Development and characterization of 24 chloroplast microsatellite markers for two species of <Emphasis Type="Italic">Eranthis</Emphasis> (Ranunculaceae)

Chloroplast microsatellites for two Korean endemic species, Eranthis byunsanensis and E. pungdoensis (Ranunculaceae), were isolated to address the questions of their distributional patterns and evolutionary relationships, using next-generation sequencing. Twenty-four polymorphic chloroplast microsatellite markers for these two species were developed, and then characterized in 65 individuals (55 individuals of E. byunsanensis and 10 individuals of E. pungdoensis). The number of alleles per locus ranged from 2 to 9; the average number of alleles across all the loci scored 4.792. The unbiased diversity per locus ranged from 0.089 to 0.880; the unbiased diversity averaged over all the loci was 0.646. The developed markers were successfully amplified for three congeneric species, E. stellata, E. pinnatifida, and E. longistipitata. The markers developed in this study can provide a valuable and important tool for understanding genetic variations, population structures, evolutionary histories and phylogeography of E. byunsanensis, E. pungdoensis, and related species.     

De novo development and characterization of polymorphic microsatellite markers in a schilbid catfish, <Emphasis Type="Italic">Silonia silondia</Emphasis> (Hamilton, 1822) and their validation for population genetic studies

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity–capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6–24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall F_ST = 0.075; P < 0.0001) with maximum variation (92.5 %) within populations. Cross-priming assessment revealed successful amplification (35–38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.     

Development of 21 polymorphic microsatellite markers for the black-banded sea krait, <Emphasis Type="Italic">Laticauda semifasciata</Emphasis> (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species

The genus Laticauda (Reptilia: Elapidae), commonly known as sea kraits, is venomous marine amphibious snakes distributed throughout the south and southeast Asian islands and mostly found in coastal waters. To facilitate genetic studies, we have developed microsatellite loci for L. semifasciata using the 454 GS-FLX pyrosequencing technique. A total of 65,680 sequences containing a minimum of five repeat motifs were identified from 451,659 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5%) produced strong PCR products, of which 21 were polymorphic among 36 samples of L. semifasciata. All loci exhibited high genetic variability, with an average of 7.38 alleles per locus, and the mean observed and expected heterozygosities were 0.73 and 0.76, respectively. The cross-species amplification of these loci in two laticaudine species, L. colubrina and L. laticaudata, revealed a high transferability (78.6%) and polymorphism (59.5%) of the loci. Our work demonstrated the utility of next-generation 454 sequencing as the rapid and cost-effective method for development of microsatellite markers. The high level of polymorphism in these microsatellite loci will be useful for the detection of population subdivision and the study of migration, gene flow, relatedness and philopatry of L. semifasciata and other laticaudine species.     

Isolation and characterization of 20 polynucleotide microsatellite markers in a vulnerable Korean snail, <Emphasis Type="Italic">Ellobium chinense,</Emphasis> using 454 pyrosequencing

A small and air-breathing snail, Ellobium chinense (Ellobiidae), is a vulnerable species by International Union for Conservation of Nature (IUCN). To protect and manage habitat and population of E. chinense, microsatellite markers were developed using 454 pyrosequencing and 20 polymorphic microsatellite markers were identified. A total of 146,704 sequences containing a minimum of four repeat motifs (mean, 631 base pairs) were identified from 499,505 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5 %) produced strong PCR products, of which 20 were polymorphic among 48 samples of E. chinense. All loci exhibited high genetic variability, with an average of 18.9 alleles per locus, and the mean observed and expected heterozygosities were 0.65 and 0.90, respectively. In addition, cross-amplification was tested for all 20 loci in the same family species, Melampus sincaporensis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 20 newly developed microsatellites will be useful in future conservation genetic studies of this species.     

Novel polymorphic microsatellite loci for distinguishing rock bass (<Emphasis Type="Italic">Ambloplites rupestris</Emphasis>), Roanoke bass (<Emphasis Type="Italic">Ambloplites cavifrons</Emphasis>), and their hybrids

The rock bass (Ambloplites rupestris) is a popular sport-fish native to the Mississippi and Great Lakes basins of North America. The species has been widely introduced outside its native range, including into Atlantic-slope streams of Virginia where it may hybridize with an imperiled, similar-looking congener, the Roanoke bass (Ambloplites cavifrons). In this study, we identified and evaluated novel molecular markers to facilitate identification of these species and study the extent of hybridization. Using molecular libraries developed from A. rupestris, we identified a suite of candidate nuclear microsatellite loci, synthesized primer sets, and tested these markers for amplification and polymorphism in populations of both species. We then calculated standard diversity statistics within and differentiation statistics between species, the latter providing an indication of marker power for distinguishing the species and their hybrids. Additionally, we evaluated our efficiency for identifying hybrids by classifying simulated genotypes of known ancestry. Eleven loci were polymorphic (2–22 alleles per locus) and reliably amplified in both species. Multilocus genetic differentiation between A. cavifrons and A. rupestris was quite high (F _ST  = 0.66; D _LR  = 19.3), indicating the high statistical power of this marker set for species and hybrid identification. Analyses of simulated data suggested these markers reliably distinguish between hybrids and non-hybrids, as well as between F1 hybrids and backcrossed individuals. This panel of 11 loci should prove useful for understanding patterns of hybridization between A. rupestris and A. cavifrons. As the first microsatellite markers developed for Ambloplites, these markers also should prove broadly useful for population genetic studies of this genus.     

Variability of microsatellite loci in <Emphasis Type="Italic">Vincetoxicum</Emphasis> Wolf species in southeastern Ukraine

Genetic diversity of 13 species of the genus Vincetoxicum Wolf found in Ukraine with the use of four of eight nuclear microsatellite markers previously developed for Vincetoxicum atratum from Japan was studied. The number of alleles in studied loci varied in the range from 8 to 25. The expected heterozygosity was 0.690–0.938; the observed heterozygosity varied in the range from 0.205 to 0.806. The total rate of genetic variability of studied species was found to be comparable to the rate of variability of Vincetoxicum atratum from Japan. Microsatellite loci Vinc5, Vinc104, Vinc123, and Vinc124 can be successfully used for estimating the intra- and interspecific polymorphism of the species of genus Vincetoxicum Wolf in Ukraine.     

Isolation and Characterization of Microsatellite Markers for <Emphasis Type="Italic">Viola websteri</Emphasis> (Violaceae) and Cross-Species Amplification within the Genus <Emphasis Type="Italic">Viola</Emphasis>

We isolated and characterized microsatellite loci in Viola websteri (Violaceae), an endangered species from Korea and endemic to Northeast Asia. A total of 27 microsatellite loci were developed and tested in Korean and Chinese populations. The number of alleles per locus varied from two to eight. The observed and expected heterozygosities within two populations were 0.000 to 1.000 and 0.080 to 0.816, respectively. Korean and Chinese populations were clearly distinguished by the private alleles from 16 loci. A total of 21 loci out of the 27 developed loci were successfully cross-amplified in 39 other Viola species. We believe that these microsatellite loci will be useful for future studies on genetic diversity and population structure of V. websteri, as well as other Viola species.     

New microsatellite markers developed from <Emphasis Type="Italic">Urochloa humidicola</Emphasis> (Poaceae) and cross amplification in different <Emphasis Type="Italic">Urochloa</Emphasis> species

Background

Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.

Conclusions

This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

     

First microsatellite markers for <Emphasis Type="Italic">Paspalum plicatulum</Emphasis> (Poaceae) characterization and cross-amplification in different <Emphasis Type="Italic">Paspalum</Emphasis> species of the Plicatula group

Background

Paspalum plicatulum is a perennial rhizomatous grass with natural diploid and polyploid cytotypes. It is a member of Plicatula, which has historically been recognized as a highly complex group containing species of ecological, ornamental and forage importance. The complex nature of the P. plicatulum genome makes it a challenging species for genetic research. This study aimed to develop and characterize microsatellite molecular markers in P. plicatulum and to evaluate their transferability to other Plicatula group species.

Findings

Microsatellite sequences were identified from three enriched libraries from P. plicatulum. Specific primers were designed, and 25 displayed polymorphism when screened across 48 polyploid Paspalum spp. genotypes. The number of bands per locus ranged from 2 to 17, with a mean of 8.65. Private bands for each species were identified; the highest number of private bands was observed for P. plicatulum in 52% of the loci analyzed. The mean polymorphism information content of all loci was 0.69, and the mean discriminatory power was 0.82. Microsatellite markers were satisfactorily cross-amplified for the eight tested Plicatula-group Paspalum species, with P. atratum exhibiting the highest transferability rate (89.86%). STRUCTURE and Discriminant Analysis of Principal Components separated accessions into three groups but did not reveal separation of the accessions according to species.

Conclusions

This study describes the first microsatellite markers in P. plicatulum, which are polymorphic, efficient for the detection and quantification of genetic variation, and show high transferability into other species of the Plicatula group. This set of markers can be used in future genetic and molecular studies necessary for the proper development of conservation and breeding programs. Private bands within the markers can be used to assist in species identification.

     

Development of microsatellite genetic markers in Siberian stone pine (<Emphasis Type="Italic">Pinus sibirica</Emphasis> Du Tour) based on the <Emphasis Type="Italic">de novo</Emphasis> whole genome sequencing

Siberian stone pine, Pinus sibirica Du Tour is one of the most economically and environmentally important forest-forming species of conifers in Russia. To study these forests a large number of highly polymorphic molecular genetic markers, such as microsatellite loci, are required. Prior to the new high-throughput next generation sequencing (NGS) methods, discovery of microsatellite loci and development of micro-satellite markers were very time consuming and laborious. The recently developed draft assembly of the Siberian stone pine genome, sequenced using the NGS methods, allowed us to identify a large number of microsatellite loci in the Siberian stone pine genome and to develop species-specific PCR primers for amplification and genotyping of 70 microsatellite loci. The primers were designed using contigs containing short simple sequence tandem repeats from the Siberian stone pine whole genome draft assembly. Based on the testing of primers for 70 microsatellite loci with tri-, tetra- or pentanucleotide repeats, 18 most promising, reliable and polymorphic loci were selected that can be used further as molecular genetic markers in population genetic studies of Siberian stone pine.     

Development and characterization of 12 polymorphic microsatellite loci in the sea sandwort, <Emphasis Type="Italic">Honckenya peploides</Emphasis>

Codominant marker systems are better suited to analyze population structure and assess the source of an individual in admixture analyses. Currently, there is no codominant marker system using microsatellites developed for the sea sandwort, Honckenya peploides (L.) Ehrh., an early colonizer in island systems. We developed and characterized novel microsatellite loci from H. peploides, using reads collected from whole genome shotgun sequencing on a 454 platform. The combined output from two shotgun runs yielded a total of 62,669 reads, from which 58 loci were screened. We identified 12 polymorphic loci that amplified reliably and exhibited disomic inheritance. Microsatellite data were collected and characterized for the 12 polymorphic loci in two Alaskan populations of H. peploides: Fossil Beach, Kodiak Island (n?=?32) and Egg Bay, Atka Island (n?=?29). The Atka population exhibited a slightly higher average number of alleles (3.9) and observed heterozygosity (0.483) than the Kodiak population (3.3 and 0.347, respectively). The overall probability of identity values for both populations was PID?=?2.892e^?6 and PID_sib?=?3.361e^?3. We also screened the 12 polymorphic loci in Wilhelmsia physodes (Fisch. ex Ser.) McNeill, the most closely related species to H. peploides, and only one locus was polymorphic. These microsatellite markers will allow future investigations into population genetic and colonization patterns of the beach dune ruderal H. peploides on new and recently disturbed islands.     

Isolation and characterization of species-specific microsatellite markers for blue and black wildebeest (<Emphasis Type="BoldItalic">Connochaetes taurinus</Emphasis> and <Emphasis Type="BoldItalic">C. gnou</Emphasis>)

The blue wildebeest (Connochaetes taurinus) is distributed throughout southern and east Africa while the black wildebeest (Connochaetes gnou) is endemic to South Africa and was driven to near extinction in the early 1900s due to hunting pressure and disease outbreaks. Extensive translocation of both species throughout South Africa is threatening the genetic integrity of blue and black wildebeest. To effectively manage these species, genetic tools that can be used to detect hybrid individuals, identify genetically unique subpopulations and determine the levels of genetic diversity are required. In this study, 11 microsatellite markers were developed for wildebeest through next-generation sequencing. The microsatellite loci displayed 2.00–4.14 alleles, unbiased heterozygosity values ranged from 0.32 to 0.60 and observed heterozygosity values ranged from 0.26 to 0.52. The comparatively high level of polymorphism observed in the microsatellite markers indicates that these markers can contribute significantly to our knowledge of population genetic structure, relatedness, genetic diversity and hybridization in these species.     

Development of a large number of SSR and InDel markers and construction of a high-density genetic map based on a RIL population of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Capsicum annuum, the most widely cultivated species of pepper, is used worldwide for its important nutritional and medicinal values. The construction of an intraspecific high-density genetic linkage map would be of practical value for pepper breeding. However, the numbers of PCR-based simple sequence repeat (SSR) and insertion/deletion (InDel) markers that are available are limited, and there is a need to develop a saturated, intraspecific linkage map. The non-redundant Capsicum species’ expressed sequence tag (EST) database from the National Center for Biotechnology Information was used in this study to develop a total of 902 usable EST-SSR markers. Additionally, 177,587 SSR loci were identified based on the pepper genomic information, including 9182 SSR loci 500 bp both upstream and downstream of coding regions. Another 4497 stable and reliable InDel loci were also developed. From 9182 SSR and 4497 InDel loci, 3356 pairs of genomic SSR primers and 1400 pairs of InDel primers that were evenly distributed in 12 chromosomes were selected. A high-density intraspecific genetic map of C. annuum was constructed using the F₁₀-generation recombinant inbred line of parents PM702 and FS871 as the mapping population, screening the selected 3356 pairs of genomic SSR primers and 1400 pairs of InDel primers and the 902 EST-SSR markers developed earlier, and 524 published SSR markers and 299 orthologous markers (including 263 COSII markers and 36 tomato-derived markers) used previously to develop an interspecific genetic map (C. annuum × C. frutescens). Eventually, a high-density complete genetic intraspecific linkage map of C. annuum containing 12 linkage groups and 708 molecular markers with a length of 1260.00 cM and an average map distance of 1.78 cM was produced. This intraspecific, high-density, complete genetic linkage map of C. annuum contains the largest number of SSR and InDel markers and the highest amount of saturation so far, and it will be of considerable significance for the breeding of improved cultivars of this important field crop in the future.     

Development of microsatellite markers for <Emphasis Type="Italic">Manilkara maxima</Emphasis> T.D. Penn. (Sapotaceae) and their use in conservation genetics

Manilkara maxima is an endemic tree species of the Atlantic Forest in southern Bahia, Brazil. It is considered important for forest conservation due to its mutualistic interactions with endemic and endangered animals. Our aim was to develop microsatellite markers to estimate genetic diversity in order to provide information for effectiveness of future conservation programs. We used next generation sequencing technology to develop the first specific microsatellite markers for M. maxima. Seventeen new microsatellite loci were applied in 72 individuals sampled in three natural populations. On average, the number of alleles per loci was 8.8. The expected heterozygosity varied between 0.72 and 0.77, indicating that the developed set of molecular markers is useful for genetic diversity studies. Additionally, the estimated value for the combined probability of exclusion (Q) was greater than 0.999, which indicates the powerful of these molecular tools for paternity and kinship analysis. Our results demonstrate that the set of microsatellites developed in this work is a powerful tool for population genetics, molecular ecology and conservation biology purposes.     

Development and characterization of twelve microsatellite markers for <Emphasis Type="Italic">Porphyra linearis</Emphasis> Greville

The genus Porphyra (and its sister genus Pyropia) contains important red algal species that are cultivated and/or harvested for human consumption, sustaining a billion-dollar aquaculture industry. A vast amount of research has been focused on species of this genus, including studies on genetics and genomics among other areas. Twelve novel microsatellite markers were developed here for Porphyra linearis. Markers were characterized using 32 individuals collected from four natural populations of P. linearis with total heterozygosity varying from 0.098 to 0.916. The number of alleles per locus ranged from 2 to 18. All markers showed cross amplification with Porphyra umbilicalis and/or Porphyra dioica. These polymorphic microsatellite markers are useful for investigating population genetic diversity and differentiation in P. linearis and may become useful for other genetic research on the reproductive biology of this important species.     

Isolation of Polymorphic RAPD-SSR Markers from Xinjiang Arctic Grayling (<Emphasis Type="Italic">Thymallus arcticus grubei</Emphasis>) and a Test of Cross-Species Amplification

A total of 18 polymorphic microsatellite loci were isolated and characterized from RAPD products in the Xinjiang Arctic Grayling (Thymallus arcticus grubei). The number of alleles (N_a) per locus varied from 2 to 10. Observed (H_o) and expected (H_e) heterozygosities ranged from 0.64 to 0.92, and from 0.63 to 0.88, respectively. Considerable differences were found among HBH, FH and FY populations in the number of alleles, effective number of alleles, number of genotypes at all of these loci. These new RAPD-SSR markers have provided a helpful tool for genetic analyses and resources conservation of T. arcticus grubei. Five additional fish species, Amur grayling (Thymallus grubii), Taimen (Hucho taimen), Sea perch (Lateolabrax japonicus), Lenok (Brachymystax lenok) and Red seam bream (Pagrosomus major) were assessed for cross-species amplification. Three of the five species showed at least one polymorphic locus. In addition, seven loci were found to be polymorphic in at least one species.     

Isolation and characterization of microsatellite markers in a highland fish, <Emphasis Type="BoldItalic">Pareuchiloglanis sinensis</Emphasis> (Siluriformes: Sisoridae) by next-generation sequencing

Pareuchiloglanis sinensis (Siluriformes: Sisoridae) is an endemic and highland fish species which occurs only in some rivers of south-west China. In this study, the isolation and characterization of polymorphic microsatellite loci of this fish species by next-generation sequencing is described. A total of 9471 simple-sequence repeats (SSRs) were observed from RNA-seq data. One hundred and twenty primer pairs were chosen randomly and validated across 48 P. sinensis individuals collected from the Dadu river (a branch of the Yangtze river) of which 28 polymorphic microsatellite loci were detected. The number of alleles ranged from 2 to 14, with an average of seven alleles per locus. Twenty loci exhibited high polymorphism with the polymorphism information content (PIC) larger than 0.5. The mean observed and expected heterozygosity varied from 0.104 to 0.958 and 0.157 to 0.844, with an average of 0.583 and 0.613, respectively. The microsatellite markers characterized in the current study serve as a useful tool for the conversation genetic studies and population evaluation of P. sinensis.     

A report on identification of sequence polymorphism in barcode region of six commercially important <Emphasis Type="Italic">Cymbopogon</Emphasis> species

Cymbopogon

is an important member of grass family Poaceae, cultivated for essential oils which have greater medicinal and industrial value. Taxonomic identification of Cymbopogon species is determined mainly by morphological markers, odour of essential oils and concentration of bioactive compounds present in the oil matrices which are highly influenced by environment. Authenticated molecular marker based taxonomical identification is also lacking in the genus; hence effort was made to evaluate potential DNA barcode loci in six commercially important Cymbopogon species for their individual discrimination and authentication at the species level. Four widely used DNA barcoding regions viz., ITS 1 & ITS 2 spacers, matK, psbA-trnH and rbcL were taken for the study. Gene sequences of the same or related genera of the concerned loci were mined from NCBI domain and primers were designed and validated for barcode loci amplification. Out of the four loci studied, sequences from matK and ITS spacer loci revealed 0.46% and 5.64% nucleotide sequence diversity, respectively whereas the other two loci i.e., psbA-trnH and rbcL showed 100% sequence homology. The newly developed primers can be used for barcode loci amplification in the genus Cymbopogon. The identified Single Nucleotide Polymorphisms from the studied sequences may be used as barcodes for the six Cymbopogon species. The information generated can also be utilized for barcode development of the genus by including more number of Cymbopgon species in future.