Transcriptomic and Genetic Analysis of Seed Germination Vigor under Low Temperature in Weedy Rice WR04-6

Low temperature is one of the major factors affecting rice germination, and low tempera-ture germination (LTG) is an important agronomic trait. Although genetic variation is abundant in rice germplasm resources, the molecular mechanism of LTG remains poorly understood. In this study, we first proved that weedy rice WR04-6 had significantly better low-temperature germination (LTG) ability at 10°C than the cultivated rice Qishanzhan (QSZ). RNA-seq was used to investigate the gene expression of WR04-6 and QSZ at 10, 12 and 14 days of seed germination at 10°C. The results of GO enrichment and KEGG en-richment revealed that the differentially expressed genes between WR04-6 and QSZ were mainly concentrated on the response to starch catabolic processes and the response to ab-scisic acid. This is consistent with the results of α-amylase activity, ABA and GA treat-ment. A recombinant inbred line (RIL) population derived from a cross between WR04-6 and QSZ and its high density SNP genetic map were used to detect quantitative trait loci (QTL) for low temperature germination rates at 10°C for 14 days. The results showed that two new QTLs were located on chromosome 3 and chromosome 12. Combined with the mapped QTLs and RNA-seq differential genes (DEGs), sixteen candidate genes potentially associated with LTG were identified. Validation of expression of the candidates by qRT-PCR were consistent with the RNA-seq data. These results will enable us to under-stand the genetic basis of LTG in weedy rice and provide new genetic resources for gener-ation of rice germplasm with LTG. An RNA-seq analysis was performed to further investigate difference between WR04-6 and QSZ. Low temperature (10°C) germinating seed of WR04-6 and QSZ were collected at 10, 12 and 14 days after imbibition, with three biological replications.

低温是影响水稻萌发的主要因素之一，低温萌发（low temperature germination, LTG）是一项重要的农艺性状。尽管水稻种质资源中存在丰富的遗传变异，但学界对LTG的分子调控机制仍知之甚少。本研究首先证实，杂草稻WR04-6在10℃条件下的低温萌发能力显著优于栽培稻齐粒占（QSZ）。本研究采用RNA测序（RNA-seq）技术，对10℃条件下萌发10、12、14天的WR04-6与QSZ的基因表达水平进行分析。基因本体（Gene Ontology, GO）富集与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析结果显示，WR04-6与QSZ间的差异表达基因主要富集于淀粉分解代谢过程响应与脱落酸（abscisic acid, ABA）响应通路，该结果与α-淀粉酶活性、ABA及赤霉素（gibberellin, GA）处理的实验结果一致。本研究以WR04-6与QSZ杂交构建的重组自交系（recombinant inbred line, RIL）群体及其高密度单核苷酸多态性（single nucleotide polymorphism, SNP）遗传图谱为材料，对10℃条件下萌发14天的低温萌发率进行数量性状位点（quantitative trait locus, QTL）定位，结果显示两个新的QTL分别位于3号染色体与12号染色体上。结合已定位的QTL与RNA-seq差异表达基因（differentially expressed genes, DEGs），本研究共筛选得到16个与LTG潜在相关的候选基因。采用实时定量反转录聚合酶链式反应（quantitative real-time reverse transcription polymerase chain reaction, qRT-PCR）对候选基因的表达量进行验证，结果与RNA-seq分析数据一致。本研究结果有助于解析杂草稻LTG的遗传基础，并为培育具备低温萌发能力的水稻种质资源提供新的遗传材料。本研究额外开展了一轮RNA-seq分析以进一步探究WR04-6与QSZ的基因表达差异：在吸胀后10、12、14天分别采集10℃低温萌发的WR04-6与QSZ种子样本，每组设置3次生物学重复。