Table_5_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（wheat coleoptile）是一类鞘状结构，可将幼苗的第一片叶从胚体推送至土壤表层。本研究以周8425B × 中国春构建的包含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（Illumina iSelect 90K assay）进行基因型分型，用于胚芽鞘长度（coleoptile length, CL）的数量性状位点（Quantitative Trait Locus, QTL）定位。本研究共定位到3个控制胚芽鞘长度的QTL，分别位于2BL、4BS和4DS染色体上。其中，2个主效QTL QCL.qau-4BS和QCL.qau-4DS被检测到，它们分别位于Rht-B1和Rht-D1位点，可解释不同环境下9.1%~22.2%的表型变异。已有多项研究表明Rht-B1b可能会缩短小麦胚芽鞘长度，但目前尚未有从分子层面开展的相关研究。为验证Rht-B1基因是4B位点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基因编辑敲除株系（Rht-B1b-KO）并开展相关实验。结果表明，相较于转基因阴性植株（TNL），Rht-B1b过表达可降低胚芽鞘长度，而Rht-B1b功能缺失则会提升胚芽鞘长度。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进行了比较转录组测序（RNA-Seq）。转录组分析结果显示，参与胚芽鞘发育调控的Rht-B1b功能相关通路主要包括植物激素、昼夜节律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，以提升干旱地区小麦幼苗早期活力，增强其穿透土壤结壳的能力。