Discordant patterns of genetic variation between mitochondrial and microsatellite markers in Acanthogobius ommaturus across the coastal areas of China

Acanthogobius ommaturus, which belongs to the family Gobiidae, is a euryhaline and demersal fish that is widely distributed in the coastal areas, harbors, and estuaries of China, D. P. R. Korea and Japan. In this study, the genetic diversity and genetic structure of five geographical populations of A. ommaturus was assessed using the mitochondrial hypervariable region gene and microsatellite markers. The results of the two genetic markers indicated that the A. ommaturus populations had a high level of genetic diversity. The mitochondrial marker detected weak genetic differentiation among populations, and the Neighbor-Joining tree showed that there was no obvious pedigree branches and geographic structure as well. However, population of Zhoushan showed significant genetic differentiation with other populations by microsatellite markers. The population of A.ommaturus has not experienced bottleneck effect recently. We speculated that the Pleistocene climate change and juvenile fish dispersal played an important role in the population differentiation of A. ommaturus.

Development and Characterization of Microsatellite Markers for Harpadon nehereus Based on High-Throughput Sequencing and Cross-Species Amplification in Three Myctophiformes Fishes

Harpadon nehereus is a widespread economical fish found in the coastal seas of China and has important ecological value in the marine ecosystem.H_(o)wever,its germplasm resources have been seriously degraded due to natural factors and anthropogenic activities.In this study,high-throughput sequencing was applied to search for microsatellite loci in H.nehereus transcriptome to provide references for its resource conservation and utilization.Polymorphic loci were developed by non-denaturing polyacrylamide gel electrophoresis,and their cross-species amplified ability was detected in three related species.A total of 5652 microsatellites were identified from 16974320 unigenes.Among the primer pairs designed for 100 SSRs for PCR amplification,80%were successfully amplified,and 26 loci were polymorphic with a high number of alleles from 3 to 11 each.The expected(H_(e))and observed(H_(o))heterozygosities were 0.355–0.885 and 0.375–0.958,respectively.Most of the loci were highly polymorphic(polymorphism information content:0.316–0.852;mean:0.713),and these markers can be applied in the population genetic diversity research of H.nehereus.H_(o)wever,the transferability of these primers was low,probably because of the close relation of the collected species.In follow-up work,simple sequence repeats will be excavated with genome-based technologies,and related species will be gathered to address the present inadequacies.

Isolation and Characterization of Nine Microsatellite Markers for Red-backed Ratsnake,Elaphe rufodorsata

The red-backed ratsnake(Elaphe rufodorsata) is widely distributed in East Asia, especially China. This species is a common snake in plain river network region. In the past several decades, E. rufodorsata has dramatically declined due to the effect of human activities and over hunting for traditional Chinese medicine. We developed nine species-specific microsatellite loci in 190 individuals collected from Huzhou, Zhejiang province in China. These markers revealed a high degree of genetic diversity(13–41 alleles per locus) and heterozygosity(H O ranged from 0.266 to 0.941, and H E ranged from 0.851 to 0.937). No locus exhibited significant deviations from Hardy–Weinberg equilibrium. There was no evidence of linkage disequilibrium among pairs of loci. These microsatellite markers were described in our study will be valuable tools for the long term management and population-level studies(e.g. the population structure, genetic diversity and variation, individual paternity and evolutionary history) of the species.

Development and characterization of novel polymorphic microsatellite markers for the Korean freshwater snail Semisulcospira coreana and cross-species amplification using next-generation sequencing

Korean freshwater snails of the genus Semisulcospira are widely distributed across East Asia.It has been a very popular nutritional food in Korea,and is an ecologically important water quality indicator because it lives only in clean water.However,no microsatellite markers have been generated to study the population genetic diversity of this genus.In the present study,we developed and characterized 18 novel microsatellite loci from Semisulcospira coreana genomic DNA.The microsatellites were isolated using 454 GS-FLX titanium sequencing and 18 markers were used for genotyping in S.coreana.In addition,we also tested the cross-species transferability of the microsatellite markers in four additional Semisulcospira spp.We identified 18 polymorphic loci and the number of alleles per loci,and their polymorphism information content values ranged from 2 to 17 and 0.203 to 0.902,respectively.The observed and expected heterozygosities of the loci ranged from 0.063 to 0.924 and 0.226 to 0.924,respectively.According to the analysis of the cross-species transferability of these markers,four species,S.forticosta,S.gottschei,S.tegulata,and S.libertina,showed a very high transferability(80%–85%).These results show that this set of nuclear markers could be useful for population genetics studies of this species and closely related species.

Morphometric analysis and fluorescent microsatellite markers to evaluate the genetic diversity of five populations of Penaeus japonicus in China

To understand the current status of germplasm resources of Penaeus japonicus,in this study,genetic diversity was analyzed based on morphometric analysis and fluorescent microsatellite markers.For morphometric analysis,the cumulative ratios of the PC1 and PC2(85.90%)were greater than 75%,which can explain the morphological variation among populations.For microsatellite analysis,the allele number in 10 microsatellite loci ranged from 27 to 53,and the effective allele number ranged from 21 to 42.The observed heterozygosity ranged from 0.463 to 0.983,whereas expected heterozygosity ranged from 0.932 to 0.966,and the average polymorphism information content was 0.947,which indicated a high level of genetic diversity.The unbiased genetic distance among populations ranged from 0.230 to 0.431,and the genetic similarity coefficient ranged from 0.654 to 0.782.Analysis of molecular variance(AMOVA)showed that the genetic variation within individuals was the main source of variation,accounting for 88.55%,and only 1.49%of the genetic variation came from among populations.

Isolation and characterization of 49 polymorphic microsatellite loci for Decapterus maruadsi using SLAF-seq, and cross-amplifi cation to related species

Decapterus maruadsi is a commercially important species in China, but has been heavily exploited in some areas. There is a growing need to develop microsatellites promoting its genetic research for the adequate management of this ?shery resources. The recently developed speci?c-locus ampli?ed fragment sequencing(SLAF-seq) is an effcient and high-resolution method for genome-wide microsatellite markers discovery. In this study, 28 905 microsatellites(mono-to hexa-nucleotide repeats) were identi?ed using SLAF-seq technology, of which di-nucleotide was the most frequent(13 590, 47.02%), followed by mono-nucleotide(8 138, 28.15%), tri-nucleotide(5 727, 19.81%), tetra-nucleotide(1 104, 3.82%), pentanucleotide(234, 0.81%), and hexa-nucleotide(112, 0.39%). One hundred and thirty-two microsatellite loci(di-and tri-nucleotide) were randomly selected for ampli?cation and polymorphism, of which 49 were highly polymorphic and well-resolved. The average number of alleles per locus was 13.63, ranging from 4 to 25, and allele sizes varied between 110 bp and 309 bp. The observed heterozygosity( Ho) and expected heterozygosity( He) ranged from 0.233 to 1.000 and from 0.374 to 0.959, with mean values of 0.738 and0.836, respectively. The polymorphism information content(PIC) ranged from 0.341 to 0.941(mean=0.806).However, 12 loci deviated from Hardy-Weinberg equilibrium. Furthermore, transferability tests were also successful in validating the utility of the developed markers in ?ve phylogenetically related species of family Carangidae. A total of 48 microsatellite markers were successfully cross-ampli?ed in Decapterus macarellus, Decapterus macrosoma, Decapterus kurroides, Trachurus japonicus, and Selaroides leptolepis.The present microsatellites provided the ?rst known set of microsatellite DNA markers for D. maruadsi,D. macarellus, D. kurroides, and D. macrosoma, and would be useful for further population genetic and molecular phylogeny studies as well as help with the ?sheries management formulation and implementation of the understudied species.

应用微卫星标记对海南和广西恒河猴遗传多样性的研究

本研究利用11个微卫星位点,对海南和广西恒河猴进行了遗传检测,并通过POPGEN32软件计算各个微卫星座位的等位基因频率、有效等位基因数目(Ne)、多态信息含量(PIC)和遗传杂合度(H)。结果表明,所选择的11个微卫星位点均存在高度的遗传多态性,H为0.6848~0.河河猴的Ne、PIC和H的平均值均高于海南猴,分别为4.2583、0.7090、0.7706和4.2054、0.7025、0.7656,这种差别可能与地域来源有关。这些研究为微卫星标记分析恒河猴遗传多样性提供理论基础。

Comparative population genetic analyses suggest hybrid origin of Rhododendron pubicostatum,an endangered plant species with extremely small populations endemic to Yunnan,China

Gene flow between sympatric congeneric plants is thought to be very common and may pose serious threats to endangered species.In the present study,we evaluate the genetic diversity and divergence of three sympatric Rhododendron species in Jiaozi Mountain using newly developed microsatellites through the Illumina MiSeq sequencing approach.Genetic diversity of all three Rhododendron species studied was moderate in comparison to genetic parameters previously reported from species of this genus.Interestingly,genetic structure analysis of the three species identified a possible hybrid origin of the threatened Rh.pubicostatum.This sympatry should be considered a unimodal hybrid zone,since Rh.pubicostatum is predominant here.Unimodal hybrid zones are uncommon in Rhododendron,despite the fact that hybridization frequently occurs in the genus.Issues pertaining to the conservation of Rh.pubicostatum resulting from admixture of genetic material from its parental species are discussed.

Genetic structure,gene flow pattern,and association analysis of superior germplasm resources in domesticated upland cotton(Gossypium hirsutum L.)

Gene flow patterns and the genetic structure of domesticated crops like cotton are not well understood.Furthermore,marker-assisted breeding of cotton has lagged far behind that of other major crops because the loci associated with cotton traits such as fiber yield and quality have scarcely been identified.In this study,we used 19 microsatellites to first determine the population genetic structure and patterns of gene flow of superior germplasm resources in upland cotton.We then used association analysis to identify which markers were associated with 15 agronomic traits(including ten yield and five fiber quality traits).The results showed that the upland cotton accessions have low levels of genetic diversity(polymorphism information content=0.427),although extensive gene flow occurred among different ecological and geographic regions.Bayesian clustering analysis indicated that the cotton resources used in this study did not belong to obvious geographic populations,which may be the consequence of a single source of domestication followed by frequent genetic introgression mediated by human transference.A total of 82 maker-trait associations were examined in association analysis and the related ratios for phenotypic variations ranged from 3.04% to 47.14%.Interestingly,nine SSR markers were detected in more than one environmental condition.In addition,14 SSR markers were co-associated with two or more different traits.It was noteworthy that NAU4860 and NAU5077 markers detected at least in two environments were simultaneously associated with three fiber quality traits(uniformity index,specific breaking strength and micronaire value).In conclusion,these findings provide new insights into the population structure and genetic exchange pattern of cultivated cotton accessions.The quantitative trait loci of domesticated cotton identified will also be very useful for improvement of yield and fiber quality of cotton in molecular breeding programs.

Genetic structure of the invasive Colorado potato beetle Leptinotarsa decemlineata populations in China

The Colorado potato beetle(CPB),Leptinotarsa decemlineata Say,is an infamous invasive species worldwide.It was first found in Xinjiang Uygur Autonomous Region of China in 1993 and spread to Northeast China in 2013.To better understand the genetic structure and the diffusion path of their populations in China,we used nine polymorphic microsatellite loci to elucidate the genetic diversity,genetic structure and gene flow among nine CPB populations across Xinjiang and Northeast China.The results show that:(1)Two genetically separated clusters were identified by phylogenetic tree,principal coordinate analysis(PCoA)and Bayesian cluster method.Cluster one contained populations from Xinjiang,China.Cluster two contained populations from Northeast China.A genetic differentiation existed between the two clusters.(2)Three populations in Northeast China hold an obvious genetic differentiation according to the phylogenetic tree and PCoA,indicating that multiple introductions may occur in Northeast China.(3)The Altay population in Xinjiang showed a closer genetic relationship with the populations in Northeast China which may be due to the fact that they collectively originated in neighboring Russia.(4)Among all populations,Mulei and Wusu had obvious gene migrations from Tacheng,indicating that the inland populations are most likely to originate from Tacheng,Xinjiang.

Morphological characteristics and genetic differentiation of Lutraria maxima in coast waters off southeast China

To explore genetic diversity and estimate the genetic differences among populations of Lutraria maxima in the coastal waters off south to southeast China,the morphology of the species of five different geographical populations(Beihai,Weizhou Island,Zhanjiang,Xiamen,and Fuzhou)in Guangxi,Guangdong,and Fujian provinces was studied statistically in combination with the microsatellite markers.As revealed by morphological principal component analysis(PCA),the cumulative contribution rate of the first three principal components was 72.596%.The discrimination accuracy ranged from 47.5%to 80.0%,and the scatter plots of principal component and discriminant analysis were consistent in overall,showing that the Xiamen and Fuzhou populations were overlapped obviously.For microsatellite markers,10 pairs of polymorphic primers were obtained by high-throughput transcriptome sequencing,and used for genetic diversity analysis.It was showed that the average number of alleles and eff ective alleles observed in each population ranged from 8.100 to 10.900,and from 3.497 to 4.228,respectively.The average observed heterozygosity(H_(o))and expected heterozygosity(H_(e))in the five populations ranged from 0.541 to 0.615,and from 0.642 to 0.733,respectively.The genetic distance(DA)ranged from 0.078 to 0.523,and the population genetic differentiation index(F_(ST))ranged from 0.027 to 0.139.The unweighted pair-population method with arithmetic means(UPGMA)and structure analysis showed that the five populations could be divided into two main clusters,the Beibu Gulf group(Beihai and Weizhou Island)and the Southeast China Sea group(Zhanjiang,Xiamen,and Fuzhou),suggesting that L.maxima has been separated geographically by the barrier of the Leizhou Peninsula into two groups in evolution,which provided us with a scientific clue to better protect the bioresource and establish an appropriate fishery management stocks for L.maxima populations in south China.

Effects of hydrological connection and human disturbance on genetic variation of submerged Vallisneria natans populations in four lakes in China

With the increase in the need for flood prevention and lake resource used by humans,the construction of floodgates and sluices has changed the hydrological connection between rivers and lakes,and between adjacent lakes.In river-disconnected lakes,exploitation and use of lake resources have resulted in water quality decline and mechanical disturbance intensification to a different degree.Of the large number of river-disconnected lakes in the middle-lower reaches of the Changjiang(Yangtze)River,the Futou Lake,and the Xiliang Lake lie close together and are,historically,directly connected,and so do Liangzi Lake and Baoan Lake.The extent of human disturbance is severe in the Futou Lake and the Baoan Lake,but relatively mild in the Xiliang Lake and Liangzi Lake.The freshwater rosette-forming submerged plant Vallisneria natans is one of the dominant species in the four lakes.Using microsatellite markers,we studied the genetic variation of V.natans subpopulations in lakes with different intensities of human disturbance and historical direct hydrological connections.Our results showed that human disturbance decreased plant density and clonal growth in V.natans,but might increase genetic and clonal diversity at a subpopulation level and enhance gene flow among subpopulations by sexual propagule movement.Under similar climatic conditions,different intensities of disturbance seem to have such a high selective potential to differentiate genetically adjacent lake populations that they outperform the forces of gene flow through historical direct hydrological interconnection,which tends to produce genetic homogeneity.Our findings imply that human disturbance has a profound eff ect on the evolutionary process of natural populations of submerged plants.Moreover,increased subpopulation genetic diversity can enhance resistance and resilience to environmental disturbances.To a certain degree,we could expect that disturbed populations have the possibility of restoring spontaneously if humans cease to perturb natural ecosystems in the future.

Constructing Genetic Linkage Maps Using the Whole Genome Sequence of Pacific Bluefin Tuna (Thunnus orientalis) and a Comparison of Chromosome Structure among Teleost Species

Pacific bluefin tuna (Thunnus orientalis) is one of the most economically important species in the Percomorpha group of teleost fishes. Their migrations are extensive and depend upon continuous swimming at a high rate of speed throughout their life. The draft genome sequence of this species has been reported but remains highly fragmented. We constructed a Pacific bluefin tuna genetic linkage map using microsatellite markers developed on each of the scaffolds from the draft genome sequence to link these genome fragments and understand the genomic structure of species in Percomorpha. Of the 606 polymerase chain reaction microsatellite primer pairs tested, 473 were polymorphic in the mapping populations for the linkage analysis. We constructed sex-specific maps for 24 linkage groups consisting of 470 markers, which allowed us to place scaffolds that cumulatively represented 20.8% (153.8 Mb) of the sequenced genome onto the linkage groups. The distribution of orthologous genes on the chromosomes of tuna and four other teleost fish species suggested that the constitution of tuna chromosomes is closest to that of medaka. Both species have the 24 chromosomes of the ancestral teleost, including several chromosomal inversions. The integrated map developed in this study will be useful to construct a complete physical map to conduct comparative teleost genomics and genetic studies on economically useful traits in Pacific bluefin tuna.

Performance and genetic diversity of 23 provenances of northern red oak(Quercus rubra L.)after 25 years of growth in South Korea

Growth traits and genetic diversity of 23 provenances of Quercus rubra introduced from North America were analyzed in a provenance trial established with a randomized block design in Hwaseong,Gyeonggi,South Korea in 1993.Growth variables and survival at age 25 were compared with results from early stages.Height,DBH,volume and stem straightness of Q.rubra was better than those of the domestic oak(Quercus accutissima).Growth of the Dunham Island provenance from New York was the best among the 23 provenances that of the Eagle River provenance from Wisconsin was poorest.Survival rate at age25 was on average 52%.The longitude of seed origin and growth of provenance were consistently significantly negatively correlated at all ages.Growth of coastal provenances was superior to that of the inland provenances,which were separated by the Appalachian Mountains.Genetic diversity and genetic distance among the provenances were evaluated using micro satellite markers.The allelic frequencies showed high polymorphism in 10 microsatellite loci,and 292 alleles were found.Of 10 loci,two were commonly found in the 23 provenances.The mean allelic diversity and heterozygosity observed among the provenances were similar to those from the native populations of Q.rubra in North America.Nei’s genetic distance among the 23 was estimated and showed that a clear trend between geographic and genetic distances,indicating that some provenances have high genetic diversity with superior growth performance.

Fine-scale analysis reveals a potential influence of forest management on the spatial genetic structure of Eremanthus erythropappus

Forest management may have significant effects on forest connectivity and natural population sizes.Harvesting old-growth single trees may also change natural patterns of genetic variation and spatial genetic structure.This study evaluated the impacts of forest management using a silvicultural system of seed trees on the genetic diversity and spatial genetic structure of Eremanthus erythropappus(DC.)MacLeish.A complete survey of 275 trees on four plots was undertaken out to compare the genetic variation of a managed stand with an unmanaged stand.We genotyped all adult and juvenile individuals 60 months after the management and compared the genetic diversity and the spatial genetic structure parameters.Genetic diversity was considered high because of an efficient gene flow between stands.There were no genetic differences between stands and no evidence of inbreeding.Genetic clustering identified a single population(K=1),indicating no genetic differentiation between managed and unmanaged stands.Adult and juvenile individuals of the unmanaged stand were more geographically structured than individuals from the managed one.There was a tendency of coancestry among juveniles at the first class of distance of the managed stand,suggesting a drift of genetic structure possibly caused by management.Understanding early responses to management on genetic diversity and stand structure is a first step to ensuring the effectiveness of conservation practices of tree species.The sustainability of forest management of E.erythropappus on genetic diversity,and more accurately,on spatial genetic structure needs evaluation over time to promote effective conservation of the population size and genetic variability.

Genetic analysis for geographic isolation comparison of brown bears living in the periphery of the Western Carpathians Mountains with bears living in other areas

Populations of the European brown bear (Ursus arctos L.) differ substantially in size, degree of geographic isolation and level of genetic diversity. Present patterns result from phylogeographic processes and profound human intervention. We assessed the genetic variability of a subpopulation of brown bears near the periphery of their range in the Western Carpathian Mountains and compared their genetic properties with those of bears in the core of the same population and elsewhere. Samples were collected non-invasively in 2007-2008 and2010 in Strázovské Vrchy Protected Landscape Area (PLA) in Slovakia (included in the NATURA 2000 networking programme). Seven polymorphic microsatellite loci (UaMU26, UaMU64, G10B, G1D, G10L, UaMU50 and UaMU51) were amplified using a nested PCR in order to assess the following parameters: variability, allelic combinations, heterozygosity, number of alleles and inbreeding coefficient. Sufficient brown bear DNA for analysis was obtained from 57 out of 140 samples (41%), among which 45 different genotypes were identified. Loci had a mean of 2.71 ± 0.76 alleles. Average observed heterozygosity was 0.59. The inbreeding coefficient was negative for all but one of the analysed loci (2007-2008). In the year 2010 was negative three of seven loci. These results imply that gene flow with other parts of the population has been maintained in the reduced level and the isolation level of bears in the study area was not so low. Nevertheless, the genetic variability of bears in Strázovské Vrchy PLA was lower than that reported from other localities in the Carpathian Mountains. The results are discussed in the context of behavioural ecology and conservation genetics.

Evidence for association of D1S249 locus on human chromosome 1 with the susceptibility to essential hypertension in Han Chinese

Essential hypertension (EH) is thought to result from theinteraction of environmental and genetic factors. The molecular genetics of EH has witnessed considerable progress during the past few years. However, the number of genes involved, their chromosomal location and the magnitude of their effect on EH susceptibility are unknown. We conducted the present study to screen susceptibility genes to essential hypertension using a genome-wide scanning method in a group of Han people from Fangshan district located in the southwest of Beijing. A case-control study and affected sibpair were performed. Genotyping was carried out using a fluorescence-based semiautomated technique on automated DNA sequencer (ABI 377, PE). The basis for the genome-screen was the ABI prism linkage mapping sets of 400 microsatellite markers (version 2, PE, Co.). PCR for amplification of markers was carried out as multiplex reactions with Ampli Taq gold (PE, Co.) following protocols developed in our laboratory. Data were exported as a text file from genotyper for subsequent two-point affected sibpair linkage analysis. The data from case-control association study showed a linkage disequilibrium between EH and marker D1S249 locus (X2 = 14.6, P = 0.002). There are 12 alleles in the D1S249 locus. The frequency of A9 allele in hypertension was higher than in normotensives, (13.6% v.s. 2.7%, X2 = 6.30, p = 0.01, OR = 4.57, 95%CI = 1.24-25.4). The data from two-point affected sibpair linkage analysis demonstrated a linkage between EH and A9 allele, P＜0.05. It suggested that microsatellite marker D1S249 locus might be associated with the genetic susceptibility to essential hypertension in Han Chinese.

Kinship analysis reveals reproductive success skewed toward overwintered Brandt’s voles in semi-natural enclosures

Age structure and seasonality influence the population fluctuations of small rodents.Age determines body weight and social experience,while seasonality regulates the duration of the breeding season and onset of sex­ual maturity in newborn offspring.Therefore,reproductive success and skew usually occur in different age groups.Brandt’s vole(Lasiopodomys brandtii)is a social,short-lived and seasonal breeding small rodent with a dramatic seasonal population fluctuation,but reproductive skew is not fully understood in this species.In the present study,we determined kinship in semi-natural enclosure populations using microsatellite markers based on genotyping,analyzed the reproductive skew between sexes and between overwintered and newborn voles,and monitored variation in male reproductive activity by testing fecal testosterone levels throughout the year.Overwintered voles had the most reproductive success along with a striking increase in the population size in the enclosures,with all biological fathers and 77.8%of biological mothers,which had 100%and 87%of the to­tal offspring,respectively.Compared to overwintered voles,reproductive skews were significantly higher in po­tential overwintered and newborn parents,implying the possible reproductive suppression of newborn voles by dominant overwintered voles.Moreover,both heavier body weight and higher testosterone levels in overwin­tered males supported their potential social status in the population.Our study provided new evidence for repro­ductive skew and differentiation of postnatal gonadal development patterns of different age groups in Brandt’s vole.