The molecular basis for allelic differences suggests Restorer-of-fertility 1 is a complex locus in sugar beet (Beta vulgaris L.)

Abstract

Background 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. Results 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. Conclusions 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.

Selection of Nuclear Genotypes Associated with the Thermo-inducibility of Owen-type Cytoplasmic Male Sterility in Sugar Beet (Beta vulgaris L.)

“…Rf1 is a principal genetic factor for Owen-type CMS expression, with several different alleles recently reported (Arakawa et al 2020b). Our previous study identified a molecular variant linked to s17 marker type p4 as the most prevalent form in maintainer lines (Ohgami et al 2016).…”

“…The TA-37-19-p3 and TA-30-37-p5 alleles were likely PI 615522 rf1 and PI 59068 rf1, respectively, whose Rf1-Oma1 coding regions were identical but s17 types differed from each other (Ohgami et al 2016). Arakawa et al (2020b) investigated the molecular function of PI 615522 rf1 and concluded that the ability to bind to the CMS-associated sugar beet protein was missing. Therefore, we infer that other genes such as Z are associated with thermo-inducible MS.…”

“…The DNA marker s17 is a cleaved amplified sequence marker closely linked to Rf1 (Taguchi et al 2014). DNA band patterns of s17 have been linked to the Rf1 alleles (Arakawa et al 2018(Arakawa et al , 2019(Arakawa et al , 2020b. A total of 169 plants were genotyped using s17 (Table 1), and we observed that these plants had band patterns 1 (hereafter abbreviated as p1), p3, p4, and p5.…”

“…Whereas little is known about Z (Honma et al 2014), X was identified as restorer-of-fertility 1 (Rf1), and its nucleotide sequence was determined (Matsuhira et al 2012). Rf1 is a complex locus consisting of a gene cluster whose constituents have sequence similarity with Oma1, a gene involved in mitochondrial quality control (Arakawa et al 2020b;Migdal et al 2017). The clustered gene copies (hereafter, each copy is referred to as Rf1-Oma1) were divided into two classes based on a single property of their encoded protein; namely, those with or without the ability to bind to a CMS-associated sugar beet mitochondrial protein (Arakawa et al 2020b).…”

“…Rf1 is a complex locus consisting of a gene cluster whose constituents have sequence similarity with Oma1, a gene involved in mitochondrial quality control (Arakawa et al 2020b;Migdal et al 2017). The clustered gene copies (hereafter, each copy is referred to as Rf1-Oma1) were divided into two classes based on a single property of their encoded protein; namely, those with or without the ability to bind to a CMS-associated sugar beet mitochondrial protein (Arakawa et al 2020b). This binding activity is crucial for altering the higher-order structure of the CMS-associated mitochondrial protein to restore male fertility (Kitazaki et al 2015).…”