Using bulked segregant analysis of exome sequence, we fine-mapped the ABA-hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A. Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat 'Zak.' ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660S allele as Zak, whereas Louise carries the PHS-tolerant MKK3-A-C660R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.

中文翻译：

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大量分离子的外显子组测序鉴定了与小麦 ERA8 ABA 超敏感萌发表型相关的新型 TaMKK3-A 等位基因。

使用外显子组序列的批量分离分析，我们将小麦回交群体中的 ABA 超敏感突变体 ERA8 精细定位到染色体 4A 的 TaMKK3-A 基因座。收获前发芽（PHS）是成熟谷物在收获前下雨时在母株上发芽。对 ABA8 (ERA8) 突变体的增强反应增加了种子休眠，因此增加了软白小麦“Zak”的 PHS 耐受性。使用外显子组测序 DNA (BSA-exome-seq) 的批量分离分析，将 ERA8 映射到 Zak/'ZakERA8' 回交群体中的 4A 号染色体。相对于诱变剂诱导的 SNP，ERA8 被精细映射到包含 70 个基因的 4.6 Mb 区域。在回交群体中，ERA8 ABA 超敏表型与 TaMKK3-A-G1093A (LOD 16.5) 中的错义突变密切相关，与大麦和小麦的天然 PHS 耐受性相关的基因。ERA8 的地图位置在“Otis”/ZakERA8 中得到确认，但在“Louise”/ZakERA8 映射群体中没有得到证实。这可能是因为 Otis 携带与 Zak 相同的天然 PHS 易感 MKK3-A-A660S 等位基因，而 Louise 携带 PHS 耐受性 MKK3-A-C660R 等位基因。因此，Louise/ZakERA8 群体中谷物休眠和 PHS 耐受性的变化可能是由于其他基因座的分离而不是 MKK3 基因座的 PHS 耐受性分离。这种无意的互补测试表明 MKK3-A-G1093A 突变导致 ERA8 表型。此外，MKK3 是 70 基因 4.6 Mb ERA8 区间中已知的 ABA 信号基因。这 70 个基因中没有一个显示出野生型 Zak 与预期启动子突变的 ERA8 的差异调节。