Isolation and characterization of microsatellite markers for the endangered <Emphasis Type="Italic">Taxus yunnanensis</Emphasis>

Eleven polymorphic microsatellite loci were characterized for the endangered conifer Taxus yunnanensis. Eight loci were isolated through SSR-anchored PCR, one locus was developed by cross-species amplification tests, while the last two loci were obtained from cross-species microsatellite sequences available in GenBank. Variability of these markers was tested in 48 individuals collected from its main distribution range in Southwest China. The number of alleles per locus ranged from 2 to 9, and the observed and expected heterozygosity varied from 0.000 to 0.625 and from 0.062 to 0.853, respectively. Ten of these eleven loci were significantly deviated from Hardy–Weinberg expectation, except TS07 which showed a distinct heterozygote excess. The availability of these new polymorphic microsatellite markers will provide an ideal marker system for detailed population genetics studies in T. yunnanensis and potentially also for closely related species.     

Polymorphic microsatellites in the Reeves's pheasant developed by cross-species amplification

Cross-species microsatellite amplification is an effective way of obtaining microsatellite loci for closely related taxa in bird species. The Reeves's pheasant, Syrmaticus reevesii, is a vulnerable species endemic to China. To improve population genetics and parentage analysis studies in this species, we obtained nine polymorphic microsatellite markers, in addition to the nine markers previously isolated, from the cross-species amplification of 52 markers. The number of alleles per locus varied between two and 12 with expected heterozygosity ranging from 0.298 to 0.714 (n = 107). The success rates of simulated paternity tests using CERVUS software improved at different confidence levels after adding these markers to the previous ones.     

Isolation and characterization of 23 microsatellite loci in the black-faced spoonbill (Platalea minor) and amplification in other Ciconiiformes waterbirds

We isolated 23 polymorphic microsatellite loci of tetranucleotide, dinucleotide or compound repeat motif in the black-faced spoonbill (Platalea minor). In a panel of up to 20 individuals, number of alleles per locus ranged from two to 19 (mean = 8) and heterozygosity from 0.278 to 0.950 (mean = 0.606). Allele frequencies of five loci deviated from that expected under Hardy–Weinberg Equilibrium, and one of them was likely Z-linked. Excluding these loci, the remaining 18 loci should provide a sufficient set of molecular markers for use in ecological and conservation genetic investigation of this globally endangered wader species, and cross-species amplification test suggests that they are potentially useful in other Ciconiiformes waterbirds.     

High degree of transferability of 86 newly developed zebra finch EST-linked microsatellite markers in 8 bird species

High-resolution analysis for population genetic and functional studies requires the use of large numbers of polymorphic markers. The recent increase of available genetic tools is facilitated by the use of publicly available expressed sequence tag (EST) sequence databases that are a valuable resource for identifying gene-linked markers. In the present study, we applied bioinformatics analyses to identify microsatellite markers present in EST sequences from a zebra finch (Taeniopgia guttata) EST database and we explore the success of cross-species amplification of EST-linked microsatellite markers in 7 passerine and 1 nonpasserine species. Eighty-six zebra finch EST-linked microsatellite loci were screened for polymorphism revealing a high amplification success rate and adequate levels of polymorphism (33.3-51%) for relatively closely related species, whereas success decreased in the most distantly related species to zebra finch. EST-linked microsatellites appear to be more highly transferable between taxa than anonymous microsatellites as they revealed higher amplification and polymorphism success between different families indicating that they will be a useful source of gene-linked polymorphic markers in a broad range of avian species.     

A wide-range survey of cross-species microsatellite amplification in birds

The possibility to perform cross-species microsatellite amplification in birds was surveyed by analysing sets of primers developed from the swallow and the pied flycatcher genomes on a panel of 48 different bird species. In total, 162 cases (species/marker combinations) of heterologous amplification were recorded. Ten amplification products were sequenced and all were found to be true homologues of the original loci. There was a significant and negative relationship between microsatellite performance and evolutionary distance between the original species and the tested species. As a rough indicator of expected cross-species microsatellite performance we estimate that 50% of markers will reveal polymorphism in a species with a DNA-DNA hybridization δT_mH value of 5 separating it from the original species. This corresponds to a divergence time of = 11 million years before present for passerine birds. The established relationship between performance and evolutionary distance agrees very well with data obtained from some mammalian species. The proportion of polymorphic loci among those markers that amplified decreased with increasing genetic distance, suggesting that few long repeats are preserved during evolution. One of the swallow markers, HrU2, amplified a specific product in all species analysed and will thus allow access to nuclear sequence data over a broad range of species. The only predictor of cross-species performance was the amount of non-specific amplification seen in the original species. An analysis of 10 species from within the family Hirundinidae with the swallow primers consistently revealed extensive polymorphism with average probabilities of identical genotypes ranging from 6 times 10^-4 to 6 times 10^-7. There were distinct allele frequency differences between the Hirundinidae species and we envisage that microsatellite cross-species amplification will be a useful tool in phylogeny construction and in species identification.     

Development of microsatellite DNA loci from the wood stork (Aves,Ciconiidae, Mycteria americana)

We isolated 11 polymorphic microsatellite loci for wood stork (Mycteria americana). Polymerase chain reaction (PCR) primers and conditions are described for the amplification of five dinucleotide, one trinucleotide and five tetranucleotide microsatellite loci. The PCR primers were tested on two wood stork populations, Fazenda Ipiranga, Mato Grosso, Brazil (n = 11) and Tamiami West, Everglades, Florida, USA (n = 20). The primers yielded two to four alleles per locus, an observed heterozygosity of 0.0–0.727 and a polymorphic information content of 0.048–0.604. The low level of polymorphism for these markers is consistent with previous studies of this species.     

Isolation and characterization of 15 microsatellite loci from mango (Mangifera indica L.) and cross‐species amplification in closely related taxa

We report here on the development and characterization of 15 microsatellite loci isolated from Mangifera indica L. These markers were evaluated using 59 Florida cultivars and four related species from the USDA germplasm collection for mango. Two loci were monomorphic and 13 polymorphic, with two to seven alleles per locus. Four loci departed significantly from Hardy–Weinberg equilibrium and have significant heterozygote deficiency. Nine loci exhibited significant linkage disequilibrium. Cross‐species amplification was successful in four related species. These loci are being used to investigate patterns of genetic variation within M. indica and between closely related species.     

Utility of pairwise mtDNA genetic distances for predicting cross-species microsatellite amplification and polymorphism success in fishes

In many studies involving microsatellites cross-species amplification, primers designed for one (source) species are used to amplify homologous loci in related (target) species. However, it is not clear how closely related the species must be to attain significant success. Genetic divergence is a clear and easy way to assess similarity between species and provides an accurate measure of their evolutionary distance. Eight Mediterranean target species of the family Serranidae were analysed using twelve primers developed for Serranus cabrilla. Additionally, two mitochondrial genes (12S rRNA and 16S rRNA) were chosen on the basis of their extensive use in phylogenetic and evolutionary analyses to compute genetic divergence between the species. Significant negative correlations were found between genetic divergence and both cross-species amplification and maintained polymorphism of microsatellite markers, which could be generalized by gathering information from different fish studies. The success of obtaining amplifiable and polymorphic microsatellite loci can be a priori approximated knowing the mtDNA genetic divergence between a given source and target species using our inferred regression equations. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development and characterization of microsatellite loci in Chinese medicinal plant Akebia trifoliate ssp. australis and cross-species amplification in closely related taxa

Eleven polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of Akebia trifoliate ssp. australis. The number of allele per locus ranged from 3 to 14. The observed heterozygosity and expected heterozygosity at population level were 0.196–1.000 and 0.522–0.902, respectively. In addition, this set of microsatellites produced robust cross-species amplification in other two related taxa, suggesting these microsatellite markers should provide a useful tool for genetic and conservation studies of the Akebia species.     

Characterization of polymorphic microsatellite loci for the Tawny Pipit Anthus campestris and their utility in other steppe birds

New microsatellite loci for Tawny Pipit Anthus campestris were isolated from a genomic library. We were able to unambiguously score six loci: two were dinucleotide, one trinucleotide, two tetranucleotide and one pentanucleotide that turned out to be sex-linked. Four out of six loci were polymorphic with 7–23 alleles in our population and an observed heterozygosity ranging between 0.286 and 0.936. Cross-utility of these markers was tested in other 17 steppe-bird species of six families. In addition, 16 microsatellite loci developed for other species were tested for cross-species amplification in A. campestris. Eight microsatellite markers were successfully amplified; seven of them were polymorphic with 2–43 alleles and an observed heterozygosity of 0.040–0.863. Overall, 14 functional locus markers have been characterized for A. campestris that could be useful for future studies of paternity, genetic variability and population structure.     

Novel Polymorphic Microsatellite Markers for Panulirus ornatus and their Cross-species Primer Amplification in Panulirus homarus

Polymorphic microsatellite loci were isolated for Panulirus ornatus using 454 GS-FLX Titanium pyrosequencing. Fifteen markers containing perfect di-, tri-, tetra-, and penta-nucleotide motifs were consistently co-amplified in five multiplexes in a panel of 91 randomly selected samples. Observed number of alleles varied from 2 to 14 per locus. Observed and expected heterozygosity ranged from 0.090 to 0.79 and 0.08 to 0.87, respectively. Ten loci deviated from Hardy-Weinberg equilibrium after sequential Bonferroni correction. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between 11 loci. The microsatellite markers were also amplified successfully in related Panulirus homarus species with adequate level of polymorphism. The successful cross-species primer amplification of the 15 microsatellites indicates the potential of the developed markers to be transferred to other Panulirus species. The 15 novel microsatellite markers reported in this work add to the previously characterized markers by our group, exhibit adequate levels of polymorphism for wide range of future studies investigating population structure, genetic diversity, and evolutionary relationships among Panulirus species.     

微卫星跨物种交叉PCR扩增的一个新问题:扩增非同源产物

微卫星已被广泛应用于群体遗传学、生态学和进化生物学研究。然而,一些物种微卫星尚未克隆。为了节省时间和经费,研究人员往往使用一个物种已发表的微卫星引物扩增其近缘物种的微卫星。该研究对属于3个不同科(Clariidae、Heteropneustidae 和Pimelodidae)的7个鲶鱼物种的微卫星跨物种PCR扩增产物进行了序列分析,研究发现扩增非同源（non-orthologous）产物是微卫星跨物种PCR扩增的一个新问题。该研究共采用4对胡子鲶微卫星座位引物对7个鲶鱼物种进行了跨物种PCR扩增。对获得的204个PCR产物的序列分析结果表明,两对微卫星座位引物扩增了所有7个物种的同源特异产物。而其他两个座位的引物扩增了特异但非同源的多态产物,对近缘物种的扩增也获得类似结果。另外,除胡子鲶等位基因大小异源同型(size homoplasy)的特征不明显外,其他物种在3个微卫星座位都具有这一非常明显的特征。这些数据表明,微卫星跨物种间交叉扩增能产生非同源产物;等位基因大小异源同型与微卫星座位本身有关,而与物种间的亲缘关系无明显的相关性。微卫星跨物种扩增产生的非同源产物和等位基因大小异源同型将使系统发育、群体遗传学和进化研究明显复杂化。因此,在应用微卫星跨物种交叉扩增数据以前,最好对跨物种交叉扩增产物进行测序验证。     

Microsatellite DNA in Actinidia chinensis: isolation, characterisation, and homology in related species

 We have isolated and sequenced 263 microsatellite-containing clones from two small insert libraries of Actinidia chinensis enriched for (AC/GT) and (AG/CT) repeats, respectively. Primer pairs were designed for 203 microsatellite loci and successfully amplified from both plasmid and A. chinensis genomic DNA. In this paper we report the sequences of 40 primer pairs for which we have demonstrated Mendelian segregation in the progeny from controlled crosses. The polymorphism of ten microsatellites of each type was evaluated in four diploid and six tetraploid genotypes of A. chinensis. All microsatellites proved to be polymorphic, the number of alleles per locus detected in polyacrylamide sequencing gels ranging from 9 to 17. The high degree of polymorphism in Actinidia renders these markers useful either for mapping in A. chinensis or for fingerprinting cultivars of both domesticated kiwifruit species (A. chinensis and A. deliciosa). While most primer pairs produced single amplification products, about 20% generated banding patterns consistent with the amplification of two different loci. This supports the hypothesis that diploid species of Actinidia (2n=2x=58) are polyploid in origin with a basic chromosome number x=14/15 and that chromosome duplication may have occurred during the evolution of the genus. Finally, we have assayed the cross-species transportability of primer pairs designed from A. chinensis sequences and have found extensive cross-species amplification within the genus Actinidia; 75% of primer pairs gave successful amplification in the eight species assayed (A. arguta, A. rufa, A. polygama, A. chrysantha, A. callosa, A. hemsleyana, A. eriantha, and A. deliciosa), which are representative of the four sections into which the genus is currently split. Received: 14 February 1998 / Accepted: 26 May 1998     

Multiplex panels of polymorphic microsatellite loci for two cryptic bat species of the genus Pipistrellus,developed by cross‐species amplification within the family Vespertilionidae

The paper presents multiplex panels of polymorphic microsatellites for two closely related cryptic species Pipistrellus pipistrellus and Pipistrellus pygmaeus. We tested the cross‐species amplification of 34 microsatellite loci, originally developed for five vespertilionid bat species. Ten and nine polymorphic loci in P. pipistrellus (mean number of alleles per locus = 10.5) and P. pygmaeus (8.1), respectively, in three multiplex polymerase chain reactions per species were amplified. All loci can be analysed in a single fragment analysis and can be used as markers to the study of evolution and the ecology of structured populations of socially living bats.     

Development of polymorphic microsatellite markers in barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) by the cross-species amplification

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two commercially important flatfish species in the Northeast Asia. In the present study, we reported polymorphic microsatellite markers in V. moseri and V. variegatus by the cross-species amplification of microsatellite primers developed previously in two other related marine fish species. A total of 244 polymorphic microsatellite markers were selected for cross-species amplification in V. moseri and V. variegatus, of which 182 markers deriving from Atlantic halibut (Hippoglossus hippoglossus) and 62 markers deriving from Japanese flounder (Paralichthys olivaceus). A sample of 10 individuals were detected. As a result, a total of 67 loci showed polymorphisms in V. moseri, and 62 loci showed polymorphisms in V. variegatus, with the observed number of alleles per locus ranging from two to five in V. moseri, and from two to seven in V. variegatus, respectively. This paper provided more candidate microsatellite markers which could be useful for construction of genetic linkage maps, evaluation of population genetic structure and stock management of V. moseri and V. variegatus.     

Characterization of microsatellite DNA markers for a mahseer species,Tor tambroides (Cyprinidae) and cross‐amplification in four congeners

This study reports the isolation and characterization of microsatellite DNA markers in a mahseer species, Tor tambroides (Pisces, Cyprinidae). Of a total of 14 loci evaluated, 10 were polymorphic in T. tambroides samples, with an average of 2.86 alleles per locus. Deviations from Hardy–Weinberg equilibrium were observed at one locus and there was no indication of linkage disequilibrium among loci. A high level of cross‐amplification among four congeners was achieved, with 12 loci successfully amplifying and 11 loci showing polymorphism in at least one other species. These markers will be a useful resource for population genetic studies and broodstock management of closely related mahseer species.     

Characterization of novel microsatellite loci in rare minnow (<Emphasis Type="Italic">Gobiocypris rarus</Emphasis>) and amplification in closely related species in Gobioninae

Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2–7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose.     

A set of microsatellite markers for population genetics of leopard cat (Prionailurus bengalensis) and cross-species amplification in other felids

We describe the isolation and characterization of novel microsatellite loci from the leopard cat, Prionailurus bengalensis Kerr, 1792 (Family Felidae). Using Illumina HiSeq2500 sequencing technology, we sequenced the leopard cat genome and identified 1.5 million loci of simple sequence repeats with di- to deca-nucleotide motifs. We developed twelve polymorphic markers with tetra-nucleotide motif types after screening 35 loci for amplification and polymorphism. The observed and expected heterozygosities of the markers were 0.438 and 0.423, respectively. The number of alleles per locus ranged from 2 to 7, with a mean polymorphism information content of 0.383. Eleven loci were at Hardy-Weinberg equilibrium and no linkage disequilibrium was detected among any pairs of loci. We tested cross-species amplification of these markers across five other felids (Panthera tigris, P. pardus, P. onca, Acinonyx jubatus, and Felis catus). All loci were transferable to at least one other feline species and four amplified all five species. The microsatellite markers developed in this study will be valuable for estimating ecological parameters of populations and to establish conservation and management strategies for feline species.     

Development and characterization of eleven microsatellite markers for an endangered cavefish (Triplophysa rosa Chen and Yang, 2005) using 454 sequencing

Triplophysa rosa is an endangered cave‐dwelling and endemic fish species found only in Chongqing, southwestern China. The genetic data available for this species is very limited. Polymorphic microsatellites were identified in the genome of T. rosa using 454 sequencing. Of the 145 loci screened, 106 were amplified successfully and 11 showed polymorphic patterns. The number of alleles ranged from 2 to 6, and the observed and expected heterozygosity varied from 0.061 to 0.543 (mean = 0.349) and from 0.248 to 0.789 (mean = 0.551), respectively. The polymorphic information content (PIC) ranged from 0.215 to 0.744 (mean = 0.486), indicating moderate levels of polymorphism. In addition, cross‐species amplification was tested for the 106 loci in Triplophysa moquensis, which showed a high level of transferability (76.4%), implying that the microsatellite markers developed here could be used effectively for other closely related species.