Genome sequencing of the staple food crop white Guinea yam enables the development of a molecular marker for sex determination.

BACKGROUND: Root and tuber crops are a major food source in tropical Africa. Among these crops are several species in the monocotyledonous genus Dioscorea collectively known as yam, a staple tuber crop that contributes enormously to the subsistence and socio-cultural lives of millions of people, principally in West and Central Africa. Yam cultivation is constrained by several factors, and yam can be considered a neglected "orphan" crop that would benefit from crop improvement efforts. However, the lack of genetic and genomic tools has impeded the improvement of this staple crop. RESULTS: To accelerate marker-assisted breeding of yam, we performed genome analysis of white Guinea yam (Dioscorea rotundata) and assembled a 594-Mb genome, 76.4% of which was distributed among 21 linkage groups. In total, we predicted 26,198 genes. Phylogenetic analyses with 2381 conserved genes revealed that Dioscorea is a unique lineage of monocotyledons distinct from the Poales (rice), Arecales (palm), and Zingiberales (banana). The entire Dioscorea genus is characterized by the occurrence of separate male and female plants (dioecy), a feature that has limited efficient yam breeding. To infer the genetics of sex determination, we performed whole-genome resequencing of bulked segregants (quantitative trait locus sequencing [QTL-seq]) in F1 progeny segregating for male and female plants and identified a genomic region associated with female heterogametic (male = ZZ, female = ZW) sex determination. We further delineated the W locus and used it to develop a molecular marker for sex identification of Guinea yam plants at the seedling stage. CONCLUSIONS: Guinea yam belongs to a unique and highly differentiated clade of monocotyledons. The genome analyses and sex-linked marker development performed in this study should greatly accelerate marker-assisted breeding of Guinea yam. In addition, our QTL-seq approach can be utilized in genetic studies of other outcrossing crops and organisms with highly heterozygous genomes. Genomic analysis of orphan crops such as yam promotes efforts to improve food security and the sustainability of tropical agriculture.

Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells.

BACKGROUND: The interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3'Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro- and eukaryotic cells without prior fixation or physical disruption of the interaction. RESULTS: Human epithelial cells were infected with Salmonella enterica Typhimurium as a model system for invasion of the intestinal epithelium, and the transcriptional response of the infected host cells together with the differential expression of invading and intracellular pathogen cells was determined by dual 3'Seq coupled with the next-generation sequencing-based transcriptome profiling technique deepSuperSAGE (deep Serial Analysis of Gene Expression). Annotation to reference transcriptomes comprising the operon structure of the employed S. enterica Typhimurium strain allowed for in silico separation of the interacting cells including quantification of polycistronic RNAs. Eighty-nine percent of the known loci are found to be transcribed in prokaryotic cells prior or subsequent to infection of the host, while 75% of all protein-coding loci are represented in the polyadenylated transcriptomes of human host cells. CONCLUSIONS: Dual 3'Seq was alternatively coupled to MACE (Massive Analysis of cDNA ends) to assess the advantages and drawbacks of a library preparation procedure that allows for sequencing of longer fragments. Additionally, the identified expression patterns of both organisms were validated by qRT-PCR using three independent biological replicates, which confirmed that RELB along with NFKB1 and NFKB2 are involved in the initial immune response of epithelial cells after infection with S. enterica Typhimurium.

Next-generation sequencing reveals novel differentially regulated mRNAs, lncRNAs, miRNAs, sdRNAs and a piRNA in pancreatic cancer.

BACKGROUND: Previous studies identified microRNAs (miRNAs) and messenger RNAs with significantly different expression between normal pancreas and pancreatic cancer (PDAC) tissues. Due to technological limitations of microarrays and real-time PCR systems these studies focused on a fixed set of targets. Expression of other RNA classes such as long intergenic non-coding RNAs or sno-derived RNAs has rarely been examined in pancreatic cancer. Here, we analysed the coding and non-coding transcriptome of six PDAC and five control tissues using next-generation sequencing. RESULTS: Besides the confirmation of several deregulated mRNAs and miRNAs, miRNAs without previous implication in PDAC were detected: miR-802, miR-2114 or miR-561. SnoRNA-derived RNAs (e.g. sno-HBII-296B) and piR-017061, a piwi-interacting RNA, were found to be differentially expressed between PDAC and control tissues. In silico target analysis of miR-802 revealed potential binding sites in the 3' UTR of TCF4, encoding a transcription factor that controls Wnt signalling genes. Overexpression of miR-802 in MiaPaCa pancreatic cancer cells reduced TCF4 protein levels. Using Massive Analysis of cDNA Ends (MACE) we identified differential expression of 43 lincRNAs, long intergenic non-coding RNAs, e.g. LINC00261 and LINC00152 as well as several natural antisense transcripts like HNF1A-AS1 and AFAP1-AS1. Differential expression was confirmed by qPCR on the mRNA/miRNA/lincRNA level and by immunohistochemistry on the protein level. CONCLUSIONS: Here, we report a novel lncRNA, sncRNA and mRNA signature of PDAC. In silico prediction of ncRNA targets allowed for assigning potential functions to differentially regulated RNAs.

SuperSAGE as an analytical tool for host and viral gene expression.

SuperSAGE is a tag-based transcript profiling method, which allows to analyze the expression of thousands of genes at a time. In SuperSAGE, 26 bp tags are extracted from cDNA using the type III restriction enzyme, EcoP15I. In SuperSAGE, the amount of transcripts was represented by tag counts. Taking advantage of uniqueness of the 26 bp tags, host and virus transcripts can be monitored in virus-infected cells. Combining next generation sequencing technology, we established High-throughput SuperSAGE (Ht-SuperSAGE), which allows the analysis of multiple samples with reduced time and cost. In this chapter, we present the protocol of Ht-SuperSAGE involving a recently available benchtop type next generation sequencer.

Genome-based analysis of the transcriptome from mature chickpea root nodules.

Symbiotic nitrogen fixation (SNF) in root nodules of grain legumes such as chickpea is a highly complex process that drastically affects the gene expression patterns of both the prokaryotic as well as eukaryotic interacting cells. A successfully established symbiotic relationship requires mutual signaling mechanisms and a continuous adaptation of the metabolism of the involved cells to varying environmental conditions. Although some of these processes are well understood today many of the molecular mechanisms underlying SNF, especially in chickpea, remain unclear. Here, we reannotated our previously published transcriptome data generated by deepSuperSAGE (Serial Analysis of Gene Expression) to the recently published draft genome of chickpea to assess the root- and nodule-specific transcriptomes of the eukaryotic host cells. The identified gene expression patterns comprise up to 71 significantly differentially expressed genes and the expression of twenty of these was validated by quantitative real-time PCR with the tissues from five independent biological replicates. Many of the differentially expressed transcripts were found to encode proteins implicated in sugar metabolism, antioxidant defense as well as biotic and abiotic stress responses of the host cells, and some of them were already known to contribute to SNF in other legumes. The differentially expressed genes identified in this study represent candidates that can be used for further characterization of the complex molecular mechanisms underlying SNF in chickpea.

Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane.

One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes ( αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFP α1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.

APADB: a database for alternative polyadenylation and microRNA regulation events.

Alternative polyadenylation (APA) is a widespread mechanism that contributes to the sophisticated dynamics of gene regulation. Approximately 50% of all protein-coding human genes harbor multiple polyadenylation (PA) sites; their selective and combinatorial use gives rise to transcript variants with differing length of their 3' untranslated region (3'UTR). Shortened variants escape UTR-mediated regulation by microRNAs (miRNAs), especially in cancer, where global 3'UTR shortening accelerates disease progression, dedifferentiation and proliferation. Here we present APADB, a database of vertebrate PA sites determined by 3' end sequencing, using massive analysis of complementary DNA ends. APADB provides (A)PA sites for coding and non-coding transcripts of human, mouse and chicken genes. For human and mouse, several tissue types, including different cancer specimens, are available. APADB records the loss of predicted miRNA binding sites and visualizes next-generation sequencing reads that support each PA site in a genome browser. The database tables can either be browsed according to organism and tissue or alternatively searched for a gene of interest. APADB is the largest database of APA in human, chicken and mouse. The stored information provides experimental evidence for thousands of PA sites and APA events. APADB combines 3' end sequencing data with prediction algorithms of miRNA binding sites, allowing to further improve prediction algorithms. Current databases lack correct information about 3'UTR lengths, especially for chicken, and APADB provides necessary information to close this gap. Database URL: http://tools.genxpro.net/apadb/.

omiRas: a Web server for differential expression analysis of miRNAs derived from small RNA-Seq data.

SUMMARY: Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA-mRNA interaction databases. AVAILABILITY AND IMPLEMENTATION: The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/.

SymGRASS: a database of sugarcane orthologous genes involved in arbuscular mycorrhiza and root nodule symbiosis.

BACKGROUND: The rationale for gathering information from plants procuring nitrogen through symbiotic interactions controlled by a common genetic program for a sustainable biofuel production is the high energy demanding application of synthetic nitrogen fertilizers. We curated sequence information publicly available for the biofuel plant sugarcane, performed an analysis of the common SYM pathway known to control symbiosis in other plants, and provide results, sequences and literature links as an online database. METHODS: Sugarcane sequences and informations were downloaded from the nucEST database, cleaned and trimmed with seqclean, assembled with TGICL plus translating mapping method, and annotated. The annotation is based on BLAST searches against a local formatted plant Uniprot90 generated with CD-HIT for functional assignment, rpsBLAST to CDD database for conserved domain analysis, and BLAST search to sorghum's for Gene Ontology (GO) assignment. Gene expression was normalized according the Unigene standard, presented as ESTs/100 kb. Protein sequences known in the SYM pathway were used as queries to search the SymGRASS sequence database. Additionally, antimicrobial peptides described in the PhytAMP database served as queries to retrieve and generate expression profiles of these defense genes in the libraries compared to the libraries obtained under symbiotic interactions. RESULTS: We describe the SymGRASS, a database of sugarcane orthologous genes involved in arbuscular mycorrhiza (AM) and root nodule (RN) symbiosis. The database aggregates knowledge about sequences, tissues, organ, developmental stages and experimental conditions, and provides annotation and level of gene expression for sugarcane transcripts and SYM orthologous genes in sugarcane through a web interface. Several candidate genes were found for all nodes in the pathway, and interestingly a set of symbiosis specific genes was found. CONCLUSIONS: The knowledge integrated in SymGRASS may guide studies on molecular, cellular and physiological mechanisms by which sugarcane controls the establishment and efficiency of endophytic associations. We believe that the candidate sequences for the SYM pathway together with the pool of exclusively expressed tentative consensus (TC) sequences are crucial for the design of molecular studies to unravel the mechanisms controlling the establishment of symbioses in sugarcane, ultimately serving as a basis for the improvement of grass crops.

Expression Analysis of Sugarcane Aquaporin Genes under Water Deficit.

The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP), already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1) showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4) were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific.

Transcriptomic analysis of oxylipin biosynthesis genes and chemical profiling reveal an early induction of jasmonates in chickpea roots under drought stress.

Drought is one of the major constraints in subtropical agriculture. Therefore improving water stress tolerance is of great importance to breed for drought tolerance in future. The first plant organ sensing dehydration is the root. Aim of the present work was to clarify the potential impact of the phyto-oxylipins pathway on drought tolerance of chickpea (Cicer arietinum), the third important legume crop worldwide. Therefore, we measured the expression of key genes involved in oxylipins metabolism by qPCR on samples from stressed and non-stressed roots of a drought-tolerant and a drought-sensitive chickpea variety using commercially available TaqMan assays. We demonstrate that the drought tolerant variety reacts to drought with sustained and earlier activation of a specific lipoxygenase (Mt-LOX 1) gene, two hydroperoxide lyases (Mt-HPL 1 and Mt-HPL 2), an allene oxide synthase (Mt-AOS), and an oxo-phytodienoate reductase (Mt-OPR). We further show that gene over-expression positively correlates with the levels of major oxylipin metabolites from the AOS branch of the pathway, which finally leads to the synthesis of jasmonates. Higher levels of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA) and the active form JA-isoleucine (JA-Ile) were especially detected in the root tissues of the tolerant variety, prompting us to assume a role of jasmonates in the early signalling of drought stress in chickpea and its involvement in the tolerance mechanism of the drought-tolerant variety.

SuperSAGE: powerful serial analysis of gene expression.

SuperSAGE is a variant of the Serial Analysis of Gene Expression (SAGE) technology, based on counting transcripts by sequencing analysis of short sequence tags. In SuperSAGE, 26 bp tags are extracted from cDNA using the Type III restriction endonuclease EcoP15I. The use of a longer tag size in SuperSAGE allows a secure tag-to-gene annotation in any eukaryotic organism. We have succeeded in combining SuperSAGE and high-throughput sequencing technology (Now- or Next-Generation Sequencing, NGS) in an approach we call High-throughput SuperSAGE (HT-SuperSAGE). This approach allows deep transcriptome analysis and multiplexing, while reducing time, cost, and effort for the analysis. In this chapter, we present the detailed HT-SuperSAGE protocol for both the Illumina Genome Analyzer and also the AppliedBiosystems SOLiD sequencer.

Novel SSR markers from BAC-end sequences, DArT arrays and a comprehensive genetic map with 1,291 marker loci for chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes.

Integration of genetic and physical maps of the chickpea (Cicer arietinum L.) genome using flow-sorted chromosomes.

Cultivated chickpea is the third most important legume after field bean and garden pea worldwide. Despite considerable breeding towards improved yield and resistance to biotic and abiotic stresses, the production of chickpea remained stagnant, but molecular tools are expected to increase the impact of current improvement programs. As a first step towards this goal, various genetic linkage maps have been established and markers linked to resistance genes been identified. However, until now, only one linkage group (LG) has been assigned to a specific chromosome. In the present work, mitotic chromosomes were sorted using flow cytometry and used as template for PCR with primers designed for genomic regions flanking microsatellites. These primers amplify sequence-tagged microsatellite site markers. This approach confirmed the assignment of LG8 to the smallest chromosome H. For the first time, LG5 was linked to the largest chromosome A, LG4 to a medium-sized chromosome E, while LG3 was anchored to the second largest chromosome B. Chromosomes C and D could not be flow-sorted separately and were jointly associated to LG6 and LG7. By the same token, chromosomes F and G were anchored to LG1 and LG2. To establish a set of preferably diagnostic cytogenetic markers, the genomic distribution of various probes was verified using FISH. Moreover, a partial genomic bacterial artificial chromosome (BAC) library was constructed and putative single/low-copy BAC clones were mapped cytogenetically. As a result, two clones were identified localizing specifically to chromosomes E and H, for which no cytogenetic markers were yet available.

Identification of plant protein kinases in response to abiotic and biotic stresses using SuperSAGE.

Plants are sessile organisms subjected to many environmental adversities. For their survival they must sense and respond to biotic and abiotic stresses efficiently. During this process, protein kinases are essential in the perception of environmental stimuli, triggering signaling cascades. Kinases are among the largest and most important gene families for biotechnological purposes, bringing many challenges to the bioinformaticians due to the combination of conserved domains besides diversified regions. Cowpea [Vigna unguiculata (L.) Walp.] is an important legume that is adapted to different agroclimatic conditions, including drought, humidity and a range of temperatures. For this crop, the association of the SuperSAGE method with high-throughput sequencing technology would generate reliable transcriptome profiles with millions of tags counted and statistically analyzed. An approach evaluating biotic and abiotic stresses was carried out generating over 13 million cowpea SuperSAGE tags available from leaves/roots of plants under abiotic (mechanical injury and salinity) or biotic (CABMV, Cowpea aphid born mosaic virus) stresses. The annotation and identification of tags linked by BlastN to previously well described ESTs, allowed the posterior identification of kinases. The annotation efficiency depended on the database used, with the KEGG figuring as a good source for annotated ESTs especially when complemented by an independent Gene Ontology categorization, as well as the Gene Index using selected species. The use of different approaches allowed the identification of 1,350 kinase candidates considering biotic libraries and 2,268 regarding abiotic libraries, based on a combination of both, adequate descriptions and GO terms. Additional searches in kinase specific databases allowed the identification of a relatively low number of additional kinases, uncovering the lack of kinase databases for non-model organisms, especially plants. Concerning the kinase families, a total of 713 potential kinases were classified into 13 families of the CMGC and STE groups. Concerning the differentially expressed kinases, 169 of the 713 potential kinases were identified (p < 0.05), 100 up- and 69 down-regulated when comparing distinct libraries, allowing the generation of a comprehensive panel of the differentially expressed kinases under biotic and abiotic stresses in a non-model plant as cowpea.

The salt-responsive transcriptome of chickpea roots and nodules via deepSuperSAGE.

BACKGROUND: The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche's 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level. RESULTS: We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26 bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26 bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress.Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways. From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available. CONCLUSIONS: This report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms. As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25 mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea. Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26 bp tags by SuperSAGE.

High-throughput SuperSAGE.

SuperSAGE is a method of digital gene expression profiling that allows isolation of 26-bp tag fragments from expressed transcripts. Combined with the ultrahigh-throughput sequencing technologies, SuperSAGE enables analysis of millions of transcripts with lower cost and reduced effort and time. In this chapter, we present an updated protocol for this High-throughput SuperSAGE method with a special emphasis on the technique of library multiplexing.

High-throughput SuperSAGE for digital gene expression analysis of multiple samples using next generation sequencing.

We established a protocol of the SuperSAGE technology combined with next-generation sequencing, coined "High-Throughput (HT-) SuperSAGE". SuperSAGE is a method of digital gene expression profiling that allows isolation of 26-bp tag fragments from expressed transcripts. In the present protocol, index (barcode) sequences are employed to discriminate tags from different samples. Such barcodes allow researchers to analyze digital tags from transcriptomes of many samples in a single sequencing run by simply pooling the libraries. Here, we demonstrated that HT-SuperSAGE provided highly sensitive, reproducible and accurate digital gene expression data. By increasing throughput for analysis in HT-SuperSAGE, various applications are foreseen and several examples are provided in the present study, including analyses of laser-microdissected cells, biological replicates and tag extraction using different anchoring enzymes.

Integration of novel SSR and gene-based SNP marker loci in the chickpea genetic map and establishment of new anchor points with Medicago truncatula genome.

This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea (Cicer arietinum ICC 4958 x C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM-ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2-21 alleles and polymorphic information content value 0.04-0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago.