Eleven novel microsatellite markers for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, we developed 11 novel microsatellite loci for this rare species and applied them to examine genetic variation of indigenous alligators from Changxing Nature Reserve and America-born Chinese alligators. The 11 polymorphic microsatellites presented a total of 31 alleles among 57 individuals scored, yielding an average of 2.82 alleles per locus. One allele was unique to the American population but four private alleles were detected in the Changxing population. The average expected and observed heterozygosities were 0.400 and 0.482 for the Changxing alligators and 0.520 and 0.621 for the America-born individuals, respectively. These microsatellite markers would be useful tools in the genetic examination of this endangered species.     

Characterization of microsatellite DNA markers for the alligator snapping turtle,Macrochelys temminckii

Two trinucleotide and seven tetranucleotide microsatellite loci were isolated from an alligator snapping turtle Macrochelys temminckii. To assess the degree of variability in these nine microsatellite loci, we genotyped 174 individuals collected from eight river drainage basins in the southeastern USA. These markers revealed a moderate degree of allelic diversity (six to 16 alleles per locus) and observed heterozygosity (0.166–0.686). These polymorphic microsatellite loci provide powerful tools for population genetic studies for a species that is afforded some level of conservation protection in every state in which it occurs.     

Isolation and characterization of polymorphic microsatellite loci in <Emphasis Type="Italic">Achnatherum sibiricum</Emphasis> (Poaceae)

Achnatherum sibiricum is a threaten and toxic perennial bunchgrass mainly in the north of China. We developed 11 polymorphic microsatellite loci from A. sibiricum by combining biotin capture method. After validating and scoring, these loci were polymorphic in a test population with the range of alleles from four to 13 per locus. The observed and expected heterozygosities ranged from 0.1649 to 0.5624 and from 0.3071 to 0.8826, respectively. All 11 microsatellite markers were in Hardy–Weinberg equilibrium. These polymorphic microsatellites will be useful for genetic diversity analysis and population structure construction for A. sibiricum.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

Polymorphic microsatellite loci for the cardinal fish (<Emphasis Type="Italic">Apogon imberbis</Emphasis>)

Eight polymorphic microsatellite loci were isolated and characterized for the Cardinal fish (Apogon imberbis), a coastal-reef fish endemic to the Mediterranean Sea. Characterization of 30 Cardinal fish individuals form the western Mediterranean showed moderate to high allelic diversity ranging from 6 to 19 alleles per locus. Two loci showed significant departures from Hardy-Weinberg equilibrium presumably due to null alleles. No evidence of linkage disequilibrium was found for any locus pairwise comparasions. This microsatellite set could be useful for any basic population genetic studies of this species.     

Polymorphic microsatellite DNA markers for the ark shell Scapharca subcrenata (bivalve: Arcidae)

We have isolated and characterized twelve novel polymorphic microsatellite loci from Scapharca subcrenata to analyse the population structure. The number of observed alleles per locus ranged from 3 to 17. Observed heterozygosity (H _O) ranged from 0.321 to 0.929. Cross-species amplification was tested successfully in three other bivalve species. These microsatellite markers will be useful for genetic diversity studies of S. subcrenata and other Lamellibranchia species.     

Isolation and characterization of 10 new compound microsatellite markers for a mangrove tree species,Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

Avicennia marina is an ecologically important mangrove tree species. We isolated 10 polymorphic microsatellite loci from this species using an improved technique. Our isolated loci provided compound microsatellite markers with polymorphism of two to six alleles per locus. The observed and expected heterozygosities ranged from 0.025 to 0.625 and from 0.096 to 0.767, respectively. These markers would be the useful tools for researching on the genetic diversity and population genetic structure of A. marina.     

Microsatellite markers for <Emphasis Type="Italic">Juglans cinerea</Emphasis> L. and their utility in other Juglandaceae species

Ten polymorphic microsatellite markers were found to amplify in butternut (Juglans cinerea; Juglandaceae). These microsatellite loci were found to amplify across most of nine other species and five hybrids examined. Loci were highly polymorphic, with 18 to 32 alleles per locus across species. These nuclear microsatellite markers will be useful in examining genetic diversity within and among populations of butternut, and in distinguishing butternut from interspecific hybrids.     

Twelve polymorphic microsatellite loci for <Emphasis Type="Italic">Achnatherum inebrians</Emphasis> (Poaceae)

Microsatellites are often highly variable and abundant in most complex genomes, therefore are widely used in population genetic studies. In this study, twelve polymorphic microsatellite loci were isolated and characterized for the Achnatherum inebrians, a plant abundant in grasslands of Northwest China. Characterization of 24 A. inebrians individuals form four geographically distant populations (Gansu, Qinghai, Xinjiang and Inner Mongolia provinces) showed moderate to high allelic diversity ranging from 3 to 13 alleles per locus, and the expected heterozygosity ranging from 0.41 to 0.67. No evidence of linkage disequilibrium was found for any locus pairwise comparisons. The markers described here will be useful for investigating the genetic diversity, genetic structure and gene flow of this species. Na Chen and Yan-Zhuo Yang contributed equally to this work.     

Isolation and characterization of twelve novel microsatellite loci in the ark shell <Emphasis Type="Italic">Scapharca broughtonii</Emphasis>

Scapharca broughtonii is an over-exploited bivalve in China. In order to promote a conservation program for this species, it is necessary to evaluate its genetic diversity and population genetics. For this purpose, we isolated twelve novel polymorphic microsatellite markers and examined their allelic variation using 40 wild individuals collected from one locality along the Chinese coast. The number of alleles ranged from 2 to 22 per locus with the expected heterozygosities ranging from 0.444 to 0.944. These markers will be useful for the assessment of genetic variation for this species.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

Development and characterization of new microsatellite markers for ginseng (<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer)

Panax ginseng C.A. Meyer, commonly known as Korean or Asian ginseng, is a perennial herb native to Korea and China. Its roots are highly prized for several medicinal properties. The present study describes development and characterization of twenty-two polymorphic microsatellite markers for this species. A total of 99 alleles were detected with an average of 4.5 alleles per locus across 20 accessions. Values for observed (H _O ) and expected (H _E ) heterozygosities ranged from 0.05 to 1.00 and from 0.18 to 0.73, respectively. Eleven loci deviated from Hardy–Weinberg equilibrium (P < 0.001). Significant (P < 0.05) heterozygote deficiency was observed at 13 loci. Exact test for linkage disequilibrium showed significant values (P < 0.05) between 12 pairs of loci. These microsatellite markers provide powerful tools for understanding population and conservation genetics of this species and also for genetic differentiation and authentication of different Panax species being used in commercial ginseng products.     

Polymorphic microsatellite loci for population studies of the razor clam, <Emphasis Type="Italic">Sinonovacula constricta</Emphasis>

The razor clam (Sinonovacula constricta) is an important aquacultured bivalve in China. The natural populations of this species are decreasing quickly. To facilitate studies on genetic diversity and population structure of wild populations, microsatellites were isolated from a CA enriched genomic library. Eight microsatellite loci were polymorphic in 30 individuals from Chongming in Shanghai, China. The number of alleles per polymorphic locus varied from 6 to 13 and the values of observed heterozygosity and expected heterozygosity ranged from 0.350 to 1.000 and from 0.602 to 0.902, respectively. These microsatellites are being used in studying population differentiation and genetic diversity for effective conservation and management genetic resources of S. constricta.     

AFLP Analysis of genetic variation on captive‐bred chinese alligators: an application to select individuals for release

The Chinese alligator (Alligator sinensis) is a critically endangered species in China. Wild populations of Chinese alligator are on the edge of extinction. Through a release program, some captive‐bred alligators will be selected and released into the wild to supplement and renew natural populations. The purpose of this study was to investigate the genetic variation of captive‐bred Chinese alligators by AFLP markers and to select individuals with maximally different genetic backgrounds for release. Forty‐three captive‐bred alligators of the second filial generation from the Anhui Research Center for Chinese Alligator Reproduction (ARCCAR) were surveyed using four primer combinations, yielding 117 AFLP markers. According to AFLP fingerprints, six samples had distinctly different band patterns compared to other samples. When the six samples were removed from the analysis, there were 19 monomorphic loci and 98 polymorphic loci yielding 84% polymorphic loci. Moreover, the genetic similarity (GS) among 37 samples varied from 0.13–0.97, and the average was 0.7503±0.0064 standard error (SE). When the six samples were included, the GS value among the 43 samples declined and varied from 0.06–0.97, and the average was 0.6523±0.0079 SE. Based on a cluster analysis using UPGMA, a dendrogram of the 43 alligators was constructed. According to the cluster analysis and gender of the 43 samples, eight Chinese alligators with very different genetic backgrounds were selected and suggested for release with two groups in the future. Zoo Biol 0:1–12, 2006. © 2006 Wiley‐Liss, Inc.     

Development of single sequence repeat markers for the ant Aphaenogaster senilis and cross-species amplification in A. iberica, A . gibbosa, A. subterranea and Messor maroccanus

Eight polymorphic microsatellite loci were isolated and characterized for Aphaenogaster senilis, a common ant species distributed in the Western Mediterranean. Characterization of 15 individuals form southern Spain showed moderate to high allelic diversity ranging from 2 to 9 alleles per locus. Cross-species tests on 10 individuals of A. iberica, A. gibbosa, A. subterranea and Messor maroccanus revealed successful amplification for most loci. This set of markers can be useful for population genetic studies and might even prove useful in other phylogenetically close species of the subfamily Myrmicinae.     

Isolation and characterization of 19 novel microsatellite loci in Rhododendron aureum and Rhododendron brachycarpum (Ericaceae)

Nineteen polymorphic microsatellite loci are described for Rhododendron aureum Georgi (Ericaceae), an endangered species in Korea, and the closely related Rhododendron brachycarpum. The sequences containing repeat motifs were identified using low-depth next generation sequencing and 148 microsatellite loci were examined for amplification success and the detection of polymorphisms. All 19 loci were polymorphic and the number of alleles for these markers varied from 4 to 25, with an average of 15 alleles per locus. Three R. aureum loci showed departure from Hardy–Weinberg equilibrium (HWE) and one R. brachycarpum locus deviated significantly from HWE. These microsatellite loci will be useful for investigating the genetic diversity and population structure of both species.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.     

Genetic diversity of Chinese alligator (Alligator sinensis) revealed by AFLP analysis: an implication on the management of captive conservation

Chinese alligator (Alligator sinensis) is one of the most critically endangered species among 23 extant crocodiles in the world. To prevent the extinction of the species, a captive propagation started at early 1980s, and the total number of alligator was brought up to 10 thousands from dozens of founder in 2000. But several genetic investigations showed those alligators were under an extremely low genetic diversity status with few detectible polymorphic loci. To get more insight into its genetic diversity for the management of captive Chinese alligator, AFLP was adopted to characterize variations in the population. Total of 347 bands were generated from 47 individuals using 3 primer combinations, of which 203 (58.50%) were polymorphic, and 35 AFLP phenotypes were revealed from those individuals. Comparing the results between RAPD and AFLP analysis on almost same sample set clearly indicated that AFLP is more efficient in revealing polymorphic loci, especially in those populations with extremely low genetic diversity. In present three assays, electrophoresis profile also displayed 3 individuals possessing very highly polymorphic AFLP phenotypes that were never been found by RAPD and mtDNA D-loop sequencing, implicating that we should offer these individuals more breeding opportunities to maintain the genetic diversity in the population and restrict those carrying few polymorphic loci from reproduction.     

Isolation and characterization of 31 polymorphic microsatellite markers in barfin flounder (Verasper moseri) and the cross-species amplification in spotted halibut (Verasper variegatus)

The construction of a genetic linkage map and evaluation of population genetic diversity both require large numbers of polymorphic molecular markers. In the present study, we report 31 polymorphic microsatellite markers, of which 28 were isolated from two repeat-enriched libraries constructed from genomic DNA, and three were detected in two genes (MCH-R1 and MCH-R2) of barfin flounder (Verasper moseri). A total of 94 alleles were detected with an average of 3.0 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to six, from 0.30 to 1.00 and from 0.33 to 0.78, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0016) and no significant linkage disequilibrum between pairs of loci was found. Cross-species amplification of these markers was evaluated using the closely related species spotted halibut (Verasper variegatus). This study will potentially be useful for stock management, constructing of a genetic linkage map, mapping economically important quantitative trait loci (QTL), and studying the population genetic diversity of barfin flounder (Verasper moseri).     

Development of nuclear microsatellite markers for the Japanese conifers Tsuga diversifolia and T. sieboldii (Pinaceae)

Nuclear microsatellite markers were developed for the two Tsuga species native to the Japanese Archipelago, Tsuga diversifolia and T. sieboldii, and a population with genetic affinities to T. diversifolia on Ulleung Island, Korea. Tsuga diversifolia and T. sieboldii are widespread dominant trees of temperate and subalpine forests in Japan but to date no genetic markers have been developed for these species. Fifteen polymorphic loci were developed and characterized, of which 14 are reliably amplified in each taxon. Across both species and the Ulleung Island population, the number of alleles per locus ranged from 3 to 26 (average = 13.93) and observed heterozygosity ranged from 0.005 to 0.935 (average = 0.535). In addition, all 15 loci were successfully amplified in a single accession of the Chinese species, T. chinensis. These markers will be useful for investigating the species’ biogeography, range‐wide genetic diversity, conservation genetic issues and potential for hybridisation.