Development and characterization of EST-SSR markers from Jatropha curcas EST database and their transferability across jatropha-related species/genus

Jatropha curcas (jatropha) is a multipurpose plant with potential as a raw material for biofuel. In the present study, a total of 43,349 expressed sequence tags (ESTs) from J. curcas were searched for type and frequency of simple sequence repeat (SSR) markers. Five thousand one hundred and seventy-five sequences were indentified to contain 6,108 SSRs with 90.8% simple and 9.2% compound repeat motifs. One hundred and sixty-three EST-SSRs were developed and used to evaluate the transferability and genetic relatedness among 4 accessions of J. curcas from China, Mexico, Thailand and Vietnam; 5 accessions of congeneric species, viz. J. gossypiifolia, dwarf J. integerrima, normal J. integerrima, J. multifida, J. podagrica; and Ricinus communis. The polymorphic markers showed 75.56–85.19% transferability among four species of Jatropha and 26.67% transferability across genera in Ricinus communis. Investigation of genetic relatedness showed that J. curcas and J. integerrima are closely related. EST-SSRs used in this study demonstrate a high efficiency of cross species/genera amplification and are useful for identifying genetic diversity of jatropha and its close taxa and to choose the desired related species for wide crossing to improve new varieties of jatropha. The markers can also be further exploited for genetic resource management and genetic improvement of related species/genera through marker-assisted breeding programs.     

Development of microsatellite markers in potato and their transferability in some members of Solanaceae

We have developed thirty new microsatellite markers in potato by screening genomic libraries and ESTs. Genomic libraries of potato cultivar Kufri Bahar were screened for sequences containing microsatellite motifs GA, GT, ACA, ATC, GAA, TAA and GATA. Using flanking sequences, PCR primers were designed for microsatellites identified from genomic libraries and ESTs. Sixteen new primer pairs from genomic libraries and fourteen from ESTs along with seven previously published primer pairs amplified PCR products in the selected genotypes comprising of 65 Solanum tuberosum lines and 14 other species of the potato gene pool. Neighbor-joining tree based on genetic distance matrix developed using microsatellite markers successfully distinguished all these genotypes in the expected size range. Seventeen microsatellites could also be cross-amplified in at least one of the five members of solanaceae, namely tomato, eggplant, pepper, petunia and tobacco. The new microsatellite markers obtained in this study will be useful in various genetic and taxonomic studies in potato and related genomes.     

Development, characterization and transferability of microsatellite markers for Cullen australasicum (Leguminosae)

The development of eight polymorphic microsatellite markers for use in the Australian native legume Cullen australasicum is described. The number of alleles per locus ranged from 3 to 8. Observed heterozygosity ranged from 0.208 to 0.667. The cross-species amplification of these eight markers and a ninth marker, which is monomorphic in the populations examined, but may be used to distinguish between species, was also tested in five other species. These microsatellite markers will be useful for investigating the population structure of natural populations of C. australasicum and other Cullen species which may be susceptible to genetic contamination via pollen mediated gene flow from planted pastures of C. australasicum.     

Development, characterization, and transferability of microsatellite markers for Kirengeshoma palmata (Hydrangeaceae)

? Premise of the study: Microsatellite markers were developed for Kirengeshoma palmata to assess the population genetics and mating pattern of this critically endangered species. ? Methods and Results: A total of 24 microsatellite markers were developed for K. palmata using an enrichment protocol. These markers were screened in 37 individuals from four populations in China and Japan, and twelve were found to be polymorphic, with the number of alleles per locus ranging from two to eight. All of these primers also amplified in K. koreana. ? Conclusions: These microsatellite markers provide a useful tool to investigate the mating system, gene flow, parentage, and population dynamics of Kirengeshoma.     

Interspecies and intergenus transferability of barley and wheat D-genome microsatellite markers

A selection of 147 wheat D-genome and 130 barley genomic simple sequence repeat (gSSR) markers were screened for their utility in Hordeum chilense, as an alien donor genome for cereal breeding. Fifty-eight wheat D-genome and 71 barley PCR primer pairs consistently amplified products from H. chilense. Nineteen wheat D-genome and 20 barley gSSR markers were polymorphic and allowed wide genome coverage of the H. chilense genome. Twenty-three of the wheat D-genome and 11 barley PCR primer pairs were suitable for studying the introgressions of H. chilense into wheat, amplifying H. chilense products of distinct size. In 88% of the markers tested, H. chilense products were maintained in the expected homeologous linkage group, as revealed by the analysis of wheat/H. chilense addition lines. Twenty-nine microsatellite markers (eight gSSRs and 21 expressed sequence tags-SSRs) uniformly distributed across the genome were tested for their utility in genetic diversity analysis within the species. Three genetic clusters are reported, in accordance with previous morphological and amplified fragment length polymorphism data. These results show that it is possible to discriminate the three previously established germplasm groups with microsatellite markers. The reported markers represent a valuable resource for the genetic characterisation of H. chilense, for the analysis of its genetic variability, and as a tool for wheat introgression. This is the first intraspecific study in a collection of H. chilense germplasm using microsatellite markers.     

EST and random genomic nuclear microsatellite markers for Streptocarpus

Microsatellite markers have been developed from standard enriched genomic libraries and a cDNA library for the genus Streptocarpus. Out of 15 loci derived from ESTs (expressed sequence tags), four gave working primer pairs, with expected heterozygosities (H_E) ranging from 0.42 to 0.86. Out of 89 genomic library derived loci, 6 gave working primer pairs, with H_E ranging from 0.63 to 0.93.     

Mapping microsatellite markers identified in porcine EST sequences

A sequence search of swine expressed sequence tags (EST) data in GenBank identified over 100 sequence files which contained a microsatellite repeat or simple sequence repeat (SSR). Most of these repeat motifs were dinucleotide (CA/GT) repeats; however, a number of tri-, tetra-, penta- and hexa-nucleotide repeats were also detected. An initial assessment of six dinucleotide and 14 higher-order repeat markers indicated that only dinucleotide markers yielded a sufficient number of informative markers (100% vs. 14% for dinucleotide and higher order repeats, respectively). Primers were designed for an additional 50 di- and one tri-nucleotide SSRs. Overall, 42 markers were polymorphic in the US Meat Animal Research Center (MARC) reference population, 17 markers were uninformative and 12 primer pairs failed to satisfactorily amplify genomic DNA. A comparison of di-nucleotide repeat vs. markers with repeat motifs of three to six bases demonstrated that 72% of dinucleotide markers were informative relative to only 7% of other repeat motifs. The difference was the result of a much higher percentage of monomorphic markers in the three to six base repeat motif markers than in the dinucleotide markers (64% vs. 14%). Either higher order repeat motifs are less polymorphic in the porcine genome or our selection criteria for repeat length of more than 17 contiguous bases was too low. The mapped microsatellite markers add to the porcine genetic map and provide valuable links between the porcine and human genome.     

Development of genomic microsatellite markers in Coffea canephora and their transferability to other coffee species.

Of the 103 accepted Coffea species, 70% are threatened with extinction but only a few of them have been studied. A set of 40 polymorphic microsatellite markers was developed using a GA/GT-enriched Coffea canephora genomic library. Amplification of these markers was tested in accessions of C. heterocalyx (a Critically Endangered species) and C. pseudozanguebariae (a Vulnerable species) belonging to different African geographical clades. All microsatellites were polymorphic in C. canephora, with a mean allele number per polymorphic locus of more than 3 (at least 9 genotypes were tested). Observed and expected heterozygosities calculated for C. canephora and C. pseudozanguebariae varied from 0.10 to 0.91 and from 0.20 to 0.77, respectively. In total, 38 primer pairs (95%) were amplified in C. heterocalyx and C. pseudozanguebariae, indicating their high level of transferability across the genus Coffea. This large marker set will be useful for more extensive genetic studies of threatened Coffea species.     

Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size

Flow cytometry analysis showed variation of nuclear DNA content among different species of Spartina. Spartina alterniflora had the biggest genome (1763.9 Mbp) and S. cynosuroides had the smallest genome (756.35 Mbp), whereas the genomes of S. patens (969.36 Mbp) and S. spartinae (979.78 Mbp) were comparable. Mining simple sequence repeats (SSR) from 1227 expressed sequence tags (EST) generated from salt stressed S. alterniflora showed an abundance of di- and tri-nucleotide repeats. Of 100 ESSR (EST-derived SSR) loci with five or more repeats, 81 loci were successfully amplified in eight S. alterniflora genotypes and 15 (22.2%) ESSR markers were polymorphic. Eleven of the 15 polymorphic ESSRs showed amplification across six different species of Spartina while 100% cross transferability was observed with at least one species of Spartina. The average number of alleles per marker was 3.9 and 5.8 within S. alterniflora and among Spartina species, respectively. The ESSR markers discriminated different members within and between species of Spartina genus.     

Cross-taxon transferability of sugarcane expressed sequence tags derived microsatellite (EST-SSR) markers across the related cereal grasses

Journal of Plant Biochemistry and Biotechnology - Sugarcane is a crop of economic importance providing sugar and bioenergy resource which has a substantial contribution to the national GDP of India...     

Development, characterization and transferability of microsatellite markers for the plant genus Phlox (Polemoniaceae)

In order to study diversification and microevolution in Phlox, we developed nine polymorphic microsatellite loci. In 20 individuals of Phlox pilosa from a single population, the average number of alleles per locus was 10.0 ± 5.1, and average observed and expected heterozygosities were 0.611 ± 0.234 and 0.769 ± 0.170, respectively. Most of these markers amplified successfully in 11 additional species of Phlox, representing a broad diversity of the genus, and some also amplified in more distantly related members of the Polemoniaceae. These microsatellite markers will be valuable for investigation of evolutionary processes in this important study system.     

Tall fescue EST-SSR markers with transferability across several grass species

Tall fescue (Festuca arundinacea Schreb.) is a major cool season forage and turf grass in the temperate regions of the world. It is also a close relative of other important forage and turf grasses, including meadow fescue and the cultivated ryegrass species. Until now, no SSR markers have been developed from the tall fescue genome. We designed 157 EST-SSR primer pairs from tall fescue ESTs and tested them on 11 genotypes representing seven grass species. Nearly 92% of the primer pairs produced characteristic simple sequence repeat (SSR) bands in at least one species. A large proportion of the primer pairs produced clear reproducible bands in other grass species, with most success in the close taxonomic relatives of tall fescue. A high level of marker polymorphism was observed in the outcrossing species tall fescue and ryegrass (66%). The marker polymorphism in the self-pollinated species rice and wheat was low (43% and 38%, respectively). These SSR markers were useful in the evaluation of genetic relationships among the Festuca and Lolium species. Sequencing of selected PCR bands revealed that the nucleotide sequences of the forage grass genotypes were highly conserved. The two cereal species, particularly rice, had significantly different nucleotide sequences compared to the forage grasses. Our results indicate that the tall fescue EST-SSR markers are valuable genetic markers for the Festuca and Lolium genera. These are also potentially useful markers for comparative genomics among several grass species.Electronic Supplementary Material Supplementary material is available for this article at .     

Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size

Flow cytometry analysis showed variation of nuclear DNA content among different species of Spartina. Spartina alterniflora had the biggest genome (1763.9 Mbp) and S. cynosuroides had the smallest genome (756.35 Mbp), whereas the genomes of S. patens (969.36 Mbp) and S. spartinae (979.78 Mbp) were comparable. Mining simple sequence repeats (SSR) from 1227 expressed sequence tags (EST) generated from salt stressed S. alterniflora showed an abundance of di- and tri-nucleotide repeats. Of 100 ESSR (EST-derived SSR) loci with five or more repeats, 81 loci were successfully amplified in eight S. alterniflora genotypes and 15 (22.2%) ESSR markers were polymorphic. Eleven of the 15 polymorphic ESSRs showed amplification across six different species of Spartina while 100% cross transferability was observed with at least one species of Spartina. The average number of alleles per marker was 3.9 and 5.8 within S. alterniflora and among Spartina species, respectively. The ESSR markers discriminated different members within and between species of Spartina genus.     

Development and characterization of microsatellite markers from tropical forage Stylosanthes species and analysis of genetic variability and cross-species transferability

A limited number of functional molecular markers has slowed the desired genetic improvement of Stylosanthes species. Hence, in an attempt to develop simple sequence repeat (SSR) markers, genomic libraries from Stylosanthes seabrana B.L. Maass & 't Mannetje (2n=2x=20) using 5' anchored degenerate microsatellite primers were constructed. Of the 76 new microsatellites, 21 functional primer pairs were designed. Because of the small number of primer pairs designed, 428 expressed sequence tag (EST) sequences from seven Stylosanthes species were also examined for SSR detection. Approximately 10% of sequences delivered functional primer pairs, and after redundancy elimination, 57 microsatellite repeats were selected. Tetranucleotides followed by trinucleotides were the major repeated sequences in Stylosanthes ESTs. In total, a robust set of 21 genomic-SSR (gSSR) and 20 EST-SSR (eSSR) markers were developed. These markers were analyzed for intraspecific diversity within 20 S. seabrana accessions and for their cross-species transferability. Mean expected (He) and observed (Ho) heterozygosity values with gSSR markers were 0.64 and 0.372, respectively, whereas with eSSR markers these were 0.297 and 0.214, respectively. Dendrograms having moderate bootstrap value (23%-94%) were able to distinguish all accessions of S. seabrana with gSSR markers, whereas eSSR markers showed 100% similarities between few accessions. The set of 21 gSSRs, from S. seabrana, and 20 eSSRs, from selected Stylosanthes species, with their high cross-species transferability (45% with gSSRs, 86% with eSSRs) will facilitate genetic improvement of Stylosanthes species globally.     

Development, characterization, and transferability to other Solanaceae of microsatellite markers in pepper (Capsicum annuum L.).

A novel set of informative microsatellite markers for pepper (Capsicum annuum L.) is provided. Screening of approximately 168 000 genomic clones and 23 174 public database entries resulted in a total of 411 microsatellite-containing sequences that could be used for primer design and functional testing. A set of 154 microsatellite markers originated from short-insert genomic libraries and 257 markers originated from database sequences. Of those markers, 147 (61 from genomic libraries and 86 from database sequences) showed specific and scoreable amplification products and detected polymorphisms between at least 2 of the 33 lines of a test panel consisting of cultivated and wild Capsicum genotypes. These informative markers were subsequently surveyed for allelic variation and information content. The usefulness of the new markers for diversity and taxonomic studies was demonstrated by the construction of consistent phylogenetic trees based on the microsatellite polymorphisms. Conservation of a subset of microsatellite loci in pepper, tomato, and potato was proven by cross-species amplification and sequence comparisons. For several informative pepper microsatellite markers, homologous expressed sequence tag (EST) counterparts could be identified in these related species that also carry microsatellite motifs. Such orthologs can potentially be used as reference markers and common anchoring points on the genetic maps of different solanaceous species.     

Tall fescue genomic SSR markers: development and transferability across multiple grass species

Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies. Currently, a very limited number of SSR markers are available for tall fescue (Festuca arundinacea Schreb.) and other forage grass species. A tall fescue genomic library enriched in (GA/CT) _n repeats was used to develop primer pairs (PPs) flanking SSRs and assess PP functionality across different forage, cereal, and turf grass species. A total of 511 PPs were developed and assessed for their utility in six different grass species. The parents and a subset of a tall fescue mapping population were used to select PPs for mapping in tall fescue. Survey results revealed that 48% (in rice) to 66% (in tall fescue) of the PPs produced clean SSR-type amplification products in different grass species. Polymorphism rates were higher in tall fescue (68%) compared to other species (46% ryegrass, 39% wheat, and 34% rice). A set of 194 SSR loci (38%) were identified which amplified across all six species. Loci segregating in the tall fescue mapping population were grouped as loci segregating from the female parent (HD28-56, 37%), the male parent (R43-64, 37%), and both parents (26%). Three percent of the loci that were polymorphic between parents were monomorphic in the pseudo F1 mapping population and the remaining loci segregated. Sequencing of amplified products obtained from PP NFFAG428 revealed a very high level of sequence similarity among the grass species under study. Our results are the first report of genomic SSR marker development from tall fescue and they demonstrate the usefulness of these SSRs for genetic linkage mapping in tall fescue and cross-species amplification.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Novel microsatellite markers for Begonia maxwelliana and transferability to 23 Begonia species of Peninsular Malaysia

Begonias are hyper-diverse and important horticultural plants. Six polymorphic microsatellite markers were developed from CT- and GT-enriched libraries of Begonia maxwelliana. The number of alleles per locus ranged from 2 to 12 and the observed heterozygosity ranged from 0.036 to 0.813. Null alleles were detected in one locus (Bma161) after Bonferroni correction. All the six markers were amplifiable in 23 selected Begonia species with the success rates of 17–100%. On average, species of the same section as B. maxwelliana (i.e. sect. Platycentrum) yielded higher transferability (91%). These markers will be useful for population genetic studies of the genus Begonia.     

Genetic diversity of Eucalyptus hybrids estimated by genomic and EST microsatellite markers

The knowledge of breeding impacts on the genetic diversity of hybrids of Eucalyptus is crucial to the exploration of genetic resources. We estimated genetic polymorphic parameters of 112 hybrids of Eucalyptus spp. using 10 genomic simple sequence repeats (SSR) markers and 10 expressed sequence tags (EST) microsatellite markers. According to Student’s t-test, there were no significant differences between genomic SSR and EST-SSR markers. Our results also revealed high polymorphism in the hybrids analyzed, indicating that both markers are appropriate for use in genetic breeding programs.     

Development and characterization of novel microsatellite markers in Puccinia striiformis f.sp. tritici and their transferability in Puccinia species

Puccinia striiformis f.sp. tritici (Pst) and P. striiformis f.sp. hordei (Psh) causing stripe rust disease in wheat and barley, respectively, are two devastating phytopathogens. Microsatellite/simple sequence repeat (SSR) markers are increasingly being utilized for analysis of genetic diversity, diagnosis, population structure and possible migratory routes of plant pathogens. In the current study, novel polymorphic SSR markers were designed for Pst using the genomic sequences of PST-78 isolate. A total of 1,191 SSR motifs, comprising 30% each of di- and tri-nucleotide type of repeats, 17% of penta-nucleotide, 15% of tetra-nucleotide and 8% of hexa-nucleotide repeats, were detected through in silico scanning of PST-78 genomic sequences. Polymorphism was detected by nine of the 50 designed SSRs (PsSSRs) in seven stripe rust pathotypes of wheat and barley. The mean number of alleles per SSR locus, mean polymorphism information content (PIC), mean heterozygosity, mean major allele frequency (MAF) and mean gene diversity were 2.33, 0.34, 0.33, 0.71 and 0.40, respectively. The dendrogram analysis suggested that newly developed PsSSR markers could distinguish stripe rust pathotypes based on their virulence phenotype. Further, the cross-genera and cross-species amplification test of these markers in 14 different rust pathotypes revealed that 9 PsSSRs are capable of amplification in Pst species infecting wild grass, followed by 6 PsSSRs in Pt, 3 PsSSRs in Pgt, 1 PsSSRs in Puccinia species on barberry and Melampsora lini. Thus, the transferability of PsSSRs to other species reduced with increasing genetic distance of target species. These newly designed SSR markers expand the available Pst SSR marker resources and allow better genetic studies.     

Characterisation of polymorphic microsatellite markers from Aegilops tauschii and transferability to the D-genome of bread wheat

Microsatellites were isolated from a Aegilops tauschii (the D-genome donor of bread wheat) library enriched for various motifs. Primers generated from the flanking region of the microsatellites were used successfully to amplify the corresponding loci in the D genome of bread wheat. Additional amplification sometimes also occurred from the A and B genomes. The majority of the microsatellites contained (GA)(n) and (GT)(n) motifs. GA and GT repeats appeared to be both more abundant in this library and more polymorphic than other types of repeats. The allele number for both types of dinucleotide repeats fitted a Poisson distribution. Deviance analysis showed that GA and GT were more polymorphic than other motifs in bread wheat. Within each motif type (di-, tri- and tetra-nucleotide repeats), repeat number has no influence on polymorphism. The microsatellites were mapped using the Triticum aestivum Courtot x Chinese Spring mapping population. A total of 100 markers was developed on this intraspecific map, mainly on the D genome. For polyploid species, isolation of microsatellites from an ancestral diploid donor seems to be an efficient way of developing markers for the corresponding genome in the polyploid plant.