| Wild rice relatives have valuable sources of genetic variation for the improvement of agronomical traits in cultivated rice. Oryza longistaminata, a perennial wild rice species with the same AA genome as O. sativa, provides a model system for genetic and molecular dissection of the rhizomatous trait in grasses. Meanwhile, O. longistaminata possesses many important adaptive traits for cultivation such as tolerance to cold and can contribute to the development of more sustainable grain, forage, and bioenergy cropping systems.In present study, functional genomic and comparative transcriptomic techniques including microarray and RNA-seq were used to discover candidate genes and putative pathways specifically responsible for rhizome development in O. longistaminata and Sorghum propinquum, as well as for chilling tolerance among O. longistaminata, LTH and IR29. All those functional candidate genes identified in this study provided a basis for future cloning of genes associated with rhizome development and chilling stress tolerance in rice. The main results are as following:1) We sequenced10.283cDNA clones from a normalized cDNA library constructed from rhizome tip tissues of O. longistaminata and generated4,419unisequences with diverse functional categories. These unisequences were mapped onto the O. sativa genome, which revealed that4,285(96.97%) and4,151(93.94%) of the unisequences were alignable to the japonica and indica genomic sequences, respectively, with≥80%sequence identity. Additionally,41unisequences showed four typical types of alternative splicing patterns. More than600simple sequence repeats were identified. A subset of unisequences were physically co-localized onto rhizome-related QTLs intervals in rice and sorghum; and one gene, OLRR1, was further confirmed to be highly enriched in the rhizome tip and young leaf by quantitative PCR and in situ hybridization. 2) We used a whole rice genome oligonucleotide microarray to obtain tissue-specific gene expression profiles of S. propinquum. A total of548tissue-enriched genes were detected, including31and114unique genes that were expressed predominantly in the rhizome tips (RT) and internodes (RI), respectively. Further GO analysis indicated that the functions of these tissue-enriched genes corresponded to their characteristic biological processes. A few distinct cis-elements, including ABA-responsive RY repeat CATGCA, sugar-repressive TTATCC, and GA-responsive TAACAA, were found to be prevalent in RT-enriched genes, implying an important role in rhizome growth and development. Comprehensive comparative analysis of these rhizome-enriched genes and rhizome-specific genes previously identified in O. longistaminata and S. propinquum indicated that phytohormones, including ABA, GA, and SA, are key regulators of gene expression during rhizome development.3) We sequenced the RNAs of S. propinquum shoot and rhizome using an Illumina platform. More than70%of the genes in the S. propinquum genome were expressed in shoot and rhizome. The expression patterns of1963and599genes, including transcription factors, were found to be specific or enriched in shoot and rhizome respectively, indicating their possible roles in physiological processes in these organs. Comparative analysis revealed several cis-elements, ACGT box, GCCAC, GATC and TGACG box, which showed significantly higher abundance in shoot-specific genes. Cis-elements including MYB and ROOTMOTIFTAPOX1motifs, and10promoters and cytokinin-responsive elements were highly enriched in rhizome-specific genes. Of the S. propinquum genes,27.9%were identified as alternatively spliced and about60%of the alternative splicing events were organ-specific, suggesting that alternative splicing played a crucial role in determining organ-specific cellular function.Comprehensive comparative analysis of rhizome-enriched genes mentioned above and previously identified ones in O. longistaminata and S. propinquum resulted in a number of111genes were found to be commonly enriched in rhizome in at least two different platforms, which mutually confirmed the rhizome-enriched genes as well as their evolutionarily conserved expression pattern in both rice and sorghum.4) The temporal gene expression patterns of two rice genotypes (chilling-tolerant LTH and chilling-sensitive IR29) in response to chilling stress were compared. A set of genes with higher basal expression were identified in chilling-tolerant LTH compared with chilling-sensitive IR29, indicating their possible role in intrinsic tolerance to chilling stress. Under chilling stress, the major effect on gene expression was up-regulated in the chilling-tolerant genotype and strongly repressed in chilling-sensitive genotype. Early responses to chilling stress in both genotypes featured commonly upregulated genes related to transcription regulation and signal transduction, while functional categories for late phase chilling regulated genes were diverse with a wide range of functional adaptations to continuous stress. Following the cessation of chilling treatments, there was quick and efficient reversion of gene expression in the chilling-tolerant genotype, while the chilling-sensitive genotype displayed considerably slower recovering capacity at the transcriptional level. In addition, the detection of differentially regulated TF genes and enriched cis-elements demonstrated that multiple regulatory pathways, including CBF and MYBS3regulons, were involved in chilling stress tolerance.5) RNA sequencing was performed to profiling the gene expression level of rhizome and shoot of O. longistaminata under long term chilling stress. The total length of the reads was over25gigabases (Gb), representing about58-fold coverage of the O. longistaminata genome. However, about10%of total reads remained unmapped, indicating the existence of gaps and diversity between the genome sequences of O. sativa and O. longistaminata. There were913and884genes differentially regulated in shoot and rhizome under chilling stress, respectively, including33up regulated transcription factors in both tissues. Further analysis found a set of chilling specific alternative splicing (AS) transcripts, which fell into very broad functional categories such as signal transduction, regulation of biological process, localization and cellular component organization, indicating AS events could play an important role in chilling stress tolerance. Besides, we identified8,005novel transcripts and3,916fusion genes in shoot and rhizome during chilling stress and control conditions. And56chilling induced genes in O. longisataminata were colocalized onto the chilling tolerant QTLs intervals in rice.Comparative analysis of transcriptomic data in O. longistaminata, LTH and IR29under chilling stress, we identified154chilling induced genes sharing in three genotypes. Among them,34are transcription factors, indicating their conserved function between cultivaty and wild rice in response to chilling stress. Besides, the promoters of56(36.4%) of these commonly chilling induced genes contained at least one CRT/DRE core motif A/GCCGAC. These chilling responsive genes in three genotypes may play an important role in responding to chilling stress and their function need further elucidated. |
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