背景:水稻的耐寒性与其生产和地理分布密切相关。耐冷相关基因的鉴定对开发耐冷水稻具有重要意义。东乡野生稻(OryzarufipogonGriff。)(DXWR)很好地适应了世界上最北纬栖息地的寒冷气候,是耐寒性改良最有价值的水稻种质之一。
结果:转录组分析显示,在室温(RT)下,谢清灶B(XB;冷敏感品种)和19H19(源自DXWR和XB之间的种间杂交)之间的基因差异表达,低温(LT),和康复治疗。结果表明,叶绿体基因可能参与了水稻耐寒性的调控。基于基因分型测序(GBS)技术,使用来自19H19和XB之间杂交的120个BC5F2系构建了高分辨率SNP遗传图谱。两个数量性状位点(QTL)在幼苗早期(CTS)耐冷,检测到qCTS12和qCTS8。此外,根据批量分离分析测序(BSA-seq),共鉴定出112个与耐寒性相关的候选基因.这些候选基因分为八个功能类别,与“氧化还原过程”和“对胁迫的响应”相关的候选基因的表达趋势在RT中XB和19H19之间有所不同,LT和恢复治疗。在这些候选基因中,在LT治疗中,L0C_Os12g18729在19H19中的表达水平(与“对应激反应”有关)降低,但在恢复治疗期间恢复并增强,而XB中L0C_Os12g18729的表达水平在恢复治疗期间下降。此外,XB在LOC_Os12g18729的第三个外显子中包含42bp的缺失,存活百分比(SP)低于15%的BC5F2个体的基因型与XB的基因型一致。加权基因共表达网络分析(WGCNA)和具有每个基因信息的模块化调控网络学习(MERLIN)算法揭示了调节水稻耐寒性的基因相互作用/共表达网络。在网络中,与“氧化还原过程”相关的差异表达基因(DEGs),“对压力的反应”和“蛋白质磷酸化”与LOC_Os12g18729相互作用。此外,与野生型相比,LOC_Os12g18729的敲除突变体在早稻苗期的耐寒性显着降低。
结论:一般来说,水稻耐寒遗传基础的研究对于开发耐寒水稻品种具有重要意义。在本研究中,QTL映射,整合BSA-seq和RNA-seq以鉴定两个CTSQTLqCTS8和qCTS12。此外,qRT-PCR,基因型测序和基因敲除分析表明LOC_Os12g18729可能是qCTS12的候选基因。本研究结果有望通过标记辅助选择从DXWR中导入耐冷基因,进一步探索水稻CTS的遗传机制,提高栽培稻的耐冷能力。
BACKGROUND: The cold tolerance of rice is closely related to its production and geographic distribution. The identification of cold tolerance-related genes is of important significance for developing cold-tolerant rice. Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) is well-adapted to the cold climate of northernmost-latitude habitats ever found in the world, and is one of the most valuable rice germplasms for cold tolerance improvement.
RESULTS: Transcriptome analysis revealed genes differentially expressed between Xieqingzao B (XB; a cold sensitive variety) and 19H19 (derived from an interspecific cross between DXWR and XB) in the room temperature (RT), low temperature (LT), and recovery treatments. The results demonstrated that chloroplast genes might be involved in the regulation of cold tolerance in rice. A high-resolution SNP genetic map was constructed using 120 BC5F2 lines derived from a cross between 19H19 and XB based on the genotyping-by-sequencing (GBS) technique. Two quantitative trait loci (QTLs) for cold tolerance at the early seedling stage (CTS), qCTS12 and qCTS8, were detected. Moreover, a total of 112 candidate genes associated with cold tolerance were identified based on bulked segregant analysis sequencing (BSA-seq). These candidate genes were divided into eight functional categories, and the expression trend of candidate genes related to \'oxidation-reduction process\' and \'response to stress\' differed between XB and 19H19 in the RT, LT and recovery treatments. Among these candidate genes, the expression level of LOC_Os12g18729 in 19H19 (related to \'response to stress\') decreased in the LT treatment but restored and enhanced during the recovery treatment whereas the expression level of LOC_Os12g18729 in XB declined during recovery treatment. Additionally, XB contained a 42-bp deletion in the third exon of LOC_Os12g18729, and the genotype of BC5F2 individuals with a survival percentage (SP) lower than 15% was consistent with that of XB. Weighted gene coexpression network analysis (WGCNA) and modular regulatory network learning with per gene information (MERLIN) algorithm revealed a gene interaction/coexpression network regulating cold tolerance in rice. In the network, differentially expressed genes (DEGs) related to \'oxidation-reduction process\', \'response to stress\' and \'protein phosphorylation\' interacted with LOC_Os12g18729. Moreover, the knockout mutant of LOC_Os12g18729 decreased cold tolerance in early rice seedling stage signifcantly compared with that of wild type.
CONCLUSIONS: In general, study of the genetic basis of cold tolerance of rice is important for the development of cold-tolerant rice varieties. In the present study, QTL mapping, BSA-seq and RNA-seq were integrated to identify two CTS QTLs qCTS8 and qCTS12. Furthermore, qRT-PCR, genotype sequencing and knockout analysis indicated that LOC_Os12g18729 could be the candidate gene of qCTS12. These results are expected to further exploration of the genetic mechanism of CTS in rice and improve cold tolerance of cultivated rice by introducing the cold tolerant genes from DXWR through marker-assisted selection.