Development and characterization of polymorphic microsatellite markers in Momordica charantia (Cucurbitaceae)

? Premise of the study: Polymorphic microsatellite markers were developed for Momordica charantia L. to investigate the genetic diversity and population structure within and between M. charantia and its four related species (Cucurbita pepo L., Luffa cylindrical L., Lagenaria siceraria L., and Cucumis sativus L.). ? Methods and Results: Using the Fast Isolation by AFLP of Sequence COntaining Repeats (FIASCO) method, 16 polymorphic microsatellite loci were identified in 36 individuals of M. charantia. Across all the M. charantia samples, the number of alleles per locus ranged from three to eight. Seven primers successfully amplified in the four related species. ? Conclusions: These markers will be useful to study population ecology and population differentiation among M. charantia species and its related species.     

Genetic diversity in european and Argentinian cultivated potatoes (Solanum tuberosum subsp. tuberosum) detected by inter-simple sequence repeats (ISSRs).

In this study, the use of inter-simple sequence repeats (ISSR) to assess genetic diversity between cultivated potatoes (Solanum tuberosum subsp. tuberosum) is reported. ISSR technology rapidly reveals high polymorphic fingerprints and thus determines the genetic diversity among potato cultivars. Nine primers were selected according to the number of amplified markers and the level of polymorphism detected. Three primers (GAG(CAA)5, CTG(AG)8, and (AG)8) were used to cluster the 28 potato accessions and 77 polymorphic markers were sufficient to identify all of the accessions. Among the 15 simple sequence repeat (SSR) motifs tested, the most abundant were CAA and AG. Argentinian- and European-grown potatoes were easily distinguished, with a higher level of genetic diversity among potatoes from Argentina. An ISSR study using a limited number of cultivars and very few primers clearly differentiated between all cultivars, thus ISSR was revealed to be a good tool for the genetic identification of potato and for future germplasm-management programs.     

Identification and characterization of 74 novel polymorphic EST-SSR markers in the tea plant, Camellia sinensis (Theaceae)

? Premise of the study: New simple sequence repeat (SSR) markers were developed in the tea plant (Camellia sinensis) using published Expressed Sequence Tag (EST) sequences for further genetic studies and breeding programs. ? Methods and Results: A total of 74 EST-based SSR markers were generated. Polymorphism and transferability validation in 45 individuals of seven species and varieties of Camellia L. sect. Thea (L.) Dyer revealed that the number of alleles (N(A)) per locus varied between one and four or five in each species or variety. The observed heterozygosity (H(O)) and expected heterozygosity (H(E)) ranged from 0.000 to 1.000 and 0.893, respectively. ? Conclusions: These new polymorphic and transferable EST-SSR markers will have potential for applications in genetic diversity evaluation, molecular fingerprinting identification, comparative genomics analysis, and genetic mapping in the tea plant.     

Development and characterization of 41 novel EST-SSR markers for Pisum sativum (Leguminosae)

? Premise of the study: Expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers were developed in Pisum sativum for further use in genetic studies and breeding programs. ? Methods and Results: Forty-one novel EST-SSR primers were developed and characterized for size polymorphism in 32 Pisum sativum individuals from four populations from China. In each population, the number of alleles per locus ranged from one to seven, with observed heterozygosity and expected heterozygosity ranging from 0 to 0.8889 and 0 to 0.8400, respectively. Furthermore, 53.7% of these markers could be transferred to the related species, Vicia faba. ? Conclusions: The developed markers have potential for application in the study of genetic diversity, germplasm appraisal, and marker-assisted breeding in pea and other legume species.     

Characterization of microsatellite loci from Litsea hypophaea (Lauraceae), a tree endemic to Taiwan

? Premise of the study: Microsatellite primers were developed for the endemic tree Litsea hypophaea (Lauraceae) in Taiwan to investigate its genetic diversity and population genetic structure and to investigate species delimitation within Litsea. ? Methods and Results: Fifteen new simple sequence repeat markers were developed from L. hypophaea with a magnetic bead enrichment method. Most loci were also amplified from three closely related species, L. coreana, L. lii, and L. acutivena. The number of alleles and observed and expected heterozygosities across loci varied with a range of 1-25, 0.000-1.000, and 0.000-0.956, respectively. ? Conclusions: The application of these microsatellite markers of L. hypophaea provides a tool for understanding genetic diversity and population differentiation. In addition, interspecific amplification suggests that these markers will also be useful for species identification of related taxa within Litsea in Taiwan.     

Development of microsatellite markers in heterostylous Hedyotis chrysotricha (Rubiaceae)

? Premise of the study: Microsatellite primers were developed for a heterostylous herb, Hedyotis chrysotricha to investigate the effect of habitat fragmentation on its genetic diversity and population structure. ? Methods and Results: Twelve primer sets were developed and their polymorphisms were tested on 47 individuals from two island populations of H. chrysotricha in Thousand Island Lake of China. The number of alleles per locus ranged from five to 10, with an average of seven alleles. Expected heterozygosity per locus ranged from 0.284 to 0.821 and observed heterozygosity ranged from 0.191 to 0.851. ? Conclusions: We showed that all of the 12 microsatellite markers developed for H. chrysotricha are polymorphic within populations, which should provide a powerful tool for assessing population structure and genetic diversity across fragmented and continuous populations, and for studying the genetic effects of habitat fragmentation on this species.     

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.     

Generation and characterization of 11 novel est derived microsatellites from Vicia faba (Fabaceae)

? Premise of the study: Simple sequence repeat (SSR) markers were developed for faba bean using expressed sequence tags (ESTs) from the NCBI database to study for genetic diversity. ? Methods and Results: A total of 11 novel EST-SSR loci were generated and characterized when tested on four populations of 29 faba bean individuals from China and Europe. The number of alleles (A) ranged from 1 to 3 in each population, and observed heterozygosity (H(O)) and expected heterozygosity (H(E)) ranged from 0 to 0.5000 and 0.6400, respectively. Furthermore, transferable analysis revealed that eight of these loci (72.73%) amplified in Pisum sativum L., six of which (75.00%) detected polymorphism. ? Conclusions: The developed markers in this study will provide valuable tools for genetic diversity, resource conservation, genetic mapping, and marker-assisted breeding of faba bean in the future.     

Development of microsatellite markers in Lupinus luteus (Fabaceae) and cross-species amplification in other lupine species

? Premise of the study: Microsatellite primers were developed in Lupinus luteus L., an emerging temperate protein crop, to investigate genetic diversity, population structure, and to facilitate the generation of better yellow lupine varieties. ? Methods and Results: Thirteen polymorphic primer sets were evaluated in a European and Eastern European accession collection of L. luteus. The primers amplified di-, tri-, and tetranucleotide repeats with 2-4 alleles per locus. These revealed a moderate to low level of genetic variation, as indicated by an average observed heterozygosity of 0.0126. Select loci also amplified successfully in the closely related species L. hispanicus Boiss. & Reut. and in the New World species L. mutabilis Sweet. ? Conclusions: These results indicate the utility of primers for the study of genetic diversity across L. luteus populations and related lupine species. The use of these microsatellite markers will facilitate the implementation of several molecular breeding strategies in yellow lupine.     

Characterization and multiplexing of EST-SSR primers in Cynodon (Poaceae) species1

? Premise of the study: Cynodon species are multiple-use grasses that display varying levels of adaptation to biotic and abiotic stress. Previously identified EST-SSR primers were characterized and multiplexed to assess the level of genetic diversity present within a collection of almost 1200 Cynodon accessions from across Australia. ? Methods and Results: Two multiplex reactions were developed comprising a total of 16 EST-SSR markers. All SSR markers amplified across different Cynodon species and different levels of ploidy. The number of alleles ranged from one to eight per locus and the total number of alleles for the germplasm collection was 79. ? Conclusions: The 16 markers show sufficient variation for the characterization of Cynodon core collections and analysis of population genetic diversity in Cynodon grasses.     

Development of microsatellite markers in a riparian shrub, Spiraea thunbergii (Rosaceae)

? Premise of the study: Microsatellite primers in the deciduous shrub Spiraea thunbergii were developed to investigate genetic diversity and population genetic structure. Cross-species transferability was assayed in four congeneric species. ? Methods and Results: Using a compound simple sequence repeat (SSR) marker method, 10 primer sets were identified in Japanese populations of S. thunbergii. The primers amplified compound SSRs with two to five alleles per locus. More than half of the primers were also amplified in S. prunifolia, S. nipponica var. nipponica, and S. japonica. ? Conclusions: These markers might be useful for future studies of population genetics of S. thunbergii and congeneric species.     

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.     

Microsatellite marker development for the threatened orchid Masdevallia solomonii (Orchidaceae)

? Premise of the study: Microsatellite markers for Masdevallia solomonii were developed to serve as a tool in future population genetic studies of this threatened species from the Bolivian Yungas. ? Methods and Results: Thirteen microsatellite primers were characterized by cloning an intersimple sequence repeat (ISSR) library. From these, 10 loci presented considerable variation in allele number (3-10), expected heterozygosity (0.537-0.865), and polymorphic information content per locus (0.500-0.848). ? Conclusions: The markers obtained for M. solomonii are the first in the genus and subtribe. The observed polymorphism will make it possible to assess genetic diversity and structure of this species and will serve to propose effective conservation actions.     

Microsatellite primers in the endangered quillwort Isoetes hypsophila (Isoetaceae) and cross-amplification in I. sinensis

? Premise of the study: The first microsatellite primers were developed for Isoetes hypsophila, an endangered quillwort species endemic to the Qinghai-Tibetan Plateau in China, to further describe its genetic variability and population structure. We also examined their cross-amplification in a congeneric species, I. sinensis. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining Repeats (FIASCO) protocol, nine microsatellite loci were isolated and characterized in 32 samples from four natural populations of I. hypsophila. The primers amplified di- and hexanucleotide repeats with three to 11 alleles per locus. Seven of nine primers were cross-amplified in I. sinensis with two to seven alleles per locus. ? Conclusion: The microsatellite loci primers will be useful for studies of genetic diversity and gene flow in natural populations of Isoetes species.     

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.     

Isolation and characterization of microsatellite loci in a clonal herb, Cardamine leucantha (Brassicaceae)

? Premise of the study: Microsatellite markers were developed to analyze the clonal diversity and population genetic structure of a clonal plant, Cardamine leucantha. ? Methods and Results: Microsatellite loci were isolated by using compound simple sequence repeat (SSR) primers, resulting in 25 pairs including 14 useful markers. Samples from 33, 20, and 17 ramets in three populations were identified as belonging to 23, 18, and 17 genotypic individuals, respectively. ? Conclusions: These SSR markers will be used for identifying genets and evaluating genetic diversity of the clonal plant C. leucantha.     

Isolation of compound microsatellite markers for the endangered plant Neolitsea sericea (Lauraceae)

? Premise of the study: The development of compound microsatellite markers was conducted in Neolitsea sericea to investigate genetic diversity and population genetic structure of this endangered insular species. ? Methods and Results: Using the compound microsatellite marker technique, 10 compound microsatellite markers that were successfully amplified showed polymorphism when assessed in 55 individuals from two populations in East China and Japan. Overall, the number of alleles ranged from 3 to 17, with an average of 7.9 alleles per locus. In addition, these primers could be easily amplified in Neolitsea aurata var. paraciculata and N. aurata var. chekiangensis. ? Conclusions: The highly polymorphic markers developed and characterized in this study will be useful for population genetic studies of N. sericea.     

Mitochondrial DNA variation in cultivated and wild potato species (Solanum spp.).

The European cultivated potato, Solanum tuberosum subsp. tuberosum, has 6 related cultivated species and more than 200 wild relatives. In Solanum spp., studies of cytoplasmic organelles have been mainly confined to the plastid DNA composition of cultivated and wild species. In this study, 53 genotypes of 30 potato species belonging to the subsections Estolonifera and Potatoe, 2 tomato species, and a black nightshade genotype were examined using PCR markers to evaluate mitochondrial DNA diversity and assess whether mtDNA variability was correlated with series classification, geographical origin, ploidy, and endosperm balance number (EBN). The markers used revealed interspecific mtDNA variability in Solanum spp. and identified 13 different haplotypes. Intraspecific variability was also observed in a few species and genomic regions. Cluster analysis allowed arrangement of the 13 haplotypes into 7 subgroups, and statistical association tests showed significant relationships between mitochondrial patterns detected by molecular analysis and ploidy, EBN, and geographical origin. On the whole, the evolutionary patterns for the genomic regions analyzed reflected the species relationships established on the basis of morphological and molecular (nuclear and plastidial DNA) data. The mtDNA variability shown is also important for better characterization of genetic resources for potato breeding.     

Microsatellite development for an endangered riparian inhabitant, Lilaeopsis schaffneriana subsp. recurva (Apiaceae)

? Premise of the study: Microsatellite markers were developed and characterized to evaluate genetic diversity and population structure in Lilaeopsis schaffneriana subsp. recurva, an endangered species endemic to wetlands dispersed throughout southeastern Arizona, USA, and northern Sonora, Mexico. ? Methods and Results: Eight loci (one of which was monomorphic) were developed and characterized in 48 individuals from two populations. The total number of alleles was 35, ranging from one to 10 per locus. Many of the primers amplified in L. carolinensis, L. chinensis, L. masonii, L. occidentalis, L. schaffneriana subsp. schaffneriana, Oxypolis fendleri, and Eryngium lemmonii. ? Conclusions: Development of these novel microsatellite loci will facilitate a deeper understanding of genetic diversity, mode of reproduction, and population structure not only in L. schaffneriana subsp. recurva, but also in apiaceous relatives.