Development and characterisation of microsatellite markers for the medicinal plant Scutellaria baicalensis (Lamiaceae)

Scutellaria baicalensis is a popular medicinal plant that is on the verge of extinction due to uncontrolled harvesting, habitat destruction and deterioration of its ecosystem. We isolated and characterised 21 microsatellite loci in this species. Ninety-four individuals from six populations were used to test the polymorphism of the microsatellite loci. The number of alleles per locus ranged from 1 to 13, with a mean of 7.2. Observed and expected heterozygosities varied from 0.000 to 1.000 and 0.000 to 0.938, respectively. Among these new microsatellite markers, only two loci showed significant deviation from Hardy–Weinberg equilibrium. No locus pairs showed significant linkage disequilibrium. The 21 primer pairs were tested in other Scutellaria species. Most of these primer pairs worked successfully, except for Scut18. These new microsatellite markers could be applied to investigate the genetic diversity and population genetic structure of S. baicalensis and its closely related species.     

Genetic diversity of wild population of Pyropia haitanensis based on SSR analysis

Genetic diversity analysis was conducted on 80 individuals of 4 populations of non-bred Pyropia haitanensis by simple sequence repeat (SSR) method. Using 15 pairs of microsatellite primers, 37 polymorphic loci were amplified, representing 94.9% of all loci. At the population level, the percentage of polymorphic bands (P) and polymorphism information content (PIC) were 66.67–84.62% and 0.481–0.488, with average value at 73.72% and 0.483, respectively. Expected heterozygosity (H_e) and Shannon's information index (I) were 0.279 and 0.434, respectively, at the species level, and 0.233 and 0.356 at population level. According to the coefficient of gene differentiation (G_ST), a large proportion of genetic variance (83.6%) of P. haitanensis was among individuals within populations, only 16.4% genetic variance was among populations, which was identified with the moderate gene flow value (N_m = 2.542). UPGMA clustered the 4 populations into 3 groups, and no significant correlation was found between the genetic distance and the corresponding geographic distance among the populations.     

Characterization of microsatellite loci in Schizopygopsis chengi chengi and their utilization in assessment of the genetic diversity in Schizopygopsis chengi baoxingensis

Schizopygopsis chengi, which belongs to the subfamily Schizothoracinae, comprises two subspecies, Schizopygopsis chengi chengi and Schizopygopsis chengi baoxingensis. S. c. chengi inhabits streams and lakes in upper reaches of the Dadu River. S. c. baoxingensis occurs as a single population endemic to the Baoxing River in China. In order to provide the reliable molecular markers for genetic investigation of S. chengi, eleven tetranucleotide microsatellite loci were developed from GATA-enriched library. The polymorphism of the loci was tested in 25 S. c. chengi individuals and the results showed that the loci were highly polymorphic. Eight loci were successfully amplified in S. c. baoxingensis. Sixty-seven different alleles were observed in the 82 S. c. baoxingensis individuals, and A_I, H_O, and PIC were 2.258, 0.549 and 0.572, respectively. The level of genetic diversity based on microsatellite loci was different from the previous studies based on mitochondrial DNA for S. c. baoxingensis, suggesting combined analysis of nuclear and mitochondrial DNA was required.     

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.     

Isolation and characterization of 16 highly polymorphic tetranucleotide microsatellite DNA markers in Paa spinosa

The giant spiny frog, Paa spinosa is a vulnerable species. By using a modified FIASCO (Fast Isolation by AFLP Sequences Containing repeats) method, we isolated and characterized 16 polymorphic tetranucleotide microsatellite loci for P. spinosa. These loci had high polymorphism when screened in 33 randomly caught individuals. The average number of alleles for 16 loci was 7.13 (range 2–14). Observed and expected heterozygosities were 0.152–0.818 and 0.144–0.918, respectively, and did not deviate from Hardy–Weinberg expectations. There was no evidence for scoring error due to stutter, null alleles, or large allele dropout. No significant association between pairs of loci was found after Bonferroni correction. These polymorphic loci will be effective for evaluating genetic diversity, population structure, gene flow and determining paternity in this frog, and also for conservation management.     

Molecular Phylogeny Analysis and Species Identification of Dendrobium (Orchidaceae) in China

Dendrobium plants are important commercial herbs in China, widely used in traditional medicine and ornamental horticulture. In this study, sequence-related amplified polymorphism (SRAP) markers were applied to molecular phylogeny analysis and species identification of 31 Chinese Dendrobium species. Fourteen SRAP primer pairs produced 727 loci, 97% of which (706) showed polymorphism. Average polymorphism information content of the SRAP pairs was 0.987 (0.982–0.991), showing that plenty of genetic diversity exists at the interspecies level of Chinese Dendrobium. The molecular phylogeny analysis (UPGMA) grouped the 31 Dendrobium species into six clusters. We obtained 18 species-specific markers, which can be used to identify 10 of the 31 species. Our results indicate the SRAP marker system is informative and would facilitate further application in germplasm appraisal, evolution, and genetic diversity studies in the genus Dendrobium.     

Development of twenty-one polymorphic tetranucleotide microsatellite loci for Schizothorax o&#x27;connori and their conservation application

Schizothorax o'connori, of the subfamily Schizothoracinae, is an endemic fish in the Yarlung Tsangpo River in Tibet, China. To evaluate the population genetic diversity as a means of devising conservation strategies, a total of 21 polymorphic tetranucleotide microsatellite loci were isolated from S. o'connori using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. The characterization of these loci was assessed against a panel of 42 individuals. The number of alleles per locus ranged from 4 to 29 with an average of 17.76. Expected locus-specific heterozygosity and the multilocus Shannon–Wiener diversity index ranged from 0.114 to 0.951 and from 0.280 to 3.131, with an average of 0.851 and 2.338, respectively. The cross-species amplification and applicability of these loci were tested against the two other Schizothorax species from the same river. These microsatellite loci will be valuable for further studies of population genetic diversity and genetic structure, and assessments of the artificial propagation release effect of S. o'connori and other related species.     

Development of thirty-five novel polymorphic microsatellite markers in Pseudosciaena polyactis (Perciformes:Sciaenidae) and cross-species amplification in closely related species,Pseudosciaena crocea

Small yellow croaker, Pseudosciaena polyactis, is an economically important marine fishery species. In this study, we isolated 35 novel polymorphic microsatellite primers in P. polyactis by using the combined biotin capture method. The polymorphism of each locus was assessed in 30 individuals from Donggang, Northern Yellow Sea, China. A total of 519 alleles were detected and the number of alleles per locus ranged from 3 to 23. The PIC values of these 35 microsatellite loci ranged from 0.367 to 0.940. The observed (H_o) and expected heterozygosity (H_e) per locus ranged from 0.233 to 1.000 and from 0.438 to 0.943, respectively. Seven loci significantly deviated from Hardy–Weinberg equilibrium (HWE) after Bonferroni correction and no significant linkage disequilibrium (LD) was found between pairs of loci. In addition, cross-species amplification was performed in Pseudosciaena crocea, a closely related species of P. polyactis, to assess the applicability of these markers. These polymorphic microsatellites will provide useful tools for the study of genetic diversity and population structure of P. polyactis and P. crocea.     

Effects of Medicago truncatula Genetic Diversity, Rhizobial Competition, and Strain Effectiveness on the Diversity of a Natural Sinorhizobium Species Community

We investigated the genetic diversity and symbiotic efficiency of 223 Sinorhizobium sp. isolates sampled from a single Mediterranean soil and trapped with four Medicago truncatula lines. DNA molecular polymorphism was estimated by capillary electrophoresis-single-stranded conformation polymorphism and restriction fragment length polymorphism on five loci (IGS_NOD, typA, virB11, avhB11, and the 16S rRNA gene). More than 90% of the rhizobia isolated belonged to the Sinorhizobium medicae species (others belonged to Sinorhizobium meliloti), with different proportions of the two species among the four M. truncatula lines. The S. meliloti population was more diverse than that of S. medicae, and significant genetic differentiation among bacterial populations was detected. Single inoculations performed in tubes with each bacterial genotype and each plant line showed significant bacterium-plant line interactions for nodulation and N₂ fixation levels. Competition experiments within each species highlighted either strong or weak competition among genotypes within S. medicae and S. meliloti, respectively. Interspecies competition experiments showed S. meliloti to be more competitive than S. medicae for nodulation. Although not highly divergent at a nucleotide level, isolates collected from this single soil sample displayed wide polymorphism for both nodulation and N₂ fixation. Each M. truncatula line might influence Sinorhizobium soil population diversity differently via its symbiotic preferences. Our data suggested that the two species did not evolve similarly, with S. meliloti showing polymorphism and variable selective pressures and S. medicae showing traces of a recent demographic expansion. Strain effectiveness might have played a role in the species and genotype proportions, but in conjunction with strain adaptation to environmental factors.     

Characterization of microsatellite markers in the endangered Sinojackia xylocarpa (Styracaceae) and cross‐species amplification in closely related taxa

Twenty‐four polymorphic microsatellite loci were isolated and characterized from an AAG‐enriched genomic library of Sinojackia xylocarpa. The average allele number of these microsatellites was 3.3 per locus, ranging from two to seven. The observed and expected heterozygosities at population level were 0.10–0.83 and 0.10–1.00, respectively. In addition, successful cross‐species amplification of this set of microsatellites in three other species of Sinojackia and a closely related taxon, Changiostyrax dolichocarpa, suggested that this set of microsatellite markers should provide a useful tool for genetic and conservation studies of Sinojackia species and other closely related taxa in the Styracaceae.     

Genetic Characterization of the Gypsy Moth from China (Lepidoptera,Lymantriidae) Using Inter Simple Sequence Repeats Markers

This study provides the first genetic characterization of the gypsy moth from China ( Lymantria dispar ), one of the most recognized pests of forests and ornamental trees in the world. We assessed genetic diversity and structure in eight geographic populations of gypsy moths from China using five polymorphic Inter simple sequence repeat markers, which produced reproducible banding patterns. We observed 102 polymorphic loci across the 176 individuals sampled. Overall genetic diversity (Nei’s, H) was 0.2357, while the mean genetic diversity within geographic populations was 0.1845 ± 0.0150. The observed genetic distance among the eight populations ranged from 0.0432 to 0.1034. Clustering analysis (using an unweighted pair-group method with arithmetic mean and multidimensional scaling), revealed strong concordance between the strength of genetic relationships among populations and their geographic proximity. Analysis of molecular variance demonstrated that 25.43% of the total variability (F _ST = 0.2543, P < 0.001) was attributable to variation among geographic populations. The results of our analyses investigating the degree of polymorphism, genetic diversity (Nei’s and Shannon) and genetic structure, suggest that individuals from Hebei may be better able to adapt to different environments and to disperse to new habitats. This study provides crucial genetic information needed to assess the distribution and population dynamics of this important pest species of global concern.     

Genetic Diversity of Grasspea and Its Relative Species Revealed by SSR Markers

The study of genetic diversity between Lathyrus sativus L. and its relative species may yield fundamental insights into evolutionary history and provide options to meet the challenge of climate changes. 30 SSR loci were employed to assess the genetic diversity and population structure of 283 individuals from wild and domesticated populations from Africa, Europe, Asia and ICARDA. The allele number per loci ranged from 3 to 14. The average gene diversity index and average polymorphism information content (PIC) was 0.5340 and 0.4817, respectively. A model based population structure analysis divided the germplasm resources into three subgroups: the relative species, the grasspea from Asia, and the grasspea from Europe and Africa. The UPGMA dendrogram and PCA cluster also demonstrated that Asian group was convincingly separated from the other group. The AMOVA result showed that the cultivated species was quite distinct from its relative species, however a low level of differentiation was revealed among their geographic origins. In all, these results provided a molecular basis for understanding genetic diversity of L. sativus and its relatives.     

Isolation and characterization of eighteen polymorphic microsatellite loci in Schizopygopsis younghusbandi Regan and cross-amplification in three other schizothoracinae species

Eighteen polymorphic microsatellite loci were isolated from Schizopygopsis younghusbandi Regan and the characterization of these loci was assessed in 46 individuals collected from the Yarlung Tsangpo River in Tibet, China. The number of alleles per locus ranged from 2 to 14. The expected heterozygosity and Shannon-Wiener diversity index ranged from 0.022 to 0.879 and from 0.059 to 2.313, respectively. The cross-species amplification and applicability of these loci were tested in three other Schizothoracinae species belonging to Schizothorax and Oxygymnocypris. These loci will be useful for the evaluation of genetic diversity and population genetic structure in S. younghusbandi and other related species.     

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.     

Genetic variation in Pueraria lobata (Fabaceae), an introduced, clonal, invasive plant of the southeastern United States

Pueraria lobata (kudzu), a clonal, leguminous vine, is invading the southeastern United States at a rate of 50 000 ha per year. Genetic variability and clonal diversity were measured in 20 southeastern U.S. populations using 14 allozyme loci. Within its U.S. range, 92.9% of the loci were polymorphic and overall genetic diversity was 0.290. Such high levels of genetic diversity are consistent with its history of multiple introductions over an extended period of time. The average proportions of polymorphic loci and genetic diversity within populations were 55.7% (range = 28.6–85.7%) and 0.213 (range = 0.114–0.317), respectively. The proportion of total genetic diversity found among populations was similar to species with equivalent life history characters (G_ST = 0.199). No regional patterns of variation were seen. The number of putative genotypes in each population ranged from 2 to 26. Mean genotypic diversity was 0.694, ranging from 0.223 to 0.955. Such high levels of genotypic diversity indicate that local sites are often colonized by several propagules (most likely seeds) and/or that sexual reproduction occurs within populations after establishment. An excess of heterozygosity was observed in populations with few unique genets, implying that selection for highly heterozygous individuals may occur in populations of P. lobata.     

Isolation and characterization of fifteen polymorphic microsatellite loci in the threatened freshwater mussel Solenaia oleivora (Bivalvia: Unionidae)

Freshwater mussels are among the most endangered fauna in the world, while little is known about their genetic diversity. We isolated 15 polymorphic microsatellite loci in a threatened freshwater mussel, Solenaia oleivora. The characteristics of these loci were tested with 46 individuals. The number of alleles per locus ranged from 2 to 15, and the PIC value ranged from 0.250 to 0.880. The observed and expected heterozygosity ranged from 0.167 to 0.929 and from 0.297 to 0.900, with an average of 0.562 and 0.647, respectively. Eight loci deviated significantly from Hardy–Weinberg equilibrium. These polymorphic microsatellites will be useful for the assessment of population genetic diversity and resource conservation of S. oleivora. Cross-species amplification demonstrated that six of 15 microsatellite loci amplified successfully in two other unionid species.     

Signatures of adaptation and genetic structure among the mainland populations of Pinus radiata (D. Don) inferred from SNP loci

Insights into the relative contributions of locus specific and genome-wide effects on population genetic diversity can be gained through separation of their resulting genetic signals. Here we explore patterns of adaptive and neutral genetic diversity in the disjunct natural populations of Pinus radiata (D. Don) from mainland California. A first-generation common garden of 447 individuals revealed significant differentiation of wood phenotypes among populations (P _ST), possibly reflecting local adaptation in response to environment. We subsequently screened all trees for genetic diversity at 149 candidate gene single nucleotide polymorphism (SNP) loci for signatures of adaptation. Ten loci were identified as being possible targets of diversifying selection following F _ST outlier tests. Multivariate canonical correlation performed on a data set of 444 individuals identified significant covariance between environment, adaptive phenotypes and outlier SNP diversity, lending support to the case for local adaptation suggested from F _ST and P _ST tests. Covariation among discrete sets of outlier SNPs and adaptive phenotypes (inferred from multivariate loadings) with environment are supported by existing studies of candidate gene function and genotype–phenotype association. Canonical analyses failed to detect significant correlations between environment and 139 non-outlier SNP loci, which were applied to estimate neutral patterns of genetic differentiation among populations (F _ST 4.3 %). Using this data set, significant hierarchical structure was detected, indicating three populations on the mainland. The hierarchical relationships based on neutral SNP markers (and SSR) were in contrast with those inferred from putatively adaptive loci, potentially highlighting the independent action of selection and demography in shaping genetic structure in this species.     

Diversity of Arabidobsis thaliana (L.) Heynh. natural populations at the northern limits of the species range: RAPD analysis

In natural populations of Arabidobsis thaliana (L.) Heynh., occupying northern limits of the species range (Karelia), the level of genetic diversity was evaluated. In two insular and one mainland population variability at 82 RAPD loci was tested. Considerable genetic diversity revealed (P = 4.5%; H _exp = 0.177) was not typical of self-pollinating plant species. It was demonstrated that genetic differentiation among the populations (G _ST = 0.680) was rather high, pointing to the low level of gene flow in the isolated insular populations. It was suggested that the high level of Arabidopsis population polymorphism in Karelia could be associated with extreme growing conditions at the northern limits of the species range.     

Identification of candidate AFLP markers for shell color of the Pacific oyster (Crassostrea gigas) under artificial selection

The shell color of the Pacific oyster (Crassostrea gigas) is a desirable trait, but only a few genetic studies on shell color have been documented. Through successive selective breeding, four shell color variants of white (W), gold (G), black (B) and purple (P) C. gigas have been developed. The amplified fragment length polymorphism (AFLP) technique was used to scan the genomes of the four variants with different shell colors and one wild population (C) to identify candidate markers for shell polymorphism. Fifteen AFLP primer combinations were used, 1079 loci were scored as polymorphic loci, and the percentage of polymorphic bands was 95.5%. In the gold, white, black, purple and wild populations, the percentages of polymorphic loci were estimated to be 90.5% (G), 90.0% (W), 91.1% (B), 95.3% (P) and 93.2% (C); the expected heterozygosity values were 0.3115 (G), 0.3044 (W), 0.3102 (B), 0.3285 (P) and 0.3105 (C). The white shell variant was observed to have slightly lower genetic diversity than others, with a F_ST value of 0.1483. These results indicated that the four different shell color variants had high genetic diversity and that the genetic differentiation of populations mostly results from genetic diversity of individuals within populations. Furthermore, 11 outlier loci were considered candidate markers for shell color. This work provides new insights on relationships among color variants of C. gigas.     

Analysis of Allelic Diversity and Genetic Relationships Among Cultivated Mangosteen (<Emphasis Type="Italic">Garcinia mangostana</Emphasis> L.) in Java,Indonesia Using Microsatellite Markers and Morphological Characters

The genetic variation and relationships of the mangosteen (Garcinia mangostana L.) were observed across mangosteen populations in Java, Indonesia using newly identified microsatellite loci and morphological characters. In this study, we developed an improved protocol to isolate microsatellite loci, named Selective Repeats from AFLP Sequence, by using a hybridized membrane. Twenty microsatellite loci were evaluated using 78 individuals from five mangosteen populations, and we successfully amplified four closely related Garcinia species, including G. malaccensis, G. hombroniana, G. celebica, and G. porrecta. Eight loci were monomorphic and the others were polymorphic. Sixty-nine alleles were found, with 3.491 per locus on an average. Genetic diversity (H?) was calculated with an average across loci within population (H?_S) as 0.39, an average loci across many populations (H?_T) as 0.444, and genetic differentiation (F?_ST) as 0.147. Furthermore, based on morphological characters, mangosteen individuals from four populations including Leuwiliang, Wanayasa, Puspahiang, and Kaligesing also had morphologically distinct fruit weight, rind weight, and rind thickness among populations. The study also elucidated the dispersal pattern of mangosteen in Java; the source of the genotype mangosteen in Java population was the Wanayasa population. In addition, we found evidence of tetraploidy in mangosteen. These results have potential applications in future breeding, conservation studies, and genetic assessment of mangosteen and their closely related species.