Characterization of polymorphic microsatellite markers and genetic diversity in wild bronze featherback, Notopterus notopterus (Pallas, 1769)

Six polymorphic microsatellite DNA loci were identified in the primitive fish, bronze featherback, Notopterus notopterus for the first time and demonstrated significant population genetic structure. Out of the six primers, one primer (NN90) was specific to N. notopterus (microsatellite sequence within the RAG1 gene) and five primers were product of successful cross-species amplification. Sixty-four primers available from 3 fish species of order Osteoglossiformes and families Notopteridae and Osteoglossidae were tested to amplify homologous microsatellite loci in N. notopterus. Fifteen primer pairs exhibited successful cross-priming PCR product. However, polymorphism was detected only at five loci. To assess the significance of these six loci (including NN90) in population genetic study, 215 samples of N. notopterus from five rivers, viz Satluj, Gomti, Yamuna, Brahmaputra and Mahanadi were analyzed. The five sample sets displayed different diversity levels and observed heterozygosity ranged from 0.6036 to 0.7373. Significant genotype heterogeneity (P < 0.0001) and high F_ST (0.2205) over all loci indicated that the samples are not drawn from the same genepool. The identified microsatellite loci are promising for use in fine-scale population structure analysis of N. notopterus.     

Cross-priming of microsatellite loci in subfamily cyprininae (family Cyprinidae): their utility in finding markers for population genetic analysis in three Indian major carps

This study is aimed to identify polymorphic microsatellite markers and establish their potential for population genetics studies in three carp (family cyprinidae; subfamily cyprininae) species, Labeo rohita, Catla catla and Cirrhinus mrigala through use of cyprinid primers. These species have high commercial value and knowledge of genetic variation is important for management of farmed and wild populations. We tested 108 microsatellite primers from 11 species belonging to three different cyprinid subfamilies, Cyprininae, Barbinae and Leuciscinae out of which 63 primers (58.33 %) successfully amplified orthologous loci in three focal species. Forty-two loci generated from 29 primers were polymorphic in these three carp species. Sequencing of amplified product confirmed the presence of SSRs in these 42 loci and orthologous nature of the loci. To validate potential of these 42 polymorphic loci in determining the genetic variation, we analyzed 486 samples of three focal species collected from Indus, Ganges and Brahmaputra river systems. Results indicated significant genetic variation, with mean number of alleles per locus ranging from 6.80 to 14.40 and observed heterozygosity ranging from 0.50 to 0.74 in the three focal species. Highly significant (P < 0.00001) allelic homogeneity values revealed that the identified loci can be efficiently used in population genetics analysis of these carp species. Further, thirty-two loci from 19 primers were useful for genotyping in more than one species. The data from the present study was compiled with cross-species amplification data from previous results on eight species of subfamily cyprininae to compare cross-transferability of microsatellite loci. It was revealed that out of 226 heterologous loci amplified, 152 loci that originated from 77 loci exhibited polymorphism and 45 primers were of multispecies utility, common for 2–7 species.     

Development of 13 microsatellite markers for <Emphasis Type="Italic">Castanopsis tribuloides</Emphasis> (Fagaceae) using next-generation sequencing

Catanopsis tribuloides is a climax tree species commonly distributed in evergreen forests and has been used to restore degraded areas in northern Thailand. To aid in study of genetic diversity of the species, microsatellite markers, which are specific to C. tribuloides, were developed using whole genome sequencing by next-generation sequencing technology. The primers for microsatellite were developed and screened for optimal annealing temperature by PCR assay. The loci primers specific with C. tribuloides, 13 polymorphic microsatellite primers were successfully developed. The results from genetic information analyzing showed the number of alleles presented were between 2 and 24. Accordingly, the expected and observed heterozygosity obtained were between 0.298 and 0.920 and 0.364 to 1.000, respectively. Null allele frequency was presented 0.000–0.199. Genetic information was generated 10 loci primers significantly deviated from Hardy–Weinberg Equilibrium. All 13 primer pairs of loci were not significant with linkage disequilibrium. A set of microsatellite markers in this study could be applied to gene flow, genetic structure and population genetic studies in the future.     

Microsatellite markers for Lycium ruthenicum (Solananeae)

We developed microsatellite markers in Lycium ruthenicum, a desert plant widely distributed in northwestern China. In order to investigate its population genetic structure, genetic diversity, and its evolutionary history, we have isolated 11 novel microsatellite loci primers and characterized them in 24 individuals from 3 populations of L. ruthenicum using the combined biotin capture technique. For these microsatellites, one to seven alleles per locus were identified. The observed heterozygosities ranged from 0 to 0.958, meanwhile the expected heterozygosities ranged from 0 to 0.841. These microsatellite markers could be first useful for population level studies like genetic diversity and structure in this species.     

Microsatellite primers for fungus‐growing ants

We isolated five polymorphic microsatellite loci from a library of two thousand recombinant clones of two fungus‐growing ant species, Cyphomyrmex longiscapus and Trachymyrmex cf. zeteki. Amplification and heterozygosity were tested in five species of higher attine ants using both the newly developed primers and earlier published primers that were developed for fungus‐growing ants. A total of 20 variable microsatellite loci, developed for six different species of fungus‐growing ants, are now available for studying the population genetics and colony kin‐structure of these ants.     

Development of new microsatellite loci for the genus <Emphasis Type="Italic">Polistes</Emphasis> from publicly available expressed sequence tag sequences

Over the last 20 years, microsatellites have revolutionized the study of cooperation in the social insects. The Polistes paper wasps have been an important model system for investigations of cooperative behavior. Recently, an expressed sequence tag (EST) library has been developed for P. metricus, allowing researchers to investigate the genetic basis of cooperative behavior in primitive social insect societies for the first time. We searched these freely available EST sequences for microsatellite motifs. This represents a relatively new approach to the development of microsatellite loci that allows for the development of a greater number of loci at less expense. We designed 32 PCR primer pairs, of which 23 amplified PCR products and 18 were polymorphic. These loci exhibited high levels of polymorphism, comparable to anonymous loci isolated via screens of partial genomic libraries. Thus, they are appropriate for population genetic studies as well as the reconstruction of colony genetic structure. A screen of the entire EST database found a total of 708 di-, tri-, tetra- and penta-nucleotide repeats with large repeat units typical of polymorphic loci and at least 30 bp of flanking sequence for primer design. This pool of potential loci represents a new genetic tool for P. metricus, as well as Polistes more generally, as there is great promise for cross amplification in other species.     

Isolation and characterization of microsatellite markers in Labiotermes labralis (Isoptera,Termitidae, Nasutitermitinae)

We report here on the development of six polymorphic microsatellite loci for Labiotermes labralis. This soil‐feeding species is restricted to the Neotropical rainforest and then could represent a major candidate for being a bio‐indicator of anthropic disturbance resulting in the forest fragmentation. The microsatellite loci were isolated and characterized using a microsatellite‐enriched genomic library. The primers were tested on a French Guyanese population of L. labralis, represented by the sampling of one soldier in 22 nests. The primers were also tested against nine species of the same Nasutitermitinae subfamily and were successfully amplified at five loci in Armitermes minutus.     

Characterization of 11 novel polymorphic microsatellite loci in the threatened Korean loach,Iksookimia koreensis,isolated using a next-generation sequencing method

To investigate the genetic diversity and population structure of Iksookimia koreensis, we characterized 11 novel polymorphic microsatellite markers developed using next-generation sequencing. The number of alleles per locus ranged from 4 to 10 (average = 6.26). Observed and expected heterozygosities ranged from 0.333 to 0.866 and from 0.375 to 0.866, respectively. No loci showed significant deviation from Hardy–Weinberg equilibrium (HWE). These loci were also used successfully to study the genetic diversity of a closely related species, Iksookimia longicorpa. Four of the 11 loci amplified in the two species showed different allele frequency and distribution, indicating deep genetic divergence between I. koreensis and I. longicorpa. The newly developed microsatellite markers reported here will provide a useful tool for examining gene flow, population genetic structure, and genetic diversity of these species.     

Development of highly conserved primers for 12 new polymorphic microsatellite loci for the genus Rorippa Scop. (Brassicaceae), yellow‐cress

We isolated 12 highly conserved polymorphic microsatellite loci for the yellow‐cress species Rorippa amphibia and Rorippa sylvestris. We used a partial genomic library enriched for several repeat motifs. Obtained sequences containing repetitive elements were blasted and aligned with the Arabidopsis thaliana sequence. We evaluated the cross‐species compatibility of primers designed from sequences either aligning strongly or weakly with A. thaliana. The former proved much more efficient in obtaining primers that worked in both species. The developed conserved primers for microsatellite loci provide excellent markers for studying segregation, gene flow and hybridization in the genus Rorippa.     

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.     

Application of microsatellite primers for the social wasp Polistes to another social wasp genus, Parapolybia, to estimate genetic relationships among nestmates

We tested 14 pairs of microsatellite primers that had been developed for the genus Polistes to assess their applicability for determining genetic structure in a colony of the genus Parapolybia. At least one of the 14 Polistes primer sets was useful for Parapolybia indica, and the locus identified by this primer set was highly polymorphic. Using this primer set, we estimated the average relatedness among female nestmates of Parapolybia indica to be 0.82 ± 0.05 (mean ± SE), indicating that in this species single mating is normal, although some cases of multiple mating was found.     

Development and characterization of microsatellite loci for Rosa odorata var. gigantea Rehder & E. H. Wilson (Rosaceae)

Eighteen microsatellite markers were developed from Rosa odorata var. gigantea (Rosaceae), including 11 new microsatellite markers and 7 modified microsatellite loci having been developed from other Rosa species. About 27 wild individuals from 3 populations were used to screen polymorphism of these 18 microsatellite makers. The average allele number of these markers was 3.9 per locus, ranging from 2 to 9. The expected and observed heterozygosities varied from 0.2711 to 0.8043 and from 0.0370 to 0.5556, respectively. Cross-species amplification in other eight Rosa species showed their potential use for congeneric species. These microsatellite primers will be used for population genetics studies, constructing genetic linkage maps or locating quantitative trait locus (QTL) of R. odorata var. gigantea and related species.     

Development of Genomic Microsatellite Markers in Carthamus tinctorius L. (Safflower) Using Next Generation Sequencing and Assessment of Their Cross-Species Transferability and Utility for Diversity Analysis

Background

Safflower (Carthamus tinctorius L.), an Asteraceae member, yields high quality edible oil rich in unsaturated fatty acids and is resilient to dry conditions. The crop holds tremendous potential for improvement through concerted molecular breeding programs due to the availability of significant genetic and phenotypic diversity. Genomic resources that could facilitate such breeding programs remain largely underdeveloped in the crop. The present study was initiated to develop a large set of novel microsatellite markers for safflower using next generation sequencing.

Principal Findings

Low throughput genome sequencing of safflower was performed using Illumina paired end technology providing ~3.5X coverage of the genome. Analysis of sequencing data allowed identification of 23,067 regions harboring perfect microsatellite loci. The safflower genome was found to be rich in dinucleotide repeats followed by tri-, tetra-, penta- and hexa-nucleotides. Primer pairs were designed for 5,716 novel microsatellite sequences with repeat length ≥ 20 bases and optimal flanking regions. A subset of 325 microsatellite loci was tested for amplification, of which 294 loci produced robust amplification. The validated primers were used for assessment of 23 safflower accessions belonging to diverse agro-climatic zones of the world leading to identification of 93 polymorphic primers (31.6%). The numbers of observed alleles at each locus ranged from two to four and mean polymorphism information content was found to be 0.3075. The polymorphic primers were tested for cross-species transferability on nine wild relatives of cultivated safflower. All primers except one showed amplification in at least two wild species while 25 primers amplified across all the nine species. The UPGMA dendrogram clustered C. tinctorius accessions and wild species separately into two major groups. The proposed progenitor species of safflower, C. oxyacantha and C. palaestinus were genetically closer to cultivated safflower and formed a distinct cluster. The cluster analysis also distinguished diploid and tetraploid wild species of safflower.

Conclusion

Next generation sequencing of safflower genome generated a large set of microsatellite markers. The novel markers developed in this study will add to the existing repertoire of markers and can be used for diversity analysis, synteny studies, construction of linkage maps and marker-assisted selection.     

Isolation and characterization of microsatellites in Aphidius ervi (Hymenoptera: Braconidae) and their applicability to related species

Aphidius ervi (Hymenoptera: Braconidae) is an important biological control agent of aphids, and a model organism for the study of host–parasitoid ecology and evolution. We isolated microsatellite loci from A. ervi to examine the genetic effects of its introduction from Europe to North America. We present primers for 11 microsatellites from A. ervi. We have assayed six of these loci for variability and found from two to 37 alleles. These six primer pairs were tested on 17 related species and appear broadly applicable. These microsatellite loci provide a new tool for research on A. ervi and its relatives.     

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.     

Genetic Diversity and Population Structure of the Rare and Endangered Plant Species Pulsatilla patens (L.) Mill in East Central Europe

Pulsatilla patens s.s. is a one of the most endangered plant species in Europe. The present range of this species in Europe is highly fragmented and the size of the populations has been dramatically reduced in the past 50 years. The rapid disappearance of P. patens localities in Europe has prompted the European Commission to initiate active protection of this critically endangered species. The aim of this study was to estimate the degree and distribution of genetic diversity within European populations of this endangered species. We screened 29 populations of P. patens using a set of six microsatellite primers. The results of our study indicate that the analyzed populations are characterized by low levels of genetic diversity (H_o = 0.005) and very high levels of inbreeding (F_IS = 0.90). These results suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species. Private allelic richness was very low, being as low as 0.00 for most populations. Average genetic diversity over loci and mean number of alleles in P. patens populations were significantly correlated with population size, suggesting severe genetic drift. The results of AMOVA point to higher levels of variation within populations than between populations.The results of Structure and PCoA analyses suggest that the genetic structure of the studied P. patens populations fall into three clusters corresponding to geographical regions. The most isolated populations (mostly from Romania) formed a separate group with a homogeneous gene pool located at the southern, steppic part of the distribution range. Baltic, mostly Polish, populations fall into two genetic groups which were not fully compatible with their geographic distribution.Our results indicate the serious genetic depauperation of P. patens in the western part of its range, even hinting at an ongoing extinction vortex. Therefore, special conservation attention is required to maintain the populations of this highly endangered species of European Community interest.     

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

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

Identification of Microsatellite Repeats in Turbot (Scophthalmus maximus) and Dover Sole (Solea solea) using a RAPD-Based Technique: Characterization of Microsatellite Markers in Dover Sole

We have used a RAPD-based technique to identify several microsatellite repeats in turbot (Scophthalmus maximus) and Dover sole (Solea solea) and report the characterization of six novel polymorphic microsatellite markers for Dover sole. These are the first such markers to be developed for this flatfish species. They exhibit much higher levels of heterozygosity than those previously observed with allozyme loci and should prove useful in addressing population genetic questions as well as more fundamental aquaculture-related questions. Received ; accepted July 13, 1999.     

Microsatellite loci for the field cricket,Gryllus bimaculatus and their cross‐utility in other species of Orthoptera

We have isolated 16 microsatellite loci in the field cricket, Gryllus bimaculatus. Nine loci were found to be polymorphic in G. bimaculatus and the number of alleles varied from seven to 14. All 16 loci were tested for amplification in nine other species. In the five species tested belonging to the same subfamily (Gryllinae), a minimum of nine loci amplified. These loci will be used to determine paternity as part of a study to investigate the genetic benefits of polyandry.     

Development of microsatellite markers for Carapa guianensis (Aublet), a tree species from the Amazon forest

Carapa guianensis is a timber species found in Central America and the north of South America. We have developed microsatellite primers which will allow analysis of gene flow and population genetic structure in natural populations of this tree species. Polymorphism of five microsatellite loci was evaluated using a total of 12 adult trees from a natural population. An average of 4.2 alleles per locus was detected, and expected heterozygosity ranging from 0.397 to 0.806. These loci are being used for genetic population analysis in a managed forest in the state of Pará, in Amazonian Brazil, as part of the Dendrogene project.