Multiple Origins of Chalcone Synthase Short Interfering RNAs in Glycine max Revealed by Genome Assemblies of Mutant Alleles

With long-read assemblies, we delineate three origins of short interfering RNAs for chalcone synthase (CHS siRNAs) in Glycine max. Mutations from ii (yellow seed coat with black hilum) to i (fully pigmented) lack either a single antisense CHS1 gene by deletion or the partial 5' subtilisin fragment-CHS1 gene region by a large inversion mutation. On the other hand, we show that the dominant I allele, with fully yellow seed coats, arose from the i black, wild soybean genome by an inverted duplication that places a partial 5' DnaJ fragment next to slightly truncated CHS genes. In a direct I to i mutation, the two CHS genes are further truncated, destroying their capacity to produce siRNAs. The two-color saddle pattern of the ik allele arose by an inverted duplication event that places a partial 5' P450 fragment in front of two inverted repeat CHS genes. Despite the importance of the three different 5' promoters that abut inverted repeat CHS genes as drivers of precursor expression, the profiles of cognate gene family members for subtilisin, DnaJ, and P450 are not always limited to the seed coat as tightly as CHS siRNAs, implying that transcriptional or processing events for CHS dsRNA or siRNAs differ in other tissues. Overall design: High-throughput RNA sequencing was performed on different tissues and genotypes of soybean as listed in each sample. High-throughput RNA sequencing was performed on dissections of the hilum versus the seed coat without the hilum for two stages of seed coat development with two biological repeats from the cultivar Williams which has black hilum on a yellow seed coat.

借助长读长组装（long-read assemblies）技术，我们解析了大豆（Glycine max）中查尔酮合酶小干扰RNA（CHS siRNAs）的三种起源模式。携带从ii（种脐黑色的黄色种皮）到i（完全色素化种皮）突变的个体，要么因缺失突变丧失单个反义CHS1基因，要么因大片段倒位突变丢失部分5'枯草杆菌蛋白酶（subtilisin）片段-CHS1基因区域。另一方面，我们证实具有完全黄色种皮的显性I等位基因，源自i型黑色种皮的野生大豆基因组，通过倒位重复将一段部分5' DnaJ片段整合至略截短的CHS基因旁侧。在直接由I突变为i的株系中，两个CHS基因发生进一步截短，彻底丧失了合成siRNA的能力。ik等位基因所呈现的双色马鞍表型，则源于一次倒位重复事件：将一段部分5'细胞色素P450（P450）片段置于两个反向重复的CHS基因上游。尽管紧邻反向重复CHS基因的三种不同5'启动子作为前体表达的驱动元件至关重要，但枯草杆菌蛋白酶、DnaJ和P450的同源基因家族成员的表达谱，并不像CHS siRNAs那样严格局限于种皮组织，这提示CHS双链RNA（dsRNA）或siRNA的转录与加工过程在其他组织中存在差异。整体实验设计：针对不同组织与基因型的大豆开展高通量RNA测序，具体样本信息参见各样品说明。针对两个发育阶段的种皮，我们分别分离了种脐与去除种脐的种皮组分，并以种皮黄色、种脐黑色的栽培品种Williams设置两次生物学重复，完成高通量RNA测序。