Development and characterization of microsatellite markers for Arenaria grandiflora L. (Caryophyllaceae)

Genomic libraries of Arenaria grandiflora enriched for di- and trinucleotide repeats were used for the development of novel microsatellite markers. The subset of 13 polymorphic markers was characterized on 40 individuals of A. grandiflora originating from lowland locations in France. The loci amplified 3 to 10 alleles per locus and expected heterozygosities ranged from 0.46 to 0.83. The newly developed markers will be used for population genetic studies and for assessing genetic composition of a restoration experiment of lowland A. grandiflora populations that are protected in France.     

Development of microsatellite markers in Dendrobium fimbriatum Hook, an endangered Chinese endemic herb

As an endangered endemic herb, Dendrobium fimbriatum, is under threat from numerous impacts. In order to analyse the genetic diversity and structure of this endangered species, we provide details of 10 microsatellite loci (out of 15 primer pairs designed) which showed polymorphic for D. fimbriatum. These loci were used to screen 25 individuals from across the species' geographical range. Ten loci were polymorphic with 2 to 19 alleles; three loci were monomorphic, while the rest produced no amplification fragments. These loci will be used to investigate population genetic structure, genetic diversity, conservation, and individual authentication in the endangered D. fimbriatum.     

Development and characterization of microsatellite markers for two dioecious Ficus species

Microsatellite markers for Ficus montana and Ficus septica were developed using genomic libraries enriched for di‐, tri‐ and tetranucleotide repeats. The subsets of five and three best scorable primer pairs were characterized on 24 F. montana and 36 F. septica individuals, respectively. For F. montana, loci showed five to 14 alleles per locus and expected heterozygosities ranged between 0.23 and 0.87. For F. septica, loci showed three to five alleles per locus and expected heterozygosities ranged between 0.36 and 0.49. Four primer pairs (two from each subset) cross‐amplified in the other species, indicating transportability of the markers within the genus Ficus.     

Development and characterization of ten new microsatellite markers in a mangrove tree species Bruguiera gymnorrhiza (L.)

Bruguiera gymnorrhiza is an ecologically and somewhat economically important mangrove tree species. We isolated 10 polymorphic microsatellite loci from B. gymnorrhiza using a dual‐suppression polymerase chain reaction (PCR) technique. These loci provided microsatellite markers with polymorphism of two to five alleles per locus within 216 individuals from nine natural populations of B. gymnorrhiza on Iriomote Island, the Sakishima Islands, Japan. The expected and observed heterozygosities ranged from 0.220 to 0.720 and from 0.104 to 0.447, respectively.     

Trinucleotide repeat microsatellite markers for black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned microsatellite repeats from black poplar (Populus nigra L.) and developed primers for microsatellite marker analysis. Ten primer pairs, mostly for trinucleotide repeats, produced polymorphic fragments in P. nigra. Some of them also showed amplification in other poplar species. (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, P. lasiocarpa). The best six loci were tested on 23 P. nigra genotypes collected across Europe. The microsatellites produced up to 12 alleles per locus in this set, with observed heterozygosity between 0.32 and 0.91.     

Isolation and characterization of microsatellite markers in Dendrobium officinale,an endangered herb endemic to China

As an invaluable herb, Dendrobium officinale has been in severe danger since 1950 because of human exploitation and habitat deterioration. In order to investigate the genetic diversity and structure of this species, we isolated and characterized 12 microsatellite loci derived from two microsatellite‐enriched libraries. Twenty‐two individuals from Leye population were analysed. These loci were polymorphic and displayed three to 12 alleles per locus. The observed and expected heterozygosities ranged from 0.150 to 0.624 and from 0.162 to 0.605, respectively. These are the first microsatellite loci characterized from D. officinale that will contribute to research on the conservation, genetic diversity, population structure and individual authentication.     

Development and characterization of microsatellite markers for Liporrhopalum tentacularis Grandi,the pollinator fig wasp of Ficus montana Blume

Microsatellite markers for the pollinator fig wasp Liporrhopalum tentacularis were developed using genomic libraries enriched for di‐, tri‐ and tetranucleotide repeats. A subset of 31 positive clones was sequenced and primers were designed. Eleven primer pairs produced polymorphic amplification products in L. tentacularis. Eight markers gave unambiguously scorable patterns and were further characterized on 29 individuals collected from different fruits of the dioecious host fig Ficus montana in Indonesia. Three to 19 alleles per locus were detected in this set of samples. The observed heterozygosity ranged between 0.10 and 0.55.     

Fifty‐three polymorphic microsatellite loci in the chestnut blight fungus,Cryphonectria parasitica

We report on 53 microsatellite loci for use in population genetic or linkage mapping studies in Cryphonectria parasitica. In 40 isolates collected from throughout the Northern Hemisphere, the number of alleles per locus ranged from two to 14 (mean 5.17) with gene diversity values ranging from 0.049 to 0.859 (mean 0.437). Samples from Asia were more diverse than those from Europe and North America. Most of the markers (48 of 53) were developed from an expressed sequence tag library, and hence, offer the opportunity to examine population structure or provide genome location information for specific expressed genes vs. anonymous genomic regions.     

Fifty‐two polymorphic microsatellite loci in the rust fungus,Cronartium quercuum f.sp. fusiforme

We report on 52 microsatellite markers for use in Cronartium quercuum f.sp. fusiforme. The markers were developed from di‐, tri‐, and tetranucleotide repeat‐enriched genomic libraries. In 46 isolates collected from two natural populations in the southeastern USA, the number of alleles per locus ranged from two to 20 (mean 6.94) with gene diversity values ranging from 0.043 to 0.933 (mean 0.537). The markers should prove highly useful for genetic ‘fingerprinting’ of single‐spore isolates commonly used in host–pathogen gene interaction studies, as marker loci for linkage mapping studies, and for examining fine‐scale population genetic structure in natural populations of the fungus.     

Development of 18 polymorphic microsatellite DNA markers of Laminaria japonica (Phaeophyceae)

Eighteen polymorphic microsatellite DNA markers were developed for Laminaria japonica, a brown alga cultured intensively in China. These markers are independent from each other and are moderately variable in L. japonica. The number of alleles and gene diversity detected in 53 gametophyte clones representing the varieties of L. japonica cultured once or currently in China range from two to nine and from 0.355 to 0.768, respectively. These markers will certainly facilitate the management and exploitation of the germplasm resource of L. japonica conserved indoor as gametophyte clones and the determination of the genetic diversity of L. japonica naturally distributed.     

PERMANENT GENETIC RESOURCES: Development of microsatellite markers for the guava rust fungus, Puccinia psidii

We developed and characterized 15 polymorphic microsatellite markers present in the genome of the guava rust fungus, Puccinia psidii. The primers for these microsatellite markers were designed by sequencing clones from a genomic DNA library enriched for a simple sequence repeat (SSR) motif of (AG). All these 15 primer pairs successfully amplified DNA fragments from a sample of 22 P. psidii isolates, revealing a total of 71 alleles. The observed heterozygosity at the 15 loci ranged from 0.05 to 1.00. The SSR markers developed would be useful for population genetics study of the rust fungus.     

Development of simple sequence repeat (SSR) markers and their use in identification of Dendrobium varieties

The aim of this study was to develop simple sequence repeat (SSR) markers for Dendrobium varieties/species, many of which have medicinal and horticultural values. Two genomic DNA libraries of Dendrobium Sonia enriched with GA repeats and CA repeats were constructed. Fourteen polymorphic SSR markers were identified when screened against 42 popular commercial Dendrobium hybrids. The average allele number was 12.0 ± 1.9 and the observed heterozyosity was averaged at 0.70. All 42 hybrids tested, except for two tissue culture mutants, were uniquely identified with the markers used. Sibling hybrids were closely clustered. Hybrids were also closer to parents. These SSR markers can be used for molecular ecology research, genetic mapping and marker‐assisted breeding. They can also help protection for new Dendrobium varieties.     

Isolation and characterization of microsatellite markers from the entomopathogenic fungus Metarhizium anisopliae

Fourteen polymorphic microsatellite markers were isolated from the entomopathogenic fungus, Metarhizium anisopliae, based on enriched genomic libraries. In order to assess allelic variability, the microsatellite loci were analysed in a collection of 34 isolates sampled from across Switzerland. The number of detected alleles in 14 loci ranged from two to eight and expected heterozygosity from 0.265 to 0.808. Because of the high expected heterozygosity, the 14 microsatellite loci are very useful for ecological studies and analysis of population diversity, and to identifying, monitoring, and tracking M. anisopliae strains applied as biological control agents.     

华仁杏EST-SSR标记引物设计与开发

随着生物科技的进步,ESTs（表达序列标签）已经成为开发SSR（简单重复序列）标记的重要资源。本文利用NCBI公共数据库下载蔷薇科EST序列22 458条,使用SSRHunter1.3软件进行了SSR搜索,从中获得22 527条SSR,应用Primer5.0软件设计并经由Oligo7.0软件检测,共得到61对EST-SSR引物。利用这些引物对8个华仁杏品种进行了PCR扩增及检测,得到10对能产生清晰多态性条带的EST-SSR标记,标明了10对引物的序列,为进一步开展华仁杏SSR分子标记辅助育种研究奠定了基础。     

Isolation and characterization of microsatellite markers from the phytopathogenic fungus Alternaria dauci

Eleven polymorphic microsatellite markers were isolated from the necrotrophic phytopathogenic fungus Alternaria dauci based on enriched genomic libraries. In order to assess allelic variability, the microsatellite loci were analysed in a collection of 43 isolates. The number of detected alleles in 11 loci ranged from two to 24 (mean 10.4). Test of cross-species amplification and sequencing of the resulting amplicons showed that some of these microsatellites could be used in different species such as Alternaria solani, Alternaria bataticola and Alternaria zinniae.     

Microsatellite markers developed using a next‐generation sequencing technique for Neotrogla spp. (Psocodea: Prionoglarididae), cave dwelling insects with sex‐reversed genitalia

The genus Neotrogla (Psocodea: Prinoglarididae) comprises four named species from Brazil. Females of this cave‐dwelling insect are characterized by a conspicuous penis‐like intromittent organ, termed a gynosome, which is inserted into the vagina‐like male genitalia during copulation. Another evolutionarily novel structure, the spermathecal plate, enables a female to simultaneously store two freshly deposited spermatophores (consisting of sperm and possibly nutritious substances) in her sperm storage organ (spermatheca). It is unknown whether the two spermatophores are derived from two different males. To investigate the mating ecology and population genetic structures of these insects with sex‐reversed genitalia, 16 novel highly polymorphic microsatellite loci were isolated and characterized based on ~2,275 Mbp genomic sequences from an undescribed Neotrogla species. Our first screening detected 99,888 candidate loci. Similar to other hemipteroid insects studied thus far, AAT motif microsatellites were conspicuously dominant. We further screened 99 sequences, for which 50 pairs of polymerase chain reaction primers were successfully designed. Sixteen of these primers successfully amplified products of the expected size in the 11 Neotrogla sp. individuals collected from two caves. The number of alleles per loci varied from two to nine, with no significant deviation from the Hardy–Weinberg equilibrium in either population. Although the caves sampled were only approximately 1 km apart, significant genetic differentiation was detected between the two populations. In total, 13, 12, 13 and 11 loci were cross‐amplified in N. aurora, N. brasiliensis, N. curvata and N. truncata, respectively, indicating the applicability of these microsatellite loci for metapopulation genetic studies in multiple Neotrogla species.     

Genetic structure of Cenococcum geophilum populations in primary successional volcanic deserts on Mount Fuji as revealed by microsatellite markers

Polymorphic simple sequence repeat (SSR) markers were used to investigate the genetic structure in a Cenococcum geophilum population associated with Salix reinii in an early successional volcanic desert at Gotenba, on the south-eastern slope of Mount Fuji in Japan, and in three other populations associated with the same host at more developed sites on the mountain, one at Fujinomiya and two at Subashiri. The genotype richness of C. geophilum tended to be higher in more developed vegetation patches as well as in more developed sites, suggesting that genotype richness increased with advanced succession because new genotypes might have been introduced into these sites over time. High genotypic similarity was observed between the Gotenba and Fujinomiya populations but not between the Gotenba and Subashiri populations, suggesting that C. geophilum genotypes in Gotenba were introduced from the direction of Fujinomiya. Genotypes in the Gotenba population were clearly distinguishable into two groups. The absence of any intermediate genotype suggests the absence of frequent recombination in this C. geophilum population associated with early successional vegetation.     

Allelic and population variation of microsatellite loci in aspen (Populus tremuloides)

To develop a robust basis for inferences about population genetics and evolution, this work assayed 192 aspens (Populus tremuloides) from 11 sites in Wisconsin, USA, for allelic and population variation at 16 microsatellite loci distributed across the Populus genome. Frequency distributions of fluorochrome-labeled alleles resolved by capillary electrophoresis were analyzed for relationships to repeat size and number. Population-level statistics were compared with those of other studies, especially in Populus. All loci were polymorphic, varying widely in the number of alleles per locus (mean = 8.25, range 2-20). Expected and observed heterozygosities were high (0.45 and 0.41, respectively), with little differentiation among populations (F(ST) = 0.006-0.045) and a moderate level of inbreeding (F(IS) = 0.09), intermediate among levels reported in studies based on isozymes. Contrary to several other reports, allele frequencies clustered tightly around the modal frequency, and the genetic diversity (measured as alleles per locus or as expected heterozygosity) was not related to either the repeat unit size or to the number of repeats.     

Development and characterization of microsatellite markers for Fagus crenata Blume

Microsatellite markers can yield high‐resolution genetic profiles for individual identification, and for parentage analysis, when evaluating gene dispersal in populations. Fagus crenata is an important dominant species in the cool temperate forests in Japan, and although many studies on the species have been conducted the patterns of gene dispersal via pollen and seeds are poorly understood. In order to be better informed about gene dispersal in Fagus crenata, we have developed 16 new microsatellite loci from an enriched library of genomic DNA. These 16 loci were highly variable, with 3–40 alleles per locus and an expected heterozygosity value of 0.11–0.98.     

Isolation of polymorphic microsatellite markers for British Euphrasia L.

Euphrasia species in Britain attract a large amount of conservation attention due to the recognition of numerous endemic taxa in what is essentially a species‐poor flora. To develop a set of research tools to investigate the evolutionary processes underlying this diversification, a membrane enrichment procedure has been used to isolate five polymorphic microsatellite loci from Euphrasia nemorosa (Pers.) Wallr. These loci amplify polymorphic products in several other British Euphrasia species.