Development Of Simple Sequence Repeats Research Articles

Development and Characterization of Novel EST-SSRs from Larix gmelinii and Their Cross-Species Transferability.

A set of 899 L. gmelinii expression sequence tags (ESTs), available at the National Center of Biotechnology Information (NCBI), was employed to address the feasibility on development of simple sequence repeat (SSR) markers for Larch species. Totally, 634 non-redundant unigenes including 145 contigs and 489 singletons were finally identified and mainly involved in biosynthetic, metabolic processes and response to stress according to BLASTX results, gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) maps. Approximately 11.7% (74) unigenes contained 90 candidate SSRs, which were mainly trinucleotides (29, 32.2%) and dinucleotides (26, 28.9%). A relatively high frequency of SSRs was respectively found in the Open Reading Frame (ORF, about 54.4%) and 5′-untranslated region (5′-UTR, 31.2%), while a low frequency was observed in the 3′-untranslated region (3′-UTR, about 14.4%). Of the 45 novel EST-SSRs markers, nine were found to be polymorphic at two L. gmelinii populations. The number of alleles per locus (Na) ranged from two to four, and the observed (Ho) and expected (He) heterozygosity values were 0.200–0.733 and 0.408–0.604, respectively. The inbreeding coefficients (FIS) for all loci were more than zero except Lg41. Most of these 9EST-SSR markers were transferable to its related species L. kaempferi, L. principis-rupprechtii and L. olgensis. These novel EST-SSRs will be useful for further research on comparative genomics, genetic resources conservation and molecular breeding in larch trees.

Development of SSR and ILP markers in horsegram (Macrotyloma uniflorum), their characterization, cross-transferability and relevance for mapping

Simple sequence repeat (SSR) and intron length polymorphism (ILP) primers were developed and characterized from public sequence data in horsegram. All characterized primers amplified alleles in a range of 1–8 with an average of 2.64 alleles per primer. Polymorphism information content ranged from 0.048 for primers MUMS-24, MUMST-21, MUMST-26 and MUMST-29 to 0.755 in MUMS-10 with an average of 0.360. Dendrogram and principal component analysis showed that newly characterized SSR and ILP markers developed in this study distinguished horsegram germplasm lines into two groups and revealed two genetic stocks for characterized germplasm. Cross-amplification of these developed markers was tested across 12 related legume species. Nineteen primers pairs showed a maximum of 100 % cross-transferability, while a minimum of 8.3 % transferability was shown by 27 primers. As no genomic resources are available in horsegram, these newly developed markers can advance molecular breeding research in this crop species and can be helpful in germplasm characterization, diversity studies, mapping and in comparative genomic studies.

Analysis of BAC-end sequences in common bean (Phaseolus vulgaris L.) towards the development and characterization of long motifs SSRs.

The increasing volume of genomic data on the Phaseolus vulgaris species have contributed to its importance as a model genetic species and positively affected the investigation of other legumes of scientific and economic value. To expand and gain a more in-depth knowledge of the common bean genome, the ends of a number of bacterial artificial chromosome (BAC) were sequenced, annotated and the presence of repetitive sequences was determined. In total, 52,270 BESs (BAC-end sequences), equivalent to 32 Mbp (~6 %) of the genome, were processed. In total, 3,789 BES-SSRs were identified, with a distribution of one SSR (simple sequence repeat) per 8.36 kbp and 2,000 were suitable for the development of SSRs, of which 194 were evaluated in low-resolution screening. From 40 BES-SSRs based on long motifs SSRs (≥ trinucleotides) analyzed in high-resolution genotyping, 34 showed an equally good amplification for the Andean and for the Mesoamerican genepools, exhibiting an average gene diversity (H E) of 0.490 and 5.59 alleles/locus, of which six classified as Class I showed a H E ≥ 0.7. The PCoA and structure analysis allowed to discriminate the gene pools (K = 2, FST = 0.733). From the 52,270 BESs, 2 % corresponded to transcription factors and 3 % to transposable elements. Putative functions for 24,321 BESs were identified and for 19,363 were assigned functional categories (gene ontology). This study identified highly polymorphic BES-SSRs containing tri- to hexanucleotides motifs and bringing together relevant genetic characteristics useful for breeding programs. Additionally, the BESs were incorporated into the international genome-sequencing project for the common bean.

Transcriptome sequencing of two parental lines of cabbage (Brassica oleracea L. var. capitata L.) and construction of an EST-based genetic map

BackgroundExpressed sequence tag (EST)-based markers are preferred because they reflect transcribed portions of the genome. We report the development of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers derived from transcriptome sequences in cabbage, and their utility for map construction.ResultsTranscriptome sequences were obtained from two cabbage parental lines, C1184 and C1234, which are susceptible and resistant to black rot disease, respectively, using the 454 platform. A total of 92,255 and 127,522 reads were generated and clustered into 34,688 and 40,947 unigenes, respectively. We identified 2,405 SSR motifs from the unigenes of the black rot-resistant parent C1234. Trinucleotide motifs were the most abundant (66.15%) among the repeat motifs. In addition, 1,167 SNPs were detected between the two parental lines. A total of 937 EST-based SSR and 97 SNP-based dCAPS markers were designed and used for detection of polymorphism between parents. Using an F2 population, we built a genetic map comprising 265 loci, and consisting of 98 EST-based SSRs, 21 SNP-based dCAPS, 55 IBP markers derived from B. rapa genome sequence and 91 public SSRs, distributed on nine linkage groups spanning a total of 1,331.88 cM with an average distance of 5.03 cM between adjacent loci. The parental lines used in this study are elite breeding lines with little genetic diversity; therefore, the markers that mapped in our genetic map will have broad spectrum utility.ConclusionsThis genetic map provides additional genetic information to the existing B. oleracea map. Moreover, the new set of EST-based SSR and dCAPS markers developed herein is a valuable resource for genetic studies and will facilitate cabbage breeding. Additionally, this study demonstrates the usefulness of NGS transcriptomes for the development of genetic maps even with little genetic diversity in the mapping population.

Novel and useful genic-SSR markers from de novo transcriptome sequencing of radish (Raphanus sativus L.)

Simple sequence repeats (SSRs) are highly polymorphic and co-dominant markers, providing an important genomic resource for genetic research. Recently, large-scale transcriptome sequencing has become a reliable and efficient approach for the identification and development of new genic-SSR markers and has been successfully conducted in a few important plant species. However, SSR development based on transcriptome sequencing remains limited in radish (Raphanus sativus L.). In the present study, from a total of 73,084 unigenes and 150,455 contigs which were assembled from 71.95 million Illumina sequence reads of a radish taproot library, a collection of 11,928 genic-SSR loci were successfully identified in 11,311 unigene sequences. Trinucleotide repeats were the most abundant repeat units, as in many other plants, with a frequency of 52 %. Furthermore, a total of 5,503 genic-SSR primers were developed, from which 1,052 SSR primers were synthesized, and a subset of 823 (78.23 %) primers could generate stable bands. Moreover, 67 selected informative genic-SSR markers were used to determine the genetic diversity of 32 radish genotypes, in which the polymorphism information content values ranged from 0.49 to 0.89. For effective cultivar identification, a novel strategy called manual cultivar identification diagram was employed. Thirty-two radish accessions were clearly separated by six genic-SSR markers. Additionally, the cross-species/genera transferability of these SSRs was further validated in nine relatives in Brassicaceae. These results suggested that the novel genic-SSR markers, as a basis for future genetic linkage and gene tagging analysis, could be very valuable in facilitating genetic mapping, marker-assisted selection and comparative genome analysis.

A genome-wide analysis of simple sequence repeats in maize and the development of polymorphism markers from next-generation sequence data

BackgroundMaize (Zea mays ssp. mays L.), as the most important plant for staple food of several million people, animal feed and bioenergy productions, is widely cultivated around the world. Simple sequence repeats (SSRs) are widely used as molecular markers in maize genetics and breeding, but only two thousands pairs of SSRs have been published currently, which hardly satisfies for the increasing needs of geneticists and breeders. Furthermore, the increasing studies have revealed that SSRs also play a vital role in functional regulation and evolution. It is fortunate that the development of sequencing technology and bio-software provides the basis for characterization and development of SSRs in maize.ResultsIn this study, MISA was applied to identify overall 179,681 SSRs in maize reference genome B73, with an average distance of 11.46 Kbp. Their distributions within the genome in different regions were non-random, and the density followed in a descending order of UTR, promotor, intron, intergenic and CDS. Meanwhile, 82,694 (46.02%) SSRs with unique flanking sequences were selected, and then applied to analyze the polymorphism of next-generation sequencing data from 345 maize inbred lines and data from maize reference genome B73. There were 58,946 SSRs with length information results in ten or more than ten genomes, accounting for 71.28% of SSRs with unique flanking sequences, while 55,621 SSRs had polymorphism, with an average PIC value of 0.498. 250 pairs of SSR primers in different genomic regions covering all maize chromosomes were randomly chosen for the experimental validation, with an average PIC value of 0.63 in 11 elite maize inbred lines.ConclusionsOur work provided insight into the non-random distribution spatterns and compositions of SSRs in different regions of maize genome, and also developed more polymorphic SSR markers using next-generation sequencing reads. The genome-wide SSRs polymorphism markers could be useful for genetic analysis and marker-assisted selection in breeding practice, and it was also proved to be high efficient for molecular marker development via next-generation sequencing reads.

Development of simple sequence repeat (SSR) markers from Paeonia ostii to study the genetic relationships among tree peonies (Paeoniaceae)

The tree peony belongs to section Moutan of Paeonia, known as the ‘King of Flowers’ in China and has great ornamental, medicinal and economic value. In this study, 12 novel polymorphic microsatellite markers from Paeonia ostii were developed and characterized by constructing a simple sequence repeat (SSR)-enriched genomic library. In total, 42 alleles across 48 tree peony accessions were detected, with an average of 3.5 alleles per locus, the number of effective alleles ranged from 1.4 to 4.7 with an average of 2.4 per locus, the observed and expected heterozygosity per locus ranged from 0.333 to 1.000 (an average of 0.785) and from 0.298 to 0.795 (an average of 0.541), respectively. The polymorphism information content (PIC) varied from 0.257 to 0.794, with an average of 0.468. Shannon Index was ranged from 0.594 to 1.771 and the mean value was 0.906. Moreover, these markers can also be amplified in herbaceous peonies Paeonia lactiflora and Paeonia veitchii of section Paeon. A neighbor-joining (NJ) tree was constructed using the proportion of shared alleles coefficient to assess the genetic relationships among species and cultivars. In the NJ tree, the accessions in the section Paeon spanned the extremes of the dendrogram and were used as an outgroup. The accessions in the section Moutan primarily grouped into two clusters. In cluster I, Paeonia rockii and Xibei Group cultivars clustered into one branch, indicating that P. rockii was the main ancestor of the Xibei cultivars. P. rockii and its cultivars were closely related to Paeonia decomposita. All Zhongyuan Group cultivars, Paeonia×lemoinei hybrids and P. ostii formed clusterII. P. ostii grouped with Zhongyuan cultivars, indicating that P. ostii contributed significant genetic information to Zhongyuan cultivars. Zhongyuan cultivars and P.×lemoinei hybrids were divided into groups with various corresponding bootstrap values. Principal component analysis (PCA) based on gene frequency further illustrated these genetic relationships. PCA supported the results of the clustering analysis, and it is easily discernable that cultivars from the same group distributed together, while wild accessions with a close relationship to cultivars were distributed with the cultivars. The results show that these SSR markers are very powerful in studying genetic relationships and that they have the ability to cross-amplify in other Paeoniaceae family members. Furthermore, the new SSR markers will be useful tools in assessing genetic diversity, detecting quantitative trait loci (QTL) and finally in facilitating marker-assisted selection (MAS).

Development and characterization of new microsatellites for walnut (Juglans regia)

The expressed sequence tag (EST) database represents a potentially valuable resource for the development of simple sequence repeat (SSR) markers for use in evolutionary studies. EST-SSRs reveal polymorphisms not only within the source taxon, but in related taxa as well. In this paper, we describe a case study in which the publicly available walnut (Juglans regia) EST database was used to develop SSR markers for use in the genetic analysis of the widespread Juglans nigra and Carya cathayensis and an endangered species Annamocarya sinensis. A total of 7262 unigenes, including 1911 contigs and 5351 singletons, were obtained from 13,559 ESTs retrieved from the NCBI database. The 7262 unigenes were further reduced to 706 EST-SSR sequences containing 805 SSR loci. Then, 309 EST-SSR primers were randomly designed, and 77 were identified with five high across-species transferability cross-species: namely, J. regia, J. nigra, C. cathayensis, Carya dabieshanensis, and A. sinensis. Thirteen highly polymorphic EST-SSRs were further used for genetic analyses in these above five species.

Development of Pineapple Microsatellite Markers and Germplasm Genetic Diversity Analysis

Two methods were used to develop pineapple microsatellite markers. Genomic library-based SSR development: using selectively amplified microsatellite assay, 86 sequences were generated from pineapple genomic library. 91 (96.8%) of the 94 Simple Sequence Repeat (SSR) loci were dinucleotide repeats (39 AC/GT repeats and 52 GA/TC repeats, accounting for 42.9% and 57.1%, resp.), and the other three were mononucleotide repeats. Thirty-six pairs of SSR primers were designed; 24 of them generated clear bands of expected sizes, and 13 of them showed polymorphism. EST-based SSR development: 5659 pineapple EST sequences obtained from NCBI were analyzed; among 1397 nonredundant EST sequences, 843 were found containing 1110 SSR loci (217 of them contained more than one SSR locus). Frequency of SSRs in pineapple EST sequences is 1SSR/3.73 kb, and 44 types were found. Mononucleotide, dinucleotide, and trinucleotide repeats dominate, accounting for 95.6% in total. AG/CT and AGC/GCT were the dominant type of dinucleotide and trinucleotide repeats, accounting for 83.5% and 24.1%, respectively. Thirty pairs of primers were designed for each of randomly selected 30 sequences; 26 of them generated clear and reproducible bands, and 22 of them showed polymorphism. Eighteen pairs of primers obtained by the one or the other of the two methods above that showed polymorphism were selected to carry out germplasm genetic diversity analysis for 48 breeds of pineapple; similarity coefficients of these breeds were between 0.59 and 1.00, and they can be divided into four groups accordingly. Amplification products of five SSR markers were extracted and sequenced, corresponding repeat loci were found and locus mutations are mainly in copy number of repeats and base mutations in the flanking region.

Development of simple sequence repeat (SSR) markers from a genome survey of Chinese bayberry (Myrica rubra).

BackgroundChinese bayberry (Myrica rubra Sieb. and Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species.ResultsThe whole genome shotgun survey of Chinese bayberry produced 9.01Gb of sequence data, about 26x coverage of the estimated genome size of 323 Mb. The genome sequences were highly heterozygous, but with little duplication. From the initial assembled scaffold covering 255 Mb sequence data, 28,602 SSRs (≥5 repeats) were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica species: 91.14%, 89.87% and 46.84% SSRs could be used in Myrica adenophora, Myrica nana and Myrica cerifera, respectively. The UPGMA dendrogram tree showed that cultivated Myrica rubra is closely related to Myrica adenophora and Myrica nana, originating in southwest China, and very distantly related to Myrica cerifera, originating in America. These markers can be used in the construction of a linkage map and for genetic diversity studies in Myrica species.ConclusionMyrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species.

Identification and Validation of a Major Quantitative Trait Locus for Slow‐rusting Resistance to Stripe Rust in WheatF

Stripe (yellow) rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriks (Pst), is one of the most important wheat (Triticum aestivum L.) diseases and causes significant yield losses. A recombinant inbred (RI) population derived from a cross between Yanzhan 1 and Xichang 76-9 cultivars was evaluated for resistance to wheat stripe rust strain CYR32 at both the seedling and adult plant stages. Four resistance quantitative trait loci (QTLs) were detected in this population, in which the major one, designated as Yrq1, was mapped on chromosome 2DS. The strategy of using the Brachypodium distachyon genome, wheat expressed sequence tags and a draft DNA sequences (scaffolds) of the D-genome (Aegilops tauschii Coss.) for the development of simple sequence repeat (SSR) markers was successfully used to identify 147 SSRs in hexaploid wheat. Of the 19 polymorphic SSRs in the RI population, 17 SSRs were mapped in the homeologous group 2 chromosomes near Yrq1 region and eight SSRs were genetically mapped in the 2.7 cM region of Yrq1, providing abundant DNA markers for fine-mapping of Yrq1 and marker-assisted selection in wheat breeding program. The effectiveness of Yrq1 was validated in an independent population, indicating that this resistance QTL can be successfully transferred into a susceptible cultivar for improvement of stripe rust resistance.

SSR FINGERPRINTING OF BLACK RASPBERRY CULTIVARS SHOWS DISCREPANCIES IN IDENTIFICATION

Breeding progress in black raspberry (Rubus occidentalis L.) has been limited by a lack of genetic diversity in elite germplasm. Black raspberry cultivars are noted for showing very few differences, and seedlings for a lack of segregation for important traits. Genetic fingerprinting using microsatellite, or simple sequence repeat (SSR) markers, can reliably identify unique clones and evaluate diversity in black raspberry cultivars. Twenty-one black raspberry cultivars were sampled from the USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon. Black raspberry clones were also sampled from nurseries, grower’s fields, and other black raspberry breeding programs for comparison. These genotypes were compared using 18 polymorphic SSR primer pairs. The black raspberries 'Bristol,' 'Jewel,' and 'Mac Black' had consistent SSR fingerprints between sources. However, plants being sold as 'Black Hawk' and 'Cumberland' had the same fingerprint as 'Jewel'. Plants of 'Bristol', 'Cumberland', 'Munger', 'New Logan', 'Plum Farmer' and 'Shuttleworth' in the NCGR collection had identical fingerprints. Eleven unique SSR fingerprints were found among plants being grown or sold as ‘Munger’, though there was one predominant fingerprint for this cultivar. 'Allen' and 'John Robertson' were each represented by three different fingerprints from three different sources, and 'Earlysweet' and 'Jewel' had SSR alleles at multiple loci that cannot be explained by their reported pedigrees. While overall genetic diversity in black raspberry cultivars is low, discrepancies in the naming of clones appear to be widespread in commercial and research plantings. Future work in this area should focus on sampling additional independent sources of plant material and evaluating clones to determine the extent of performance differences. Further SSR development in black raspberry may be needed to fingerprint some unique clones.

Analysis of expressed sequence tags from grapevine flower and fruit and development of simple sequence repeat markers

A total of 6,230 EST sequences were produced from 7,561 clones in a cDNA library generated from grapevine (Vitis vinifera cv. 'Summer Black') flower and fruit tissues in this study. After cluster and assembly analysis of the datasets, 3,582 unigenes (GenBank accession numbers GW836604-GW840185) were established, among which 381 were new grapevine EST sequences. Out of the 381 new ESTs, 289 could be mapped on the 19 grapevine chromosomes. 913 unique ESTs with known or putative functions were assigned to 11 putative cellular roles. 540 potentially workable grapevine EST-SSRs were developed from 3,582 unigenes and about 42.6% of these unigenes were identified as true-to-type SSR loci and could amplify polymorphic bands from 22 individual plants of V. vinifera L, indicating that grapevine EST datasets are a valuable source for the development of functional simple sequence repeat (SSR) markers.

Development of simple sequence repeat (SSR) markers that are polymorphic between cultivars in Brassica rapa subsp. rapa

Simple sequence repeats (SSRs) markers were developed through data mining of 3,803 expressed sequence tags (ESTs) previously published. A total of 144 di- to penta-type SSRs were identified and they were screened for polymorphism between two turnip cultivars, ‘Tsuda’ and ‘Yurugi Akamaru’. Out of 90 EST-SSRs for which polymerase chain reaction (PCR) primers were designed, 85 could be successfully amplified, 13 showed polymorphism and eight were regarded as valid markers based on Hardy-Weinberg equilibrium (HWE) in the F 2 population developed from a cross between the two cultivars. Additionally, 1551 SSR markers reported in public database were also subjected to polymorphism survey. Among these SSR markers, 66 showed polymorphism between the two cultivars, while 28 showed HWE with Ho in a range of 0.4 to 0.6. A linkage map was constructed using these eight EST-SST markers and 28 published SSR markers in the F 2 population. Key words: EST-SSR, linkage map, polymorphism, single sequence repeats, turnip.

Using next‐generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.

In silico polymorphism analysis for the development of simple sequence repeat and transposon markers and construction of linkage map in cultivated peanut

BackgroundPeanut (Arachis hypogaea) is an autogamous allotetraploid legume (2n = 4x = 40) that is widely cultivated as a food and oil crop. More than 6,000 DNA markers have been developed in Arachis spp., but high-density linkage maps useful for genetics, genomics, and breeding have not been constructed due to extremely low genetic diversity. Polymorphic marker loci are useful for the construction of such high-density linkage maps. The present study used in silico analysis to develop simple sequence repeat-based and transposon-based markers.ResultsThe use of in silico analysis increased the efficiency of polymorphic marker development by more than 3-fold. In total, 926 (34.2%) of 2,702 markers showed polymorphisms between parental lines of the mapping population. Linkage analysis of the 926 markers along with 253 polymorphic markers selected from 4,449 published markers generated 21 linkage groups covering 2,166.4 cM with 1,114 loci. Based on the map thus produced, 23 quantitative trait loci (QTLs) for 15 agronomical traits were detected. Another linkage map with 326 loci was also constructed and revealed a relationship between the genotypes of the FAD2 genes and the ratio of oleic/linoleic acid in peanut seed.ConclusionsIn silico analysis of polymorphisms increased the efficiency of polymorphic marker development, and contributed to the construction of high-density linkage maps in cultivated peanut. The resultant maps were applicable to QTL analysis. Marker subsets and linkage maps developed in this study should be useful for genetics, genomics, and breeding in Arachis. The data are available at the Kazusa DNA Marker Database (http://marker.kazusa.or.jp).

Transferability of Sorghum Genic Microsatellite Markers to Peanut

Currently development of new marker types has shifted from anonymous DNA fragments to gene-based markers. Simple Sequence Repeats (SSRs) are useful DNA markers in plant genetic research including in peanut. However, de novo development of SSRs is expensive and time consuming. Gene-based DNA markers are transferable among related species owing to the conserved nature of genes. In this study transferability of sorghum EST-SSR (SbEST-SSR) markers to peanut was prospected. A set of 411 SbEST-SSR primer pairs were used to amplify peanut genomic DNA extracted from cultivated peanut where 39% of them successfully amplified. A comparison of amplification patterns between sorghum and peanut showed similar banding pattern with majority of transferable SbEST-SSRs. Among these transferable SSR markers, 14% have detected polymorphism among 4 resistant and 4 susceptible peanut lines for rust and late leaf spot diseases. These transferable markers will benefit peanut genome research by not only providing additional DNA markers for population genetic analyses, but also allowing comparative mapping to be possible between peanut and sorghum—a possible monocot-dicot comparison.

SSR-Primer Generator: A Tool for Finding Simple Sequence Repeats and Designing SSR-Primers

Abstract Simple sequence repeats (SSRs) are ubiquitous short tandem duplications found within eukaryotic genomes. Their length variability and abundance throughout the genome has led them to be widely used as molecular markers for crop-breeding programs, facilitating the use of marker-assisted selection as well as estimation of ge-netic population structure. Here, we report a software application, “SSR-Primer Generator” for SSR discovery, SSR-primer design, and homology-based search of in silico amplicons from a DNA sequence dataset. On sub-mission of multiple FASTA-format DNA sequences, those analyses are batch processed in a Java runtime environment (JRE) platform, in a pipeline, and the result-ing data are visualized in HTML tabular format. This ap-plication will be a useful tool for reducing the time and costs associated with the development and application of SSR markers.Keywords: EST clustering and assembly, homology- based search, in silico amplicon, PCR, simple sequence repeat (SSR), SSR-primerAvailability: SSR-Primer Generator is available at http:// 168.188.15.158:8080/ssrpg/. A source code for SSR- Primer Generator is also available from the authors upon request (yplim@cnu.ac.kr).

Development of simple sequence repeat (SSR) markers to study diversity in the herbaceous peony (Paeonia lactiflora)

The herbaceous peony (Paeonia lactiflora Pall.) is a well-known herbaceous flowering plant that yields high medicinal and economic value in Korea and China. In this study, 14 novel polymorphic microsatellite markers were developed and characterized through the construction of a simple sequence repeat (SSR)-enriched library from genomic deoxyribonucleic acid (DNA) of P. lactiflora. In total, 103 alleles across 85 P. lactifloraaccessions were detected, with an average of 7.4 alleles per locus. The values of the major allele frequency, genetic diversity, and polymorphism information content (PIC) ranged from 0.39 to 0.88 (mean = 0.61), 0.21 to 0.71 (mean = 0.50), and 0.19 to 0.67 (mean = 0.43), respectively. The mean genetic distance among the P. lactiflora accessions was 0.5559, indicating wide variation. These new SSR markers will be useful tools for assessing genetic diversity, understanding population structure, identifying genotypes, and breeding of P. lactiflora. Key words: Genetic diversity, microsatellites, Paeonia lactiflora, simple sequence repeat (SSR)-enriched library.

Development of EST–SSR markers in castor bean (Ricinus communisL.) and their utilization for genetic purity testing of hybrids

Expressed sequence tag (EST) databases offer opportunity for the rapid development of simple sequence repeat (SSR) markers in crops. Sequence assembly and clustering of 57 895 ESTs of castor bean resulted in the identification of 10 960 unigenes (6459 singletons and 4501 contigs) having 7429 SSRs. On an average, the unigenes contained 1 SSR for every 1.23kb of unigene sequence. The identified SSRs mostly consisted of dinucleotide (62.4%) and trinucleotide (33.5%) repeats. The AG class was the most common among the dinucleotide motifs (68.9%), whereas the AAG class (25.9%) was predominant among the trinucleotide motifs. A total of 611 primer pairs were designed for the SSRs, having repeat length more than or equal to 20 nucleotides, of which a set of 130 markers were tested and 92 of these yielding robust amplicons were analyzed for their utility in genetic purity assessment of castor bean hybrids. Nine markers were able to detect polymorphism between the parental lines of nine commercial castor bean hybrids (DCH-32, DCH-177, DCH-519, GCH-2, GCH-4, GCH-5, GCH-6, GCH-7, and RHC-1), and their utility in genetic purity testing was demonstrated. These novel EST-SSR markers would be a valuable addition to the growing molecular marker resources that could be used in genetic improvement programmes of castor bean.