Development of SSR markers derived from SSR-enriched genomic library of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Eggplant (Solanum melongena L.), also known as aubergine or brinjal, is an important vegetable in many countries. Few useful molecular markers have been reported for eggplant. We constructed simple sequence repeat (SSR)-enriched genomic libraries in order to develop SSR markers, and sequenced more than 14,000 clones. From these sequences, we designed 2,265 primer pairs to flank SSR motifs. We identified 1,054 SSR markers from amplification of 1,399 randomly selected primer pairs. The markers have an average polymorphic information content of 0.27 among eight lines of S. melongena. Of the 1,054 SSR markers, 214 segregated in an intraspecific mapping population. We constructed cDNA libraries from several eggplant tissues and obtained 6,144 expressed sequence tag (EST) sequences. From these sequences, we designed 209 primer pairs, 7 of which segregated in the mapping population. On the basis of the segregation data, we constructed a linkage map, and mapped the 236 segregating markers to 14 linkage groups. The linkage map spans a total length of 959.1 cM, with an average marker distance of 4.3 cM. The markers should be a useful resource for qualitative and quantitative trait mapping and for marker-assisted selection in eggplant breeding.     

Development and characterization of EST‐SSR markers in an East Asian temperate plant genus Diabelia (Caprifoliaceae)

A set of expressed sequence tag (EST) simple sequence repeat (SSR) markers were developed and characterized using next‐generation sequencing technology for the genus Diabelia (Caprifoliaceae). De novo assembly of RNA‐seq reads resulted in 58 669 contigs with the N50 length of 1211 bp. A total of 2746 contigs were identified to harbor SSR motifs, of which 48 primer pairs were designed and 11 were shown to be polymorphic across three morphospecies of Diabelia. When evaluated with 30 individuals, the number of alleles per locus ranged from 2 to 11 and the expected heterozygosity varied from 0.399 to 0.873, respectively. Distance‐based clustering indicated that the EST‐SSR markers can provide sufficient power to distinguish the three species (or populations). These markers will be useful for evaluating the range‐wide genetic diversity of each species and examining genetic divergence and gene flow between the three species.     

Characterization of RFLP probe sequences for gene discovery and SSR development in Sorghum bicolor (L.) Moench

In this study, we collected and analyzed DNA sequence data for 789 previously mapped RFLP probes from Sorghum bicolor (L.) Moench. DNA sequences, comprising 894 non-redundant contigs and end sequences, were searched against three GenBank databases, nucleotide (nt), protein (nr) and EST (dbEST), using BLAST algorithms. Matching ESTs were also searched against nt and nr. Translated DNA sequences were then searched against the conserved domain database (CDD) to determine if functional domains/motifs were congruent with the proteins identified in previous searches. More than half (500/894 or 56%) of the query sequences had significant matches in at least one of the GenBank searches. Overall, proteins identified for 148 sequences (17%) were consistent among all searches, of which 66 sequences (7%) contained congruent coding domains. The RFLP probe sequences were also evaluated for the presence of simple sequence repeats (SSRs) and 60 SSRs were developed and assayed in an array of sorghum germplasm comprising inbreds, landraces and wild relatives. Overall, these SSR loci had lower levels of polymorphism ( D = 0.46, averaged over 51 polymorphic loci) compared with sorghum SSRs that were isolated by library hybridization screens ( D = 0.69, averaged over 38 polymorphic loci). This result was probably due to the relatively small proportion of di-nucleotide repeat-containing markers (42% of the total SSR loci) obtained from the DNA sequence data. These di-nucleotide markers also contained shorter repeat motifs than those isolated from genomic libraries. Based on BLAST results, 24 SSRs (40%) were located within, or near, previously annotated or hypothetical genes. We determined the location of 19 of these SSRs relative to putative coding regions. In general, SSRs located in coding regions were less polymorphic ( D = 0.07, averaged over three loci) than those from gene flanking regions, UTRs and introns ( D = 0.49, averaged over 16 loci). The sequence information and SSR loci generated through this study will be valuable for application to sorghum genetics and improvement, including gene discovery, marker-assisted selection, diversity and pedigree analyses, comparative mapping and evolutionary genetic studies.     

Development of simple sequence repeat markers from bacterial artificial chromosomes without subcloning

Simple sequence repeats (SSRs) were isolated from pearl millet bacterial artificial clones (BACs) without any subcloning steps. SSR sequences were targeted using 3' end-anchored SSR primers. Flanking sequences were isolated by suppression PCR. In this pilot study, 25 SSR markers have been developed from 40 BAC pools, comprising a total of 384 clones. This novel way to develop new markers has the added advantage that mapping the SSR markers will anchor individual BACs to the genetic maps and, thus, facilitate the construction of BAC contigs.     

SSR-based linkage map with new markers using an intraspecific population of common wheat

Simple sequence repeats (SSRs) are valuable molecular markers in many plant species. In common wheat (Triticum aestivum L.), which is characteristic of its large genomes and alloploidy, SSRs are one of the most useful markers. To increase SSR marker sources and construct an SSR-based linkage map of appropriate density, we tried to develop new SSR markers from SSR-enriched genomic libraries and the public database. SSRs having (GA)n and (GT)n motifs were isolated from enriched libraries, and di- and tri-nucleotide repeats were mined from expressed sequence tags (ESTs) and DNA sequences of Triticum species in the public database. Of the 1,147 primer pairs designed, 842 primers gave accurate amplification products, and 478 primers showed polymorphism among the nine wheat lines examined. Using a doubled haploid (DH) population from an intraspecific cross between Kitamoe and Münstertaler (KM), we constructed an SSR-based linkage map that consisted of 464 loci: 185 loci from genomic libraries, 65 loci from the sequence database including ESTs, 213 loci from the SSR markers already reported, and 1 locus of morphological marker. Although newly developed SSR loci were distributed throughout all chromosomes, clustering of them around putative centromeric regions was found on several chromosomes. The total length of the KM map spanned 3,441 cM and corresponded to approximately 86% genome coverage. The KM map comprised of 23 linkage groups because two gaps of over 50 cM distance remained on chromosome 6A. This is a first report of SSR-based linkage map using single intraspecific population of common wheat. This mapping result suggests that it becomes possible to construct linkage maps with sufficient genome coverage using only SSR markers without RFLP markers, even in an intraspecific population of common wheat. Moreover, the new SSR markers will contribute to the enrichment of molecular marker resources in common wheat.     

Genetic Diversity of Buckwheat Cultivars (<Emphasis Type="Italic">Fagopyrum tartaricum</Emphasis> Gaertn.) Assessed with SSR Markers Developed from Genome Survey Sequences

Tartary buckwheat is an important edible crop as well as medicinal plant in China. More and more research is being focused on this minor grain crop because of its medicinal functions, but there is a paucity of molecular markers for tartary buckwheat due to the lack of genomics. In this study, a genome survey was carried out in tartary buckwheat, from which SSR markers were developed for analysis of genetic diversity. The survey generated 21.9 Gb raw sequence reads which were assembled into 348.34 Mb genome sequences included 204,340 contigs. The genome size was estimated to be about 497 Mb based on K-mer analysis. In total, 24,505 SSR motifs were identified and characterised from this genomic survey sequence. Most of the SSR motifs were di-nucleotide (67.14 %) and tri-nucleotide (26.05 %) repeats. AT/AT repeat motifs were the most abundant, accounting for 78.60 % of di-nucleotide repeat motifs. SSR fingerprinting of 64 accessions yielded 49.71 effective allele loci from a total of 63 with the 23 polymorphic SSR primer combinations. Analyses of the population genetic structure using SSR data strongly suggested that the 64 accessions of tartary buckwheat clustered into two separate subgroups. One group was mainly distributed in Nepal, Bhutan and the Yunnan-Guizhou Plateau regions of China; the other group was mainly derived from the Loess Plateau regions, Hunan and Hubei of China and USA. The cluster analysis of these accession’s genetic similarity coefficient by UPMGA methods strongly supported the two subgroup interpretation. However accessions from Qinghai of China could be grouped into either of the two subgroups depending on which classification method was used. This region is at the intersection of the two geographical regions associated with the two subgroups. These results and information could be used to identify and utilize germplasm resources for improving tartary buckwheat breeding.     

Development of genomic simple sequence repeat markers for linseed using next-generation sequencing technology

Linseed (Linum usitatissimum L.) is regarded as a cash crop of tomorrow because of the presence of nutraceutically important ??-linolenic acid (ALA) and lignan. However, only limited breeding progress has been made in this crop, mainly due to the lack of sufficient genetic and genomic resources. Among these, simple sequence repeats (SSR) are useful DNA markers for diversity analysis, genetic mapping and tagging traits because of their co-dominant and highly polymorphic nature. In order to develop SSR markers for linseed, we used three microsatellite isolation methods, viz., PCR Isolation of Microsatellite Arrays (PIMA), 5??-anchored PCR method, and Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). The amplified products from these methods were pooled and sequenced using the 454 GS-FLX platform. A total of 36,332 reads were obtained, which assembled into 2,183 contigs and 2,509 singlets. The contigs and the singlets contained 1,842 microsatellite motifs, with dinucleotide motifs as the most abundant repeat type (54%) followed by trinucleotide motifs (44%). Based on this, 290 SSR markers were designed, 52 of which were evaluated using a panel of 27 diverse linseed genotypes. Among the three enrichment methods, the 5??-anchored PCR method was most efficient for isolation of microsatellites, while FIASCO was most efficient for developing SSR markers. We show the utility of next-generation sequencing technology for efficiently discovering a large number of microsatellite markers in non-model plants.     

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.     

Genome‐wide microsatellite characterisation and marker development in Chenopodium quinoa

Microsatellites, as the tracts of repetitive DNA, are an essential constituent of the plant genome that holds important evolutionary significance, and have been extensively used to develop molecular makers for genetic analysis. To understand the microsatellite dynamics of quinoa genome and its relatives, in this study we performed a genome‐wide analysis of microsatellites in five Amaranthaceae species using available genome sequences. The results demonstrated that the microsatellites of the five Amaranthaceae species were characterised by relatively high proportions of mono‐, di‐ and trinucleotide repeats with A/T rich motifs, implying conservative organisation and composition of microsatellites in this family. Furthermore, a significant negative correlation between microsatellite frequencies and GC contents (r = ?.87) were observed. In total, 533,961 (89.57%) and 542,601 (89.86%) microsatellite loci could be used to develop simple sequence repeat (SSR) molecular markers, of which 7,178 were found to be polymorphic between the two sequenced quinoa cultivars, QQ74 and Real Blanca, through in silico PCR analysis. Finally, 15 SSR markers were randomly selected to validate their polymorphism across 12 quinoa accessions by wet‐lab PCR amplification. The newly developed genome‐wide SSR markers provide a useful resource for population genetics, gene mapping and molecular breeding studies in quinoa and beyond.     

Mining and validating grape (<Emphasis Type="Italic">Vitis</Emphasis> L.) ESTs to develop EST-SSR markers for genotyping and mapping

Grape expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping. An integrated pipeline including several computational tools for SSR identification and functional annotation was developed to identify 6,447 EST-SSR sequences from a total collection of 215,609 grape ESTs retrieved from NCBI. The 6,447 EST-SSRs were further reduced to 1,701 non-redundant sequences via clustering analysis, and 1,037 of them were successfully designed with primer pairs flanking the SSR motifs. From them, 150 pairs of primers were randomly selected for PCR amplification, polymorphism and heterozygosity analysis in V. vinifera cvs. Riesling and Cabernet Sauvignon, and V. rotundifolia (muscadine grape) cvs. Summit and Noble, and 145 pairs of these primers yielded PCR products. Pairwise comparisons of loci between the parents Riesling and Cabernet Sauvignon showed that 72 were homozygous in both cultivars, while 70 loci were heterozygous in at least one cultivar of the two. Muscadine parents Noble and Summit had 90 homozygous SSR loci in both parents and contained 50 heterozygous loci in at least one of the two. These EST-SSR functional markers are a useful addition for grape genotyping and genome mapping.     

Development and analysis of EST-SSRs for flax (Linum usitatissimum L.)

A set of 146,611 expressed sequence tags (ESTs) were generated from 10 flax cDNA libraries. After assembly, a total of 11,166 contigs and 11,896 singletons were mined for the presence of putative simple sequence repeats (SSRs) and yielded 806 (3.5%) non-redundant sequences which contained 851 putative SSRs. This is equivalent to one EST-SSR per 16.5 kb of sequence. Trinucleotide motifs were the most abundant (76.9%), followed by dinucleotides (13.9%). Tetra-, penta- and hexanucleotide motifs represented <10% of the SSRs identified. A total of 83 SSR motifs were identified. Motif (TTC/GAA)n was the most abundant (10.2%) followed by (CTT/AAG)n (8.7%), (TCT/AGA)n (8.6%), (CT/AG)n (6.7%) and (TC/GA)n (5.3%). A total of 662 primer pairs were designed, of which 610 primer pairs yielded amplicons in a set of 23 flax accessions. Polymorphism between the accessions was found for 248 primer pairs which detected a total of 275 EST-SSR loci. Two to seven alleles were detected per marker. The polymorphism information content value for these markers ranged from 0.08 to 0.82 and averaged 0.35. The 635 alleles detected by the 275 polymorphic EST-SSRs were used to study the genetic relationship of 23 flax accessions. Four major clusters and two singletons were observed. Sub-clusters within the main clusters correlated with the pedigree relationships amongst accessions. The EST-SSRs developed herein represent the first large-scale development of SSR markers in flax. They have potential to be used for the development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping and fingerprinting cultivars for example. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mining and gene ontology based annotation of SSR markers from expressed sequence tags of Humulus lupulus

Humulus lupulus is commonly known as hops, a member of the family moraceae. Currently many projects are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. The genetically characterized domains in these databases are limited due to non-availability of reliable molecular markers. The large data of EST sequences are available in hops. The simple sequence repeat markers extracted from EST data are used as molecular markers for genetic characterization, in the present study. 25,495 EST sequences were examined and assembled to get full-length sequences. Maximum frequency distribution was shown by mononucleotide SSR motifs i.e. 60.44% in contig and 62.16% in singleton where as minimum frequency are observed for hexanucleotide SSR in contig (0.09%) and pentanucleotide SSR in singletons (0.12%). Maximum trinucleotide motifs code for Glutamic acid (GAA) while AT/TA were the most frequent repeat of dinucleotide SSRs. Flanking primer pairs were designed in-silico for the SSR containing sequences. Functional categorization of SSRs containing sequences was done through gene ontology terms like biological process, cellular component and molecular function.     

Development of a set of public SSR markers derived from genomic sequence of a rapid cycling <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. genotype

The traditional development of simple sequence repeat (SSR) or microsatellite markers by probe hybridization can be time-consuming and requires the use of specialized laboratory equipment. In this study, probe hybridization was circumvented by using sequence information on 3,500 genomic clones mainly from Brassica oleracea to identify di, tri, tetra and penta-nucleotide repeats. A total of 587 primer pairs flanking SSR were developed using this approach. From these, 420 SSR markers amplified DNA in two parental lines of B. rapa (26% were polymorphic) and 523 in two parental lines of B. oleracea (32% were polymorphic). A diverse array of motif types was identified, characterized and compared with traditional SSR detection methods. The most abundant motifs found were di- (38%) and trinucleotides (33%) followed by penta- (16%) and tetranucleotide (13%) motifs. The type of motif class, motif length and repeat were not indicative of polymorphisms. The frequency of B. oleracea SSRs in genomic shotgun sequence was estimated to be 1 every 4 Kb. In general, the average motif length and repeat numbers were shorter than those obtained previously by probe hybridization, and they contained a more balanced representation of SSR motif types in the genome by identifying those that do not hybridize well to DNA probes. Brassica genomic DNA sequence information is a promising resource for developing a large number of SSR molecular markers in Brassica species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development and mapping of a public reference set of SSR markers in Lolium perenne L.

We report on the characterization and mapping of 76 simple sequence repeat (SSR) markers for Lolium perenne. These markers are publicly available or obtained either from genomic libraries enriched for SSR motifs or L. perenne expressed sequence tag (EST) clones. Four L. perenne mapping populations were used to map the SSR markers. A consensus linkage map of the four mapping populations containing 65 of the SSR markers is presented, together with primer information and a quality score indicating the usefulness of the SSR marker in different populations. The SSR markers identified all seven L. perenne linkage groups.     

Efficiency of microsatellite enrichment in<Emphasis Type="Italic">Prosopis chilensis</Emphasis> using magnetic capture

Microsatellites (i.e., simple sequence repeats [SSRs]) are highly variable genetic markers that are widely used at an intraspecific level in population genetic studies. Here we employed an enrichment strategy for microsatellite isolation by using microsatellite oligoprobes and magnetic capture of the fragments (Fischer and Bachmann, 1998) inProsopis chilensis (Mol.) Stuntz (Fabaceae). We analyzed the obtained level of enrichment by sequencing 120 enriched genomic fragments. A total of 521 SSR motives were detected. According to specific search criteria (SSR motifs ≥3 repeat units and ≥6 bp length), 95.8% of the clones contained SSR motifs. Of these, 7.8% showed homology to chloroplast sequences and 92.2% to nuclear sequences. When regarding only nuclear SSRs with 5 or more repeat units and a minimum length of 10 bp, the level of enrichment was 30.8%. A FASTA search against the European Molecular Biology Laboratory (EMBL) database univocally revealed 4 clones in transcribed regions, 102 clones in genomic regions with unknown function, and 9 clones in chloroplast regions. Among the loci with longer repeat units (≥10 bp, ≥5 repeat units), 3 were in transcribed regions and 65 were in other genomic regions. We discuss the applicability of these markers for population genetic studies.     

Identification and characterization of SSRs from soybean (Glycine max) ESTs

Microsatellites, or simple sequence repeats (SSRs), are very useful molecular markers for a number of plant species. We used a new publicly available module (TROLL) to extract microsatellites from the public database of soybean expressed sequence tag (EST) sequences. A total of 12,833 sequences containing di- to penta-type SSRs were identified from 200,516 non-redundant soybean ESTs. On average, one SSR was found per 7.25?kb of EST sequences, with the tri-nucleotide motifs being the most abundant. Primer sequences flanking the SSR motifs were successfully designed for 9,638 soybean ESTs using the software primer3.0 and only 59 pairs of them were found in earlier studies. We synthesized 124 pairs of the primers to determine the polymorphism and heterozygosity among eight genotypes of soybean cultivars, which represented a wide range of the cultivated soybean cultivars. PCR amplification products with anticipated SSRs were obtained with 81 pairs of primers; 36 PCR products appeared to be homozygous and the remaining 45 PCR products appeared to be heterozygous and displayed polymorphism among the eight cultivars. We further analysed the EST sequences containing 45 polymorphic EST-SSR markers using the programs BLASTN and BLASTX. Sequence alignment showed that 29 ESTs have homologous sequences and 15 ESTs could be classified into a Uni-gene cluster with comparatively convincing protein products. Among these 15 ESTs belonging to a Uni-gene cluster, 9 SSRs were located in 3'-UTR, 4 SSRs were located in the intron region and 2 SSRs were located in the CDS region. None of these SSRs was located in the 5'-UTR. These novel SSRs identified in the ESTs of soybean provide useful information for gene mapping and cloning in future studies.     

Development of Gene-Based SSR Markers in Rice Bean (Vigna umbellata L.) Based on Transcriptome Data

Rice bean (Vigna umbellata (Thunb.) Ohwi & Ohashi) is a warm season annual legume mainly grown in East Asia. Only scarce genomic resources are currently available for this legume crop species and no simple sequence repeat (SSR) markers have been specifically developed for rice bean yet. In this study, approximately 26 million high quality cDNA sequence reads were obtained from rice bean using Illumina paired-end sequencing technology and assembled into 71,929 unigenes with an average length of 986 bp. Of these unigenes, 38,840 (33.2%) showed significant similarity to proteins in the NCBI non-redundant protein and nucleotide sequence databases. Furthermore, 30,170 (76.3%) could be classified into gene ontology categories, 25,451 (64.4%) into Swiss-Prot categories and 21,982 (55.6%) into KOG database categories (E-value < 1.0E-5). A total of 9,301 (23.5%) were mapped onto 118 pathways using the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database. A total of 3,011 genic SSRs were identified as potential molecular markers. AG/CT (30.3%), AAG/CTT (8.1%) and AGAA/TTCT (20.0%) are the three main repeat motifs. A total of 300 SSR loci were randomly selected for validation by using PCR amplification. Of these loci, 23 primer pairs were polymorphic among 32 rice bean accessions. A UPGMA dendrogram revealed three major clusters among 32 rice bean accessions. The large number of SSR-containing sequences and genic SSRs in this study will be valuable for the construction of high-resolution genetic linkage maps, association or comparative mapping and genetic analyses of various Vigna species.