Development of microsatellite markers for Anadenanthera colubrina var. cebil (Fabaceae), a native tree from South America

? Premise of the study: Microsatellite primers were developed in the native legume tree Anadenanthera colubrina var. cebil to study the genetic diversity and genetic structure in natural populations in Argentina. ? Methods and Results: Nine microsatellite markers were identified using a genomic library enriched for tandemly repeated motifs, eight of which markers were polymorphic. The polymorphism of these markers was assessed by investigating 20 individuals for fragment polymorphism; three to 13 alleles were observed for each locus. Observed and expected heterozygosities ranged from 0.300 to 1.000 and from 0.463 to 0.900, respectively. ? Conclusions: These results confirm that these primers will be useful for investigating the genetic diversity and genetic structure of natural populations of A. colubrina var. cebil in future studies.     

Development of EST-SSR markers for Elaeocarpus photiniifolia (Elaeocarpaceae), an endemic taxon of the Bonin Islands

? Premise of the study: Expressed sequence tag (EST)-derived microsatellite markers were developed for Elaeocarpus photiniifolia, an endemic taxon of the Bonin Islands. ? Methods and Results: Initially, a complementary DNA (cDNA) library was constructed by de novo pyrosequencing of total RNA extracted from a seedling. A total of 267 primer pairs were designed from the library. Of the 48 tested loci, 25 loci were polymorphic among 41 individuals representing the entire geographical range of the species, with the number of alleles per locus and expected heterozygosity ranging from two to 14 and 0.09 to 0.86, respectively. Most loci were transferable to a related species, E. sylvestris. ? Conclusions: The developed markers will be useful for evaluating the genetic structure of E. photiniifolia.     

Isolation and characteristics of eight novel polymorphic microsatellite loci in Lippia alba (Verbenaceae)

? Premise of the study: A set of eight microsatellite (simple sequence repeat [SSR]) markers for Lippia alba, an important medicinal and cosmetic plant, was developed to aid studies of genetic diversity and to define efficient strategies for breeding programs. ? Methods and Results: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 11 SSR loci were developed and optimized in L. alba. Of the 11 loci, eight were found to be polymorphic after screening 61 accessions from two populations. The parameters used to characterize loci were expected heterozygosity (H(e)) and number of alleles. A total of 44 alleles were identified, with an average of 5.5 alleles per loci, which were moderately to highly informative according to H(e). ? Conclusions: These new SSR markers have potential for informing genetic diversity, allele mining, and mapping studies and will be used to generate information for breeding programs of L. alba.     

Development of microsatellite markers for Qualea grandiflora (Vochysiaceae), a typical species of the Brazilian cerrado

? Premise of the study: Microsatellite primers were developed to investigate genetic diversity and population structure of Qualea grandiflora, a typical species of the Brazilian cerrado. ? Methods and Results: Eight microsatellite loci were isolated using an enrichment cloning protocol. These loci were tested on a population of 110 individuals of Q. grandiflora collected from a cerrado fragment in S?o Paulo State, Brazil. The loci polymorphism ranges from seven to 19 alleles and the average heterozygosity value is 0.568, while the average polymorphic information content is 0.799. ? Conclusions: The developed markers were found to be highly polymorphic, indicating their applicability to studies of population genetic diversity in Q. grandiflora.     

Development of novel chloroplast microsatellite markers for Miscanthus species (Poaceae)

? Premise of the study: A set of novel chloroplast microsatellite markers (cpSSRs) was developed for the bioenergy crop Miscanthus, and their utility in cross-species amplification was evaluated. ? Methods and Results: Twenty-eight novel primers flanking cpSSR loci were designed from a complete chloroplast genome sequence of Saccharum officinarum, a species closely related to Miscanthus. These primers were then tested on eight Miscanthus species, among which 16 cpSSR loci were found to be polymorphic. The number of alleles per polymorphic locus ranged from two to seven, with an average 3.94 alleles. ? Conclusions: These cpSSR markers can be applied to all Miscanthus species and will be useful for studying Miscanthus population structure, diversity, and phylogeography.     

Characterization of 35 novel microsatellite DNA markers from the duck (Anas platyrhynchos) genome and cross-amplification in other birds

In order to study duck microsatellites, we constructed a library enriched for (CA)n, (CAG)n, (GCC)n and (TTTC)n. A total of 35 pairs of primers from these microsatellites were developed and used to detect polymorphisms in 31 unrelated Peking ducks. Twenty-eight loci were polymorphic and seven loci were monomorphic. A total of 117 alleles were observed from these polymorphic microsatellite markers, which ranged from 2 to 14 with an average of 4.18 per locus. The frequencies of the 117 alleles ranged from 0.02 to 0.98. The highest heterozygosity (0.97) was observed at the CAUD019 microsatellite locus and the lowest heterozygosity (0.04) at the CAUD008 locus, and 11 loci had heterozygosities greater than 0.50 (46.43%). The polymorphism information content (PIC) of 28 loci ranged from 0.04 to 0.88 with an average of 0.42. All the above markers were used to screen the polymorphism in other bird species. Two markers produced specific monomorphic products with the chicken DNA. Fourteen markers generated specific fragments with the goose DNA: 5 were polymorphic and 9 were monomorphic. But no specific product was detected with the peacock DNA. Based on sequence comparisons of the flanking sequence and repeat, we conclude that 2 chicken loci and 14 goose loci were true homologous loci of the duck loci. The microsatellite markers identified and characterized in the present study will contribute to the genetic map, quantitative traits mapping, and phylogenetic analysis in the duck and goose.     

Development and characterization of microsatellite markers for Actaea racemosa (black cohosh, Ranunculaceae)

? Premise of the study: Microsatellite markers were developed in Actaea racemosa to analyze population genetic structure, compare genetic diversity across the species' range, and provide a genetic context for studies of phytochemical variation. ? Methods and Results: A total of seven polymorphic loci were screened in 60 individuals from 12 localities. The number of alleles per locus ranged from three to six, and observed heterozygosity ranged from 0.133 to 0.900. Most of the loci tested cross-amplified in A. pachypoda, A. podocarpa, and A. rubra, indicating the utility of these markers for the genus. ? Conclusions: These new loci will provide tools for population genetics studies, including the characterization of genetic variation in A. racemosa and other eastern North American species of Actaea.     

Microsatellite markers for population studies of Phytophthora megakarya (Pythiaceae), a cacao pathogen in Africa

? Premise of the study: Phytophthora megakarya is the agent of black pod disease of cacao and is the main pathogen of this crop in Africa. Population genetic studies are required to investigate how this pathogen emerged. To this end, we developed 12 novel polymorphic microsatellite markers for P. megakarya. ? Methods and Results: Microsatellite sequences were obtained by pyrosequencing of multiplex-enriched libraries. Candidate loci with di- or trinucleotide motifs were selected, and primer pairs were tested with nine P. megakarya isolates. The 12 most polymorphic and unambiguous loci were selected to develop three multiplex PCR pools. The total number of alleles varied from two to nine, depending on loci, and higher than expected heterozygosity was observed. ? Conclusions: These markers were used for population genetic studies of P. megakarya in Cameroon and for comparison with reference strains from West Africa. This is the first time that microsatellite markers have been developed for P. megakarya.     

Microsatellite markers for the endangered root holoparasite Dactylanthus taylorii (Balanophoraceae) from 454 pyrosequencing

? Premise of the study: Microsatellite loci were isolated and developed as polymorphic markers for the New Zealand endemic root holoparasite Dactylanthus taylorii for use in population and conservation genetics studies. ? Methods and Results: Shotgun 454 pyrosequencing was performed on genomic DNA pooled from three individuals of D. taylorii. From 61709 individual sequence reads, primers for 753 microsatellite loci were developed in silico and 72 of these were tested for consistent amplification and variability. Ten microsatellite loci were found to be polymorphic and consistently scorable when screened in 44 individuals from five geographically distant populations. The number of alleles per locus ranged from four to 16 with an average of 9.7, and average observed heterozygosity per locus was between 0.182 and 0.634. ? Conclusions: These polymorphic microsatellite markers establish an important resource for ongoing conservation initiatives and planned population genetic studies of D. taylorii.     

Isolation and characterization of microsatellite markers from Tiarella polyphylla (Saxifragaceae)

? Premise of the study: Microsatellite markers were developed for Tiarella polyphylla in Korea and Japan to study the genetic diversity and genetic structure of the species. ? Methods and Results: We successfully isolated a total of nine polymorphic simple sequence repeat loci. These nine loci contained from two to eight alleles per locus. The observed and expected heterozygosities ranged from 0.000 to 0.250 and 0.000 to 0.974, respectively. ? Conclusions: The markers developed in this study may be used to estimate genetic diversity, genetic structure, and historical and recent gene flow in T. polyphylla, which requires conservation and management.     

Development and characterization of 38 polymorphic microsatellite markers from an economically important fruit tree, the Indian jujube

? Premise of the study: A total of 38 polymorphic microsatellite loci of the Indian jujube (Ziziphus mauritiana), an economically important fruit tree, were developed to evaluate genetic diversity and aid in the identification of cultivars. ? Methods and Results: The 38 microsatellite markers were isolated from the Indian jujube using a magnetic bead enrichment method, and polymorphisms were identified in 24 Indian jujube cultivars. The number of alleles ranged from two to 13, with expected heterozygosity ranging from 0.261 to 0.898. The polymorphism information content ranged from 0.248 to 0.889, with a mean of 0.616. Of these 38 simple sequence repeat loci, 20 loci from Z. jujuba (Chinese jujube) were successfully amplified using the simple sequence repeat primer sets. ? Conclusions: These polymorphic loci should be useful in further studies of the genetic diversity and the identification of cultivars of both the Indian jujube and the Chinese jujube.     

Isolation and characterization of novel microsatellite markers for Avena sativa (Poaceae) (oat)

? Premise of the study: A new set of microsatellite primers was developed for Avena sativa and characterized to assess the level of genetic diversity among cultivars and wild genotypes. ? Methods and Results: Using an enrichment genomic library, 14 simple sequence repeat markers were identified. The loci of these markers were characterized and found to be polymorphic in size among 48 genotypes of oat from diverse geographical locations. The number of alleles per locus ranged from two to eight, while the observed heterozygosity ranged from 0.031 to 0.75. ? Conclusions: These newly identified microsatellite markers will facilitate genetic diversity studies, fingerprinting, and genetic mapping of oat. Moreover, these new primers for A. sativa will aid future studies of polyploidy and hybridization in other species in this genus.     

Characterization of novel microsatellite markers for Eschscholzia californica (Papaveraceae), an invasive species in central Chile

? Premise of the study: We isolated and characterized microsatellite markers for the California poppy, Eschscholzia californica, which is an invasive species in central Chile. ? Methods and Results: A total of eight polymorphic and six monomorphic loci were developed for the species. Between one and 12 alleles were observed per locus. Polymorphic loci showed heterozygosity ranging from 0 to 0.875 in a sample of 96 individuals obtained from four populations. Only one locus showed significant departures from Hardy-Weinberg equilibrium at all sites. ? Conclusions: The successful microsatellite amplification makes this set of primers an important tool for understanding the recent and future patterns of invasion and adaptation of E. californica into the new Chilean geographic area. The variation detected is currently being used in a more inclusive study that assesses population expansion in central Chile.     

Development of microsatellite markers for Ophiocordyceps sinensis (Ophiocordycipitaceae) Using an ISSR-TAIL-PCR method

? Premise of the study: Microsatellite primers were developed for Ophiocordyceps sinensis, an endangered medicinal fungus endemic to the Tibetan Plateau, to investigate its genetic diversity and population structure. ? Methods and Results: An inter-simple sequence repeat (ISSR)-thermal asymmetric interlaced (TAIL)-PCR method was established to develop microsatellite markers. A total of 30 perfect and imperfect microsatellites were identified in 48 individuals of O. sinensis from five provinces within China representing different populations. Seventeen loci were polymorphic with two to four alleles per locus, while 13 were monomorphic. ? Conclusions: The results indicate that the microsatellite markers developed here may be used in studies of population genetics and conservation biology of O. sinensis. Furthermore, the ISSR-TAIL-PCR method is a simple strategy for microsatellite marker development.     

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

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

Nuclear and chloroplast SSR markers in Paeonia delavayi (Paeoniaceae) and cross-species amplification in P. ludlowii

? Premise of the study: Microsatellite primers were developed for Paeonia delavayi and P. ludlowii (Paeoniaceae) to study their population genetics and phytogeography. ? Methods and Results: Nine polymorphic nuclear microsatellite loci were isolated from an enriched library of P. delavayi and primers were designed. The number of alleles per locus ranged from two to 16; the observed and expected heterozygosities ranged from 0.014 to 0.687 and 0.042 to 0.875, respectively. Six polymorphic chloroplast microsatellite loci were identified in P. delavayi and primers were provided. The number of alleles per locus ranged from two to six and the polymorphic information content ranged from 0.08 to 0.716. Both nuclear and chloroplast primers were successfully applicable to P. ludlowii. ? Conclusions: The markers developed here will facilitate analyses of genetic diversity, population genetic structure, phytogeographical patterns, and conservation for P. delavayi and P. ludlowii.     

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 loci for the endangered species Pityopsis ruthii (Asteraceae)

? Premise of the study: Microsatellite loci were developed for the endangered species Pityopsis ruthii and will permit genetic and conservation studies of the species. ? Methods and Results: A microsatellite-enriched library was used to develop 12 polymorphic microsatellite loci for P. ruthii. The loci amplified perfect and imperfect repeats with three to seven alleles per locus. Observed heterozygosity ranged from 0.05 to 0.80 and expected heterozygosity ranged from 0.23 to 0.75. ? Conclusions: These microsatellite loci provide a sufficient set of markers for further investigation of population genetics of P. ruthii.     

Isolation and strategies of novel tetranucleotide microsatellites with polymorphisms from different chromosomes of the rhesus monkey (Macaca mulatta)

A total of 45 tetranucleotide chromosome-specific microsatellite markers with polymorphism were developed successfully based on three reference rhesus monkey genomes and on In-silico PCR prescreening. The polymorphic information content (PIC) values of 45 polymorphic microsatellite loci ranged from 0.487 to 0.879, with an average of 0.715, which were proven to be moderate to highly polymorphic. We detected 315 alleles on 45 microsatellite loci in 24 Rhesus monkeys. The number of alleles ranged from 3 to 15 and the mean number of alleles was 7 for each locus. Accordingly, the observed and expected heterozygosities obtained were between 0.417 and 1.0 and between 0.550 and 0.908, with an average value of 0.736 and 0.767, respectively. Genetic information demonstrated that 10 loci significantly deviated from Hardy–Weinberg equilibrium (P?<?0.05). All 45 primers were not significant with regard to linkage disequilibrium (P?>?0.001). Pearson correlation indicated that the PIC value exhibited a significant negative correlation with the loci number (r?=?? 0.741, P?=?0.022), whereas the positive correlation with the number of the samples (r?=?0.847, P?=?0.070) was not significant. This may be attributed to the presence of random particularities within the loci. The T test of the sample groups indicated that the PIC difference was not significant when the number of samples was set at 10 and/or?≥?15 (P?=?0.7472?~?0.8564). These polymorphic and valuable microsatellite loci will facilitate further conservation genetics studies for rhesus monkeys and can be further applied to develop novel genetic markers for other species.

     

Nuclear microsatellite loci for Arabidopsis halleri (Brassicaceae), a model species to study plant adaptation to heavy metals

? Premise of the study: Arabidopsis halleri is a model species to study the adaptation of plants to soils contaminated by zinc, cadmium, and lead. To provide a neutral genetic background with which adaptive genetic markers could be compared, we developed highly polymorphic neutral microsatellite markers. ? Methods and Results: Using a microsatellite-enriched library method, we identified 120 microsatellite loci for quantitative trait locus (QTL) mapping analysis, of which eight primer pairs were developed in a single multiplex for population genetic studies. Analyses were performed on 508 individuals from 26 populations. All loci were polymorphic with six to 23 alleles per locus. Genetic diversity varied between 0.56 and 0.76. ? Conclusions: Our results demonstrated the value of these eight microsatellite markers to investigate neutral population genetic structure in A. halleri. To increase the resolution of population genetic analyses, we suggest adding them to the 11 markers previously developed independently.