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).     

Development of genetic markers in abalone through construction of a SNP database

In the absence of a reference genome, single-nucleotide polymorphisms (SNP) discovery in a group of abalone species was undertaken by random sequence assembly. A web-based interface was constructed, and 11 932 DNA sequences from the genus Haliotis were assembled, with 1321 contigs built. Of these, 118 contigs that consisted of at least ten annotation groups were selected. The 1577 putative SNPs were identified from the 118 contigs, with SNPs in several HSP70 gene contigs confirmed by PCR amplification of an 809-bp DNA fragment. SNPs in the HSP70 gene were compared across eight abalone species. A total of 129 polymorphic sites, including heterozygote sites within and among species, were observed. Phylogenetic analysis of the partial HSP70 gene region showed separation of the tested abalone into two groups, one reflecting the southern hemisphere species and the other the northern hemisphere species. Interestingly, Haliotis iris from New Zealand showed a closer relationship to species distributed in the northern Pacific region. Although HSP genes are known to be highly conserved among taxa, the validation of polymorphic SNPs from HSP70 in this mollusc demonstrates the applicability of cross-species SNP markers in abalone and the first step towards universal nuclear markers in Haliotis.     

Isolation and cross-amplification of microsatellites in pink abalone (Haliotis corrugata)

Ten novel microsatellite loci were isolated in pink abalone, Haliotis corrugata, using (GT)₁₅ and (CT)₁₅ enriched genomic libraries. Two previously reported Haliotis kamtschatkana microsatellites cross‐amplified in H. corrugata. A set of 12 polymorphic microsatellites were evaluated in a wild population sample (N = 49). The number of alleles ranged from two to 55, and the observed and expected heterozygosities ranged from 0.104 to 0.939 and from 0.213 to 0.982, respectively. Significant deviations from Hardy–Weinberg equilibrium at three loci and no linkage disequilibrium were observed. Haliotis corrugata microsatellites cross‐amplified in other abalone species, two in H. fulgens, and seven in H. rufescens.     

Isolation and characterization of eleven microsatellite loci in small abalone, Haliotis diversicolor Reeve

Eleven novel microsatellite markers were isolated from small abalone, Haliotis diversicolor Reeve. These loci were tested on 22 individuals from two different geographic populations. We identified a total of 162 alleles from the 11 microsatellite loci. All of the loci were highly polymorphic. Polymorphism information content (PIC) is ranging from 0.7276 to 0.9163. Observed and expected heterozygosities ranged from 0.2727 to 1.0000 and from 0.7738 to 0.9429, respectively. Three loci deviated significantly from Hardy–Weinberg equilibrium. No pairs of loci displayed linkage disequilibrium. These polymorphic markers will be used to analyze population structure, genetic diversity and construct a genetic linkage map. Xin Zhan and Hai-Yan Hu contribute equally to this study.     

Development of 15 polymorphic genic microsatellite DNA markers of Pacific abalone Haliotis discus hannai

Fifteen polymorphic genic microsatellite DNA markers were developed based on the expressed sequence tags of Pacific abalone Haliotis discus hannai we ourselves generated, which were used to type 32 individuals representing a cultured population. The number of alleles each locus ranged from two to 12. The observed and expected heterozygosities ranged from 0.094 to 0.969 and from 0.091 to 0.878, respectively. Among 15 loci, four were found to deviate significantly from Hardy–Weinberg equilibrium (P_HW < 0.001). No linkage disequilibrium was found between these loci. It is certain that these markers will facilitate the management and exploitation of the genetic resource of Pacific abalone.     

Genetic diversity and species identification in the endangered white abalone (<Emphasis Type="Italic">Haliotis sorenseni</Emphasis>)

In 2001, the white abalone Haliotis sorenseni became the first marine invertebrate in United States waters to receive federal protection as an endangered species. Prior to the endangered species listing, 20 abalone were collected as potential broodstock for a captive rearing program. Using DNA from these animals, we have developed genetic markers, including five nuclear microsatellite loci and partial sequences of one nuclear (VERL) and two mitochondrial (COI and CytB) genes, to assess genetic variability in the species, aid in species identification, and potentially track the success of future outplanting of captive-reared animals. All five microsatellite loci were polymorphic and followed expectations of simple Mendelian inheritance in laboratory crosses. Each of the wild-caught adult abalone exhibited a unique composite microsatellite genotype, suggesting that significant genetic variation remains in natural populations. A combination of nuclear and mitochondrial gene sequencing demonstrated that one of the original wild-caught animals was, in fact, not a white abalone, but H. kamtschatkana (possibly subspecies assimilis). Similarly, another animal of uncertain identity accidentally collected by dredging was also shown to be H. kamtschatkana. Inclusion of these two animals as broodstock could have resulted in unintentional hybridizations detrimental to the white abalone recovery program. Molecular genetic identifications will be useful both in preventing broodstock contamination and as markers for future restocking operations.     

Development and characterization of 60 microsatellite markers in the abalone Haliotis diversicolor

The abalone, Haliotis diversicolor, is one of the most important mariculture species in southern China. We developed 60 new polymorphic microsatellite markers for H. diversicolor and characterized them in 30 individuals from a cultured population in Sanya, China. All 60 markers were found to be polymorphic. The number of alleles ranged from two to nine per locus, with an average of 4.12/locus. The expected and observed heterozygosities ranged from 0.10 to 0.88 and from 0.07 to 0.87, respectively. Forty-four loci were in Hardy-Weinberg equilibrium. These 44 microsatellite markers should be useful for genome mapping and population genetic studies.     

Isolation and characterization of microsatellite DNA loci in sea bass,Lates calcarifer Bloch

Polymerase chain reaction (PCR)‐based isolation of microsatellite arrays (PIMA) technique was used to isolate seven polymorphic microsatellite loci in sea bass, Lates calcarifer Bloch. A total of 62 samples of wild and cultivated sea bass collected from a few populations within Peninsular Malaysia were used in the study. For seven polymorphic loci, the number of alleles ranged from four to nine and locus heterozygosities ranged from 0.710 to 1.000. The loci will be useful for studying population structure, genetic variability of wild and hatchery stocks of L. calcarifer and broodstock management purposes.     

Development of microsatellite markers for the bracken fern, Pteridium aquilinum

We isolated eight novel polymorphic microsatellite loci from Pteridium aquilinum. These loci were characterized in 30 individuals, one from Bolivia, two from Peru, one from the USA, one from Japan, and 25 from Northeast China to Southwest China. The number of alleles per locus ranged from two to seven. The observed heterozygosity (H(O) ) ranged from 0.000 to 0.600 with an average of 0.3051, and the expected heterozygosity (H(E) ) ranged from 0.0966 to 0.7780 with an average of 0.4267. One locus deviated from Hardy-Weinberg equilibrium and four pairs of loci were found to be in linkage disequilibrium. These polymorphic loci will be useful in the study of the population genetic structure of Pteridium.     

Population genetics of black abalone, Haliotis cracherodii, along the central California coast

Over the past three decades, the black abalone, Haliotis cracherodii, has experienced precipitous declines in abundance over portions of its range in southern and central California. The potential for recovery of these populations is dependent in part on dispersal processes; that is, can distant populations serve as sources of recruits to locales that no longer harbor H. cracherodii? Here we use population genetic analysis to assess levels of population subdivision and infer recruitment processes. Epipodial tissue samples were obtained from over 400 black abalone from seven geographic sites between Santa Cruz and Santa Barbara counties in central California. Allelic frequencies were determined for each population at three polymorphic enzyme-encoding loci (GPI, AAT-1 and PGM). Significant allelic frequency differentiation among sites was observed at all three loci. Genetic distance was found to be independent of geographic distance over the approximately 300-km sampling range. In addition, a limited number of DNA sequences (total N=51) were obtained for the mitochondrial cytochrome oxidase subunit I gene (COI) from five of the populations. Since the same common COI haplotype dominated each population, this analysis had little statistical power and failed to detect population structure. The observed level of population differentiation at allozyme loci was three-fold higher than that observed in California red abalone, H. rufescens. The species differ in their breeding period and it is suggested that the relatively short, summer breeding season of black abalone limits dispersal because larvae experience reduced variance in oceanographic conditions relative to red abalone that spawn year-round. Based on these results, rates of recolonization and recovery of locally depressed or extirpated black abalone populations are likely to be slow despite harvest restrictions.     

Nuclear and chloroplast SSR markers in Paeonia delavayi (Paeoniaceae) and cross-species amplification in P. ludlowii

? Premise of the study: Microsatellite primers were developed for Paeonia delavayi and P. ludlowii (Paeoniaceae) to study their population genetics and phytogeography. ? Methods and Results: Nine polymorphic nuclear microsatellite loci were isolated from an enriched library of P. delavayi and primers were designed. The number of alleles per locus ranged from two to 16; the observed and expected heterozygosities ranged from 0.014 to 0.687 and 0.042 to 0.875, respectively. Six polymorphic chloroplast microsatellite loci were identified in P. delavayi and primers were provided. The number of alleles per locus ranged from two to six and the polymorphic information content ranged from 0.08 to 0.716. Both nuclear and chloroplast primers were successfully applicable to P. ludlowii. ? Conclusions: The markers developed here will facilitate analyses of genetic diversity, population genetic structure, phytogeographical patterns, and conservation for P. delavayi and P. ludlowii.     

Isolation of eight microsatellite markers from Moina macrocopa for assessing cryptic genetic structure in the wild

We isolated eight polymorphic microsatellite loci from the zooplankton Moina macrocopa (Straus), which is sensitive to pollutants such as insecticides and heavy metals. The isolated loci were polymorphic, with three to seven alleles among 23 individuals. Expected heterozygosities ranged from 0.167 to 0.787. These loci can be used to examine cryptic genetic structure and to infer the connectivity among metapopulations.     

Characterization and cross-amplification of 13 microsatellite loci in the northern pine processionary moth, Thaumetopoea pinivora (Lepidoptera: Notodontidae)

Thirteen polymorphic microsatellite loci were developed for the northern pine processionary moth (Thaumetopoea pinivora) and tested for cross-amplification in seven other species within the Thaumetopoea family. Number of alleles ranged from two to 10 when at least 28 individuals from one population were screened and one locus, Thapin06, appears to be sex linked. Expected heterozygosity ranged from 0.094 to 0.856 and observed heterozygosity ranged from 0.097 to 0.806. Amplification success varied between sister species, with two up to seven loci being successfully amplified. The described loci will be valuable for studying the population genetic structure and dispersal behaviour of this forest pest.     

Novel microsatellite loci in the grass snake (Natrix natrix) and cross-amplification in the dice snake (Natrix tessellata)

Six novel polymorphic microsatellite loci are presented for the grass snake (Natrix natrix), a species with declining populations in many regions. The number of alleles per locus ranged from two to seven. Four dice snake (Natrix tessellata) microsatellites were polymorphic in the grass snake with three to four alleles. At two loci, the expected heterozygosity differed significantly from observed heterozygosity. Cross-amplification of the grass snake markers in the dice snake showed two polymorphic microsatellites with two and four alleles.     

Isozyme polymorphism and genetic structure of the population of Sorbus torminalis (L.) Crantz from the Bytyń Forest (Poland)

Dormant buds collected from 35 wild service trees (Sorbus torminalis) in the Bytyń Forest were tested with horizontal gel electrophoresis to assess the genetic structure of the population. Among 16 investigated isozyme loci, seven loci (ADH-A, 6PGD-A, GDH-B, ME-A, SOD-A, PGM-A, PGM-B) proved to be polymorphic, whereas the other nine loci (SDH-A, SDH-B, DIA-C, DIA-D, FLE-A, FLE-B, GOT-B, IDH-A, IDH-B) were monomorphic. The number of alleles per polymorphic locus ranged from two to three, with a mean of 2.29. The average observed and expected heterozygosities were 0.2665 and 0.3462, respectively. The combined FIS value over all polymorphic loci was 0.2179, which reflects a substantial deficit of heterozygotes. Two polymorphic loci (SOD-A, PGM-A) were identified in S. torminalis for the first time.     

Isolation and characterization of polymorphic microsatellite markers in Iberolacerta monticola, and cross-species amplification in Iberolacerta galani and Zootoca vivipara

Fourteen polymorphic microsatellite loci are described for the Iberian rock lizard, Iberolacerta monticola. Genetic variation in a sample of 20 individuals from Piornedo (northwestern Spain) was quantified both by the number of alleles per locus, which ranged from six to 13, and by the expected frequency of heterozygotes under random mating (heterozygosity), which ranged from 0.761 to 0.902. Single locus and global exclusion probabilities were also computed, and indicate a high power of these markers for paternity assignments and mating system studies of I. monticola. All the analysed loci were also polymorphic in Iberolacerta galani, but only seven in Zootoca vivipara.     

Characterization of 13 polymorphic microsatellite loci in the Japanese land leech

We developed 13 polymorphic microsatellite loci of the Japanese land leech (Haemadipsa japonica; Haemadipsidea) using an Illumina MiSeq sequencing approach. A total of 42,064 nuclear DNA contigs were filtered for microsatellite motifs, among which 30,873 simple sequence repeat loci were identified. From these sequences, we selected 30 primer sets, and 13 of these loci were successfully amplified. Polymorphism of the 13 loci was tested using 16 individuals sampled from sixteen populations across Japan. The number of alleles and polymorphism information content varied from 5 to 17 and 0.335 to 0.883, respectively, and observed and expected heterozygosity values ranged from 0.143 to 0.875 and 0.349 to 0.893, respectively, indicating that these loci are polymorphic. Furthermore, we established useful multiplex PCR using these loci. The 13 microsatellite loci described in this paper are the first nuclear microsatellite markers for a land leech species.     

Development and characterization of microsatellite loci for lotus (Nelumbo nucifera)

This paper reports the development of microsatellite primers for Nelumbo nucifera Gaerten. By screening genomic libraries enriched with 10 kinds of probes, Seventeen polymorphic loci were isolated and primers were designed. Polymorphism of these 17 loci was assessed in 24 individuals. All the 17 loci are polymorphic and the number of alleles ranged from two to seven. Observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.9176 and from 0.2837 to 0.7917 respectively. These microsatellite loci should be useful for studying the genetic diversity of N. nucifera.     

Genotyping single nucleotide polymorphisms (SNPs) across species in Old World Monkeys

The development of DNA markers is becoming increasingly useful in the field of primatology for studies on paternity, population history, and biomedical research. In this study, we determine the efficacy of using cross-species amplification to identify single nucleotide polymorphisms (SNPs) in closely related species. The DNA of 93 individuals representing seven Old World Monkey species was analyzed to identify SNPs using cross-species amplification and genotyping. The loci genotyped were 653 SNPs identified and validated in rhesus macaques. Of the 653 loci analyzed, 27% were estimated to be polymorphic in the samples studied. SNPs identified at the same locus among different species (coincident SNPs) were found in six of the seven species studied with longtail macaques exhibiting the highest number of coincident SNPs (84). The distribution of coincident SNPs among species is not biased based on proximity to genes in the samples studied. In addition, the frequency of coincident SNPs is not consistent with expectations based on their phylogenetic relationships. This study demonstrates that cross-species amplification and genotyping using the Illumina Golden Gate Array is a useful method to identify a large number of SNPs in closely related species, although issues with ascertainment bias may limit the type of studies where this method can be applied.