Molecular evidence of a reed warbler × great reed warbler hybrid ( Acrocephalus scirpaceus × A. arundinaceus) in Belgium

We report on the second case of a reed warbler × great reed warbler hybrid (Acrocephalus scirpaceus and A. arundinaceus). The bird was captured during a standardised ringing session in Belgium in autumn 1999, and fell between the parental species in all measurements. Molecular analyses of two microsatellite loci verified that the bird was a female that had a reed warbler father and a great reed warbler mother.     

Microsatellite markers for <Emphasis Type="Italic">Pseudopanax</Emphasis> trees and their cross-species amplification in the Araliaceae

In order to study hybridisation, taxonomic boundaries and phylogeography in the genus Pseudopanax (Araliaceae), we developed seven novel microsatellite loci from enriched genomic libraries of P. lessonii and P. crassifolius. These loci were characterised in 16 individuals from a single population of P. crassifolius, and displayed 2–11 alleles per locus. Observed heterozygosities ranged from 0.063 to 0.688. Most loci were polymorphic in the closely related Pseudopanax species, and several loci amplified widely across the Araliaceae.     

Isolation and characterization of polymorphic microsatellite loci in Candidia barbata (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Candidia barbata is an endemic cyprinid fish in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, a total of 14 polymorphic microsatellite loci from C. barbata were isolated and characterized using an optimized protocol to construct a microsatellite-enriched genomic library. The analysis of variability was performed in 30 specimens of Taiwan. The mean number of alleles across loci was 4.92 ± 1.44. The levels of expected and observed heterozygosities varied from 0.1266 to 0.5079, and from 0.0667 to 0.9667, respectively. Frequencies of null alleles of the 14 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population.     

Integrative placement and orientation of non-redundant SSR loci in cotton linkage groups by deficiency analysis

A combination of previously mapped and unmapped non-redundant SSR loci, using 381 primer pairs were chromosomally and sub-chromosomally localized by deficiency analysis of two sets of quasi-isogenic interspecific Gossypium hirsutum L. hypoaneuploid F₁ hybrids involving Gossypium barbadense L. and Gossypium tomentosum (Nuttall ex Seemann). Polymorphisms were detected for 369 SSR primer pairs. A total of 318 SSR loci were rendered deficient by the available hypoaneuploid stocks, which included primary monosomics (2n = 51), monotelodisomics and duplication-deficient (segmental trisomic–monosomic) (2n = 52) types. Chromosomal associations were newly determined for 123 SSR loci, of which 90, 106 and 73 were polymorphic in G. tomentosum, G. barbadense, and both sets, respectively. The deficiency tests independently confirmed the recent identifications of linkage groups (LG) A01, A02, A03 and D08 to be chromosome (Chr)-13, Chr-8, Chr-11 and Chr-19, respectively, and collectively delimited LG D02 and D03 to Chr-21 and 24, and their homeologs to Chr-8 and 11. Segmental homeology was detected between Chr-2 and Chr-17 loci, adding to evidence of segmental homeology between Chr-2 and 3 versus Chr-14 and 17. The 318 non-redundant SSR loci localized in this study will enhance the construction of linkage maps and QTL identification in molecular marker assisted selection since the confirmed and newly discovered SSR loci can serve as anchor loci for their respective chromosomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of EST-SSRs in scallop (Patinopecten yessoensis) from sequence database

Patinopecten yessoensis is a kind of cold water shellfish, and is an important economic species in China. In our study, we developed and evaluated simple sequence repeat (SSR) markers of P. yessoensis. Characteristics of 11 EST-SSR loci were investigated using 40 P. yessoensis individuals. The number of alleles per locus ranged from two to five. The observed heterozygosity ranged from 0.1026 to 0.9487, while the expected heterozygosity ranged from 0.1865 to 0.7433. All loci except P4 departed from Hardy–Weinberg equilibrium (P < 0.05) significantly. There was no LD observed between all pairs of EST-SSRs loci. These loci and markers will be useful for population genetics and systemic evolution of scallop.     

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.     

Diversity, host, and habitat specificity of oomycete communities in declining reed stands (Phragmites australis) of a large freshwater lake

We studied the diversity of oomycetes in a declining reed belt (Phragmites australis) of Lake Constance, Germany, using conventional baiting with specific reed and standard oak baits, accompanied by molecular techniques. Apart from an Aphanomyces sp. and a Phytophthora sp., baiting from reed rhizosphere samples from flooded, as well as drier, littoral sites revealed only Pythium spp. A total of 67 oomycete isolates was classified according to PCR-RFLP banding patterns and ITS sequencing, and 18 different sequence types could be separated. The majority of these seemed previously unknown species, as indicated by the degree of similarity to those deposited in nucleotide databases. Species communities in both flooded and drier habitats or both reed and oak baits clearly differed from one another, and only few species occurred in both dry and flooded sites, or in both oak and reed baits. A frequently occurring group of related Pythium species appeared to be specifically associated with reed, and these were the only species that proved pathogenic towards this host in vitro. Our study proved that unexplored natural ecosystems harbour diverse communities of oomycete species with specific habitat and host preferences within close-by, but ecologically contrasting, sites. Among the species isolated, those associated with the predominating plant might accumulate and thus may be reed pathogens of considerable importance.     

Plant regeneration from callus cultures in two ecotypes of reed (Phragmites communis Trinius)

Summary Different ecotypes of reed (Phragmites communis Trinius) provide an ideal resource for studies on plant environmental adaptations and presence of genes relating to stress resistance. Dune reed is a drought-tolerant reed ecotype growing in the desert regions of north-west China. In this work, in vitro culture systems of dune reed and local swamp reed (as control) were established by optimizing the culture conditions for each of them. Bright yellow calluses were induced on a Murashige and Skoog medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.4 μM naphthaleneacetic acid and 2.2μM benzyladenine. Benzyladenine promoted callus induction, but was not required for callus maintenance. Four types of callus have been identified from each of the reed ecotypes. Two types of callus, i.e. type A (formed normal green shoots) and type C (formed albino plants), were both found as embryogenic calluses. The optimal concentrations of 2,4-D to maintain embryogenic callus were 2.3–4.5 μM for dune reed and 9.0–13.5 μM for swap reed. Plant regeneration was achieved from types A and C callus in a hormone-free medium. The embryogenic calluses of swamp reed have been maintained for over 2 yr and still retain their strong embryogenic potential; however, those of dune reed gradually lost their embryogenic potential after only 7 mo. of culture. Regenerated plants from the two reed ecotypes showed, after a growth season, similar morphology and the same chromosome number (2n=8x=96, octoploid) as the wild plants.     

Temporal patterns of occurrence and transmission of the blood parasite Haemoproteus payevskyi in the great reed warbler Acrocephalus arundinaceus

We studied the prevalence and intensity of the haemosporidian blood parasite Haemoproteus payevskyi in great reed warblers at Lake Kvismaren (6 years) and Lake Segersj? (3 years) in Sweden. Based on microscopic inspection of slides from 282 adult birds, 20.6% showed infection of H. payevskyi in circulating red blood cells in at least 1 year. For parasite prevalence, there was no difference between years, sex, and age classes. However, parasite intensity was higher in females than in males, and this was most pronounced in 1-year-old birds. Individuals scored to carry parasites in year _n were more likely to show parasite infection year _n + 1 than birds scored to be parasite-free in year _n . None of 99 juvenile birds examined at the breeding site in late summer, 4–9 weeks after hatching, showed infection of H. payevskyi. Parasite intensity in infected adult birds decreased in the course of the breeding season and no new or relapse infections were observed during this period. Thus, our data imply that in the great reed warbler, a long-distance migrant to tropical Africa, transmission of H. payevskyi occurs on wintering sites or at stopover sites during migration.     

Genetic diversity in the blackberry rust pathogen, Phragmidium violaceum, in Europe and Australasia as revealed by analysis of SAMPL

Indigenous to Europe, the blackberry rust fungus Phragmidium violaceum was introduced to Australia and subsequently appeared in New Zealand, with the most recent authorised introductions to Australia specifically for the biological control of European blackberry. Markers for ‘selective amplification of microsatellite polymorphic loci’ (SAMPL) were developed for studying the population genetics of P. violaceum. Modification of one of the two SAMPL primers with a HaeIII adapter (H) revealed significantly greater levels of genetic variation than primers used to generate AFLPs, the latter revealing little or no variation among 25 Australasian and 19 European isolates of P. violaceum. SAMPL was used to describe genetic variation among these 44 isolates of P. violaceum from 51 loci generated using primer pairs (GACA)₄ + H–G and R1 + H–G. The European isolates were more diverse than Australasian isolates, with 37 and 22 % polymorphic loci, respectively. Cluster analysis revealed geographic clades, with Australasian isolates forming one cluster separated from two clusters comprising the European isolates. However, low bootstrap support at these clades suggested that Australian isolates had not differentiated significantly from European isolates since the first record of P. violaceum in Australia in 1984. In general, the results support two hypotheses. First, that the population of P. violaceum in Australia was founded from a subset of individuals originating from Europe. Second, that P. violaceum in New Zealand originated from the Australian population of P. violaceum, probably by wind dispersal of urediniospores across the Tasman Sea. The application of SAMPL markers to the current biological control programme for European blackberry is discussed.     

Isolation of microsatellite loci from the endemic and endangered Adriatic sturgeon (<Emphasis Type="Italic">Acipenser naccarii</Emphasis>)

With the aim of elaborating a breeding plan on a captive stock of the highly endangered Adriatic sturgeon (Acipenser naccarii), a total of 10 polymorphic microsatellite loci were isolated from an enriched library. The results of cross amplification of additional 8 loci previously isolated from A. oxyrinchus, A. fulvescens and Scaphyrinchus platorhynchus are also reported. Given the tetraploid condition of the species the genetic variability was estimated basing on the number of alleles per individuals and the average band sharing.     

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).     

Analysis of genetic diversity in the endangered Hucho taimen from China

Hucho taimen are listed as endangered in China. The population size has declined recently, prompting an increase in the level of listing from grade three in 2002 to grade five in 2006. We analyzed the genetic diversity of wild populations using 17 microsatellite markers to establish a scientific basis for conservation of this species. We collected tissue samples from four populations in the Heilongjiang River basin: Huma River (HM), Hutou (HT), Haiqing (HQ), and Zhuaji (ZJ). A total of 21 loci were amplified, 18 of which were polymorphic. The number of alleles per locus ranged from 2 to 9 (mean: 4.1905). There were 13 highly polymorphic loci and 5 moderately polymorphic loci. Analysis of five genetic diversity parameters (Na, Ne, Ho, He, and PIC) suggested moderate levels of diversity within the populations. The populations were ranked HT > HQ > ZJ > HM, but the differences in diversity were not statistically significant (P > 0.05). A comparison of variation among all four populations suggested Hardy–Weinberg disequilibrium at 20% of the loci. Genetic differentiation (Fst) was 0.0644 and the gene flow among populations was estimated at 3.36 individuals per generation. The majority of diversity (93.88%) occurred among individuals within a population. In contrast, relatively little (6.12%) of the genetic diversity was distributed between the populations. An analysis of genetic differentiation and genetic distance between pairs of populations revealed that both parameters were higher in comparisons of the HM population to the HT, HQ, and ZJ populations than among the three latter populations. This suggests that the HM population has a distinct genetic structure. We hypothesize that habitat degradation and excessive fishing, not low genetic diversity, has caused the decline in H. taimen populations. However, this species should be protected from further declines in genetic diversity.     

Development and characterization of 13 novel microsatellite loci from the flat-headed bat (Tylonycteris pachypus) with cross-species amplification in closely related taxa

13 novel microsatellite loci was isolated using the enriched library method from genomic DNA of the flat-headed bat (Tylonycteris pachypus). These loci showed high levels of genetic polymorphism testing on 54 individuals sampled from Guangxi province, China. The number of observed alleles per locus ranged from 2 to 15. The expected and observed heterozygosity values ranged from 0.170 to 0.900 and from 0.185 to 0.944, respectively. One loci departed significantly from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction and no Linkage disequilibrium was found between any pairs of loci. In addition, these loci were tested in the sister species, Tylonycteris robustula, seven loci amplified successfully and were also polymorphic.     

Low levels of quantitative and molecular genetic differentiation among natural populations of Medicago ciliaris Kroch. (Fabaceae) of different Tunisian eco-geographical origin

In this paper, we analyze the genetic variability in four Tunisian natural populations of Medicago ciliaris using 19 quantitative traits and six polymorphic microsatellite loci. We investigated the amplification transferability of 30 microsatellites developed in the model legume M. truncatula to M. ciliaris. Results revealed that about 56.66% of analyzed markers are valuable genetic markers for M. ciliaris. The most genetic diversity at quantitative traits and microsatellite loci was found to occur within populations (>80%). Low differentiations among populations at quantitative traits Q _ST  = 0.146 and molecular markers F _ST  = 0.18 were found. The majority of measured traits exhibited no significant difference in the level of Q _ST and F _ST . Furthermore, significant correlations established between these traits and eco-geographical factors suggested that natural selection should be invoked to explain the level of phenotypic divergence among populations rather than drift. There was no significant correlation between population differentiation at quantitative traits and molecular markers. Significant spatial genetic structure consistent with models of isolation by distance was detected within all studied populations. The site-of-origin environmental factors explain about 9.07% of total phenotypic genetic variation among populations. The eco-geographical factors that influence more the variation of measured traits among populations are the soil texture and altitude. Nevertheless, there were no consistent pattern of associations between gene diversity (He) and environmental factors.     

Isolation and characterization of microsatellite markers for <Emphasis Type="Italic">Cedrela odorata</Emphasis> L. (Meliaceae), a high value neotropical tree

We describe 9 primers for amplification of microsatellite loci for the Neotropical tree Cedrela odorata L. (Meliaceae). Loci were isolated from an enriched library derived from a single DNA sample from a tree in Costa Rica. Levels of polymorphism were determined using samples from a large progeny trial. Across loci, the number of alleles ranged from 14 to 30. Observed heterozygosity levels ranged from 0.61 to 0.88. No linkage disequilibria were detected although some departures from Hardy-Weinberg equilibrium (HWE) were found, probably due to a Wahlund effect.     

Isolation and characterization of 11 polymorphic microsatellite loci from <Emphasis Type="Italic">Fatsia polycarpa</Emphasis> (Araliaceae), an element of evergreen forests in Taiwan

Fatsia polycarpa Hayata (Araliaceae) is an element of evergreen forests at middle elevations in Taiwan, also representing the southern limitation in distribution of the genus. In this study, we described the development of 11 microsatellite loci from F. polycarpa in Taiwan for genetic studies. These new markers were tested in 20 individuals of 5 populations of the woody species. The number of alleles ranged from 9 to 18. The expected (H _E ) and observed (H _O ) heterozygosities were 0.80–0.95 and 0.10–0.75, respectively. Six of the 11 microsatellite loci are significantly deviated from Hardy–Weinberg expectations likely due to the population structure within samples. No linkage disequilibrium were observed from pairwise comparisons of loci. Chi-Chun Huang, Kuo-Hsiang Hung, and Tsai-Wen Hsu contributed equally to this article.     

Characterization of eight microsatellite loci in Sea Otter, Enhydra lutris, and cross-species amplification in other Mustelidae

Herein we describe the development of eight microsatellite markers for the northern sea otter, Enhydra lutris kenyoni. A total of 45 primer pairs were developed and screened from enriched AAAT, CATC, TACA, and TAGA libraries derived from genomic DNA of E. lutris kenyoni. Of these, eight amplified successfully. The average observed heterozygosity, expected heterozygosity, and number of alleles per locus was 0.506, 0.537, and 3.4, respectively. These eight loci were tested across three additional genra; Vulpes lagopus, Martes americana, and Mustela nivalis. Based on the success of our results these loci will be useful for future studies across all sub-species of E. lutris.     

Genetic analyses of the federally endangered Echinacea laevigata using amplified fragment length polymorphisms (AFLP)—Inferences in population genetic structure and mating system

Echinacea laevigata (Boynton and Beadle) Blake is a federally endangered flowering plant species restricted to four states in the southeastern United States. To determine the population structure and outcrossing rate across the range of the species, we conducted AFLP analysis using four primer combinations for 22 populations. The genetic diversity of this species was high based on the level of polymorphic loci (200 of 210 loci; 95.24%) and Nei’s gene diversity (ranging from 0.1398 to 0.2606; overall 0.2611). There was significant population genetic differentiation (G_ST = 0.294; Ө^II = 0.218 from the Bayesian f = 0 model). Results from the AMOVA analysis suggest that a majority of the genetic variance is attributed to variation within populations (70.26%), which is also evident from the PCoA. However, 82% of individuals were assigned back to the original population based on the results of the assignment test. An isolation by distance analysis indicated that genetic differentiation among populations was a function of geographic distance, although long-distance gene dispersal between some populations was suggested from an analysis of relatedness between populations using the neighbor-joining method. An estimate of the outcrossing rate based on genotypes of progenies from six of the 22 populations using the multilocus method from the program MLTR ranged from 0.780 to 0.912, suggesting that the species is predominantly outcrossing. These results are encouraging for conservation, signifying that populations may persist due to continued genetic exchange sustained by the outcrossing mating system of the species.     

Isolation and characterization of polymorphic microsatellite loci from a repeat-enriched genomic library of stone flounder (Kareius bicoloratus) and cross-species amplification

Stone flounder (Kareius bicoloratus) is a rare fish species in China. Here, we reported 11 polymorphic microsatellite loci isolated from a repeat-enriched genomic library of stone flounder. The number of alleles, observed and expected heterozygosity per locus in 32 individuals ranged from 3 to 6, from 0.1613 to 0.8667 and from 0.1549 to 0.7932, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Kareius bicoloratus. Yong-Sheng Tian and Gui-Dong Miao are contributed equally.