Non-redundant Expressed Sequence Tags Research Articles

Jasmonic acid and ethylene signaling pathways participate in the defense response of Chinese cabbage to Pectobacterium carotovorum infection

Chinese cabbage (Brassica rapa subsp. pekinensis) suffers from soft rot disease caused by Pectobacterium carotovorum (Pc). To uncover the mechanisms underlying the defense response of Chinese cabbage to Pc, we constructed a suppression subtractive hybridization (SSH) library from Pc-infected cabbage and obtained 1 919 non-redundant expressed sequence tags (ESTs), which were used for cDNA microarray. We detected 800 differentially expressed genes (DEGs) in cabbage at different time points post-Pc inoculation, which were further confirmed by quantitative real-time PCR. One quarter of these DEGs were involved in the biotic stress pathways visualized by MapMan. Among them, 8, 8, 1, 3, and 2 DEGs were related to jasmonic acid (JA), ethylene (ET), JA+ET, auxin, and abscisic acid (ABA) signaling pathways, respectively, while no DEG was detected for salicylic acid (SA) signaling. Assessment of phytohormone production in the Pc-infected leaves showed that JA and ET production was increased, while SA production was decreased. Treatment with JA, methyl jasmonate (MeJA), the ET precursor 1-aminocyclopropane-1-carboxylate (ACC), or combinations thereof, reduced the disease severity, and the JA and JA+ACC treatments were superior and performed equally well. Our findings suggest that JA and ET may act synergistically against Pc infection in Chinese cabbage, and JA-mediated signaling might be the most significant.

In silico identification of conserved miRNAs in the genome of fibre biogenesis crop Corchorus capsularis

Corchorus capsularis, commonly known as jute occupies the leading position in the production of natural fibre alongside lower environmental threat. Small noncoding ~21 to 24 nucleotides long microRNAs play significant roles in regulating the gene expression as well as different functions in cellular growth and development. Here, the study adopted a comprehensive in silico approach to identify and characterize the conserved miRNAs in the genome of C. capsularis including functional annotation of specific gene targets. Expressed Sequence Tags (ESTs) based homology search of 3350 known miRNAs of dicotyledons were allowed against 763 non-redundant ESTs of jute genome, resulted in the prediction of 5 potential miRNA candidates belonging five different miRNA families (miR1536, miR9567-3p, miR4391, miR11300, and miR8689). The putative miRNAs were composed of 18 nucleotides having a range of -0.49 to -1.56 MFEI values and 55%–61% of (A + U) content in their pre-miRNAs. A total of 1052 gene targets of putative miRNAs were identified and their functions were extensively analyzed. Most of the gene targets were involved in plant growth, cell cycle regulation, organelle synthesis, developmental process and environmental responses. Five gene targets, namely, NAC Domain Containing Protein, WRKY DNA binding protein, 3-dehydroquinate synthase, S-adenosyl-L-Met–dependent methyl transferase and Vascular-related NAC-Domain were found to be involved in the lignin biosynthesis, phenylpropanoid pathways and secondary wall formation. The present study might accelerate the more miRNA discovery, strengthening the complete understanding of miRNAs association in the cellular basis of lignin biosynthesis towards the production of high standard jute products.

In Silico Investigation of Conserved miRNAs and Their Targets From the Expressed Sequence Tags in Neospora Caninum Genome.

Neospora caninum is a protozoan parasite, the etiologic agent of Neosporosis—a common cause of abortion in cattle worldwide. Herd level prevalence of Neosporosis could be as high as 90%. However, there is no approved treatment and vaccines available for Neosporosis. MicroRNA (miRNA) based prophylaxis and therapeutics could be options for Neosporosis in cattle and other animals. The current study aimed to investigate the genome of Neospora caninum to identify and characterize the conserved miRNAs through Expressed Sequence Tags (ESTs) dependent homology search. A total of 1,041 mature miRNAs of reference organisms were employed against 336 non-redundant ESTs available in the genome of Neospora caninum. The study predicted one putative miRNA “nca-miR-9388-5p” of 19 nucleotides with MFEI value -1.51 kcal/mol and (A + U) content% 72.94% corresponding with its pre-miRNA. A comprehensive search for specific gene targets was performed and discovered 16 potential genes associated with different protozoal physiological functions. Significantly, the gene “Protein phosphatase” was found responsible for the virulence of Neospora caninum. The other genes were accounted for gene expression, vesicular transport, cell signaling, cell proliferation, DNA repair mechanism, and different developmental stages of the protozoon. Therefore, this study finding will provide pivotal information to future aspirants upon Bovine Neosporosis. It will also serve as the baseline information for further studies of the bioinformatics approach to identify other protozoal miRNAs.

Development of EST-derived microsatellite markers to investigate the population structure of sparganum - the causative agent of zoonotic sparganosis.

The plerocercoid (sparganum) of Spirometra erinaceieuropaei is the main aetiological agent of human sparganosis. To improve the current knowledge on S. erinaceieuropaei evolution, we performed multi-locus microsatellite typing of sparganum isolates from China for the first time. All available expressed sequence tag (EST) sequences for the Spirometra were downloaded from the GenBank. The identification and localization of microsatellites in ESTs was accomplished by MISA. Based on the selected microsatellites, the genetic structure of 64 sparganum isolates collected from 11 geographical locations in southwest China were investigated through principal component analysis, STRUCTURE analysis and neighbour-joining clustering. A total of 522 non-redundant ESTs containing 915 simple sequence repeats were identified from 12 481 ESTs screened. Five primer pairs were finally selected. Using these loci, a total of 12 alleles were detected in 64 sparganum isolates. Little variability was observed within each of geographical population, especially among isolates derived from Kunming of Yunnan (YN-KM) province. Both STRUCTURE analysis and the clustering analysis supported that two genotypes existed among the sparganum isolates from southwest China. In conclusion, five microsatellite markers were successfully developed, and sparganum population was observed to harbour low genetic variation, further investigation with deeper sampling was needed to elucidate the population structure.

Exploiting the transcriptome of Euphrates Poplar, Populus euphratica (Salicaceae) to develop and characterize new EST-SSR markers and construct an EST-SSR database.

BackgroundMicrosatellite markers or Simple Sequence Repeats (SSRs) are the most popular markers in population/conservation genetics. However, the development of novel microsatellite markers has been impeded by high costs, a lack of available sequence data and technical difficulties. New species-specific microsatellite markers were required to investigate the evolutionary history of the Euphratica tree, Populus euphratica, the only tree species found in the desert regions of Western China and adjacent Central Asian countries.Methodology/Principal FindingsA total of 94,090 non-redundant Expressed Sequence Tags (ESTs) from P. euphratica comprising around 63 Mb of sequence data were searched for SSRs. 4,202 SSRs were found in 3,839 ESTs, with 311 ESTs containing multiple SSRs. The most common motif types were trinucleotides (37%) and hexanucleotides (33%) repeats. We developed primer pairs for all of the identified EST-SSRs (eSSRs) and selected 673 of these pairs at random for further validation. 575 pairs (85%) gave successful amplification, of which, 464 (80.7%) were polymorphic in six to 24 individuals from natural populations across Northern China. We also tested the transferability of the polymorphic eSSRs to nine other Populus species. In addition, to facilitate the use of these new eSSR markers by other researchers, we mapped them onto Populus trichocarpa scaffolds in silico and compiled our data into a web-based database (http://202.205.131.253:8080/poplar/resources/static_page/index.html).ConclusionsThe large set of validated eSSRs identified in this work will have many potential applications in studies on P. euphratica and other poplar species, in fields such as population genetics, comparative genomics, linkage mapping, QTL, and marker-assisted breeding. Their use will be facilitated by their incorporation into a user-friendly web-based database.

In silico identification and validation of microsatellite markers from onion EST sequences

SummaryMicrosatellites or simple sequence repeats (SSRs) play an important role in plant genetics and breeding. The development of microsatellites is a time-consuming and expensive process. In silico mining of microsatellites from expressed sequence tags (ESTs), which are available in electronic molecular databases, is a cheaper alternative. In the present study, we used a computational approach for mining SSRs from 20,159 ESTs in Allium cepa. These onion ESTs represented a total length of 13.2 Mb and were downloaded from the dbEST database of the National Center for Biotechnology Information (NCBI) and subjected to various pre-processing steps. The pre-processed ESTs were clustered, resulting in non-redundant unigenes. These unigenes were analysed for their SSR content and distribution. In all, 1,464 SSRs consisting of di-, tri-, tetra-, penta- and hexa-nucleotide repeats were mined from the non-redundant ESTs (contigs and singletons). Tri-nucleotide SSRs were the most abundant, followed by tetra-, di-, hexa- and penta-nucleotide SSRs. Among the tri-coding repeats, leucine and serine codons were more abundant. The SSR-containing sequences were annotated and grouped into their respective functional categories. The predominant functional group among the annotated unigenes was “metabolism”, followed by “transcription factors” and “transporter proteins”. Primer pairs could be designed for 1,092 SSR-containing sequences. Of these, 51 primer pairs were validated in the laboratory. A database has been developed to store the unigenes, primer pairs, putative annotations, and BLAST results. After validation, the EST-derived microsatellite (SSR) markers can be used in studies related to marker-assisted selection, detection of polymorphism, DNA fingerprinting, and diversity analysis in onion.

Cross‐genera transferable e‐microsatellite markers for 12 genera of the Lamiaceae family

The Lamiaceae family contains many high-valued medicinal, aromatic and ornamental plant species. Several members of the genera in this family are under heavy pressure of collection for commercial use. DNA markers such as microsatellites could be used to identify commercially important genotypes and to select high-yielding ones for development of new varieties. A total of 12,432 expressed sequence tags (ESTs) from Salvia fruticosa, S. miltiorrhiza, S. sclarea and Stenogyne rugosa were analyzed. A total of 6216 ESTs were found to be unique according the redundancy test used. Results of this study indicated that the use of redundant ESTs in comparison to non-redundant ESTs was advantageous in terms of higher cross-genera transferability of the markers. A total of 75 EST-microsatellite primer pairs were tested using two different polymerase chain reaction amplification profiles and 52 were found to be cross-genera transferable. Cross-genera transferability of the e-microsatellite primer pairs varied from one species to 12 species tested. It was noted that cross-genera transferability of e-microsatellite primer pairs decreased as the evolutionary distance between the sources and target species increased. This study indicated that EST resources from Salvia spp. and Stenogyne rugosa could be successfully used to identify cross-genera transferable e-microsatellite markers for uncharacterized genomes of the genera in the Lamiaceae family. These e-microsatellite markers could allow one to perform comparative analyses of population structure and genomic studies, and facilitate comparative linkage mapping in the genera studied. E-microsatellite primer pairs reported in this manuscript are equivalent to a total of 135 e-microsatellite primer pairs since many e-microsatellite primer pairs show cross-genera transferability.

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 7.25kb 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.

Gene expression and proteomic analysis of the formation of Phakopsora pachyrhizi appressoria.

BackgroundPhakopsora pachyrhizi is an obligate fungal pathogen causing Asian soybean rust (ASR). A dual approach was taken to examine the molecular and biochemical processes occurring during the development of appressoria, specialized infection structures by which P. pachyrhizi invades a host plant. Suppression subtractive hybridization (SSH) was utilized to generate a cDNA library enriched for transcripts expressed during appressoria formation. Two-dimensional gel electrophoresis and mass spectroscopy analysis were used to generate a partial proteome of proteins present during appressoria formation.ResultsSequence analysis of 1133 expressed sequence tags (ESTs) revealed 238 non-redundant ESTs, of which 53% had putative identities assigned. Twenty-nine of the non-redundant ESTs were found to be specific to the appressoria-enriched cDNA library, and did not occur in a previously constructed germinated urediniospore cDNA library. Analysis of proteins against a custom database of the appressoria-enriched ESTs plus Basidiomycota EST sequences available from NCBI revealed 256 proteins. Fifty-nine of these proteins were not previously identified in a partial proteome of P. pachyrhizi germinated urediniospores. Genes and proteins identified fell into functional categories of metabolism, cell cycle and DNA processing, protein fate, cellular transport, cellular communication and signal transduction, and cell rescue. However, 38% of ESTs and 24% of proteins matched only to hypothetical proteins of unknown function, or showed no similarity to sequences in the current NCBI database. Three novel Phakopsora genes were identified from the cDNA library along with six potentially rust-specific genes. Protein analysis revealed eight proteins of unknown function, which possessed classic secretion signals. Two of the extracellular proteins are reported as potential effector proteins.ConclusionsSeveral genes and proteins were identified that are expressed in P. pachyrhizi during appressoria formation. Understanding the role that these genes and proteins play in the molecular and biochemical processes in the infection process may provide insight for developing targeted control measures and novel methods of disease management.

A comparative transcriptomic analysis of the extremely boron tolerant plant Puccinellia distans with the moderately boron tolerant Gypsophila arrostil

The Turkish ecotype of Puccinellia distans displays exceptional boron (B) tolerance, >1,250 mg B L⁻¹, compared to <50 mg B L⁻¹ for Gypsophila arrostil. In the present study, we compare the molecular basis for the difference in B tolerance between the two species by constructing high B-responsive suppression subtractive hybridization libraries to identify the upregulated genes. A total of 219 and 113 unique non-redundant expressed sequence tags (ESTs) were identified and functionally classified in P. distans and G. arrostil, respectively. In addition, 63 ESTs were down-regulated in P. distans in response to high B. The majority of the high B upregulated genes belong to four categories: metabolism, protein synthesis, cellular organization, and stress/defense. We hypothesize that the superior B tolerance exhibited by P. distans may be due to its ability to restrict the accumulation of B in plant tissues through the upregulated expression of efflux transporters comparable to the Bot1 transporter of barley. In addition, our results are consistent with the view that other molecular mechanisms involved in stress/defense, such as detoxification, anti-oxidative, and signaling pathways, are needed to tolerate B-toxicity stress. The molecular basis of boron tolerance of two plant species (Puccinellia distans and Gypsophila arrostil) that differ greatly in their boron tolerance was studied in this manuscript.

EST-SSR markers derived from Laminaria digitata and its transferable application in Saccharina japonica

The public availability of numerous expressed sequence tag (EST) enables EST-based SSR (simple sequence repeat) markers to be widely used for genetics and breeding studies. In the present study, EST-SSR markers were developed from ESTs of and were transferred to the non-congeneric species . Among the 2,668 non-redundant ESTs, 83 (3.1%) ESTs containing SSR were identified totally, with an average of one SSR per 13.6 kb. Analysis of SSR motifs revealed that the trinucleotide and tetranucleotide were major motifs, accounted for 44.58% and 16.87%, respectively. Based on the 83 ESTs containing SSR, we designed 45 pairs of primers in the flanking regions of the SSR, of which 13 pairs showed polymorphism in a wild population, and the mean alleles per locus was 3.6 (ranging from 2 to 6). The observed (Ho) and expected (He) heterozygosities of these EST-SSRs were 0.234-0.632 and 0.260-0.635, respectively. All loci were in Hardy-Weinberg equilibrium in the wild population and no linkage disequilibrium was detected among loci. The obtained EST-SSR markers can facilitate and promote related research such as ecological investigation, genetic diversity assessment and breeding practice of as well.

Development and utilization of novel intron length polymorphic markers in foxtail millet (Setaria italica (L.) P. Beauv.)

Introns are noncoding sequences in a gene that are transcribed to precursor mRNA but spliced out during mRNA maturation and are abundant in eukaryotic genomes. The availability of codominant molecular markers and saturated genetic linkage maps have been limited in foxtail millet (Setaria italica (L.) P. Beauv.). Here, we describe the development of 98 novel intron length polymorphic (ILP) markers in foxtail millet using sequence information of the model plant rice. A total of 575 nonredundant expressed sequence tag (EST) sequences were obtained, of which 327 and 248 unique sequences were from dehydration- and salinity-stressed suppression subtractive hybridization libraries, respectively. The BLAST analysis of 98 EST sequences suggests a nearly defined function for about 64% of them, and they were grouped into 11 different functional categories. All 98 ILP primer pairs showed a high level of cross-species amplification in two millets and two nonmillets species ranging from 90% to 100%, with a mean of ∼97%. The mean observed heterozygosity and Nei's average gene diversity 0.016 and 0.171, respectively, established the efficiency of the ILP markers for distinguishing the foxtail millet accessions. Based on 26 ILP markers, a reasonable dendrogram of 45 foxtail millet accessions was constructed, demonstrating the utility of ILP markers in germplasm characterizations and genomic relationships in millets and nonmillets species.

In silico mining for simple sequence repeat loci in a pineapple expressed sequence tag database and cross-species amplification of EST-SSR markers across Bromeliaceae

A collection of 5,659 expressed sequence tags (ESTs) from pineapple [Ananas comosus (L.) Merr.] was screened for simple sequence repeats (EST-SSRs) with motif lengths between 1 and 6bp. Lower thresholds of 15, 7 and 5 repeat units were used to define microsatellites of the mono-, di-, and tri- to hexanucleotide repeat type, respectively. Based on these criteria, 696 SSRs were identified among 3,389 EST unigenes, together representing 2,840kb. This corresponds to an average density of one SSR every 4.1kb of non-redundant EST sequences. Dinucleotide repeats were most abundant (38.4% of all SSRs) followed by trinucleotide repeats (38.1%). Flanking primer pairs were designed for 537 EST-SSR loci, and 49 of these were screened for their functionality in 12 accessions of A. comosus, 14 accessions of 5 additional Ananas species and 1 species of Pseudananas. Distinct PCR products of the expected size range were obtained with 36 primer pairs. Eighteen loci analyzed in more detail were all polymorphic in pineapple, and primer pairs flanking these loci also generated PCR products from a wide range of genera and species from six subfamilies of the Bromeliaceae. The potential to reveal polymorphism in a heterologous target species was demonstrated in Deuterocohnia brevifolia (subfamily Pitcairnioideae).

Identification of Differentially Expressed Genes During the Brachyurization of the Chinese Mitten Crab Eriocheir japonica sinensis

Expressed sequence tags (ESTs) obtained from two-directional suppression subtractive hybridization (SSH) of cDNA libraries derived from the pleon of the Chinese mitten crab were analyzed using mRNA subtractive hybridization. After cDNA cloning and sequencing, gene expression profiles were examined, focusing on brachyurization. We assembled 211 non-redundant ESTs from SSH library 1 (yielding 130 unique transcripts: 25 consensi, 105 singletons) and 669 from SSH library 2 (195: 51 consensi, 144 singletons). Functional analysis shows that these genes are putatively involved in various cellular processes (33%), ribosomal RNA/proteins (30%), molting (12%), signal transduction (6%), immune factor and stress proteins (6%), development (4%), energy metabolism (3%), and mitochondrial membrane protein, hormone metabolizing, and chaperone (1% each) of other organisms. Some 3% are of unknown function in SSH library 1. The results facilitate the functional study of candidate genes involved in early developmental processes, especially the regulation of brachyurization in this crab.

Microsatellite DNA in genomic survey sequences and UniGenes of loblolly pine

Genomic DNA sequence databases are a potential and growing resource for simple sequence repeat (SSR) marker development in loblolly pine (Pinus taeda L.). Loblolly pine also has many expressed sequence tags (ESTs) available for microsatellite (SSR) marker development. We compared loblolly pine SSR densities in genome survey sequences (GSSs) to those in non-redundant EST and cDNA sequences (UniGenes) and designed SSR primer pairs from both sequence types. Overall SSR densities were 96 SSR/Mb in GSSs and 38 SSR/Mb in UniGenes. Loblolly pine had the lowest transcriptome SSR density when compared to 49 other species in the NCBI UniGene database. Among the five different GSS genome fractions of loblolly pine analyzed, methylation-filtered DNA had the highest SSR density at 145 SSR/Mb. The most abundant loblolly pine SSR motif was AT in both GSSs (34 SSR/Mb) and UniGenes (7.6 SSR/Mb). Among the trinucleotide SSR motifs, the most abundant was AAT (9.5 SSR/Mb) in GSSs and AGC (4.9 SSR/Mb) in UniGenes. We designed PCR primer pairs for 120 genomic SSRs and 315 EST-SSRs and evaluated PCR amplification for 108 (25%). We identified 21 primer pairs that reliably amplified polymorphic loci from 31 loblolly pine individuals and estimated that at least 60 additional polymorphic marker loci could be developed from available P. taeda GSS and UniGene resources.

Suppression subtractive hybridization reveals differential gene expression in sunflower grown in high P

Sunflower (Helianthus annuus L.) is a commercially important oilseed crop. Previous studies proved that this crop is a promising plant species for phytoextraction of excess soil phosphorus (P) because of its superior P accumulating characteristics. Suppression subtractive hybridization (SSH) strategy was employed to isolate and characterize genes that are induced in response to high P in this crop. SSH library was prepared using cDNA generated from plants treated with high P as the 'tester'. Based on the results of dot blot analysis, 360 positive cDNA clones were selected from the SSH library for sequencing. A total of 89 non-redundant expressed sequence tags (ESTs) were identified as high P-responsive genes and they were classified into 6 functional groups. Several genes involved in metabolism showed markedly preferential expression in the library. For further confirmation, thirteen of the representative ESTs were selected from all categories for RT-PCR analysis and the results showed up-regulation of these genes in response to high P-treatment. The gene expression data derived from this study suggested that several of the up-regulated genes identified under high P-treatment might be involved in P-accumulation and tolerance in this plant.

Differential expression of expressed sequence tags in alfalfa roots under aluminum stress

To gain a better understanding of differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance and to investigate the molecular mechanisms of Al toxicity, cDNA subtraction libraries were generated from Al-stressed roots of alfalfa (Medicago sativa L.) compared with no Al-stressed ones, employing suppression subtractive hybridization. Using differential screening technique in which the probes were labeled with DIG, we identified 45 non-redundant ESTs in Al-stressed alfalfa root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding identified proteins, including malate dehydrogenase, 6-phosphogluconate dehydrogenase, peroxidase, and an ABC transporter, while the down-regulate genes included ATPase, secretory carrier membrane protein 2, pectinesterase inhibitor. In addition, two novel ESTs, EW678752 and EY976957, up- and down-regulated by Al stress were sequenced. Analyzed by real-time PCR, the expressions of EST EW678718, EW678739, EY976969 and EW678728, which encode for ABC transporter, malate dehydrogenase, peroxidase and 6-phosphogluconate dehydrogenase correspondingly, increased 1.64-, 2.75-, 3.27- and 6.54-folds, respectively, and the expression of EY976957 encoding for ATPase decreased 3.27 folds. The expression of EST EW678752 increased 34.54-fold, while that of EY976957 decreased 16.68 folds. It suggested that the two novel ESTs maybe play a significant role in the aluminum tolerance of alfalfa.

Differential profiles of gene expression in grouper Epinephelus coioides, infected with Singapore grouper iridovirus, revealed by suppression subtractive hybridization and DNA microarray

Suppression subtractive hybridization (SSH) was used to generate a subtracted cDNA library enriched with gene transcripts differentially expressed in the spleen of orange-spotted grouper Epinephelus coioides after 5 days of infection with Singapore grouper iridovirus (SGIV). In the forward and reverse-subtracted libraries, 260 and 153 non-redundant expressed sequence tags (EST), respectively, were identified. These annotated genes responding to SGIV infection were grouped into eight gene categories related to immunity, cell structure, transcription-translation, cell signalling, metabolism, mitochondrial proteins, ribosomal proteins and unknown or hypothetical proteins. A DNA microarray containing all the differentially expressed genes was constructed, and the gene expression patterns in different tissues were investigated in virus-infected E. coioides. Of these genes, four associated with the infection processes were identified and further investigated by quantitative real-time PCR. These results provide new insights into the molecular basis of host-pathogen interactions in E. coioides, and will help the development of control strategies against SGIV infection.

Mining, characterization, and exploitation of EST-derived microsatellites in Gossypium barbadense

Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. However, the number of expressed sequence tags (ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species. In this study, EST-SSR distribution from G. barbadense was characterized and new G. barbadense-derived EST-SSR markers were determined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G. barbadense. By mining 9697 non-redundant ESTs, a total of 638 SSR loci derived from 595 ESTs were observed. In G. barbadense, the frequency of ESTs containing SSRs was 6.13%, with an average of 1 SSR in every 10.4 kb of EST sequence. Furthermore, trinucleotide was found to be the most abundant repeat type among 2–6-nucleotide repeat types. It accounted for 26.6% of the total, followed by the hexanucleotide (26.0%) and pentanucleotide repeats (25.9%). Among all the repeat motifs, (AAG)n accounted for the highest proportion. EST-SSR primer pairs were developed using the Primer3 program, and the redundant primers were removed using the virtual PCR approach. As a result, 380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G. hirsutum ‘TM-1’ and G. barbadense ‘Hai7124’ for constructing linkage groups in cultivated allotetraploid cotton. Out of these, 98 (25.8%) primer pairs detected polymorphisms. Finally, 95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map; of these, 42 were mapped into the A subgenome and 53 into the D subgenome. The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.

Normalized embryoid cDNA library of oil palm ( Elaeis guineensis )

A normalized embryoid cDNA library (EON) was constructed based on reassociation kinetics reaction. Results from dot blot hybridization and sequencing of EON cDNA clones clearly indicated that the normalization process reduced the frequency of high abundance transcripts and increased the frequency of low abundance gene transcripts. A total of 553 non-redundant expressed sequence tags (ESTs) were identified, 325 of these were not observed in the standard oil palm cDNA libraries sequenced previously. A total of 10 EON cDNA clones were chosen for expression profiling across samples from different stages of the tissue culture process. Two of the genes exhibited promising expression patterns for predicting the embryogenic potential in callus. Some of these genes were also differentially expressed in the various tissues of oil palm. This study showed that normalization of the existing embryoid library improved the chances of identifying transcripts not captured in the standard libraries, some of which could be associated with embryogenesis. This collection of ESTs is particularly well suited for use as candidate genes for development of an oil palm DNA chip, which can be used to obtain a more comprehensive view of the molecular mechanism associated with oil palm tissue culture.