Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo, 060-8589, Japan.
Gifu Prefectural Research Institute for Agricultural Technology in Hilly and Mountainous Areas, Nakatsugawa, 508-0203, Japan.
BMC Plant Biol. 2020 Nov 3;20(1):503. doi: 10.1186/s12870-020-02721-9.
Cytoplasmic male sterility (CMS) is a widely used trait for hybrid seed production in many crops. Sugar beet CMS is associated with a unique mitochondrial protein named preSATP6 that forms a 250-kDa complex. Restorer-of-fertility 1 (Rf1) is a nuclear gene that suppresses CMS and is, hence, one of the targets of sugar beet breeding. Rf1 has dominant, semi-dominant and recessive alleles, suggesting that it may be a multi-allelic locus; however, the molecular basis for differences in genetic action is obscure. Molecular cloning of Rf1 revealed a gene (orf20) whose protein products produced in transgenics can bind with preSATP6 to generate a novel 200-kDa complex. The complex is also detected in fertility-restored anthers concomitant with a decrease in the amount of the 250-kDa complex. Molecular diversity of the Rf1 locus involves organizational diversity of a gene cluster composed of orf20-like genes (RF-Oma1s). We examined the possibility that members of the clustered RF-Oma1 in this locus could be associated with fertility restoration.
Six yet uncharacterized RF-Oma1s from dominant and recessive alleles were examined to determine whether they could generate the 200-kDa complex. Analyses of transgenic calli revealed that three RF-Oma1s from a dominant allele could generate the 200-kDa complex, suggesting that clustered RF-Oma1s in the dominant allele can participate in fertility restoration. None of the three copies from two recessive alleles was 200-kDa generative. The absence of this ability was confirmed by analyzing mitochondrial complexes in anthers of plants having these recessive alleles. Together with our previous data, we designed a set of PCR primers specific to the 200-kDa generative RF-Oma1s. The amount of mRNA measured by this primer set inversely correlated with the amount of the 250-kDa complex in anthers and positively correlated with the strength of the Rf1 alleles.
Fertility restoration by sugar beet Rf1 can involve multiple RF-Oma1s clustered in the locus, implying that stacking 200-kDa generative copies in the locus strengthens the efficacy, whereas the absence of 200-kDa generative copies in the locus makes the allele recessive irrespective of the copy number. We propose that sugar beet Rf1 is a complex locus.
细胞质雄性不育（CMS）是许多作物杂种种子生产中广泛应用的特性。甜菜 CMS 与一种名为 preSATP6 的独特线粒体蛋白有关，该蛋白形成 250kDa 的复合物。育性恢复基因 1（Rf1）是一种核基因，它抑制 CMS，因此是甜菜育种的目标之一。Rf1 具有显性、半显性和隐性等位基因，表明它可能是一个多等位基因座；然而，遗传作用差异的分子基础尚不清楚。Rf1 的分子克隆揭示了一个基因（orf20），其在转基因中产生的蛋白质产物可以与 preSATP6 结合，产生一种新的 200kDa 复合物。在恢复育性的花药中也检测到该复合物，同时 250kDa 复合物的量减少。Rf1 基因座的分子多样性涉及由 orf20 样基因（RF-Oma1s）组成的基因簇的组织多样性。我们研究了该基因座中聚集的 RF-Oma1 成员是否与育性恢复有关的可能性。
从显性和隐性等位基因中检查了六个尚未表征的 RF-Oma1，以确定它们是否能够产生 200kDa 复合物。对转基因愈伤组织的分析表明，来自显性等位基因的三个 RF-Oma1 可以产生 200kDa 复合物，这表明显性等位基因中的聚集 RF-Oma1 可以参与育性恢复。来自两个隐性等位基因的三个拷贝都没有 200kDa 生成能力。通过分析具有这些隐性等位基因的植物花药中的线粒体复合物来证实了这种能力的缺失。结合我们以前的数据，我们设计了一组针对产生 200kDa 的 RF-Oma1 的特定 PCR 引物。通过该引物组测量的 mRNA 量与花药中 250kDa 复合物的量呈负相关，与 Rf1 等位基因的强度呈正相关。
甜菜 Rf1 的育性恢复可能涉及聚集在基因座中的多个 RF-Oma1，这表明在基因座中堆积产生 200kDa 的拷贝可以增强效力，而基因座中缺乏产生 200kDa 的拷贝会使等位基因无论拷贝数如何都呈隐性。我们提出甜菜 Rf1 是一个复杂的基因座。
