Whole Genome Bisulfite Sequencing in TaMET1-1 mutants

Mutants in TaMET1-1 were obtained from the Cadenza TILLING population. The parental lines were Cadenza0465 and C0884. We collected leaf samples from the fourth leaf at Zadok's growth stage Z15.6/23 for wild type segregant, single and double mutants and Cadenza WT, or from the sixth leaf at Z16 for the Aabbdd mutant. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from three biological replicates per genotype, excluding the Aabbdd genotype, of which only one plant was recovered. DNA concentration was estimated for each sample using an Invitrogen Qubit 4 fluorometer (Thermo Fisher Scientific, Massachusetts, USA) and the three replicates per genotype were pooled in equimolar ratios. Library preparation, bisulfite conversion, and sequencing by Illumina NovaSeq 6000 was performed by BMKgene (Münster, Germany), to obtain 150 bp paired-end reads.

TaMET1-1突变体源自Cadenza定向诱导基因组局部突变（TILLING）群体，其亲本株系为Cadenza0465与C0884。我们针对不同实验材料采集叶片样本：对于野生型分离株、单突变体、双突变体及Cadenza野生型（WT），取样于扎多克（Zadok）生长阶段Z15.6/23的第四片叶；对于Aabbdd突变体，则取样于Z16生长阶段的第六片叶。采用德国希尔登市Qiagen公司的DNeasy植物微量提取试剂盒（DNeasy Plant Mini Kit），对每个基因型的三份生物学重复样本提取基因组DNA；但Aabbdd基因型仅存活1株，未设置生物学重复。采用美国马萨诸塞州赛默飞世尔科技公司的Invitrogen Qubit 4荧光计对每份样本的DNA浓度进行定量，随后将每个基因型的三份生物学重复样本按等摩尔浓度混合。文库构建、亚硫酸氢盐转化及Illumina NovaSeq 6000测序均由德国明斯特市的BMKgene公司完成，最终获得150 bp双端测序读段。