Genome-Wide Comparative Analyses of Microsatellites in Papaya

Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic and universally distributed in eukaryotes. SSRs have been used extensively as sequence tagged markers in genetic studies. Recently, the functional and evolutionary importance of SSRs has received considerable attention. Here we report the mining and characterization of the SSRs in papaya genome. We analyzed SSRs from 277.4 Mb of whole genome shotgun (WGS) sequences, 51.2 Mb bacterial artificial chromosome (BAC) end sequences (BES), and 13.4 Mb expressed sequence tag (EST) sequences. The papaya SSR density was one SSR per 0.7 kb of DNA sequence in the WGS, which was higher than that in BES and EST sequences. SSR abundance was dramatically reduced as the repeat length increased. According to SSR motif length, dinucleotide repeats were the most common motif in class I, whereas hexanucleotides were the most copious in class II SSRs. The tri- and hexanucleotide repeats of both classes were greater in EST sequences compared to genomic sequences. In class I SSR, AT and AAT were the most frequent motifs in BES and WGS sequences. By contrast, AG and AAG were the most abundant in EST sequences. For SSR marker development, 9,860 primer pairs were surveyed for amplification and polymorphism. Successful amplification and polymorphic rates were 66.6% and 17.6%, respectively. The highest polymorphic rates were achieved by AT, AG, and ATG motifs. The genome wide analysis of microsatellites revealed their frequency and distribution in papaya genome, which varies among plant genomes. This complete set of SSRs markers throughout the genome will assist diverse genetic studies in papaya and related species.     

Sixteen new simple sequence repeat markers from Brassica juncea expressed sequences and their cross‐species amplification

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 16 expressed sequence tags (EST)‐SSR markers from Brassica juncea and their cross‐amplification across Brassica species. Sixteen primer pairs were assessed for polymorphism in all genomes of the diploid and amphidiploid Brassica species. The markers show reliable amplification, considerable polymorphism and high transferability across species, demonstrating the utility of EST‐SSRs for genetic analysis of brassicas.     

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley ( Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3'-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5'-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 +/- 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.     

Mining and charactering microsatellites in Cucumis melo expressed sequence tags from sequence database

Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) are valuable markers because they represent transcribed regions and often have putative functions. We mined and characterized microsatellites in melon ESTs. Three hundred and eighty‐three SSR loci were identified in 309 of 3188 unigenes assembled by 5747 EST and mRNA sequences in GenBank with occurring frequency of 1/4.7 kb. Twenty‐two polymorphic EST‐SSR markers were developed with the mean allele number of 2.9 per locus and mean expected heterozygosity of 0.442. Amplification products were also detected by 15 pairs of primer in Cucumis sativus. Those informative EST‐SSR markers can be used in melon genetic improvement projects.     

Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)‐SSR markers from Brassica napus and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST‐SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.     

Characterization and compilation of polymorphic simple sequence repeat (SSR) markers of peanut from public database

ABSTRACT: BACKGROUND: There are several reports describing thousands of SSR markers in the peanut (Arachis hypogaea L.) genome. There is a need to integrate various research reports of peanut DNA polymorphism into a single platform. Further, because of lack of uniformity in the labeling of these markers across the publications, there is some confusion on the identities of many markers. We describe below an effort to develop a central comprehensive database of polymorphic SSR markers in peanut. FINDINGS: We compiled 1,343 SSR markers as detecting polymorphism (14.5%) within a total of 9,274 markers. Amongst all polymorphic SSRs examined, we found that AG motif (36.5%) was the most abundant followed by AAG (12.1%), AAT (10.9%), and AT (10.3%).The mean length of SSR repeats in dinucleotide SSRs was significantly longer than that in trinucleotide SSRs. Dinucleotide SSRs showed higher polymorphism frequency for genomic SSRs when compared to trinucleotide SSRs, while for EST-SSRs, the frequency of polymorphic SSRs was higher in trinucleotide SSRs than in dinucleotide SSRs. The correlation of the length of SSR and the frequency of polymorphism revealed that the frequency of polymorphism was decreased as motif repeat number increased. CONCLUSIONS: The assembled polymorphic SSRs would enhance the density of the existing genetic maps of peanut, which could also be a useful source of DNA markers suitable for high-throughput QTL mapping and marker-assisted selection in peanut improvement and thus would be of value to breeders. KEYWORDS: SSR, motif, polymorphism, cultivated peanut.     

Development and Characterization of Polymorphic EST-SSR and Genomic SSR Markers for Tibetan Annual Wild Barley

Tibetan annual wild barley is rich in genetic variation. This study was aimed at the exploitation of new SSRs for the genetic diversity and phylogenetic analysis of wild barley by data mining. We developed 49 novel EST-SSRs and confirmed 20 genomic SSRs for 80 Tibetan annual wild barley and 16 cultivated barley accessions. A total of 213 alleles were generated from 69 loci with an average of 3.14 alleles per locus. The trimeric repeats were the most abundant motifs (40.82%) among the EST-SSRs, while the majority of the genomic SSRs were di-nuleotide repeats. The polymorphic information content (PIC) ranged from 0.08 to 0.75 with a mean of 0.46. Besides this, the expected heterozygosity (He) ranged from 0.0854 to 0.7842 with an average of 0.5279. Overall, the polymorphism of genomic SSRs was higher than that of EST-SSRs. Furthermore, the number of alleles and the PIC of wild barley were both higher than that of cultivated barley, being 3.12 vs 2.59 and 0.44 vs 0.37. Indicating more polymorphism existed in the Tibetan wild barley than in cultivated barley. The 96 accessions were divided into eight subpopulations based on 69 SSR markers, and the cultivated genotypes can be clearly separated from wild barleys. A total of 47 SSR-containing EST unigenes showed significant similarities to the known genes. These EST-SSR markers have potential for application in germplasm appraisal, genetic diversity and population structure analysis, facilitating marker-assisted breeding and crop improvement in barley.     

Identification and mapping of polymorphic SSR markers from expressed gene sequences of barley and wheat

The growing availability of EST sequences from a range of crop plantsprovides a potentially valuable source of new DNA markers. We have examined theInternational Triticeae EST Cooperative database for the presence ofdinucleotide and trinucleotide simple sequence repeats. Analysis of 24,344 ESTsidentified 388 dinucleotide repeats and 978 trinucleotide repeats in ESTs,representing 1.6% and 4.0% of the total number of ESTs, respectively. To testthe utility and cross-species transferability of EST-derived SSR markers,primers were designed to the flanking regions of 41 barley SSRs and used toscreen 11 barley and 15 wheat varieties. Sixteen of the barley SSR markers werepolymorphic in barley and five were polymorphic in wheat. This represents arelatively high level of transferability of SSR markers between barley andwheat, which has important implications for the development of new markers andcomparative mapping of barley, wheat and other cereals. An additional 56 SSRsfrom wheat ESTs were tested in the same barley and wheat varieties. Four wheatEST SSR markers were polymorphic in wheat and one in barley. Chromosomallocations in barley and wheat were determined for the majority of polymorphicmarkers.     

Identification and characterization of simple sequence repeat (SSR) markers from Fragaria×ananassa expressed sequences

The availability of expressed sequence data derived from gene discovery programmes enables mining for simple sequence repeats (SSRs), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study reports on the development and characterization of expressed sequence tag (EST)–SSR markers in the cultivated strawberry, Fragaria×ananassa. Fourteen primer pairs were assessed for polymorphism in 13 F.×ananassa genotypes. The markers show reliable amplification and considerable polymorphism, demonstrating the utility of EST–SSRs for genetic analysis of commercial strawberry germplasm.     

Development of 28 EST‐SSR markers based on transcriptome sequences of Gobiobotia filifer and cross‐species amplification

Gobiobotia filifer is a small benthic fish distributed in Yangtze River Basin. The abundance of G. filifer increased after impoundment of Xiluodu Dam and Xiangjiaba Dam. The state of population structure and changes of genetic diversity before and after impoundment of Xiluodu Dam and Xiangjiaba Dam were interesting issues. However, efficient molecular markers were rare, which will limit us to solve above problems. Twenty‐eight expressed sequence tag SSRs (EST‐SSRs) were successfully identified and verified as stable amplification and polymorphic loci by polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The number of alleles at these EST‐SSR loci ranged from 3 to 14, the polymorphism information content values were 0.125–0.897, and the observed and expected heterozygosities were 0.0–0.857 and 0.132–0.928, respectively. Cross‐species amplification of the 28 loci developed in this study was examined in seven individuals of each of the 7 taxa. The amplification efficiency of 28 EST‐SSRs primer pairs is related to the distance of genetic relationship between cross‐species with G. filifer, and same subfamily species (Xenophysogobio boulengeri and Xenophysogobio nudicorpa) showed the highest (50%) amplification efficiency. These EST‐SSR markers could be used to analyse genetic diversity and population structure of G. filifer and related species.     

Development of simple sequence repeat DNA markers and their integration into a barley linkage map

Simple sequence repeats (SSRs), or microsatellites, are a new class of PCR-based DNA markers for genetic mapping. The objectives of the present study were to develop SSR markers for barley and to integrate them into an existing barley linkage map. DNA sequences containing SSRs were isolated from a barley genomic library and from public databases. It is estimated that the barley genome contains one (GA)_n repeat every 330 kb and one (CA)_n repeat every 620 kb. A total of 45 SSRs were identified and mapped to seven barley chromosomes using doubled-haploid lines and/or wheat-barley addition-line assays. Segregation analysis for 39 of these SSRs identified 40 loci. These 40 markers were placed on a barley linkage map with respect to 160 restriction fragment length polymorphism (RFLP) and other markers. The results of this study demonstrate the value of SSRs as markers in genetic studies and breeding research in barley.     

Large-scale identification of polymorphic microsatellites using an <Emphasis Type="Italic">in silico</Emphasis> approach

Background  

Simple Sequence Repeat (SSR) or microsatellite markers are valuable for genetic research. Experimental methods to develop SSR markers are laborious, time consuming and expensive. In silico approaches have become a practicable and relatively inexpensive alternative during the last decade, although testing putative SSR markers still is time consuming and expensive. In many species only a relatively small percentage of SSR markers turn out to be polymorphic. This is particularly true for markers derived from expressed sequence tags (ESTs). In EST databases a large redundancy of sequences is present, which may contain information on length-polymorphisms in the SSR they contain, and whether they have been derived from heterozygotes or from different genotypes. Up to now, although a number of programs have been developed to identify SSRs in EST sequences, no software can detect putatively polymorphic SSRs.     

Level of genetic differentiation affects relative performances of expressed sequence tag and genomic SSRs

Microsatellites, also called simple sequence repeats (SSRs), are markers of choice to estimate relevant parameters for conservation genetics, such as migration rates, effective population size and kinship. Cross‐amplification of SSRs is the simplest way to obtain sets of markers, and highly conserved SSRs have recently been developed from expressed sequence tags (EST) to improve SSR cross‐species utility. As EST‐SSRs are located in coding regions, the higher stability of their flanking regions reduces the frequency of null alleles and improves cross‐species amplification. However, EST‐SSRs have generally less allelic variability than genomic SSRs, potentially leading to differences in estimates of population genetic parameters such as genetic differentiation. To assess the potential of EST‐SSRs in studies of within‐species genetic diversity, we compared the relative performance of EST‐ and genomic SSRs following a multispecies approach on passerine birds. We tested whether patterns and levels of genetic diversity within and between populations assessed from EST‐ and from genomic SSRs are congruent, and we investigated how the relative efficiency of EST‐ and genomic SSRs is influenced by levels of differentiation. EST‐ and genomic SSRs ensured comparable inferences of population genetic structure in cases of strong genetic differentiation, and genomic SSRs performed slightly better than EST‐SSRs when differentiation is moderate. However and interestingly, EST‐SSRs had a higher power to detect weak genetic structure compared to genomic SSRs. Our study attests that EST‐SSRs may be valuable molecular markers for conservation genetic studies in taxa such as birds, where the development of genomic SSRs is impeded by their low frequency.     

SSR mining in coffee tree EST databases: potential use of EST-SSRs as markers for the Coffea genus

Expressed sequence tags (ESTs) from Coffea canephora leaves and fruits were used to search for types and frequencies of simple sequence repeats (EST–SSRs) with a motif length of 1–6 bp. From a non-redundant (NR) EST set of 5,534 potential unigenes, 6.8% SSR-containing sequences were identified, with an average density of one SSR every 7.73 kb of EST sequences. Trinucleotide repeats were found to be the most abundant (34.34%), followed by di- (25.75%) and hexa-nucleotide (22.04%) motifs. The development of unique genic SSR markers was optimized by a computational approach which allowed us to eliminate redundancy in the original EST set and also to test the specificity of each pair of designed primers. Twenty-five EST–SSRs were developed and used to evaluate cross-species transferability in the Coffea genus. The orthology was supported by the amplicon sequence similarity and the amplification patterns. The >94% identity of flanking sequences revealed high sequence conservation across the Coffea genus. A high level of polymorphic loci was obtained regardless of the species considered (from 75% for C. liberica to 86% for C. canephora). Moreover, the polymorphism revealed by EST–SSR was similar to that exposed by genomic SSR. It is concluded that Coffea ESTs are a valuable resource for microsatellite mining. EST-SSR markers developed from C. canephora sequences can be easily transferred to other Coffea species for which very little molecular information is available. They constitute a set of conserved orthologous markers, which would be ideal for assessing genetic diversity in coffee trees as well as for cross-referencing transcribed sequences in comparative genomics studies.     

Developing and characterising Ricinus communis SSR markers by data mining of whole-genome sequences

Ricinus communis is a versatile industrial oil crop that is cultivated worldwide. Genetic improvement and marker-assisted breeding of castor bean have been slowed owing to the lack of abundant and efficient molecular markers. As co-dominant markers, simple sequence repeats (SSRs) are useful for genetic evaluation and molecular breeding. The recently released whole-genome sequence of castor bean provides useful genomic resources for developing markers on a genome-wide scale. In the present study, the distribution and frequency of microsatellites in the castor bean genome were characterised and numerous SSR markers were developed using genomic data mining. In total, 18,647 SSR loci at a density of one SSR per 18.89 Kb in the castor bean genome sequence (representing approximately 352.27 Mb) were identified. Dinucleotide repeats were the most frequently observed microsatellites, although the AAT repeat motif was also prevalent. Using six cultivars as screening samples, 670 polymorphic SSR markers from 1,435 primer pairs (46.7 %) were developed. Trinucleotide motif loci contained a higher proportion of polymorphisms (48.5 %) than dinucleotide motif loci (39.2 %). The polymorphism level in the SSR loci was positively correlated with the increasing number of repeat units in the microsatellites. The phylogenetic relationship among 32 varieties was evaluated using the developed SSR markers. Cultivars developed at the same institute clustered together, suggesting that these cultivars have a narrow genetic background. The large number of SSR markers developed in this study will be useful for genetic mapping and for breeding improved castor-oil plants. These markers will also facilitate genetic and genomic studies of Euphorbiaceae.     

ESMP: A high-throughput computational pipeline for mining SSR markers from ESTs

With the advent of high-throughput sequencing technology, sequences from many genomes are being deposited to public databases at a brisk rate. Open access to large amount of expressed sequence tag (EST) data in the public databases has provided a powerful platform for simple sequence repeat (SSR) development in species where sequence information is not available. SSRs are markers of choice for their high reproducibility, abundant polymorphism and high inter-specific transferability. The mining of SSRs from ESTs requires different high-throughput computational tools that need to be executed individually which are computationally intensive and time consuming. To reduce the time lag and to streamline the cumbersome process of SSR mining from ESTs, we have developed a user-friendly, web-based EST-SSR pipeline "EST-SSR-MARKER PIPELINE (ESMP)". This pipeline integrates EST pre-processing, clustering, assembly and subsequently mining of SSRs from assembled EST sequences. The mining of SSRs from ESTs provides valuable information on the abundance of SSRs in ESTs and will facilitate the development of markers for genetic analysis and related applications such as marker-assisted breeding. AVAILABILITY: The database is available for free at http://bioinfo.aau.ac.in/ESMP.     

Development of microsatellite markers in autopolyploid sugarcane and comparative analysis of conserved microsatellites in sorghum and sugarcane

Sugarcane has become an increasingly important first-generation biofuel crop in tropical and subtropical regions. It has a large, complex, polyploid genome that has hindered the progress of genomic research and marker-assisted selection. Genetic mapping and ultimately genome sequence assembly require a large number of DNA markers. Simple sequence repeats (SSRs) are widely used in genetic mapping because of their abundance, high rates of polymorphism, and ease of use. The objectives of this study were to develop SSR markers for construction of a saturated genetic map and to characterize the frequency and distribution of SSRs in a polyploid genome. SSR markers were mined from expressed sequence tag (EST), reduced representation library genomic sequences, and bacterial artificial chromosome (BAC) sequences. A total of 5,675 SSR markers were surveyed in a segregating population. The overall successful amplification and polymorphic rates were 87.9 and 16.4%, respectively. The trinucleotide repeat motifs were most abundant, with tri- and hexanucleotide motifs being the most abundant for the ESTs. BAC and genomic SSRs were mostly AT-rich while the ESTs were relatively GC-rich due to codon bias. These markers were also aligned to the sorghum genome, resulting in 1,203 markers mapped in the sorghum genome. This set of SSRs conserved in sugarcane and sorghum would be the most informative for mapping quantitative trait loci in sugarcane and for comparative genomic analyses. This large collection of SSR markers is a valuable resource for sugarcane genomic research and crop improvement.     

Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum

Analysis of a sugarcane (Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG)_n trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5′ untranslated region. No primer pairs producing a polymorphic product were found in the 3′ untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera (Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.     

基于转录组测序信息的水生植物莕菜SSR标记开发

利用软件MISA对水生植物莕菜转录组测序所获得的79536条EST序列进行分析,共检测出12319个EST-SSR位点。在莕菜的EST-SSR中,二核苷酸和三核苷酸重复单元是主导类型,分别占总SSR的57.31%和30.87%,其中AG/CT、AAG/CTT分别是二、三重复单元类型的优势重复基元,分别占总SSR的29.76%和8.66%。随机挑选了130对EST-SSR引物对莕菜两个居群进行遗传多样性检测,结果发现：78对引物能扩增出清晰可分辨的条带,其中37对能成功检测出多态性,引物多态率为 47.44%。这些多态性引物共检测出114个等位基因,每个位点2~6个,平均3.08个。观测杂合度（H_o）及预期杂合度（H_e）分别在0.229~1.000和0.351~0.756之间,多态信息含量PIC值在0.286~0.698之间,平均达0.495。以上研究结果表明,通过莕菜转录组测序产生的EST数据来开发SSR标记是一种简单而高效的途径,这些新的SSR分子标记为研究莕菜的居群遗传多样性及其遗传结构提供了工具。