic mechanism of population recovery. In this study, we isolated 18 novel polymorphic microsatellite loci from a dinucleotide-enriched library. This suit of markers presented 2–3 alleles for each locus and their observed and expected heterozygosities were 0.057–0.610 and 0.056–0.598, respectively. These new microsatellite loci had an average of 2.12 alleles and thus contributed to relatively low exclusion probabilities of parentage and paternity testing (0.768 and 0.921). However, when these loci were examined in combination with previous microsatellite markers, overall probabilities of parentage and paternity exclusion went up to 0.905 and 0.990, respectively, showing that these 26 microsatellite loci should be adopted together in future genetic analyses for this highly inbred species.     

Bovine microsatellite loci are highly conserved in red deer (Cervus elaphus), sika deer (Cervus nippon) and Soay sheep (Ovis aries)

We tested 174 bovine microsatellite primer pairs for use in a primitive breed of sheep and two species of deer. Of 173 markers, 127 (73·4%) gave a product in Soay sheep ( Ovis aries ) of which 54 (42·5%) were polymorphic. One hundred and twenty-nine of 174 (74·1%) markers gave a product in red deer ( Cervus elaphus ) of which 72 (55·8%) were polymorphic. In sika deer ( Cervus nippon ) 126 of 171 (73·7%) microsatellite primers gave a product with 47 (37·3%) polymorphic. The proportion of bovine microsatellite loci conserved across artiodactyl species was significantly greater in this study than previously reported. Reasons for this high degree of microsatellite conservation are discussed. We suggest that a high resolution comparative map of the artiodactyls can be constructed using microsatellites.     

Introgression of exotic Cervus (nippon and canadensis) into red deer (Cervus elaphus) populations in Scotland and the English Lake District

Since the mid‐19th century, multiple introductions of Japanese sika deer (Cervus nippon nippon) and North American wapiti (C. canadensis) have taken place in the British Isles. While wapiti have generally been unsuccessful, sika have been very successful, especially in Scotland where they now overlap at least 40% of the range of native red deer (C. elaphus). Hybridization between these two species and red deer has been demonstrated in captivity and in the wild. Using a panel of 22 microsatellite loci that are highly diagnostic between red deer and sika, and moderately diagnostic between red deer and wapiti, we investigated the extent of introgression between these species in 2,943 deer sampled from around Scotland and from the English Lake District using the Bayesian clustering software STRUCTURE. We also used a diagnostic mitochondrial marker for red deer and sika. Our survey extends previous studies indicating little introgression of wapiti nuclear alleles into red deer, in particular in Northern Scotland, Kintyre, and the Lake District. We found a new area of extensive sika introgression in South Kintyre. In the North Highlands, we show for the first time geographically scattered evidence of past hybridization followed by extensive backcrossing, including one red‐like individual with sika introgression, two sika‐like individuals with red deer introgression, and six individuals that were apparently pure sika at the nuclear markers assessed but which carried red deer mitochondria. However, there has not been a collapse of assortative mating in this region. Similarly, in the English Lake District red deer, we found only traces of past sika introgression. No sika alleles were detected in the Central Highlands or the Hebridean red deer refugia. We make suggestions for management to prevent further spread of sika alleles into red deer and vice versa.     

Genetic diversity analysis by microsatellite markers in four captive populations of the sika deer (Cervus nippon)

The Chinese sika deer (Cervus nippon) is a rare and vulnerable animal in China for medical use. In this study, the genetic diversity and population genetic structure of 113 Chinese sika deer from 4 populations (Linyi Farm, LF; Linyi Park, LP, Yangzhou Farm, YF; Yangzhou Zoo, YZ) were investigated with 14 microsatellite loci. Eighty-three alleles were detected at the 14 loci in all populations. The expected heterozygosity ranged from 0.257 to 0.863 and the observed heterozygosity from 0.226 to 0.821. The polymorphism information content at different loci ranged from 0.217 to 0.825. The results of the HWE (Hardy–Weinberg equilibrium) tests indicated that only four loci (CEH-5, BL42, Mber70, and CEH-2) were in HWE (P > 0.01). The mean number of alleles per population ranged from 3.21 to 5.64, observed and expected heterozygosities ranged from 0.568 to 0.685, respectively. Positive inbreeding coefficient (F_IS) values were found in every population. F_ST values ranged from 0.101 in the LF to 0.155 in the YZ. The genetic identity ranged from 0.1236 to 0.1645. The genetic distance ranged from 0.4746 to 0.6025. The results of this study indicate moderate genetic variation and polymorphism across the loci. Appropriate breeding strategies should be designed for deer in captivity.     

Establishment of paternity testing system using microsatellite markers in Chinese Holstein

To estimate the efficiency of microsatellite markers in paternity testing among Chinese Holstein, 30 microsatellite loci were used to differentiate 330 Chinese Holstein genotypes, according to the calculation of the allele frequency, number of alleles, effective number of alleles, genetic heterozygosity, polymorphic information content (PIC), and the exclusion probability in this cattle population. The results demonstrated that the exclusion probability ranged from 0.620 in locus BM1818 to 0.265 in locus INRA005 with the average of 0.472 and 11 microsatellite markers exceeding 0.5. The combined exclusion probability of nine microsatellite markers was over 0.99. The result showed that paternity testing of Chinese Holstein was basically resolved using the nine microsatellite markers selected.     

Ten polymorphic microsatellite DNA loci for paternity and population genetics analysis in the fen raft spider (Dolomedes plantarius)

Ten polymorphic di‐ and trinucleotide microsatellite loci were developed in the fen raft spider Dolomedes plantarius from a partial phagemid genomic library enriched for microsatellite inserts. The expected heterozygosity at these loci ranges from 0.62 to 0.9, with the observed allele numbers varying from four to 15 in the 22 individuals tested. Average paternity exclusion probabilities ranged between 0.290 and 0.686. In combination, the 10 polymorphic loci elicit an exclusion probability of 0.999. The high level of polymorphism of these microsatellite loci makes them ideal genetic markers for paternity and population genetics analysis in this endangered species.     

Characterization of 22 microsatellites loci from the endangered Houbara bustard (Chlamydotis undulata undulata)

Here we present a new set of 22 microsatellite loci isolated from Chlamydotis undulata undulata, an endangered Houbara bustard found across North Africa. The number of alleles per locus ranged from one to nine, and heterozygosities ranged from 0.167 to 0.944. Total exclusionary probabilities using these loci for the first and the second parent were 0.992932 and 0.999915, respectively. Successful cross‐amplification was observed in eight other Otididae species (12–22 of the 22 loci). These microsatellite markers are powerful tools for genetic identification, paternity assignment and population genetic studies.     

微卫星在种公牛个体识别与亲缘鉴定方面的应用

采用美国ABI公司牛亲子鉴定试剂盒(Bovine Paternity PCR Typing Kit, 包括11个常染色体)和3个自选的Y染色体微卫星座位, 检测我国部分种公牛站肉用种公牛14个微卫星座位的多态性分布, 评估其遗传多样性, 并探讨其用于个体识别与亲缘鉴定的可行性。结果表明: 种公牛在14个微卫星座位中遗传多样性均较高, 其中MCM158座位的平均多态信息含量最高达到0.888, ETH10座位的最低, 为0.482。单个座位的个体识别能力在0.715~0.968之间, 累积个体识别能力为99.99%, 累计非父排除率达到99.99%, 表明采用的14个位点适用于个体识别和亲缘鉴定。     

Isolation and characterization of eight microsatellite loci for the vulnerable Hainan Eld’s deer (<Emphasis Type="Italic">Cervus eldi hainanus</Emphasis>) in China

We report on the isolation and characterization of eight microsatellite markers in the Hainan Eld’s deer (Cervus eldi hainanus) from genomic DNA-enriched libraries. Thirty-three microsatellites were screened from the libraries, and 8 of the screened microsatellites were polymorphic. The number of observed alleles for each locus in 47 individuals ranged from 2 to 9, and the expected and observed heterozygosity was 0.141–0.792 and 0.128–0.957, respectively. Three loci (CEH-2, CEH-6 and CEH-8) of eight deviated from Hardy-Weinberg expectation and no significant linkage association was found among all these loci. These microsatellite markers provide useful tool for population genetic studies of the Eld’s deer.     

The establishment of a hybrid zone between red and sika deer (genus Cervus)

Japanese sika deer ( Cervus nippon nippon ) were introduced to Scotland around 80 years (20 generations) ago. The sika phenotype is expanding its range and hybridizing extensively with native red deer ( Cervus elaphus ) leading to the establishment of a hybrid zone. This zone is currently moving and cannot be considered to be at equilibrium. Cervid genotypes and mitochondrial haplotypes were mapped across the sika phenotype range, using diagnostic protein isozymes, microsatellite nuclear DNA markers and RFLPs in mtDNA. These were analysed to estimate heterozygote deficits and nuclear linkage disequilibria and cytonuclear disequilibria in relation to gene frequencies and time since contact. Introgression was found in both taxa and strong linkage disequilibria and heterozygote deficits characterize the populations longest exposed to hybridization. Populations further from the introduction site, where hybridization is facilitated by the dispersal of sika-like stags, show low values for linkage disequilibria and heterozygote deficit. The observed patterns in genotype are explained in terms of assortative mating and a selective advantage of the sika genotype. The genetic integrity of the Scottish mainland red deer is shown to be at risk from the invasion of sika.     

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 20 microsatellite loci for an important tropical tree Shorea leprosula (Dipterocarpaceae) and their applicability to S. parvifolia

Shorea leprosula is an important timber tree in Southeast Asia. From a genomic library enriched for dinucleotide (CT) repeats, 20 polymorphic microsatellite markers were developed for this species. Polymorphism of these loci was evaluated in a sample of 24 adult individuals from a natural population. The number of alleles ranged from five to 15, and the observed heterozygosity ranged from 0.333 to 0.875. Probability of paternity exclusion, in the case where the mother is known, ranged from 0.285 to 0.784. These markers were also characterized for applicability in S. parvifolia; 16 loci were successfully cross‐amplified and showed high levels of polymorphism.     

Characterization of microsatellite loci for the Australian sea urchin Heliocidaris erythrogramma

We report 16 polymorphic microsatellite loci from Heliocidaris erythrogramma, a common sea urchin endemic to temperate Australian waters. These microsatellites were tested in a minimum of 30 individuals, which yielded between five and 14 alleles per locus. Expected heterozygosity ranged from 0.52 to 0.92 with four loci deviating from Hardy-Weinberg expectations. These markers are expected to be useful for experimental studies involving paternity analysis and for quantifying population structure in H. erythrogramma across its geographic range.     

Characterization of 13 polymorphic microsatellite loci for an evergreen tree,Myrica rubra

We developed 13 microsatellite markers from a genomic library enriched for dinucleotide (CT) repeats in a dioecious evergreen tree, Myrica rubra. We screened loci from 32 adult trees. The number of alleles ranged from two to 14, and the expected heterozygosity ranged from 0.324 to 0.884. The total paternity exclusionary power where the mother was known equalled 0.99965. These loci are of clear value in studying mating system and parentage analysis of this species.     

Genetic differentiation between introduced Central European sika and source populations in Japan: effects of isolation and demographic events

Japanese sika deer (Cervus nippon nippon) were introduced at the turn of nineteenth and twentieth century to many countries in Eurasia, North America and Australasia. Subsequently, free-living invasive populations have become established in several countries, including the Czech Republic, where the expanding sika population causes serious problems through overgrazing, damage through browsing and through competition and hybridisation with native red deer. 122 Japanese and 221 Czech samples were used to examine the genetic diversity, genetic structure, and the level of genetic differentiation between native populations and those introduced to the Czech Republic. Analyses of 22 microsatellite loci revealed, for both countries, evidence of isolation by distance and clear sub-structuring of populations, different from patterns previously revealed by mtDNA markers. The high number of private alleles (58 within the Czech Republic and 84 within Japan), the Fst values, factorial correspondence analysis and Bayesian clustering support a high level of divergence between the source and introduced populations. Genetic variability was generally low due to recent demographic events (founder effect in the Czech population, bottlenecks in Japanese populations); however, the values of expected heterozygosity differed greatly between subpopulations and were not the lowest in the introduced Czech populations. Multiple introductions, rapid population growth, and possible hybridisation with red deer seem to have helped the successful expansion of sika within the Czech Republic. The results also indicate that male-mediated gene flow and human-mediated translocations have significantly influenced the current genetic structure of native sika populations in Japan.     

Analysis of microsatellites and parentage testing in saltwater crocodiles

Fifteen microsatellite loci were evaluated in farmed saltwater crocodiles for use in parentage testing. One marker (C391) could not be amplied. For the remaining 14, the number of alleles per locus ranged from two to 16, and the observed heterozygosities ranged from 0.219 to 0.875. The cumulative exclusion probability for all 14 loci was .9988. the 11 loci that showed the greatest level of polymorphism were used for parentage testing, with an exclusion probability of .9980. With these 11 markers on 107 juveniles from 16 known breeding pairs, a 5.6% pedigree error rate was detected. This level of pedigree error, if consistent, could have an impact on the accuracy of gentic parameter and breeding value estimation. The usefulness of these markers was also evaluated for assigning parentage in situations where maternity, paternity, or both may not be known. In these situations, a 2% error in parentage assignment was predicted. It is therefore recommended that more micro-satellite markers be used in these situations. The use of these microsatellite markers will broaden the scope of a breeding program, allowing progeny to be tested from adults maintained in large breeding lagoons for selection as future breeding animals.     

Bottlenecks, drift and differentiation: the population structure and demographic history of sika deer (Cervus nippon) in the Japanese archipelago

We assessed genetic differentiation and diversity in 14 populations of sika deer (Cervus nippon) from Japan and four populations of sika deer introduced to the UK, using nine microsatellite loci. We observed extreme levels of differentiation and significant differences in diversity between populations. Our results do not support morphological subspecies designations, but are consistent with previous mitochondrial DNA analyses which suggest the existence of two genetically distinct lineages of sika deer in Japan. The source of sika introduced to the UK was identified as Kyushu. The underlying structure of Japanese populations probably derives from drift in separate glacial refugia and male dispersal limited by distance. This structure has been perturbed by bottlenecks and habitat fragmentation, resulting from human activity from the mid-nineteenth century. Most current genetic differentiation and differences in diversity among populations probably result from recent drift. Coalescent model analysis suggests sika on each of the main Japanese islands have experienced different recent population histories. Hokkaido, which has large areas of continuous habitat, has maintained high levels of gene flow. In Honshu the population is highly fragmented and is likely to have been evolving by drift alone. In Kyushu there has been a balance between gene flow and drift but all the populations have experienced high levels of drift. Habitat fragment size was not significantly associated with genetic diversity in populations but there was a significant correlation between habitat fragment size and effective population size.     

Characterization of microsatellites in wild and sweet cherry (Prunus avium L.)--markers for individual identification and reproductive processes.

Nuclear microsatellites were characterized in Prunus avium and validated as markers for individual and cultivar identification, as well as for studies of pollen- and seed-mediated gene flow. We used 20 primer pairs from a simple sequence repeat (SSR) library of Prunus persica and identified 7 loci harboring polymorphic microsatellite sequences in P. avium. In a natural population of 75 wild cherry trees, the number of alleles per locus ranged from 4 to 9 and expected heterozygosity from 0.39 to 0.77. The variability of the SSR markers allowed an unambiguous identification of individual trees and potential root suckers. Additionally, we analyzed 13 sweet cherry cultivars and differentiated 12 of them. An exclusion probability of 0.984 was calculated, which indicates that the seven loci are suitable markers for paternity analysis. The woody endocarp was successfully used for resolution of all microsatellite loci and exhibited the same multilocus genotype as the mother tree, as shown in a single seed progeny. Hence, SSR fingerprinting of the purely maternal endocarp was also successful in this Prunus species, allowing the identification of the mother tree of the dispersed seeds. The linkage of microsatellite loci with PCR-amplified alleles of the self-incompatibility locus was tested in two full-sib families of sweet cherry cultivars. From low recombination frequencies, we inferred that two loci are linked with the S locus. The present study provides markers that will significantly facilitate studies of spatial genetic variation and gene flow in wild cherry, as well as breeding programs in sweet cherry.