Characterization of nine polymorphic microsatellite markers in sprat (Sprattus sprattus L.)

Nine polymorphic microsatellites were isolated from sprat (Sprattus sprattus) using a microsatellite enrichment protocol and selective hybridization with a biotinylated (AC)(12) probe. The loci showed different variation patterns in a Baltic Sea population (44 individuals) with mean number of alleles at 12.7 and mean observed heterozygosity at 0.78. These microsatellite loci are expected to be used for taxonomic considerations in sprat, stock differentiation and population genetic analysis.     

Isolation and characterization of 12 dinucleotide microsatellite loci in the anadromous fish Osmerus eperlanus (L.)

A subtractive hybridization method was used to isolate 12 dinucleotide microsatellite loci for the anadromous European smelt, Osmerus eperlanus (L.). Three to 17 (mean 8.08) alleles per locus were identified in the two populations screened, and observed heterozygosity ranged from 0.067 to 0.933. Loci in both populations showed significant deviations from Hardy-Weinberg expected frequencies. These 12 loci provide a good basis for investigation of O. eperlanus population structure.     

Isolation and characterization of 12 polymorphic microsatellite markers for the frog Pelophylax hubeiensis

Twelve polymorphic microsatellite loci were isolated from an (AC)n- and (AG)n-enriched DNA library for the endemic Chinese frog Pelophylax hubeiensis (Ranidae). The number of alleles per locus ranged from two to eight, with a mean of 5.17. The observed and expected heterozygosities ranged from 0.226 to 0.839 and from 0.204 to 0.826, with means of 0.568 and 0.656, respectively. No significant linkage disequilibrium was detected among these loci. However, two significant deviations from HWE were discovered at loci Pehu-11 and Pehu-12 (P<0.05). MICRO-CHECKER tests showed that null alleles could be present at locus Pehu-12. These polymorphic microsatellite loci can be employed for exploring mating mechanisms, population genetic structure and other relevant genetic investigations of P. hubeiensis.     

梨小食心虫微卫星标记的扩增稳定性及多态性分析

【目的】筛选适合我国梨小食心虫Grapholita molesta种群遗传学研究的微卫星位点,并依据所筛选的微卫星位点进行梨小食心虫地理种群的遗传多样性分析。【方法】利用欧洲梨小食心虫和苹果蠹蛾Cydia pomonella种群中已报道的11个微卫星位点, 分析各位点在我国12个种群257头梨小食心虫样本中的扩增稳定性,再进行其多态性分析,筛选适合的位点,然后进行种群遗传多态性分析。【结果】在分析的11个微卫星位点中, 位点Gm01, Gm03, Gm04和Cyd15无法稳定扩增; 位点Gm05扩增成功率较低, 位点Gm07遗传多态性较低; 而位点Gm02, Gm06, Gm08, Gm09和Gm10等扩增效果稳定且遗传多态性丰富。这5个稳定扩增的微卫星位点平均等位基因数量（N_A）为7.417~12.500, 平均观察杂合度（H_o）为0.366~0.655, 平均期望杂合度（H_e）为0.642~0.846, 多态信息含量（PIC）为0.800~0.935。【结论】本研究成功筛选出位点Gm02, Gm06, Gm08, Gm09和Gm10等5个微卫星位点。基于这5个微卫星位点标记的结果显示, 山东和陕西不同梨小食心虫地理种群均具有丰富的遗传多样性。 这5个位点可以适用于我国梨小食心虫种群的进一步遗传分析研究。     

Polymorphic CA/GT and GA/CT microsatellite loci for Ostrinia nubilalis (Lepidoptera: Crambidae)

Ten polymorphic dinucleotide (CA/GT and GA/CT) microsatellite loci suitable for population genetic screening were characterized from enriched partial Ostrinia nubilalis genomic libraries. Sequence from 126 enriched small insert genomic library clones identified 25 CA/GT and 58 GA/CT loci that were unique. Perfect repeats tended to be short (n = 10–12). Ten microsatellites, PCR amplified from a Crawfordsville Iowa population showed a mean of 10 alleles per locus (range six to 20), and six of 10 loci showed heterozygote deficiency. Amplification of eight loci was observed in the sister species O. furnicalis.     

Characterisation of 12 microsatellite loci in the Vietnamese commercial clam <Emphasis Type="Italic">Lutraria rhynchaena</Emphasis> Jonas 1844 (Heterodonta: Bivalvia: Mactridae) through next-generation sequencing

The marine clam Lutraria rhynchaena is gaining popularity as an aquaculture species in Asia. Lutraria populations are present in the wild throughout Vietnam and several stocks have been established and translocated for breeding and aquaculture grow-out purposes. In this study, we demonstrate the feasibility of utilising Illumina next-generation sequencing technology to streamline the identification and genotyping of microsatellite loci from this clam species. Based on an initial partial genome scan, 48 microsatellite markers with similar melting temperatures were identified and characterised. The 12 most suitable polymorphic loci were then genotyped using 51 individuals from a population in Quang Ninh Province, North Vietnam. Genetic variation was low (mean number of alleles per locus = 2.6; mean expected heterozygosity = 0.41). Two loci showed significant deviation from Hardy–Weinberg equilibrium (HWE) and the presence of null alleles, but there was no evidence of linkage disequilibrium among loci. Three additional populations were screened (n = 7–36) to test the geographic utility of the 12 loci, which revealed 100 % successful genotyping in two populations from central Vietnam (Nha Trang). However, a second population from north Vietnam (Co To) could not be successfully genotyped and morphological evidence and mitochondrial variation suggests that this population represents a cryptic species of Lutraria. Comparisons of the Qang Ninh and Nha Trang populations, excluding the 2 loci out of HWE, revealed statistically significant allelic variation at 4 loci. We reported the first microsatellite loci set for the marine clam Lutraria rhynchaena and demonstrated its potential in differentiating clam populations. Additionally, a cryptic species population of Lutraria rhynchaena was identified during initial loci development, underscoring the overlooked diversity of marine clam species in Vietnam and the need to genetically characterise population representatives prior to microsatellite development. The rapid identification and validation of microsatellite loci using next-generation sequencing technology warrant its integration into future microsatellite loci development for key aquaculture species in Vietnam and more generally, aquaculture countries in the South East Asia region.     

Microsatellite markers for assessing genetic diversity of the medicinal plant Paris polyphylla var. chinensis (Trilliaceae)

Paris polyphylla var. chinensis is a perennial herb with medicinal properties that is widely used in traditional Chinese medicine. However, this plant has been on the edge of extinction during the last few decades because of excessive deforestation based on the intense ethnopharmaceutical interest. We isolated 12 microsatellite loci from a (CT)(n)-enriched genomic library of P. polyphylla var. chinensis. The polymorphism at each locus was analyzed by screening 30 individuals from a natural population. The number of alleles ranged from 2 to 5. The observed and expected heterozygosities ranged from 0.000 to 0.467, with a mean of 0.247, and from 0.383 to 0.662 with a mean of 0.537, respectively. Six loci (Pp1, Pp3, Pp6, Pp7, Pp9, and Pp12) were found to significantly deviate from Hardy-Weinberg equilibrium. This may be due to the small population size, inbreeding or null alleles. Five of the pairwise comparisons (Pp1 and Pp4, Pp2 and Pp5, Pp2 and Pp9, Pp2 and Pp12, Pp11 and Pp12) exhibited significant linkage disequilibrium (P < 0.05). We conclude that these microsatellite markers will be useful for population genetic studies of P. polyphylla var. chinensis.     

DNA microsatellite characterization of the jaguar (Panthera onca) in Colombia

The Colombian jaguar population is thought to contain two different subspecies, Panthera onca centralis and Panthera onca onca. The genetic structure of this population was evaluated using 12 microsatellite loci (n = 62 samples). In addition, 22 jaguar DNA samples from Guatemala, Paraguay, Perú, Bolivia, Venezuela and Brazil were analyzed for these microsatellite loci (n = 84 samples). The results of this study indicate six primary themes. First, the levels of gene diversity were very high. Second, the majority of the loci analyzed showed an absence of Hardy-Weinberg equilibrium, probably due to the Wahlund effect (= population subdivision). Third, several microsatellite loci showed significant heterogeneity between the two supposed subspecies in the country. Nevertheless, gene flow was present between them, and heterogeneity was relatively low, although the assignment analyses showed good classification of the jaguars studied into their respective subspecies. Fourth, the long-term historical effective population sizes were calculated through a maximum likelihood procedure for single and multi-step mutation models. Fifth, seven out of twelve DNA microsatellites studied significantly deviated from a single-step mutation model. However, the overall mean multi-step mutation percentage for these 12 DNA microsatellites was only 6%. Therefore, 94% of mutations were uni-step. Sixth, no bottleneck events were detected in the Colombian jaguar population overall.     

Temporal changes in allele frequencies in two reciprocally selected maize populations

The effects of breeding on allele frequency changes at 82 restriction fragment length polymorphism (RFLP) loci were examined in two maize (Zea mays L.) populations undergoing reciprocal recurrent selection, Iowa Stiff Stalk Synthetic and Iowa Corn Borer Synthetic #1. After 12 cycles of selection, approximately 30% of the alleles were extinct and 10% near fixation in each population. A test of selective neutrality identified several loci in each population whose allele frequency changes cannot be explained by genetic drift; interpopulation mean expected heterozygosity increased for that subset of 28 loci but not for the remaining 54 loci. Mean expected heterozygosity within the two subpopulations decreased 39%, while the between-population component of genetic variation increased from 0.5% to 33.4% of the total. Effective population size is a key parameter for discerning allele frequency changes due to genetic drift versus those resulting from selection and genetic hitchhiking. Empirical estimates of effective population size for each population were within the range predicted by the breeding method. Received: 10 June 1998 / Accepted: 29 April 1999     

Novel microsatellite loci in the threatened European long-snouted seahorse (<Emphasis Type="Italic">Hippocampus guttulatus</Emphasis>) for genetic diversity and parentage analysis

The long-snouted seahorse Hippocampus guttulatus is one of the two European seahorse species. We describe the isolation of the first 12 microsatellite loci in this threatened species. These new markers were tested in non-invasive samples of 32 seahorses from NW Spain. The number of alleles ranged from 2 to 15 (mean: 6.3) and expected heterozygosity from 0.031 to 0.912 (mean: 0.500). All loci conformed to Hardy–Weinberg expectations and no genotypic disequilibrium was observed between any pair of loci. The theoretical exclusion probabilities for this set of loci, when no parental information exists or when one parent is known, were 0.973 and 0.998, respectively. This study indicates the usefulness of these novel loci for population analysis and kinship studies in Hippocampus guttulatus. Their potential application is extended to the other European seahorse species, since all loci were successfully cross-amplified in H. hippocampus.     

Characterization of 12 new microsatellite loci in Aenictus and Neivamyrmex army ants

Here we describe 12 polymorphic microsatellite loci that were cloned and characterized for three species of army ants: the North American Neivamyrmex nigrescens, and the Asian Aenictus laeviceps and Aenictus dentatus. Observed and expected heterozygosities ranged from 0.37 to 0.97 (mean 0.70), and from 0.48 to 0.95 (mean 0.72), respectively. We observed 2–30 (mean 12) alleles per locus. These new genetic markers will be useful for studies of overall population structure and the complex pedigrees in colonies of army ants.     

A panel of polymorphic microsatellite markers in Himalayan monal Lophophorus impejanus developed by cross-species amplification and their applicability in other Galliformes

Isolation and development of new microsatellite markers for any species is still labour-intensive and requires substantial inputs of time, money and expertise. Therefore, cross-species microsatellite amplification can be an effective way in obtaining microsatellite loci for closely related taxa in bird species. We have reported microsatellite loci for Himalayan monal for the first time. Fifteen microsatellite markers developed for chicken were cross-amplified in Himalayan monal. All the tested 15 microsatellite markers were polymorphic, with mean (± s.e.) allelic number of 4 ± 1.51, ranging 2–7 per locus. The observed heterozygosity in the population ranged between 0.285 and 0.714, with mean (± s.e.) of 0.499 ± 0.125, indicating considerable genetic variation in this population. While 12 loci conformed to Hardy–Weinberg equilibrium (P > 0.05), 3 loci, i.e. MCW0295, MCW0081, MCW0330 deviated from it (P < 0.05). No evidence for linkage disequilibrium was observed among pair of loci. Our study show that these 15 microsatellites loci could be employed in population genetic studies for Himalayan monal and their applicability in Jungle Bush Quail, Grey francolin and Kalij pheasant.

     

Isolation and characterization of polymorphic microsatellites in a Yangtze River fish,brass gudgeon (Coreius heterodon Bleeker)

The brass gudgeon (Coreius heterodon) is a fish of economic importance in the Yangtze River. From a (GATA)_n‐enriched genomic library, 25 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Nine loci exhibited polymorphism with two to 12 alleles (mean 3.9 alleles/locus) in a test population, and observed heterozygosity ranged from 0.1111 to 0.9630 (mean 0.4426). Three of the nine loci showed polymorphism in a congeneric species, the largemouth bronze gudgeon Coreius guichenoti. These loci should provide sufficient level of genetic diversity to evaluate the fine‐scale population structure of C. heterodon.     

Evolutionary relationships among 12 species belonging to three genera of the family Microhylidae in Papua New Guinea revealed by allozyme analysis

To elucidate the potential of electrophoretic analysis for understanding relationships among microhylid frogs in Papua New Guinea, an allozyme analysis was conducted. A total of 119 individuals from nine species of Cophixalus, two species of Sphenophryne and one species of Barygenys, all of which belong to the family Microhylidae, were studied. Fourteen enzymes extracted from skeletal muscles and livers were analyzed by starch-gel electrophoresis. These enzymes were encoded by genes at 20 loci. There were 2-15 phenotypes produced by 2-12 alleles at these loci. The mean proportion of heterozygous loci per individual, mean proportion of polymorphic loci per population, and mean number of alleles per locus in 12 species were 6.1%, 17.1% and 1.17a on average, respectively. The NJ and ML trees constructed from Nei's genetic distances showed that the genus Sphenophryne can be distinguished biochemically from Cophixalus and Barygenys, and that the species groups of Cophixalus, which are similar in external morphology, can be divided biochemically into several species.     

Short tandem repeat based analysis of genetic variability in Kanarese buffalo of South India

The goal of the present study was assessing genetic diversity within Kanarese buffalo, the dual purpose breed of South India. A total of 48 unrelated animals were genotyped at 23 short tandem repeat (STR markers)loci. The total number of observed alleles was 180 with a mean of 7.83 per locus, which varied from 3 to 12 across different loci. The mean observed and expected heterozygosity in South Kanara buffaloes was estimated to be 0.518 and 0.712 respectively. Within population inbreeding estimate (F _IS) was significantly positive in most of the investigated loci which resulted in significant deviation from Hardy-Weinberg equilibrium at 19 of 23 loci analyzed. Evaluation of South Kanara buffalo population for mutation drift equilibrium revealed no significant heterozygosity excess under three different models of evolution viz. infinite alleles model (IAM), step-wise mutation model (SMM) and two phase model (TPM), thus indicating the absence of any recent genetic bottleneck. The results of the present study will help in formulating rational breeding strategies as well as conservation of this important germplasm.     

Development of twelve novel microsatellite markers in the medicinal mushroom Cordyceps militaris (Ascomycota: Clavicipitaceae) for its strain identification and genetic analysis

Cordyceps militaris (a caterpillar fungus), which belongs to the class Ascomycetes, has extensively been used for medicinal purposes in East Asia. Here, we isolated and characterized 12 microsatellite loci from the medicinal mushroom, C. militaris. Twenty‐nine individual samples were taken from a single locality in southwestern Korea and used to characterize the developed markers. The number of alleles of these loci ranged from two to 13 (mean allelic richness = 6.44). Observed heterozygosity and expected heterozygosity in the population ranged from 0.035 to 0.880 and from 0.035 to 0.886, respectively. Five of 12 loci significantly deviated from Hardy–Weinberg Equilibrium, most likely due to heterozygote deficiency caused by inbreeding. Tests of genotypic linkage disequilibrium between the 12 loci showed no significant association of alleles. These microsatellite markers will provide valuable tools for genetic analyses for the strain identification of C. militaris as well as for its resource conservation.     

秦川母牛群体遗传特性的微卫星标记研究

为了从DNA分子水平揭示秦川牛群体遗传多态性和群体遗传结构,寻找可用于秦川牛的微卫星标记,本研究选择了12个普通牛（Bos taurus）微卫星标记检测了90头秦川母牛各微卫星位点的遗传变异及多态性。结果表明,在秦川母牛群体中,12个微卫星位点共检测到了247个等位基因,各位点的等位基因数在13（INRA005）～33个（HEL13）之间,平均每个微卫星位点的等位基因数为21个;总有效等位基因数和平均每个位点平均有效等位基因数（Ne）分别分为142．6229和11．8852。各位点平均基因频率取样方差（V（pij））为2．6036×10^-4。12个微卫星位点平均观察杂合度（Ho）和平均期望杂合度（He）在0．7842（INRA005）～0．9775（BM315）和0．7952（BM315）～0．9446（HEL13）之间。12个位点平均多态信息含量（PIC）在0．7653（INRA005）～0．9420（HEL13）之间,平均为0．8965．12个微卫星位点均属于高度多态位点,这表明秦川母牛群体中所检测各微卫星位点具有丰富的遗传多态性,具备较大的选择潜力。12个微卫星位点的平均固定指数（F）为-0．0076,即各位点杂合子的缺陷度不高,即偏离Hardy—Weinberg平衡的程度不大。     

Development and testing of 13 polymorphic microsatellite markers in Larimichthys polyactis (Sciaenidae) using 5' anchored PCR

Larimichthys polyactis is a commercially important marine fish species in southeast Asia. The population crashed due to overfishing in the 1970s, but has since recovered. We developed 13 novel polymorphic microsatellite markers in L. polyactis using 5' anchored PCR. The characteristics of these loci were estimated by analyzing a sample of 30 individuals. A total of 74 alleles were detected, with a mean of 5.7 alleles per locus. There were 2 to 12 alleles, 0.2760 to 0.8247 polymorphism information content, and 0.3214 to 1.000 observed and 0.3097 to 0.8567 expected heterozygosity per locus. The mean observed and expected heterozygosity was 0.6816 and 0.6724, respectively. Three loci deviated significantly from Hardy-Weinberg equilibrium after Bonferroni's correction, and no significant linkage disequilibrum between pairs of loci was found. This information will be useful for the analysis of population genetic diversity, and the management of this important fish resource.     

Microsatellite loci for the Chinese bamboo rat Rhizomus sinensis

We describe 19 polymorphic microsatellite loci for the bamboo rat Rhizomus sinensis. When tested with 20 samples from a single population, these loci exhibited a mean of 4.5 alleles per locus and a mean expected heterozygosity of 0.612. All loci were in Hardy-Weinberg equilibrium and no evidence for linkage disequilibrium was detected between any loci. These loci will be useful for studying population genetic diversity and differentiation in the Chinese bamboo rat.     

利用微卫星技术分析不同类群鸡的遗传多样性

利用8个微卫星标记分析了6个生产类群鸡的遗传多样性。计算了各群体在各位点上的等位基因频率,并据此计算出各群体的平均遗传杂合度(h)、多态信息含量(PIC)和有效等位基因数(Ne)。结果表明:6个鸡群在8个微卫星座位上的基因频率存在明显的差异,其平均基因杂合度为0.7317,平均多态信息含量为0.6815。其中,群体平均杂合度最高的是安卡红鸡,为0.7716;平均杂合度最低的是新罗曼鸡,为0.7073。所选的8个微卫星座位均为高度多态,可作为有效的遗传标记用于鸡群体遗传多样性的分析。