Microsatellite markers for mungbean developed from sequence database

A novel set of microsatellite markers for mungbean [Vigna radiata (L.) Wilczek] was developed from the public sequence database. Seventy-eight primers were designed and evaluated for polymorphism among 22 cultivated accessions. Eight polymorphic loci detected two to three alleles per locus with an average of 2.25. The observed heterozygosity varied from 0.00 to 0.18, while the expected heterozygosity ranged from 0.09 to 0.46. Among them, all eight loci showed significant departuring from Hardy-Weinberg equilibrium, while four pairs of loci displayed significant pairwise linkage disequilibrium values. All eight loci except DMB-SSR1 showed heterozygote deficiency.     

Development and characterization of 15 microsatellite loci for Cariniana estrellensis and transferability to Cariniana legalis, two endangered tropical tree species

From a genomic library enriched for GA/CA repeats, 15 highly polymorphic microsatellite markers were developed for Cariniana estrellensis, a tropical forest tree. The microsatellite loci were screened in 49 mature trees found between Pardo river and Mogi-Guaçu river basins, in the state of São Paulo, Brazil. A total of 140 alleles were detected with an average of 9.33 alleles per locus. The expected heterozygosity ranged from 0.37 to 0.88. These loci showed a high probability of paternity exclusion. Additionally, 12 loci were effectively transferred to Cariniana legalis. High levels of polymorphism make the present SSR markers useful for population genetic studies.     

Development of microsatellite marker loci for European hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis> L.) from ISSR fragments

Polymorphic microsatellite markers were developed for European hazelnut (Corylus avellana L.) from the sequences of inter-simple sequence repeat (ISSR) fragments and flanking regions. Twenty-five ISSR primers were used to generate fragments for cloning. Of the 520 unique sequences obtained, 41 contained long internal repeats (≥20 bp) with flanking sequences sufficient for primer design. From these, we developed 23 new polymorphic microsatellite loci. The flanking sequences were obtained for fragment ends by chromosome walking, and an additional 47 polymorphic markers were developed. Two additional polymorphic markers were developed from a GA-enriched library. The 72 new marker loci were characterized using 50 diverse hazelnut accessions. For the internal repeat loci, the number of alleles per locus ranged from 2 to 16, with a mean of 7.52. Mean values for expected heterozygosity (H_e), observed heterozygosity (H_o), and polymorphism information content (PIC) were 0.62, 0.59, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.05 at six of the 23 loci. For the 47 marker loci developed from fragment ends, the number of alleles per locus ranged from 2 to 16, with a mean of 7.30. Mean values for H_e, H_o, and PIC were 0.62, 0.47, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.10 at 18 of the 47 loci. Of the 70 loci developed from ISSR and flanking sequences, 50 segregated in our mapping population and were assigned to linkage groups.     

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

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

Isolation and characterization of 13 microsatellite markers for the viviparous surfperch Ditrema temmincki (Embiotocidae) and cross-species amplification

Thirteen microsatellite loci were isolated from a size-selected genomic library of the surfperch (Ditrema temmincki Bleeker). All loci displayed a high degree of length polymorphism, as observed in the total number of alleles per locus (two to 23) and a high degree of estimated heterozygosity, ranging from 0.080 to 0.893. The primers developed for D. temmincki were also tested for their ability to amplify homologous sequences in D. viride and Neoditrema ransonetii. Distinct differences were observed among three species of surfperches, in both genetic variability and the frequency distribution of the alleles.     

Isolation and characterization of polymorphic microsatellites in a Yangtze River fish,brass gudgeon (Coreius heterodon Bleeker)

The brass gudgeon (Coreius heterodon) is a fish of economic importance in the Yangtze River. From a (GATA)_n‐enriched genomic library, 25 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Nine loci exhibited polymorphism with two to 12 alleles (mean 3.9 alleles/locus) in a test population, and observed heterozygosity ranged from 0.1111 to 0.9630 (mean 0.4426). Three of the nine loci showed polymorphism in a congeneric species, the largemouth bronze gudgeon Coreius guichenoti. These loci should provide sufficient level of genetic diversity to evaluate the fine‐scale population structure of C. heterodon.     

Characterization of eight microsatellite loci for the sea urchin Meoma ventricosa (Spatangoida,Brissidae) through Next Generation Sequencing

Eight microsatellite loci were characterized for Meoma ventricosa (Lamarck, 1816), a burrowing sea urchin that can be afflicted by a bacterial disease causing localized mass mortality. For the analyzed population (29 individuals from St. Croix, US Virgin Islands), we observed 8.125 mean number of alleles, 0.640 mean observed heterozygosity (H_o) and 0.747 mean expected heterozygosity (H_e). Two loci showed significant deviations from Hardy–Weinberg equilibrium. Overall, the described loci were characterized by a moderately high level of polymorphism suggesting that these markers are useful for a population genetic study in the Caribbean Sea.     

Isozyme analysis of genetic variability and population structure of Lactuca L. germplasm

Isozymes were used to investigate the genetic variability, population structure, and relationships of Lactuca germplasm. The isozyme systems revealed 16 putative loci of a total of 31 alleles. Out of these 16 loci, 11 were polymorphic. The average values of expected heterozygosity (H_e), observed heterozygosity (H_o), mean number of alleles per locus (A) and effective number of alleles per locus (A_e) were 0.2227, 0.266, 1.3005 and 1.369, respectively. The average fixation indices were lower than zero for most of the accessions studied, indicating an excess of heterozygotes. Genetic differentiation among accessions (F_ST) exhibited that 51.3% of the isozyme variation was recorded among accessions, and 48.7% of the genetic variation resided within accessions. The average values of total heterozygosity (H_T) and intra-accessional genetic diversity (H_S) were 0.352 and 0.171, respectively. Moreover, the inter-accessional genetic diversity (D_ST) ranged from 0 to 0.424 with an average of 0.18. Cluster analysis revealed that L. sativa cultivars were distributed throughout different Lactuca species. Thereby, isozymes results confirms the hypothesis of the polyphyletic origin of L. sativa. This high level of genetic variation proved that isozymes are efficient for polymorphism analysis of Lactuca germplasm.     

Identification of unique alleles and assessment of genetic diversity of rabi sorghum accessions using simple sequence repeat markers

Genetic diversity among 42 sorghum accessions representing landraces (19), advanced breeding lines (16), local cultivars (2) and release varieties (5) with 30 simple sequence repeat (SSR) markers revealed 7.6 mean number of alleles per locus showing 93.3% polymorphism and an average polymorphism information content of 0.78 which range from 0.22 (Xtxp12) and 0.91(Xtxp321). The average heterozygosity and effective number of alleles per locus were 0.8 and 6.65 respectively. Cluster analysis based on microsatellite allelic diversity clearly demarcated the accessions into ten clusters. A total of 24 unique alleles were obtained from seven SSR loci in 23 accessions in a size range of 110–380 bp; these unique alleles may serve as diagnostic tools for particular region of the genome of respective genotypes. Selected SSR markers from different linkage groups provided an accurate way of determining genetic diversity at the molecular level.     

Evaluation of the genetic diversity and population structure of sesame (Sesamum indicum L.) using microsatellite markers

Sixteen polymorphic microsatellite (SSR) markers, developed from an SSR-enriched genomic DNA library of sesame (Sesamum indicum L.), were used to assess genetic diversity, phylogenetic relationships, and population structure among 150 sesame accessions collected from 22 countries. A total of 121 alleles were detected among the sesame accessions. The number of detected alleles varied from 2 to 18, with an average of 7.6 alleles per locus. Polymorphism information content values ranged from 0.03 to 0.79, with an average of 0.42. These values indicated an excess of heterozygous individuals at 16 loci and an excess of homozygous individuals at three loci. Of these, 32 genotype-specific alleles were identified at 11 of 16 polymorphic SSR markers. Cluster analyses were performed by accession and population, revealing a complex accession distribution pattern with mean genetic similarity coefficient of 0.45 by accession and 0.52 by population. The wide variation in genetic similarity among the accessions revealed by SSRs reflected a high level of polymorphism at the DNA level. Model-based structure analysis revealed the presence of three groups that were basically consistent with the clustering results based on genetic distance. These findings may be used to augment the sesame germplasm and to increase the effectiveness of sesame breeding.     

Isolation and characterization of 20 microsatellite loci for an important tropical tree Shorea leprosula (Dipterocarpaceae) and their applicability to S. parvifolia

Shorea leprosula is an important timber tree in Southeast Asia. From a genomic library enriched for dinucleotide (CT) repeats, 20 polymorphic microsatellite markers were developed for this species. Polymorphism of these loci was evaluated in a sample of 24 adult individuals from a natural population. The number of alleles ranged from five to 15, and the observed heterozygosity ranged from 0.333 to 0.875. Probability of paternity exclusion, in the case where the mother is known, ranged from 0.285 to 0.784. These markers were also characterized for applicability in S. parvifolia; 16 loci were successfully cross‐amplified and showed high levels of polymorphism.     

Microsatellite markers reveal high genetic diversity in date palm (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) germplasm from Sudan

Genetic diversity in date palm germplasm from Sudan representing 37 female and 23 male accessions was investigated using 16 loci of microsatellite (SSR) primers. Eight female accessions from Morocco were included as reference material. The tested SSR markers showed a high level of polymorphism. A total of 343 alleles were detected at the 16 loci. The number of alleles per marker ranged from 14 to 44 with an average of 21.4 per locus. A high level of expected heterozygosity was observed among Sudan cultivars (0.841), Morocco cultivars (0.820) and male accessions (0.799). The results indicate that the genetic groups of the Sudan cultivars and/or males do not follow a clear geographic pattern. However, the morocco group showed significant differentiation in relation to the Sudan groups, as measured by F (ST) values and genetic distances. The effect of the methods of pollination and cultivar selection on the genetic structure was clearly detected by the weak clustering association that was observed for the majority of accessions originating from Sudan and Morocco as well. This suggests the need for further investigation on the genetic diversity of Sudanese date palm germplasm. A deeper insight will be revealed by a detailed analysis of populations originating from different geographic locations.     

Isolation and characterization of polymorphic tetranucleotide microsatellite loci in the pelagic perciform fish Pomatomus saltatrix (Linnaeus, 1766) from South Africa

Eight polymorphic microsatellite loci, containing simple tetranucleotide repeats, were isolated de novo from a Pomatomus saltatrix partial genomic library using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. These loci were further characterized in 100 individuals from two putative populations off the South African east coast. The loci are highly polymorphic with 18-37 alleles (on average 24 alleles/locus) and the observed heterozygosity in both populations was high (0.79). These loci will be used to assess population structuring in P. saltatrix along the southern African coast with consideration of implications for future management of this important linefish species.     

Development and use of novel SSR markers for molecular genetic diversity in Italian millet (Setaria italica L.)

Italian millet is a commercially important grain crop. Nineteen polymorphic simple sequence repeat (SSR) markers, developed through construction of an SSR-enriched library from genomic DNA of Italian millet (Setaria italica L., P. Beauv.), were used for assessment of molecular genetic diversity against 40 accessions of S. italica. In total, 85 alleles were detected, with an average of 4.5 alleles per locus. The average gene diversity and polymorphism information content (PIC) values were 0.412 and 0.376, ranging from 0.02 to 0.88 and from 0.02 to 0.87, respectively. Values for observed (H _O) and expected (H _E) heterozygosities ranged from 0 to 0.73 and from 0.03 to 0.89, respectively. Nine loci deviated from Hardy-Weinberg equilibrium. The mean similarity coefficient among accessions was 0.6593. Based on the UPGMA algorithm, six different groups were successfully identified. In this clustering analysis, all Korean accessions grouped in one cluster, indicating that Korean accessions are genetically quite distinct from other introduced accessions. These newly developed microsatellite markers should be very useful tools for several genetic studies, including an assessment of diversity and population structure in Italian millet.     

Characterization of novel microsatellite loci in the great leaf-nosed bat, Hipposideros armiger and cross-amplification in other related species

Eight microsatellite loci were isolated from an enriched genomic library of the great leaf-nosed bat, Hipposideros armiger. The polymorphism of these loci was tested on a population of 48 individuals from Anhui Province, China. All loci revealed the polymorphism ranging from three to 12 alleles. The observed heterozygosity values were from 0.213 to 0.875 and expected heterozygosity from 0.232 to 0.820. No significant linkage disequilibrium was detected. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. In addition, successful cross-amplification also suggested that these microsatellite markers will facilitate research on the population genetics and gene flow of H. armiger and other related species.     

Isolation and characterization of polymorphic microsatellite markers from the mosquito Anopheles moucheti,malaria vector in Africa

Anopheles moucheti is a major human malaria vector in Equatorial Africa. The screening of an Anopheles moucheti genomic microsatellite library allowed us to select 36 sequences with AC/GT dinucleotide tandem repeats. Primer pairs were designed to amplify the loci and 25 out of 36 gave a repeatable and scorable amplification. In total, 17 loci were selected for their high degree of polymorphism (the number of alleles per locus ranged from four to 16, and observed heterozygosity from 0.43 to 0.87) and suspicion of absence of null alleles, using 30 wild females from South‐Cameroon. No linkage disequilibrium was found between the loci.     

Isolation and characterization of 12 tetra‐repeated microsatellite loci from the Formosan grass lizard (Takydromus formosanus)

Twelve tetra‐repeated microsatellite loci were developed for the island‐endemic grass lizard Takydromus formosanus (Squamata: Lacertidae). We characterized these loci by genotyping 51 individuals sampled from six localities. The number of alleles per locus ranged between 12 and 23, whereas the observed heterozygosity ranged between 0.686 and 0.941. None of these loci showed gametic disequilibrium or sex linkage. With such high polymorphism, we believe that these loci should be suitable for fine‐scaled analysis of population structure, hybridizations or individual identification of grass lizards.     

Isolation and characterization of 15 polymorphic microsatellite markers for the fig-pollinating wasp,Blastophaga nipponica (Hymenoptera: Agaonidae)

We developed microsatellite markers for the fig-pollinating wasp Blastophaga nipponica Grandi using a dual-suppression-PCR technique. Twenty-one candidates of microsatellite loci were obtained, of which 15 yielded scorable patterns. The degree of polymorphism for the 15 loci was further characterized using summary statistics describing the genetic variation in 60 individuals from three natural populations in Japan. All 15 loci were polymorphic and yielded 2–27 alleles per locus. Overall observed heterozygosity (H _O) and expected heterozygosity (H _E) were 0.465 and 0.631, respectively. As expected, based on the inbreeding tendency of this species, the mean inbreeding coefficient (F _IS) was high (= 0.255). These markers will contribute to studies on the population structure of this species.     

Isolation and characterization of 21 microsatellite loci in Lycium chinense and cross-amplification in Lycium barbarum

The present study reports isolation and characterization of 21 polymorphic microsatellite markers developed from Lycium chinense Mill. These markers produced a total of 86 alleles across 30 L. chinense accessions, with an average of 4.1 alleles per locus. Values for observed heterozygosity and polymorphism information content ranged from 0.03 to 0.81 (mean = 0.35) and from 0.03 to 0.78 (mean = 0.31), respectively. At the significance threshold (P < 0.05), 12 loci deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibrium values were observed between 52 pairs of loci. All loci were successfully amplified for all L. barbarum accessions. These newly developed polymorphic microsatellite markers will be very useful for programming in the genetic conservation and classification of L. chinense and L. barbarum.     

Assessment of genetic diversity and population structure in mungbean

This study was carried out to assess the genetic diversity and to analyze the population genetic structure for a total of 692 mungbean accessions preserved at National Agrobiodiversity Center (NAC) of the Rural Development Administration (RDA), Korea. Mungbean accessions were collected from 27 countries in nine different geographic regions, and were genotyped using 15 microsatellite markers, which were developed in our previous study. A total of 66 alleles were detected among 692 accessions at all the loci with an average of 4.4 alleles per locus. All the microsatellite loci were found to be polymorphic. The expected heterozygosity (H _E ) and polymorphism information content (PIC) ranged from 0.081 to 0.588 (mean = 0.345) and from 0.080 to 0.544 (mean = 0.295), respectively. Of the 66 alleles, 17 (25.8%) were common (frequency range between 0.05 and 0.5), 15 (22.7%) were abundant (frequency range > 0.5), and 34 (51.5%) were rare (frequency range < 0.05). Locus GB-VR-7 provided the highest number of rare alleles(eight), followed by GB-VR-91(six) and GB-VR-113(four). Country-wide comparative study on genetic diversity showed that accessions from the USA possessed the highest genetic diversity (PIC) followed by Nepal, Iran, and Afghanistan. And region-wide showed that accessions from Europe possessed the highest average genetic diversity, followed by accessions from the USA, South Asia, West Asia, and Oceania. Twenty-seven countries were grouped into seven clades by phylogenetic relationship analysis, but clustering pattern did not strictly follow their geographical origin because of extensive germplasm exchange between/among countries and regions. As a result of a model-based analysis (STRUCTURE) of microsatellite data, two distinct genetic groups were identified which shared more than 75% membership with one of the two genetic groups. However the genetic group pattern did not reflect their geographical origin. The Duncan’s Multiple Range Test among these two genetic groups and an admixed group, with a mean of 16 phenotypic traits, showed significant difference in 12 quantitative and qualitative traits on the basis of ANOVA. These 15 newly developed SSR markers proved to be useful as DNA markers to detect genetic variation in mungbean germplasm for reasonable management and crossbreeding purposes.