亚麻EST-SSR信息分析与标记开发

与基因组SSR相比,以EST为基础的EST-SSR分子标记具有自身的优点。本研究从11240条亚麻（Linum sitatissmum L.）EST序列中检索出877条含有SSR的序列,其出现频率为7.8%。其中以三核苷酸重复出现的频率最高,占总SSR序列的60.1%;其次是二核苷酸重复,占21.9%;四、五和六核苷酸重复占18%。根据这些含SSR的EST序列共设计了73对SSR引物,在8份亚麻材料间通过PCR扩增检测,有63对引物扩增出清晰条带,引物可用率86.3%;有17对引物在8份亚麻材料间显现出多态性,占可扩增引物的26.3%。     

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 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.     

In silico mining and characterization of simple sequence repeats from gilthead sea bream (Sparus aurata) expressed sequence tags (EST-SSRs); PCR amplification, polymorphism evaluation and multiplexing and cross-species assays

We screened for simple sequence repeats (SSRs) found in ESTs derived from an EST-database development project ('Marine Genomics Europe' Network of Excellence). Different motifs of di-, tri-, tetra-, penta- and hexanucleotide SSRs were evaluated for variation in length and position in the expressed sequences, relative abundance and distribution in gilthead sea bream (Sparus aurata). We found 899 ESTs that harbor 997 SSRs (4.94%). On average, one SSR was found per 2.95 kb of EST sequence and the dinucleotide SSRs are the most abundant accounting for 47.6% of the total number. EST-SSRs were used as template for primer design. 664 primer pairs could be successfully identified and a subset of 206 pairs of primers was synthesized, PCR-tested and visualized on ethidium bromide stained agarose gels. The main objective was to further assess the potential of EST-SSRs as informative markers and investigate their cross-species amplification in sixteen teleost fish species: seven sparid species and nine other species from different families. Approximately 78% of the primer pairs gave PCR products of expected size in gilthead sea bream, and as expected, the rate of successful amplification of sea bream EST-SSRs was higher in sparids, lower in other perciforms and even lower in species of the Clupeiform and Gadiform orders. We finally determined the polymorphism and the heterozygosity of 63 markers in a wild gilthead sea bream population; fifty-eight loci were found to be polymorphic with the expected heterozygosity and the number of alleles ranging from 0.089 to 0.946 and from 2 to 27, respectively. These tools and markers are expected to enhance the available genetic linkage map in gilthead sea bream, to assist comparative mapping and genome analyses for this species and further with other model fish species and finally to help advance genetic analysis for cultivated and wild populations and accelerate breeding programs.     

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.     

Distribution and characterization of simple sequence repeats in Gossypium raimondii genome

Simple sequence repeats (SSRs) can be derived from the complete genome sequence. These markers are important for gene mapping as well as marker-assisted selection (MAS). To develop SSRs for cotton gene mapping, we selected the complete genome sequence of Gossypium raimondii, which consisted of 4447 non-redundant scaffolds. Out of 775.2 Mb sequence examined, a total of 136,345 microsatellites were identified with a density of 5.69 kb per SSR in the G. raimondii genome leading to development of 112,177 primer pairs. The distributions of SSRs in the genome were non-random. Among the different motifs ranging from 1 to 6 bp, penta-nucleotide repeats were most abundant (30.5%), followed by tetra-nucleotide repeats (18.2%) and di-nucleotide repeats (16.9%). Among all identified 457 motif types, the most frequently occurring repeat motifs were poly-AT/TA, which accounted for 79.8% of the total di-nt SSRs, followed by AAAT/TTTA with 51.5% of the total tetra-nucleotede. Further, 18,834 microsatellites were detected from the protein-coding genes, and the frequency of gene containing SSRs was 46.0% in 40,976 genes of G. raimondii. These genome-based SSRs developed in the present study will lay the groundwork for developing large numbers of SSR markers for genetic mapping, gene discovery, genetic diversity analysis, and MAS breeding in cotton.     

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.     

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).     

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.     

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.     

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.     

Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium

Although lily is the second largest flower crop in cutting flower commodity, only six simple sequence repeats SSRs have been reported. Thus, we developed expressed sequence tag derived-SSRs (EST-SSRs) for the Lilium genus. Among 2,235 unique ESTs, 754 ESTs contained SSR motifs, among which 165 ESTs were amenable to primer design. Among these 165 EST-SSRs, 131 EST-SSRs showed amplification in at least one Lilium species, and 76 EST-SSRs showed amplification in at least nine species. Of the 76 EST-SSRs, 47 showed amplification in all Lilium species analyzed. Using 10 breeding lines, we selected 21 EST-SSRs that had the highest number of alleles and polymorphism information content. The polymorphism information content values of these selected EST-SSRs ranged from 0.49 to 0.94 with an average of 0.76, which are higher than other plant species. The phylogenetic dendrogram derived from the amplification profiles of the 21 high polymorphic EST-SSRs was congruent with the genetic background of the 84 selected lily accessions and hybrids, which are available in commerce. Thus, the developed EST-SSRs will be very useful in germplasm management, genetic diversity analysis, cultivar finger printing, and molecular breeding in the lily.     

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers for genetic studies in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most popular non-alcoholic beverage crops worldwide. The availability of complete genome sequences for the Camellia sinensis var. ‘Shuchazao’ has provided the opportunity to identify all types of simple sequence repeat (SSR) markers by genome-wide scan. In this study, a total of 667,980 SSRs were identified in the ~?3.08 Gb genome, with an overall density of 216.88 SSRs/Mb. Dinucleotide repeats were predominant among microsatellites (72.25%), followed by trinucleotide repeats (15.35%), while the remaining SSRs accounted for less than 13%. The motif AG/CT (49.96%) and AT/TA (40.14%) were the most and the second most abundant among all identified SSR motifs, respectively; meanwhile, AAT/ATT (41.29%) and AAAT/ATTT (67.47%) were the most common among trinucleotides and tetranucleotides, respectively. A total of 300 primer pairs were designed to screen six tea cultivars for polymorphisms of SSR markers using the five selected repeat types of microsatellite sequences. The resulting 96 SSR markers that yielded polymorphic and unambiguous bands were further deployed on 47 tea cultivars for genetic diversity assessment, demonstrating high polymorphism of these SSR markers. Remarkably, the dendrogram revealed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or places of origin. The identified genome-wide SSRs and newly developed SSR markers will provide a powerful means for genetic researches in tea plant, including genetic diversity and evolutionary origin analysis, fingerprinting, QTL mapping, and marker-assisted selection for breeding.     

Characterization of masson pine (Pinus massoniana Lamb.) microsatellite DNA by 454 genome shotgun sequencing

Microsatellites (simple sequence repeats, SSRs) are important genetic markers in tree breeding and conservation. Here we utilized high-throughput 454 sequencing technology to mine microsatellites from masson pine (MP) genomic DNA. First, we analyzed the characteristics of SSRs in all nonredundant MP reads (genome survey sequences, GSSs) and compared them with loblolly pine (LP) GSSs and BACs (bacterial artificial chromosome clone sequences), and three other nonconiferous species GSSs. Second, a set of MP GSS–SSR primer pairs were designed. There were extremely low overall GSS–SSR densities (28 SSR/Mb) in MP when compared with LP (48 SSR/Mb) and the other species. AT, AAT, AAAT, and AAAAAT were the richest motifs in di-, tri-, tetra-, and hexanucleotides, respectively. Two hundred forty GSS–SSR primer pairs were designed in total, and 20 novel polymorphic markers were identified using three populations (two natural and one clonal seed orchard) as evaluating samples. These markers should be useful for future MP population genetics studies.     

茶树EST-SSRs分布特征及引物开发

为了在茶树中开发EST-SSRs功能性标记,利用生物信息学方法对NCBI网上公开的3288奈茶树（Camellia subebsus）ESTs序列进行EST-SSRs特征分析。剔除冗余序列,得到非冗余序列2083条。在非冗余序列中发现含不同重复基元SSRs的EST序列有385条,共486个EST-SSRs,平均相隔2．10kb出现1个SSR。在2～6bp的重复基元中,二核苷酸重复基元的SSRs出现频率最高（51．97％）,其次是三核苷酸（19．55％）。对所有的重复基元类型进行统计分析发现,所占比例最高的是AG／CT（47．74％）,其次分别是AT／TA（4．73％）和AAG／CTT（4．73％）。利用Prime5软件,设计了206对EST-SSRs引物,随机选用72对引物进行SSR扩增,发现31对引物可以扩增出条带,其中29对引物具有多态性,多态性比率为93．5％。这些EST-SSRs将有助于茶树基因组学方面的研究。