Project description:‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea accessions tolerant and susceptible to high-salinity stress. Two groups of a tolerant and susceptible accession were challenged with high-salinity stress. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaf/shoot and root tissues were collected at 24 and 48 h post-treatment (hpt) and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (shoot and root tissues separate for each time point) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1. The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress. Globally, the level of 409 transcripts was affected in response to high-salinity stress in all the genotypes and tissue types studied. Of the transcripts consistently expressed in tolerant genotypes in response to high-salinity stress, the annotation of transcripts at 24 hpt suggest a reduction in energy production in shoots and roots by repression of putative genes including P700 chlorophyll a-apoprotein (DY475501) and NADH-plastoquinone oxidoreductase subunit I (DY475287), cytosolic fructose 1,6-bisphosphatase (DY475548) and splicing factor-like protein (DY396290). The ATHP3 (DY396300) and protein kinase (DY475077) that are potentially involved in signalling cascades responsible for sensing and relaying osmotic stress signals, were consistently repressed in tolerant genotypes in response to high-salinity. Additionally a glycine rich protein (DY396342) that is associated with lignification of cell walls in response to wounding and pathogen attack was repressed in tolerant genotypes. In tolerant genotypes at 48 hpt, two energy and metabolic-related transcripts were consistently repressed in roots, including a carbonic anhydrase (DY475403) and putative thiazole biosynthetic enzyme (DY475242). In shoots, only a pathogenesis-related protein (DY396301) was consistently repressed at 48 hpt, but a similar transcript (DY396281) was induced in roots of tolerant genotypes at the same time. Only four transcripts were DE in susceptible genotypes at 24 hpt, all occurring in root tissue. Two of these were unknown/unclear, but the others included a proline oxidase transcript (DY475225) and a nuclear transport factor 2 (DY396436). At 48 hpi, a putative splicing factor-like protein (DY396290) and polyubiquitin (DY396328) were repressed in roots of susceptible genotypes. Interstingly, a putative xylosidase (DY475408) was induced in susceptible roots at 48 hpi. Finally, several unknown/unclear transcripts were DE in both tolerant and susceptible genotypes. Of interest were two unknowns (DY475293 and DY475521) that were consistently expressed in tolerant genotypes and may be important for high-salinity tolerance. Keywords: Chickpea, High-salinity stress, tolerant, susceptible, cDNA microarray

Project description:Background and Aims: In the interleukin-10-deficient (Il10-/-) mouse model of IBD, 10 quantitative trait loci (QTL) have been shown to be associated with colitis susceptibility by linkage analyses on experimental crosses of highly susceptible C3H/HeJBir (C3Bir)-Il10-/- and partially resistant C57BL/6J (B6)-Il10-/- mice. The strongest locus (C3Bir-derived cytokine deficiency-induced colitis susceptibility [Cdcs]1 on Chromosome [Chr] 3) controlled multiple colitogenic subphenotypes and contributed the vast majority to the phenotypic variance in cecum and colon. This was demonstrated by interval-specific Chr 3 congenic mice wherein defined regions of Cdcs1 from C3Bir or B6 were bred into the IL-10-deficient reciprocal background and altered the susceptible or resistant phenotype. Furthermore, this locus likely acts by inducing innate hypo- and adaptive hyperresponsiveness, associated with impaired NFΚB responses of macrophages. The aim of the present study was to dissect the complexity of Cdcs1 by further development and characterization of reciprocal Cdcs1 congenic strains and to identify potential candidate genes in the congenic interval. Material and Methods: In total, 15 reciprocal congenic strains were generated from Il10-/- mice of either C3H/HeJBir or C57BL/6J backgrounds by 10 cycles of backcrossing. Colitis activity was monitored by histological grading. Candidate genes were identified by fine mapping of congenic intervals, sequencing, microarray analysis and a high-throughput real-time RT-PCR approach using bone marrow-derived macrophages. Results: Within the originally identified Cdcs1-interval, three independent regions were detected that likely contain susceptibility-determining genetic factors (Cdcs1.1, Cdcs1.2, and Cdcs1.3). Combining results of candidate gene approaches revealed Fcgr1, Cnn3, Larp7, and Alpk1 as highly attractive candidate genes with polymorphisms in coding or regulatory regions and expression differences between susceptible and resistant mouse strains. Conclusions: Subcongenic analysis of the major susceptibility locus Cdcs1 on mouse chromosome 3 revealed a complex genetic structure. Candidate gene approaches revealed attractive genes within the identified regions with homologs that are located in human susceptibility regions for IBD.

Project description:‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea accessions tolerant and susceptible to cold stress. Two groups of a tolerant and susceptible accession were challenged with cold stress. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaves and flowers/buds/early pods tissues were collected and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (leaf and flower tissues separate) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1. The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress. Globally, the level of 221 transcripts was affected in response to cold stress in all the genotypes and tissue types studied. The DE transcripts in response to cold stress fell into various functional categories, indicating a broad response. Sixteen out of the 221 DE transcripts were consistently expressed in cold tolerant/susceptible genotypes. All these transcripts were repressed and none was found to be consistently induced in response to cold-stress. Most of the putative genes were identified in leaves of tolerant genotypes, and included a beta-galactosidase (DY475141) transcript that was possibly indicative of disaccharide (e. g. sucrose) retention with the effect of protecting cell membranes during cold stress. Several protein synthesis/modification and energy/metabolism transcripts were also repressed (e.g. DY475282, DY396371 and DY475555), which was likely due to the impairment of photosynthesis and respiration at low temperature. Other consistently repressed transcripts in tolerant genotypes included putative signalling (DY396262, DY475384 and DY396307) and defence-related proteins (CV793589 and DY396343), which may be involved in the repression of cell death mechanisms that are absent in tolerant genotypes. In susceptible genotypes, a putative superoxide dismutase precursor protein (DY475397) and sorting nexin protein (DY475523) were the only known transcripts to be consistently repressed. Keywords: Chickpea, Cold stress, Tolerant, Susceptible, cDNA microarray

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