Project description:Among Brassica rapa, rapid cycling Brassica rapa and Brassica rapa inbred line Kenshin showed contrasting leaf morphology. To identify genes associated with leaf morphology, four distinct F2 progeny of RcBr X Kenshin cross and their parents were selected. Leaf samples were collected from 6 materials, isolated total RNA, and subjected to newly developved 135K microarray. Experiments were performed with three or two biologic Overall design: A total of 6 chips were used for the microarray experiment. Experiments were performed with three or two biological replicates.
Project description:The mapping and functional analysis of quantitative traits in Brassica rapa can be greatly improved with the availability of physically positioned, gene-based genetic markers and accurate genome annotation. In this study, deep transcriptome RNA sequencing (RNA-Seq) of Brassica rapa was undertaken with two objectives: SNP detection and improved transcriptome annotation. We performed SNP detection on two varieties that are parents of a mapping population to aid in development of a marker system for this population and subsequent development of high-resolution genetic map. An improved Brassica rapa transcriptome was constructed to detect novel transcripts and to improve the current genome annotation. Deep RNA-Seq of two Brassica rapa genotypes—R500 (var. trilocularis, Yellow Sarson) and IMB211 (a rapid cycling variety)—using eight different tissues (root, internode, leaf, petiole, apical meristem, floral meristem, silique, and seedling) grown across three different environments (growth chamber, greenhouse and field) and under two different treatments (simulated sun and simulated shade) generated 2.3 billion high-quality Illumina reads. In this experiment, two pools were made, with one pool consisting of 66 samples collected from growth chamber and another pool consisting of 60 samples collected from greenhouse and field. Each pool was sequenced on eight lanes (total 16 lanes) of an Illumina Genome Analyzer (GAIIx) as 100-bp paired end reads.
Project description:Root and leaf samples from Brassica rapa line R-O-18 were compared. The results will be compared to the same samples hybridised to the Affymetrix Brassica Exon 1.0 ST array. Overall design: 6 samples were hybridised. Triplcate samples of 11 day old roots and 2 semi-expanded leaves from 23 day old Brassica rapoa R-O-18 plants.
Project description:Root and leaf samples from Brassica rapa line R-O-18 were compared. The results will be compared to the same samples hybridised to the Affymetrix Brassica Exon 1.0 ST array. 6 samples were hybridised. Triplcate samples of 11 day old roots and 2 semi-expanded leaves from 23 day old Brassica rapoa R-O-18 plants.
Project description:An allopolyploid formation consists of the two processes of hybridisation and chromosome doubling. Hybridisation makes a different genome combined in the same cell, and genome “shock” and instability occur during this process, whereas chromosome doubling results in doubling and reconstructing the genome dosage. Recent studies have demonstrated that small RNAs, mainly siRNAs and miRNAs, play an important role in maintaining the genome reconstruction and stability. However, to date, little is known regarding the role of small RNAs during the process of wide hybridisation and chromosome doubling, which is essential to elucidate the mechanism of polyploidisation. Therefore, the genetic and DNA methylation alterations and changes in the siRNA and miRNA were assessed during the formation of an allodiploid (genome: AB) and its allotetraploid (genome: AABB) between Brassica rapa (♀) and Brassica nigra (♂) in the present study.The phenotypic analysis exhibited that the allotetraploid had high heterosis compared with their parents and the allodiploid. The methylation-sensitive amplification polymorphism (MSAP) analysis indicated that the proportion of changes in the methylation pattern of the allodiploid was significantly higher than that found in the allotetraploid, while the DNA methylation ratio was higher in the parents than the allodiploid and allotetraploid. The high-throughput sequencing results obtained for the small RNAs showed that the expression levels of miRNAs increased in the allodiploid and allotetraploid compared with the parents, and the expression levels of siRNAs increased and decreased compared with the parents B. rapa and B. nigra, respectively. Moreover, the percentages of miRNAs increased with an increase in the polyploidy levels, but the percentages of siRNAs and DNA methylation alterations decreased with an increase in the polyploidy levels. Furthermore, 320 known and 52 novel miRNAs were obtained from the parents in both the allodiploid and allotetraploid. However, quantitative real-time polymerase chain reaction (qRT PCR) analysis showed that the expression levels of the targets genes were negatively corrected with the expressed miRNAs.The study showed that siRNAs and DNA methylation play an important role in maintaining the genome stability in the formation of an allotetraploid. The miRNAs regulate gene expression and induce the phenotype variation, which may play an important role in the occurrence of heterosis in the allotetraploid. The findings of this study may provide new information for elucidating that the allotetraploids have a growth advantage over the parents and the allodiploids. High throughput sequence of the parents (Brassica rapa and Brassica nigra) and their hybrids (allodiploid and allotetraploid)
Project description:In this study genome-wide gene expression profiling was used to analyze mechanisms of drought tolerance in Brassica rapa. Using an Illumina Mi-Seq platform we sequenced RNA from shoot tissues of drought tolerant and drought sensitive B. rapa genotypes in control conditions and after application of osmotic stress. Differentially expressed genes between the different conditions and genotypes were used to identify drought relevant gene networks. Overall design: Two accessions of Brassica rapa with previously-identified contrasting drought responses were exposed to osmotic stress in a hydroponic growth system. Polyethylene glycol (PEG) simulated osmotic stress applied to half of the plants of each accession when plants had 4-5 true leaves while the other half remained with Murashige and Skoog (MS) medium as control for PEG. Total RNA isolated from shoot tissue was used for RNA-sequencing. For each genotype and condition three biological replicates were taken at 4h, 8h and 12h after stress treatment. cDNA libraries were prepared with the TruSeq Stranded Total RNA with Ribo-ZeroTM Plant kit from Illumina. Sequencing was carried out on each library to generate 150 bp PE reads using an Illumina Mi-Seq platform. The transcriptome reads were mapped to the B. rapa cv. Chiifu reference genome v1.5 (Wang et al., 2011) allowing 3 mismatches, then normalised and quantified as FPKM values using the spliced aligner TopHat/Cufflinks (http://ccb.jhu.edu/software/tophat/index.shtml,http://cole-trapnell-lab.github.io/cufflinks/).
Project description:Purpose: Zinc deficiency (ZnD) and iron deficiency (FeD), excess Zn (ZnE) and cadmium exposure (CdE) are major environmental problems for crop cultivation. Methods: Applying Tag-Seq technology to leaves of Brassica rapa grown under FeD, ZnD, ZnE or CdE conditions, with normal conditions as a control, we examined global gene expression changes and compared the expression patterns of multiple paralogs. Results: We identified 812, 543, 331 and 447 differentially expressed genes under FeD, ZnD, ZnE and CdE conditions, respectively, in B. rapa leaves.Further analysis revealed that genes associated with Zn, Fe and Cd responses tended to be over-retained in the B. rapa genome. Most of these multiple-copy genes showed the same direction of expression change under stress conditions. Conclusion: We conclude that the duplicated genes involved in trace element responses in B. rapa are functionally redundant, making the regulatory network more complex in B. rapa than in Arabidopsis thaliana. In total, there were 15 Digital gene expression libraries, one for each of the three replicates under the four trace metal element treatments and normal nutrient supply conditions as a control.
Project description:The aim of this study was to identify eQTL in Brassica rapa grown under altered soil phosphorus (P) supply, to understand better the genetic architecture of P-use efficiency (PUE) in plants. Recombinant inbred lines (RILs) of the BraIRRI mapping population were grown at adequate and growth-limiting soil P. Variation in leaf gene expression was quantified using an Agilent Brassica 95k oligonucleotide array. Informative gene expression markers (GEMs) were used to map eQTL and PUE-related QTL. Gene expression was highly dependent on soil P supply. However, the altered expression of many genes, including known P-responsive genes, was highly heritable. Interval mapping using P supply as a covariate revealed 18,876 eQTL, representing 15,912 unique probes. Notable trans-eQTL hotspots occurred on chromosomes A06 and A01; these were enriched with protein modification and phosphorus metabolism-related (A06), as well as chloroplast and photosynthesis-related (A01) transcripts. Regulatory loci and genes associated with P-use efficiency identified through eQTL analysis are potential targets for further characterisation and may have potential for crop improvement. Availability of the annotated B. rapa genome sequence will facilitate their study, including the separation of cis- and trans- effects. Overall design: The experiment was designed to identify expression QTL associated with availability of phosphorus in Brassica rapa. Seventy-eight informative lines from the “BraIRRI” mapping population of Brassica rapa L. (2n = 2x = 10; A-genome) and the two parent lines (IMB211, female; R500, male) were selected for study. The establishment of the BraIRRI population is described by Iniguez-Luy et al. (2009). Plants were grown from seed in compost, under two [P]ext treatments of 9 mg L-1 (low) or 30 mg L-1 (optimal) Olsen extractable P. RNA was extracted from leaf samples from one experimental run (78 lines at low and optimal [P]ext, with one line duplicated i.e. 158 samples), and from leaf samples from the parent lines at low and optimal P in all three experimental runs (12 samples) using a modified TRIzol extraction method (Hammond et al., 2006).