Differences in the frequency of restorer-of-fertility 1 haplotype are associated with crop history of garden beet and sugar beet (Beta vulgaris L.)

Differences in the frequency of restorer-of-fertility 1 haplotype are associated with crop history of garden beet and sugar beet (Beta vulgaris L.) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Differences in the frequency of restorer-of-fertility 1 haplotype are associated with crop history of garden beet and sugar beet (Beta vulgaris L.) Eigo Taniguchi, Yohei Kanomata, Haruto Tanaka, Mion Oishi, Ryo Hayakawa, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5424865/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Feb, 2025 Read the published version in Genetic Resources and Crop Evolution → Version 1 posted 9 You are reading this latest preprint version Abstract Cytoplasmic male sterility (CMS) is used in breeding to facilitate hybrid seed production. Restorer of fertility ( Rf ), the suppressor of CMS, consists of a gene cluster with multiple haplotypes. Selection of restoring or non-restoring alleles depends on the discrimination of Rf haplotypes using DNA markers. The efficacy of this system is decreased if the Rf haplotype differs within the population of interest, which can occur during the crop evolution. In sugar beet, the Rf1 gene has multiple haplotypes that are grouped by a linked polymorphic region (s17), with one group, termed p4, uniquely linked to the recessive rf1 haplotype. Garden beet is the predecessor cultivar group of the sugar beet group. We questioned whether Rf1 haplotypes differ between these two cultivar groups to assess the utility of marker-assisted selection. We analyzed 48 garden beet landraces and observed differences in the s17 polymorphism compared to sugar beet, suggesting that the Rf1 haplotype frequency has changed during evolution of the crop. We next selected non-restoring genotypes from the garden beet landraces through test crosses and identified three recessive rf1 haplotypes: one is identical to the p4 haplotype and the others are novel haplotypes. The p4 haplotype occurs in a few accessions and its frequency in garden beet is approximately 0.01. We analyzed the s17 polymorphism in modern garden beet hybrids and their constituents. We identified the p4 haplotype and suggest the presence of other recessive rf1 haplotypes. Selection of p4 haplotype was efficient on the identification of non-restoring genotype in garden beet. Crop domestication Cultivar group divergence Cytoplasmic male sterility DNA marker assisted selection Hybrid breeding Standing genetic variation Figures Figure 1 Figure 2 Figure 3 Introduction Cytoplasmic male sterility (CMS) is used for hybrid seed production in a broad range of crops as it confers male-specific sterility while leaving other organs unaffected (reviewed in Xu et al. 2022 ). Expression of CMS is genetically conditioned by the presence of male sterility-inducing cytoplasm and the absence of its suppressor gene, known as restorer of fertility ( Rf ) (reviewed in Kitazaki et al. 2023 ). Plants with the desired combination of these genetic factors should be efficiently selected in hybrid breeding program, but this remains a practical challenge. DNA marker assisted selection is expected to address this issue, but the rationale for this approach is yet to be established. As our understanding of Rf genetics improves, we can elaborate a selection system for Rf alleles (see below). Molecular cloning of Rf has facilitated advances in Rf genetics. While Rf gene products include pentatricopeptide repeat (PPR) proteins and other proteins, it has been observed that some Rf loci exhibit similar polymorphic patterns regardless of their gene products (Kubo et al. 2020 ). In these Rf loci, open reading frames (ORFs) that resemble each other form gene clusters, which are polymorphic in terms of ORF copy number and nucleotide sequence (Kato et al. 2007 ; Arakawa et al. 2020a ). In sugar beet ( Beta vulgaris ), citrus ( Citrus sp.) and rice ( Oryza sativa ), Rf gene clusters are considered as haplotypes, and attempts have been made to assign them to Rf allelomorphs (Arakawa et al. 2019 ; Goto et al. 2023 ; Zhao et al. 2023 ). The results lead to the discovery of weaker Rf alleles (semidominant Rf ), revealing multiple allelism at some Rf loci, a concept that has been previously suggested from the perspective of classical genetics (Duvick 1965 ; Wise et al. 1996 ; Lee et al. 2008 ). In both sugar beet and rice, variations in the copy number of the ORFs within Rf haplotypes have been associated with the differences in allelomorphs (Arakawa et al. 2019 ; Zhao et al. 2023 ), suggesting that Rf loci are complex loci and involve multiple ORFs in fertility restoration (Arakawa et al. 2020a ). This implies that a specific ORF within a haplotype may not be the sole determinant of the allele's function; rather, the haplotype as a whole determines its function. If the Rf locus of interest is highly polymorphic and numerous Rf haplotypes are found in breeding materials, DNA marker-assisted selection of Rf alleles will be challenging without a clear rationale to differentiate between Rf haplotypes. If the molecular basis of fertility restoration is well-established and allows to predict the function of the haplotype of interest, the nucleotide sequence of the haplotype could help elucidate its functionality (c.f. Yamagishi et al. 2021 ). Another strategy consists in characterizing haplotype(s) preferred by the breeders in advance, and then selecting them from the breeding materials. This strategy is used in sugar beet breeding. Since the yield of sugar beet comes from the vegetative organ (root), hybrid sugar beet does not need to have male fertility restored, and the breeders' interest is on the recessive rf allele. Sugar beet breeders have made tremendous efforts to select genotypes unable to restore male fertility (i.e., lacking the dominant Rf allele), as the frequency of such genotypes is generally 3–5% (Bosemark 2006 ). These selected genotypes are termed “maintainers”. One of the sugar beet Rf genes, Rf1 , has been cloned and encodes a protein similar to OMA1 in budding yeast, which is involved in quality control of mitochondria (Matsuhira et al. 2012 ). At the Rf1 locus, ORFs encoding OMA1-like proteins (hereafter referred to as orf20 -like ORFs) are clustered, with one to four (or possibly more) copies, and a large number of haplotypes have been identified (Moritani et al. 2013 ). Practically, Rf1 haplotypes are grouped into five categories based on polymorphisms in a physically linked non-coding region targeted by the DNA marker s17 (Taguchi et al. 2014 ). Analysis of maintainers from Japan, the United States, and Europe has shown that breeders have selected three haplotypes as the recessive rf1 (Moritani et al. 2013 ; Ohgami et al. 2016 ). One of the three recessive rf1 haplotypes is uniquely associated with a specific s17 pattern, making DNA marker-assisted selection of maintainers feasible in sugar beet (Moritani et al. 2013 ; Taguchi et al. 2014 ). From the perspective of molecular evolution, beet Rf1 and PPR-type Rf in other plants display a similar evolutionary pattern, generating a large number of haplotypes (Fujii et al. 2011 ; Arakawa et al. 2020b ). Given that crop domestication and subsequent selection were accompanied by a genetic bottleneck, the Rf haplotypes observed in a cultivar group represent only a subset of the entire Rf haplotypes in the gene pool. This raises the question of whether marker assisted selection of Rf (or rf ) is also effective for other groups in the same species. For example, sugar beet originated from a few fodder beet cultivars at the end of the 18th century (Fischer 1989 ), while garden beet, the first cultivated root-type beet, appeared in the 16th century (Goldman and Janick 2021 ). We expected differences in the Rf1 haplotype between garden beet and sugar beet, both in terms of haplotype repertoire and frequency. Hybrid garden beet is gaining popularity, but selecting maintainers for this crop remains very challenging (Goldman and Navazio 2008 ). The CMS- Rf system has been introduced from sugar beet into garden beet to facilitate hybrid breeding in this crop (Bliss and Gabelman 1965 ). Therefore, some modern garden beet varieties carry the sugar beet rf1 haplotype, but it remains unknown whether garden beet possesses another rf1 haplotypes absent in sugar beet. It is also necessary to examine whether, and how, DNA marker-assisted selection of the recessive rf1 allele can be effectively applied in garden beet hybrid breeding. In the present study, we demonstrate the differences in the Rf1 haplotype between garden beet and sugar beet. Despite these differences, the DNA marker-assisted selection adopted for sugar beet has also proven effective for garden beet. Additionally, our study also suggests that Rf haplotype polymorphism is not only useful for selection but also provides insights into crop evolution. Materials and methods Plant materials Garden beet and sugar beet accessions used in this study are summarized in Table 1. Accessions prefixed by ‘BETA’ or ‘K’ were obtained from The Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany. Garden beet lines W357B and W446B are maintainers developed at the University of Wisconsin (Goldman 1996; https://www.warf.org/technologies/agriculture/plant-varieties/summary/inbred-table-beet-w446a-and-w446b-p01012us.cmsx). Four garden beet hybrids, ‘F1 Solo’ (Mr. Fothergill's), ‘F1 Pablo’ (Johnsons Seeds), ‘F1 Cardeal’ (Johnsons Seeds) and ‘F1 Red Titan’ (Johnsons Seeds) were purchased online. All sugar beet accessions were obtained from the Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization, Japan. The sugar beet cultivar ‘Donyu-2’ is a selection derived from the US cultivar ‘GW359’ and was released in 1954. Sugar beet cultivar ‘Hon-iku 192’ is derived from the German cultivar ‘Kleinwanzlebener’ and the French cultivar ‘Virmolin White French’ and was released in 1935. For the test cross to examine the genotype, we used the sugar beet line TA-33BB-CMS, which possesses a male sterility inducing cytoplasm (Matsuhira et al. 2022). TA-33BB-O line shares the same nuclear genotype as TA-33BB-CMS but differs in having non-sterility inducing cytoplasm. The procedures for crossing using paper bags is detailed in Ohgami et al. (2016). Plants were grown in a greenhouse from the seedling stage to the young plant stage, after which they were transplanted into the experimental field of the Field Science Center for Northern Biosphere, Hokkaido University. Male fertility was evaluated according to Matsuhira et al. (2022) and Moritani et al. (2013). Genotyping by DNA markers Total cellular DNA was isolated according to a procedure described in Matsuhira et al. (2022). The cleaved amplified polymorphic sequence (CAPS) marker s17 is detailed in Taguchi et al. (2014). Oligonucleotide primers for s17 amplification are s17-Fw and s17-Rv (Table S1). PCR products were digested with the restriction endonucleases HapII and HindIII (Takara Bio, Kusatsu, Japan), and electrophoresed in a 1.5% agarose gel. For genotyping using dCAPS-p5, total cellular DNA was PCR amplified with the primers dCAPS-p5-Fw and dCAPS-p5-Rv (Table S1). The PCR products were then digested with HindIII. Molecular cloning and nucleotide sequencing For nucleotide sequencing, the s17 region was PCR amplified as three overlapping segments using three pairs of primers (s17-1-Fw and s17-1-Rv, s17-2-Fw and s17-2-Rv, and s17-3-Fw and s17-3-Rv) (Table S1). PCR products for sequencing were treated with Illustra Alkaline Phosphatase (0.1 unit) and Illustra Exonuclease Ⅰ (1 unit) (GE Healthcare Life Sciences, Marlborough, MA, USA) at 37˚C for 30 min followed by incubation at 80˚C for 15 min. Alternatively, PCR products were electrophoresed in an agarose gel, and the PCR fragment was excised and purified by using the QIAquick Gel Extraction Kit (Qiagen, Tokyo, Japan). orf20-like ORFs were PCR amplified from total cellular DNA using the primer pairs orf20-infu-Fw / orf20-infu-Rv or orf20-infu2-Fw / orf20-infu2-Rv (Table S1). PCR products were purified via agarose gel electrophoresis as described above. The purified products were then mixed with a reaction buffer containing In-Fusion Snap Assembly Master Mix and linearized pUC19 plasmid vector DNA (Takara Bio). The mixture was incubated at 50˚C for 15 min, then chilled on ice before being used to transform E. coli strain DH5α. Transformants were selected on LB plate containing ampicillin (50 mg/L) and then cultured in liquid LB medium with the same amount of ampicillin (Sambrook et al. 1989). Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen). Plasmid DNA sequencing was performed using an ABI3130 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) with primers M13-Fw, M13-Rv, MPL-intron-Fw, and MPL-intron-Rv (Table S1). PCR amplified orf20-like ORF segments were directly sequenced with primers p4-1-Fw, p4-1-Rv, p4-2-Fw, p4-2-Rv, p4-3-Fw and p4-3-Rv (Table S1). Nucleotide sequence analyses were performed using Clustalw (https://www.genome.jp/tools-bin/clustalw) or MEGA 11 (Tamura et al. 2021). Phylogenetic trees were constructed using the neighbor-joining method with default parameters. Statistical analysis Fisher's exact test was performed using a webtool (http://aoki2.si.gunma-u.ac.jp/exact/fisher/getpar.html) or the fisher.multcomp function from the R package RVAideMemoire (https://cran.r-project.org). The P value adjustment method used was Holm. Results Polymorphic patterns of s17 differ between garden beet and sugar beet To examine the Rf1 haplotype polymorphism in garden beet, we analyzed the polymorphism of s17, a multiallelic CAPS marker linked to Rf1 (Taguchi et al. 2014) (Fig. 1). The s17 marker targets a noncoding region located 3.9 kbp away and its polymorphism serves as a useful proxy for estimating Rf1 polymorphism, as s17 can differentiate Rf1 haplotypes into several distinct groups (Taguchi et al. 2014). We selected 48 garden beet accessions that are registered as landraces in the genebank of IPK. Although five alleles of s17 are known (p1 to p5), our initial analysis revealed that the frequency was highly skewed towards p5 (>0.9). To determine whether p5 is monomorphic, we sought novel polymorphisms within the p5 allele. We selected one to two plants homozygous for p5 from each accession and determined the nucleotide sequences of their s17 target regions. Comparison of the sequences obtained from 55 plants revealed ten single nucleotide polymorphic sites (SNPs) which allowed us to group the sequences into three distinct groups (Fig. S1). As two of the three groups are nearly identical, differing by only one nucleotide, we classify p5 into two subtypes: p5A and p5B (Fig. S1). We developed a dCAPS marker (dCAPS-p5) that targets one of the SNPs to differentiate between p5A and p5B (Fig. 1 and detailed in Fig. S1). Plants with p5 were further analyzed using dCAPS-p5. A total of 485 plants from 48 garden beet accessions were genotyped (Table S2). s17 allelic frequencies of garden beet accessions are shown in Table 1. The frequency is highest in p5A, followed by p5B, p1, p2, p3, and p4, in this order. The combined frequency of p5A and p5B exceeds 0.95. Every garden beet accession contains either p5A (7 accessions), p5B (8 accessions), or both (33 accessions). Generally, the frequencies of each p1 through p4 are very low (~0.01) and they are absent in 31 accessions. We next aimed to compare these results with those from sugar beet. Although Taguchi et al. (2014) have analyzed several open-pollinated cultivars (OPCs) of sugar beet they did not distinguish between p5A and p5B. For this study, we selected two old Japanese OPC, ‘Donyu-2’ and ‘Hon-iku 192’, and genotyped them using the same method as for garden beet. The genotypes and their allelic frequencies are shown in Table S2 and Table 1, respectively. The allelic frequencies in ‘Donyu-2’ and ‘Hon-iku 192’ are similar to each other except for p4, which is decreased in ‘Hon-iku 192’. No plants were found to have the p2 allele. Taguchi et al. (2014) reported the allelic frequencies of seven sugar beet OPCs as follows: p1, 0-0.19 (mean 0.10); p2, 0 (0); p3, 0.04-0.31 (0.14); p4, 0-0.17 (0.09); and p5 (which can be considered as the sum of p5A and p5B), 0.41-0.88 (0.67). Therefore, the data from ‘Donyu-2’ and ‘Hon-iku 192’ are consistent with the findings of Taguchi et al. (2014). Comparison of the allelic frequencies of ‘Donyu-2’ and ‘Hon-iku 192’ with those of garden beet reveals that p5A is significantly reduced in sugar beet while p4 is increased. The difference in the allelic frequencies of s17 between garden beet and sugar beet is statistically significant (Fisher's exact test; p = 4.14 x 10 -35 ) Maintainer selection from garden beet genetic resources: Is p4 selection also effective in garden beet? To identify maintainers (i.e., genotypes without the restoring Rf allele) from garden beet genetic resources, we conducted test crosses. We selected 31 plants from 17 accessions, each one with six s17 alleles, and used them as pollen parent to cross with a sugar beet CMS line TA-33BB-CMS. Since the s17 genotype of TA-33BB-CMS is p4p4, the F1 plants should have one p4 allele. The F1 plants were grown and flowered in the field and phenotyped for pollen fertility. The results are shown in Table 2. We examined the male fertility of a total of 317 F1 plants. Of these, 52 were completely sterile, while the remaining were either male-fertile or semi-sterile (Table 2). The relationship between the pollen parental s17 allele and male fertility of the F1 plants is summarized in Figure 2A. Combining male-fertile and semi-sterile phenotypes as “fertility restored”, the ratios of fertility restored to complete sterility varies among the s17 alleles (Fig. 2B). We conducted pairwise comparisons of Fisher's exact test to determine if the occurrence of complete sterility differs among the alleles (Table S3 and Fig. 2B). The results suggest that complete sterility occurs most frequently in p4, followed by p3 and p5B, whereas very few or none in p1, p2 and p5A. Of the 31 cross combinations, #4, #6 and #24 resulted in F1 plants with complete sterility. In cross combination #6, one of the expected F1 genotype (p4p5) was missing for unknown reason, so we could not definitively conclude that the pollen parent of #6 was a maintainer. Since the number of F1 plants from cross #24 was rather small, we performed additional crosses. One of the F1plants from #24 was used as a seed parent to cross with TA-33BB-O, a maintainer with the same nuclear genotype as TA-33BB-CMS (hence this cross is effectively a backcross). The six BC1 plants obtained were completely sterile in the field. We focused on the accession BETA 1229 that provided the p4 allele in our test crosses (Table 2). Because p4 is uniquely linked to a recessive rf1 haplotype preferred by sugar beet breeders (Taguchi et al. 2014), p4 selection has been adopted in sugar beet maintainer selection. Using BETA 1229, we tested whether p4 selection is also effective in garden beet. We genotyped 253 plants of BETA 1229 and identified a p4p4 homozygous plant. This plant was used as the pollen parent for test crosses, and seven F1 plants were obtained. All the seven plants were p4p4 and completely male sterile in the field. Garden beet recessive rf1 haplotypes are either identical or different from sugar beet We wanted to know whether the recessive rf1 haplotypes in garden beet are the same as those in sugar beet. We first examined p4 of BETA 1229. As rf1 haplotypes are composed of orf20 -like ORFs (e. g., Arakawa et al. 2020a) (see Fig. 1), we investigated the nucleotide sequence of BETA 1229 p4. The p4 homozygote identified in the previous section was subjected to PCR amplification of orf20 -like ORFs. The PCR products yielded a single nucleotide sequence (Fig. S2) which is identical to the one found in the recessive rf1 haplotype marked by p4 (TK-81mm-O rf1 in Arakawa et al. 2020a). As no trace of other orf20 -like ORF was observed, we concluded that this haplotype is identical to TK-81mm-O rf1 . We next investigated the haplotype marked by p5B in cross combination #4. One of the F1 plants in #4 was subjected to the molecular cloning of orf20 -like ORFs. All the orf20 -like ORFs in this plant were PCR amplified and cloned into a plasmid vector. Multiple recombinant plasmids were analyzed, and five orf20 -like ORF sequences were obtained, one of which appeared to be derived from the seed parent TA-33BB-CMS. The remaining four are therefore derived from the haplotype marked by p5B. We compared these sequences to the orf20 -like ORFs identified so far in sugar beet. For this comparison, we chose a total of eleven orf20 -like ORFs of sugar beet: four from dominant NK-198 Rf1 , two from semidominant NK-305 Rf1 , one from recessive TK-81mm-O rf1 , three from recessive NK-219mm-O rf1 , and one from recessive PI 615522 rf1 . No sugar beet copies were found to be identical to the orf20 -like ORF of p5B of cross #4. We referred the orf20 -like ORF copies of p5B of #4 to BETA 1229 p5B Copies 1 to 4. Nucleotide identities of coding regions and gene regions (exons and introns) are shown in Tables S3 and S4, respectively. We analyzed p3 in cross #24. Copies of the orf20 -like ORF were sequenced using a complete sterile F1 plant from #24. Aside from those derived from TA-33BB-CMS, we obtained one orf20 -like ORF sequence. As this sequence was not identical to any sugar beet copies identified so far, it was referred to as BETA 223 p3 Copy 1. Nucleotide identities of coding regions and gene regions with other orf20 -like ORFs are shown in Tables S3 and S4, respectively. We explored the relationship between the novel orf20 -like ORFs and the sugar beet copies by constructing phylogenetic trees of 16 orf20 -like ORFs including those from sugar beet and garden beet. The results were consistent across both coding regions (exons) and entire gene regions (exons and introns) (Fig. 3 and Fig. S3). A notable cluster includes orf20 -like ORFs from NK-198 Rf1 and NK-305 Rf1 . However, some of the orf20 -like ORFs from these Rf1 alleles are outside of this cluster (i. e., orf20 NK-198-2 and orf20 NK-305-2 ). Similarly, not all the orf20 -like ORFs from recessive rf1 alleles are clustered. The novel orf20 -like ORFs identified from garden beet are clustered with the sugar beet copies. For example, BETA 223 p3 Copy 1 is clustered with the copy of PI 615522 rf1 (Fig. 3). Note that they share features such as being marked by p3 and unable to restore male fertility (Ohgami et al. 2016), suggesting that PI 615522 rf1 and BETA223 p3 haplotypes are close relatives. Two copies of BETA 1229 p5B cluster with those from recessive rf1 haplotype and dominant Rf1 haplotype, respectively. The remaining two copies (Copies 2 and 4) are relatively isolated. Haplotypes used in hybrid garden beet We investigated the recessive rf1 haplotypes currently used in garden beet breeding. First, we genotyped two garden beet maintainer lines W357B and W446B, that were developed in the United States, using s17 and dCAPS-p5 markers. The results are shown in Table S2 (genotypes) and Table 1 (allelic frequencies). We found that W357B is fixed with p4 whereas p4 and p5B were found in W446B. To examine whether p5B in W446B is linked to recessive rf1 , we selected one of the W446B plants with the p4p5B genotype and crossed it with TA-33BB-CMS. Of the ten F1 plants obtained, six were completely male sterile with a p4p4 genotype and four were male fertile with a p4p5B genotype. Therefore, p5B in W446B is linked to the dominant Rf1 . To determine the identity of p4 in W357B, we selected one p4p4 homozygote and determined the nucleotide sequence of its orf20 -like ORF. The sequence obtained was identical to that of TK-81mm-O rf1 . We next investigated the s17 genotype of four commercial hybrid garden beets developed in the UK. Although we do not have detailed information about their breeding methods (e. g., whether they are developed by single cross or three-way cross, or whether the pollen parent of the hybrid was a maintainer or not), the hybrids are expected to contain the recessive rf1 allele. We found that p5B is prevalent in ‘F1 Solo’ and ‘F1 Pablo’, that four s17 alleles (p3, p4, p5A and p5B) were found in ‘F1 Cardeal’, and all the examined plants of ‘F1 Red Titan’ had the p4p5A genotype. Therefore, the recessive rf1 haplotype linked to p4 is used in current garden beet hybrid breeding. We cannot rule out that other recessive haplotypes are also utilized. Discussion Hybrid breeding programs of garden beet are prevalent and will continue to develop high performance varieties (Goldman and Navazio 2003 ). Therefore, understanding the polymorphism of the garden beet Rf1 locus is crucial for expediating breeding, particularly when CMS is used for hybrid seed production. In the present study, we evaluated the Rf1 haplotype polymorphism based on the alleles of s17. Our analysis of garden beet genetic resources indicates that the allelic frequency of s17 is different between garden beet and sugar beet, despite both belonging to B. vulgaris . Wu et al. ( 2024 ) reported the prevalence of p5 in some garden beet varieties in China, the United States, the United Kingdom and the Netherlands. The present study provides a good example of how frequency of the Rf haplotype can differ between cultivar groups within a crop species. It is likely that this difference originated during the establishment of the garden beet and sugar beet groups. Garden beet is the first beet cultivar group with swollen roots, from which fodder beet originated (Ford-Lloyd 1995 ). Sugar beet was selected from fodder beet, but unintentional crosses with other beets, such as leaf beet, is associated with the initial breeding material (Fischer 1989 ). Such events invoke decrease in population size and the introduction of foreign germplasm, mechanisms that could explain the changes in s17 allelic frequencies. We may focus on p1, p3 and p4 because their allelic frequencies are increased in sugar beet. Although these alleles occur sporadically in European garden beet, they appeared to be slightly more frequent in the East Mediterranean and Black Sea coasts, including regions such as Georgia (p1 and p3), Romania (p1 and p4), Greece (p3) and Turkey (p4). Wascher et al. ( 2022 ) analyzed European wild beet ( B. vulgaris ssp. maritima ) genomes and found the closest relative of sugar beet in Greece. The role of wild and cultivated beets in this region in the domestication of sugar beet is an interesting question that warrants further investigation. We conducted test crosses to identify maintainers from the garden beet genetic resources. Although alleles of s17 do not necessarily correspond to a single Rf1 haplotype but rather to groups of haplotypes, our data reveals certain trends that can assist in selecting maintainers in garden beet. We observed that no or few male sterile F1 plants occurred in certain p17 alleles, such as p1, p2 and p5A, in stark contrast to p4. The alleles of p5A and p3 gave rise to some male sterile plants, but the occurrence frequencies were significantly lower compared to p4. Considering that the majority of garden beets possess p5A or p5B, and that the occurrence of male sterile F1 with these alleles is 0.06 and 0.24, respectively, we believe that, as a general rule, selecting garden beet maintainers from these genetic resources is difficult. This is consistent with Goldman and Navazio ( 2008 ) assessment that some garden beet genetic backgrounds are much more recalcitrant sources for maintainers. P4 is a potentially promising s17 allele; unfortunately, its allelic frequency is very low in garden beet genetic resources. We obtained maintainers from BETA 1229 (Turkey origin) and BETA 223 (Georgia origin) which possess p3, p4 and p5B. We investigated the identity of these recessive rf1 haplotypes, and to our surprise, BETA 1229 p4 is linked to a recessive rf1 haplotype identical to that of sugar beet (i.e., TK-81mm-O rf1 ). In sugar beet, rf1 haplotypes linked to p4 have been sequenced from several different sources and were found to be identical to TK-81mm-O rf1 at the nucleotide sequence level (Moritani et al. 2013 ; Ohgami et al. 2016 ). On the other hand, morphologically, BETA 1229 appears to be a typical garden beet and different from sugar beet (Fig. S4 ). The TK-81mm-O rf1 haplotype may be key to understanding the genetic origin of sugar beet. The rf1 haplotype linked to BETA 223 p3 is similar to one of the previously identified recessive rf1 haplotype PI 615522 rf1 . Although these two alleles are apparently unable to restore male fertility, it is too early to conclude on their usefulness because, for unknown reasons, not all sugar beet breeders have selected PI 615522 rf1 as maintainer (Ohgami et al. 2016 ). It is possible that PI 615533 rf1 and BETA 223 p3 are associated with an undesirable trait. The utility of BETA 1229 p5B should also be investigated in detail. Our study revealed the usefulness of p4 (i.e., TK-81mm-O rf1 ) in the current garden beet hybrid breeding. Garden beet maintainers W357B and W446B have complex genealogies involving several lines and cultivars (Goldman 1996 ). It is possible that a garden beet like BETA 1229 did provide p4 to these maintainer lines. Another possibility is that Warren H. Gabelman, who introduced the CMS system into garden beet (Bliss and Gabelman 1965 ), may have introduced p4 from sugar beet. The genealogy of W357B includes sugar beet and US sugar beet maintainers frequently possess the p4 allele (Ohgami et al. 2016 ). We favor the latter possibility because p4 is rare in garden beet landraces. Altogether, garden beet breeders consider the recessive rf1 haplotype linked to p4 as a reliable and practical allele. Since no dominant Rf1 haplotype linked to p4 has been found in garden beet so far, selection of a p4p4 homozygote is one of the most effective methods to identify maintainers in garden beet breeding programs, as demonstrated in the case of BETA 1229. It is clear that several recessive rf1 haplotypes, in addition to p4, are used in garden beet hybrids as some commercial hybrids lack the p4 allele. It has been pointed out that the repertoire of recessive rf1 haplotypes in sugar beet is limited, and genetic vulnerability is a potential concern (Taguchi et al. 2014 ). Novel rf1 haplotypes could widen the choice for sugar beet breeder. However, it should be kept in mind that the seed production methods used in the breeding stations (e.g., field or green house) may impact CMS expression (c. f., Matsuhira et al. ( 2022 )), thus requiring careful evaluation. Our study focused on rf1 without considering other factors, such as the presence of other Rf genes or environmental conditions (Theurer and Ryser 1969 ; Matsuhira et al. 2022 ; Honma et al. 2014 ; Arakawa et al. 2018 ). The impact of these factors on garden beet is still unknown. Selection of p4 is recommended as a means to expedite maintainer identification in this crop, but it is necessary to investigate the selected plants in detail. Declarations Competing interests The authors have no relevant financial or non-financial interest to disclose. Funding This work was supported in part by NARO Bio-oriented Technology Research Advancement Institution (BRAIN) (Research program on development of innovative technology, Grant Number 30001A), and the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (21H02159 and 24K01726 to H. Matsuhira, K. Kitazaki and T. Kubo, 22K05569 to H. Matsuhira and K. Kitazaki, and 22H02267 to Y. Kuroda, K. Kitazaki and T. Kubo). Author Contribution Conceptualization: Tomohiko Kubo; Methodology: Tomohiko Kubo, and Kazuyoshi Kitazaki; Formal analysis and investigation: Eigo Taniguchi, Yohei Kanomata, Haruto Tanaka, Mion Oishi, Ryo Hayakawa, Hiroyo Kagami-Katsuyama, Tomohiko Kubo, and Kazuyoshi Kitazaki; Resources: Hiroaki Matsuhira, Tsubasa Narihiro, and Yosuke Kuroda; Supervision: Tomohiko Kubo, and Kazuyoshi Kitazaki; Writing - original draft preparation: Eigo Taniguchi, Yohei Kanomata, and Haruto Tanaka; Writing - review and editing: Tomohiko Kubo, and Kazuyoshi Kitazaki: Funding acquisition: Hiroaki Matsuhira, Yosuke Kuroda, Tomohiko Kubo, and Kazuyoshi Kitazaki. All authors read and approved the final manuscript. Acknowledgement We thank Prof. Dr. Irwin L. Goldman for his help to obtain W357B and W446B. Part of this study was conducted at the Field Science Center for the Northern Biosphere, Hokkaido University. Data Availability Nucleotide sequences reported in this study are available under DDBJ/EMBL/GenBank accession numbers AB646133, AB646135, AB646136, LC085626, LC085628, LC385768, LC849784, LC849785, LC849786, LC849787, LC849788, LC849789, LC849790 and LC849791. References Arakawa T, Uchiyama D, Ohgami T, Ohgami R, Murata T, et al. (2018) A fertility-restoring genotype of beet (Beta vulgaris L.) is composed of a weak restorer-of-fertility gene and a modifier gene tightly linked to the Rf1 locus. PLoS ONE 13: e0198409 Arakawa T, Matsunaga M, Matsui K, Itoh K, Kuroda Y, Matsuhira H, Kitazaki K, Kubo T (2020a) The molecular basis for allelic differences suggests Restorer-of-fertility 1 is a complex locus in sugar beet (Beta vulgaris L.). BMC Plant Biol 20:503 Arakawa T, Kagami H, Katsuyama T, Kitazaki K, Kubo T (2020b) A lineage-specific paralogue of Oma1 evolved into a gene family from which a suppressor of male sterility-inducing mitochondria emerged in plants. Genome Biol Evol 12:2314-2327 Arakawa T, Ue S, Sano C, Matsunaga M, Kagami H, Yoshida Y, Kuroda Y, Taguchi K, Kitazaki K, Kubo T (2019) Identification and characterization of a semi-dominant restorer-of-fertility 1 allele in sugar beet (Beta vulgaris). Theor Appl Genet 132: 227-240 Bliss FA, Gabelman WH (1965) Inheritance of male sterility in beets, Beta vulgaris L. Crop Sci 5: 403-406 Bosemark NO (2006) Genetics and breeding. In: Draycott AP (ed) Sugar beet. Blackwell, Oxford, pp 50–88 Duvick DN (1965) Cytoplasmic pollen sterility in corn. Adv Genet 13:1–56 Fischer HE (1989) Origin of the ‘Weisse Schlesische Rübe’ (white Silesian beet) and resynthesis of sugar beet. Euphytica 41: 75–80 Ford-Lloyd BV (1995) 11 Sugarbeet, and other cultivated beets. In: Smartt J and Simmonds NW (eds) Evolution of crop plants. Wiley, New York, pp. 35-40 Fujii S, Bond CS, Small ID (2011) Selection patterns on restorer-like genes reveal a conflict between nuclear and mitochondrial genomes throughout angiosperm evolution. Proc Natl Acad Sci USA 108: 1723-1728. Goldman IL (1996) A list of germplasm releases from the University of Wisconsin table beet breeding program, 1964-1992. Hort Science 31: 880-881. Goldman IL, Navazio JP (2003) History and breeding of table beet in the United States. In: Janick J, editor. Plant Breeding Reviews, volume 22. Hoboken: John Wiley and Sons. pp. 357-388. Goldman IL, Navazio JP (2008) Table beet. In: Prohens J and Nuez F, editors. Asteraceae, Brassicaceae, Chenopodiaceae, and Cucurbitaceae. Vegetables I. Handbook of plant breeding. Heidelberg: Springer. pp. 219-238. Goldman, IL, Janick J (2021) Evolution of Root Morphology in Table Beet: Historical and Iconographic. Front Plant Sci 12: 689926 Goto S, Fujii H, Hamada H, Ohta S, Endo T, et al. (2023) Allelic haplotype combinations at the MS-P1 region, including P-class pentatricopeptide repeat family genes, influence wide phenotypic variation in pollen grain number through a cytoplasmic male sterility model in citrus. Front Plant Sci 14:1163358 Honma Y, Taguchi K, Hiyama H, Yui-Kurino R, Mikami T, et al. (2014) Molecular mapping of restorer-of-fertility 2 gene identified from a sugar beet (Beta vulgaris L. ssp. vulgaris) homozygous for the non-restoring restorer-of-fertility 1 allele. Theor Appl Genet 127: 2567-2574 Kato H, Tezuka K, Feng YY, Kawamoto T, Takahashi H, Mori K, Akagi H (2007) Structural diversity and evolution of the Rf-1 locus in the genus Oryza. Heredity 99: 516–524 Kitazaki K, Oda K, Akazawa A, Iwahori R (2023) Molecular genetics of cytoplasmic male sterility and restorer-of-fertility for the fine tuning of pollen production in crops. Theor Appl Genet 136: 156 Kubo T, Arakawa T, Honma Y, Kitazaki K (2020) What does the molecular genetics of different types of restorer-of-fertility genes imply? Plants, 9: 361 Lee J, Yoon JB, Park HG (2008) Linkage analysis between the partial restoration (pr) and the restorer-of-fertility (Rf) loci in pepper cytoplasmic male sterility. Theor Appl Genet 117:383–389 Matsuhira H, Kagami H, Kurata M, Kitazaki K, Matsunaga M, et al. (2012) Unusual and typical features of a novel restorer-of-fertility gene of sugar beet (Beta vulgaris L.), Genetics, 192: 1347-1358 Matsuhira H, Kitazaki K, Matsui K, Kubota K, Kuroda Y, et al. (2022) Selection of nuclear genotypes associated with the thermo-sensitivity of Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.). Theor Appl Genet 135:1457-1466 Moritani M, Taguchi K, Kitazaki K et al (2013) Identification of the predominant nonrestoring allele for Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.): development of molecular markers for the maintainer genotype. Mol Breed 32:91–100 Ohgami T, Uchiyama D, Ue S, Yui-Kurino R, Yoshida Y, et al. (2016) Identification of molecular variants of the nonrestoring restorer-of-fertility 1 allele in sugar beet (Beta vulgaris L.), Theor Appl Genet 129: 675-688 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). New York: Cold Spring Harbor Laboratory Press. Taguchi K, Hiyama H, Yui-Kurino R, Muramatsu A, Mikami T, Kubo T (2014) Hybrid breeding skewed the allelic frequencies of molecular variants derived from the restorer-of-fertility 1 locus for cytoplasmic male sterility in sugar beet (Beta vulgaris L.). Crop Sci 54:1407–1412 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol Biol and Evol 38:3022-3027 Theurer JC, Ryser GK (1969) Inheritance studies with a pollen fertility restorer sugarbeet inbred. J ASSBT 15:538-345 Wascher FL, Stralis-Pavese N, McGrath JM, Schulz B, Himmelbauer H, et al. (2022) Genomic distances reveal relationships of wild and cultivated beets. Nat Commun. 13: 2021. Wise RP, Dill CL, Schnable PS (1996) Mutator-induced mutations of the rf1 nuclear fertility restorer of T-cytoplasm maize alter the accumulation of T-urf13 mitochondrial transcripts. Genetics 143:1383–1394 Yamagishi H, Jikuya M, Okushiro K, Hashimoto A, Fukunaga A, et al. (2021) A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish. Mol Genet Genomics 296: 705-717 Xu F, Yang X, Zhao N, Hu Z, Mackenzie A, et al. (2022) Exploiting sterility and fertility variation in cytoplasmic male sterile vegetable crops. Hort Res 9: uhab039 Wu X, Pi Z, Li S, Wu Z (2024) Identification of the fertility types of red beet varieties (lines) using molecular-marker technology. Sugar Tech, https://doi.org/10.1007/s12355-024-01373-5 Zhao Z, Ding Z, Huang J, Meng H, Zhang Z, et al. (2023) Copy number variation of the restorer Rf4 underlies human selection of three-line hybrid rice breeding. Nat Commun 14: 7333 Additional Declarations No competing interests reported. Supplementary Files FigS1.pdf FigS2.pdf FigS3.pdf FigS4.pdf TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx Cite Share Download PDF Status: Published Journal Publication published 08 Feb, 2025 Read the published version in Genetic Resources and Crop Evolution → Version 1 posted Editorial decision: Revision requested 09 Jan, 2025 Reviews received at journal 09 Jan, 2025 Reviews received at journal 20 Dec, 2024 Reviewers agreed at journal 24 Nov, 2024 Reviewers agreed at journal 14 Nov, 2024 Reviewers invited by journal 14 Nov, 2024 Editor assigned by journal 13 Nov, 2024 Submission checks completed at journal 13 Nov, 2024 First submitted to journal 10 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5424865","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381944484,"identity":"2f16f15a-24f4-4a90-a107-e44c21b6f53c","order_by":0,"name":"Eigo Taniguchi","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Eigo","middleName":"","lastName":"Taniguchi","suffix":""},{"id":381944485,"identity":"748bd266-91c6-4982-bb4b-4faf2372792e","order_by":1,"name":"Yohei Kanomata","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Yohei","middleName":"","lastName":"Kanomata","suffix":""},{"id":381944486,"identity":"894c4373-126e-4042-a3f0-dbf200e2e648","order_by":2,"name":"Haruto Tanaka","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Haruto","middleName":"","lastName":"Tanaka","suffix":""},{"id":381944487,"identity":"f116ed0a-d968-4e05-9005-ebe68148358e","order_by":3,"name":"Mion Oishi","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Mion","middleName":"","lastName":"Oishi","suffix":""},{"id":381944488,"identity":"d313cfdd-82ca-4083-b725-f86b2fbf7329","order_by":4,"name":"Ryo Hayakawa","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Ryo","middleName":"","lastName":"Hayakawa","suffix":""},{"id":381944489,"identity":"9fe25e1b-a045-44f2-aab5-304f111c4543","order_by":5,"name":"Hiroaki Matsuhira","email":"","orcid":"","institution":"National Agriculture and Food Research Organization","correspondingAuthor":false,"prefix":"","firstName":"Hiroaki","middleName":"","lastName":"Matsuhira","suffix":""},{"id":381944490,"identity":"0700be8e-5b69-4524-a75a-757d99211912","order_by":6,"name":"Tsubasa Narihiro","email":"","orcid":"","institution":"National Agriculture and Food Research Organization","correspondingAuthor":false,"prefix":"","firstName":"Tsubasa","middleName":"","lastName":"Narihiro","suffix":""},{"id":381944491,"identity":"9226f893-d678-48b2-9a7e-8b538c2b085a","order_by":7,"name":"Yosuke Kuroda","email":"","orcid":"","institution":"National Agriculture and Food Research Organization","correspondingAuthor":false,"prefix":"","firstName":"Yosuke","middleName":"","lastName":"Kuroda","suffix":""},{"id":381944492,"identity":"1cef15af-8ea9-426c-8b13-a31a547167ed","order_by":8,"name":"Hiroyo Kagami-Katsuyama","email":"","orcid":"","institution":"Hokkaido Information University","correspondingAuthor":false,"prefix":"","firstName":"Hiroyo","middleName":"","lastName":"Kagami-Katsuyama","suffix":""},{"id":381944493,"identity":"a4604b87-2f51-4dfa-8bbc-a012e91e6396","order_by":9,"name":"Tomohiko Kubo","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Tomohiko","middleName":"","lastName":"Kubo","suffix":""},{"id":381944494,"identity":"ebd3e49c-ed40-42d0-b8ef-f18459185d24","order_by":10,"name":"Kazuyoshi Kitazaki","email":"data:image/png;base64,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","orcid":"","institution":"Hokkaido University","correspondingAuthor":true,"prefix":"","firstName":"Kazuyoshi","middleName":"","lastName":"Kitazaki","suffix":""}],"badges":[],"createdAt":"2024-11-10 08:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5424865/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5424865/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10722-025-02353-8","type":"published","date":"2025-02-08T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70932087,"identity":"24e84bde-2935-433b-9c43-836e4b9281b1","added_by":"auto","created_at":"2024-12-09 10:18:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56912,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the s17 target region and its alleles. Scale bars are shown below each panel. These panels are based on Figure 1 of Taguchi et al. (2014) with some modifications. \u003cstrong\u003eA.\u003c/strong\u003eOrganization of chromosomal segment containing the \u003cem\u003eRf1\u003c/em\u003e locus and the s17 target region. The \u003cem\u003eRf1\u003c/em\u003e locus consists of \u003cem\u003eorf20\u003c/em\u003e-like ORFs, which have slightly differing nucleotide sequences. Each \u003cem\u003eorf20\u003c/em\u003e-like ORF consists of three exons (open boxes) and two introns (wedges). The copy number of \u003cem\u003eorf20\u003c/em\u003e-like ORFs varies among the haplotypes (indicated by dashed line). The direction of transcription is indicated by arrows. s17 (shown as a red bold line) is ~3.9 kbp away from the \u003cem\u003eRf1\u003c/em\u003e locus. \u003cstrong\u003eB. \u003c/strong\u003eOrganization of six s17 alleles, p1 to p5B. Wedges with dotted lines indicate deletions. Positions of HindIII and HapII recognition sites are shown by filled- and open triangles, respectively. The polymorphic site to distinguish p5A and p5B alleles is shown (A:T or G:C).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5424865/v1/19b4a360c286e23fa5f2f051.jpg"},{"id":70931923,"identity":"0521ad3b-93ac-4650-9845-a4ac3785b19e","added_by":"auto","created_at":"2024-12-09 10:10:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":52207,"visible":true,"origin":"","legend":"\u003cp\u003eInherited s17 allele from pollen parent and its impact on male fertility phenotype in the F1 progeny. \u003cstrong\u003eA.\u003c/strong\u003eNumber of plants with male fertile (MF), semi-sterility (SS), fertility restored, and complete sterility (CS) phenotypes. The F1 plants with unknown p5 identity are omitted. \u003cstrong\u003eB.\u003c/strong\u003e Comparison of ratios of fertility restored and complete sterile plants among s17 alleles. ns, nonsignificant pair as determined by Fisher's exact test (Table S3).\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5424865/v1/329e1790ea5c41c936220080.jpg"},{"id":70931928,"identity":"98849be0-3cb0-431d-9b3f-b866c44b9c63","added_by":"auto","created_at":"2024-12-09 10:10:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60223,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-Joining tree of \u003cem\u003eorf20\u003c/em\u003e-like ORFs constructed using the nucleotide sequence of amino acid coding regions. Labels are as follows (see also Arakawa et al. 2020a): NK-198-1, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK198-1\u003c/sub\u003e; NK-198-2, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK198-2\u003c/sub\u003e; NK-198-3, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK198-3\u003c/sub\u003e; NK-198-4, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK198-4\u003c/sub\u003e; NK-219-1, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK219-1\u003c/sub\u003e; NK-219-2, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK219-2\u003c/sub\u003e; NK-219-3, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK219-3\u003c/sub\u003e; NK-305-1, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK305-1\u003c/sub\u003e; NK-305-2, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK305-2\u003c/sub\u003e; PI 615522, \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e PI 615522\u003c/sub\u003e; and TK-81, \u003cem\u003eorf129\u003c/em\u003e\u003csub\u003e TK-81\u003c/sub\u003e. Copies from dominant- and semidominant \u003cem\u003eRf1\u003c/em\u003e haplotypes are highlighted in red. Copies from recessive \u003cem\u003erf1\u003c/em\u003e haplotypes are highlighted in blue. The following labels denote copies identified in this study: BETA 1229 Copy 1, BETA 1229 p5B Copy 1; BETA 1229 Copy 2, BETA 1229 p5B Copy 2; BETA 1229 Copy 3, BETA 1229 p5B Copy 3; BETA 1229 Copy 4, BETA 1229 p5B Copy 4; and BETA 223 Copy 1, BETA 223 p3 Copy1.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5424865/v1/30521c59a8aad0ea6a65c8cb.jpg"},{"id":75930446,"identity":"f561b3f7-fabe-44c5-b041-929f53cedc63","added_by":"auto","created_at":"2025-02-10 16:11:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":902929,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5424865/v1/5b05d51a-06b5-451c-a264-74b1f0707afe.pdf"},{"id":70931917,"identity":"658050bc-5cd5-46cf-bb98-31b161fa6e03","added_by":"auto","created_at":"2024-12-09 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10:10:41","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":10785,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5424865/v1/1069e6d6928dfb9702da4316.xlsx"},{"id":70932088,"identity":"7ad2c942-efbd-4bef-a4ec-32fa71634e0a","added_by":"auto","created_at":"2024-12-09 10:18:41","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":12462,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5424865/v1/d62dc38bc7fd62406130aae0.xlsx"},{"id":70931922,"identity":"b8f1a5eb-7a40-4e1f-8cf5-f8705c9568e5","added_by":"auto","created_at":"2024-12-09 10:10:41","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":12593,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5424865/v1/fa717fd4b09ccaae9e5a1d03.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Differences in the frequency of restorer-of-fertility 1 haplotype are associated with crop history of garden beet and sugar beet (Beta vulgaris L.)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCytoplasmic male sterility (CMS) is used for hybrid seed production in a broad range of crops as it confers male-specific sterility while leaving other organs unaffected (reviewed in Xu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Expression of CMS is genetically conditioned by the presence of male sterility-inducing cytoplasm and the absence of its suppressor gene, known as restorer of fertility (\u003cem\u003eRf\u003c/em\u003e) (reviewed in Kitazaki et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Plants with the desired combination of these genetic factors should be efficiently selected in hybrid breeding program, but this remains a practical challenge. DNA marker assisted selection is expected to address this issue, but the rationale for this approach is yet to be established. As our understanding of \u003cem\u003eRf\u003c/em\u003e genetics improves, we can elaborate a selection system for \u003cem\u003eRf\u003c/em\u003e alleles (see below).\u003c/p\u003e \u003cp\u003eMolecular cloning of \u003cem\u003eRf\u003c/em\u003e has facilitated advances in \u003cem\u003eRf\u003c/em\u003e genetics. While \u003cem\u003eRf\u003c/em\u003e gene products include pentatricopeptide repeat (PPR) proteins and other proteins, it has been observed that some \u003cem\u003eRf\u003c/em\u003e loci exhibit similar polymorphic patterns regardless of their gene products (Kubo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In these \u003cem\u003eRf\u003c/em\u003e loci, open reading frames (ORFs) that resemble each other form gene clusters, which are polymorphic in terms of ORF copy number and nucleotide sequence (Kato et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Arakawa et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). In sugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e), citrus (\u003cem\u003eCitrus\u003c/em\u003e sp.) and rice (\u003cem\u003eOryza sativa\u003c/em\u003e), \u003cem\u003eRf\u003c/em\u003e gene clusters are considered as haplotypes, and attempts have been made to assign them to \u003cem\u003eRf\u003c/em\u003e allelomorphs (Arakawa et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Goto et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The results lead to the discovery of weaker \u003cem\u003eRf\u003c/em\u003e alleles (semidominant \u003cem\u003eRf\u003c/em\u003e), revealing multiple allelism at some \u003cem\u003eRf\u003c/em\u003e loci, a concept that has been previously suggested from the perspective of classical genetics (Duvick \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Wise et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn both sugar beet and rice, variations in the copy number of the ORFs within \u003cem\u003eRf\u003c/em\u003e haplotypes have been associated with the differences in allelomorphs (Arakawa et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), suggesting that \u003cem\u003eRf\u003c/em\u003e loci are complex loci and involve multiple ORFs in fertility restoration (Arakawa et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). This implies that a specific ORF within a haplotype may not be the sole determinant of the allele's function; rather, the haplotype as a whole determines its function. If the \u003cem\u003eRf\u003c/em\u003e locus of interest is highly polymorphic and numerous \u003cem\u003eRf\u003c/em\u003e haplotypes are found in breeding materials, DNA marker-assisted selection of \u003cem\u003eRf\u003c/em\u003e alleles will be challenging without a clear rationale to differentiate between \u003cem\u003eRf\u003c/em\u003e haplotypes.\u003c/p\u003e \u003cp\u003eIf the molecular basis of fertility restoration is well-established and allows to predict the function of the haplotype of interest, the nucleotide sequence of the haplotype could help elucidate its functionality (c.f. Yamagishi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Another strategy consists in characterizing haplotype(s) preferred by the breeders in advance, and then selecting them from the breeding materials. This strategy is used in sugar beet breeding. Since the yield of sugar beet comes from the vegetative organ (root), hybrid sugar beet does not need to have male fertility restored, and the breeders' interest is on the recessive \u003cem\u003erf\u003c/em\u003e allele. Sugar beet breeders have made tremendous efforts to select genotypes unable to restore male fertility (i.e., lacking the dominant \u003cem\u003eRf\u003c/em\u003e allele), as the frequency of such genotypes is generally 3\u0026ndash;5% (Bosemark \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). These selected genotypes are termed \u0026ldquo;maintainers\u0026rdquo;. One of the sugar beet \u003cem\u003eRf\u003c/em\u003e genes, \u003cem\u003eRf1\u003c/em\u003e, has been cloned and encodes a protein similar to OMA1 in budding yeast, which is involved in quality control of mitochondria (Matsuhira et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). At the \u003cem\u003eRf1\u003c/em\u003e locus, ORFs encoding OMA1-like proteins (hereafter referred to as \u003cem\u003eorf20\u003c/em\u003e-like ORFs) are clustered, with one to four (or possibly more) copies, and a large number of haplotypes have been identified (Moritani et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Practically, \u003cem\u003eRf1\u003c/em\u003e haplotypes are grouped into five categories based on polymorphisms in a physically linked non-coding region targeted by the DNA marker s17 (Taguchi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Analysis of maintainers from Japan, the United States, and Europe has shown that breeders have selected three haplotypes as the recessive \u003cem\u003erf1\u003c/em\u003e (Moritani et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ohgami et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). One of the three recessive \u003cem\u003erf1\u003c/em\u003e haplotypes is uniquely associated with a specific s17 pattern, making DNA marker-assisted selection of maintainers feasible in sugar beet (Moritani et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Taguchi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom the perspective of molecular evolution, beet \u003cem\u003eRf1\u003c/em\u003e and PPR-type \u003cem\u003eRf\u003c/em\u003e in other plants display a similar evolutionary pattern, generating a large number of haplotypes (Fujii et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Arakawa et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). Given that crop domestication and subsequent selection were accompanied by a genetic bottleneck, the \u003cem\u003eRf\u003c/em\u003e haplotypes observed in a cultivar group represent only a subset of the entire \u003cem\u003eRf\u003c/em\u003e haplotypes in the gene pool. This raises the question of whether marker assisted selection of \u003cem\u003eRf\u003c/em\u003e (or \u003cem\u003erf\u003c/em\u003e) is also effective for other groups in the same species. For example, sugar beet originated from a few fodder beet cultivars at the end of the 18th century (Fischer \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), while garden beet, the first cultivated root-type beet, appeared in the 16th century (Goldman and Janick \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We expected differences in the \u003cem\u003eRf1\u003c/em\u003e haplotype between garden beet and sugar beet, both in terms of haplotype repertoire and frequency.\u003c/p\u003e \u003cp\u003eHybrid garden beet is gaining popularity, but selecting maintainers for this crop remains very challenging (Goldman and Navazio \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The CMS-\u003cem\u003eRf\u003c/em\u003e system has been introduced from sugar beet into garden beet to facilitate hybrid breeding in this crop (Bliss and Gabelman \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). Therefore, some modern garden beet varieties carry the sugar beet \u003cem\u003erf1\u003c/em\u003e haplotype, but it remains unknown whether garden beet possesses another \u003cem\u003erf1\u003c/em\u003e haplotypes absent in sugar beet. It is also necessary to examine whether, and how, DNA marker-assisted selection of the recessive \u003cem\u003erf1\u003c/em\u003e allele can be effectively applied in garden beet hybrid breeding.\u003c/p\u003e \u003cp\u003eIn the present study, we demonstrate the differences in the \u003cem\u003eRf1\u003c/em\u003e haplotype between garden beet and sugar beet. Despite these differences, the DNA marker-assisted selection adopted for sugar beet has also proven effective for garden beet. Additionally, our study also suggests that \u003cem\u003eRf\u003c/em\u003e haplotype polymorphism is not only useful for selection but also provides insights into crop evolution.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGarden beet and sugar beet accessions used in this study are summarized in Table 1. Accessions prefixed by \u0026lsquo;BETA\u0026rsquo; or \u0026lsquo;K\u0026rsquo; were obtained from The Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany. Garden beet lines W357B and W446B are maintainers developed at the University of Wisconsin (Goldman 1996; https://www.warf.org/technologies/agriculture/plant-varieties/summary/inbred-table-beet-w446a-and-w446b-p01012us.cmsx). Four garden beet hybrids, \u0026lsquo;F1 Solo\u0026rsquo; (Mr. Fothergill\u0026apos;s), \u0026lsquo;F1 Pablo\u0026rsquo; (Johnsons Seeds), \u0026lsquo;F1 Cardeal\u0026rsquo; (Johnsons Seeds) and \u0026lsquo;F1 Red Titan\u0026rsquo; (Johnsons Seeds) were purchased online. All sugar beet accessions were obtained from the Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization, Japan. The sugar beet cultivar \u0026lsquo;Donyu-2\u0026rsquo; is a selection derived from the US cultivar \u0026lsquo;GW359\u0026rsquo; and was released in 1954. Sugar beet cultivar \u0026lsquo;Hon-iku 192\u0026rsquo; is derived from the German cultivar \u0026lsquo;Kleinwanzlebener\u0026rsquo; and the French cultivar \u0026lsquo;Virmolin White French\u0026rsquo; and was released in 1935. For the test cross to examine the genotype, we used the sugar beet line TA-33BB-CMS, which possesses a male sterility inducing cytoplasm (Matsuhira et al. 2022). TA-33BB-O line shares the same nuclear genotype as TA-33BB-CMS but differs in having non-sterility inducing cytoplasm. The procedures for crossing using paper bags is detailed in Ohgami et al. (2016). Plants were grown in a greenhouse from the seedling stage to the young plant stage, after which they were transplanted into the experimental field of the Field Science Center for Northern Biosphere, Hokkaido University. \u0026nbsp;Male fertility was evaluated according to Matsuhira et al. (2022) and Moritani et al. (2013). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping by DNA markers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal cellular DNA was isolated according to a procedure described in Matsuhira et al. (2022). The cleaved amplified polymorphic sequence (CAPS) marker s17 is detailed in Taguchi et al. (2014). Oligonucleotide primers for s17 amplification are s17-Fw and s17-Rv (Table S1). PCR products were digested with the restriction endonucleases HapII and HindIII (Takara Bio, Kusatsu, Japan), and electrophoresed in a 1.5% agarose gel. For genotyping using dCAPS-p5, total cellular DNA was PCR amplified with the primers dCAPS-p5-Fw and dCAPS-p5-Rv (Table S1). The PCR products were then digested with HindIII. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular cloning and nucleotide sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor nucleotide sequencing, the s17 region was PCR amplified as three overlapping segments using three pairs of primers (s17-1-Fw and s17-1-Rv, s17-2-Fw and s17-2-Rv, and s17-3-Fw and s17-3-Rv) (Table S1). PCR products for sequencing were treated with Illustra Alkaline Phosphatase (0.1 unit) and Illustra Exonuclease Ⅰ (1 unit) (GE Healthcare Life Sciences, Marlborough, MA, USA) at 37˚C for 30 min followed by incubation at 80˚C for 15 min. \u0026nbsp;Alternatively, PCR products were electrophoresed in an agarose gel, and the PCR fragment was excised and purified by using the QIAquick Gel Extraction Kit (Qiagen, Tokyo, Japan). orf20-like ORFs were PCR amplified from total cellular DNA using the primer pairs orf20-infu-Fw / orf20-infu-Rv or orf20-infu2-Fw / orf20-infu2-Rv (Table S1). PCR products were purified via agarose gel electrophoresis as described above. The purified products were then mixed with a reaction buffer containing In-Fusion Snap Assembly Master Mix and linearized pUC19 plasmid vector DNA (Takara Bio). The mixture was incubated at 50˚C for 15 min, then chilled on ice before being used to transform E. coli strain DH5\u0026alpha;. Transformants were selected on LB plate containing ampicillin (50 mg/L) and then cultured in liquid LB medium with the same amount of ampicillin (Sambrook et al. 1989). Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen). Plasmid DNA sequencing was performed using an ABI3130 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) with primers M13-Fw, M13-Rv, MPL-intron-Fw, and MPL-intron-Rv (Table S1). PCR amplified orf20-like ORF segments were directly sequenced with primers p4-1-Fw, p4-1-Rv, p4-2-Fw, p4-2-Rv, p4-3-Fw and p4-3-Rv (Table S1). Nucleotide sequence analyses were performed using Clustalw (https://www.genome.jp/tools-bin/clustalw) or MEGA 11 (Tamura et al. 2021). Phylogenetic trees were constructed using the neighbor-joining method with default parameters. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFisher\u0026apos;s exact test was performed using a webtool (http://aoki2.si.gunma-u.ac.jp/exact/fisher/getpar.html) or the fisher.multcomp function from the R package RVAideMemoire (https://cran.r-project.org). The P value adjustment method used was Holm.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePolymorphic patterns of s17 differ between garden beet and sugar beet\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the \u003cem\u003eRf1\u003c/em\u003e haplotype polymorphism in garden beet, we analyzed the polymorphism of s17, a multiallelic CAPS marker linked to \u003cem\u003eRf1\u003c/em\u003e (Taguchi et al. 2014) (Fig. 1). The s17 marker targets a noncoding region located 3.9 kbp away and its polymorphism serves as a useful proxy for estimating \u003cem\u003eRf1\u003c/em\u003e polymorphism, as s17 can differentiate \u003cem\u003eRf1\u003c/em\u003e haplotypes into several distinct groups (Taguchi et al. 2014). We selected 48 garden beet accessions that are registered as landraces in the genebank of IPK. Although five alleles of s17 are known (p1 to p5), our initial analysis revealed that the frequency was highly skewed towards p5 (\u0026gt;0.9). To determine whether p5 is monomorphic, we sought novel polymorphisms within the p5 allele. We selected one to two plants homozygous for p5 from each accession and determined the nucleotide sequences of their s17 target regions. Comparison of the sequences obtained from 55 plants revealed ten single nucleotide polymorphic sites (SNPs) which allowed us to group the sequences into three distinct groups (Fig. S1). As two of the three groups are nearly identical, differing by only one nucleotide, we classify p5 into two subtypes: p5A and p5B (Fig. S1). We developed a dCAPS marker (dCAPS-p5) that targets one of the SNPs to differentiate between p5A and p5B (Fig. 1 and detailed in Fig. S1). Plants with p5 were further analyzed using dCAPS-p5. \u003c/p\u003e\n\u003cp\u003eA total of 485 plants from 48 garden beet accessions were genotyped (Table S2). s17 allelic frequencies of garden beet accessions are shown in Table 1. The frequency is highest in p5A, followed by p5B, p1, p2, p3, and p4, in this order. The combined frequency of p5A and p5B exceeds 0.95. Every garden beet accession contains either p5A (7 accessions), p5B (8 accessions), or both (33 accessions). Generally, the frequencies of each p1 through p4 are very low (~0.01) and they are absent in 31 accessions.\u003c/p\u003e\n\u003cp\u003eWe next aimed to compare these results with those from sugar beet. Although Taguchi et al. (2014) have analyzed several open-pollinated cultivars (OPCs) of sugar beet they did not distinguish between p5A and p5B. For this study, we selected two old Japanese OPC, \u0026lsquo;Donyu-2\u0026rsquo; and \u0026lsquo;Hon-iku 192\u0026rsquo;, and genotyped them using the same method as for garden beet. The genotypes and their allelic frequencies are shown in Table S2 and Table 1, respectively. The allelic frequencies in \u0026lsquo;Donyu-2\u0026rsquo; and \u0026lsquo;Hon-iku 192\u0026rsquo; are similar to each other except for p4, which is decreased in \u0026lsquo;Hon-iku 192\u0026rsquo;. No plants were found to have the p2 allele. Taguchi et al. (2014) reported the allelic frequencies of seven sugar beet OPCs as follows: p1, 0-0.19 (mean 0.10); p2, 0 (0); p3, 0.04-0.31 (0.14); p4, 0-0.17 (0.09); and p5 (which can be considered as the sum of p5A and p5B), 0.41-0.88 (0.67). Therefore, the data from \u0026lsquo;Donyu-2\u0026rsquo; and \u0026lsquo;Hon-iku 192\u0026rsquo; are consistent with the findings of Taguchi et al. (2014). Comparison of the allelic frequencies of \u0026lsquo;Donyu-2\u0026rsquo; and \u0026lsquo;Hon-iku 192\u0026rsquo; with those of garden beet reveals that p5A is significantly reduced in sugar beet while p4 is increased. The difference in the allelic frequencies of s17 between garden beet and sugar beet is statistically significant (Fisher\u0026apos;s exact test; \u003cem\u003ep \u003c/em\u003e= 4.14 x 10\u003csup\u003e -35\u003c/sup\u003e)\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMaintainer selection from garden beet genetic resources: Is p4 selection also effective in garden beet?\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify maintainers (i.e., genotypes without the restoring \u003cem\u003eRf\u003c/em\u003e allele) from garden beet genetic resources, we conducted test crosses. We selected 31 plants from 17 accessions, each one with six s17 alleles, and used them as pollen parent to cross with a sugar beet CMS line TA-33BB-CMS. Since the s17 genotype of TA-33BB-CMS is p4p4, the F1 plants should have one p4 allele. The F1 plants were grown and flowered in the field and phenotyped for pollen fertility. The results are shown in Table 2. \u003c/p\u003e\n\u003cp\u003eWe examined the male fertility of a total of 317 F1 plants. Of these, 52 were completely sterile, while the remaining were either male-fertile or semi-sterile (Table 2). The relationship between the pollen parental s17 allele and male fertility of the F1 plants is summarized in Figure 2A. Combining male-fertile and semi-sterile phenotypes as \u0026ldquo;fertility restored\u0026rdquo;, the ratios of fertility restored to complete sterility varies among the s17 alleles (Fig. 2B). We conducted pairwise comparisons of Fisher\u0026apos;s exact test to determine if the occurrence of complete sterility differs among the alleles (Table S3 and Fig. 2B). The results suggest that complete sterility occurs most frequently in p4, followed by p3 and p5B, whereas very few or none in p1, p2 and p5A. \u003c/p\u003e\n\u003cp\u003eOf the 31 cross combinations, #4, #6 and #24 resulted in F1 plants with complete sterility. In cross combination #6, one of the expected F1 genotype (p4p5) was missing for unknown reason, so we could not definitively conclude that the pollen parent of #6 was a maintainer. Since the number of F1 plants from cross #24 was rather small, we performed additional crosses. One of the F1plants from #24 was used as a seed parent to cross with TA-33BB-O, a maintainer with the same nuclear genotype as TA-33BB-CMS (hence this cross is effectively a backcross). The six BC1 plants obtained were completely sterile in the field.\u003c/p\u003e\n\u003cp\u003eWe focused on the accession BETA 1229 that provided the p4 allele in our test crosses (Table 2). Because p4 is uniquely linked to a recessive \u003cem\u003erf1\u003c/em\u003e haplotype preferred by sugar beet breeders (Taguchi et al. 2014), p4 selection has been adopted in sugar beet maintainer selection. Using BETA 1229, we tested whether p4 selection is also effective in garden beet. We genotyped 253 plants of BETA 1229 and identified a p4p4 homozygous plant. This plant was used as the pollen parent for test crosses, and seven F1 plants were obtained. All the seven plants were p4p4 and completely male sterile in the field. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGarden beet recessive rf1 haplotypes are either identical or different from sugar beet\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe wanted to know whether the recessive \u003cem\u003erf1\u003c/em\u003e haplotypes in garden beet are the same as those in sugar beet. We first examined p4 of BETA 1229. As \u003cem\u003erf1\u003c/em\u003e haplotypes are composed of \u003cem\u003eorf20\u003c/em\u003e-like ORFs (e. g., Arakawa et al. 2020a) (see Fig. 1), we investigated the nucleotide sequence of BETA 1229 p4. The p4 homozygote identified in the previous section was subjected to PCR amplification of \u003cem\u003eorf20\u003c/em\u003e-like ORFs. The PCR products yielded a single nucleotide sequence (Fig. S2) which is identical to the one found in the recessive \u003cem\u003erf1\u003c/em\u003e haplotype marked by p4 (TK-81mm-O \u003cem\u003erf1\u003c/em\u003e in Arakawa et al. 2020a). As no trace of other \u003cem\u003eorf20\u003c/em\u003e-like ORF was observed, we concluded that this haplotype is identical to TK-81mm-O \u003cem\u003erf1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eWe next investigated the haplotype marked by p5B in cross combination #4. One of the F1 plants in #4 was subjected to the molecular cloning of \u003cem\u003eorf20\u003c/em\u003e-like ORFs. All the \u003cem\u003eorf20\u003c/em\u003e-like ORFs in this plant were PCR amplified and cloned into a plasmid vector. Multiple recombinant plasmids were analyzed, and five \u003cem\u003eorf20\u003c/em\u003e-like ORF sequences were obtained, one of which appeared to be derived from the seed parent TA-33BB-CMS. The remaining four are therefore derived from the haplotype marked by p5B. We compared these sequences to the \u003cem\u003eorf20\u003c/em\u003e-like ORFs identified so far in sugar beet. For this comparison, we chose a total of eleven \u003cem\u003eorf20\u003c/em\u003e-like ORFs of sugar beet: four from dominant NK-198 \u003cem\u003eRf1\u003c/em\u003e, two from semidominant NK-305 \u003cem\u003eRf1\u003c/em\u003e, one from recessive TK-81mm-O \u003cem\u003erf1\u003c/em\u003e, three from recessive NK-219mm-O \u003cem\u003erf1\u003c/em\u003e, and one from recessive PI 615522 \u003cem\u003erf1\u003c/em\u003e. No sugar beet copies were found to be identical to the \u003cem\u003eorf20\u003c/em\u003e-like ORF of p5B of cross #4. We referred the \u003cem\u003eorf20\u003c/em\u003e-like ORF copies of p5B of #4 to BETA 1229 p5B Copies 1 to 4. Nucleotide identities of coding regions and gene regions (exons and introns) are shown in Tables S3 and S4, respectively.\u003c/p\u003e\n\u003cp\u003eWe analyzed p3 in cross #24. Copies of the \u003cem\u003eorf20\u003c/em\u003e-like ORF were sequenced using a complete sterile F1 plant from #24. Aside from those derived from TA-33BB-CMS, we obtained one \u003cem\u003eorf20\u003c/em\u003e-like ORF sequence. As this sequence was not identical to any sugar beet copies identified so far, it was referred to as BETA 223 p3 Copy 1. Nucleotide identities of coding regions and gene regions with other \u003cem\u003eorf20\u003c/em\u003e-like ORFs are shown in Tables S3 and S4, respectively.\u003c/p\u003e\n\u003cp\u003eWe explored the relationship between the novel \u003cem\u003eorf20\u003c/em\u003e-like ORFs and the sugar beet copies by constructing phylogenetic trees of 16 \u003cem\u003eorf20\u003c/em\u003e-like ORFs including those from sugar beet and garden beet. The results were consistent across both coding regions (exons) and entire gene regions (exons and introns) (Fig. 3 and Fig. S3). A notable cluster includes \u003cem\u003eorf20\u003c/em\u003e-like ORFs from NK-198 \u003cem\u003eRf1\u003c/em\u003e and NK-305 \u003cem\u003eRf1\u003c/em\u003e. However, some of the \u003cem\u003eorf20\u003c/em\u003e-like ORFs from these \u003cem\u003eRf1\u003c/em\u003e alleles are outside of this cluster (i. e., \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK-198-2\u003c/sub\u003e and \u003cem\u003eorf20\u003c/em\u003e\u003csub\u003e NK-305-2\u003c/sub\u003e). Similarly, not all the \u003cem\u003eorf20\u003c/em\u003e-like ORFs from recessive \u003cem\u003erf1\u003c/em\u003e alleles are clustered. The novel \u003cem\u003eorf20\u003c/em\u003e-like ORFs identified from garden beet are clustered with the sugar beet copies. For example, BETA 223 p3 Copy 1 is clustered with the copy of PI 615522 \u003cem\u003erf1\u003c/em\u003e (Fig. 3). Note that they share features such as being marked by p3 and unable to restore male fertility (Ohgami et al. 2016), suggesting that PI 615522 \u003cem\u003erf1\u003c/em\u003e and BETA223 p3 haplotypes are close relatives. Two copies of BETA 1229 p5B cluster with those from recessive \u003cem\u003erf1\u003c/em\u003e haplotype and dominant \u003cem\u003eRf1\u003c/em\u003e haplotype, respectively. The remaining two copies (Copies 2 and 4) are relatively isolated. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHaplotypes used in hybrid garden beet\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the recessive \u003cem\u003erf1\u003c/em\u003e haplotypes currently used in garden beet breeding. First, we genotyped two garden beet maintainer lines W357B and W446B, that were developed in the United States, using s17 and dCAPS-p5 markers. The results are shown in Table S2 (genotypes) and Table 1 (allelic frequencies). We found that W357B is fixed with p4 whereas p4 and p5B were found in W446B. To examine whether p5B in W446B is linked to recessive \u003cem\u003erf1\u003c/em\u003e, we selected one of the W446B plants with the p4p5B genotype and crossed it with TA-33BB-CMS. Of the ten F1 plants obtained, six were completely male sterile with a p4p4 genotype and four were male fertile with a p4p5B genotype. Therefore, p5B in W446B is linked to the dominant \u003cem\u003eRf1\u003c/em\u003e. To determine the identity of p4 in W357B, we selected one p4p4 homozygote and determined the nucleotide sequence of its \u003cem\u003eorf20\u003c/em\u003e-like ORF. The sequence obtained was identical to that of TK-81mm-O \u003cem\u003erf1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eWe next investigated the s17 genotype of four commercial hybrid garden beets developed in the UK. Although we do not have detailed information about their breeding methods (e. g., whether they are developed by single cross or three-way cross, or whether the pollen parent of the hybrid was a maintainer or not), the hybrids are expected to contain the recessive \u003cem\u003erf1\u003c/em\u003e allele. We found that p5B is prevalent in \u0026lsquo;F1 Solo\u0026rsquo; and \u0026lsquo;F1 Pablo\u0026rsquo;, that four s17 alleles (p3, p4, p5A and p5B) were found in \u0026lsquo;F1 Cardeal\u0026rsquo;, and all the examined plants of \u0026lsquo;F1 Red Titan\u0026rsquo; had the p4p5A genotype. Therefore, the recessive \u003cem\u003erf1\u003c/em\u003e haplotype linked to p4 is used in current garden beet hybrid breeding. We cannot rule out that other recessive haplotypes are also utilized. \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHybrid breeding programs of garden beet are prevalent and will continue to develop high performance varieties (Goldman and Navazio \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Therefore, understanding the polymorphism of the garden beet \u003cem\u003eRf1\u003c/em\u003e locus is crucial for expediating breeding, particularly when CMS is used for hybrid seed production. In the present study, we evaluated the \u003cem\u003eRf1\u003c/em\u003e haplotype polymorphism based on the alleles of s17. Our analysis of garden beet genetic resources indicates that the allelic frequency of s17 is different between garden beet and sugar beet, despite both belonging to \u003cem\u003eB. vulgaris\u003c/em\u003e. Wu et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported the prevalence of p5 in some garden beet varieties in China, the United States, the United Kingdom and the Netherlands. The present study provides a good example of how frequency of the \u003cem\u003eRf\u003c/em\u003e haplotype can differ between cultivar groups within a crop species. It is likely that this difference originated during the establishment of the garden beet and sugar beet groups. Garden beet is the first beet cultivar group with swollen roots, from which fodder beet originated (Ford-Lloyd \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Sugar beet was selected from fodder beet, but unintentional crosses with other beets, such as leaf beet, is associated with the initial breeding material (Fischer \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Such events invoke decrease in population size and the introduction of foreign germplasm, mechanisms that could explain the changes in s17 allelic frequencies. We may focus on p1, p3 and p4 because their allelic frequencies are increased in sugar beet. Although these alleles occur sporadically in European garden beet, they appeared to be slightly more frequent in the East Mediterranean and Black Sea coasts, including regions such as Georgia (p1 and p3), Romania (p1 and p4), Greece (p3) and Turkey (p4). Wascher et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) analyzed European wild beet (\u003cem\u003eB. vulgaris\u003c/em\u003e ssp. \u003cem\u003emaritima\u003c/em\u003e) genomes and found the closest relative of sugar beet in Greece. The role of wild and cultivated beets in this region in the domestication of sugar beet is an interesting question that warrants further investigation.\u003c/p\u003e \u003cp\u003eWe conducted test crosses to identify maintainers from the garden beet genetic resources. Although alleles of s17 do not necessarily correspond to a single \u003cem\u003eRf1\u003c/em\u003e haplotype but rather to groups of haplotypes, our data reveals certain trends that can assist in selecting maintainers in garden beet. We observed that no or few male sterile F1 plants occurred in certain p17 alleles, such as p1, p2 and p5A, in stark contrast to p4. The alleles of p5A and p3 gave rise to some male sterile plants, but the occurrence frequencies were significantly lower compared to p4. Considering that the majority of garden beets possess p5A or p5B, and that the occurrence of male sterile F1 with these alleles is 0.06 and 0.24, respectively, we believe that, as a general rule, selecting garden beet maintainers from these genetic resources is difficult. This is consistent with Goldman and Navazio (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) assessment that some garden beet genetic backgrounds are much more recalcitrant sources for maintainers. P4 is a potentially promising s17 allele; unfortunately, its allelic frequency is very low in garden beet genetic resources.\u003c/p\u003e \u003cp\u003eWe obtained maintainers from BETA 1229 (Turkey origin) and BETA 223 (Georgia origin) which possess p3, p4 and p5B. We investigated the identity of these recessive \u003cem\u003erf1\u003c/em\u003e haplotypes, and to our surprise, BETA 1229 p4 is linked to a recessive \u003cem\u003erf1\u003c/em\u003e haplotype identical to that of sugar beet (i.e., TK-81mm-O \u003cem\u003erf1\u003c/em\u003e). In sugar beet, \u003cem\u003erf1\u003c/em\u003e haplotypes linked to p4 have been sequenced from several different sources and were found to be identical to TK-81mm-O \u003cem\u003erf1\u003c/em\u003e at the nucleotide sequence level (Moritani et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ohgami et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). On the other hand, morphologically, BETA 1229 appears to be a typical garden beet and different from sugar beet (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). The TK-81mm-O \u003cem\u003erf1\u003c/em\u003e haplotype may be key to understanding the genetic origin of sugar beet.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003erf1\u003c/em\u003e haplotype linked to BETA 223 p3 is similar to one of the previously identified recessive \u003cem\u003erf1\u003c/em\u003e haplotype PI 615522 \u003cem\u003erf1\u003c/em\u003e. Although these two alleles are apparently unable to restore male fertility, it is too early to conclude on their usefulness because, for unknown reasons, not all sugar beet breeders have selected PI 615522 \u003cem\u003erf1\u003c/em\u003e as maintainer (Ohgami et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is possible that PI 615533 \u003cem\u003erf1\u003c/em\u003e and BETA 223 p3 are associated with an undesirable trait. The utility of BETA 1229 p5B should also be investigated in detail.\u003c/p\u003e \u003cp\u003eOur study revealed the usefulness of p4 (i.e., TK-81mm-O \u003cem\u003erf1\u003c/em\u003e) in the current garden beet hybrid breeding. Garden beet maintainers W357B and W446B have complex genealogies involving several lines and cultivars (Goldman \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). It is possible that a garden beet like BETA 1229 did provide p4 to these maintainer lines. Another possibility is that Warren H. Gabelman, who introduced the CMS system into garden beet (Bliss and Gabelman \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1965\u003c/span\u003e), may have introduced p4 from sugar beet. The genealogy of W357B includes sugar beet and US sugar beet maintainers frequently possess the p4 allele (Ohgami et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We favor the latter possibility because p4 is rare in garden beet landraces. Altogether, garden beet breeders consider the recessive \u003cem\u003erf1\u003c/em\u003e haplotype linked to p4 as a reliable and practical allele. Since no dominant \u003cem\u003eRf1\u003c/em\u003e haplotype linked to p4 has been found in garden beet so far, selection of a p4p4 homozygote is one of the most effective methods to identify maintainers in garden beet breeding programs, as demonstrated in the case of BETA 1229.\u003c/p\u003e \u003cp\u003eIt is clear that several recessive \u003cem\u003erf1\u003c/em\u003e haplotypes, in addition to p4, are used in garden beet hybrids as some commercial hybrids lack the p4 allele. It has been pointed out that the repertoire of recessive \u003cem\u003erf1\u003c/em\u003e haplotypes in sugar beet is limited, and genetic vulnerability is a potential concern (Taguchi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Novel \u003cem\u003erf1\u003c/em\u003e haplotypes could widen the choice for sugar beet breeder. However, it should be kept in mind that the seed production methods used in the breeding stations (e.g., field or green house) may impact CMS expression (c. f., Matsuhira et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)), thus requiring careful evaluation.\u003c/p\u003e \u003cp\u003eOur study focused on \u003cem\u003erf1\u003c/em\u003e without considering other factors, such as the presence of other \u003cem\u003eRf\u003c/em\u003e genes or environmental conditions (Theurer and Ryser \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Matsuhira et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Honma et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Arakawa et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The impact of these factors on garden beet is still unknown. Selection of p4 is recommended as a means to expedite maintainer identification in this crop, but it is necessary to investigate the selected plants in detail.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interest to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported in part by NARO Bio-oriented Technology Research Advancement Institution (BRAIN) (Research program on development of innovative technology, Grant Number 30001A), and the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (21H02159 and 24K01726 to H. Matsuhira, K. Kitazaki and T. Kubo, 22K05569 to H. Matsuhira and K. Kitazaki, and 22H02267 to Y. Kuroda, K. Kitazaki and T. Kubo).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Tomohiko Kubo; Methodology: Tomohiko Kubo, and Kazuyoshi Kitazaki; Formal analysis and investigation: Eigo Taniguchi, Yohei Kanomata, Haruto Tanaka, Mion Oishi, Ryo Hayakawa, Hiroyo Kagami-Katsuyama, Tomohiko Kubo, and Kazuyoshi Kitazaki; Resources: Hiroaki Matsuhira, Tsubasa Narihiro, and Yosuke Kuroda; Supervision: Tomohiko Kubo, and Kazuyoshi Kitazaki; Writing - original draft preparation: Eigo Taniguchi, Yohei Kanomata, and Haruto Tanaka; Writing - review and editing: Tomohiko Kubo, and Kazuyoshi Kitazaki: Funding acquisition: Hiroaki Matsuhira, Yosuke Kuroda, Tomohiko Kubo, and Kazuyoshi Kitazaki. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Prof. Dr. Irwin L. Goldman for his help to obtain W357B and W446B. Part of this study was conducted at the Field Science Center for the Northern Biosphere, Hokkaido University.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eNucleotide sequences reported in this study are available under DDBJ/EMBL/GenBank accession numbers AB646133, AB646135, AB646136, LC085626, LC085628, LC385768, LC849784, LC849785, LC849786, LC849787, LC849788, LC849789, LC849790 and LC849791.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArakawa T, Uchiyama D, Ohgami T, Ohgami R, Murata T, et al. (2018) A fertility-restoring genotype of beet (Beta vulgaris L.) is composed of a weak restorer-of-fertility gene and a modifier gene tightly linked to the Rf1 locus. PLoS ONE 13: e0198409\u003c/li\u003e\n \u003cli\u003eArakawa T, Matsunaga M, Matsui K, Itoh K, Kuroda Y, Matsuhira H, Kitazaki K, Kubo T (2020a) The molecular basis for allelic differences suggests Restorer-of-fertility 1 is a complex locus in sugar beet (Beta vulgaris L.). BMC Plant Biol 20:503\u003c/li\u003e\n \u003cli\u003eArakawa T, Kagami H, Katsuyama T, Kitazaki K, Kubo T (2020b) A lineage-specific paralogue of Oma1 evolved into a gene family from which a suppressor of male sterility-inducing mitochondria emerged in plants. Genome Biol Evol 12:2314-2327\u003c/li\u003e\n \u003cli\u003eArakawa T, Ue S, Sano C, Matsunaga M, Kagami H, Yoshida Y, Kuroda Y, Taguchi K, Kitazaki K, Kubo T (2019) Identification and characterization of a semi-dominant restorer-of-fertility 1 allele in sugar beet (Beta vulgaris). Theor Appl Genet 132: 227-240\u003c/li\u003e\n \u003cli\u003eBliss FA, Gabelman WH (1965) Inheritance of male sterility in beets, Beta vulgaris L. Crop Sci 5: 403-406\u003c/li\u003e\n \u003cli\u003eBosemark NO (2006) Genetics and breeding. In: Draycott AP (ed) Sugar beet. Blackwell, Oxford, pp 50\u0026ndash;88\u003c/li\u003e\n \u003cli\u003eDuvick DN (1965) Cytoplasmic pollen sterility in corn. Adv Genet 13:1\u0026ndash;56\u003c/li\u003e\n \u003cli\u003eFischer HE (1989) Origin of the \u0026lsquo;Weisse Schlesische R\u0026uuml;be\u0026rsquo; (white Silesian beet) and resynthesis of sugar beet. Euphytica 41: 75\u0026ndash;80\u003c/li\u003e\n \u003cli\u003eFord-Lloyd BV (1995) 11 Sugarbeet, and other cultivated beets. In: Smartt J and Simmonds NW (eds) Evolution of crop plants. Wiley, New York, pp. 35-40\u003c/li\u003e\n \u003cli\u003eFujii S, Bond CS, Small ID (2011) Selection patterns on restorer-like genes reveal a conflict between nuclear and mitochondrial genomes throughout angiosperm evolution. 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(2023) Allelic haplotype combinations at the MS-P1 region, including P-class pentatricopeptide repeat family genes, influence wide phenotypic variation in pollen grain number through a cytoplasmic male sterility model in citrus. Front Plant Sci 14:1163358\u003c/li\u003e\n \u003cli\u003eHonma Y, Taguchi K, Hiyama H, Yui-Kurino R, Mikami T, et al. (2014) Molecular mapping of restorer-of-fertility 2 gene identified from a sugar beet (Beta vulgaris L. ssp. vulgaris) homozygous for the non-restoring restorer-of-fertility 1 allele. Theor Appl Genet 127: 2567-2574\u003c/li\u003e\n \u003cli\u003eKato H, Tezuka K, Feng YY, Kawamoto T, Takahashi H, Mori K, Akagi H (2007) Structural diversity and evolution of the Rf-1 locus in the genus Oryza. Heredity 99: 516\u0026ndash;524\u003c/li\u003e\n \u003cli\u003eKitazaki K, Oda K, Akazawa A, Iwahori R (2023) Molecular genetics of cytoplasmic male sterility and restorer-of-fertility for the fine tuning of pollen production in crops. Theor Appl Genet 136: 156\u003c/li\u003e\n \u003cli\u003eKubo T, Arakawa T, Honma Y, Kitazaki K (2020) What does the molecular genetics of different types of restorer-of-fertility genes imply? Plants, 9: 361\u003c/li\u003e\n \u003cli\u003eLee J, Yoon JB, Park HG (2008) Linkage analysis between the partial restoration (pr) and the restorer-of-fertility (Rf) loci in pepper cytoplasmic male sterility. Theor Appl Genet 117:383\u0026ndash;389\u003c/li\u003e\n \u003cli\u003eMatsuhira H, Kagami H, Kurata M, Kitazaki K, Matsunaga M, et al. (2012) Unusual and typical features of a novel restorer-of-fertility gene of sugar beet (Beta vulgaris L.), Genetics, 192: 1347-1358\u003c/li\u003e\n \u003cli\u003eMatsuhira H, Kitazaki K, Matsui K, Kubota K, Kuroda Y, et al. (2022) Selection of nuclear genotypes associated with the thermo-sensitivity of Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.). 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Nat Commun. 13: 2021.\u003c/li\u003e\n \u003cli\u003eWise RP, Dill CL, Schnable PS (1996) Mutator-induced mutations of the rf1 nuclear fertility restorer of T-cytoplasm maize alter the accumulation of T-urf13 mitochondrial transcripts. Genetics 143:1383\u0026ndash;1394\u003c/li\u003e\n \u003cli\u003eYamagishi H, Jikuya M, Okushiro K, Hashimoto A, Fukunaga A, et al. (2021) A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish. Mol Genet Genomics 296: 705-717\u003c/li\u003e\n \u003cli\u003eXu F, Yang X, Zhao N, Hu Z, Mackenzie A, et al. (2022) Exploiting sterility and fertility variation in cytoplasmic male sterile vegetable crops. Hort Res 9: uhab039\u003c/li\u003e\n \u003cli\u003eWu X, Pi Z, Li S, Wu Z (2024) Identification of the fertility types of red beet varieties (lines) using molecular-marker technology. Sugar Tech, https://doi.org/10.1007/s12355-024-01373-5\u003c/li\u003e\n \u003cli\u003eZhao Z, Ding Z, Huang J, Meng H, Zhang Z, et al. (2023) Copy number variation of the restorer Rf4 underlies human selection of three-line hybrid rice breeding. Nat Commun 14: 7333\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Crop domestication, Cultivar group divergence, Cytoplasmic male sterility, DNA marker assisted selection, Hybrid breeding, Standing genetic variation","lastPublishedDoi":"10.21203/rs.3.rs-5424865/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5424865/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCytoplasmic male sterility (CMS) is used in breeding to facilitate hybrid seed production. \u003cem\u003eRestorer of fertility\u003c/em\u003e (\u003cem\u003eRf\u003c/em\u003e), the suppressor of CMS, consists of a gene cluster with multiple haplotypes. Selection of restoring or non-restoring alleles depends on the discrimination of \u003cem\u003eRf\u003c/em\u003e haplotypes using DNA markers. The efficacy of this system is decreased if the \u003cem\u003eRf\u003c/em\u003e haplotype differs within the population of interest, which can occur during the crop evolution. In sugar beet, the \u003cem\u003eRf1\u003c/em\u003egene has multiple haplotypes that are grouped by a linked polymorphic region (s17), with one group, termed p4, uniquely linked to the recessive \u003cem\u003erf1\u003c/em\u003ehaplotype. Garden beet is the predecessor cultivar group of the sugar beet group. We questioned whether \u003cem\u003eRf1\u003c/em\u003e haplotypes differ between these two cultivar groups to assess the utility of marker-assisted selection. We analyzed 48 garden beet landraces and observed differences in the s17 polymorphism compared to sugar beet, suggesting that the \u003cem\u003eRf1\u003c/em\u003e haplotype frequency has changed during evolution of the crop. We next selected non-restoring genotypes from the garden beet landraces through test crosses and identified three recessive \u003cem\u003erf1\u003c/em\u003e haplotypes: one is identical to the p4 haplotype and the others are novel haplotypes. The p4 haplotype occurs in a few accessions and its frequency in garden beet is approximately 0.01. We analyzed the s17 polymorphism in modern garden beet hybrids and their constituents. We identified the p4 haplotype and suggest the presence of other recessive \u003cem\u003erf1\u003c/em\u003e haplotypes. Selection of p4 haplotype was efficient on the identification of non-restoring genotype in garden beet.\u003c/p\u003e","manuscriptTitle":"Differences in the frequency of restorer-of-fertility 1 haplotype are associated with crop history of garden beet and sugar beet (Beta vulgaris L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-09 10:10:36","doi":"10.21203/rs.3.rs-5424865/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-09T15:37:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-09T12:53:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-20T20:18:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127758555526565530037828039815331206539","date":"2024-11-24T16:03:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101641086460517882484746366100722174132","date":"2024-11-14T08:22:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-14T08:10:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-13T07:46:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-13T07:45:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genetic Resources and Crop Evolution","date":"2024-11-10T07:54:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cca3dfaa-8d31-4f66-b048-56d7d5358405","owner":[],"postedDate":"December 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T16:02:55+00:00","versionOfRecord":{"articleIdentity":"rs-5424865","link":"https://doi.org/10.1007/s10722-025-02353-8","journal":{"identity":"genetic-resources-and-crop-evolution","isVorOnly":false,"title":"Genetic Resources and Crop Evolution"},"publishedOn":"2025-02-08 15:57:46","publishedOnDateReadable":"February 8th, 2025"},"versionCreatedAt":"2024-12-09 10:10:36","video":"","vorDoi":"10.1007/s10722-025-02353-8","vorDoiUrl":"https://doi.org/10.1007/s10722-025-02353-8","workflowStages":[]},"version":"v1","identity":"rs-5424865","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5424865","identity":"rs-5424865","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}