Isolation Of Microsatellite Loci Research Articles

Analysis of Allelic Diversity and Genetic Relationships Among Cultivated Mangosteen (Garcinia mangostana L.) in Java, Indonesia Using Microsatellite Markers and Morphological Characters

The genetic variation and relationships of the mangosteen (Garcinia mangostana L.) were observed across mangosteen populations in Java, Indonesia using newly identified microsatellite loci and morphological characters. In this study, we developed an improved protocol to isolate microsatellite loci, named Selective Repeats from AFLP Sequence, by using a hybridized membrane. Twenty microsatellite loci were evaluated using 78 individuals from five mangosteen populations, and we successfully amplified four closely related Garcinia species, including G. malaccensis, G. hombroniana, G. celebica, and G. porrecta. Eight loci were monomorphic and the others were polymorphic. Sixty-nine alleles were found, with 3.491 per locus on an average. Genetic diversity (Hʹ) was calculated with an average across loci within population (HʹS) as 0.39, an average loci across many populations (HʹT) as 0.444, and genetic differentiation (FʹST) as 0.147. Furthermore, based on morphological characters, mangosteen individuals from four populations including Leuwiliang, Wanayasa, Puspahiang, and Kaligesing also had morphologically distinct fruit weight, rind weight, and rind thickness among populations. The study also elucidated the dispersal pattern of mangosteen in Java; the source of the genotype mangosteen in Java population was the Wanayasa population. In addition, we found evidence of tetraploidy in mangosteen. These results have potential applications in future breeding, conservation studies, and genetic assessment of mangosteen and their closely related species.

Isolation and characterization of microsatellite loci in the Chinese water snake Enhydris chinensis (Colubridae)

The Chinese water snake Enhydris chinensis is an ovoviviparous colubrid snake distributed in China and Vietnam. It is commonly used in the production of Chinese snake oil, which is known for treating ailments such as fever, joint pain, and headache. It is listed as a vulnerable species according to the China red data book of endangered animals. Specific molecular markers such as mitochondrial DNA and microsatellite loci are helpful for us to get a better understanding of population structure and evolutionary history of the snake and take effective conservation action for this species. Here, we characterize eight polymorphic microsatellite loci isolated from E. chinensis genomic libraries. Forty-seven individuals were collected from Ji’an population in Jiangxi, China. These markers revealed a high degree of genetic diversity (3–10 alleles per locus) and heterozygosity (H O ranged from 0.532 to 0.957, and H E ranged from 0.565 to 0.806). No locus exhibited significant deviations from Hardy–Weinberg equilibrium. There was no evidence of linkage disequilibrium among pairs of loci. The results of this paper will be useful for future study of population structure and genetic diversity of E. chinensis.

Extent and ecological consequences of hunting in Central African rainforests in the twenty-first century

Extent and ecological consequences of hunting in Central African rainforests in the twenty-first century

Isolation and characterization of microsatellite loci for the analysis of genetic diversity in Whitmania pigra

The objective of this study was to isolate microsatellite loci to analyze the genetic diversity of Whitmania pigra. Four new microsatellite markers of W. pigra were developed from an enriched library and ten from a modified SAMPL assay. A total of 127 alleles were detected, with an average of 9.1 alleles per locus. The expected heterozygosity (He) of each microsatellite locus varied from 0.451 to 0.857, with an average of 0.688. The polymorphism information content (PIC) of each microsatellite locus ranged from 0.361 to 0.838, with an average of 0.640. Analysis of molecular variance showed that the main variation component existed within the populations (81.64%) rather than among the populations (18.36%). Phylogenetic tree for 15 populations of Hirudo using the NJ method by MEGA 5.1 software were divided into two major clusters. These microsatellite markers will contribute to research on the individual identification, genetic diversity, population structure, genome mapping and conservation biology of Hirudo.

Isolation and characterization of microsatellite loci in the short-tailed pit viper Gloydius brevicaudus (Viperidae)

The short-tailed pit viper Gloydius brevicaudus is a medium-sized, viviparous, highly venomous snake that has an exclusively temperate distribution ranging from eastern China to Korea. The snake is listed as a vulnerable species according to the China Red Data Book of Endangered Animals. To develop effective conservation strategies for the snake, we need a better understanding of gene flow, population structure and evolutionary history of this species. One approach for achieving this goal is to use microsatellites. Here, we characterize 8 polymorphic microsatellite loci isolated from G. brevicaudus genomic libraries. Twelve individuals were collected from Xiaoshan population in Zhejiang, China. These markers revealed a high degree of genetic diversity (7–16 alleles per locus) and heterozygosity (HO ranged from 0.667–1.000, and HE ranged from 0.822 to 0.960). No locus exhibited significant deviations from Hardy–Weinberg equilibrium. There was no evidence of linkage disequilibrium among pairs of loci. These microsatellite markers will be useful for future studies focusing on paternity test, population structure and evolutionary history of G. brevicaudus.

Isolation of Microsatellite Loci from the Onion Thrips,Thrips tabaci

Thrips tabaci Lindeman (Thysanoptera: Thripidae), a cosmopolitan pest insect, is subdivided into at least three genetic lineages that have different biological characters, such as reproductive mode and resistibility to insecticides. Since the lineages are discriminated only by mitochondrial DNA, there is a possibility of gene flow among lineages at the genomic level. Nine polymorphic microsatellite loci were newly isolated from the taxon. Moderate to high levels of polymorphism were observed, with numbers of alleles ranging from 6 to 12 in 51 individuals. The mean observed and expected heterozygosities ranged from 0.1373 to 0.3725 and 0.5381 to 0.7748, respectively. Contrary to the expectation under Hardy-Weinberg's equilibrium, six of the nine loci exhibited a reduction to homozygosities. However, we confirmed that alleles in all the loci were inherited as Mendeilan characteristics. These new loci will be useful to explore discrimination of lineages and population genetics in this species.

Characterization of 34 polymorphic EST-SSR markers in Tegillarca granosa and their transferability in Anadara craticulata

EST-SSR is one of important methods to isolate microsatellite loci,which is low cost,high transferability among phylogenetically related species and a probability of being associated with functional regions of the genome.34 polymorphic EST-SSR markers were isolated and characterized in the blood clam,Tegillarca granosa,from the ESTs generated by 454 sequencing.The number of alleles for the 34 SSR markers in 30 individuals of Zhejiang Fenghua population varied from 2 to 7,with an average of 3.59 alleles per locus.The observed heterozygosity ranged from 0.000 to 0.600,while the expected heterozygosity varied from 0.078 to 0.771.Polymorphic information content ranged from 0.106 to 0.718,and 13 SSR loci deviated from Hardy-Weinberg equilibrium.25 EST-SSR identified from annotated genes were expected to be useful for mapping these genes in linkage maps.Interspecific transferability of the 34 markers revealed that 8 were polymorphic in Anadara craticulata,resulting in a transferability rate of 23.53%.To our knowledge,this is the first report on the survey of SSR transferability in blood clams.These novel polymorphic EST-SSR markers should be particularly useful for comparative mapping,gene tagging and QTL mapping among blood clams.

Isolation of microsatellite loci for the predatory fish Hoplias microlepis (Characiformes: Erythrinidae) from a highly impacted river system in western Ecuador

We developed 14 polymorphic, dinucleotide microsatellite loci for Hoplias microlepis, a large predatory fish from a highly impacted river in western Ecuador. These were isolated using a next-generation pyrosequencing approach and tested in a sample of 32 fish. None of the loci were in linkage disequilibrium and all appeared to be in Hardy–Weinberg. Nine of the loci exhibited greater variability, having 3–8 alleles (average = 5.22), observed heterozygosities between 0.22 and 0.81 (average = 0.58), and expected heterozygosities between 0.20 and 0.83 (average = 0.56). The other five loci had 2 alleles and heterozygosity values generally close to 0. This is the first study dedicated to isolating microsatellites for a freshwater fish from the highly impacted aquatic ecosystems of western Ecuador.

Development of polymorphic microsatellite markers issued from pyrosequencing technology for the medicinal mushroom Agaricus subrufescens

The recently described procedure of microsatellite-enriched library pyrosequencing was used to isolate microsatellite loci in the gourmet and medicinal mushroom Agaricus subrufescens. Three hundred and five candidate loci containing at least one simple sequence repeats (SSR) locus and for which primers design was successful, were obtained. From a subset of 95 loci, 35 operational and polymorphic SSR markers were developed and characterized on a sample of 14 A.subrufescens genotypes from diverse origins. These SubSSR markers each displayed from two to 10 alleles with an average of 4.66 alleles per locus. The observed heterozygosity ranged from 0 to 0.71. Several multiplex combinations can be set up, making it possible to genotype up to six markers easily and simultaneously. Cross-amplification in some closely congeneric species was successful for a subset of loci. The 35 microsatellite markers developed here provide a highly valuable molecular tool to study genetic diversity and reproductive biology of A.subrufescens.

Isolation and characterization of microsatellite markers and analysis of genetic variability in Curculigo latifolia Dryand

Curculin, a sweet protein found in Curculigo latifolia fruit has great potential for the pharmaceutical industry. This protein interestingly has been found to have both sweet taste and taste-modifying capacities comparable with other natural sweeteners. According to our knowledge this is the first reported case on the isolation of microsatellite loci in this genus. Hence, the current development of microsatellite markers for C. latifolia will facilitate future population genetic studies and breeding programs for this valuable plant. In this study 11 microsatellite markers were developed using 3′ and 5′ ISSR markers. The primers were tested on 27 accessions from all states of Peninsular Malaysia. The number of alleles per locus ranged from three to seven, with allele size ranging from 141 to 306 bp. The observed and expected heterozygosity ranged between 0.00–0.65 and 0.38–0.79, respectively. The polymorphic information content ranged from 0.35 to 0.74 and the Shannon’s information index ranged from 0.82 to 1.57. These developed polymorphic microsatellites were used for constructing a dendrogram by unweighted pair group method with arithmetic mean cluster analysis using the Dice’s similarity coefficient. Accessions association according to their geographical origin was observed. Based on characteristics of isolated microsatellites for C. latifolia accessions all genotype can be distinguished using these 11 microsatellite markers. These polymorphic markers could also be applied to studies on uniformity determination and somaclonal variation of tissue culture plantlets, varieties identification, genetic diversity, analysis of phylogenetic relationship, genetic linkage maps and quantitative trait loci in C. latifolia. Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-012-1853-z) contains supplementary material, which is available to authorized users.

Using next-generation DNA sequencing for rapid microsatellite discovery in Mexican leaf-toed geckos (Phyllodactylus tuberculosus)

We use a combination of 454 shotgun pyrose- quencing and traditional molecular cloning to isolate microsatellite loci for the Mexican yellow-bellied leaf-toed gecko (Phyllodactulus tuberculosus). A pyrosequencing run on a 454 GS Junior yields 62 Mbp of data composed of 150 k fragments with an average length of 412 bp. Among the fragments, 18 144 (12 %) contain a suitable microsat- ellite. Dinucleotides and tetranucleotides are most fre- quently encountered. We then genotype 30 individuals of P. tuberculosus using 12 polymorphic loci. We find mod- erate allelic diversity and heterozygosity. No loci are in linkage disequilibrium and only two loci show deviations from Hardy-Weinberg expectations. Our results further highlight the utility of next-generation sequencing for discovering molecular markers in non-model taxa and suggest that sequencing on a 454 GS Junior system is a rapid and cost-effective approach for microsatellite characterization.

Isolation and Characterization of Sixteen Polymorphic Microsatellite Loci in the Golden Apple Snail Pomacea canaliculata

We report the characterization of 16 polymorphic microsatellite markers in the golden apple snail, Pomacea canaliculata, a pest registered in the list of “100 of the world’s worst invasive alien species”. The fast isolation by AFLP (Amplified Fragment Length Polymorphism) of sequences containing repeats (FIASCO) method was used to isolate microsatellite loci, and polymorphism was explored with 29 individuals collected in an invasive region from China. These primers showed a number of alleles per locus ranging from three to 13. The ranges of observed and expected heterozygosity were 0.310–0.966 and 0.523–0.898, respectively. These microsatellite markers described here will be useful for population genetic studies of P. canaliculata.

Development of microsatellite markers for the yellow-legged Asian hornet, Vespa velutina, a major threat for European bees

We report the characterization of fifteen microsatellite markers in Vespa velutina, an invasive hornet from Asia that has been unintentionally introduced in France before 2005. It is expanding rapidly, covering one third of the French territory and northern Spain, and causes severe losses to honeybee colonies. An enrichment protocol was used to isolate microsatellite loci, and polymorphism was explored in an invasive population from France and in a population from the native mainland location in China. These markers showed a number of alleles per locus and per population ranging from 1 to 11, and expected heterozygosities ranging from 0.151 to 0.891. These polymorphic markers will be useful to identify the source of the invading population and to discover the invasion pathways.

Isolation and characterization of polymorphic microsatellite loci from elongate loach (Leptobotia elongate), a threatened fish species endemic to the Yangtze River

Elongate loach (Leptobotia elongate) is a threatened fish species endemic to the Yangtze River in China. A first set of 22 polymorphism microsatellite loci isolated from this species was characterized. The number of alleles, observed and expected heterozygosity ranged from 2 to 12, from 0.2667 to 1.000 and from 0.2391 to 0.9011, respectively. Seven loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. The polymorphism information content ranged from 0.204 to 0.857. Ten loci were highly informative, 11 were moderately informative, and one was slightly informative. These polymorphic microsatellite markers will be useful for genetic monitoring of natural populations and management of breeding stocks in hatcheries.

Isolation of microsatellite loci from the millipede, Brachycybe nodulosa Verhoeff

Brachycybe nodulosa is a fungivorous millipede that distributed in Japan and Korea. This species shows patchy distributions and the exclusive paternal care of eggs, and thus is an important subject for both conservation and evolutionary biology. Thirteen polymorphic microsatellite loci were newly isolated from the taxon. Moderate to high levels of polymorphism were observed, with numbers of alleles ranged from 2 to 10. The mean observed and expected heterozygosities ranged from 0.200 to 1.000 and 0.405 to 0.886, respectively. Two loci showed lower heterozygosities than the expectation under HWE. No linkage disequilibrium was detected for any pair of the loci. These new loci will be useful in conservation genetics and evolutionary ecology studies of this species.

Isolation and Characterization of Microsatellite Loci in the Chinese Cobra Naja atra (Elapidae)

We characterize thirteen polymorphic microsatellite loci isolated from Naja atra genomic libraries, which were enriched for AC-motif microsatellites. The thirteen loci were screened on a group of 48 individuals from two populations, one in Yong’an and the other in Ganzhou. These markers revealed a relatively high degree of genetic diversity (4–12 alleles per locus) and heterozygosity (Ho ranged from 0.213–0.854 and He ranged from 0.301–0.838). Tests for departure from Hardy-Weinberg equilibrium and for linkage disequilibrium were conducted for each of the two populations separately. After sequential Bonferroni correction, none of the 13 loci showed significant departures from Hardy-Weinberg equilibrium. Hierarchical analysis of molecular variance indicated that a small but significant (P < 0.001) proportion (16.0%) of the total variation in the microsatellite DNA data were attributable to differences among populations, indicating geographical structuring and restricted gene flow. It could be attributable to the Wuyi mountains in the area having a sufficiently isolating effect to significantly reduce gene flow. Our microsatellite data also showed a low Nm (1.31) value in the two populations from mainland China. Thus, the Yong’an and Ganzhou populations could be treated as distinct evolutionarily significant units (ESUs). The high level of polymorphism revealed by these microsatellite markers will be useful for the study of gene flow, population structure and evolutionary history of N. atra.

Using the 454 pyrosequencing‐based technique in the development of nuclear microsatellite loci in the alpine plant Arabis alpina (Brassicaceae)

Polymorphic microsatellite markers were developed for the inbred alpine perennial plant Arabis alpine to infer life-history parameters and measure patterns of contemporary gene fl ow within populations. Using the 454 pyrosequencing technique, 19 microsatellite primer sets were developed for A. alpina . The primer sets were tested on 60 individuals sampled from three sub-populations in the Swiss Alps. The primers amplifi ed di- and trinucleotide repeats with two to fi ve alleles per locus. Previous attempts to isolate microsatellite loci in A. alpina using enrichment libraries and cross-amplifi cation were diffi cult and produced an insufficient number of polymorphic microsatellite loci. In contrast, next-generation sequencing technology was successful in identifying microsatellite repeats in A. alpina. These newly developed microsatellite primers will be useful to further develop A. alpina into a model species for eco-genomic studies.

Isolation and characterization of microsatellite loci for white-lipped peccaries (Tayassu pecari) and cross-amplification in collared peccaries (Pecari tajacu)

White-lipped peccaries, Tayassu pecari, are neotropical ungulates whose populations have been declining in numerous locations within their geographical distribution. Here we describe 16 microsatellite loci isolated from T. pecari and their cross-amplification in collared peccaries, Pecari tajacu. In 30 individuals of T. pecari, a total of 32 alleles were found in ten polymorphic loci, ranging from 2 to 8 alleles per locus with a mean of 3.2. The expected and observed heterozygosity ranged from 0.143 to 0.802 and from 0 to 0.704, respectively. Two loci deviated from Hardy–Weinberg equilibrium. In P. tajacu, nine loci were polymorphic with a mean of 3.2 alleles per locus. These molecular markers will be useful to study the genetic status of peccary populations and, consequently, to help their conservation.

Isolation of microsatellite loci from endangered members of Lotus (Fabaceae) subgenus Syrmatium

Lotus subgenus Syrmatium is a group of 11 plant species that exhibit extensive ecological and morphological diversity throughout the California floristic province. Fifteen polymorphic microsatellite loci were isolated from two taxa, Lotus argophyllus var. adsurgens and L. dendroideus var. traskiae, and were screened for variability in 15 additional taxa within Lotus subgenus Syrmatium. Moderate levels of variability were observed with mean numbers of alleles per locus ranging from 1.3 to 7.3. The mean observed and expected heterozygosities ranged from 0.09 to 0.47 and 0.10 to 0.79, respectively. These new loci will be useful in conservation genetic and evolutionary studies within Lotus subgenus Syrmatium.

Microsatellite markers for the ectomycorrhizal basidiomycete Lactarius mammosus

We report the characterization of polymorphic microsatellite markers in the ectomycorrhizal fungus Lactarius mammosus Fr. Two enrichment protocols were used to isolate microsatellite loci and polymorphism was explored within 31 sporocarps originating from a forest site in northern Sweden. We found nine variable microsatellite loci with the number of alleles per locus varying between 2 and 5, and expected heterozygosities ranging from 0 to 0.84. These loci are available for the analysis of genetic structure and gene flow in L. mammosus populations.