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.     

Utilization of in silico EST–SSR markers for diversity studies in castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.)

Castor (Ricinus communis L.) a chief non-edible oilseed crop has numerous industrial applications. Systematic genetic diversity analysis utilizing DNA based markers has been quick and reliable method that ensures selection of diverse parents for exploitation of higher levels of heterosis in breeding programs. From NCBI database, 63,852 EST sequences of castor were mined. One thousand one hundred and five (1105) EST–SSRs and 1652 repeat motifs sequences were identified from 20,495 non-redundant unigene sequences. Repeat motifs consisted of 29.7 % mono nucleotide repeats, 24.8 % di nucleotide repeats, 27.27 % tri nucleotide repeats and 3.94 % tetra nucleotide repeats. Twenty eight primer pairs were chosen from SSR-containing ESTs to determine genetic diversity among 27 castor accessions. Twelve EST–SSRs showed polymorphism. Number of alleles detected were 2–3 with an average of 2.33 per locus. 150–400 bp was the size of an allele. Dendrogram analysis grouped the 27 accessions into two separate clusters. Genetic similarity coefficient of dendrogram ranged from 0.24 to 0.83. The polymorphic information content value of 0.28–0.49 revealed medium level of diversity in castor. Results of present study indicated that EST–SSRs to be efficient markers for genetic diversity studies. Knowledge on level of diversity existing in castor genotypes would be useful for breeders to plan efficient hybrid breeding programme.     

EST-SSR development from 5 Lactuca species and their use in studying genetic diversity among L. serriola biotypes

Prickly lettuce (Lactuca serriola L.) is a problematic weed of Pacific Northwest and recently developed resistance to the auxinic herbicide 2,4-D. There are no publically available simple sequence repeat (SSR) markers to tag 2,4-D resistance genes in L. serriola. Therefore, a study was conducted to develop SSR markers from expressed sequence tags (ESTs) of 5 Lactuca species. A total of 15,970 SSRs were identified among 57,126 EST assemblies belonging to 5 Lactuca species. SSR-containing ESTs (SSR-ESTs) ranged from 6.23% to 7.87%, and SSR densities ranged from 1.28 to 2.51 kb(-1) among the ESTs of 5 Lactuca species. Trinucleotide repeats were the most abundant SSRs detected during the study. As a representative sample, 45 ESTs carrying class I SSRs (≥ 20 nucleotides) were selected for designing primers and were also searched against the dbEST entries for L. sativa and Helianthus annuus (≤ 10(-50); score ≥ 100). In silico analysis of 45 SSR-ESTs showed 82% conservation across species and 68% conservation across genera. Primer pairs synthesized for the above 45 EST-SSRs were used to study genetic diversity among a collection of 22 L. serriola biotypes. Comparison of the resultant dendrogram to that developed using phenotypic evaluation of the same subset of lines showed limited correspondence. Taken together, this study reported a collection of useful SSR markers for L. serriola, confirmed transferability of these markers within and across genera, and demonstrated their usefulness in studying genetic diversity.     

In silico mining of EST-SSRs in <Emphasis Type="Italic">Arachis hypogaea</Emphasis> L. and their utilization for genetic structure and diversity analysis in cultivars/breeding lines in Odisha,India

A total of 26,685 unutilized public domain expressed sequence tags (ESTs) of Arachis hypogaea L. were analyzed to give a total of 4442 EST-SSRs, in which 517 ESTs contained more than one simple sequence repeat (SSR). Of these EST-SSRs, 2542 were mononucleotide repeats (MNRs), 803 were dinucleotide repeats (DNRs), 1043 were trinucleotide repeats (TNRs), 40 were tetranucleotide repeats (TtNRs), six were pentanucleotide repeats (PNRs) and eight were hexanucleotide repeats (HNRs). Out of these 4442 EST-SSRs, only 1160 were found to be successful in non-redundant primer design; 1060 were simple SSRs, while the remaining 100 were compound forms. Among all the motifs, MNRs were abundant, followed by TNRs and DNRs. The AAG/CTT motif was the most abundant (~33 %) TNR, while AG/CT was the most abundant DNR. For redundancy and novelty, a stringent criterion deploying three different strategies was used and a total of 782 novel EST-SSRs were added to the public domain of peanut. These novel EST-SSR markers will be useful for qualitative and quantitative trait mapping, marker-assisted selection and genetic diversity studies in cultivated peanut as well as related Arachis species. A subset of 30 novel EST-SSRs was further randomly selected for validation and genotyping studies with eight well-known cultivars and 32 advanced breeding lines (ADBX lines, ADBY lines and ADBZ lines) from Odisha state, India. The number of polymorphic markers among accessions of A. hypogaea was low; however, a set of informative EST-SSR markers detected considerable levels of genetic variability in peanut cultivars and uncharacterized breeding lines collected from Odisha. The 30 newly developed EST-SSRs from Arachis spp. showed ~97 % amplification in Cicer arientinum and 93 % in pigeon pea. Thus, the EST-SSRs developed in this study will be a very useful asset for genetic analysis, comparative genome mapping, population genetic structure and phylogenetic inferences among wild and allied species of Arachis.     

Identification, characterization and mapping of EST-derived SSRs from the cacao–Ceratocystis cacaofunesta interaction

Ceratocystis cacaofunesta is an ascomycete responsible for the lethal wilt disease of cacao (Theobroma cacao L.). Marker-assisted selection combined with conventional breeding is one powerful approach to improve cacao resistance to Ceratocystis wilt. In this study we screened a set of ESTs obtained from cacao elicited with C. cacaofunesta to identify EST-SSRs and test their efficacy for mapping. Among the 3,432 ESTs analysed, 384 contained SSRs and 428 EST-SSRs were identified, mainly dinucleotides (78.5 %), with four repeats (75.23 %), and preferentially AG/CT motif (25.47 %). Gene ontology function was assigned to the ESTs containing SSRs: 4.04 % belonged to “defense response” category, with 20.69 % of them to the sub-category “defense response to fungi”. In relation to the ORF, the same quantity of EST-SSRs was observed in the 5′ UTR, ORF and the 3′ UTR (about 30 %). From the 428 EST-SSRs identified, 12 were polymorphic, revealing a total of 41 alleles. The number of alleles per locus ranged from 2 to 6, with an average of 3.41. Four EST-SSRs were mapped on the F₂ Sca 6 × ICS 1 population segregating for Ceratocystis wilt, and were distributed on the 2, 3, 4 and 8 linkage groups. These markers will have potential applications in linkage mapping and will be valuable for the research community to improve the cacao breeding program.     

Genetic mapping and BAC assignment of EST-derived SSR markers shows non-uniform distribution of genes in the barley genome

A set of 111,090 barley expressed sequence tags (ESTs) was searched for the presence of microsatellite motifs [simple sequence repeat (SSRs)] and yielded 2,823 non-redundant SSR-containing ESTs (SSR–ESTs). From this, a set of 754 primer pairs was designed of which 525 primer pairs yielded an amplicon and as a result, 185 EST-derived microsatellite loci (EST–SSRs) were placed onto a genetic map of barley. The markers show a uniform distribution along all seven linkage groups ranging from 21 (7H) to 35 (3H) markers. Polymorphism information content values ranged from of 0.24 to 0.78 (average 0.48). To further investigate the physical distribution of the EST–SSRs in the barley genome, a bacterial artificial chromosomes (BAC) library was screened. Out of 129 markers tested, BAC addresses were obtained for 127 EST–SSR markers. Twenty-seven BACs, forming eight contigs, were hit by two or three EST–SSRs each. This unexpectedly high incidence of EST–SSRs physically linked at the sub-megabase level provides additional evidence of an uneven distribution of genes and the segmentation of the barley genome in gene-rich and gene-poor regions.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Primer sequences for developed SSR markers are available upon request from the corresponding author (A. Graner).     

A database of simple sequence repeats from cereal and legume expressed sequence tags mined in silico: survey and evaluation

Simple sequence repeats (SSRs) or microsatellites are an important class of molecular markers for genome analysis and plant breeding applications. In this paper, the SSR distributions within ESTs from the legumes soybean (Glycine max, representing 135.86 Mb), medicago (Medicago truncatula, 121.1 Mb) and lotus (Lotus japonicus, 45.4 Mb) have been studied relative to the distributions in cereals such as sorghum (Sorghum bicolor, 98.9 Mb), rice (Oryza sativa, 143.9 Mb) and maize (Zea mays, 183.7 Mb). The relative abundance, density, composition and putative annotations of di-, tri-, tetra- and penta-nucleotide repeats have been compared and SSR containing ESTs (SSR-ESTs) have been clustered to give a non-redundant set of EST-SSRs, available in a database. Further, a subset of such candidate EST-SSRs from sorghum have been tested for their ability to detect polymorphism between Striga-susceptible, stay-green drought tolerant mapping population parent 'E 36-1' and its Striga-resistant, non-stay-green counterpart 'N13'. Primer sets for 64% of the EST-SSRs tested produced a clear and specific PCR product band and 34% of these detected scorable polymorphism between the N13 and E 36-1 parental lines. Over half of these markers have been genotyped on 94 RILs from the (N13 x E 36-1)-based mapping population, with 42 markers mapping onto the ten sorghum linkage groups. This establishes the value of this database as a resource of molecular markers for practical applications in cereal and legume genetics and breeding. The primer pairs for non-redundant EST-SSRs have been designed and are freely available through the database (http://intranet.icrisat.org/gt1/ssr/ssrdatabase.html).     

番红花EST资源的SSR信息分析

从NCBI公共数据库下载得到6745条番红花EST,通过前处理得到全长为612.01 kb的无冗余EST 1431条.在这些序列中搜索出108个SSR,分布于103条EST中,出现频率为7.55%.这些EST-SSR的平均分布距离是5.67 kb.二核苷酸重复和三核苷酸重复是番红花主要的重复类型,分别占总EST-SSR...     

An in silico mining for simple sequence repeats from expressed sequence tags of zebrafish, medaka, Fundulus, and Xiphophorus

Teleost fish genome projects involving model species are resulting in a rapid accumulation of genomic and expressed DNA sequences in public databases. The expressed sequence tags (ESTs) collected in the databases can be mined for the analysis of both structural and functional genomics. In this study, we in silico analyzed 49,430 unigenes representing a total of 692,654 ESTs from four model fish for their potential use in developing simple sequence repeats (SSRs), or microsatellites. After bioinformatical mining, a total of 3,018 EST derived SSRs (EST-SSRs) were identified for 2,335 SSR containing ESTs (SSR-ESTs). The frequency of identified SSR-ESTs ranged from 1.5% for Xiphophorus to 7.3% for zebrafish. The dinucleotide repeat motif is the most abundant SSR, accounting for 47%, 52%, 64%, and 78% for medaka, Fundulus, zebrafish, and Xiphophorus, respectively. Simulation analysis suggests that a majority of these EST-SSRs have sufficient flanking sequences for polymerase chain reaction (PCR) primer design. Comparative DNA sequence analyses of SSR-ESTs identified several cross-species SSRs and sequences that may be used as cross-reference genes in comparative studies. For example, the flanking sequences of one SSR (CTG)n within the pituitary tumor-transforming gene (PTTG) 1 interacting protein (PTTGIP), showed conservation spanning the medaka, Fundulus, human, and mouse genomes. This study provides a large body of information on EST-SSRs that can be useful for the development of polymorphic markers, gene mapping, and comparative genome analysis. Functional analysis of these SSR-ESTs may reveal their role in metabolism and gene evolution of these model species.     

Exploitation of pepper EST–SSRs and an SSR-based linkage map

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F₂ individuals from an interspecific cross of TF68 × Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.Gibum Yi and Je Min Lee equally contributed to this work.     

Genome-Wide Comparative Analysis of Microsatellites in Pineapple

Pineapple (Ananas comosus (L.) Merrill) is the second most important tropical fruit in term of international trade. The availability of whole genomic sequences and expressed sequence tags (ESTs) offers an opportunity to identify and characterize microsatellite or simple sequence repeat (SSR) markers in pineapple. A total of 278,245 SSRs and 41,962 SSRs with an overall density of 728.57 SSRs/Mb and 619.37 SSRs/Mb were mined from genomic and ESTs sequences, respectively. 5′-untranslated regions (5′-UTRs) had the greatest amount of SSRs, 3.6–5.2 fold higher SSR density than other regions. For repeat length, 12 bp was the predominant repeat length in both assembled genome and ESTs. Class I SSRs were underrepresented compared with class II SSRs. For motif length, dinucleotide repeats were the most abundant in genomic sequences, whereas trinucleotides were the most common motif in ESTs. Tri- and hexanucleotides of total SSRs were more prevalent in ESTs than in the whole genome. The SSR frequency decreased dramatically as repeat times increased. AT was the most frequent single motif across the entire genome while AG was the most abundant motif in ESTs. Across six examined plant species, the pineapple genome displayed the highest density, substantially more than the second-place cucumber. Annotation and expression analyses were also conducted for genes containing SSRs. This thorough analysis of SSR markers in pineapple provided valuable information on the frequency and distribution of SSRs in the pineapple genome. This genomic resource will expedite genomic research and pineapple improvement.     

Development and integration of EST–SSR markers into an established linkage map in switchgrass

Switchgrass (Panicum virgatum L.) is a model cellulosic biofuel crop in the United States. Simple sequence repeat (SSR) markers are valuable resources for genetic mapping and molecular breeding. A large number of expressed sequence tags (ESTs) of switchgrass are recently available in our sequencing project. The objectives of this study were to develop new SSR markers from the switchgrass EST sequences and to integrate them into an existing linkage map. More than 750 unique primer pairs (PPs) were designed from 243,600 EST contigs and tested for PCR amplifications, resulting in 538 PPs effectively producing amplicons of expected sizes. Of the effective PPs, 481 amplifying informative bands in NL94 were screened for polymorphisms in a panel consisting of NL94 and its seven first-generation selfed (S1) progeny. This led to the selection of 117 polymorphic EST–SSRs to genotype a mapping population encompassing 139 S1 individuals of NL94. Of 83 markers demonstrating clearly scorable alleles in the mapping population, 79 were integrated into a published linkage map, with three linked to accessory loci and one unlinked. The newly identified EST–SSR loci were distributed in 17 of 18 linkage groups with 27 (32.5 %) exhibiting distorted segregations. The integration of EST–SSRs aided in reducing the average marker interval (cM) to 3.7 from 4.2, and reduced the number of gaps (each >15 cM) to 10 from 23. Developing new EST–SSRs and constructing a higher density linkage map will facilitate quantitative trait locus mapping and provide a firm footing for marker-assisted breeding in switchgrass.     

Development of EST-SSR markers in castor bean (Ricinus communis L.) 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 1.23?kb 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.     

EST-SSR markers derived from Laminaria digitata and its transferable application in Saccharina japonica

The public availability of numerous expressed sequence tag (EST) enables EST-based SSR (simple sequence repeat) markers to be widely used for genetics and breeding studies. In the present study, EST-SSR markers were developed from ESTs of Laminaria digitata and were transferred to the non-congeneric species Saccharina japonica. Among the 2,668 non-redundant ESTs, 83 (3.1%) ESTs containing SSR were identified totally, with an average of one SSR per 13.6 kb. Analysis of SSR motifs revealed that the trinucleotide and tetranucleotide were major motifs, accounted for 44.58% and 16.87%, respectively. Based on the 83 ESTs containing SSR, we designed 45 pairs of primers in the flanking regions of the SSR, of which 13 pairs showed polymorphism in a wild S. japonica population, and the mean alleles per locus was 3.6 (ranging from 2 to 6). The observed (Ho) and expected (He) heterozygosities of these EST-SSRs were 0.234–0.632 and 0.260–0.635, respectively. All loci were in Hardy–Weinberg equilibrium in the wild population and no linkage disequilibrium was detected among loci. The obtained EST-SSR markers can facilitate and promote related research such as ecological investigation, genetic diversity assessment and breeding practice of S. japonica as well.     

Development of EST-SSR markers through data mining and their use for genetic diversity study in Indian accessions of Jatropha curcas L.: a potential energy crop

Jatropha curcas L. is gaining importance as a potential energy crop. However, lack of sufficient numbers of molecular markers hinder current research on crop improvement in Jatropha. The expressed sequences tags (EST) sequences deposited in public databases, offers an excellent opportunity to identify simple sequence repeats (SSRs) through data mining, for further research on molecular breeding. In the present study 42,477 ESTs of J. curcas were screened, out of which 5,673 SSRs were identified with 48.8 % simple (excluding mononucleotide repeats) and 52.2 % compound repeat motifs. Amongst these repeat motifs, dinucleotide repeats were abundant (26.5 %), followed by trinucleotide (23.1 %) and tetranucleotide repeats (0.8 %). From these microsatellites, 32 EST-SSR (genic microsatellite) primer pairs were designed. These primers were used to analyze the genetic diversity among 42 accessions collected from different parts of India. Out of the 32 EST-SSR primers, 24 primer pairs exhibited polymorphism among the genotypes, with amplicons varying from one to eight, giving an average of 2.33 alleles per polymorphic marker. Polymorphic information content value ranged from 0.02 to 0.5 with an average of 0.402 indicating moderate level of informativeness within these EST-SSRs markers. The EST-SSR markers developed here will serve as a valuable resource for genetic studies, like linkage mapping, diversity analysis, quantitative trait locus/association mapping, and molecular breeding. The current study also revealed low diversity in the screened Indian Jatropha germplasm. Therefore, the future efforts must be made to broaden the gene pool of Jatropha for the creation of genetic diversity that can be further used for crop improvement through breeding.     

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and ‘Dulce’ (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.     

An Improved Brassica rapa Genetic Linkage Map and Locus-specific Variations in a Doubled Haploid Population

In this study, we describe the construction of an improved Chinese cabbage genetic linkage map by integrating simple sequence repeats (SSRs) and insertion/deletion polymorphisms (InDels) into a previously published map of a doubled haploid (DH) population. The population was derived from a cross between the Chinese cabbage line BY (Brassica rapa ssp. pekinensis) and a European turnip line MM (Brassica rapa L. ssp. rapifera). A total of 629 markers were aligned to ten linkage groups, with a total map length of 1,173.8 cM, and an average distance between markers of 1.87 cm. Of the 126 SSRs and 133 InDels mapped, 46 and 34 were novel, respectively. A comparison of the linkage map with the B. rapa genome showed that more than 93 % of the markers, including 112 SSRs and 129 InDels, could be anchored unambiguously to a specific location on one of the ten chromosomes. In most cases, the order of markers on the linkage map and physical map was similar; however, the majority of linkage groups contained a number of markers whose positions were either transposed or had moved slightly forwards or backwards. During microspore culture, it was observed that 11 SSRs and one InDel showed either variation in size, or the appearance of new marker bands in the DH lines. As a first step to addressing this SSR/InDel marker instability, six SSR and one InDel loci were sequenced, which revealed that the size variation was due mainly to changes in repeat-motif number or to the insertion/deletion of new fragments of DNA.     

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.