Analysis of simple sequence repeats (SSRs)dynamics in fungus Fusarium graminearum

The abundance and inherent potential for variations in simple sequence repeats (SSRs) or microsatellites resulted in valuable source for genetic markers in eukaryotes. We describe the organization and abundance of SSRs in fungus Fusarium graminearum (causative agent for Fusarium head blight or head scab of wheat). We identified 1705 SSRs of various nucleotide repeat motifs in the sequence database of F. graminearum. It is observed that mononucleotide repeats (62%) were most abundant followed by di- (20%) and trinucleotide repeats (14%). It is noted that tetra-, penta- and hexanucleotide repeats accounted for only 4% of SSRs. The estimated frequency of Class I SSRs (perfect repeats ≥20 nucleotides) was one SSR per 124.5 kb, whereas the frequency of Class II (perfect repeats >10 nucleotides and ≫20 nucleotides) was one SSR per 25.6 kb. The dynamics of SSRs will be a powerful tool for taxonomic, phylogenetic, genome mapping and population genetic studies as SSR based markers show high levels of allelic variation, codominant inheritance and ease of analysis.     

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,Characterization and Cross Species Amplification of Polymorphic Microsatellite Markers from Expressed Sequence Tags of Turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.)

Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST–SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.     

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.     

Development,characterization and utilization of GenBank microsatellite markers in <Emphasis Type="Italic">Phyllostachys pubescens</Emphasis> and related species

Public sequence databases provide a rapid, simple and cost-effective source of microsatellite markers. We analyzed 1,532 bamboo (Phyllostachys pubescens) sequences available in public domain DNA databases, and found 3,241 simple sequence repeat (SSR) loci comprising repeats of two or more nucleotides in 920 genomic survey sequences (GSSs) and 68 cDNA sequences. This corresponded to one SSR per 336 bp of GSS DNA and one SSR per 363 bp of cDNA. The SSRs consisted of 76.6 and 74.5% dinucleotide repeats, 20.0 and 22.3% trinucleotide repeats, and 3.4 and 3.2% higher-number repeats in the GSS DNA and cDNA sequences, respectively. The repeat motif AG/CT (or GA/TC) was the most abundant. Nineteen microsatellite markers were developed from Class I and Class II SSRs, showing that the limited polymorphism in Ph. pubescens cultivars and provenances could be attributed to clonal propagation of the bamboo plant. The transferability of the microsatellites reached 75.3%, and the polymorphism of loci successfully transferred was 66.7% for six additional Phyllostachys species. Microsatellite PBM014 transferred successfully to all six species, showed rich polymorphism, and could serve as species-specific alleles for the identification of Phyllostachys interspecies hybrids.     

柔嫩艾美尔球虫EST序列中SSR的获取及分析

对柔嫩艾美尔球虫EST—SSR进行生物信息学分析,共获取Eimeria tenella EST序列34074条,总长度为16．45Mb,小于12bpSSR的ESTs达7651条,从中获得SSR序列19576条、总长度为0．35Mb,EST—SSRs的频率是48．00％,平均相隔S40bp出现一个长度不小于12bp的SSR。在E．tenella的核苷酸重复基元中,2、3、4、5、6和7bp重复序列在基因组中出现的种类分别有11种472条、49种14710条、31种525条、13种25条、21种43条和15种400条,3碱基重复序列是最丰富的重复单元,占总数的75．14％。各种SSRs中富含G、C碱基的重复单元以GCA出现频率最多（28．63％）,次为AGC（17．59％）,GCT（8．76％）,TGC（7．62％）,CTG（7．15％）。     

EST-SSRs characterization and in-silico alignments with linkage map SSR loci in grape (Vitis L.) genome

11,581 grape (Vitis L.) EST-SSRs were produced and characterized from a total of 381,609 grape ESTs. Among the EST-SSRs, the tri repeat (5,560, 45.4%) represented the most abundant class of microsatellites in grape EST. Most of grape EST-SSR motifs fall within 18-24 bps in length. The EST-SSRs tri-repeats occurred a higher percentage in 5??-end (59.3%) than in 3??-end (48.3%). And EST-SSR tri-repeats had abundant codon repeats for putative amino acid runs as Proline, Arginine in grape ESTs. To better utilizing these markers, 142 of newly developed and validated EST SSR loci as well as 223 linkage map SSR loci were in silico aligned and mapped in grape genome. The orders of these SSR loci in the chromosomal physical locations and in the linkage groups were compared, and about twenty linkage map loci positions were switched or rearranged in grape genome. The EST-SSR markers extended the linkage map in grape genome. The method of in silico mapping reported in this study provided an initial collection for grape mapping resources. This approach offers great opportunities to understand the genetic variations in nucleotide sequences differences in physical map, and genetic recombination in linkage maps, as well as benefits for markers enrichment in a specific grape genome region for fine mapping or QTL mapping.     

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.     

Isolation and Enrichment of Abundant Microsatellites from a Channel Catfish (Ictalurus punctatus) Brain cDNA Library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid, single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.     

Isolation and enrichment of abundant microsatellites from a channel catfish (Ictalurus punctatus) brain cDNA library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid; single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.     

Analysis on Frequency and Density of Microsatellites in Coding Seauences of Several Eukarvotic Genomes

Microsatellites or simple sequence repeats (SSRs) have been found in most organisms during the last decade. Since large-scale sequences are being generated, especially those that can be used to search for microsatellites, the development of these markers is getting more convenient. Keeping SSRs in viewing the importance of the application, available CDS (coding sequences) or ESTs (expressed sequence tags) of some eukaryotic species were used to study the frequency and density of various types of microsatellites. On the basis of surveying CDS or EST sequences amounting to 66.6 Mb in silkworm, 37.2 Mb in fly, 20.8 Mb in mosquito, 60.0 Mb in mouse, 34.9 Mb in zebrafish and 33.5 Mb in Caenorhabditis elegans, the frequency of SSRs was 1/1.00 Kb in silkworm, 1/0.77 Kb in fly, 1/1.03 Kb in mosquito, 1/1.21 Kb in mouse, 1/1.25 Kb in zebrafish and 1/1.38 Kb in C. elegans. The overall average SSR frequency of these species is 1/1.07 Kb. Hexanucleotide repeats (64.5%-76.6%) are the most abundant class of SSR in th     

Development of transcript-associated microsatellite markers for diversity and linkage mapping studies in hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.)

Data mining of gene sequences available from various projects dealing with the development of expressed sequence tags (ESTs) can contribute to the discovery of new microsatellite markers. Our aim was to develop new microsatellite markers in hop isolated from an enriched cDNA library and from coding GenBank sequences and to test their suitability in hop diversity studies and for construction of a linkage map. In a set of 614 coding GenBank sequences, 72 containing microsatellites were found (11.7%); the most frequent were trinucleotide repeats (54.0%) followed by dinucleotide repeats (34.5%). Additionally, 11 sequences containing microsatellites were isolated from an enriched cDNA library. A total of 34 primer pairs were designed, 29 based on GenBank sequences and five on sequences from the cDNA enriched library. Twenty-seven (79.4%) coding microsatellites were successfully amplified and used in diversity and linkage mapping studies. Eleven primer pairs amplified 12 coding microsatellite loci suitable for mapping and were placed on female and male linkage maps. We were able to extend previous simple sequence repeat (SSR) female, male and integral maps by 38.8, 25.8 and 40.0 cM, respectively. In the diversity study, 36 diverse hop genotypes were analyzed. Twenty-four coding microsatellites were polymorphic, 17 showing co-dominant behavior and 7 primer pairs amplifying three or more bands in some hop genotypes. Altogether, 143 microsatellite DNA fragments were amplified and they revealed a clear separation of hop genotypes according to geographical region, use or breeding history. In addition, a discussion and comparison of results with other plant coding/EST SSR studies is presented. Our results showed that these microsatellite markers can enhance hop diversity and linkage mapping studies and are a comparable marker system to non-coding SSRs.     

Analysis on frequency and density of microsatellites in coding sequences of several eukaryotic genomes

Microsatellites or simple sequence repeats (SSRs) have been found in most organisms during the last decade. Since large-scale sequences are being generated, especially those that can be used to search for microsatelUtes, the development of these markers is getting more convenient. Keeping SSRs in viewing the importance of the application, available CDS (coding sequences) or ESTs (expressed sequence tags) of some eukaryotic species were used to study the frequency and density of various types of microsatellites. On the basis of surveying CDS or EST sequences amounting to 66.6 Mb in silkworm, 37.2 Mb in fly, 20.8 Mb in mosquito, 60.0 Mb in mouse, 34.9 Mb in zebrafish and 33.5 Mb in Caenorhabditis elegans, the frequency of SSRs was 1/1.00 Kb in silkworm, 1/0.77 Kb in fly, 1/1.03 Kb in mosquito, 1/1.21 Kb in mouse, 1/1.25 Kb in zebrafish and 1/1.38 Kb in C. elegans. The overall average SSR frequency of these species is 1/1.07 Kb. Hexanucleotide repeats (64.5%-76.6%) are the most abundant class of SSR in the investigated species, followed by trimeric, dimeric, tetrameric, monomeric and pentameric repeats. Furthermore, the A-rich repeats are predominant in each type of SSRs, whereas G-rich repeats are rare in the coding regions.     

The development of BAC-end sequence-based microsatellite markers and placement in the physical and genetic maps of soybean

The composite map of soybean shared among Soybase, LIS and SoyGD (March 2006) contained 3,073 DNA markers in the "Locus" class. Among the markers were 1,019 class I microsatellite markers with 2-3 bp simple sequence repeats (SSRs) of >10 iterations (BARC-SSR markers). However, there were few class II SSRs (2-5 bp repeats with <10 iterations; mostly SIUC-Satt markers). The aims here were to increase the number of classes I and II SSR markers and to integrate bacterial artificial chromosome (BAC) clones onto the soybean physical map using the markers. Used was 10 Mb of BAC-end sequence (BES) derived from 13,473 reads from 7,050 clones constituting minimum tile path 2 of the soybean physical map ( http://www.soybeangenome.siu.edu ; SoyGD). Identified were 1,053 1-6 bp motif, repeat sequences, 333 from class I (>10 repeats) and 720 from class II (<10 repeats). Potential markers were shown on the MTP_SSR track at Gbrowse. Primers were designed as 20-24 bp oligomers that had Tm of 55 +/- 1 C that would generate 100-500 bp amplicons. About 853 useful primer pairs were established. Motifs were not randomly distributed with biases toward AT rich motifs. Strong biases against the GC motif and all tetra-nucleotide repeats were found. The markers discovered were useful. Among the first 135 targeted for use in genetic map improvement about 60% of class II markers and 75% of class I markers were polymorphic among on the parents of four recombinant inbred line (RIL) populations. Many of the BES-based SSRs were located on the soybean genetic map in regions with few BARC-SSR markers. Therefore, BES-based SSRs represent useful tools for genetic map development in soybean. New members of a consortium to map the markers in additional populations are invited.     

Eucalyptus microsatellites mined in silico: survey and evaluation

Eucalyptus is an important short rotation pulpy woody plant, grown widely in the tropics. Recently, many genomic programmes are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. These sequences can be utilized for analysis of simple sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) available in the transcribed genes. In this study, in silico analysis of 15,285 sequences representing partial and full-length mRNA from Eucalyptus species for their use in developing SSRs or microsatellites were carried out. A total of 875 EST-SSRs were identified from 772 SSR containing ESTs. Motif size of 6 for dinucleotide and 5 for trinucleotide, tetranucleotide, and pentanucleotides were considered in locating the microsatellites. The average frequency of identified SSRs was 12.9%. The dinucleotide repeats were the most abundant among the dinucleotide, trinucleotide and tetranucleotide motifs and accounted for 50.9% of the Eucalyptus genome. Primer designing analysis showed that 571 sequences with SSRs had sufficient flanking regions for polymerase chain reaction (PCR) primer synthesis. Evaluation of the usefulness of the SSRs showed that EST-derived SSRs can generate polymorphic markers as all the primers showed allelic diversity among the 16 provenances of E. tereticornis.     

甜瓜EST序列中微卫星的分布特征

GenBank中35547条甜瓜EST经去冗余处理后,得到总长度为250.3Mb的无冗余EST34438条。这些序列中有2813个微卫星简单重复序列（Simple sequence repeat,SSR）,分布于2107条EST中,出现频率为8.16%,平均分布距离为8.90kb。三核苷酸重复是主导重复类型,占SSR总数的47.14%;其次是二核苷酸和单核苷酸重复,分别占SSR总数的20.72%和16.99%。AAG/TTC是优势重复基元,占微卫星总数的29.26%,AG/CT和A/T分别占14.61%和16.25%。在所有的SSR中,重复次数为4～10次的占70.32%,长度为12～20bp的占51.12%。并对这些SSR的多态性潜能进行了评价。     

Mining and survey of simple sequence repeats in expressed sequence tags of dicotyledonous species.

Simple sequence repeat (SSR) markers are widely used in many plant and animal genomes due to their abundance, hypervariability, and suitability for high-throughput analysis. Development of SSR markers using molecular methods is time consuming, laborious, and expensive. Use of computational approaches to mine ever-increasing sequences such as expressed sequence tags (ESTs) in public databases permits rapid and economical discovery of SSRs. Most of such efforts to date focused on mining SSRs from monocotyledonous ESTs. In this study, we have computationally mined and examined the abundance of SSRs in more than 1.54 million ESTs belonging to 55 dicotyledonous species. The frequency of ESTs containing SSRs among species ranged from 2.65% to 16.82%. Dinucleotide repeats were found to be the most abundant followed by tri- or mono-nucleotide repeats. The motifs A/T, AG/GA/CT/TC, and AAG/AGA/GAA/CTT/TTC/TCT were the predominant mono-, di-, and tri-nucleotide SSRs, respectively. Most of the mononucleotide SSRs contained 15-25 repeats, whereas the majority of the di- and tri-nucleotide SSRs contained 5-10 repeats. The comprehensive SSR survey data presented here demonstrates the potential of in silico mining of ESTs for rapid development of SSR markers for genetic analysis and applications in dicotyledonous crops.     

Mining of SSR markers from Expressed Sequence Tags of bamboo species

With the ever increasing number of Expressed Sequence Tags (ESTs) from various sequencing projects, ESTs have become valuable and first-hand source of in-silico mining of simple sequence repeats (SSR) markers. We examined a total of 3419 EST sequences from three bamboo species, namely, Phyllostachys edulis, Bambusa oldhamii and Dendrocalamus sinicus for the presence of di- to hexa- microsatellites. The frequency of SSR containing ESTs varied from 5.36% in B. oldhamii to 13.05% in P. edulis. No SSRs were found in D. sinicus. Tri-nucleotide repeats (49.34%) were most frequent in P. edulis, while not much comparable difference in repeats was found in B. oldhamii. Flanking primer pairs were also designed in-silico for the sequences containing SSRs and their position on the genome hypothesized using similarity searching. SSRs located in open reading frame (ORF) were given functional annotation using Gene Ontology. Polymorphic SSRs were also detected using new pipeline- polySSR. Polymorphism level was very low (2.43%) and the position of the polymorphic SSRs was determined. The development of SSRs and the study of polymorphism will help in the further study of intra- and inter- gene flow, genetic structure, variability, linkage mapping and evolutionary relationships in bamboo.