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.     

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.     

Characteristics and transferability of new apple EST-derived SSRs to other Rosaceae species

Genic microsatellites or simple sequence repeat markers derived from expressed sequence tags (ESTs), referred to as EST–SSRs, are inexpensive to develop, represent transcribed genes, and often have assigned putative function. The large apple (Malus × domestica) EST database (over 300,000 sequences) provides a valuable resource for developing well-characterized DNA molecular markers. In this study, we have investigated the level of transferability of 68 apple EST–SSRs in 50 individual members of the Rosaceae family, representing three genera and 14 species. These representatives included pear (Pyrus communis), apricot (Prunus armeniaca), European plum (P. domestica), Japanese plum (P. salicina), almond (P. dulcis), peach (P. persica), sour cherry (P. cerasus), sweet cherry (P. avium), strawberry (Fragaria vesca, F. moschata, F. virginiana, F. nipponica, and F. pentaphylla), and rose (Rosa hybrida). All 68 primer pairs gave an amplification product when tested on eight apple cultivars, and for most, the genomic DNA-derived amplification product matched the expected size based on EST (in silico) data. When tested across members of the Rosaceae, 75% of these primer pairs produced amplification products. Transferability of apple EST–SSRs across the Rosaceae ranged from 25% in apricot to 59% in the closely related pear. Besides pear, the highest transferability of these apple EST–SSRs, at the genus level, was observed for strawberry and peach/almond, 49 and 38%, respectively. Three markers amplified in at least one genotype within all tested species, while eight additional markers amplified in all species, except for cherry. These 11 markers are deemed good candidates for a widely transferable Rosaceae marker set provided their level of polymorphism is adequate. Overall, these findings suggest that transferability of apple EST–SSRs across Rosaceae is varied, yet valuable, thereby providing additional markers for comparative mapping and for carrying out evolutionary studies.     

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 expressed sequence tags‐derived simple sequence repeats markers from robusta coffee variety ‘CxR’ (an interspecific hybrid of Coffea canephora × Coffea congensis)

SSR (simple sequence repeats) markers derived from ESTs (expressed sequence tags), commonly called EST‐SSRs or genic SSRs provide useful genetic markers for crop improvement. These are easy and economical to develop as by‐products of large‐scale EST resources that have become available as part of the functional genomic studies in many plant species. Here, we describe for the first time, nine genic‐SSRs of coffee that are developed from the microsatellite containing ESTs from a cDNA library of moisture‐stressed leaves of coffee variety, ‘CxR’ (a commercial interspecific hybrid between Coffea congensis and Coffea canephora). The markers show considerable allelic diversity with PIC values up to 0.70 and 0.75 for Coffea arabica and Coffea canephora, respectively, and robust cross‐species amplification in 16 other related taxa of coffee. The validation studies thus demonstrate the potential utility of the EST‐SSRs for genetic analysis of coffee germplasm.     

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.     

Microsatellite markers spanning the apple (Malus x domestica Borkh.) genome

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database () to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15 cM.E. Silfverberg-Dilworth and C. L. Matasci contributed equally to this work.     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

烟草EST-SSR位点分析

利用MISA软件对烟草EST公共数据库中的简单重复序列(SSRs)进行了分析。结果表明,在133523条EST序列中,共获得81757条SSR序列,SSRs之间的距离约为0.92 kb。其中,六碱基重复丰度最大,占60.3%,而单碱基、三碱基、四碱基、二碱基和五碱基重复丰度分别为20.0%、11.0%、4.2%、2.8%和1.7%。在单碱基、二碱基、三碱基和四碱基重复模体中,丰度最大的分别是A/T、AG、AAG和AAAT,而CG在编码区内丰度很低。用CAP3软件进行冗余分析表明,在这6种类型的重复模体中,冗余与非冗余的烟草EST之间没有显著差异。在得到的SSR序列中随机选择10个序列设计引物,在7个烟草品种中进行PCR扩增。结果表明,10对引物全部扩增出PCR产物,其中8对引物扩增出预期片段。用这8组扩增出预期片段的PCR产物进行变性PAGE凝胶电泳检测,结果表明,其中有4对引物(EB4、EB5、EB6和EB8)扩增出多态性条带。     

Direct determination of the chromosomal location of bunching onion and bulb onion markers using bunching onion–shallot monosomic additions and allotriploid-bunching onion single alien deletions

To determine the chromosomal location of bunching onion (Allium fistulosum L.) simple sequence repeats (SSRs) and bulb onion (A. cepa L.) expressed sequence tags (ESTs), we used a complete set of bunching onion–shallot monosomic addition lines and allotriploid bunching onion single alien deletion lines as testers. Of a total of 2,159 markers (1,198 bunching onion SSRs, 324 bulb onion EST–SSRs and 637 bulb onion EST-derived non-SSRs), chromosomal locations were identified for 406 markers in A. fistulosum and/or A. cepa. Most of the bunching onion SSRs with identified chromosomal locations showed polymorphism in bunching onion (89.5%) as well as bulb onion lines (66.1%). Using these markers, we constructed a bunching onion linkage map (1,261 cM), which consisted of 16 linkage groups with 228 markers, 106 of which were newly located. All linkage groups of this map were assigned to the eight basal Allium chromosomes. In this study, we assigned 513 markers to the eight chromosomes of A. fistulosum and A. cepa. Together with 254 markers previously located on a separate bunching onion map, we have identified chromosomal locations for 766 markers in total. These chromosome-specific markers will be useful for the intensive mapping of desirable genes or QTLs for agricultural traits, and to obtain DNA markers linked to these.     

Intron-flanking EST–PCR markers: from genetic marker development to gene structure analysis in Rhododendron

With a long-term goal of constructing a linkage map of Rhododendron enriched with gene-specific markers, we utilized Rhododendron catawbiense ESTs for the development of high-efficiency (in terms of generating polymorphism frequency) PCR-based markers. Using the gene-sequence alignment between Rhododendron ESTs and the genomic sequences of Arabidopsis homologs, we developed ‘intron-flanking‘ EST–PCR-based primers that would anneal in conserved exon regions and amplify across the more highly diverged introns. These primers resulted in increased efficiency (61% vs. 13%; 4.7-fold) of polymorphism-detection compared with conventional EST–PCR methods, supporting the assumption that intron regions are more diverged than exons. Significantly, this study demonstrates that Arabidopsis genome database can be useful in developing gene-specific PCR-based markers for other non-model plant species for which the EST data are available but genomic sequences are not. The comparative analysis of intron sizes between Rhododendron and Arabidopsis (made possible in this study by aligning of Rhododendron ESTs with Arabidopsis genomic sequences and the sequencing of Rhododendron genomic PCR products) provides the first insight into the gene structure of Rhododendron. Electronic Supplementary Material Supplementary material is available for this article at     

Type I microsatellite markers from Atlantic salmon (Salmo salar) expressed sequence tags

The detection of simple sequence repeats (SSRs) within expressed sequence tags (ESTs) connects potential microsatellite markers with specific genes, generating Type I markers. Using an in silico approach, we identified 1975 SSRs from the Genome Research on Atlantic Salmon Project EST database. We designed primers to amplify 158 SSRs, of which 65 amplified 76 loci (including 11 duplicated loci). Sixty‐one of the 76 loci were variable in 24 Atlantic salmon from seven populations, and 96% of these markers also amplify DNA from other salmonids. Functions for 16 of the SSR associated ESTs have been determined, confirming them as Type I markers.     

Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat

Nearly 900 SSRs (simple sequence repeats) were identified among 15,000 ESTs (expressed sequence tags) belonging to bread wheat ( Triticum aestivum L.). The SSRs were defined by their minimum length, which ranged from 14 to 21 bp. The maximum length ranged from 24 to 87 bp depending upon the length of the repeat unit itself (1–7 bp). The average density of SSRs was one SSR per 9.2 kb of EST sequence screened. The trinucleotide repeats were the most abundant SSRs detected. As a representative sample, 78 primer pairs were designed, which were also used to screen the dbEST entries for Hordeum vulgare and Triticum tauschii (donor of the D-genome of cultivated wheat) using a cut-off E (expectation) value of 0.01. On the basis of in silico analysis, up to 55.12% of the primer pairs exhibited transferability from Triticum to Hordeum, indicating that the sequences flanking the SSRs are not only conserved within a single genus but also between related genera in Poaceae. Primer pairs for the 78 SSRs were synthesized and used successfully for the study of (1) their transferability to 18 related wild species and five cereal species (barley, oat, rye, rice and maize); and (2) polymorphism between the parents of four mapping populations available with us. A subset of 20 EST-SSR primers was also used to assess genetic diversity in a collection of 52 elite exotic wheat genotypes. This was done with a view to compare their utility relative to other molecular markers (gSSRs, AFLPs, and SAMPL) previously used by us for the same purpose with the same set of 52 bread wheat genotypes. Although only a low level of polymorphism was detected, relative to that observed with genomic SSRs, the study suggested that EST-SSRs can be successfully used for a variety of purposes, and may actually prove superior to SSR markers extracted from genomic libraries for diversity estimation and transferability.Communicated by R. Hagemann     

Frequency, type, distribution and annotation of simple sequence repeats in Rosaceae ESTs

Genomic resources for peach, a model species for Rosaceae, are being developed to accelerate gene discovery in other Rosaceae species by comparative mapping. Simple sequence repeats (SSRs) are an important tool for comparative mapping because of their high polymorphism and transportability. To accelerate the development of SSR markers, we analyzed publicly available Rosaceae expressed sequence tags (ESTs) for SSRs. A total of 17,284 ESTs from almond, peach and rose were assembled into putatively non-redundant EST sets. For comparison, 179,099 ESTs from Arabidopsis were also used in the analysis. About 4% of the assembled ESTs contained SSRs in Rosaceae, which was higher than the 2.4% found in Arabidopsis. About half of the SSRs were found in the putative UTR, and the estimated average distance between SSRs in the UTR was 5.5 kb in rose, 5.1 kb in almond, 7 kb in peach and 13 kb in Arabidopsis. In the putative coding region, the estimated average distance was two to four times longer than in the UTR. Rosaceae ESTs containing SSRs were functionally annotated using the GenBank nr database and further classified using the gene ontology terms associated with the matching sequences in the SwissProt database. The detailed data including the sequences and annotation results are available from .     

Development and Characterization of EST-SSR Markers in the Eastern Oyster <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) in the eastern oyster (Crassostrea virginica). ESTs of the eastern oyster were downloaded from GenBank and screened for SSRs with at least eight units of dinucleotide or five units of tri-, tetra-, penta-, and hexa-nucleotide repeats. The screening of 9101 ESTs identified 127 (1.4%) SSR-containing sequences. Primers were designed for 88 SSR-containing ESTs with good and sufficient flanking sequences. Polymerase chain reaction (PCR) amplification was successful for 71 primer pairs, including 19 (27%) pairs that amplified fragments longer than expected sizes, probably due to introns. Sixty-six pairs that produced fragments shorter than 800 bp were screened for polymorphism in five oysters from three populations via polyacrylamide gels, and 53 of them (80%) were polymorphic. Fifty-three polymorphic SSRs were labeled and genotyped in 30 oysters from three populations via an automated sequencer. Five of the SSRs amplified more than two fragments per oyster, suggesting locus duplication. The remaining 48 SSRs had 2 alleles per individual, including 11 with null alleles. In the 30 oysters analyzed, the SSRs had an average of 9.3 alleles per locus, ranging from 2 to 24. Forty-three loci segregated in a family with 100 progeny, with nine showing significant deviation from Mendelian ratios (three after Bonferroni correction). Seventy percent of the loci were successfully amplified in C. rhizophorae and 34% in C. gigas. This study demonstrates that ESTs are valuable resources for the development of SSR markers in the eastern oyster, and EST-derived SSRs are more transferable across species than genomic SSRs.     

Analysis of Microsatellites in Citrus Unigenes

Simple sequence repeats (SSRs) were investigated in the unigene sequences from expressed sequence tags (EST) of sweet orange (Citrus sinensis osbeck), trifoliate orange (Poncirus trifoliata Raf.) and other citrus species and cultivars. A total of 37 802 citrus unigene sequences corresponding to 23.29 Mb were searched, resulting in the identification of 8 218 SSRs. Among them there were 4 913 (59.8%) mono-, 1 419 (17.3%) di-, 1 709 (20.8%) tri-, 114 (1.39%) tetra-, 23 (0.28%) penta- and 40 (0.49%) hexa-nucleotide SSRs. The estimated frequency of SSRs was approximately 1/2.8 kb, which could be extrapolated to 1 SSR-containing unigene in 4.6 unigenes. The maximum length of the SSR ranged from 40 to 105 bp depending on the repeating numbers of the motif in the SSR. The overall average length of SSRs was 20.9 bp. The frequencies of different SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) were very similar between sweet orange and trifoliate orange. The mononucelotide repeats appeared to be the most abundant SSRs within sweet orange and trifoliate orange, followed by trimeric repeats. The adenine rich repeats such as A/T, AG, AT, AAG, AAAT, AAAG, AAAT, AAAAG, AAAAT etc. were predominant in each type of SSRs (mono-, di-, tri-, tetra-, and penta-), whereas the C/G, CG, CCG repeats were less abundant. Twenty-five primer pairs flanking EST-SSR loci were designed to detect the possible polymorphism of six citrus cultivars including sweet orange and trifoliate orange. The PCR result with all these 25 primer pairs revealed the existence of polymorphism within six citrus cultivars confirming that citrus EST database could be efficiently exploited for the development of gene-derived SSR markers.     

EST–SSR markers for asparagus genetic diversity evaluation and cultivar identification

Simple sequence repeat (SSR) markers generated from expressed sequence tag (EST) sequences represent useful tools for genotyping and their development is relatively easy because of the public availability of EST databases. We report design and application of EST–SSRs to assess the level of genetic diversity among thirty-five asparagus cultivars and to fingerprint DePaoli, a new variety released by University of California, Riverside. DNA was isolated from bulks of pooled cladophylls coming from five plants of each variety to reduce the number of DNA extractions and PCR reactions. Allele frequencies were estimated from the intensity of the bands in two bulks and two individual plant samples for each variety. Although asparagus varieties derive from a limited germplasm pool, eight EST–SSR loci differentiated all of the analyzed cultivars. Moreover, UPGMA (unweighted pair group method with arithmetic mean) and neighbor-joining trees, as well as principal components analysis separated the cultivars into clusters corresponding to the geographical areas where they originated.     

芦笋EST资源的SSR信息分析

为了在芦笋中开发EST-SSR功能性标记,对来源于NCBI公共数据库的8590条芦笋(AsparagusofficinalisL.)EST序列进行简单重复序列SSR搜索。剔除冗余序列,得到非冗余序列8377条。在非冗余序列中共挖掘出469个EST-SSR,平均相隔14.80kb出现1个SSR。在所有的重复基序中,二核苷酸重复基序的SSR所占比例最高40.51%(190/469),其次是三核苷酸34.97%(164/469),六核苷酸21.11%(99/469)。在所有基序里,CT/AG出现的频率最高有62次,占全部重复基序的13.22%(62/469)。选取含SSR的EST序列30条,并利用primer5软件设计引物,进行SSR位点的扩增,其中27对引物扩增产物,24对有较清晰可靠的目标扩增条带,占引物数的80%,且所检测出的芦笋等位基因数量较丰富,平均4.93个/对。这些EST-SSR标记的开发将有助于芦笋群体遗传多样性、遗传图谱构建、基因定位、分子标记和系谱分析等方面的研究。     

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.