Project description:Background: The fertile and sterile plants are derived from the self-pollinated offspring of the F1 hybrid between novel restorer line NR1 and Nsa CMS line in Brassica napus, which possess the identical cytoplasmic genetic material arising from Nsa CMS line. As far as the nuclear genetic background is concerned, both fertile and sterile plants have the complete set of chromosomes from Brassica napus, except one or two members of the added Sinapis arvensis chromosome pair in the fertile plant. To elucidate gene expression and regulation caused by the A and C subgenomes, the alien chromosome and cytoplasm from S. arvensis during the development of young floral buds, we performed genome-widely high-throughput transcriptomic sequencing between young floral buds of sterile and fertile plants. Results: In this study, equal amount of RNA taken from young floral buds of sterile and fertile plants were sequenced using Illumina/Solexa platform. A total of 4,415,866 and 4,244,140 raw tags were obtained in sterile plant (Ste) and fertile plant (Fer) libraries, respectively. After filtering out low quality data, a total of 2,760,574 and 2,714,441 clean tags remained from the two libraries, from which 242,163 (Ste) and 253,507 (Fer) distinct tags were obtained. To identify the genes corresponding to the distinct tags in each library, all distinct sequencing tags were annotated using all possible CATG+17-nt sequences of the genome and transcriptome of Brassica rapa and those of Brassica oleracea as the reference sequences, respectively. Many genes showed substantial differences in expression between the two libraries. In total, there were 3231 genes of B. rapa and 3371 genes of B. oleracea which were detected with significant differential expression levels. GO and pathway-based analyses were performed to determine and further understand the biological functions of differentially expressed genes (DEGs). In addition, there were 1089 specially expressed unknown tags in Fer, which were neither mapped to B. oleracea nor mapped to B. rapa, and these unique tags were presumed to arise basically from the added alien chromosome of S. arvensis. Fifteen genes were randomly selected and confirmed their expression levels by quantitative RT-PCR, fourteen of the fifteen genes showed expression patterns consistent with the digital gene expression (DGE) data. Conclusions: A number of genes were differentially expressed between the young floral buds of sterile and fertile plants. Some of these genes may be candidates for future research on CMS in Nsa line, fertility restoration and improved agronomic traits in NR1 line. Further study of the unknown tags which were specially expressed in Fer will help to dig those genes with desirable agronomic traits from wild species. mRNA profiles of fertile buds (Fer) and sterile buds (Ste) were generated by deep sequencing.

Project description:Background: The fertile and sterile plants are derived from the self-pollinated offspring of the F1 hybrid between novel restorer line NR1 and Nsa CMS line in Brassica napus, which possess the identical cytoplasmic genetic material arising from Nsa CMS line. As far as the nuclear genetic background is concerned, both fertile and sterile plants have the complete set of chromosomes from Brassica napus, except one or two members of the added Sinapis arvensis chromosome pair in the fertile plant. To elucidate gene expression and regulation caused by the A and C subgenomes, the alien chromosome and cytoplasm from S. arvensis during the development of young floral buds, we performed genome-widely high-throughput transcriptomic sequencing between young floral buds of sterile and fertile plants. Results: In this study, equal amount of RNA taken from young floral buds of sterile and fertile plants were sequenced using Illumina/Solexa platform. A total of 4,415,866 and 4,244,140 raw tags were obtained in sterile plant (Ste) and fertile plant (Fer) libraries, respectively. After filtering out low quality data, a total of 2,760,574 and 2,714,441 clean tags remained from the two libraries, from which 242,163 (Ste) and 253,507 (Fer) distinct tags were obtained. To identify the genes corresponding to the distinct tags in each library, all distinct sequencing tags were annotated using all possible CATG+17-nt sequences of the genome and transcriptome of Brassica rapa and those of Brassica oleracea as the reference sequences, respectively. Many genes showed substantial differences in expression between the two libraries. In total, there were 3231 genes of B. rapa and 3371 genes of B. oleracea which were detected with significant differential expression levels. GO and pathway-based analyses were performed to determine and further understand the biological functions of differentially expressed genes (DEGs). In addition, there were 1089 specially expressed unknown tags in Fer, which were neither mapped to B. oleracea nor mapped to B. rapa, and these unique tags were presumed to arise basically from the added alien chromosome of S. arvensis. Fifteen genes were randomly selected and confirmed their expression levels by quantitative RT-PCR, fourteen of the fifteen genes showed expression patterns consistent with the digital gene expression (DGE) data. Conclusions: A number of genes were differentially expressed between the young floral buds of sterile and fertile plants. Some of these genes may be candidates for future research on CMS in Nsa line, fertility restoration and improved agronomic traits in NR1 line. Further study of the unknown tags which were specially expressed in Fer will help to dig those genes with desirable agronomic traits from wild species.

Project description:Purpose: Brassica. juncea is vulnerable to abiotic stresses at specific stages in its life cycle. However, till date no attempts have been made to elucidate the genome-wide changes in the transcriptome of B. juncea subjected to either high temperature or drought stress. Hence, to gain global insights into genes, transcription factors and kinases regulated by these stresses and to provide basic information on coding transcripts that are associated with traits of agronomic importance, we utilized a combinatorial approach of next generation sequencing and de novo assembly to discover B. juncea transcriptome associated with high temperature and drought. Results: We constructed and sequenced three transcriptome libraries namely Brassica control (BC), Brassica high temperature stress (BHS) and Brassica drought stress (BDS) from control, high temperature treated and drought treated seedlings of Brassica juncea. More than 180 million purity filtered reads were generated which were processed through quality parameters and high quality reads were assembled de-novo using SOAPde-novo assembler. A total of 77750 unique transcripts were identified out of which 69,245 (89%) were annotated with high confidence. We established a subset of 19110 transcripts, which were differentially regulated by either high temperature and/or drought stress. Furthermore, 886 and 2834 transcripts that code for transcription factors and kinases, respectively, were also identified. Investigation of identified transcription factors revealed that 92 responded to high temperature, 72 exhibited alterations in expression during drought stress, and 60 were commonly associated with both the stresses. Similarly, 217, 259 and 193 kinases were responsive to high temperature, drought or both stresses, respectively. Maximum number of up-regulated transcription factors in high temperature and drought stress belonged to heat shock factors (HSFs) and dehydration responsive element-binding (DREB) families respectively. We also identified 239 metabolic pathways, which were perturbed during high temperature and drought treatments. Analysis of gene ontologies associated with differentially regulated genes forecasted their involvement in diverse biological processes. Conclusions: Our study provides first comprehensive discovery of B. juncea transcriptome under high temperature and drought stress conditions. Transcriptome resources generated in this study will enhance our understanding on the molecular mechanisms involved in defining the response of B. juncea against two important abiotic stresses. Furthermore this information would benefit designing of efficient crop improvement strategies for tolerance against conditions of high temperature regimes and water scarcity. Total three RNA-Seq libraries were prepared and sequenced independently [B. juncea control (BC), B. juncea high temperature stressed (BHS) and B. juncea drought stressed (BDS) on Illumina GAIIx sequencer].

Project description:Purpose: Brassica. juncea is vulnerable to abiotic stresses at specific stages in its life cycle. However, till date no attempts have been made to elucidate the genome-wide changes in the transcriptome of B. juncea subjected to either high temperature or drought stress. Hence, to gain global insights into genes, transcription factors and kinases regulated by these stresses and to provide basic information on coding transcripts that are associated with traits of agronomic importance, we utilized a combinatorial approach of next generation sequencing and de novo assembly to discover B. juncea transcriptome associated with high temperature and drought. Results: We constructed and sequenced three transcriptome libraries namely Brassica control (BC), Brassica high temperature stress (BHS) and Brassica drought stress (BDS) from control, high temperature treated and drought treated seedlings of Brassica juncea. More than 180 million purity filtered reads were generated which were processed through quality parameters and high quality reads were assembled de-novo using SOAPde-novo assembler. A total of 77750 unique transcripts were identified out of which 69,245 (89%) were annotated with high confidence. We established a subset of 19110 transcripts, which were differentially regulated by either high temperature and/or drought stress. Furthermore, 886 and 2834 transcripts that code for transcription factors and kinases, respectively, were also identified. Investigation of identified transcription factors revealed that 92 responded to high temperature, 72 exhibited alterations in expression during drought stress, and 60 were commonly associated with both the stresses. Similarly, 217, 259 and 193 kinases were responsive to high temperature, drought or both stresses, respectively. Maximum number of up-regulated transcription factors in high temperature and drought stress belonged to heat shock factors (HSFs) and dehydration responsive element-binding (DREB) families respectively. We also identified 239 metabolic pathways, which were perturbed during high temperature and drought treatments. Analysis of gene ontologies associated with differentially regulated genes forecasted their involvement in diverse biological processes. Conclusions: Our study provides first comprehensive discovery of B. juncea transcriptome under high temperature and drought stress conditions. Transcriptome resources generated in this study will enhance our understanding on the molecular mechanisms involved in defining the response of B. juncea against two important abiotic stresses. Furthermore this information would benefit designing of efficient crop improvement strategies for tolerance against conditions of high temperature regimes and water scarcity.

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Brassica juncea

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