Table_2_Impact of “Green Revolution” gene Rht-B1b on coleoptile length of wheat.xlsx

Wheat coleoptile is a sheath-like structure that helps to deliver the first leaf from embryo to the soil surface. Here, a RIL population consisting of 245 lines derived from Zhou 8425B × Chinese Spring cross was genotyped by the high-density Illumina iSelect 90K assay for coleoptile length (CL) QTL mapping. Three QTL for CL were mapped on chromosomes 2BL, 4BS and 4DS. Of them, two major QTL QCL.qau-4BS and QCL.qau-4DS were detected, which could explain 9.1%–22.2% of the phenotypic variances across environments on Rht-B1 and Rht-D1 loci, respectively. Several studies have reported that Rht-B1b may reduce the length of wheat CL but no study has been carried out at molecular level. In order to verify that the Rht-B1 gene is the functional gene for the 4B QTL, an overexpression line Rht-B1b-OE and a CRISPR/SpCas9 line Rht-B1b-KO were studied. The results showed that Rht-B1b overexpression could reduce the CL, while loss-of-function of Rht-B1b would increase the CL relative to that of the null transgenic plants (TNL). To dissect the underlying regulatory mechanism of Rht-B1b on CL, comparative RNA-Seq was conducted between Rht-B1b-OE and TNL. Transcriptome profiles revealed a few key pathways involving the function of Rht-B1b in coleoptile development, including phytohormones, circadian rhythm and starch and sucrose metabolism. Our findings may facilitate wheat breeding for longer coleoptiles to improve seedling early vigor for better penetration through the soil crust in arid regions.

小麦胚芽鞘是一类鞘状结构，可将幼苗的第一片叶从胚部输送至土壤表面。本研究以周8425B与中国春杂交构建的包含245个株系的重组自交系（Recombinant Inbred Line, RIL）群体为材料，采用高密度Illumina iSelect 90K基因分型芯片对该群体进行基因分型，用于胚芽鞘长度（coleoptile length, CL）的数量性状基因座（quantitative trait locus, QTL）定位。研究在2BL、4BS和4DS染色体上共定位到3个调控胚芽鞘长度的QTL。其中两个主效QTL QCL.qau-4BS与QCL.qau-4DS被成功检测，二者分别位于Rht-B1和Rht-D1基因座，可解释不同环境下9.1%~22.2%的表型变异。已有多项研究指出Rht-B1b可能缩短小麦胚芽鞘长度，但尚未有基于分子水平的相关机制研究。为验证Rht-B1基因即为4B染色体QTL的功能基因，本研究构建了Rht-B1b过表达株系（Rht-B1b-OE）以及CRISPR/SpCas9介导的Rht-B1b敲除株系（Rht-B1b-KO）并开展功能验证实验。结果显示，与空载转基因植株（null transgenic plants, TNL）相比，Rht-B1b过表达可显著缩短胚芽鞘长度，而Rht-B1b功能缺失则会使胚芽鞘长度显著增加。为解析Rht-B1b调控胚芽鞘长度的潜在分子机制，本研究对Rht-B1b-OE与TNL进行了比较转录组RNA测序（RNA-Seq）分析。转录组数据揭示了Rht-B1b参与胚芽鞘发育的关键调控通路，包括植物激素信号通路、昼夜节律通路以及淀粉与蔗糖代谢通路。本研究结果可为培育长胚芽鞘小麦品种提供理论支撑，以提升幼苗早期活力，增强干旱地区小麦幼苗穿透土壤结壳的能力。