Table5_Genome wide association study reveals new genes for resistance to striped stem borer in rice (Oryza sativa L.).xlsx

Rice is one of the most important food crops in the world and is important for global food security. However, damage caused by striped stem borer (SSB) seriously threatens rice production and can cause significant yield losses. The development and use of resistant rice varieties or genes is currently the most effective strategy for controlling SSB. We genotyped 201 rice samples using 2849855 high-confidence single nucleotide polymorphisms (SNPs). We conducted a genome-wide association study (GWAS) based on observed variation data of 201 rice cultivars resistant to SSB. We obtained a quantitative trait locus (QTL)-qRSSB4 that confers resistance to SSB. Through annotation and analysis of genes within the qRSSB4 locus, as well as qRT-PCR detection in resistant rice cultivars, we ultimately selected the candidate gene LOC_Os04g34140 (named OsRSSB4) for further analysis. Next, we overexpressed the candidate gene OsRSSB4 in Nipponbare through transgenic methods, resulting in OsRSSB4 overexpressing lines (OsRSSB4OE). In addition, we evaluated the insect resistance of OsRSSB4OE lines using wild type (Nipponbare) as a control. The bioassay experiment results of live plants showed that after 20 days of inoculation with SSB, the withering heart rate of OsRSSB4OE-34 and OsRSSB4OE-39 lines was only 8.3% and 0%, with resistance levels of 1 and 0, respectively; however, the withering heart rate of the wild-type reached 100%, with a resistance level of 9. The results of the in vitro stem bioassay showed that, compared with the wild-type, the average corrected mortality rate of the SSB fed on the OsRSSB4OE line reached 94.3%, and the resistance reached a high level. In summary, we preliminarily confirmed that OsRSSB4 positively regulates the defense of rice against SSB. This research findings reveal new SSB resistance gene resources, providing an important genetic basis for SSB resistance breeding in rice crops.

水稻是全球最重要的粮食作物之一，对全球粮食安全具有关键意义。然而，二化螟（striped stem borer, SSB）造成的危害严重威胁水稻生产，可引发显著的产量损失。目前，培育和应用抗虫水稻品种或基因是防控二化螟的最有效策略。本研究利用2849855个高可信度单核苷酸多态性（single nucleotide polymorphisms, SNPs）对201份水稻样本进行基因分型。基于201份抗二化螟水稻品种的变异观测数据，开展了全基因组关联分析（genome-wide association study, GWAS），获得了一个赋予水稻抗二化螟特性的数量性状位点（quantitative trait locus, QTL）——qRSSB4。通过对qRSSB4位点内的基因进行注释与分析，并结合抗虫水稻品种的实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）检测，最终筛选出候选基因LOC_Os04g34140（命名为OsRSSB4）开展后续研究。随后，本研究通过转基因方法在日本晴（Nipponbare）中过表达候选基因OsRSSB4，获得了OsRSSB4过表达株系（OsRSSB4OE）。此外，本研究以野生型（日本晴，Nipponbare）为对照，对OsRSSB4OE株系的抗虫性进行了评价。活体植株生测实验结果显示，在接种二化螟20天后，OsRSSB4OE-34和OsRSSB4OE-39株系的枯心率仅为8.3%和0%，抗性等级分别为1和0；而野生型株系的枯心率高达100%，抗性等级为9。离体茎秆生测实验结果表明，相较于野生型，取食OsRSSB4OE株系的二化螟平均校正死亡率达到94.3%，抗性达到高抗水平。综上，本研究初步证实OsRSSB4正向调控水稻对二化螟的防御反应。本研究发现了新的二化螟抗性基因资源，为水稻作物的抗二化螟育种提供了重要的遗传基础。