Project description:We identified 9325 reliable single nucleotide polymorphisms (SNPs) distributed throughout the genome, of which nearly 68% were represented in R9308 versus Xieqingzao B by CT and GA SNPs. This may indicate that DNA methylation, a heritable epigenetic mark, exists in the parents and their F1 hybrid. Of 2793 identified transcripts with consistent allelic biases, only 480 (17%) showed significant allelic biases during at least one stage, suggesting that trans effects may mediate most transcriptional differences in hybrid offspring. Approximately 67% and 62% of the 480 transcripts showed allelic expression biases towards the R9308 allele at tillering and heading stages, respectively. Transcripts with higher levels of gene expression in R9308 also exhibited R9308 allelic biases in the hybrid. In addition, 125 transcripts were identified with significant allelic expression biases at both stages, of which 74% showed allele expression biases towards the R9308 allele. R9308 alleles may tend to preserve their characteristic states of activity in the hybrid and may play an important role in hybrid vigor at both stages. The allelic expression of 355 transcripts was highly stage-specific, with divergent allelic expression patterns observed at different developmental stages. Many transcripts associated with stress resistance were differently regulated in the F1 hybrid. The results of this study would provide valuable information for further understanding molecular mechanisms of heterosis. Root mRNA profiles of a super-hybrid rice Xieyou 9308 and its parents at tillering and heading stages were generated by deep sequencing, in duplicate, on Illumina Hiseq 2000 platform.
Project description:A High Density Rice Array (HDRA) was developed as an Affymetrix Custom GeneChip Array by the McCouch Rice Lab at Cornell University. The HDRA assays 700,000 SNPs, or approximately one SNP every 0.54 Kb across the rice genome (genome size = 380 Mb). It was designed to capture most of the haplotype variation observed in a discovery panel consisting of 16M SNPs (generated by sequencing 125 rice genomes at ~7X genome coverage) and to maximize the inclusion of non-synonymous SNPs. Six probes per SNP target were designed as 3 A-allele and 3 B-allele probes at offsets from center ranging from -6 to +6. A small fraction of SNPs have only 4 probes (2-A, 2-B). For all SNPs, the “A” allele is the reference allele (Os-Nipponbare-Reference-IRGSP-1.0 assembly). Additionally, we designed 23,656 x 25-bp probes complimentary to invariant regions of the genome that were used to normalize systematic differences between samples. An estimated 45% of HDRA SNPs map within genes, hitting all 39,045 unique, non-TE rice gene models (MSUv7 rice genome annotation, GFF3 file, Feb. 7, 2012, http://rice.plantbiology.msu.edu/), while 55% of SNPs map to intergenic regions. Non-synonymous are found in 91% of unique, non-TE gene models, and 57% of genic SNPs are distributed within exons, 36% within introns, 5% within 5’ UTRs and 2% within 3’ UTRs. Of the intergenic SNPs, 40% are located in putative regulatory regions within 2 Kb of a transcriptional start site.
Project description:Global Epigenomic Analysis Indicates that Epialleles Contribute to Allele-specific Expression via Allele-specific Histone Modifications in Hybrid Rice
Project description:Heterosis is an important biological phenomenon; however, the role of small RNA (sRNA) in heterosis of hybrid rice remains poorly described. Here, we performed sRNA profiling of F1 super-hybrid rice LYP9 and its parents using high-throughput sequencing technology, and identified 355 distinct mature microRNAs and trans-acting small interfering RNAs, 69 of which were differentially expressed sRNAs (DES) between the hybrid and the mid-parental value. Among these, 34 DES were predicted to target 176 transcripts, of which 112 encoded 94 transcription factors. Further analysis showed that 67.6% of DES expression levels were negatively correlated with their target mRNAs either in flag leaves or panicles. The target genes of DES were significantly enriched in some important biological processes, including the auxin signalling pathway, in which existed a regulatory network mediated by DES and their targets, closely associated with plant growth and development. Overall, 20.8% of DES and their target genes were significantly enriched in quantitative trait loci of small intervals related to important rice agronomic traits including growth vigour, grain yield, and plant architecture, suggesting that the interaction between sRNAs and their targets contributes to the heterotic phenotypes of hybrid rice. Our findings revealed that sRNAs might play important roles in hybrid vigour of super-hybrid rice by regulating their target genes, especially in controlling the auxin signalling pathway. The above finding provides a novel insight into the molecular mechanism of heterosis. We constructed six sRNA sequencing libraries and six mRNA sequencing libraries of flag leaves and panicles of the super-hybrid rice Liangyou-pei9 (LYP9) combination at the grain-filling stage. The above hybrid rice combination includes F1 hybrid LYP9 and its parental lines including the male-sterile line Peiai64s (PA64s) and the restorer line 93-11.
Project description:A biological phenomenon in which hybrids exhibit superior phenotypes from its parental inbred lines known as heterosis, has been widely exploited in plant breeding and extensively used in crop improvement. Hybrid rice has immense potential to increase yield over other rice varieties and hence is crucial in meeting increasing demand of rice globally. Moreover, the molecular basis of heterosis is still not fully understood and hence it becomes imperative to unravel its genetic and molecular basis. In this context, RNA sequencing technology (RNA-Seq) was employed to sequence transcriptomes of two rice hybrids, Ajay and Rajalaxmi, their parental lines, CRMS31A (sterile line, based on WA-CMS) and CRMS32A (sterile line based on Kalinga-CMS) respectively along with the common restorer line of both hybrids, IR-42266-29-3R at two critical rice developmental stages viz., panicle initiation (PI) and grain filling (GF). Identification of differentially expressed genes (DEGs) at PI and GF stages will further pave the way for understanding heterosis. In addition, such kind of study would help in better understanding of heterosis mechanism and genes up-regulated and down-regulated during the critical stages of rice development for higher yield.
Project description:In the study of gene expression regulation in plants, it is often desirable to distinguish transcript pools derived from different alleles present in the same organism. We report here an oligonucleotide tiling microarray designed to specifically target 518 single nucleotide polymorphisms (SNPs) between the two sequenced rice (Oryza sativa) subspecies indica and japonica. The tiling array includes all pairs of 25-mer allele-specific probes interrogating each SNP by placing the polymorphic site at all 25 possible positions within the probe. Hybridization of the tiling array to a titration series in which the japonica- and indica-derived cDNA templates are mixed with altering proportions and development of a novel bioinformatic methodology allowed us to screen for diagnostic probe pairs for each SNP. Our result indicates that 284 (55%) SNPs have at least one diagnostic probe pair suitable for distinguishing and quantifying the relative abundance of allele-specific transcripts. As a proof-of-concept, we analyzed allele-specific expression in reciprocal indica × japonica F1 hybrids and detected imbalanced expression at 95 (33%) and 71 (25%) SNPs, respectively. Comparison results from RNA-sequencing and allele-specific real-time PCR experiment validates the sensitivity and reliability of the tiling array method. Together, our results demonstrate the advantages of the tiling array method in interrogating large numbers of SNPs and for the reliability of the experimental and analytical techniques used for quantifying allele-specific gene expression.
Project description:We identified 9325 reliable single nucleotide polymorphisms (SNPs) distributed throughout the genome, of which nearly 68% were represented in R9308 versus Xieqingzao B by CT and GA SNPs. This may indicate that DNA methylation, a heritable epigenetic mark, exists in the parents and their F1 hybrid. Of 2793 identified transcripts with consistent allelic biases, only 480 (17%) showed significant allelic biases during at least one stage, suggesting that trans effects may mediate most transcriptional differences in hybrid offspring. Approximately 67% and 62% of the 480 transcripts showed allelic expression biases towards the R9308 allele at tillering and heading stages, respectively. Transcripts with higher levels of gene expression in R9308 also exhibited R9308 allelic biases in the hybrid. In addition, 125 transcripts were identified with significant allelic expression biases at both stages, of which 74% showed allele expression biases towards the R9308 allele. R9308 alleles may tend to preserve their characteristic states of activity in the hybrid and may play an important role in hybrid vigor at both stages. The allelic expression of 355 transcripts was highly stage-specific, with divergent allelic expression patterns observed at different developmental stages. Many transcripts associated with stress resistance were differently regulated in the F1 hybrid. The results of this study would provide valuable information for further understanding molecular mechanisms of heterosis.