跨种扩增筛选大鳄龟微卫星标记及遗传多样性分析

跨种扩增是一种能够快速、有效地获得物种微卫星标记的方法。本研究利用在近缘种中已发表的微卫星DNA引物,对大鳄龟（Macroclemys temminckii）进行跨种PCR扩增,在合成的69对引物中获得8对具有多态性的微卫星位点。对PCR扩增产物进行统计,得出观测杂合度（H_o）的范围是0.041 7~0.954 5,平均为0.384 8;期望杂合度（H_E）的范围为0.041 7~0.811 8,平均为0.479 1;多态信息含量范围为0.040 0~0.759 2,平均为0.423 2;经过卡方检验后,部分微卫星位点符合哈迪-温伯格平衡。总体来说,这些位点是研究大鳄龟遗传结构的良好分子标记。     

Limited genetic differentiation in Labeo rohita (Hamilton 1822) populations as revealed by microsatellite markers

Labeo rohita, popularly known as rohu is a widely cultured species in the whole Indian subcontinent. Knowledge of the genetic diversity of this species is important to support management and conservation programs which will subsequently help in sustainable production of this species. DNA markers, mostly microsatellite markers are excellent tool to evaluate genetic variation of populations. Genetic variation of three wild and one farm population was assessed using eleven microsatellite loci. In analyzing 192 samples, the number of alleles ranged from 4 to 23; observed heterozygosity 0.500 to 0.870 and expected heterozygosity from 0.389 to 0.878. Exact test for Hardy Weinberg disequilibrium revealed that each riverine sample had at least one locus not in equilibrium except one river. Negative inbreeding coefficients (F_IS) were observed across populations indicating very high level of genetic diversity but little genetic differentiation among populations.     

Microsatellite development for a tetrodotoxin-containing sea slug (Pleurobranchaea maculata)

Using 454 pyrosequencing data, 24 polymorphic microsatellite markers were identified for the grey side-gilled sea slug, Pleurobranchaea maculata. The grey side-gilled sea slug is found throughout the western and south Pacific and is known to contain high concentrations of tetrodotoxin. Polymorphism was assessed in 20 individuals obtained from geographically distinct locations within New Zealand. Between 2 and 15 alleles were identified at each locus. The observed heterozygosity (H_o) and expected heterozygosity (H_e) ranged from 0.10 to 1 and 0.10–0.94, respectively. No significant linkage disequilibrium between pairs of loci or deviations from the Hardy–Weinberg proportions were observed. The markers are central to understanding the population biology and genetic structure of P. maculata.     

Genetic diversity and parentage assignment in Dojo loach,Misgurnus anguillicaudatus based on microsatellite markers

In this study, genetic diversity and structure of three Misgurnus anguillicaudatus populations from three different geographical locations in China (Hunan, Hubei and Henan province) were investigated using microsatellite markers. High level of genetic diversity of all three populations was revealed by expected heterozygosity (H_e), observed heterozygosity (H_o) and allele number. Significant genetic differentiations were found between all pairs of populations. The efficiency of eight microsatellite markers in parentage assignment of 540 progeny from twenty full-sib families was evaluated. Simulation based on allele frequency data demonstrated that probabilities of exclusion per locus range from 0.313 to 0.825 when no parent information is available and 0.504 to 0.904 when one parent is known. The assignment success rate based on the real data using eight markers was 96.85%. This study indicates that these M. anguillicaudatus resources are valuable genetic and breeding material for aquaculture and the microsatellite markers will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this species.     

Isolation and characterization of microsatellite loci from three cryptic species of Ceratosolen emarginatus

From an enriched (AG)_n/(AC)_n library, we developed nine polymorphic microsatellite loci for three cryptic species of the fig‐pollinating wasp Ceratosolen emarginatus. We genotyped one population for each of the three cryptic species across all the nine loci. In total, 204 alleles were detected from the three cryptic species of C. emarginatus. The observed heterozygosity was 0.755 ± 0.034, 0.653 ± 0.030 and 0.603 ± 0.073 in C. emarginatus populations A, B and C, respectively; the expected heterozygosity was 0.850 ± 0.031, 0.724 ± 0.035 and 0.702 ± 0.104, respectively. No linkage disequilibrium was found between any two loci of all three cryptic species. The newly isolated microsatellite markers will be very useful for estimating the genetic variation within and among the cryptic species and for revealing the mechanisms of speciation and inbreeding coexistence hypothesis of the cryptic species.     

Development of thirty-five novel polymorphic microsatellite markers in Pseudosciaena polyactis (Perciformes:Sciaenidae) and cross-species amplification in closely related species,Pseudosciaena crocea

Small yellow croaker, Pseudosciaena polyactis, is an economically important marine fishery species. In this study, we isolated 35 novel polymorphic microsatellite primers in P. polyactis by using the combined biotin capture method. The polymorphism of each locus was assessed in 30 individuals from Donggang, Northern Yellow Sea, China. A total of 519 alleles were detected and the number of alleles per locus ranged from 3 to 23. The PIC values of these 35 microsatellite loci ranged from 0.367 to 0.940. The observed (H_o) and expected heterozygosity (H_e) per locus ranged from 0.233 to 1.000 and from 0.438 to 0.943, respectively. Seven loci significantly deviated from Hardy–Weinberg equilibrium (HWE) after Bonferroni correction and no significant linkage disequilibrium (LD) was found between pairs of loci. In addition, cross-species amplification was performed in Pseudosciaena crocea, a closely related species of P. polyactis, to assess the applicability of these markers. These polymorphic microsatellites will provide useful tools for the study of genetic diversity and population structure of P. polyactis and P. crocea.     

Genetic diversity of nine faba bean (Vicia faba L.) populations revealed by isozyme markers

Seven isozyme systems (Sod, 6-Pgd, Me, Est, Skdh, Fdh and Gdh) representing nine loci were used to study the genetic diversity of nine faba bean populations. Seven loci revealed polymorphic bands and showed the same quaternary structure as that found in several species. They revealed a high number of phenotypes. Indeed, from 3 to 9 phenotypes per locus were investigated in this study. The percentage of polymorphic loci (P = 59.3 %) was higher than that mentioned in the autogamous species (P = 20.3 %) and less than the optimum (P=96 %) indicated for allogamous plants. Total genetic diversity (H _T) and within population genetic diversity (H _S) were estimated with the isozyme markers. The contribution of among population genetic diversity (D _ST) to total genetic diversity was 22%. Enzyme markers pointed out an average inbreeding level for whole population (F _IT) and within population (F _IS). Within population genetic diversity represents 78% of total diversity. Intra-population genetic diversity (H _S = 0.206) was ranged with the respect of allogamous species and was clearly higher than that of among population genetic diversity (D _ST = 0.057) indicating an out-crossing predominance in the studied populations. The expected heterozygosity was higher than that observed heterozygosity at the allogamous species was confirmed in this study. Although, the mean estimated gene flow was less than 1(Nm=0.814), the dendrogram based on Nei’s genetic distance of the 9 populations using UPGMA method showed some genetic drift between populations.     

Development of microsatellite markers for <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> (F.F. &amp; M.F. Allemão), a highly endangered species from tropical forest based on next-generation sequencing

Myracrodruon urundeuva is a tree species of high economic importance due the strength and durability of its wood. Threatened of extinction in Brazil, it is present only in a few forest remnants, mostly in conservation units. Currently, there is little information on the genetic diversity of natural populations in Brazil and even less information about the genome of this species. Here, new species-specific microsatellite loci were developed based on next-generation sequencing (Illumina). More than 100,000 loci were identified in the run, with di- to hexanucleotides motifs. Of these, 20 loci were selected for validation in 30 individuals, with 15 successfully polymorphic loci detected. The number of alleles ranged among loci from 3 to 16, with an average of 7.73, expected (H _e ) and observed (H _o ) heterozygosity ranged from 0.246 to 0.902 and from 0.103 to 0.867, respectively. These results point out that these new set of markers has a great potential for use in population genetic studies for genetic conservation of the species.     

Development and Characterization of Microsatellite Markers for Genetic Analysis of the Swimming Crab, <Emphasis Type="Italic">Portunus trituberculatus</Emphasis>

This study isolated and characterized 11 novel microsatellite markers for the commercially important swimming crab species, Portunus trituberculatus. Genetic diversity and population structure of two populations of P. trituberculatus in the East China Sea were assessed using these loci. The microsatellite markers produced 242 alleles, varying from 17 to 26 alleles per locus. In all the samples, the range of heterozygosity was 0.6324–0.9403 (observed) and 0.8998–0.9547 (expected). An F-statistic analysis revealed low genetic differentiation between the populations (mean F _ST = 0.0197), with 98% of the variation resulting from the within-population component. In addition, cross-amplification was tested in two other portunid species, and we found that many loci yielded useful information. The high degree of polymorphism exhibited by the 11 microsatellites suggests that these markers will be useful for both aquaculture and studies of natural populations of the genus.     

Genetic Diversity and Natural Population Structure of Cacao (Theobroma cacao L.) from the Brazilian Amazon Evaluated by Microsatellite Markers

A sample of 94 accessions of Theobroma cacao L. (cacao), representing four populations from the Brazilian Amazon (Acre, Rondônia, lower Amazon and upper Amazon) were analyzed using microsatellite markers to assess the genetic diversity and the natural population structure. From the 19 microsatellite loci tested, 11 amplified scorable products, revealing a total of 49 alleles, including two monomorphic loci. The Brazilian upper Amazon population contained the largest genetic diversity, with the most polymorphic loci, the highest observed heterozygosity; and the majority of rare alleles, thereby this region might be considered part of the center of diversity of the species. The observed heterozygosity for all the Brazilian populations (H _o = 0.347) was comparable with values reported for other similar upper Amazon Forastero cacao populations, with the Acre and Rondônia displaying the lowest values. The lower Amazon population, traditionally defined as highly homozygous, presented an unexpectedly high observed heterozygosity (H _o = 0.372), disclosing rare and distinct alleles, with large identity with the upper Amazon population. It was hypothesized that part of the lower Amazon population might derive from successive natural or intentional introduction of planting material from other provenances, mainly upper Amazon. Most of the loci exhibited a lower observed heterozygosity than expected, suggesting that self-pollination might be more common than usually assumed in cacao, but excess of homozygotes might also derive from sub-grouping (Wahlund effect) or from sampling related individuals. Most of the gene diversity was found to occur within groups, with small differentiation between the four Brazilian Amazon populations, typical of species with high gene flow.     

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.     

Development of microsatellite markers for the endangered freshwater pearl mussel Margaritifera margaritifera L. (Bivalvia: Unionoidea)

Freshwater pearl mussels (Margaritifera margaritifera L.) are among the most critically endangered freshwater invertebrates. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4900 recombinant clones from two genomic libraries. Thirteen loci revealed polymorphisms as demonstrated on 42 tested individuals from four river drainages. Allelic richness ranged from two to 12 alleles and averaged 6.8 alleles per locus with heterozygosity levels varying from 0 to 0.850 for observed heterozygosity (H_O) and from 0.174 to 0.850 for expected heterozygosity (H_E). Deficiency of heterozygous genotypes was observed in eight of 13 loci.     

Case study of microsatellite polymorphism of European perch in selected commercially important lakes of Latvia

Polymorphism of selected microsatellite markers was described for European perch populations of inland Lake Kala and costal Lake Babites in Latvia. The data consisted of ten microsatellite loci analysed for 90 individuals from the Lake Babites (n?=?45) and the Lake Kala (n?=?45). Both lakes differ in water area, connection with the Gulf of Riga (Baltic Sea), lake type, and fish species. In the Lake Kala population, the average number of alleles per locus varied from 4 to 15 and in the population of Lake Babites from 4 to 12. Low but significant genetic differentiation was detected between populations (F_ST?=?0.046). Observed and expected heterozygosity in both lakes was similar (Kala: H_o?=?0.680 and H_e?=?0.816; Babites: H_o?=?0.693 and H_e?=?0.815). The number of unique alleles per locus was 2.3 in Lake Kala and 0.6 in Lake Babites. The present study showed relatively high polymorphism of analysed microsatellite markers.     

Characterization of eight microsatellite loci for the sea urchin Meoma ventricosa (Spatangoida,Brissidae) through Next Generation Sequencing

Eight microsatellite loci were characterized for Meoma ventricosa (Lamarck, 1816), a burrowing sea urchin that can be afflicted by a bacterial disease causing localized mass mortality. For the analyzed population (29 individuals from St. Croix, US Virgin Islands), we observed 8.125 mean number of alleles, 0.640 mean observed heterozygosity (H_o) and 0.747 mean expected heterozygosity (H_e). Two loci showed significant deviations from Hardy–Weinberg equilibrium. Overall, the described loci were characterized by a moderately high level of polymorphism suggesting that these markers are useful for a population genetic study in the Caribbean Sea.     

Characterization of microsatellite markers in the mosquito Ochlerotatus caspius (Diptera: Culicidae)

Seven polymorphic microsatellite loci were developed for the mosquito species Ochlerotatus caspius, using an enriched genomic library. The number of alleles per locus varied between two and 11; the expected heterozygosity (H_E) ranged from 0.18 to 0.77. These microsatellite primers should prove useful for population genetic studies of this mosquito species.     

Population genetic structure of the noxious weed Amaranthus retroflexus in Central Europe

Genetic variation was assessed in a range of populations of Amaranthus retroflexus using isoenzyme analysis. Population genetic diversity was measured by evaluating patterns of variation at six putatively neutral isoenzyme loci (comprising 24 putative alleles) within and among 20 populations of A. retroflexus collected in different habitats: ruderal habitats, cereal fields and hop gardens. Amaranthus retroflexus is a noxious weed of North American origin that infests various crops. Overall, A. retroflexus displayed moderate levels of genetic diversity in comparison with other herbaceous plants. The percentage of polymorphic loci was 50.0%, with mean values of 2.01, 0.142 and 0.227 for the average number of alleles per polymorphic locus (A), observed heterozygosity (H_o) and expected heterozygosity (H_e), respectively. A discrepancy between observed and expected heterozygosity and significant differences from H-W expectation indicate that there is an excess of homozygotes in many populations. As a result, there is strong evidence of inbreeding within populations (F_IS = 0.382) and significant population differentiation (F_ST = 0.270). Even though the species is partly autogamous, inbreeding does not lead to strong inbreeding depression resulting from self-pollination, as inbreeding has no effect on the success of the species in today's countryside. Moreover, allele frequencies detected in agricultural habitats (i.e., cereal fields and hop gardens) differed from those detected in populations collected from ruderal habitats, which is probably caused by systematic application of herbicides in agricultural ecosystems.     

Genetic Structure of Wild Rice Oryza Glumaepatula Populations in Three Brazilian Biomes Using Microsatellite Markers

The existence of Oryza glumaepatula is threatened by devastation and, thus, the implementation of conservation strategies is extremely relevant. This study aimed to characterize the genetic variability and estimate population parameters of 30 O. glumaepatula populations from three Brazilian biomes using 10 microsatellite markers. The levels of allelic variability for the SSR loci presented a mean of 10.3 alleles per locus and a value of 0.10 for the average allelic frequency value. The expected total heterozygosity (H_e) ranged from 0.63 to 0.86. For the 30 populations tested, the mean observed (H_o) and expected heterozygosities (H_e) were 0.03 and 0.11within population, respectively, indicating an excess of homozygotes resulting from the preferentially self-pollinating reproduction habit. The estimated fixation index ( _IS ) was 0.79 that differed significantly from zero, indicating high inbreeding within each O. glumaepatula population. The total inbreeding of the species (_IT ) was 0.98 and the genetic diversity indexes among populations, _ST and _ST, were 0.85 and 0.90, respectively, indicating high genetic variability among them. Thus, especially for populations located in regions threatened with devastation, it is urgent that in situ preservation conditions should be created or that collections be made for ex situ preservation to prevent loss of the species genetic variability.     

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.     

Limited genetic impacts of habitat fragmentation in an “old rare” relict tree, Euptelea pleiospermum (Eupteleaceae)

Euptelea pleiospermum is an ??old rare?? tree species distributed along the high-elevation streamsides in Burma, China, and India. Deforestation and construction of roads for timber transport have highly fragmented the natural habitats of this species in the Shennongjia Forestry District. In this study, we used this fragmentation to test the hypothesis that ??old rare?? tree species are insusceptible to the genetic consequences of habitat fragmentation. Using eight microsatellite loci, we estimated allelic richness (A _R), observed heterozygosity (H _O), expected heterozygosity (H _E), Wright??s inbreeding coefficient (F _IS), and genetic differentiation (F _ST and D _EST) between pre- and post-fragmentation cohorts. We found no significant differences in either genetic diversity or genetic differentiation between the two cohorts. The limited genetic effects of fragmentation may result from too few fragmented generations, because the time between the start of fragmentation (year 1970) and our study (year 2008) was less than one generation of this tree species. It should be mentioned that clonal reproduction by sprouting, a common phenomenon in many ??old rare?? tree species, can help E. pleiospermum buffer the genetic impacts of fragmentation by delaying the time between generations. Therefore, we conclude that this ??old rare?? tree species show limited genetic impacts from recent habitat fragmentation. However, the elimination of rare alleles and increase of inbreeding coefficient in the post-fragmentation cohort are early warnings of deleterious genetic consequences of fragmentation. Our results provide valuable information to formulate conservation and restoration guidelines for E. pleiospermum.     

Population genetic characterization of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) using novel microsatellite markers

The dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) has long been considered an endangered insect in South Korea; the detection of recent population increases leaves its endangered status uncertain. Population genetic analysis subsequent to development of molecular markers is essential for establishing proper conservation strategies. In this study, we developed ten microsatellite markers specifically for C. tripartitus. Sixty-eight individuals of C. tripartitus collected from six South Korean localities were genotyped to validate these markers and preliminarily assess population genetic characteristics. Per-locus observed number of alleles, observed heterozygosity (H_O), and expected heterozygosity (H_E) ranged from 5–12, 0.499–0.958, and 0.54–0.743, respectively. All populations showed higher H_O than H_E, negative values of inbreeding coefficient, and, overall, no sign of recent population bottlenecks (excluding one population, Seosan). This suggests that C. tripartitus did not suffer from genetic drift and inbreeding, which are typically severe in small, isolated populations. Nevertheless, detection of only one of the two gene pools in some populations and resultant genetic subdivision into two population groups may suggest that the population size is not enough to cover both gene pools. Thus, a more extended period of protection may be required to ensure higher genetic diversity of widespread populations and achieve the long-term conservation goal.