Isolation and characterization of 16 tetranucleotide microsatellite loci in the red panda (Ailurus fulgens)

A total of 15 polymorphic tetranucleotide microsatellite loci in the red panda, Ailurus fulgens, were characterized in this study. Based on evaluations of 33 red pandas, the number of observed alleles for each locus ranged from seven to 17 and the expected and observed heterozygosities were 0.412–0.897 and 0.121–0.909, respectively. The mean polymorphic information content was 0.721. These markers would greatly strengthen the utilization of microsatellite tools in genetic variation studies in red panda populations.     

Patterns of genetic diversity and differentiation in the tsetse fly <Emphasis Type="Italic">Glossina morsitans morsitans</Emphasis> Westwood populations in East and southern Africa

Genetic diversity and differentiation within and among nine G. morsitans morsitans populations from East and southern Africa was assessed by examining variation at seven microsatellite loci and a mitochondrial locus, cytochrome oxidase (COI). Mean COI diversity within populations was 0.63 ± 0.33 and 0.81 taken over all populations. Diversities averaged over microsatellite loci were high (mean number of alleles/locus ≥7.4; mean H _E ≥ 65%) in all populations. Diversities averaged across populations were greater in East Africa (mean number of alleles = 22 ± 2.6; mean h _e = 0.773 ± 0.033) than in southern Africa (mean number of alleles = 18.7 ± 4.0; mean h _e = 0.713 ± 0.072). Differentiation among all populations was highly significant (R _ST = 0.25, F _ST = 0.132). Nei’s G _ij statistics were 0.09 and 0.19 within regions for microsatellites and mitochondria, respectively; between regions, G _ij was 0.14 for microsatellites and 0.23 for mitochondria. G _ST among populations was 0.23 for microsatellite loci and 0.40 for mitochondria. The F, G and R statistics indicate highly restricted gene flow among G. m. morsitans populations separated over geographic scales of 12–917 km.     

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.     

Patterns of allozyme variation in relation to population size of the threatened plantMegaleranthis saniculifolia (Ranunculaceae) in Korea

Patterns of variation at 27 allozyme loci were investigated in the endangered endemic plantMegaleranthis saniculifolia. Levels of allozyme variation (A = 1.47,P = 40%,He = 0.088) were also compared with other endemic plant species. Genetic divergence between populations was very high (G ^st = 0.271 ), with moderate to high interpopulation differentiation, which probably arose through historical bottlenecks in a landscape of habitat fragmentation and/or human influence. The percentage of polymorphic loci, heterozygosity, and mean number of alleles per locus were positively related to population size, probably due to the stochastic loss of rare alleles in the smaller populations. Individuals in the small and marginal populations (TB, KD, and CJ) showed higher proportions of fixed loci. These ecologically marginal populations were typically more distant from the nearest neighboring population and were more genetically distinct from one another. The genetic structure of the current population ofM. saniculifolia is probably the result of local extinctions of intervening populations. This, in turn, is due to the Pleistocene climatic change and increased habitat destruction. A positive association appears to exist between genetic diversity and population size. Although these small population sizes are more sensitive to stochastic events, securing a certain number of individuals from the three larger populations (SB, JB, and TG) could be accomplished as part of a conservation strategy. In addition, it is important to prioritize populations in different regions in order to limit population declines caused by large-scale environmental catastrophes.     

Development of microsatellite DNA loci for <Emphasis Type="Italic">Pugionium dolabratum</Emphasis> (Brassicaceae), an endangered psammophyte

Pugionium (Brassicaceae) is a small Central-Asian endemic genus with four tentative species described before. All of them grow in the desert habitat and have been considered as important psammophytes for evolutionary studies and ecological restorations. However, the wild resources of these species have been declined because of overexploitation. In this study, 12 polymorphic microsatellite loci have been developed for P. dolabratum Maxim, and the performance of primer pairs amplifying these loci in related three species has also been tested. The number of alleles in 18 individuals from 13 geographically distant populations ranged from three to seven per locus. The observed and expected heterozygosities varied from 0.17 to 0.30 and from 0.32 to 0.48, respectively. We further performed cross-priming tests in the remaining three species of this genus, P. cornutum, P. calcaratum and P. cristatum. These newly recovered microsatellite loci will be useful for studying the population genetics and adaptive evolution of these endangered psammophytes in the desert.     

Isolation and characterization of first microsatellite markers for the noble crayfish, <Emphasis Type="Italic">Astacus astacus</Emphasis>

Noble crayfish (Astacus astacus) is listed as vulnerable by the IUCN Red List of Threatened Species in Europe. However, very little is known about genetic diversity and structuring of noble crayfish populations, mainly because of the lack of informative genetic markers. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4,000 recombinant clones. Eight loci revealed polymorphisms in a panel of 172 individuals from seven populations in Northern Europe. Number of alleles per locus ranged from two to 10 (average 4.4) and heterozygosity levels among populations varied from 0 to 0.80 for H _o and from 0 to 0.72 for H _e.     

Genetic diversity following demographic recovery in the insular endemic plant <Emphasis Type="Italic">Galium catalinense</Emphasis> subspecies <Emphasis Type="Italic">acrispum</Emphasis>

Galium catalinense (Rubiaceae) is a perennial shrub consisting of two subspecies endemic to California’s Channel Islands: Galium catalinense subsp. catalinense on Santa Catalina Island, and G. catalinense subsp. acrispum, a state-endangered taxon on San Clemente Island. A long history of overgrazing by introduced herbivores has contributed to population declines in G. catalinense subsp. acrispum. We surveyed 12 populations throughout the taxon’s range for genetic variation using eight polymorphic microsatellite loci to determine the genetic impact of this demographic bottleneck. At the taxon level, 65 alleles were identified with an average of 8.1 alleles per locus, although many alleles were rare; the effective number of alleles per locus averaged 2.6. Expected heterozygosity was 0.550. Individual populations had between six and eight polymorphic loci, with expected heterozygosities ranging from 0.36 to 0.60, and effective numbers of alleles ranging from 1.8 to 3.5 per locus. Populations fell into three or four genetic clusters, depending on type of analysis, which may represent refugia where the populations persisted during intense herbivory. There is little evidence of genetic bottlenecks or substantial inbreeding within populations. These findings, coupled with indications of recent migration between populations, suggest that G. catalinense subsp. acrispum is currently unlikely to be endangered by genetic factors, but small population sizes make the taxon vulnerable to future loss of genetic diversity. Management strategies based on these genetic data, population sizes, and the spatial distribution of populations are discussed.     

Population Genetic Structure of Pacific White Shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>) from Mexico to Panama: Microsatellite DNA Variation

Genetic variation and population structure of wild white shrimp (Litopenaeus vannamei) from 4 geographic locations from Mexico to Panama were investigated using 5 microsatellite DNA loci. The genetic diversity between populations was indicated by the mean number of alleles per locus and mean observed heterozygosity, which ranged from 7.4 to 8.6 and from 0.241 to 0.388, respectively. Significant departures from Hardy-Weinberg equilibrium were found at most locations at each locus, with the exception Guatemala at Pvan0013, and were caused by high heterozygote deficiencies. Genetic differences between localities were detected by pairwise comparison based on allelic and genotypic frequencies, with the exception of locus Pvan1003. Significant pairwise F _ST values between locations and total F _ST showed that the white shrimp population is structured into subpopulations. However, population differentiation does not follow an isolation-by-distance model. Knowledge of the genetic diversity and structure of L.vannamei populations will be of interest for aquaculture and fisheries management to utilize and preserve aquatic biodiversity.     

Biochemical genetics of some seed proteins of Pinus radiata

In a high-salt soluble fraction of the total protein from single seeds of Pinus radiata, up to 45 polypeptides were resolved on SDS-polyacrylamide gels. At least one-fifth of these polypeptides showed variation between seeds. In the 27,000–29,000 dalton region, two polypeptides were inherited as codominant alleles at a single locus and were shown to assort independently of another seed protein locus and three allozyme loci. A survey of 120 individuals from the five known native populations of P. radiata in California detected only the 27K and 29K alleles at the locus. In all populations, the 29K allele predominated, and the two island populations were monomorphic for the 29K allele. The 27 and 29 kdalton polypeptides were shown to have very similar amino acid sequences, and the allelic difference at this locus is most probably in the gene sequence for the polypeptide.     

<Emphasis Type="Italic">HLA-A</Emphasis> allele associations with viral <Emphasis Type="Italic">MER9-LTR</Emphasis> nucleotide sequences at two distinct loci within the <Emphasis Type="Italic">MHC alpha</Emphasis> block

The study of the association of the Human Leukocyte Antigen (HLA) alleles and polymorphic retrotransposons such as Alu, HERV, and LTR at various loci within the Major Histocompatibility Complex allows for a better identification and stratification of disease associations and the origins of HLA haplotypes in different populations. This paper provides sequence and association data on two structurally polymorphic MER9-LTR retrotransposons that are located 54 kb apart and in close proximity to the multiallelic HLA-A gene involved in the regulation of the human immune system. Direct DNA sequencing and analysis of the PCR products identified DNA nucleotide variations between the MER9-LTR sequences at the two loci and their associations with HLA-A alleles as potential haplotype and evolutionary markers. All MER9-LTR sequences were haplotypic when associated with common HLA-A alleles. The number of SNP loci was 2.5 times greater for the solo LTR at the AK locus, which is located closer to the HLA-A gene than the solo or 3′ LTR at the HG locus. Our study shows that the nucleotide variations of the MER9-LTR DNA sequences are additional informative markers in fine mapping HLA-A genomic haplotypes for future population, evolutionary, and disease studies.     

Polymorphism of hordein-coding loci in cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) in Afghanistan

Polymorphism of hordeins encoded by the Hrd A, Hrd B, and Hrd F loci was analyzed in 84 accessions of local barley (Hordeum vulgare L.) varieties from major agricultural regions of Afghanistan using starch gel electrophoresis. Forty alleles of the Hrd A locus with the frequencies from 0.12 to 32.73%, 62 alleles of the Hrd B locus with the frequencies from 0.12 to 14.29%, and five alleles of the Hrd F locus with the frequencies from 0.59 to 32.15% have been identified. The conclusion about genetic similarity of barley populations from different regions of Afghanistan is made on the basis of cluster analysis of the matrix of allele frequencies in barley populations from 31 localities. The local barley populations form four unequal clusters. The largest cluster I includes populations from 14 localities of Afghanistan. The second largest cluster IV consists of populations from ten localities, and clusters II and III comprise populations from four and three localities, respectively. Each of the four clusters includes populations from different regions of northern and southern Afghanistan. Based on our results, we conclude that the diversity of hordein-coding loci and the distribution of their alleles among different regions of Afghanistan are the consequences of introduction of barley landraces and their distribution over trade routes.     

Microsatellite loci development in endangered pamphagid grasshoppers endemic to the Canary Islands (Orthoptera)

The five recognized endemic pamphagid species (Orthoptera) of the Canary Islands have restricted and fragmented ranges due to habitat decline. Seven polymorphic microsatellite markers have been developed for Acrostira tamarani, and the performance of primer pairs amplifying these loci in related taxa has been tested. The number of alleles in A. tamarani samples taken from two distant localities in the island of Gran Canaria ranged from two to eight per locus. Observed heterozygosities were from 0.151 to 0.559. Up to four primer pairs amplified in related species with moderate heterozigosities (maximum of 0.687 and 10 alleles for Ata67 locus in Purpuraria erna). These markers could be useful tools to study the population structure and management of endemic threatened pamphagids of the Canary archipelago.     

Microsatellite DNA loci from the typical halophyte <Emphasis Type="Italic">Thellungiella salsuginea</Emphasis> (Brassicaceae)

Thellungiella salsuginea (Brassiaceae) is a typical halophyte which can tolerate extreme cold, drought, and salinity. In order to understand the adaptive evolution of this species in the arid habitats, it is important to know its genetic structure. In this study, 17 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of this species. We further assessed the polymorphisms of each locus in 18 individuals from nine geographically distant populations. The number of alleles per locus ranged from six to fourteen. The observed and expected heterozygosity ranged from 0.17 to 0.28 and 0.32 to 0.45, respectively. These markers have been crossly checked in another congeneric species, T. halophila. These microsatellite markers will be useful for investigating population genetics and adaptive evolution of this species and morphological divergence between and it and the closely related species.     

Segregation analysis and RFLP mapping of the R1 and R3 alleles conferring race-specific resistance to Phytophthora infestans in progeny of dihaploid potato parents

Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato (Solanum tuberosum). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles (R genes) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F₁ populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F₁ populations analysed, whereas the RI allele segregated with a 1:1 ratio as expected in five F₁ populations. The mode of inheritance of the R10 allele could not be deduced as only very few F₁ hybrids bearing R10 were obtained. Linkage analysis in two F₁ populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3, to a distal position on chromdsome XI.     

DOES MATE LIMITATION IN SELF‐INCOMPATIBLE SPECIES PROMOTE THE EVOLUTION OF SELFING? THE CASE OF LEAVENWORTHIA ALABAMICA

Genetic diversity at the S‐locus controlling self‐incompatibility (SI) is often high because of negative frequency‐dependent selection. In species with highly patchy spatial distributions, genetic drift can overwhelm balancing selection and cause stochastic loss of S‐alleles. Natural selection may favor the breakdown of SI in populations with few S‐alleles because low S‐allele diversity constrains the seed production of self‐incompatible plants. We estimated S‐allele diversity, effective population sizes, and migration rates in Leavenworthia alabamica, a self‐incompatible mustard species restricted to discrete habitat patches in rocky glades. Patterns of polymorphism were investigated at the S‐locus and 15 neutral microsatellites in three large and three small populations with 100‐fold variation in glade size. Populations on larger glades maintained more S‐alleles, but all populations were estimated to harbor at least 20 S‐alleles, and mate availabilities typically exceeded 0.80, which is consistent with little mate limitation in nature. Estimates of the effective size (N_e) in each population ranged from 600 to 1600, and estimated rates of migration (m) ranged from 3 × 10⁻⁴ to nearly 1 × 10⁻³. According to theoretical models, there is limited opportunity for genetic drift to reduce S‐allele diversity in populations with these attributes. Although pollinators or resources limit seed production in small glades, limited S‐allele diversity does not appear to be a factor promoting the incipient breakdown of SI in populations of this species that were studied.     

Microsatellite markers for the common vole (<Emphasis Type="Italic">Microtus arvalis</Emphasis>) and their cross-species utility

Fragmentation of natural habitats of the common vole (Microtus arvalis) provides an excellent model system to study the consequences of restricted gene flow and small population sizes for isolated populations. Here we describe the isolation and characteristics of 10 autosomal and one X-linked microsatellite marker. These new markers were tested in 24 voles from a natural population in eastern Germany. Loci were highly polymorphic with numbers of alleles per locus ranging from three to 26 and expected heterozygosities from 0.51 to 0.97. All loci except for the X-linked locus Mar105 followed Hardy–Weinberg expectations. Cross-species amplifications revealed that most loci were polymorphic as well in M. agrestis, M. thomasi, and M. pennsylvanicus.     

Genetic variation and population structure of endemic yellow catfish, <Emphasis Type="Italic">Horabagrus</Emphasis><Emphasis Type="Italic">brachysoma</Emphasis> (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.     

Hybridization and introgression across different ploidy levels in the Neotropical orchids Epidendrum fulgens and E. puniceoluteum (Orchidaceae)

The hypothesis of gene flow between species with large differences in chromosome numbers has rarely been tested in the wild, mainly because species of different ploidy are commonly assumed to be reproductively isolated from each other because of instantaneous and strong postzygotic barriers. In this study, a broad‐scale survey of molecular variation was carried out between two orchid species with different ploidy levels: Epidendrum fulgens (2n = 2x = 24 chromosomes) and Epidendrum puniceoluteum (2n = 4x = 52 chromosomes). To test the strength of their reproductive barriers, we investigated the distribution of genetic variation in sympatric and allopatric populations of these two species and conducted crossing experiments. Nuclear and plastid microsatellite loci were used to genotype 463 individuals from eight populations across the geographical range of both species along the Brazilian coastal plain. All six sympatric populations analysed presented hybrid zones, indicating that hybridization between E. fulgens and E. puniceoluteum is a common phenomenon. Bayesian assignment analysis detected the presence of F₁ and F₂ individuals and also signs of introgression, demonstrating a high potential for interspecific gene flow. Introgression occurs preferentially from E. fulgens to E. puniceoluteum. Pure parental individuals of both species display strong genotype–habitat associations, indicating that environment‐dependent selection could be acting in all hybrid zones. This study suggests that hybridization and introgression are evolutionary processes playing a role in the diversification of Epidendrum and indicates the importance of investigations of hybrid zones in understanding reproductive barriers and speciation processes in Neotropical orchid species.     

Genetic diversity analysis of a world-wide collection of <Emphasis Type="Italic">Ascochyta rabiei</Emphasis> isolates using sequence tagged microsatellite markers

A previously characterized compound microsatellite locus ARMS1, containing penta- and decameric repeat units, has been reported to reveal genetic diversity in Ascochyta rabiei (Pass.) Labr. isolates. Therefore, 37 isolates of Ascochyta rabiei collected from different states of India and 38 isolates from fifteen other countries in the world were examined for their diversity at this locus. Twenty-six alleles on the basis of size (228--451 base pairs) were detected in the world isolates examined, while 15 alleles (287--418 base pairs) were observed in isolates from the Indian subcontinent. To the best of our knowledge, this study is the first to demonstrate diversity in representative Ascochyta rabiei isolates from different parts of the world at the ARMS1 locus.     

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.