Project description:Common bean (Phaseolus vulgaris) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and specificity in common bean cross-species hybridization (CSH) GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV) regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and specificity. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by about 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR analysis of a total of 20 randomly selected genes and purine-ureides pathway genes demonstrated an increased specificity after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The results from this study suggested that transcript profiling in common bean can be done using the soybean GeneChip. However, a significant decrease in sensitivity and specificity can be expected. Problems associated with CSH GeneChip data can be mitigated by masking probes targeting ISV regions. In addition to transcript profiling CSH of the GeneChip in combination with masking probes in the ISV regions can be used for comparative ecological and/or evolutionary genomics studies.
Project description:Common bean (Phaseolus vulgaris) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and specificity in common bean cross-species hybridization (CSH) GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV) regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and specificity. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by about 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR analysis of a total of 20 randomly selected genes and purine-ureides pathway genes demonstrated an increased specificity after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The results from this study suggested that transcript profiling in common bean can be done using the soybean GeneChip. However, a significant decrease in sensitivity and specificity can be expected. Problems associated with CSH GeneChip data can be mitigated by masking probes targeting ISV regions. In addition to transcript profiling CSH of the GeneChip in combination with masking probes in the ISV regions can be used for comparative ecological and/or evolutionary genomics studies. We hybridized cRNA purified from nodule, leaf, and root of common bean and soybean in, triplicate, to the soybean GeneChip (18 GeneChip hybridizations = 2 species x 3 organs x 3 replicates).
Project description:To investigate graft conferred resistance against viral diseases a novel hetero-grafting system was developed using Nicotiana benthamiana scions grafted onto different tomato rootstocks. RNAseq analysis was used to identify mobile tomato mRNAs within N. benthamiana scions
Project description:A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using high throughput sequencing as a sensitive detection method and mutants to block small RNA (sRNA) biogenesis in either source or recipient tissue, we detected endogenous and transgene specific sRNA that moved across the graft union. Surprisingly we found that the mobile endogenous sRNAs account for a substantial proportion of the sRNA in roots and we provide evidence that 24nt mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defence and responses to external stimuli that have persistent effects in plants. Keywords: Small RNA Analysis, Epigenetics 34 unique samples, 15 Biological Replicates
Project description:Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide 'phased' intervals. TasiRNAs have not been extensively described in many plant species. We identified dozens of new miRNAs in Medicago and soybean and confirmed 119 Medicago targets. A search for phased tasiRNA-like small RNAs ('phasiRNAs') found at least 114 Medicago loci, the majority of which were NB-LRR encoding genes. Notably, conserved domains in NB-LRR-encoding RNAs are targeted by several 22-nt miRNA families to trigger phasiRNA production. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phased small RNAs, suggesting synchronization between silencing and pathogen defense pathways. A second example of 'two-hit' phasiRNA processing was identified, utilizing miR156-miR172 sites. Our data illustrate a complex tasiRNA-mediated regulatory circuit that potentially modulates plant-microbe interactions. A few miRNA triggers regulate an extremely large gene family by targeting highly conserved protein motif-encoding sequences, representing a new paradigm for miRNA function. Examination of different tissue types in legumes (Medicago, soybean, common bean, peanut) by high throughput sequencing for small RNA profiling
Project description:Gene expression profiles in soybean seeds at 4 developmental stages, pod, bean 2 mm, bean 5 mm, and full-sized bean, were examined by DNA microarray analysis. Total genes of each samples were classified into 4 clusters according to developmental stages. Differentially expressed genes (DEGs) were extracted by comparing their expression in two adjacent stages, by using the rank product method. To characterize the gene expression during seed development, DEGs were sorted into 8 clusters by the hclust function, according to gene expression patterns. Keywords: time course Soybean seeds were selected at successive stages of early development for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain transitional changes in gene expression during seed develpment.
Project description:A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using high throughput sequencing as a sensitive detection method and mutants to block small RNA (sRNA) biogenesis in either source or recipient tissue, we detected endogenous and transgene specific sRNA that moved across the graft union. Surprisingly we found that the mobile endogenous sRNAs account for a substantial proportion of the sRNA in roots and we provide evidence that 24nt mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defence and responses to external stimuli that have persistent effects in plants. Keywords: Small RNA Analysis, Epigenetics
Project description:Gene expression profiles in soybean seeds at 4 developmental stages, pod, bean 2 mm, bean 5 mm, and full-sized bean, were examined by DNA microarray analysis. Total genes of each samples were classified into 4 clusters according to developmental stages. Differentially expressed genes (DEGs) were extracted by comparing their expression in two adjacent stages, by using the rank product method. To characterize the gene expression during seed development, DEGs were sorted into 8 clusters by the hclust function, according to gene expression patterns. Keywords: time course
Project description:rasiRNA (rasiRNAs, repeat-associated short interfering RNAs) system is a mechanism of silencing of mobile element transpositions in germline of a number of species including Drosophila melanogaster. rasiRNA itself is a short RNAs which participate in transposon transcription repression and mRNA degradation. Defects in rasiRNA system lead to increased transposition rate and developmental abnormalities due to accumulation of double-strand DNA breaks in fruitfly testes and ovaries. A number of proteins participate in rasiRNA-mediated repression including SPN-E (homeless), PIWI and ARMI. Mutations in the genes of these proteins lead to significant mobile element mRNA accumulation. We performed microarray-based study of effects of spn-E mutation on expression in fruitfly ovaries - one of the organs where rasiRNA system work. Our goal was the identification of other (besides mobile elements) targets of rasiRNA system regulation
Project description:Food proteins differ in their allergenic potential. Currently, there is no predictive and validated bio-assay to evaluate the allergenicity of novel food proteins. The objective of this study was to investigate the potential of a human peripheral blood mononuclear cell (PBMC) gene expression assay to identify biomarkers to predict the allergenicity of legume proteins. PBMCs from healthy donors were exposed to weakly and strongly allergenic legume proteins. Possible biomarkers for allergenicity were investigated by exposing PBMCs to a protein pair of weakly (white bean) and strongly allergenic (soybean) 7S globulins in a pilot experiment. Gene expression was measured by RNA-sequencing and differentially expressed genes were selected as biomarkers. 153 genes were identified as having significantly different expression levels to the 7S globulin of white bean compared to soybean.