Thousands of barley (Hordeum vulgare L.) mutants have been isolated over the last century, and many are stored in gene banks across various countries. In the present work, we developed a pipeline to efficiently identify causal mutations in barley. The pipeline is also efficient for mutations located in centromeric regions. Through bulked-segregant analyses using whole genome sequencing of pooled F2 seedlings, we mapped two mutations and identified a limited number of candidate genes. We applied the pipeline on F2-mapping populations made from xan-j.59 (unknown mutation) and xan-l.82 (previously known). The Xantha-j (xan-j) gene was identified as encoding chlorophyll synthase, which catalyzes the last step in the chlorophyll biosynthetic pathway: the addition of a phytol moiety to the propionate side chain of chlorophyllide. Key amino-acid residues in the active site, including the binding sites of the isoprenoid and chlorophyllide substrates, were analyzed in an AlphaFold2-generated structural model of the barley chlorophyll synthase. Three allelic mutants, xan-j.19, xan-j.59, and xan-j.64 were characterized. While xan-j.19 is a one-base pair deletion and xan-j.59 is a nonsense mutation, xan-j.64 causes an S212F substitution in chlorophyll synthase. Our analyses of xan-j.64 and treatment of growing barley with clomazone, an inhibitor of chloroplastic isoprenoid biosynthesis, suggest that binding of the isoprenoid substrate is a prerequisite for the stable maintenance of chlorophyll synthase in the plastid. We further suggest that chlorophyll synthase is a sensor for coordinating chlorophyll and isoprenoid biosynthesis.

中文翻译：

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用于识别大麦因果突变的管道将 Xantha-j 识别为叶绿素合酶基因

上个世纪，数千个大麦（Hordeum vulgare L.）突变体被分离出来，其中许多突变体储存在各国的基因库中。在目前的工作中，我们开发了一种有效识别大麦因果突变的管道。该管道对于位于着丝粒区域的突变也有效。通过使用合并的 F2 幼苗的全基因组测序进行批量分离分析，我们绘制了两个突变图谱并确定了有限数量的候选基因。我们将管道应用于由 xan-j.59（未知突变）和 xan-l.82（先前已知）制成的 F2 映射群体。 Xantha-j (xan-j) 基因被鉴定为编码叶绿素合成酶，该酶催化叶绿素生物合成途径的最后一步：将植醇部分添加到叶绿素内酯的丙酸酯侧链上。在 AlphaFold2 生成的大麦叶绿素合酶结构模型中分析了活性位点的关键氨基酸残基，包括类异戊二烯和叶绿素内酯底物的结合位点。对三个等位基因突变体 xan-j.19、xan-j.59 和 xan-j.64 进行了表征。 xan-j.19 是单碱基对缺失，xan-j.59 是无义突变，而 xan-j.64 会导致叶绿素合酶中的 S212F 取代。我们对 xan-j.64 的分析以及用异异戊二烯生物合成抑制剂异异戊二烯对生长中的大麦的处理表明，类异戊二烯底物的结合是质体中叶绿素合酶稳定维持的先决条件。我们进一步认为叶绿素合酶是协调叶绿素和类异戊二烯生物合成的传感器。