Eleven polymorphic microsatellite loci in Lindera benzoin,Lauraceae

Microsatellite markers were developed for studies of the genetic diversity and population substructure of Lindera benzoin, Lauraceae (spicebush). Nuclear microsatellite sequences were obtained from DNA libraries that were enriched for (CA), (GA), (AAG) and (ATG) repeat motifs. From 69 microsatellite sequences, 20 primer sets were developed. Of these, 11 primer pairs resulted in amplified polymorphic loci. In 29 samples of eastern Pennsylvania spicebush plants, the number of microsatellite alleles ranged from two to 16 per locus with observed heterozygosity values ranging from 0.10 to 0.82.     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.     

Development of microsatellite markers from tartary buckwheat

A genomic library enriched with (gT)_n repeats from tartary buckwheat (Fagopyrum tataricum) was constructed using 5′-anchored PCR for the development of microsattellite markers. Sequencing analysis of 5 clones from the library showed that they all contained microsatellites (totally 10 loci), and each was unique. An additional locus-specific primer was designed according to flanking sequence. Two of the microsatellite loci of 10 tartary buckwheat varieties were amplified using an anchored primer and a locus-specific primer, which revealed a clear polymorphic pattern. The data confirmed that the degenerate primer was reliably anchoring at the 5′-end of the microsatellite, and the primers developed based on this technology could be used for diversity analysis of tartary buckwheat.     

Isolation and characterization of microsatellite loci in a polyploid alpine herb,Callianthemum miyabeanum (Ranunculaceae)

• Premise of the study: Nuclear microsatellite primers were developed to analyze the clonal diversity and population genetic structure of the endemic polyploid herb Callianthemum miyabeanum. • Methods and Results: Using a protocol for constructing microsatellite-enriched libraries, 15 primer sets were developed for use in C. miyabeanum. The number of alleles found ranged from five to 22. The estimated range of expected heterozygosities was 0.574 to 0.907, and the Shannon–Weiner diversity index ranged from 1.061 to 2.733. Cross-amplification of all loci was also successful in the closely related endemic species C. kirigishiense and C. hondoense. • Conclusions: The development of these microsatellite loci will facilitate a deeper understanding of the genetic diversity, mode of reproduction, and population structure of not only C. miyabeanum, but also the other Callianthemum species endemic to Japan.     

Isolation and charaterization of polymorphic microsatellite loci from so-iuy mullet (Mugil soiuy Basilewsky 1855)

The first set of polymorphic microsatellite markers were developed from so-iuy mullet (Mugil soiuy Basilewsky 1855). From a (GT)n-enriched genomic library, 53 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. Ten of these loci were polymorphic in a test population of 24 individuals with alleles ranging from 3 to 9, and observed and expected heterozygosities from 0.2083 to 0.9167 and from 0.2651 to 0.8812, respectively. No significant linkage disequilibrium between pairs of loci was found, but two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in Mullet. Genbo Xu and Changwei Shao Contributed equally.     

Development of thirty new polymorphic microsatellite primers for <Emphasis Type="Italic">Paeonia suffruticosa</Emphasis>

Four new microsatellite primer pairs were developed in tree peony (Paeonia suffruticosa) based on the database mining and other twenty-six primer pairs by fast isolation by AFLP of sequences containing repeats (FIASCO) method. The polymorphism of each locus was further evaluated in 40 individuals of one population plus 5 tree peony related species. The number of alleles per locus ranged from 3 to 7 and the expected (H_e) and observed (H_o) heterozygosity at each locus ranged from 0.42 to 0.78 and 0.28 to 0.59, respectively. These microsatellite markers will be useful for investigating genetic diversity and studies of population genetic structure of tree peony.     

Development and characterization of microsatellite markers in a clonal plant,Dioscorea japonica Thunb.

We developed 10 microsatellite loci from genomic DNA of a dioecious clonal plant, Dioscorea japonica. Out of 384 clones, 148 contained microsatellite repeats. Polymerase chain reaction primer pairs were designed for 95 of these clones from their sequence data, of which, 10 pairs produced successful amplification. Thirty‐eight individuals were genotyped for allelic diversity. We detected three to nine alleles per locus, and the expected heterozygosity ranged from 0.461 to 0.851.     

Isolation and characterization of microsatellite markers for the clam Ruditapes philippinarum and cross-species amplification with the clam Ruditapes variegate

Ruditapes philippinarum and R. variegate are commercially important shellfish in Korea. In order to understand the processes and organization of genetic diversity and genetic resources for the sustainable management of fisheries resources we developed 13 primer pairs for microsatellite loci in R. philippinarum and tested their cross-amplification in R. variegate. Twelve primers amplified in both species. Nine loci were polymorphic in R. philippinarum and eight in R. variegate, each with nine to 26 alleles per locus. Unbiased expected heterozygosity levels varied from 0.73 to 0.94 in R. philippinarum. All polymorphic loci possessed species-specific alleles. No linkage disequilibrium was found. Results indicated that these microsatellite were highly polymorphic and will be useful for conservation genetics of both species.     

Assessment of genetic diversity and relationships of Krachaai Sayam,an endemic plant in Thailand using microsatellite markers

Nineteen microsatellite markers were developed from Boesenbergia siamensis (Gagnep.) P. Sirirugsa, an endemic plant of Kanchanaburi Province, Thailand and used to determine genetic diversity of this plant. The number of alleles of each locus varied from 4 to 16 in 25 individual samples. The average values of observed and expected heterozygosities were 0.577 and 0.804, respectively. The polymorphic information content value ranged between 0.486 and 0.912. Ten loci significantly deviated from Hardy–Weinberg equilibrium, and eight pairs of loci showed significant evidence of linkage disequilibrium. Moreover, these newly developed microsatellite markers were used to determine cross-species amplification with three samples each of B. thorelii (Gagnep.) Loes., B. rotunda (L.) Mansf. and Kaempferia parviflora Wall. Of the 19 markers developed, 13 (68.42%) primer pairs were able to be used with B. thorelii (Gagnep.) Loes. and K. parviflora Wall., while 12 (63.16%) primer pairs were able to be used with B. rotunda (L.) Mansf. These microsatellite markers will be useful for further genetic analysis of this endemic plant as well as related species.     

Characterization of polymorphic microsatellite loci in a traditional Chinese medicinal plant,Gastrodia elata

Genetic characterization of germplasm resource is essential for the conservation and efficient utilization of a traditional Chinese medicinal plant, Gastrodia elata. Thirty‐two primer pairs flanking microsatellite repeats were designed and tested using 32 individuals from eight wild populations. A total of 13 microsatellite loci were found highly polymorphic, with three to 10 alleles per locus and gene diversity ranging from 0.400 to 0.841. These microsatellites have been directly applied to the ongoing population and conservation genetics studies.     

Isolation of 39 polymorphic microsatellite loci and the development of a fluorescently labelled marker set for the Eurasian badger (Meles meles) (Carnivora: Mustelidae)

We have isolated 78 microsatellite loci from the Eurasian badger (Meles meles). Of the 52 loci characterized, 39 were found to be polymorphic. A fluorescently labelled primer set was developed to enable individual‐specific 17‐locus genotypes to be obtained efficiently.     

Development of a real-time PCR assay for the detection of Cladosporium fulvum in tomato leaves

Aims: The aim of this study was to develop a sensitive real-time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves. Methods and Results: Three PCR primer pairs were designed based on the nucleotide sequences of: (i) the internal transcribed spacer regions of ribosomal RNA; (ii) a microsatellite region amplified by the microsatellite primer M13; and (iii) the β-tubulin gene of C. fulvum. Each primer pair amplified the expected target DNA fragment from geographically diverse isolates of C. fulvum. No PCR products were amplified with these primer pairs from DNA of other fungal species. Among the three pairs of primers, the primer pair CfF1/CfR1 developed based on the microsatellite region was the most sensitive. Using this sensitive primer pair, a real-time PCR assay was developed to detect early infection of C. fulvum in tomato leaves. Significance and Impact of the Study: DNA regions amplified by the microsatellite primer M13 have a high potential for developing highly sensitive species-specific PCR primers for the detection of phytopathogenic fungi. The real-time PCR assay developed in this study is useful in monitoring early infection of C. fulvum, and can help growers make timely decisions on fungicide application.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

Isolation and characterization of microsatellite loci from the rust pathogen,Melampsora lini

We developed and characterized primers for 11 variable microsatellite loci present in the genome of the flax rust, Melampsora lini. The microsatellite loci were identified by sequencing clones from a library of EcoRI DNA fragments enriched for four simple sequence repeat motifs (AAG, AAT, TC and TG). All 11 primer pairs successfully amplified DNA fragments from a sample of 102 M. lini isolates (98 isolated from Linum marginale and four from Linum usitatissimum), revealing a total of 32 alleles. Allelic diversity at the 11 loci ranged from 0.030 to 0.449.     

Genetic Analysis of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Using Microsatellite Markers

Microsatellites are repetitive genomic elements that show high levels of variation and therefore are useful tools for studying genetic polymorphism and constructing genetic linkage maps of eukaryotic organisms. Porphyra yezoensis Ueda is an economically important seaweed that is being targeted for genetic improvement using marker-assisted breeding. Hence, in an attempt to develop microsatellite markers for P. yezoensis, a microsatellite (or simple sequence repeat)-enriched library was constructed to identify (GA)n and (CA)n motifs. A total of 71 perfect microsatellite clones were identified, of which 30 simple sequence repeat primer pairs were developed. Of these, 24 (80%) amplified polymerase chain reaction products of expected sizes. Twelve primer pairs amplified two to four bands, whereas another 12 primer pairs produced monomorphic banding patterns. Data for 12 loci were analyzed using POPGENE software version 1.32. A total of 29 alleles were produced at 12 loci, with an average of 2.42 alleles (Na) and 1.81 effective alleles (Ne) per locus. These markers were used to analyze the genetic diversity within 11 geographically different lines of P. yezoensis. Overall, these lines were clustered into two divisions with those from close geographic locations clustering together. Further cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of motif repeats in different alleles were major sources of polymorphisms.     

Isolation and charaterization of 12 dinucleotide microsatellite loci from Belenger’s jewfish (Johnius belengerii Cuvier 1830)

We describe the first isolation of 12 polymorphic microsatellite markers from Belenger’s jewfish (Johnius belengnerii Cuvier 1830). From a (GT)n-enriched genomic library, 54 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. 12 of these loci were polymorphic in a test population of 21 individuals with alleles ranging from 3 to 18, and expected and observed heterozygosities from 0.5772 to 0.9449 and from 0.4286 to 0.9231, respectively. No significant linkage disequilibrium between pairs of loci was found, however, loci Jobe24 significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in Belenger’s jewfish.     

Characterization of microsatellite loci in Primula modesta Bisset et Moore (Primulaceae)

Eleven microsatellite loci were isolated from alpine plant Primula modesta. Enriched repeat libraries were constructed and screened by colony hybridization. Forty‐eight polymerase chain reaction (PCR) primer pairs were designed, of which 11 pairs producted successful amplification. A total of 31–35 adult individuals were genotyped for allelic diversity. The polymorphism ranged from three to 14 alleles, and the heterozygosity ranged from 0.161 to 0.828. No linkage disequilibrium was found between these 11 loci.     

Development and characterization of microsatellite markers in black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned and sequenced microsatellite loci from black poplar (Populus nigra L.) and developed primers for sequence-tagged microsatellite (STMS) analysis. Twelve primer pairs for dinucleotide repeats produced fragments of sufficient quality which were polymorphic in P. nigra. Some of them also showed amplification in other Populus species (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, and/or P. lasiocarpa). The best nine and (GT) (GA) microsatellite markers were tested on a set of 23 P. nigra genotypes from all over Europe. The microsatellites were highly polymorphic, with 10–19 different alleles per microsatellite locus among these 23 genotypes. WPMS08 sometimes amplified three fragments. Using the other eight marker loci, the level of heterozygosity among the plants was on average 0.71 (range 0.25–1.00). The microsatellite markers developed will be useful for screening the genetic diversity in natural populations and in gene bank collections. Received: 21 October 1999 / Accepted: 24 November 1999     

Development and characterization of simple sequence repeat DNA markers for Zelkova serrata

We developed 14 microsatellite loci from an enriched genomic DNA library of a broad‐leaved deciduous tree, Zelkova serrata. Of 198 clones from the library, 112 contained microsatellite repeat regions. The M13‐tailed primer method was used for economy. Sequence‐specific primer pairs were designed for 58 of 76 candidate clones. Fourteen of these primer pairs successfully amplified polymorphic single loci among 34 individuals collected from the Kanto breeding region in Japan. The expected heterozygosity for the 14 microsatellite markers ranged from 0.378 to 0.876, suggesting that these will prove valuable for breeding and ecological studies on Z. serrata.     

Characterization of microsatellite loci in Lychnis flos‐cuculi (Caryophyllaceae)

We cloned microsatellite repeats from ragged robin Lychnis flos‐cuculi (Caryophyllaceae) and developed 20 primer pairs for microsatellite marker analysis. We used 18 individuals of a large Swiss population to screen microsatellites. Seven loci were polymorphic. Between seven and 11 alleles were found per locus and the observed heterozygosity was between 0.308 and 0.813. Observed heterozygote deficits were significant in five of the seven loci, in line with the mixed mating system of L. flos‐cuculi.