Analysis of genetic diversity and population structure of Chinese yak breeds (Bos grunniens) using microsatellite markers

Nine Chinese yak breeds （Maiwa, Tianzhu White, Qinghai Plateau, Sibu, Zhongdian, Pali, Tibetan High Mountain, Jiulong, and Xinjiang） and Gayal were analyzed by means of 16 microsatellite markers to determine the level of genetic variation within populations, genetic relationship between populations, and population structure for each breed. A total of 206 microsatellite alleles were observed. Mean F-statistics （0.056） for 9 yak breeds indicated that 94.4% of the genetic variation was observed within yak breeds and 5.6% of the genetic variation existed amongst breeds. The Neighbor-Joining phylogenetic tree was constructed based on Nei＇s standard genetic distances and two clusters were obtained. The Gayal separated from the yaks far away and formed one cluster and 9 yak breeds were grouped together. The analysis of population structure for 9 yak breeds and the Gayal showed that they resulted in four clusters; one cluster includes yaks from Tibet Autonomous Region and Qinghai Province, one cluster combines Zhongdian, Maiwa, and Tianzhu White, and Jiulong and Xinjiang come into the third cluster. Pali was mainly in the first cluster （90%）, Jiulong was mainly in the second cluster （87.1%）, Zhongdian was primarily in the third cluster （83%）, and the other yak breeds were distributed in two to three clusters. The Gayal was positively left in the fourth cluster （99.3%）.

Development of microsatellite markers and their utilization in genetic diversity analysis of cultivated and wild populations of the mud carp (Cirrhina molitorella)

Microsatellite markers have been increasingly used in genetic studies on fishery species because of their high applicability in selective breeding programs. Here we reported the development of microsatellite markers and their utilization in mud carp （Cirrhina molitorella）. An （CA）15 enriched library has been constructed for mud carp, using the magnetic beads enrichment procedure. Sequence analysis of 60 randomly picked positive colonies indicate that 56 （93.3%） of the colonies contain microsatellites. Microsatellite polymorphism was assessed using 10 mud carp individuals, and 12 microsatellite loci turned out to be polymorphic. We utilized these loci to study the genetic diversity of a wild population （WM） and a cultured population （CM） of the mud carp. A total of 109 alleles were detected with an average of 9.08 alleles per locus. The mean value of the observed heterozygosity of WM and CM was 0.6361 and 0.6417, respectively, and significant decrease of genetic diversity in CM was not observed. The genetic distance between the two populations was 0.1546 and the value of Gsr was 0.0473. This showed that there existed a slight genetic differentiation between WM and CM.

The analysis of genetic diversity and differentiation of six Chinese cattle populations using microsatellite markers

A total of 321 individuals from six cattle populations of four species in a bovine subfamily in China were studied using 12 pairs of microsatellite markers. The genetic diversities within and between populations were calculated. The phylogenetic trees were constructed by （δμ）^2 and DA distances, and the divergence times between populations were estimated by （δμ）^2. Altogether, 144 microsatellite alleles were detected including 24 private alleles and nine shared alleles. Chinese Holstein had the largest number of private alleles （10）, whereas Bohai black and Buffalo had the smallest number of private alleles （2）. Chinese Holstein showed the highest genetic variability. Its observed number of alleles （Na）, mean effective number of alleles （MNA）, and mean heterozygosity （He） were 7.7500, 4.9722, and 0.7719, respectively, whereas, the Buffalo and Yak showed low genetic variability. In the phylogenetic trees, Luxi and Holstein grouped first, followed by Bohai and Minnan. Yak branched next and buffalo emerged as the most divergent population from other cattle populations. Luxi and Bohai were estimated to have diverged 0.039-0.105 million years ago （MYA）, however, buffalo and Holstein diverged 0.501-1.337 MYA. The divergence time of Yak versus Minnan, Holstein and buffalo was 0.136-0.363, 0.273-0.729, and 0.326-0.600 MYA, respectively.

Assessment by microsatellite analysis of genetic diversity and population structure of Enhalus acoroides from the coast of Khanh Hoa Province, Vietnam

Seagrass beds degraded significantly since the last century on both, global and local scale. The seagrass species Enhalus acoroides(Linnaeus f.) Royle is a common species found in almost all marine ecosystems including bays,lagoons and around offshore islands in tropical regions of the West Pacific. It was shown that genetic diversity is an essential indicator of the conditions of ecosystems. In the present study, microsatellite markers were used to assess the genetic diversity and population structure of six distinct seagrass beds along the coast of the Khanh Hoa Province, Vietnam. The results indicate that the genetic diversity of the populations in the open sea is higher than in the lagoon. Seagrass beds occurring in disturbed sites show reduced genetic diversity. The fixing index value(FST) depicts a relatively high genetic structure among populations. Structure analysis clusters the populations into open sea and lagoon populations and cluster analysis and AMOVA indicate a significant difference between the two groups. There are low but non-significant positive correlations between geographic and genetic distances.The different habitats of the open sea and the lagoon are probably responsible for forming two groups.

Inbreeding and genetic diversity analysis in a hatchery release population and clones of Rhopilema esculentum based on microsatellite markers

Ten microsatellite markers were used to analyze the levels of genetic diversity and inbreeding in a hatchery release population of Rhopilema esculentum Kishinouye （Scypbozoa： Rhizostomatidae）. A total of 85 alleles were detected in 600 individuals. Within-population levels of observed （Ho） and expected （He） heterozygosity ranged from 0.152 to 0.839 （mean=0.464） and from 0.235 to 0.821 （mean=0.618）, respectively. The polymorphism information content （PIC） of each marker ranged from 0.207 to 0.795 with an average of 0.580, indicating that the hatchery population maintained a high level of genetic diversity. Inbreeding levels were estimated in the hatchery population and the inbreeding coefficient was 0.203. This result revealed that a certain level of inbreeding occurred within the population. Meanwhile, we also determined genetic diversity at the clone level. Several polyps from the same scyphistomae were genotyped at the ten microsatellite loci and there was virtually no difference in their genotypes. Furthermore, we calculated the probabilities of exclusion. When both parents were known, the average exclusion probability often loci was 99.99%. Our data suggest that the ten microsatellite markers can not only be used to analyze the identity of individuals but they can also be applied to parentage identification. Our research provides a theoretical basis and technical support for genetic diversity detection and reasonable selection of R. esculentum hatchery populations. These findings support the use of releasing studies and conservation of R. esculentum germplasm resources.

Analysis of Genetic Diversity and Differentiation of Seven Stocks of Litopenaeus vannamei Using Microsatellite Markers

Seven microsatellite markers were used to evaluate the genetic diversity and differentiation of seven stocks of Litopenaeus vannamei, which were introduced from Central and South America to China. All seven microsatellite loci were polymorphic, with polymorphism information content(PIC) values ranging from 0.593 to 0.952. Totally 92 alleles were identified, and the number of alleles(Na) and effective alleles(Ne) varied between 4 and 21 and 2.7 and 14.6, respectively. Observed heterozygosity(Ho) values were lower than the expected heterozygosity(He) values(0.526–0.754), which indicated that the seven stocks possessed a rich genetic diversity. Thirty-seven tests were detected for reasonable significant deviation from Hardy-Weinberg equilibrium. Fis values were positive at five loci, suggesting that there was a relatively high degree of inbreeding within stocks. Pairwise Fst values ranged from 0.0225 to 0.151, and most of the stock pairs were moderately differentiated. Genetic distance and cluster analysis using UPGMA revealed a close genetic relationship of L. vannamei between Pop2 and Pop3. AMOVA indicated that the genetic variation among stocks(11.3%) was much lower than that within stocks(88.7%). Although the seven stocks had a certain degree of genetic differentiation and a rich genetic diversity, there is an increasing risk of decreased performance due to inbreeding in subsequent generations.

Molecular phylogenetics and population demographic history of Amphioctopus fangsiao,inferred from mitochondrial and microsatellite DNA markers

Amphioctopus fangsiao(Cephalopoda:Octopodidae)is an important commercial species in the coastal waters of China.In recent years,however,the resource of A.fangsiao have declined because of habitat destruction and overfishing.To analyze the genetic variations of A.fangsiao caused by the fluctuation of resources,the population genetic structure of nine sampling locations collected from the Bohai Sea to the South China Sea were investigated,using mtDNA COI fragments and microsatellite DNA.The results of F-statistics,AMOVA,STRUCTURE and PCA analyses showed three phylogeographic clades(Clades A,B and C),revealing limited genetic exchange between north and south populations.These clades diverged in 2.23(Clades A and B)and 3.67(Clades A,B and C)million years ago,during the dramatic environmental fluctuations,such as sea level and temperature changes,have exerted great influence on the survival distribution pattern of global organisms.Our results for low genetic connectivity among A.fangsiao populations provide insights into the development of management strategies,that is,to manage this species as separate management unit.

Screening and application of microsatellite markers for genetic diversity analysis of Oriental White Stork (Ciconia boyciana)

The Oriental White Stork (Ciconia boyciana) is a large and endangered waterbird in East Asia.Research on conservation genetics of this species is urgently needed.In this study,microsatellite marking technology was used for screening and analysis of genetic diversity of microsatellite markers in Oriental White Storks.A total of 36 pairs of microsatellite primers were used,of which 7 pairs came from Ciconia ciconia,12 pairs from Nipponia nippon and 17 pairs from Ardea herodias.Microsatellite loci were screened from 23 individuals of the Oriental White Stork and 11 microsatellite loci were found with high polymorphism.The number of population alleles ranged from 3 to 11,averaging 7.09.The average expected heterozygosity (H e) was 0.7816 and the average polymorphism information content (PIC) 0.7172,suggesting a relatively high genetic diversity in the population.

A Review of Microsatellite Markers and their Application on Genetic Diversity Studies in Parrots

The ability of a population to adapt to a changing environment depends on its genetic variation. Thus, the study of genetic diversity within and among species or populations is especially important on conservation biology scopes. One way to assess the genetic diversity is through the use of microsatellite molecular markers. Microsatellites have been widely used to answer population genetics issues as gene flow, parentage, and population structure, mostly resulting in data on the distribution of genetic variability within and among natural populations, which are essential for ex situ and in situ conservation procedures. As the Psittacidae family comprehends one of the birds group with the largest number of endangered species, studies that aim to investigate the genetic diversity of these animals may support their conservation. This article is a review of genetic data on parrots, through the use of microsatellite markers, that have been published since 2004.

Assessment of Genetic Diversity of Moroccan Cultivated Almond(Prunus dulcis Mill.DA Webb)in Its Area of Extreme Diffusion,Using Nuclear Microsatellites

Assessment of genetic diversity of Moroccan cultivated almond (Prunus dulcis Mill.) grown from seed and cultivated at four eco-geographical regions was performed using 16 nuclear SSRs. 238 alleles were detected with an average of 14.88 alleles per locus, ranging from 4 (locus BPPCT027) to 24 (locus CPSCT018). The size of alleles ranged from 84 bp (locus UDP96-003) to 253 bp (locus UDP96-018). A high genetic diversity of the local almonds is apparent and structured into three major clusters (Oasis cluster, High and Anti Atlas cluster, and Middle Atlas cluster). Compared to the Mediterranean genetic pools, from the East to West, the genetic diversity tends to be limited in Morocco which is the area of its extreme diffusion.

Assessment of Genetic Diversity of Drought Tolerant and Susceptible Rice Genotypes Using Microsatellite Markers

The introgression of wild chromosomal segments into popular rice varieties is one of the potential approaches for developing varieties for drought stress condition. Sixteen genotypes, including nine indica, two tropical japonica and five chromosome segments substitution lines (CSSLs) with different levels of tolerance/susceptibility to drought stress, were selected for diversity study. Sixty-three microsatellite markers were utilized for assessing genetic diversity. A total of 95 alleles were amplified, and out of them, 60 were polymorphic. Six unique alleles, amplified by the microsatellite loci RM276, RM472, RM488, RM537, RM541 and RM28089, were identified in six genotypes, namely FR13A, Brahamanakhi, RUF44, Swarna-sub1, Brahamanakhi and Satyabhama. The highest genetic similarity was found among CSSLs. Polymorphism information content (PIC) value varied from 0 to 1.00 with an average of 0.66 per locus. Twenty-eight microsatellites were found to be polymorphic, which could be used in marker-assisted selection programme. All the sixteen genotypes were grouped into two major clusters at genetic similarity of 0.64. In the cluster I, five CSSLs identified as diverse genotypes had wild ancestor segments responsible for drought tolerance, and hence they could be utilized as potential donors. The popular Indian varieties, Swarna-sub1 and IR64-sub1, could be used as recurrent parents in the future breeding program for developing varieties for abiotic stresses such as submergence and drought.

Characterization of Microsatellite Markers and Their Application to Genetic Diversity Analysis of <i>Brachypodium sylvaticum</i>var. <i>breviglume</i>from Yunnan, China

Southwestern China is abundant in a large species diversity of grasses and Brachypodium sylvaticum var. breviglume is unquestionably appropriate to serve as an ideal model to investigate the adaptation, evolution and diversification of grasses in this region. In this study, a total of 37 microsatellite markers were screened and genetic variation was estimated for 100 individuals from the five natural populations of the species. Our results showed that seven of them were polymorphic for the five studied populations, while the other thirty were monomorphic. These seven SSR loci exhibited a high level of genetic diversity among populations, i.e. allele number varied from 5 to 24, with an average of 13.29;expected heterozygosity (He) ranged from 0.439 to 0.561, with an average of 0.488;percentage of polymorphic loci (PPL) ranged from 85.71% to 100%, with an average of 97. 14%. The FCA and UPGMA analyses revealed that the five populations were divided into three clusters. Our results indicate that these newly characterized SSR markers are useful for the exploration of genetic diversity and evolutionary history of the B. sylvaticum var. breviglume populations in Yunnan, China.

Assessment of Genetic Diversity of NIFOR Oil Palm Main Breeding Parent Genotypes Using Microsatellite Markers

The genetic diversity among 15 NIFOR breeding parents was assessed using 10 microsatellite markers. A high genetic diversity was observed with a total of 64 alleles including 23 rare alleles or alleles at frequencies less than 0.05. The NIFOR tenera parents recorded the highest number of rare alleles. The average observed heterozygosity and mean gene diversity across all parental groups were 0.6889 and 0.7029, respectively. Higher genetic diversity was detected among the NIFOR dura and tenera parents compared to that of the Deli dura parents in absolute terms. Analysis of molecular variance (AMOVA) showed that 87% of the total variation (p < 0.001) observed was due to differences among parents. Rogers’ genetic distance ranged from 0.2988 to 0.8000 (mean = 0.5570). The dendrogram constructed on the basis of Rogers’ genetic distance clustered the parents in three groups. They generally clustered in heterotic manner rather than by geographic origins. The groupings obtained through PCoA confirmed the results obtained by cluster analysis. The results obtained are strong assets for NIFOR breeding programme.

Analysis of genetic diversity and population structure in sweetpotato using SSR markers

Sweetpotato, Ipomoea batatas(L.) Lam., is an important food crop worldwide. Large scale evaluation of sweetpotato germplasm for genetic diversity is necessary to determine the genetic relationships between them and effectively use them in the genetic improvement. In this study, the genetic diversity of 617 sweetpotato accessions, including 376landraces and 162 bred varieties from China and 79 introduced varieties from 11 other countries, was assessed using 30 simple sequence repeat(SSR) primer pairs with high polymorphism. Based on the population structure analysis,these sweetpotato accessions were divided into three groups, Group 1, Group 2 and Group 3, which included 228, 136and 253 accessions, respectively. Consistent results were obtained by phylogenic analysis and principal coordinate analysis(PCoA). Of the three groups, Group 2 showed the highest level of genetic diversity and its accessions were mainly distributed in low-latitude regions. The accessions from South China exhibited the highest level of genetic diversity, which supports the hypothesis that Fujian and Guangdong were the first regions where sweetpotato was introduced to China. Analysis of molecular variance(AMOVA) indicated significant genetic differentiations between the different groups, but low levels of genetic differentiation existed between the different origins and accession types.These results provide valuable information for the better utilization of these accessions in sweetpotato breeding.

Microsatellite analysis of genetic diversity and population structure of Chinese mitten crab (Eriocheir sinensis)

Chinese mitten crab （Eriocheir sinensis） has higher commercial value as food source than any other species of Eriocheir in China. To evaluate the germplasm resources and characterize the genetic diversity and population structure of the crabs in different water systems, two stocks and two farming populations were assessed with 25 polymorphic microsallite loci available in public GenBank. Basic statistics showed that the average observed heterozygosity （Ho） amongst populations ranged from 0.5789 to 0.6824. However, a remarkable presence of inbreeding and heterozygote deficiencies were observed. To analyze population structure, pairwise FST coefficients explained only -10.3% variability from the subdivision of mitten crab populations, the remaining variability stems from the subdivision within subpopulations. Although the four populations had slight differentiation, different allelic frequencies resulted in distinct population structures. Two stocks and one farming population were clustered together to the phylogenetic branch of Yangtze crab, with an approximate membership of 95%. Whereas, another farming population was clustered singly to the phylogenetic branch of the Liaohe crab, with a membership of 97.1%. The tests for individual admixture showed that Yangtze crab had probably been contaminated with individuals from other water systems. Genetic relationships between populations also supported the conclusion that Yangtze crab and Liaohe crab had different gene pools in spite of the origins of the same species.

Genetic diversity and differentiation of masu salmon(Oncorhynchus masou masou) between and within cultured populations inferred from microsatellite DNA analysis

Masu salmon,Oncorhynchus masou masou,is one of the most valuable fishery species that has been introduced to China,though to date no studies on the genetic diversity and genetic relationship among hatchery populations has been performed with molecular markers.We undertook such a study and sampled 120 individuals from three hatchery stocks and analyzed 20 microsatellite loci.All loci were polymorphic and a total of 91 alleles were detected.A relatively low level of genetic diversity was revealed with effective number of allele of 3.1094,3.3299 and 3.1894 and expected heterozygosity of 0.6600,0.6648 and 0.6638 in the three stocks,respectively.Deviations from Hardy-Weinberg equilibrium were found due to heterozygote deficit.Accordingly,evidence of genetic bottlenecks were found in the three stocks.An individual assignment test demonstrated that 85% of individuals were correctly assigned into their original stocks.Pairwise Fst revealed that significant differentiation occurred between these three stocks.The results of the study indicated that disequilibrium of genetic structure and differentiation has occurred in all three stocks.This information collectively provides a basis for measures to avoid of loss of genetic diversity and introgression in Chinese aquaculture.

Genetic diversity and population structure of a Sichuan sika deer(Cervus sichuanicus) population in Tiebu Nature Reserve based on microsatellite variation

Cervus sichuanicus is a species of sika deer （Cervus nippon Group）. To date, research has mainly focused on quantity surveying and behavior studies, with genetic information on this species currently deficient. To provide scientific evidence to assist in the protection of this species, we collected Sichuan sika deer fecal samples from the Sichuan Tiebu Nature Reserve （TNR） and extracted DNA from those samples. Microsatellite loci of bovine were used for PCR amplification. After GeneScan, the genotype data were used to analyze the genetic diversity and population structure of the Sichuan sika deer in TNR. Results showed that the average expected heterozygosity of the Sichuan sika deer population in TNR was 0.562, equivalent to the average expected heterozygosity of endangered animals, such as Procapra przewalsldi. Furthermore, 8 of 9 microsatellite loci significantly deviated from the Hardy-Weinberg equilibrium and two groups existed within the Sichuan sika deer TNR population. This genetic structure may be caused by a group of Manchurian sika deer （Cervus hortulorum） released in TNR.

Genetic diversity in two Japanese flounder populations from China seas inferred using microsatellite markers and COI sequences

Japanese flounder is one of the most important commercial species in China; however, information on the genetic background of natural populations in China seas is scarce. The lack of genetic data has hampered fishery management and aquaculture development programs for this species. In the present study, we have analyzed the genetic diversity in natural populations of Japanese flounder sampled from the Yellow Sea （Qingdao population, QD） and East China Sea （Zhoushan population, ZS） using 10 polymorphic mierosatellite loci and cytochrome c oxidase subunit I （COI） sequencing data. A total of 68 different alleles were observed over 10 microsatellite loci. The total number of alleles per locus ranged from 2 to 9, and the number of genotypes per locus ranged from 3 to 45. The observed heterozygosity and expected heterozygosity in QD were 0.733 and 0.779, respectively, and in ZS the heterozygosity values were 0.708 and 0.783, respectively. Significant departures from Hardy-Weinberg equilibrium were observed in 7 of the 10 microsatellite loci in each of the two populations. The COI sequencing analysis revealed 25 polymorphic sites and 15 haplotypes in the two populations. The haplotype diversity and nucleotide diversity in the QD population were 0.746±0.072 8 and 0.003 34±0.001 03 respectively, and in ZS population the genetic diversity values were 0.712±0.047 0 and 0.003 18±0.000 49, respectively. The microsatellite data （Fst=0.048 7, P〈0.001） and mitochondrial DNA data （Fst=0.128, P〈0.001） both revealed significant genetic differentiation between the two populations. The information on the genetic variation and differentiation in Japanese flounder obtained in this study could be used to set up suitable guidelines for the management and conservation of this species, as well as for managing artificial selection programs. In future studies, more geographically diverse stocks should be used to obtain a deeper understanding of the population structure of Japanese flounder in the China seas and adjacent regions.

Genetic Diversity of Cameroon Native Goat Populations Revealed by Caprine Microsatellites

A total of seventeen caprine microsatellite markers were used on 169 goats to investigate genetic diversity of eight Cameroon native goat populations and to assess genetic differentiation with the East African small goat. All microsatellites showed a high polymorphic content （PIC） of more than 0.5 in almost all ecotypes. These microsatellite markers were useful for native goat ecotypes variability assessment. Expected heterozygosity of all ecotypes was above 0.5, varying from 0.2 to 0.7. Only goats from Eastern rainforest ecotype deviated from Hardy-Weinberg equilibrium test （P 〉 0.001）. Although geographic distribution was a good indication of differentiation, there appeared a tendency of genetic exchange between various ecotypes in Cameroon native goats.

Population genetic structure and diversity of high value vulnerable medicinal plant Acorus calamus in India using RAPD and chloroplast microsatellite markers

Acorus calamus is a highly valued medicinal plant with global distribution used in several drugs of health care systems. We evaluated the genetic diversity and population structure of 50 populations of A. calamus from different geographical regions in India through RAPD and chloroplast microsatellite markers. From the total screened 82 RAPD primers and 18 cpSSR primers, 10 RAPD and nine cpSSRs were found polymorphic. The selected 10 RAPD primers produced a total of 96 reproducible bands, out of which 65 were polymorphic （67.70%）. Whereas, the selected nine cpSSR markers produced 26 alleles and all of them were polymorphic. The mean genetic diversity （H T ） among populations using RAPD （H T = 0.263） and cpSSR （H T = 0.530） markers was higher in comparison to the mean genetic diversity within populations. Mean coefficient of gene differentiation （G ST ） between the populations was also high for both RAPD （G ST = 0.830） and cpSSR markers （G ST = 0.735）, whereas the estimated gene flow was very low for RAPD （Nm = 0.102） and for cpSSR （Nm = 0.179）. AMOVA analysis revealed that more genetic variation resided among the populations than within populations. Significant differences （p 0.001） were observed between the populations and individuals within the populations. Cluster analysis of RAPD and cpSSR data using UPGMA algorithm based on Nei’s genetic similarity matrix placed the 50 populations into two main clusters. The implication of the results of this study in devising strategy for conservation of A. calamus is discussed.