Development and characterization of Novel EST-based single-copy genic microsatellite DNA markers in white spruce and black spruce.

Due mainly to large genome size and prevalence of repetitive sequences in the nuclear genome of spruce (Picea Mill.), it is very difficult to develop single-copy genomic microsatellite markers. We have developed and characterized 25 polymorphic, single-copy genic microsatellites from white spruce (Picea glauca (Moench) Voss) EST sequences and determined their informativeness in white spruce and black spruce (Picea mariana (Mill.) B.S.P.) and inheritance in black spruce. White spruce EST sequences from NCBI dbEST were searched for the presence of microsatellite repeats. Forty-seven sequences containing dinucleotide, trinucleotide, tetranucleotide and compound repeats were selected to develop primers. Twenty-five of the designed primer pairs yielded scorable amplicons, with single-locus patterns, and were characterized in 20 individuals each of white spruce and black spruce. All 25 microsatellites were polymorphic in white spruce and 24 in black spruce. The number of alleles at a locus ranged from two to 18, with a mean of 8.8 in white spruce, and from one to 17, with a mean of 7.6 in black spruce. The expected heterozygosity/polymorphic information content ranged from 0.10 to 0.92, with a mean of 0.67 in white spruce, and from 0 to 0.93, with a mean of 0.59 in black spruce. Microsatellites with dinucleotide and compound repeats were more informative than those with trinucleotide and tetranucleotide repeats. Eighteen microsatellite markers polymorphic between the parents of a black spruce controlled cross inherited in a single-locus Mendelian fashion. The microsatellite markers developed can be applied for various genetics, genomics, breeding, and conservation studies and applications.

EST-SSR marker development based on transcriptome sequencing and genetic analyses of Phoebe bournei (Lauraceae).

High-throughput sequencing of the Phoebe bournei transcriptome was performed, and novel SSR markers were identified. A total of 73,518 nonredundant unigenes were assembled and annotated by sequence similarity searching in diverse public databases. A total of 40,853 SSRs were identified from 73,518 unigenes. Twenty-three pairs of polymorphic EST-SSR markers were selected from 98 markers and used for genetic analyses in 75 individuals from three P. bournei populations. The 23 pairs of markers could detect abundant genetic information from the samples (PIC = 0.769), and cross-species amplification was successfully performed in other related species. Three populations had high level of genetic diversity (He = 0.658 in average), of which the population YS from Jiangxi province had the most abundant genetic diversity (He = 0.722). The results of genetic structure analyses showed that the population YS from Jiangxi province had obvious genetic differences from the other two populations, and the genetic information of the population SX from Fujian province was related to that of the population LC from Guangdong province and the population YS. The transcriptomic resources and EST-SSR markers are valuable tools not only for the ecological conservation of P. bournei but also for phylogenetic studies.

Bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase.

Human serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide content hamper the expression and production of rHSA using bacterial hosts. Hence, a strategy that uses a fusion technique and engineered Escherichia coli strains was employed to improve the expression of soluble rHSA in the bacterial cytoplasm. The solubilities of the b'a' domain of human protein disulphide isomerase (PDIb'a')- and maltose-binding protein (MBP)-tagged rHSA expressed in Origami 2 at 18 °C were notably increased by up to 90.1% and 96%, respectively. A simple and efficient protocol for rHSA purification was established and approximately 9.46 mg rHSA was successfully obtained from a 500-mL culture at 97% purity. However, rHSA was mostly obtained in soluble oligomeric form. By introducing a simple refolding and size-exclusion chromatography step, monomeric rHSA was obtained at 34% yield. Native polyacrylamide gel electrophoresis confirmed the similarity in the molecular weights between E. coli-derived monomeric rHSA and commercial monomeric HSA.

Development of polymorphic EST-SSR markers and their applicability in genetic diversity evaluation in Rhododendron arboreum.

The genus Rhododendron, known for large impressive flowers is widely distributed throughout the world. Rhododendrons have limited genetic information, despite of comprising high species diversity, morphological overlap and weak genetic barrier. In present study, expressed sequence tag (EST) data from Rhododendron catawbiense Michx (Subgenus Hymenanthes, Section Ponticum) and Rhododendron mucronatum var. ripense (Makino) E.H. Wilson (Subgenus Tsutsusi, Section Tsutsusi) were utilized for mining and identification of the SSRs for genetic diversity analysis of R. arboreum Smith (Subgenus Tsutsusi, Section Tsutsusi). A total of 249 SSRs were developed from 1767 contigs. Di-nucleotide was found to be most abundant repeat followed by tri- and tetra-nucleotide repeats. The motif AG/CT was most common di-nucleotide motif (31.73%), whereas, AAC/GTT (8.43%), ACG/CGT (8.03%), AAG/CTT (7.23%) and AGG/CCT (6.43%) were most abundant tri-nucleotide repeat motif. Among these SSRs, 168 sequences were only fit into the criteria to design flanking primer pairs. A total of 30 randomly selected primer pairs were utilized for validation and genetic diversity study in 36 genotypes of R. arboreum collected from western Himalayan region. In aggregate, 26 SSR markers (86.66%) produced good and repeatable amplifications. Expected heterozygosity (HE) ranged from 0.322 to 0.841 and observed heterozygosity (HO) ranged from 0.327 to 1.000 and PIC value ranged from 0.008 to 0.786. These primers were able to distinguish the geographic differences of occurrence based on cluster analysis. These developed EST-SSRs can be useful in future population genetics analysis and micro-evolutionary studies in Rhododendron species.

Single Nucleotide Polymorphisms as Practical Molecular Tools to Support European Chestnut Agrobiodiversity Management.

European chestnut orchards are multifunctional agroforestry systems with a key role in environmental management. Their biodiversity is at risk of erosion and farmers do not have enough tools to protect and valorize traditional ecotypes. In particular, cost effective and reliable molecular markers for cultivar identification are lacking. The aim of this research was to develop a new molecular tool for varietal identification in European chestnuts. A set of cultivars was preliminarily characterized to evaluate the range of genetic diversity using random amplified polymorphic DNA (RAPD) markers. The genetic distances indicated a sufficiently wide variability range among tested genotypes and confirmed they were suitable for our goal. A single nucleotide polymorphism (SNP) mining within 64 expressed sequence tags (EST), covering all the linkage groups, was performed by high-resolution melting (HRM) and validated by target resequencing. Fifty-six SNPs were retrieved by monitoring the variability present on the whole set of considered cultivars in loci uniformly distributed on the genome. A subset of 37 SNPs was finally transformed into kompetitive allele-specific PCR (KASP) markers that were successfully evaluated for varietal discrimination. Three assays (C1083, G0115 and A5096) were identified as necessary and sufficient for distinguishing among the tested cultivars. The developed tools can be effectively exploited by stakeholders for improving the management of the European chestnut genetic resources.

Development and application of EST-SSRs markers for analysis of genetic diversity in erect milkvetch (Astragalus adsurgens Pall.).

Erect milkvetch (Astragalus adsurgens Pall.) is a major legume forage plant widely grown in Northern China. However, the lack of molecular markers has limited its research into its genetic diversity and work on germplasm improvement. In this study, a total of 39,163 EST-SSR loci were identified from 30,262 unigene sequences in the erect milkvetch transcriptome using Illumina sequencing. Moreover, 22,367 EST-SSR primer pairs (PPs) were successfully designed. In addition, 100 PPs were synthesized and preliminarily screened in two accessions; of these, 90 were determined to be clear and stable EST-SSR markers. Fifty-one PPs were randomly selected in order to assess the genetic diversity of 27 erect milkvetch accessions. The average polymorphism information content of the 51 PPs was 0.682. Greater genetic diversity was detected in accessions from Inner Mongolia and in the group of landrace and wild erect milkvetch accessions. This study provides an important resource for germplasm improvement and genetic diversity analysis in erect milkvetch.

A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing.

BACKGROUND: Despite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms. RESULTS: The sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes. CONCLUSIONS: The transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane.

Transcriptomic resources for the medicinal legume Mucuna pruriens: de novo transcriptome assembly, annotation, identification and validation of EST-SSR markers.

BACKGROUND: The medicinal legume Mucuna pruriens (L.) DC. has attracted attention worldwide as a source of the anti-Parkinson's drug L-Dopa. It is also a popular green manure cover crop that offers many agronomic benefits including high protein content, nitrogen fixation and soil nutrients. The plant currently lacks genomic resources and there is limited knowledge on gene expression, metabolic pathways, and genetics of secondary metabolite production. Here, we present transcriptomic resources for M. pruriens, including a de novo transcriptome assembly and annotation, as well as differential transcript expression analyses between root, leaf, and pod tissues. We also develop microsatellite markers and analyze genetic diversity and population structure within a set of Indian germplasm accessions. RESULTS: One-hundred ninety-one million two hundred thirty-three thousand two hundred forty-two bp cleaned reads were assembled into 67,561 transcripts with mean length of 626 bp and N50 of 987 bp. Assembled sequences were annotated using BLASTX against public databases with over 80% of transcripts annotated. We identified 7,493 simple sequence repeat (SSR) motifs, including 787 polymorphic repeats between the parents of a mapping population. 134 SSRs from expressed sequenced tags (ESTs) were screened against 23 M. pruriens accessions from India, with 52 EST-SSRs retained after quality control. Population structure analysis using a Bayesian framework implemented in fastSTRUCTURE showed nearly similar groupings as with distance-based (neighbor-joining) and principal component analyses, with most of the accessions clustering per geographical origins. Pair-wise comparison of transcript expression in leaves, roots and pods identified 4,387 differentially expressed transcripts with the highest number occurring between roots and leaves. Differentially expressed transcripts were enriched with transcription factors and transcripts annotated as belonging to secondary metabolite pathways. CONCLUSIONS: The M. pruriens transcriptomic resources generated in this study provide foundational resources for gene discovery and development of molecular markers. Polymorphic SSRs identified can be used for genetic diversity, marker-trait analyses, and development of functional markers for crop improvement. The results of differential expression studies can be used to investigate genes involved in L-Dopa synthesis and other key metabolic pathways in M. pruriens.

Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

BACKGROUND: Chrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits. RESULTS: To obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3'-UTR, and 5'-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums. CONCLUSION: We confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

Molecular marker development from transcript sequences and germplasm evaluation for cultivated peanut (Arachis hypogaea L.).

Molecular markers are important tools for genotyping in genetic studies and molecular breeding. The SSR and SNP are two commonly used marker systems developed from genomic or transcript sequences. The objectives of this study were to: (1) assemble and annotate the publicly available ESTs in Arachis and the in-house short reads, (2) develop and validate SSR and SNP markers, and (3) investigate the genetic diversity and population structure of the peanut breeding lines and the U.S. peanut mini core collection using developed SSR markers. An NCBI EST dataset with 252,951 sequences and an in-house 454 RNAseq dataset with 288,701 sequences were assembled separately after trimming. Transcript sequence comparison and phylogenetic analysis suggested that peanut is closer to cowpea and scarlet bean than to soybean, common bean and Medicago. From these two datasets, 6455 novel SSRs and 11,902 SNPs were identified. Of the discovered SSRs, 380 representing various SSR types were selected for PCR validation. The amplification rate was 89.2 %. Twenty-two (6.5 %) SSRs were polymorphic between at least one pair of four genotypes. Sanger sequencing of PCR products targeting 110 SNPs revealed 13 true SNPs between tetraploid genotypes and 193 homoeologous SNPs within genotypes. Eight out of the 22 polymorphic SSR markers were selected to evaluate the genetic diversity of Florida peanut breeding lines and the U.S. peanut mini core collection. This marker set demonstrated high discrimination power by displaying an average polymorphism information content value of 0.783, a combined probability of identity of 10(-11), and a combined power of exclusion of 0.99991. The structure analysis revealed four sub-populations among the peanut accessions and lines evaluated. The results of this study enriched the peanut genomic resources, provided over 6000 novel SSR markers and the credentials for true peanut SNP marker development, and demonstrated the power of newly developed SSR markers in genotyping peanut germplasm and breeding materials.

Transcriptome analysis in Coffea eugenioides, an Arabica coffee ancestor, reveals differentially expressed genes in leaves and fruits.

Studies in diploid parental species of polyploid plants are important to understand their contributions to the formation of plant and species evolution. Coffea eugenioides is a diploid species that is considered to be an ancestor of allopolyploid Coffea arabica together with Coffea canephora. Despite its importance in the evolutionary history of the main economic species of coffee, no study has focused on C. eugenioides molecular genetics. RNA-seq creates the possibility to generate reference transcriptomes and identify coding genes and potential candidates related to important agronomic traits. Therefore, the main objectives were to obtain a global overview of transcriptionally active genes in this species using next-generation sequencing and to analyze specific genes that were highly expressed in leaves and fruits with potential exploratory characteristics for breeding and understanding the evolutionary biology of coffee. A de novo assembly generated 36,935 contigs that were annotated using eight databases. We observed a total of ~5000 differentially expressed genes between leaves and fruits. Several genes exclusively expressed in fruits did not exhibit similarities with sequences in any database. We selected ten differentially expressed unigenes in leaves and fruits to evaluate transcriptional profiles using qPCR. Our study provides the first gene catalog for C. eugenioides and enhances the knowledge concerning the mechanisms involved in the C. arabica homeologous. Furthermore, this work will open new avenues for studies into specific genes and pathways in this species, especially related to fruit, and our data have potential value in assisted breeding applications.

Marker development, saturation mapping, and high-resolution mapping of the Septoria nodorum blotch susceptibility gene Snn3-B1 in wheat.

Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum, is a severe foliar and glume disease on durum and common wheat. Pathogen-produced necrotrophic effectors (NEs) are the major determinants for SNB on leaves. One such NE is SnTox3, which evokes programmed cell death and leads to disease when recognized by the wheat Snn3-B1 gene. Here, we developed saturated genetic linkage maps of the Snn3-B1 region using two F2 populations derived from the SnTox3-sensitive line Sumai 3 crossed with different SnTox3-insensitive lines. Markers were identified and/or developed from various resources including previously mapped simple sequence repeats, bin-mapped expressed sequence tags, single nucleotide polymorphisms, and whole genome survey sequences. Subsequent high-resolution mapping of the Snn3-B1 locus in 5600 gametes delineated the gene to a 1.5 cM interval. Analysis of micro-colinearity of the Snn3-B1 region indicated that it was highly disrupted compared to rice and Brachypodium distachyon. The screening of a collection of durum and common wheat cultivars with tightly linked markers indicated they are not diagnostic for the presence of Snn3-B1, but can be useful for marker-assisted selection if the SnTox3 reactions of lines are first determined. Finally, we developed an ethyl methanesulfonate-induced mutant population of Sumai 3 where the screening of 408 M2 families led to the identification of 17 SnTox3-insensitive mutants. These mutants along with the markers and high-resolution map developed in this research provide a strong foundation for the map-based cloning of Snn3-B1, which will broaden our understanding of the wheat-P. nodorum system and plant-necrotrophic pathogen interactions in general.

Development of EST-SSR markers in the relict tree Davidia involucrata (Davidiaceae) using transcriptome sequencing.

Davidia involucrata, reputed to be a "living fossil" in the plant kingdom, is a relict tree endemic to China. Extant natural populations are diminishing due to anthropogenic disturbance. In order to understand its ability to survive in a range of climatic conditions and to design conservation strategies for this endangered species, we developed genic simple sequence repeats (SSRs) from mRNA transcripts. In total, 142,950 contigs were assembled. Of these, 30,411 genic SSR loci were discovered and 12,208 primer pairs were designed. Dinucleotides were the most common (77.31%) followed by trinucleotides (16.44%). Thirteen randomly selected primers were synthesized and validated using 24 individuals of D. involucrata. The markers displayed high polymorphism with the number of alleles per locus ranging from 3 to 12 and the observed and expected heterozygosities ranging from 0.083 to 1.0 and 0.102 to 0.69, respectively. This large expressed sequence tag dataset and the novel SSR markers will be key tools in comparative studies that may reveal the adaptive evolution, population structure, and resolve the genetic diversity in this endangered species.

Identification and characterization of expressed retrotransposons in the genome of the Paracoccidioides species complex.

BACKGROUND: Species from the Paracoccidioides complex are thermally dimorphic fungi and the causative agents of paracoccidioidomycosis, a deep fungal infection that is the most prevalent systemic mycosis in Latin America and represents the most important cause of death in immunocompetent individuals with systemic mycosis in Brazil. We previously described the identification of eight new families of DNA transposons in Paracoccidioides genomes. In this work, we aimed to identify potentially active retrotransposons in Paracoccidioides genomes. RESULTS: We identified five different retrotransposon families (four LTR-like and one LINE-like element) in the genomes of three Paracoccidioides isolates. Retrotransposons were present in all of the genomes analyzed. P. brasiliensis and P. lutzii species harbored the same retrotransposon lineages but differed in their copy numbers. In the Pb01, Pb03 and Pb18 genomes, the number of LTR retrotransposons was higher than the number of LINE-like elements, and the LINE-like element RtPc5 was transcribed in Paracoccidioides lutzii (Pb01) but could not be detected in P. brasiliensis (Pb03 and Pb18) by semi-quantitative RT-PCR. CONCLUSION: Five new potentially active retrotransposons have been identified in the genomic assemblies of the Paracoccidioides species complex using a combined computational and experimental approach. The distribution across the two known species, P. brasiliensis and P. lutzii, and phylogenetics analysis indicate that these elements could have been acquired before speciation occurred. The presence of active retrotransposons in the genome may have implications regarding the evolution and genetic diversification of the Paracoccidioides genus.

Deep developmental transcriptome sequencing uncovers numerous new genes and enhances gene annotation in the sponge Amphimedon queenslandica.

BACKGROUND: The demosponge Amphimedon queenslandica is amongst the few early-branching metazoans with an assembled and annotated draft genome, making it an important species in the study of the origin and early evolution of animals. Current gene models in this species are largely based on in silico predictions and low coverage expressed sequence tag (EST) evidence. RESULTS: Amphimedon queenslandica protein-coding gene models are improved using deep RNA-Seq data from four developmental stages and CEL-Seq data from 82 developmental samples. Over 86% of previously predicted genes are retained in the new gene models, although 24% have additional exons; there is also a marked increase in the total number of annotated 3' and 5' untranslated regions (UTRs). Importantly, these new developmental transcriptome data reveal numerous previously unannotated protein-coding genes in the Amphimedon genome, increasing the total gene number by 25%, from 30,060 to 40,122. In general, Amphimedon genes have introns that are markedly smaller than those in other animals and most of the alternatively spliced genes in Amphimedon undergo intron-retention; exon-skipping is the least common mode of alternative splicing. Finally, in addition to canonical polyadenylation signal sequences, Amphimedon genes are enriched in a number of unique AT-rich motifs in their 3' UTRs. CONCLUSIONS: The inclusion of developmental transcriptome data has substantially improved the structure and composition of protein-coding gene models in Amphimedon queenslandica, providing a more accurate and comprehensive set of genes for functional and comparative studies. These improvements reveal the Amphimedon genome is comprised of a remarkably high number of tightly packed genes. These genes have small introns and there is pervasive intron retention amongst alternatively spliced transcripts. These aspects of the sponge genome are more similar unicellular opisthokont genomes than to other animal genomes.

Transcriptome assembly, profiling and differential gene expression analysis of the halophyte Suaeda fruticosa provides insights into salt tolerance.

BACKGROUND: Improvement of crop production is needed to feed the growing world population as the amount and quality of agricultural land decreases and soil salinity increases. This has stimulated research on salt tolerance in plants. Most crops tolerate a limited amount of salt to survive and produce biomass, while halophytes (salt-tolerant plants) have the ability to grow with saline water utilizing specific biochemical mechanisms. However, little is known about the genes involved in salt tolerance. We have characterized the transcriptome of Suaeda fruticosa, a halophyte that has the ability to sequester salts in its leaves. Suaeda fruticosa is an annual shrub in the family Chenopodiaceae found in coastal and inland regions of Pakistan and Mediterranean shores. This plant is an obligate halophyte that grows optimally from 200-400 mM NaCl and can grow at up to 1000 mM NaCl. High throughput sequencing technology was performed to provide understanding of genes involved in the salt tolerance mechanism. De novo assembly of the transcriptome and analysis has allowed identification of differentially expressed and unique genes present in this non-conventional crop. RESULTS: Twelve sequencing libraries prepared from control (0 mM NaCl treated) and optimum (300 mM NaCl treated) plants were sequenced using Illumina Hiseq 2000 to investigate differential gene expression between shoots and roots of Suaeda fruticosa. The transcriptome was assembled de novo using Velvet and Oases k-45 and clustered using CDHIT-EST. There are 54,526 unigenes; among these 475 genes are downregulated and 44 are upregulated when samples from plants grown under optimal salt are compared with those grown without salt. BLAST analysis identified the differentially expressed genes, which were categorized in gene ontology terms and their pathways. CONCLUSIONS: This work has identified potential genes involved in salt tolerance in Suaeda fruticosa, and has provided an outline of tools to use for de novo transcriptome analysis. The assemblies that were used provide coverage of a considerable proportion of the transcriptome, which allows analysis of differential gene expression and identification of genes that may be involved in salt tolerance. The transcriptome may serve as a reference sequence for study of other succulent halophytes.

High-level expression of LMW-GS and α-gliadin genes promoted by the expressed tag sequence of 5' end in Escherichia coli.

Wheat storage protein genes, especially low molecular weight glutenin subunit (LMW-GS) and gliadin genes are difficult to be expressed in Escherichiacoli, mainly due to the presence of highly repetitive sequences. In order to establish a high efficiency expression system for these genes, five different expression plasmids combining with 9 genes, viz. 6 LMW-GS and 3 α-gliadin genes isolated from common wheat and related species, were studied for heterologous expression in E. coli. In this study, when an expressed tag sequence encoding signal peptide, His-S or GST-tag was fused to the 5' end of LMW-GS or gliadin gene as the leading sequence, all recombination genes could be stably expressed at a high level. On the contrast, as expected, the inserted genes encoding mature protein failed without an expressed tag sequence. This result indicated that using expressed tag sequences as leading sequences could promote LMW-GS and gliadin genes to be well expressed in E. coli. Further transcriptional analysis by quantitative real-time PCR (qRT-PCR) showed transcription levels of recombination genes (e.g. GST-Glutenin, His-S-Glutenin and SP(∗)-His-Glutenin) were 4-fold to 33-fold higher than those of the LMW-GS genes, which suggested these expressed tag sequences might play an important role in stimulating transcription. The possible molecular mechanism under this phenomenon was discussed.

Gene expression profiling of coelomic cells and discovery of immune-related genes in the earthworm, Eisenia andrei, using expressed sequence tags.

The coelomic cells of the earthworm consist of leukocytes, chlorogocytes, and coelomocytes, which play an important role in innate immunity reactions. To gain insight into the expression profiles of coelomic cells of the earthworm, Eisenia andrei, we analyzed 1151 expressed sequence tags (ESTs) derived from the cDNA library of the coelomic cells. Among the 1151 ESTs analyzed, 493 ESTs (42.8%) showed a significant similarity to known genes and represented 164 unique genes, of which 93 ESTs were singletons and 71 ESTs manifested as two or more ESTs. From the 164 unique genes sequenced, we found 24 immune-related and cell defense genes. Furthermore, real-time PCR analysis showed that levels of lysenin-related proteins mRNA in coelomic cells of E. andrei were upregulated after the injection of Bacillus subtilis bacteria. This EST data-set would provide a valuable resource for future researches of earthworm immune system.

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation.

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.

Development of expressed sequence tag-simple sequence repeat markers for Chrysanthemum morifolium and closely related species.

With the development of chrysanthemum breeding in recent years, an increasing number of wild species in genera related to Chrysanthemum were introduced to extend the genetic resources and facilitate the genetic improvement of chrysanthemums via hybridization. However, few simple sequence repeat (SSR) markers are available for marker-assisted breeding and population genetic studies of chrysanthemum and closely related species. Expressed sequence tags (ESTs) in public databases and cross-species transferable markers are considered to be a cost-effective means for developing sequence-based markers. In this study, 25 EST-SSRs were successfully developed from Chrysanthemum EST sequences for Chrysanthemum morifolium and closely related species. In total, 4164 unigene sequences were assembled from 7180 ESTs of chrysanthemum in GenBank, which were subsequently used to screen for the presence of microsatellites with the SSRIT software. The screening criteria were 8, 5, 4, and 3 repeating units for di-, tri-, tetra-, and penta- and higher-order nucleotides, respectively. Moreover, 310 SSR loci from 296 sequences were identified, and 198 primer pairs for SSR amplification were designed with the Primer Premier 5.0 software, of which 25 SSR loci showed polymorphic amplification in 52 species and varieties belonging to Chrysanthemum, Ajania, and Opisthopappus. The application of EST-SSR markers to the identification of intergeneric hybrids between Chrysanthemum and Ajania was demonstrated. Therefore, EST-SSRs can be developed for species that lack gene sequences or ESTs by utilizing ESTs of closely related species.