Loss-of-Function Mutations in Three Homoeologous PHYTOCLOCK 1 Genes in Common Wheat Are Associated with the Extra-Early Flowering Phenotype

Triticum aestivum L. cv ‘Chogokuwase’ is an extra-early flowering common wheat cultivar that is insensitive to photoperiod conferred by the photoperiod insensitive alleles at the Photoperiod-B1 (Ppd-B1) and Ppd-D1loci, and does not require vernalization for flowering. This reduced vernalization requirement is likely due to the spring habitat allele Vrn-D1 at the VERNALIZATION-D1 locus. Genotypes of the Ppd-1 loci that determine photoperiod sensitivity do not fully explain the insensitivity to photoperiod seen in ‘Chogokuwase’. We detected altered expression patterns of clock and clock-output genes including Ppd-1 in ‘Chogokuwase’ that were similar to those in an einkorn wheat mutant that lacks the clock-gene homologue, wheat PHYTOCLOCK 1 (WPCL1). Presumptive loss-of-function mutations in all WPCL1 homoeologous genes were found in ‘Chogokuwase’ and ‘Geurumil’, one of the parental cultivars. Segregation analysis of the two intervarietal F2 populations revealed that all the examined F2 plants that headed as early as ‘Chogokuwase’ had the loss-of-function wpcl1 alleles at all three homoeoloci. Some F2 plants carrying the wpcl1 alleles at three homoeoloci headed later than ‘Chogokuwase’, suggesting the presence of other loci influencing heading date. Flowering repressor Vrn-2 was up-regulated in ‘Chogokuwase’ and ‘Geurumil’ that had the triple recessive wpcl1 alleles. An elevated transcript abundance of Vrn-2 could explain the observation that ‘Geurumil’ and some F2 plants carrying the three recessive wpcl1 homeoealleles headed later than ‘Chogokuwase’. In spite of the up-regulation of Vrn-2, ‘Chogokuwase’ may have headed earlier due to unidentified earliness genes. Our observations indicated that loss-of-function mutations in the clock gene wpcl1 are necessary but are not sufficient to explain the extra-early heading of ‘Chogokuwase’.普通小麦（Triticum aestivum L.）品种‘Chogokuwase’是一个特早花型普通小麦品种，其光周期（photoperiod）不敏感性状由位于光周期-B1（Photoperiod-B1, Ppd-B1）与Ppd-D1位点的光周期不敏感等位基因介导，且开花无需经历春化作用（vernalization）。该品种对春化需求较低的原因，可能与其春化-D1（VERNALIZATION-D1）位点上的春生型等位基因Vrn-D1有关。仅通过调控光周期敏感性的Ppd-1位点基因型，无法完全解释‘Chogokuwase’所表现出的光周期不敏感特性。研究团队在‘Chogokuwase’中检测到了生物钟基因（clock gene）及生物钟下游调控基因（包括Ppd-1）的表达模式改变，该特征与缺失生物钟同源基因小麦PHYTOCLOCK 1（WPCL1）的一粒小麦（einkorn wheat）突变体相似。在‘Chogokuwase’及其亲本栽培品种之一‘Geurumil’中，均发现了所有WPCL1部分同源基因（homoeologous gene）的推定功能丧失型突变（loss-of-function mutation）。对两个品种间F₂群体的分离分析结果显示，所有表现出与‘Chogokuwase’一致早抽穗表型的F₂植株，在全部三个部分同源位点上均携带功能丧失型wpcl1等位基因。部分携带三个部分同源位点wpcl1等位基因的F₂植株，其抽穗时间晚于‘Chogokuwase’，这表明存在其他影响抽穗期（heading date）的位点。携带有三重隐性wpcl1等位基因的‘Chogokuwase’与‘Geurumil’中，开花抑制因子Vrn-2的转录本丰度（transcript abundance）均显著上调。Vrn-2转录本丰度的升高，可解释‘Geurumil’以及部分携带三个隐性wpcl1部分同源等位基因的F₂植株抽穗晚于‘Chogokuwase’的现象。尽管Vrn-2表达上调，但‘Chogokuwase’仍能提早抽穗，这可能得益于未被鉴定的早熟基因。本研究结果表明，wpcl1生物钟基因的功能丧失型突变，是‘Chogokuwase’特早抽穗的必要非充分条件。