Novel tetranucleotide microsatellite markers from the Del Norte salamander (Plethodon elongatus) with application to its sister species the Siskiyou Mountain salamander (P. stormi)

Eleven tetranucleotide microsatellite loci were developed for the Del Norte salamander (Plethodon elongatus). The loci were variably polymorphic, ranging from two to 20 alleles per locus, with expected heterozygosities ranging from 0.07 to 0.86. The loci also amplified in a congener, the Siskiyou Mountain salamander (P. stormi). The microsatellite loci will be used to assess the utility of highly polymorphic markers to assay within‐ and between‐species differentiation between these two closely related species.     

Landscape genetics of the blotched tiger salamander (Ambystoma tigrinum melanostictum)

The field of landscape genetics has great potential to identify habitat features that influence population genetic structure. To identify landscape correlates of genetic differentiation in a quantitative fashion, we developed a novel approach using geographical information systems analysis. We present data on blotched tiger salamanders (Ambystoma tigrinum melanostictum) from 10 sites across the northern range of Yellowstone National Park in Montana and Wyoming, USA. We used eight microsatellite loci to analyse population genetic structure. We tested whether landscape variables, including topographical distance, elevation, wetland likelihood, cover type and number of river and stream crossings, were correlated with genetic subdivision (F(ST)). We then compared five hypothetical dispersal routes with a straight-line distance model using two approaches: (i) partial Mantel tests using Akaike's information criterion scores to evaluate model robustness and (ii) the BIOENV procedure, which uses a Spearman rank correlation to determine the combination of environmental variables that best fits the genetic data. Overall, gene flow appears highly restricted among sites, with a global F(ST) of 0.24. While there is a significant isolation-by-distance pattern, incorporating landscape variables substantially improved the fit of the model (from an r2 of 0.3 to 0.8) explaining genetic differentiation. It appears that gene flow follows a straight-line topographic route, with river crossings and open shrub habitat correlated with lower F(ST) and thus, decreased differentiation, while distance and elevation difference appear to increase differentiation. This study demonstrates a general approach that can be used to determine the influence of landscape variables on population genetic structure.     

Polymorphic tetranucleotide microsatellites for Cope's giant salamander (Dicamptodon copei) and Pacific giant salamander (Dicamptodon tenebrosus)

We present primers and amplification conditions for 15 microsatellite loci developed for the Cope's giant salamander (Dicamptodon copei), 14 of which are tetranucleotide repeats. Cross-species amplification revealed 10 of these loci to also be polymorphic in the Pacific giant salamander (Dicamptodon tenebrosus). Several loci produced nonoverlapping allelic ranges between the two species and may be useful in species identification. These polymorphic microsatellite loci are potentially useful for future studies of population genetics in dicamptodontid salamanders.     

Tetranucleotide microsatellites in the gecko Oedura reticulata isolated from an enriched library

Ten tetranucleotide microsatellite loci were isolated from an enriched library for the gecko Oedura reticulata. The species is endemic to the southwest of Western Australia, known to be a habitat specialist, and exposed to severe habitat fragmentation in the Western Australian wheatbelt. These highly polymorphic markers (two to 25 alleles) will facilitate the population genetic analyses of this species. In particular, they will enable estimates of gene flow between remnant populations — a critical element in assessing extinction dynamics in fragmented populations.     

The influence of altitude and topography on genetic structure in the long-toed salamander (Ambystoma macrodactulym)

A primary goal of molecular ecology is to understand the influence of abiotic factors on the spatial distribution of genetic variation. Features including altitudinal clines, topography and landscape characteristics affect the proportion of suitable habitat, influence dispersal patterns, and ultimately structure genetic differentiation among populations. We studied the effects of altitude and topography on genetic variation of long-toed salamanders (Ambystoma macrodactylum), a geographically widespread amphibian species throughout northwestern North America. We focused on 10 low altitude sites (< 1200 m) and 11 high-altitude sites in northwestern Montana and determined multilocus genotypes for 549 individuals using seven microsatellite loci. We tested four hypotheses: (1) gene flow is limited between high- and low-altitude sites; and, (2) gene flow is limited among high-altitude sites due to harsh habitat and extreme topographical relief between sites; (3) low-altitude sites exhibit higher among-site gene flow due to frequent flooding events and low altitudinal relief; and (4) there is a negative correlation between altitude and genetic variation. Overall F(ST) values were moderate (0.08611; P < 0.001). Pairwise F(ST) estimates between high and low populations and a population graphing method supported the hypothesis that low-altitude and high-altitude sites, taken together, are genetically differentiated from each other. Also as predicted, gene flow is more prominent among low-altitude sites than high-altitude sites; low-altitude sites had a significantly lower F(ST) (0.03995; P < 0.001) than high altitude sites (F(ST) = 0.10271; P < 0.001). Use of Bayesian analysis of population structure (BAPS) resulted in delineation of 10 genetic groups, two among low-altitude populations and eight among high-altitude populations. In addition, within high altitude populations, basin-level genetic structuring was apparent. A nonequilibrium algorithm for detecting current migration rates supported these population distinctions. Finally, we also found a significant negative correlation between genetic diversity and altitude. These results are consistent with the hypothesis that topography and altitudinal gradients shape the spatial distribution of genetic variation in a species with a broad geographical range and diverse life history. Our study sheds light on which key factors limit dispersal and ultimately species' distributions.     

Microsatellite markers isolated from the wild medicinal plant Centella asiatica (Apiaceae) from an enriched genomic library

? Premise of the study: Microsatellite markers for Centella asiatica, an important medicinal herb, were developed and characterized to promote genetic and molecular studies. ? Methods and Results: A GA/GT-enriched genomic library was constructed from an accession from Madagascar. Roughly 75% of the 768 clones of the enriched library contained microsatellites. Eighty sequences containing microsatellites were obtained from 96 positive clones. Specific primers were designed for 20 loci, and 17 of them displayed polymorphism when screened across 17 C. asiatica accessions, with an average of 4.3 alleles per locus. The observed and expected heterozygosity values averaged 0.114 and 0.379, respectively. ? Conclusions: This is the first report constructing an enriched genomic library and identifying microsatellite markers from C. asiatica. These 17 polymorphic microsatellite markers are a useful resource for this plant, applicable for diversity studies, pedigree analyses, and genetic mapping.     

Isolation of dinucleotide microsatellite loci from red‐backed salamander (Plethodon cinereus)

We isolated 13 variable dinucleotide microsatellites from red‐backed salamanders (Plethodon cinereus). After generating fragments using degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR), AC repeats were captured using biotinylated probes and streptavidin‐coated magnetic particles. Captured fragments were cloned into plasmids, screened for microsatellites with a simple PCR reaction, and select plasmids then sequenced. PCR primers were designed and optimized for robust amplification, and nine primers have been further optimized for multiplex reactions with fluorescent primers. These nine loci are variable with an average of 6.11 alleles per locus and an average heterozygosity of 0.61.     

Development of microsatellite markers in polyploid persimmon (Diospyros kaki Lf) from an enriched genomic library

The oriental persimmon (Diospyros kaki Lf) is believed to have originated in China with subsequent introduction into Japan and Korea in ancient times. The species was then brought to Europe, Brazil and the USA from Japan in the 19th century. Recent studies highlighted the poor state of identification of cultivars in these countries due to incorrect labelling and presence of synonyms among local varieties. Thus, molecular marker characterization of germplasm resources is of great value for genetic resource preservation and plant breeding of persimmon. Therefore, to identify accessions for further plant breeding and germplasm management, 37 microsatellite loci were developed from a CT/AG‐enriched persimmon genomic library.     

Eleven new microsatellite markers in jack mackerel (Trachurus japonicus) derived from an enriched genomic library

Jack mackerel (Trachurus japonicus, Carangidae) are a commercially important fisheries resource in Korea. To understand patterns of genetic variation for conservation and management efforts, we developed microsatellite DNA markers fromT. japonicus. We report the isolation and characterization of eleven microsatellite loci isolated using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. To characterize each locus, 50 individuals from a naturalT. japonicus population in southern Korea were genotyped. All loci except one, KTJ38, were polymorphic with an average of 14 alleles per locus (range 6–23). The mean observed and expected heterozygosities were 0.70 (range 0.46–0.92) and 0.81 (range 0.49–1.00), respectively. Significant deviation from Hardy-Weinberg equilibrium was observed at three loci, KTj3, KTJ20 and KTJ28. Such high variability indicates that these microsatellites are useful markers for high-resolution analysis for population gemetic studies.     

New microsatellite markers in turbot (Scophthalmus maximus) derived from an enriched genomic library and sequence databases

Scophthalmus maximus is an important commercially aquaculture fish species. We tackle the search for new microsatellites using two different approaches: an enriched partial genomic library and a screening of all turbot DNA sequences deposited in GenBank. Out of 15 genomic library derived loci, five gave working primer pairs, with expected heterozygosities (H_E) ranging from 0.13 to 0.91. Out of seven loci derived from database sequences, two amplified successfully with an H_E ranging from 0.56 to 0.63. The average allele number of these microsatellites was 5.7 per locus with a range between two and 15.     

患病中国大鲵中分离到一株虹彩病毒及其特性的研究

从陕西某大鲵养殖场患病的大鲵体内分离到一株病毒。患病大鲵以体表溃疡,特别是肢体远端溃烂为主要临床特征。该病毒于10℃~30℃能在BF-2(Caudal trunk cells of blue-gillfry)、CO(Gorad cells of grass carp)、CHSE(Embryo cells of Chinook salmon)、FHM(cells of fathed minnow)等细胞中较好地增殖,最适生长温度为25℃~30℃。病毒对氯仿、热、pH3、pH10敏感,DNA抑制剂5-氟-2′-脱氧尿苷(5-fluro-2-′deoxyuridine,FUDR)能抑制病毒在细胞中的增殖,提示该病毒是有囊膜的DNA病毒。经电镜观察,在感染了病毒的细胞切片中可见到大量直径约130~150 nm有囊膜的六角形病毒颗粒成晶格排列在细胞质里,病毒呈典型的虹彩病毒形态。抽提病毒核酸后进行PCR扩增,用已知蛙病毒主要衣壳蛋白(MCP)基因的保守序列设计的引物能扩增出431bp的片段。扩增的片段测序后,和已知的几种蛙病毒属成员的主要衣壳蛋白基因中的相应片段进行比对,相似性在96%以上。血清学试验结果显示该病毒和IPNV(Infectious pancreatic necrosis virus,IPNV)、GCRV(Grass carp reovirus,GCRV)、SVCV(Spring viraemia of carp virus,SVCV)I、HNV(Infectious hematopoietic necrosis virus,IHNV)在血清学上没有相关性。以上结果提示该病毒可能是虹彩病毒科蛙病毒属的成员,暂时命名为大鲵虹彩病毒(Andrias davidianus iridovirus,ADIV)。该病毒与大鲵发病的关系有待进一步研究。     

Multiplex genotyping of novel tetranucleotide microsatellites from a marine foodfish species crimson red snapper (Lutjanus erythropterus)

Crimson red snapper (Lutjanus erythropterus) is an important marine foodfish species in Asia with great potentials for aquaculture. We isolated nine polymorphic microsatellites, among which eight were tetranucleotide repeats, and one was CA‐microsatellite. The average allele number present in 48 individuals was 11.1/locus, ranging from three to 33. The expected heterozygosity ranged from 0.49 to 0.97 with an average of 0.74. Six of the nine markers conformed to the Hardy–Weinberg expectations. No pairwise markers showed the possibility of linkage. Protocols for two multiplex polymerase chain reactions (PCRs), each amplifying two and seven markers, respectively, were presented. The developed microsatellites and optimized multiplex PCRs will be useful for studying population structure of wild stocks and parentage assignment for cultured populations.     

Polymorphic tetranucleotide microsatellite DNA loci from the southern dusky salamander (Desmognathus auriculatus)

We describe polymerase chain reaction (PCR) primers and amplification conditions for seven tetranucleotide microsatellite DNA loci isolated from the southern dusky salamander (Desmognathus auriculatus). Primers were tested on 16 individuals from one population in Aiken County, South Carolina. We detected an average of 6.57 alleles per locus, an observed heterozygosity range of 0.44–0.94, and high polymorphic information contents (mean of 0.68).     

Isolation and strategies of novel tetranucleotide microsatellites with polymorphisms from different chromosomes of the rhesus monkey (Macaca mulatta)

A total of 45 tetranucleotide chromosome-specific microsatellite markers with polymorphism were developed successfully based on three reference rhesus monkey genomes and on In-silico PCR prescreening. The polymorphic information content (PIC) values of 45 polymorphic microsatellite loci ranged from 0.487 to 0.879, with an average of 0.715, which were proven to be moderate to highly polymorphic. We detected 315 alleles on 45 microsatellite loci in 24 Rhesus monkeys. The number of alleles ranged from 3 to 15 and the mean number of alleles was 7 for each locus. Accordingly, the observed and expected heterozygosities obtained were between 0.417 and 1.0 and between 0.550 and 0.908, with an average value of 0.736 and 0.767, respectively. Genetic information demonstrated that 10 loci significantly deviated from Hardy–Weinberg equilibrium (P?<?0.05). All 45 primers were not significant with regard to linkage disequilibrium (P?>?0.001). Pearson correlation indicated that the PIC value exhibited a significant negative correlation with the loci number (r?=?? 0.741, P?=?0.022), whereas the positive correlation with the number of the samples (r?=?0.847, P?=?0.070) was not significant. This may be attributed to the presence of random particularities within the loci. The T test of the sample groups indicated that the PIC difference was not significant when the number of samples was set at 10 and/or?≥?15 (P?=?0.7472?~?0.8564). These polymorphic and valuable microsatellite loci will facilitate further conservation genetics studies for rhesus monkeys and can be further applied to develop novel genetic markers for other species.

     

Eight new microsatellite markers in Atlantic cod (Gadus morhua L.) derived from an enriched genomic library

One hundred and fifty random clones from an enriched genomic library of Atlantic cod were sequenced. Primer pairs were designed for 15 microsatellites containing perfect di‐, tri‐, tetra‐ and hexanucleotide repeats and characterized in 96 unrelated fish. Eight markers were successfully amplified, with the number of alleles ranging from two to nine per locus and observed heterozygosity ranging from 0.341 to 0.977. Loci Gmo‐G13 and Gmo‐G14 had a significant excess of homozygotes. All loci conformed to the Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

Isolation, characterization and cross-species testing of microsatellites obtained from a sand smelt (Atherina boyeri) genomic library

This study reports the isolation and characterization of 11 polymorphic microsatellites from a sand smelt (Atherina boyeri) genomic library. Enrichment was performed with di-, tri- and tetranucleotide motifs following the FIASCO procedure (fast isolation by AFLP of sequences containing repeats). All loci were found to be in linkage and in Hardy-Weinberg equilibrium. This represents the first microsatellite isolation for the family Atherinidae and the isolated loci were accordingly tested on four additional species of the family: two recognized (A. presbyter and A. hepsetus) and two proposed ('punctata' and 'non-punctata' forms). Moreover their cross-species suitability on Menidia menidia, belonging to the same order but to the family Atherinopsidae, was also tested.     

Tandem repeat markers for population genetic studies of the protected California tiger salamander (<Emphasis Type="Italic">Ambystoma californiense</Emphasis>)

Habitat loss is the single greatest threat to persistence of the critically threatened California tiger salamander (Ambystoma californiense). To aid management plans that designate critical habitat for this species, I developed and characterized 21 tetranucleotide microsatellite markers using two native populations in Santa Barbara and Alameda Counties. Allelic variation and average heterozygosities were lower in the endangered Santa Barbara population (allele range 1–4, mean 2.4; H _O = 0.308 H _E = 0.288) compared with the threatened Alameda population (allele range 2–10, mean 6.7; H _O = 0.712, H _E = 0.722). In-depth population studies using these markers will provide vital information for plans to assign critical habitat that optimize gene flow among breeding populations, as well as for identifying non-native hybrid genotypes that threaten native A. californiense stocks. Beyond the conservation goals for A. californiense, the close phylogenetic relationships within the tiger salamander complex also suggest a broad utility for population studies using these markers.     

Development of microsatellite markers from an enriched genomic library for genetic analysis of melon (Cucumis melo L.)

Background  

Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species.