Isolation and characterization of eight microsatellite loci from the endangered plant species Hypochaeris salzmanniana (Asteraceae)

We report the isolation and characterization of eight microsatellite loci for Hypochaeris salzmanniana, an endangered species endemic to the southwestern coast of Spain and the Atlantic coast of Morocco. A total of 32 alleles were detected across a sample of 45 individuals, with an average number of 4.0 alleles per locus. The average polymorphic information content (PIC) was 0.533 and the observed (H _O) and expected (H _E) heterozygosity values varied from 0.022 to 0.978 and from 0.434 to 0.759, respectively. Five loci exhibited significant deviation from Hardy–Weinberg equilibrium (P ≤ 0.001) and three pairs of loci showed significant linkage disequilibrium (P ≤ 0.01). The eight loci were tested for transferability in three others species (H. arachnoidea, H. glabra, and H. radicata) belonging to the same section of H. salzmanniana. With the exception of locus Hsalz7, all loci successfully amplified in the three species. These preliminary data confirm the usefulness of microsatellite markers for assessing the ecology and genetic structure of H. salzmanniana and to understand the evolution of species within the section Hypochaeris.     

Eight SSR loci from Oyster Crassostrea gigas EST database and cross-species amplification in C. plicatula

Oyster (Crassostrea plicatula) is widely distributed in coastal areas of China. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Crassostrea gigas and to amplify EST-SSR in C. plicatula. Characteristics of eight EST-SSR loci were investigated using 37 wild-type C. plicatula individuals. The number of alleles per locus ranged from four to six. The observed heterozygosity (H _O) ranged from 0.1892 to 0.7027 (0.3919 on average) and the expected heterozygosity (H _E) ranged from 0.6068 to 0.7656 (0.7039 on average) (P < 0.05). The average observed heterozygosity was much lower than the average expected heterozygosity. All loci except C15 departed from Hardy-Weinberg equilibrium (P < 0.05) significantly. Contribution of the eight EST-SSR primers presented here will provide necessary and powerful molecular tools for management and conservation studies on the species of oysters in the future.     

Isolation and characterization of 18 microsatellites in the Peking duck (Anas platyrhynchos) and their application in other waterfowl species

We have isolated and characterized 18 microsatellite loci in the Peking duck (Anas platyrhynchos). The average number of alleles per locus was 3.5, ranging from one to six in domestic Peking ducks (n = 40). All of the markers were polymorphic in a sample of five mallards (A. platyrhynchos; two to eight alleles). Seventeen of the 18 markers amplified in Muscovy ducks (Cairina moschata) with 11 being polymorphic in our sample (n = 14). Amplification of the markers in different species comprising the subfamilies Anatinae and Anserinae indicates their potential value for population genetic applications in a wide range of waterfowl species.     

Genetic variation amongst viviparid snails in the genus Mekongia (Prosobranchia: Viviparidae) in Thailand

Allozyme variation in viviparid snails from the genus Mekongia in Thailand were examined across the different species, subspecies and geographical locations (river drainage systems). Using horizontal starch gel electrophoresis, 11 presumed allozyme loci (eight polymorphic) from eight enzyme systems were screened. Heterozygosity was moderately low (Hexp = 0.000–0.109, mean = 0.037). One population of Mekongia pongensis (Nong Khai) was monomorphic at all 11 examined loci in contrast to the other two populations, suggesting bottleneck within this population. Populations were more differentiated in the Mekongia sphaericula complex (F_ST = 0.587) than in either the Mekongia swainsoni complex (F_ST = 0.161) or M. pongensis species (F_ST = 0.073). Mekongia sphaericula sphaericula and Mekongia sphaericula extensa exhibited fixed allele differences at two loci, a high genetic distance (D = 0.265–0.300) and a potential polyphyletic relationship, suggesting two distinct lineages (species).     

Polymorphic microsatellite loci isolated from the marine sponge Scopalina lophyropoda (Demospongiae: Halichondrida)

A total of seven microsatellites out of 88 isolated from a genomic library enriched for (CA)_n and (GA)_n repeats were characterized in the Mediterranean marine sponge Scopalina lophyropoda. The microsatellite motifs were large (34.81 ± 13.9 bp) and imperfect. The seven microsatellite loci were screened in 30 individuals collected from Blanes, northwestern Mediterranean. All of them were polymorphic (allele numbers and observed heterozygosities ranged from 3 to 6 and from 0.16 to 0.76, respectively). No significant linkage disequilibrium between pairs of loci and no departure from Hardy–Weinberg equilibrium were found. These markers are therefore promising for studies of the population structure of the species.     

Isolation and characterization of eight microsatellite loci for the Neotropical freshwater catfish Pimelodella chagresi (Teleostei: Pimelodidae)

We report eight (CA)_10?35 unlinked microsatellite loci from the Neotropical freshwater catfish, Pimelodella chagresi (Siluriformes: Pimelodidae). These loci were characterized with 23 individuals collected in Panama. Number of alleles per locus varied from 7 to 23 (mean = 12.9) and observed heterozygosity ranged from 0.522 to 0.909 (mean = 0.732). These loci will be used to investigate the existence of cryptic species within the P. chagresi clade, and to study fine‐scale population structure.     

Isolation and characterization of eight microsatellite loci from <Emphasis Type="Italic">Lycorma delicatula</Emphasis> (White) (Hemiptera: Fulgoridae) for population genetic analysis in Korea

Lycorma delicatula (White) is native to China but is becoming an important insect pest in Korea. Polymorphic DNA markers like microsatellites are widely used for characterizing dispersal patterns and capacity of invasive insect pests which can contribute to designing effective management of the species. To facilitate such population genetic studies of L. delicatula in Korea, we isolated and characterized eight microsatellite loci for L. delicatula using a hybridization-biotin enrichment method. We further used these novel microsatellite loci to determine population genetic parameters for 33 L. delicatula specimens collected from Cheonan, South Korea where outbreaks of this species were first reported in Korea. The number of alleles per locus ranged from three to ten, with an average of 6.25. The mean expected (H _E) and observed heterozygosities (H _O) were 0.575 and 0.626, respectively. The eight loci showed no deviation from Hardy–Weinberg equilibrium according to the adjusted significance threshold (P = 0.00625), and there was no linkage disequilibrium between each pair of these eight markers. Bayesian cluster analysis using the program structure revealed no evidence of genetic structuring in L. delicatula samples from Cheonan. These new microsatellite markers will be widely applicable to future ecological genetic studies of L. delicatula, including assessment of the level of gene flow and genetic connectivity among populations that are necessary for effective management and monitoring of the species.     

Isolation and characterization of microsatellite loci in the Cape fynbos heath Erica coccinea (Ericaceae)

Enriched genomic libraries were used to isolate and characterize dinucleotide microsatellite loci in Erica coccinea, a South African Cape fynbos heath species with distinct resprouter and seeder populations. Microsatellites were required to investigate the effect of the contrasting demographic pattern driven by these two post-fire responses in the population genetic structure of seeder and resprouter forms within this species. Eight microsatellite loci were characterised and amplified a total of 106 alleles in 2 samples each of 30 individuals from 1 resprouter and 1 seeder population. Mean allele numbers were 7.88 and 11.0 for the resprouter and seeder population, respectively. Both populations showed similar average observed and expected heterozygosity levels, H _O(resprouter) = 0.683, H _O(seeder) = 0.696; H _E(resprouter) = 0.726, H _E(seeder) = 0.756, and average positive inbreeding coefficients F _IS(resprouter) = 0.058, F _IS(seeder) = 0.080. This set of microsatellite loci will be used to conduct a population genetic survey of seeder and resprouter populations throughout the range of the species. Cross-species transferability was also assayed in four other South African and four European species of the genus Erica, supporting their potential use for population genetic analyses.     

Isolation and characterization of six microsatellite loci in the drywood termite Cryptotermes secundus (Kalotermitidae)

The Kalotermitidae is a family of lower termites and its species are ideal to study the evolution and maintenance of eusociality within the Isoptera. Co‐dominant genetic markers are an essential tool to elucidate the putatively complex colony and population structure. Six polymorphic microsatellite loci were identified in the drywood termite Cryptotermes secundus (Kalotermitidae). To test the variability of the markers, 18 colonies (n = 18) from Darwin (Australia) were assayed. We found two to eight alleles per locus with the level of observed heterozygosity at each locus ranging from 0.00 to 0.53. The significantly positive F_IT value suggests that some kind of inbreeding does occur in this population.     

Isolation and cross‐species amplification of microsatellite loci in the freshwater minnow Zacco pachycephalus (Teleostei: Cyprinidae) for diversity and conservation genetic analysis

Asian minnows of the genus Zacco are dominant fish in various freshwater ecosystems. Two species, Zacco pachycephalus and Z. platypus, occur in Taiwan and are favourite targets for local sport anglers. The introduction of Zacco fish into nonindigenous habitats in Taiwan has become a conservation issue. We developed eight polymorphic microsatellites for Z. pachycephalus (average H_E = 0.779) and these microsatellites were applicable to Z. platypus, which showed a comparable polymorphic level (average H_E = 0.784). These loci can be used as genetic markers for identifying conservation units and studying population differentiation for both Zacco species.     

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

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

Isolation and characterization of polymorphic microsatellite markers in Anthyllis vulneraria

To study the reproductive system of the putative autogamous species Anthyllis vulneraria in Belgian calcareous grasslands, eight polymorphic microsatellite loci were developed using the sequence‐tagged microsatellite profiling technique and 35 plants were typed for all loci. No linkage disequilibrium was found between any pair of loci. Over the loci, the number of alleles ranged from two to five. Observed and expected heterozygosities ranged from 0.053 to 0.688 and from 0.053 to 0.631, respectively. The estimated F_IS values (F_IS = 0.031 and 0.122) are not in accordance with a predominantly selfing reproductive system.     

Isolation of 22 new Haliaeetus microsatellite loci and their characterization in the critically endangered Madagascar fish‐eagle (Haliaeetus vociferoides) and three other Haliaeetus eagle species

We isolated a total of 22 microsatellite loci from two Haliaeetus species: the Madagascar fish‐eagle (Haliaeetus vociferoides) and the bald eagle (Haliaeetus leucocephalus). Five loci were monomorphic in both the Madagascar fish‐eagle (n = 24–43) and the bald eagle (n = 2–8) but were found to be polymorphic in other Haliaeetus species. Haliaeetus loci have proved useful for investigating gene flow in Haliaeetus and Aquila eagles. Ten loci were polymorphic in the critically endangered Madagascar fish‐eagle and will be used to investigate the genetic population structure and mating system in this species.     

Isolation and characterization of microsatellite markers in the southern emu‐wren (Stipiturus malachurus: Aves)

We isolated and characterized eight novel microsatellite loci in the southern emu‐wren (Stipiturus malachurus). We used nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic DNA library. Based on genotypes from a single population, six loci showed no evidence of null alleles and were polymorphic (allele range = 2–9, mean heterozygosity = 0.57), and one locus was sex‐linked (N_A = 4). These loci were variable and had different allele size ranges in three other populations of southern emu‐wrens, and are therefore useful for determining levels of genetic diversity within and between populations of the species.     

Isolation and characterization of polymorphic microsatellite loci for the study of paternity and population structure in the little penguin Eudyptula minor

We isolated and characterized eight novel microsatellite loci in the little penguin Eudyptula minor, using nonradioactive polymerase chain reaction‐based techniques to screen GA and GAAA repeats from enriched genomic DNA libraries. All eight loci were polymorphic and seven were variable in our main study population (mean H_E = 0.613, mean N_A = 7.14). Cross‐amplification using a microsatellite primer developed in Spheniscus demersus (African penguin) yielded one additional polymorphic locus. This locus combined with six of the little penguin loci is suitable for paternity assignment in little penguins (exclusion probability for seven unlinked loci = 0.993).     

Genetic variation of wild litchi (<Emphasis Type="Italic">Litchi chinensis</Emphasis> Sonn. subsp. <Emphasis Type="Italic">chinensis</Emphasis>) revealed by microsatellites

Understanding the amount and distribution of genetic diversity in natural populations can inform the conservation strategy for the species in question. In this study, genetic variation at eight nuclear microsatellite loci was used to investigate genetic diversity and population structure of wild litchi (Litchi chinensis Sonn. subsp. chinensis). Totally 215 individuals were sampled, representing nine populations of wild litchi. All eight loci were polymorphic, with a total of 51 alleles. The expected heterozygosity in the nine populations ranged from 0.367 to 0.638 with an average value of 0.526. Inbreeding within wild litchi populations was indicated by a strong heterozygote defect. Significant bottleneck events were detected in the populations from Yunnan and Vietnam, which could be responsible for lower levels of genetic diversity in these populations. Measures of genetic differentiation (F _ST = 0.269) indicated strong differentiation among wild litchi populations. Significant correlation was found between genetic differentiation and geographical distance (r = 0.655, P = 0.002), indicating a strong isolation by distance in these populations. Bayesian clustering suggested genetic separation among three regional groups, namely, the western group, the central group and the eastern group. Some conservation strategies for wild litchi populations were also proposed based on our results.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Characterization and the broad cross‐species applicability of 20 anonymous nuclear loci isolated from the Taiwan Hwamei (Garrulax taewanus)

We isolated 20 anonymous nuclear loci (8556 bp in total) from the Taiwan Hwamei (Garrulax taewanus), an endemic songbird of Taiwan. A panel of nine to 15 individuals with unknown relationship was used to characterize polymorphism of these loci. We identified 46 single nucleotide polymorphic sites (SNPs) in 15 polymorphic loci. Frequency of SNPs was one per every 186 bp in average. Nucleotide diversity, θ, ranged from 0.00054 to 0.00371 per locus. We also tested cross‐species applicability of these loci on 17 species from eight different passerine families. All 20 loci could be successfully amplified (ranged from one to 16 species, mean = 7.9 species).     

Polymorphic microsatellites in a mangrove species,Rhizophora mangle L. (Rhizophoraceae)

Twenty‐six microsatellite loci were isolated and characterized from the mangrove species Rhizophora mangle using (GT)_n and (CT)_n repeats. Eighty‐four per cent of the clones contained microsatellite sequences; the most common dinucleotides were the (GA/CT) and (CA/GT) repeats. Ten primers were selected to investigate the polymorphism among individuals of R. mangle from two natural populations of the Colombian Pacific Coast. The observed heterozygosity per locus varied from 0.20 to 0.80, the power of discrimination was 0.32–0.84 and the power of exclusion was 0.03–0.75. This set of microsatellites offers an efficient tool for population genetics studies on this species.     

Isolation and characterization of fourteen novel microsatellite loci in the Hong Kong oyster, Crassostrea hongkongensis

Fourteen polymorphic microsatellite markers were isolated from the Hong Kong oyster, Crassostrea hongkongensis, with a partial genomic library enriched for tandem repeat sequences of (CA)₁₂, (GA)₁₂, (ATG)₆ and (TAGA)₄. Polymorphism of these loci was assessed in a sample of 48 wild unrelated individuals. The average allelic number of these polymorphic loci was 6.36 per locus, with a range of 4–16. The observed and expected heterozygosity varied from 0.208 to 0.729 (averaging 0.502) and from 0.193 to 0.789 (averaging 0.615), respectively. After Bonferroni correction (P > 0.0036), 11 of the 14 loci accorded with Hardy–Weinberg equilibrium, and the rest three were detected significant departure from HWE. Additionally, two loci (Ch103 and Ch104) showed significant linkage disequilibrium (P < 0.01). This is the first set of microsatellite loci developed in this species and would be useful for studies of population genetics, stock management and other relevant research in C. hongkongensis.