Non-redundant ESTs Research Articles

Characterization of leaf transcriptome, development and utilization of unigenes-derived microsatellite markers in sugarcane (Saccharum sp. hybrid).

Sugarcane (Saccharum species hybrid) is the major source of sugar (> 80% sugar) in the world and is cultivated in more than 115 countries. It has recently gained attention as a source of biofuel (ethanol). Due to genomic complexity, the development of new genomic resources is imperative in understanding the gene regulation and function, and to fine tune the genetic improvementof sugarcane. In this study, a cDNA library was constructed from mature leaves so as to develop ESTs resources which were further compared with nucleotide and protein databases to explore the functional identity of sugarcane genes. The non-redundant ESTs (unigenes) were categorized into 18 metabolic functions. The major categories were bioenergetics and photosynthesis (4%), cell metabolism (5%), development related protein (3%), membrane-related, mobile genetic elements (5%), signal transduction (2%), DNA (1%), RNA (1%) and protein (2%) metabolism, other metabolic processes (3%), transcription factors (1%), transport (4%) and proteinsrelated to stress/defense (4%). From 540 unique ESTs, 212 simple sequence repeats were identified, of which 206 were from 463 singlets and six were mined from 77 contig sequences. A total of 540 unique EST sequences were used for SSR search of which 97 (17.9%) contained specified SSR motifs, generating 212 unique SSRs. The genes characterized in this study and the EST-derived microsatellite markers identified from the cDNA library will enrich genomic resources for association- and linkage-mapping studies in sugarcane.

Isolation and expression analysis of differentially expressed genes in stem tissue of the Greek lemon cv. Adamopoulou

ABSTRACTLemon (Citrus limon (L.) Burm. f.) is susceptible to mal secco, a serious vascular disease caused by the fungus Phoma tracheiphila (Petri) Kant. and Gik., as well as low temperatures. The greek lemon cultivar Adamopoulou, thought to be derived from the Portuguese cultivar Lisbon, exhibits enhanced resistance to mal secco and cold as opposed to cv. Lisbon. Suppression subtractive hybridization (SSH) was employed for the isolation of differentially expressed genes in lemon stem tissue. A subtractive cDNA library was constructed and a total of 296 clones were sequenced. The obtained sequences were edited, resulting in 56 non-redundant ESTs. Sequence analysis revealed homology to previously identified genes involved in defense mechanisms against biotic and abiotic stresses, as well as sequences with no significant similarity in the GenBank. Selected ESTs were analyzed by real-time PCR for confirmation purposes. This analysis revealed significant expression differences between the two cultivars for genes expressing allantoinase, ultraviolet-B-repressible protein, 4-coumarate:CoA ligase and other proteins that are known to be upregulated under biotic and abiotic stress conditions.

Development of EST-SSR markers and construction of a linkage map in faba bean (Vicia faba).

To develop a high density linkage map in faba bean, a total of 1,363 FBES (Faba bean expressed sequence tag [EST]-derived simple sequence repeat [SSR]) markers were designed based on 5,090 non-redundant ESTs developed in this study. A total of 109 plants of a ‘Nubaria 2’ × ‘Misr 3’ F2 mapping population were used for map construction. Because the parents were not pure homozygous lines, the 109 F2 plants were divided into three subpopulations according to the original F1 plants. Linkage groups (LGs) generated in each subpopulation were integrated by commonly mapped markers. The integrated ‘Nubaria 2’ × ‘Misr 3’ map consisted of six LGs, representing a total length of 684.7 cM, with 552 loci. Of the mapped loci, 47% were generated from multi-loci diagnostic (MLD) markers. Alignment of homologous sequence pairs along each linkage group revealed obvious syntenic relationships between LGs in faba bean and the genomes of two model legumes, Lotus japonicus and Medicago truncatula. In a polymorphic analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES markers showed polymorphisms. Along with the EST-SSR markers, the dense map developed in this study is expected to accelerate marker assisted breeding in faba bean.

De novo transcriptome profiling uncovers a drastic downregulation of photosynthesis upon nitrogen deprivation in the nonmodel green alga Botryosphaerella sudeticus

BackgroundNeutral lipid storage is enhanced by nitrogen deprivation (ND) in numbers of green microalgal species. However, little is known about the metabolic pathways whose transcription levels are most significantly altered following ND in green microalgae, especially the nonmodel species.ResultsTo start gaining knowledge on this, we performed transcriptome profiling of the nonmodel green microalga Botryosphaerella sudeticus cells in response to ND. Transcriptome of B. sudeticus is de novo assembled based on millions of HiSEQ short sequence reads using CLC Genomics Workbench software. The resulting non-redundant ESTs are annotated based on the best hits generated from the BLASTX homology comparison against the “best” proteins in the model microalgae Chlamydomonas reinhardtii and Chlorella variabilis. By using a pathway-based approach according to KEGG databases, we show that ESTs encoding ribosomal proteins and photosynthetic functions are the most abundantly expressed ESTs in the rapidly growing B. sudeticus cells. We find that ESTs encoding photosynthetic function but not the ribosomal proteins are most drastically downregulated upon ND. Notably, ESTs encoding lipid metabolic pathways are not significantly upregulated. Further analyses indicate that chlorophyll content is markedly decreased by 3-fold and total lipid content is only slightly increased by 50%, consistent with the transcriptional profiling. On the other hand, carbon content and photosynthesis efficiency are only marginally decreased by 7% and 20%, respectively, indicating that photosynthesis is only slightly reduced upon drastic downregulation of photosynthetic ESTs and chlorophyll content upon ND. In addition, TAG content is found to be greatly increased by 50-fold, though total lipid content is only slightly increased by 1.5-fold.ConclusionsTaken together, our results suggest that light-harvesting proteins and chlorophylls are in excess in B. sudeticus. Degradation of excess photosynthesis proteins is most likely a mechanism for recycling of nitrogen-rich molecules to synthesize new proteins for preparation of gametogenesis and zygospore formation in adaptation and survival upon ND. Furthermore, our analyses indicate that TAG accumulation is largely attributed to the modification of other pre-existing lipid molecules, rather than de novo synthesis. We propose that this is likely an evolutionarily conserved mechanism in many green microalgae species.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-715) contains supplementary material, which is available to authorized users.

아카풀코나리에서 Differential Slot Blot을 이용한 약발현 유전자 목록작성

Anther is the major organ of flower in responsible to reproduction and outward appearance. From anther-specific cDNA library of Lilium Oriental Hybrid 'Acapulco', 2000 expressed sequence tags were selected randomly. Differential slot blot analysis with cDNA probes from the anther and leaf was used to get anther-expressed clone and 570 non-redundant ESTs were obtained and sequenced. Compared to the GenBank database using BLASTX algorithm, 191 clones showed significant similarity but others (66.5%) did not measured to known sequence. Functional categories according to gene ontology (GO) annotation included sequence representing a significant portion of protein in cell and cell part respectively. A transcriptional analysis at 7 different organs and developmental stage was performed using northern blot with thirty ESTs as putative anther specific gene. This report suggest that selection of anther expressed clone using differential slot blot was considered as very effective tool and our current study can provide fundamental information on the lily anther including pollen furthermore.

Development of EST-SSR marker and genetic diversity analysis in Sorghum bicolor×Sorghum sudanenes

A total of 57 498 non-redundant ESTs were identified from 210 878 ESTs of Sorghum in NCBI by sequence analysis. In all, 3 338 SSRs were distributed in 3 116 ESTs with an average frequency of one SSR per 11.28 kb, which included 215 SSR motifs. Analysis of SSR motifs revealed that the trinucleotides were major motifs, accounting for 68.33%.The dinucleotides motifs accounted for 17.97%. There were 1 694 sequences from 3 338 EST-SSR sequences could be designed into primers and the proportion was 50.75%. Fourteen primers were selected to amplify EST-SSR loci with 50 collections of Sorghum bicolor × S. sudanenes, 7 collections of S. bicolor and 3 collections of S. sudanenes. Seventy-two allele variations were detected and the frequency was 5.14 gene loci per primer. The polymorphism index of each primer was in the range of 0.54-0.93. The genetic distance ranged from 0.1646 to 0.6398. This showed abundant genetic diversity in the materials. The materials were divided into 5 groups with clustering analysis of EST-SSR data. Each group included the varieties with similar parents or similar regional distribution. Meanwhile, 4 specific molecular markers were found. Primer D1763 was specific in the registered variety GB-4-2 which was the progeny of S. bicolor 314A × S. sudanenes White Skull. The marker was specific in justification of the germ difference. These results showed that the EST-SSR was an effective marker for genetic diversity analysis and specificity studies on S. bicolor × S. sudanenes.

Data Mining for Simple Sequence Repeats in Expressed Sequence Tags from Japanese Flounder (Paralichthys olivaceus)

Based on ESTs of Japanese flounder (Paralichthys olivaceus) in the public database, EST-SSR makers were developed after mining and evaluating SSRs in them by bioinformatics methods. 5 927 non-redundant ESTs of Japanese flounder were screened and 390 SSRs were mined out. The frequency of these EST-SSRs was 7.95% and the average distance of distribution was 7.9 kb in non-redundant ESTs. The dinucleotide repest motif was dominant type (59.02%) with repeat motif AC being the most common (16.91%). The distribution of trinucleotide, tetranucleotide and hexanucleotide repeats were dispersive. 30 primer pairs for EST-SSRs were designed, 27 primer pairs showed the amplification, and 17 primer pairs showed polymorphisms, the rates of polymorphic EST-SSRs were 62.96% with the alleles per locus ranging from 2 to 6 (mean 3.5). The observed (HO) and expected (HE) heterozygosities of these EST-SSRs were 0.280.92 and 0.31550.8033, respectively. Two EST-SSR loci significantly deviated from the HardyWeinberg equilibrium (HWE) expectation, and theremaining 15 loci were in HWE. These new EST-SSR markers would provide sufficient polymorphism for population genetic studies and genome mapping of Japanese flounder.

Development of eSSR-Markers in Setaria italica and Their Applicability in Studying Genetic Diversity, Cross-Transferability and Comparative Mapping in Millet and Non-Millet Species

Foxtail millet ( Setaria italica L.) is a tractable experimental model crop for studying functional genomics of millets and bioenergy grasses. But the limited availability of genomic resources, particularly expressed sequence-based genic markers is significantly impeding its genetic improvement. Considering this, we attempted to develop EST-derived-SSR (eSSR) markers and utilize them in germplasm characterization, cross-genera transferability and in silico comparative mapping. From 66,027 foxtail millet EST sequences 24,828 non-redundant ESTs were deduced, representing ~16 Mb, which revealed 534 (~2%) eSSRs in 495 SSR containing ESTs at a frequency of 1/30 kb. A total of 447 pp were successfully designed, of which 327 were mapped physically onto nine chromosomes. About 106 selected primer pairs representing the foxtail millet genome showed high-level of cross-genera amplification at an average of ~88% in eight millets and four non-millet species. Broad range of genetic diversity (0.02–0.65) obtained in constructed phylogenetic tree using 40 eSSR markers demonstrated its utility in germplasm characterizations and phylogenetics. Comparative mapping of physically mapped eSSR markers showed considerable proportion of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (~68%), maize (~61%) and rice (~42%) chromosomes. Synteny analysis of eSSRs of foxtail millet, rice, maize and sorghum suggested the nested chromosome fusion frequently observed in grass genomes. Thus, for the first time we had generated large-scale eSSR markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.

Identification of Differentially Expressed Genes in Sweetpotato Storage Roots Between Kokei No. 14 and Its Mutant Nongdafu 14 Using PCR-Based cDNA Subtraction

The contents of carotenoids in the storage root of sweetpotato, Ipomoea batatas (L.) Lam. vary dramatically among different cultivars. However, so far little is known about the regulation of carotenoids synthesis in sweetpotato. In our laboratory, we identified a novel sweetpotato mutant, Nongdafu 14, which is a homogenous mutant derived from the wild type Kokei No. 14. The contents of carotenoids in the storage root of Nongdafu 14 were analyzed using high performance liquid chromatography (HPLC), and it was found that the amount of carotenoids, b-carotene, lutein and zeaxantion, three major types of carotenoids in sweetpotato storage roots, increased 2–26 folds in Nongdafu 14 compared to Kokei No. 14. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed in Nongdafu 14, and a differentially expressed cDNA library was constructed using the cDNA of Nongdafu 14 storage roots as tester and that of Kokei No. 14 storage roots as driver. Out of the 1 530 clones sequenced, we identified 292 nonredundant ESTs. GO and KEGG analyses of these differentially expressed ESTs indicated that diverse metabolism pathways were affected and candidate genes involved in regulation of carotenoids synthesis are suggested.

CDNA-SCoT Analysis of Differentially Expressed Genes in Sugarcane Induced by Leifsonia xyli subsp. xyli

Ratoon stunting disease (RSD) is one of the major diseases, which is harmful to sugarcane in the world. To investigate the molecular mechanism of gene differential expression in sugarcane under ratoon stunting disease stress, we inoculated the stem of sugarcane variety ROC22 with cane juice with RSD pathogen Leifsonia xyli subsp. xyli to induce resistance, then took stem samples at 2, 4, 6, 8, and 10 days, respectively, after inoculation, and established different cDNA mixture pools transcribed from the stem RNA mixture pools of RSD treatment and control for cDNA-SCoT analysis to detect the gene differential expressions. More than 500 bands with length of 100–1800 bp were obtained using 80 SCoT primers. A total of 30 differentially expressed EST sequences were screened out, and 22 non-redundant ESTs with high quality were obtained by cluster analyses of the ESTs sequencing. The results of BlastN showed that the differentially expressed genes of ROC22 mainly related to energy metabolism, disease defense, signal transduction, and protein metabolism. Further analysis of gene function indicated that brassinosteroid biosynthesis-like proteins, NBS-LRR type disease resistance protein, jasmonic acid induced protein, α-tubulin, abscisic stress ripen-ing protein, proline-rich protein, and translation initiation factor eif-2b α subunit may be involved in the process of the incompatible interaction between plants and Leifsonia xyli subsp. xyli.

SSR markers in transcripts of genes linked to post-transcriptional and transcriptional regulatory functions during vegetative and reproductive development of Elaeis guineensis

The oil palm (Elaeis guineensis Jacq.) is a perennial monocotyledonous tropical crop species that is now the world's number one source of edible vegetable oil, and the richest dietary source of provitamin A. While new elite genotypes from traditional breeding programs provide steady yield increases, the long selection cycle (10-12 years) and the large areas required to cultivate oil palm make genetic improvement slow and labor intensive. Molecular breeding programs have the potential to make significant impacts on the rate of genetic improvement but the limited molecular resources, in particular the lack of molecular markers for agronomic traits of interest, restrict the application of molecular breeding schemes for oil palm. In the current study, 6,103 non-redundant ESTs derived from cDNA libraries of developing vegetative and reproductive tissues were annotated and searched for simple sequence repeats (SSRs). Primer pairs from sequences flanking 289 EST-SSRs were tested to detect polymorphisms in elite breeding parents and their crosses. 230 of these amplified PCR products, 88 of which were polymorphic within the breeding material tested. A detailed analysis and annotation of the EST-SSRs revealed the locations of the polymorphisms within the transcripts, and that the main functional category was related to transcription and post-transcriptional regulation. Indeed, SSR polymorphisms were found in sequences encoding AP2-like, bZIP, zinc finger, MADS-box, and NAC-like transcription factors in addition to other transcriptional regulatory proteins and several RNA interacting proteins. The identification of new EST-SSRs that detect polymorphisms in elite breeding material provides tools for molecular breeding strategies. The identification of SSRs within transcripts, in particular those that encode proteins involved in transcriptional and post-transcriptional regulation, will allow insight into the functional roles of these proteins by studying the phenotypic traits that cosegregate with these markers. Finally, the oil palm EST-SSRs derived from vegetative and reproductive development will be useful for studies on the evolution of the functional diversity within the palm family.

Study of EST-SSR marker system of Cordyceps

To establish the EST-SSR marker system for Cordyceps by using ESTs of C. bassiana and C. militaris. The ESTs of Cordyceps were downloaded from the public database of NCBI, and the redundant ESTs with low quality were removed. The EST-SSR primers were designed by Sequece Seiner 1. 2. And the primers were screened through PAGE-Electrophoresis. The 4 556 non-redundant ESTs which from C. bassiana with total length of 2 953 173 bp were selected. 718 EST-SSRs distributed in 616 ESTs were totally screened out, accounting for 15.8% of the non-redundant ESTs. It was discovered that the average distance of EST-SSSR was 1/4 096 bp in EST-SSRs distribution of C. bassiana. Trinucleotide repeats were the most abundant types with 419 repeated sequences. Regarding to C. militaris, totally 1 363 non-redundant ESTs were acquired, from which 1 117 EST-SSRs were screened, and rate of SSR sites in ESTs was 81.95%. The leading motif of SSR was nucleotide A. The 50 pairs of EST-SSR primers were designed according to the ESTs of C. bassiana, and preliminary test showed the 34 pairs of primers amplified clear fragments,accounting for 68% of all primers. Furthermore, the 39 of the 40 pairs of primers from the ESTs of C. militaris were found to be amplified as the clear fragments, accounting for 97.5%. The phylogenetic analysis revealed that different anamorph of Cordyceps spieces were divided into four branches. The EST-SSR of Cordyceps had comparably higher utility value. The EST-SSR markers developed from ESTs of C. bassiana and C. militaris had well transferability in Cordyceps. And it was suggested that the EST-SSR markers should be an easy and effective way to assay molecular genetic structure of Cordyceps.

Identification of Copper-Induced Genes in the Marine Alga Ulva compressa (Chlorophyta)

In order to identify genes/proteins involved in copper tolerance, the marine alga Ulva compressa was cultivated with 10μM copper for 3days. The activities of antioxidant enzymes ascorbate peroxidase (AP), peroxiredoxin (PRX), thioredoxin (TRX), and glutathione-S-transferase (GST) and the level of lipoperoxides were determined in the alga cultivated with and without copper addition. Antioxidant enzyme activities and lipoperoxides level increased in response to copper excess, indicating that the alga was under oxidative stress. A cDNA library was prepared using U. compressa cultivated with 10μM copper for 3days. A total of 3 × 10(4) clones were isolated and 480 clones were sequenced, resulting in 235 non-redundant ESTs, of which 104 encode proteins with known functions. Among them, we identified proteins involved in (1) antioxidant metabolism such as AP, PRX, TRX, GST, and metalothionein (MET), (2) signal transduction, such as calmodulin (CAM), (3) calcium-dependent protein kinase (CDPK) and nucleoside diphosphate kinase (NDK), (4) gene expression, (5) protein synthesis and degradation, and (6) chloroplast and mitochondria electron transport chains. Half of the identified proteins are potentially localized in organelles. The relative level of 18 genes, including those coding for AP, PRX, TRX, GST, MET, CAM, CDPK, and NDK were determined by quantitative RT-PCR in the alga cultivated with 10μM copper for 0 to 7days. Transcript levels increased in response to copper stress and most of them reached a maximum at days3 and 5. Thus, the selected genes are induced by copper stress and they are probably involved in copper acclimation and tolerance.

Comparative transcriptome analysis of contrasting foxtail millet cultivars in response to short-term salinity stress

Soil salinity represents a major abiotic stress that adversely affects crop growth and productivity. In this study, 21-day-old seedlings of two foxtail millet ( Setaria italica) cultivars differing in salt tolerance were found to also differ in lipid peroxidation, ion balance and activity of antioxidative enzymes (glutathione reductase and catalase) under short-term salinity stress (250 mM NaCl for 1–48 h). With the aim of better understanding the molecular mechanisms underlying plant responses to short-term salinity stress, two suppression subtractive hybridization cDNA libraries (forward and reverse) were constructed of these cultivars. A total of 249 non-redundant ESTs was identified by random EST sequencing and grouped into 11 functional categories. Macroarray analysis of these clones showed that 159 (63.9%) were differentially expressed (≥2-fold) in response to salinity stress, with 115 (72.3%) up and 44 (27.7%) down-regulated. A data search of transcriptional profiling under salinity stress in other species revealed that 81 (51%) of the 159 differentially expressed transcripts found in foxtail millet have not been reported in previous studies. Hence, these new transcripts may represent untapped gene sources allowing specific responses to short-term salt-stress in an orphan crop known to possess a natural adaptation capacity to abiotic stress. Quantitative real-time PCR of 21 highly up-regulated (≥2.5-fold) transcripts showed temporal variation in expression in both cultivars under salinity. Among them, several transcription factors and signalling genes were preferentially expressed in the tolerant cultivar. These results suggest that the tolerant cultivar possesses more effective signal-perception mechanisms for metabolic adjustments in plants under harsh saline conditions. Our findings provide evidence that the unknown genes identified in this study, in addition to several known genes, may play important roles in stress tolerance mechanisms present in foxtail millet.

Development and Characterization of EST-SSR Markers from NCBI and cDNA Library in Cultivated Peanut (<i>Arachis hypogaea L.</i>)

86 132 ESTs downloaded from GenBank in NCBI and 12 501 ESTs from cDNA library constructed by high-oil linoleic acid accession E12 were analysed. After the preprocession, there were 18 051 singletons and 9 972 contigs in the GenBank of NCBI and cDNA library. Totally 3 104 SSR locis had been screened by MISA software, accounting for 11.08% for these non-redundant ESTs. All SSR locis are divided into di-nucleotide, thi-nucleotide, tetra-nucleotide, penta-nucleotide, hexa-nucleotide and multi-nucleotide etc., and thi-nucleotide motif is the most motifs and the frequency was 43.0% and 56.8% in NCBI and cDNA libraray, respectively. The number of di- and penta-nucleotide motifs were second and third in all motifs. And the hexa-nucleotide was the least motif both in NCBI and cDNA library. In all repeat motifs nucleotide, AG/TC was the most motifs and accounted for 8.65% and 13.42% in NCBI and cDNA library respectively. Among the tri-nucleotide repeats, CTT/GAA was the most frequent motif, accounting for 6.7% and 13.42%, respectively. The repeat unit number of SSR locis is between 4 and 51.

ESTs from Seeds to Assist the Selective Breeding of Jatropha curcas L. for Oil and Active Compounds.

We report here on the characterization of a cDNA library from seeds of Jatropha curcas L. at three stages of fruit maturation before yellowing. We sequenced a total of 2200 clones and obtained a set of 931 non-redundant sequences (unigenes) after trimming and quality control, ie, 140 contigs and 791 singlets with PHRED quality ≥10. We found low levels of sequence redundancy and extensive metabolic coverage by homology comparison to GO. After comparison of 5841 non-redundant ESTs from a total of 13193 reads from GenBank with KEGG, we identified tags with nucleotide variations among J. curcas accessions for genes of fatty acid, terpene, alkaloid, quinone and hormone pathways of biosynthesis. More specifically, the expression level of four genes (palmitoyl-acyl carrier protein thioesterase, 3-ketoacyl-CoA thiolase B, lysophosphatidic acid acyltransferase and geranyl pyrophosphate synthase) measured by real-time PCR proved to be significantly different between leaves and fruits. Since the nucleotide polymorphism of these tags is associated to higher level of gene expression in fruits compared to leaves, we propose this approach to speed up the search for quantitative traits in selective breeding of J. curcas. We also discuss its potential utility for the selective breeding of economically important traits in J. curcas.

Identification of genes related to root shape in radish (Raphanus sativus) using suppression subtractive hybridization

Radish is an important vegetable, and displays a wide variation in root shape. To understand the mechanisms controlling development and/or inheritance of different root shapes, we attempted to identify the genes expressed differentially in roots of two cultivars with different root shapes; long and thick type (Lt) cv. Taibyousoubutori and skinny type (St) cv. Kosena. Morphological studies revealed that significant differences in thickness of roots began at 4 weeks after sowing. By suppression subtractive hybridization using roots at this stage, 140 and 70 non-redundant ESTs were identified from subtraction of St (tester) × Lt (driver) and reverse subtraction, respectively. Of these ESTs, 102 and 52 showed high similarity to previously identified genes, and the remaining 38 and 18 showed no matches to known genes. Quantitative RT-PCR analysis for selected eleven genes demonstrated that they are differentially expressed in roots of two radish cultivars according to the direction of subtraction. These results will contribute to a better understanding of the mechanisms and inheritance of radish root shape.

EST-PCR markers representing watermelon fruit genes are polymorphic among watermelon heirloom cultivars sharing a narrow genetic base

To date, there are only a few sequenced-tagged site (STS) markers associated with genes controlling fruit quality in watermelon. A normalized cDNA library for watermelon fruit (Citrullus lanatusvar. lanatus) was constructed. Sequence analysis of the cDNA clones resulted in the development of 4700 non-redundant ESTs (EST unigenes) expressed in watermelon fruit (http://www.ncbi.nlm.nih.gov,http://www.icugi.org). One hundred of these EST unigenes [including 40 EST unigenes that contain simple sequence repeat (SSR) motives (EST-SSRs) and 60 customary EST unigenes (not containing SSR motives)] were used for designing primer pairs for PCR experiments. The EST primer pairs were tested in PCR experiments with genomic DNA of 25 watermelon heirloom cultivars and 13 United States Plant Introductions (US PIs) ofCitrullussp., including fourC. lanatusvar.lanatus, fiveC. lanatusvar.citroidesPIs and fourCitrullus colocynthisPIs. The 40 EST-SSR and 60 EST primer pairs produced 108 and 142 EST-PCR markers, respectively, among theCitrullusPIs and watermelon cultivars. A large number of the EST-PCR markers were polymorphic between theCitrullusPIs and the watermelon cultivars, but significantly less polymorphic among the cultivars. Of the 108 EST-PCR markers associated with EST-SSRs, 103 exist in theCitrullusPIs and 64 in the cultivars. Of these 64 markers, 45 (70.3%) were polymorphic among the cultivars. Of the 142 EST-PCR markers associated with customary ESTs, 134 exist in theCitrullusPIs and 108 in the cultivars. Of these 108 markers, 86 (79.6%) were polymorphic among cultivars. The results in this study indicate that polymorphism exists in coding regions of genes expressed in fruits of watermelon cultivars. In total, 131 polymorphic EST-PCR markers (45 associated with EST-SSRs and 86 associated with customary ESTs) related to watermelon fruit genes were generated using the above data. These markers should be useful for DNA fingerprinting of cultivars and breeding lines, for assessing genetic relationships and for genetic mapping of watermelon.

Exploration and mapping of microsatellite markers from subtracted drought stress ESTs in Sorghum bicolor (L.) Moench

Molecular variation within defined genes underlying specific biochemical or physiological functions provide candidate gene-based markers which show very close association with the trait of interest and thus should enable to design superior genotypes. We explored microsatellite loci in a total of 9,892 subtracted drought stress ESTs of sorghum (6,295 after flowering ESTs and 3,597 before flowering ESTs) available in the NCBI dbEST database. Analysis of 9,892 ESTs identified 221 non-redundant ESTs with SSRs, from which 109 functional SSRs were developed. Among them 62 EST-microsatellites (56.8%) exhibited polymorphism for at least one sorghum genotype among the five tested and yielded a total of 161 alleles, with an average of 2.59 alleles per marker. We present a microsatellite linkage map using a RIL population derived from the cross 296B and IS18551. The map contains 128 microsatellite loci distributed over 15 linkage groups, and spanning a genetic distance of 1,074.5 cM. The map includes map positions of 28 drought EST-microsatellites developed and seven new genomic-SSRs, and are distributed throughout the map. The developed EST markers include genes coding for important regulatory proteins and functional proteins that are involved in stress related metabolism. The drought EST-microsatellites will have applications in functional diversity studies, association studies, QTL studies for drought, and other agronomically important traits in sorghum, and comparative genomics studies between sorghum and other members of the Poaceae family.

Development of a cDNA array for chicken gene expression analysis

BackgroundThe application of microarray technology to functional genomic analysis in the chicken has been limited by the lack of arrays containing large numbers of genes.ResultsWe have produced cDNA arrays using chicken EST collections generated by BBSRC, University of Delaware and the Fred Hutchinson Cancer Research Center. From a total of 363,838 chicken ESTs representing 24 different adult or embryonic tissues, a set of 11,447 non-redundant ESTs were selected and added to an existing collection of clones (4,162) from immune tissues and a chicken bursal cell line (DT40). Quality control analysis indicates there are 13,007 useable features on the array, including 160 control spots. The array provides broad coverage of mRNAs expressed in many tissues; in addition, clones with expression unique to various tissues can be detected.ConclusionsA chicken multi-tissue cDNA microarray with 13,007 features is now available to academic researchers from genomics@fhcrc.org. Sequence information for all features on the array is in GenBank, and clones can be readily obtained. Targeted users include researchers in comparative and developmental biology, immunology, vaccine and agricultural technology. These arrays will be an important resource for the entire research community using the chicken as a model.