2005年, 平成17年度日本育種学会奨励賞　　　　　　　　　　　　　　　
日本国

Mitochondrial phylogeny and distribution of cytoplasmic male sterility-associated genes in Beta vulgaris
Keishi Kubota, Mion Oishi, Eigo Taniguchi, Akiho Akazawa, Katsunori Matsui, Kazuyoshi Kitazaki, Atsushi Toyoda, Hidehiro Toh, Hiroaki Matsuhira, Yosuke Kuroda, Tomohiko Kubo
PLOS ONE, 19, 9, e0308551, e0308551, Public Library of Science (PLoS), 2024年09月27日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Cytoplasmic male sterility (CMS) is a mitochondrial-encoded trait that confers reproductive defects in males but not in females or any vegetative function. Why CMS is so often found in plants should be investigated from the viewpoint of mitochondrial phylogeny. Beta vulgaris, including the wild subspecies maritima and cultivated subspecies vulgaris (e.g., sugar beet), is known to be mitochondrially polymorphic, from which multiple CMS mitochondria have been found, but their evolutionary relationship has been obscure. We first refined the B. vulgaris reference mitochondrial genome to conduct a more accurate phylogenetic study. We identified mitochondrial single-nucleotide polymorphic sites from 600 B. vulgaris accessions. Principal component analysis, hierarchical clustering analysis, and creation of a phylogenetic tree consistently suggested that B. vulgaris mitochondria can be classified into several groups whose geographical distribution tends to be biased toward either the Atlantic or Mediterranean coasts. We examined the distribution of CMS-associated mitochondrial genes from Owen, E- and G-type CMS mitochondria. About one-third of cultivated beets had Owen-type CMS, which reflects the prevalence of using Owen-type CMS in hybrid breeding. Occurrence frequencies for each of the three CMS genes in wild beet were less than 4%. CMS genes were tightly associated with specific mitochondrial groups that are phylogenetically distinct, suggesting their independent origin. However, homologous sequences of the Owen type CMS gene occurred in several different mitochondrial groups, for which an intricate explanation is necessary. Whereas the origin of cultivated beet had been presumed to be Greece, we found an absence of Owen-type mitochondria in Greek accessions.

Two cytoplasmic male sterility phenotypes in beet (Beta vulgaris L.): implications of their simultaneous onset and divergent paths
Naoyuki Katsura, Kanna Itoh, Hiroaki Matsuhira, Yosuke Kuroda, Tomohiko Kubo, Kazuyoshi Kitazaki
Euphytica, Springer Nature, 2023年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Nuclear DNA segments homologous to mitochondrial DNA are obstacles for detecting heteroplasmy in sugar beet (Beta vulgaris L.)
Taniguchi E, Satoh K, Ohkubo M, Ue S, Matsuhira H, Kuroda Y, Kubo T, Kitazaki K
PLoS ONE, 18, e0285430, 2023年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Selection of nuclear genotypes associated with the thermo-sensitivity of Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.)
Hiroaki Matsuhira, Kazuyoshi Kitazaki, Katsunori Matsui, Keisi Kubota, Yosuke Kuroda, Tomohiko Kubo
Theoretical and Applied Genetics, 135, 5, 1457, 1466, Springer Science and Business Media LLC, 2022年02月11日, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, KEY MESSAGE: Cytoplasmic male sterility in sugar beet becomes thermo-sensitive when combined with specific genotypes, potentially offering a means to environmentally control pollination by this trait. The stability of cytoplasmic male sterility expression in several genetic backgrounds was investigated in sugar beet (Beta vulgaris L.). Nine genetically heterogenous plants from open-pollinated varieties were crossed with a cytoplasmic male sterile line to obtain 266 F1 plants. Based on marker analysis using a multiallelic DNA marker linked to restorer-of-fertility 1 (Rf1), we divided the F1 plants into 15 genotypes. We evaluated the phenotypes of the F1 plants under two environmental conditions: greenhouse rooms with or without daytime heating during the flowering season. Three phenotypic groups appeared: those consistently expressing male sterility, those consistently having restored pollen fertility, and those expressing male sterility in a thermo-sensitive manner. All plants in the consistently male sterile group inherited a specific Rf1 marker type named p4. We tested the potential for thermo-sensitive male sterile plants to serve as seed parents for hybrid seed production, and three genotypes were selected. Open pollination by a pollen parental line with a dominant trait of red-pigmented hypocotyls and leaf veins resulted in seed setting on thermo-sensitive male sterile plants, indicating that their female organs were functional. More than 99.9% of the progeny expressed the red pigmentation trait; hence, highly pure hybrids were obtained. We determined the nucleotide sequences of Rf1 from the three genotypes: One had a novel allele and two had known alleles, of which one was reported to have been selected previously as a non-restoring allele at a single U.S. breeding station but not at other stations in the U.S., or in Europe or Japan, suggesting environmental sensitivity.

Nuclear and mitochondrial DNA polymorphisms suggest introgression contributed to garden beet (Beta vulgaris L.) domestication
Yohei Kanomata, Ryo Hayakawa, Jun Kashikura, Kosuke Satoh, Hiroaki Matsuhira, Yosuke Kuroda, Kazuyoshi Kitazaki, Tomohiko Kubo
Genetic Resources and Crop Evolution, 69, 1, 271, 283, Springer Science and Business Media LLC, 2022年01月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Garden beet is the ancestor of fodder beets and sugar beets, but the origin of garden beet's genetic potential to evolve novel beet types is debatable. In this study, we analyzed nuclear and mitochondrial DNAs in 47 garden beet accessions using DNA markers. Multiple analytical methods revealed a unified population structure with subpopulations evident in the European and Caucasian accessions. We diagnosed mitochondrial genome types (mitotypes) based on mitochondrial minisatellite loci in 541 plants from the 47 accessions, revealing a major mitotype and 11 minor mitotypes in garden beets from Europe and the Caucasus region that were also present in endemic leaf beets and wild beets. Our data indicate that European and Caucasian garden beets include genetically differentiated subpopulations. Provided that the occurrence of minor mitotypes is a vestige from crosses with leaf beets and wild beets, the notion that introgression contributed to increasing the genetic diversity in the garden beet gene pool is substantiated at the molecular level.

A Lineage-Specific Paralog of Oma1 Evolved into a Gene Family from Which a Suppressor of Male Sterility-Inducing Mitochondria Emerged in Plants
Takumi Arakawa, Hiroyo Kagami, Takaya Katsuyama, Kazuyoshi Kitazaki, Tomohiko Kubo
Genome Biology and Evolution, 12, 12, 2314, 2327, Oxford University Press (OUP), 2020年12月06日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）,
Cytoplasmic male sterility (MS) in plants is caused by MS-inducing mitochondria, which have emerged frequently during plant evolution. Nuclear restorer-of-fertility (Rf)genes can suppress their cognate MS-inducing mitochondria. Whereas many Rfs encode a class of RNA-binding protein, the sugar beet (Caryophyllales) Rf encodes a protein resembling Oma1, which is involved in the quality control of mitochondria. In this study, we investigated the molecular evolution of Oma1 homologs in plants. We analyzed 37 plant genomes and concluded that a single copy is the ancestral state in Caryophyllales. Among the sugar beet Oma1 homologs, the orthologous copy is located in a syntenic region that is preserved in Arabidopsis thaliana. The sugar beet Rf is a complex locus consisting of a small Oma1 homolog family (RF-Oma1 family) unique to sugar beet. The gene arrangement in the vicinity of the locus is seen in some but not all Caryophyllalean plants and is absent from Ar. thaliana. This suggests a segmental duplication rather than a whole-genome duplication as the mechanism of RF-Oma1 evolution. Of thirty-seven positively selected codons in RF-Oma1, twenty-six of these sites are located in predicted transmembrane helices. Phylogenetic network analysis indicated that homologous recombination among the RF-Oma1 members played an important role to generate protein activity related to suppression. Together, our data illustrate how an evolutionarily young Rf has emerged from a lineage-specific paralog. Interestingly, several evolutionary features are shared with the RNA-binding protein type Rfs. Hence, the evolution of the sugar beet Rf is representative of Rf evolution in general.

The molecular basis for allelic differences suggests Restorer-of-fertility 1 is a complex locus in sugar beet (Beta vulgaris L.)
Takumi Arakawa, Muneyuki Matsunaga, Katsunori Matsui, Kanna Itoh, Yosuke Kuroda, Hiroaki Matsuhira, Kazuyoshi Kitazaki, Tomohiko Kubo
BMC Plant Biology, 20, 1, 503, 503, Springer Science and Business Media LLC, 2020年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）,


Cytoplasmic male sterility (CMS) is a widely used trait for hybrid seed production in many crops. Sugar beet CMS is associated with a unique mitochondrial protein named preSATP6 that forms a 250-kDa complex. Restorer-of-fertility 1 (Rf1) is a nuclear gene that suppresses CMS and is, hence, one of the targets of sugar beet breeding. Rf1 has dominant, semi-dominant and recessive alleles, suggesting that it may be a multi-allelic locus; however, the molecular basis for differences in genetic action is obscure. Molecular cloning of Rf1 revealed a gene (orf20) whose protein products produced in transgenics can bind with preSATP6 to generate a novel 200-kDa complex. The complex is also detected in fertility-restored anthers concomitant with a decrease in the amount of the 250-kDa complex. Molecular diversity of the Rf1 locus involves organizational diversity of a gene cluster composed of orf20-like genes (RF-Oma1s). We examined the possibility that members of the clustered RF-Oma1 in this locus could be associated with fertility restoration.





Six yet uncharacterized RF-Oma1s from dominant and recessive alleles were examined to determine whether they could generate the 200-kDa complex. Analyses of transgenic calli revealed that three RF-Oma1s from a dominant allele could generate the 200-kDa complex, suggesting that clustered RF-Oma1s in the dominant allele can participate in fertility restoration. None of the three copies from two recessive alleles was 200-kDa generative. The absence of this ability was confirmed by analyzing mitochondrial complexes in anthers of plants having these recessive alleles. Together with our previous data, we designed a set of PCR primers specific to the 200-kDa generative RF-Oma1s. The amount of mRNA measured by this primer set inversely correlated with the amount of the 250-kDa complex in anthers and positively correlated with the strength of the Rf1 alleles.





Fertility restoration by sugar beet Rf1 can involve multiple RF-Oma1s clustered in the locus, implying that stacking 200-kDa generative copies in the locus strengthens the efficacy, whereas the absence of 200-kDa generative copies in the locus makes the allele recessive irrespective of the copy number. We propose that sugar beet Rf1 is a complex locus.

How did a duplicated gene copy evolve into a restorer-of-fertility gene in a plant? The case of Oma1
Takumi Arakawa, Hajime Sugaya, Takaya Katsuyama, Yujiro Honma, Katsunori Matsui, Hiroaki Matsuhira, Yosuke Kuroda, Kazuyoshi Kitazaki, Tomohiko Kubo
Royal Society Open Science, 6, 11, 190853, 190853, The Royal Society, 2019年11月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Restorer-of-fertility
(
Rf
) is a suppressor of cytoplasmic male sterility (CMS), a mitochondrion-encoded trait that has been reported in many plant species. The occurrence of CMS is considered to be independent in each lineage; hence, the question of how
Rf
evolved was raised. Sugar beet
Rf
resembles
Oma1
, a gene for quality control of the mitochondrial inner membrane.
Oma1
homologues comprise a small gene family in the sugar beet genome, unlike Arabidopsis and other eukaryotes. The sugar beet sequence that best matched Arabidopsis
atOma1
was named
bvOma1
; sugar beet
Rf
(
RF1-Oma1
) was another member. During anther development,
atOma1
mRNA was detected from the tetrad to the microspore stages, whereas
bvOma1
mRNA was detected at the microspore stage and
RF1-Oma1
mRNA was detected during the meiosis and tetrad stages. A transgenic study revealed that, whereas
RF1-Oma1
can bind to a CMS-specific protein and alter the higher-order structure of the CMS-specific protein complex, neither
bvOma1
nor
atOma1
show such activity. We favour the hypothesis that an ancestral
Oma1
gene duplicated to form a small gene family, and that one of the copies evolved and acquired a novel expression pattern and protein function as an
Rf
, i.e.
RF1-Oma1
evolved via neofunctionalization.

Identification and characterization of a semi-dominant restorer-of-fertility 1 allele in sugar beet (Beta vulgaris)
Takumi Arakawa, Sachiyo Ue, Chihiro Sano, Muneyuki Matsunaga, Hiroyo Kagami, Yu Yoshida, Yosuke Kuroda, Kazunori Taguchi, Kazuyoshi Kitazaki, Tomohiko Kubo
Theoretical and Applied Genetics, 132, 1, 227, 240, Springer Science and Business Media LLC, 2019年01月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The sugar beet Rf1 locus has a number of molecular variants. We found that one of the molecular variants is a weak allele of a previously identified allele. Male sterility (MS) caused by nuclear-mitochondrial interaction is called cytoplasmic male sterility (CMS) in which MS-inducing mitochondria are suppressed by a nuclear gene, restorer-of-fertility. Rf and rf are the suppressing and non-suppressing alleles, respectively. This dichotomic view, however, seems somewhat unsatisfactory to explain the recently discovered molecular diversity of Rf loci. In the present study, we first identified sugar beet line NK-305 as a new source of Rf1. Our crossing experiment revealed that NK-305 Rf1 is likely a semi-dominant allele that restores partial fertility when heterozygous but full fertility when homozygous, whereas Rf1 from another sugar beet line appeared to be a dominant allele. Proper degeneration of anther tapetum is a prerequisite for pollen development; thus, we compared tapetal degeneration in the NK-305 Rf1 heterozygote and the homozygote. Degeneration occurred in both genotypes but to a lesser extent in the heterozygote, suggesting an association between NK-305 Rf1 dose and incompleteness of tapetal degeneration leading to partial fertility. Our protein analyses revealed a quantitative correlation between NK-305 Rf1 dose and a reduction in the accumulation of a 250 kDa mitochondrial protein complex consisting of a CMS-specific mitochondrial protein encoded by MS-inducing mitochondria. The abundance of Rf1 transcripts correlated with NK-305 Rf1 dose. The molecular organization of NK-305 Rf1 suggested that this allele evolved through intergenic recombination. We propose that the sugar beet Rf1 locus has a series of multiple alleles that differ in their ability to restore fertility and are reflective of the complexity of Rf evolution.

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
Takumi Arakawa, Daisuke Uchiyama, Takashi Ohgami, Ryo Ohgami, Tomoki Murata, Yujiro Honma, Hiroyuki Hamada, Yosuke Kuroda, Kazunori Taguchi, Kazuyoshi Kitazaki, Tomohiko Kubo
PLOS ONE, 13, 6, e0198409, e0198409, Public Library of Science (PLoS), 2018年06月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）

Efficient callus formation and plant regeneration are heritable characters in sugar beet (Beta vulgaris L.)
Kagami Hiroyo, Taguchi Kazunori, Arakawa Takumi, Kuroda Yosuke, Tamagake Hideto, Kubo Tomohiko
Hereditas, 153, 1, BioMed Central, 2016年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Obtaining dedifferentiated cells (callus) that can regenerate into whole plants is not always feasible for many plant species. Sugar beet is known to be recalcitrant for dedifferentiation and plant regeneration. These difficulties were major obstacles for obtaining transgenic sugar beets through an Agrobacterium-mediated transformation procedure. The sugar beet line 'NK-219mm-O' is an exceptional line that forms callus efficiently and is easy to regenerate, but the inheritance of these characters was unknown. Another concern was whether these characters could coexist with an annual habitat that makes it possible to breed short life-cycle sugar beet suitable for molecular genetic analysis. Findings: Five sugar beet lines including NK-219mm-O were crossed with each other and subjected to in vitro culture to form callus. F1s with a NK-219mm-O background generally formed callus efficiently compared to the others, indicating that efficient callus formation is heritable. The regeneration potential was examined based on the phenotypes of calli after placement on regeneration medium. Five phenotypes were observed, of which two phenotypes regenerated shoots or somatic embryo-like structures. Vascular differentiation was evident in regenerable calli, whereas non-regenerable calli lacked normally developed vascular tissues. In a half-diallel cross,the callus-formation efficiency and the regeneration potential of reciprocal F1s progeny having a NK-219mm-O background were high. Finally, we crossed NK-219mm-O with an annual line that had a poor in vitro performance. The callus-formation efficiency and the regeneration potential of reciprocal F1 were high. The regenerated plants showed an annual habitat. Conclusions: Efficient callus formation and the high plant regeneration potential of NK-219mm-O were inherited and expressed in the F1. The annual habitat does not impair these high in vitro performances.

Identification of molecular variants of the nonrestoring restorer-of-fertility 1 allele in sugar beet (Beta vulgaris L.)
Takashi Ohgami, Daisuke Uchiyama, Sachiyo Ue, Rika Yui-Kurino, Yu Yoshida, Yoko Kamei, Yosuke Kuroda, Kazunori Taguchi, Tomohiko Kubo
Theoretical and Applied Genetics, 129, 4, 675, 688, Springer Science and Business Media LLC, 2016年04月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Only three variants of nonrestoring alleles for sugar beet Rf1 were found from the US maintainer lines which were the selections from a broad range of genetic resources.Cytoplasmic male sterility is widely used for hybrid breeding of sugar beets. Specific genotypes with a nonsterility-inducing cytoplasm and a nonrestoring allele of restorer-of-fertility gene (rf) are called maintainers. The infrequent occurrence of the maintainer genotype evokes the need to diagnose rf alleles. Molecular analysis of Rf1, one of the sugar beet Rfs, revealed a high level of nucleotide sequence diversity, but three variants were tightly associated with maintainer selection in Japan. The question was raised whether this small number of variants would be seen in cases where a wider range of genetic resources was used for maintainer selection. Fifty-seven accessions registered as maintainers in the USDA germplasm collection were characterized in this study. Mitochondrial DNA types (mitotypes) of 551 plants were diagnosed based on minisatellite polymorphism. A mitotype associated with sterility-inducing (S) cytoplasm was identified in 58 plants, indicating S-cytoplasm contamination. The organization of rf1 was investigated by two PCR markers and DNA gel blot analysis. Eight haplotypes were found among the US maintainers, but subsequently two haplotypes were judged as restoring alleles after a test cross and another haplotype was not inherited by the progeny. Nucleotide sequences of rf1 regions in the remaining five haplotypes were compared, and despite the sequence diversity of the gene-flanking regions, the gene-coding regions were identified to be three types. Therefore, there are three rf1 variants in US maintainers, the same number as in the Japanese sugar beet germplasm collection. The implications of having a small repertoire of rf1 variants are discussed.

Two male sterility-inducing cytoplasms of beet (Beta vulgaris) are genetically distinct but have closely related mitochondrial genomes: implication of a substoichiometric mitochondrial DNA molecule in their evolution
Yasuyuki Onodera, Takumi Arakawa, Rika Yui-Kurino, Masayuki P. Yamamoto, Kazuyoshi Kitazaki, Shigehiko Ebe, Muneyuki Matsunaga, Kazunori Taguchi, Yosuke Kuroda, Shiko Yamashita, Tomoyuki Sakai, Toshiro Kinoshita, Tetsuo Mikami, Tomohiko Kubo
EUPHYTICA, 206, 2, 365, 379, SPRINGER, 2015年11月, ［査読有り］
英語, 研究論文（学術雑誌）, I-12CMS(2) and I-12CMS(3) are sugar beet lines with different sources of cytoplasmic male sterility (CMS) derived from wild beets in Turkey and Pakistan, respectively. We established that I-12CMS(2) has a genetically distinct cytoplasm, but its mitochondrial genome is very similar to I-12CMS(3). Male fertility was assessed in F-1 hybrids produced with a common pollen parent. Fertility in the F-1's carrying the I-12CMS(3) cytoplasm exceeded that of the F-1's with the I-12CMS(2) cytoplasm. Organization of the I-12CMS(2) and I-12CMS(3) mitochondrial genomes were compared based on their physical maps. Mitochondrial genomes of the two strains were largely collinear, except for a large deletion in the noncoding region of I-12CMS(2). Because a mitochondrial orf129 in the I-12CMS(3) cytoplasm is associated with a male sterility phenotype and preservation of orf129 was evident in I-12CMS(2), I-12CMS(2) orf129 was investigated in detail. I-12CMS(2) plants contained three to five times more ORF129 protein than did I-12CMS(3) plants. A single nucleotide substitution, present in the putative promoter region of orf129, appeared to be responsible for the differential accumulation of orf129 transcript. A long N-terminal extension of atp6 is a common feature of some beet CMSs and is found in I-12CMS(2), but the amino acid sequence is unique. I-12CMS(3) mitochondria, but not I-12CMS(2) mitochondria, were found to be heteroplasmic. This heteroplasmy is characterized by a substoichiometric DNA molecule(s) that has at least two I-12CMS(2)-type mitochondrial loci, suggesting the possibility that the I-12CMS(2) mitochondrial genome might have evolved from such a substoichiometric DNA molecule in I-12CMS(3) mitochondria.

Post-translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet (Beta vulgaris)
Kazuyoshi Kitazaki, Takumi Arakawa, Muneyuki Matsunaga, Rika Yui-Kurino, Hiroaki Matsuhira, Tetsuo Mikami, Tomohiko Kubo
PLANT JOURNAL, 83, 2, 290, 299, WILEY-BLACKWELL, 2015年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Genetic conflict between cytoplasmically inherited elements and nuclear genes arising from their different transmission patterns can be seen in cytoplasmic male sterility (CMS), the mitochondrion-encoded inability to shed functional pollen. CMS is associated with a mitochondrial open reading frame (ORF) that is absent from non-sterility inducing mitochondria (S-orf). Nuclear genes that suppress CMS are called restorer-of-fertility (Rf) genes. Post-transcriptional and translational repression of S-orf mediates the molecular action of Rf that encodes a class of RNA-binding proteins with pentatricopeptide repeat (PPR) motifs. Besides the PPR-type of Rfs, there are also non-PPR Rfs, but the molecular interactions between non-PPR Rf and S-orf have not been described. In this study, we investigated the interaction of bvORF20, a non-PPR Rf from sugar beet (Beta vulgaris), with preSatp6, the S-orf from sugar beet. Anthers expressing bvORF20 contained a protein that interacted with preSATP6 protein. Analysis of anthers and transgenic calli expressing a FLAG-tagged bvORF20 suggested the binding of preSATP6 to bvORF20. To see the effect of bvORF20 on preSATP6, which exists as a 250-kDa protein complex in CMS plants, signal bands of preSATP6 in bvORF20-expressing and non-expressing anthers were compared by immunoblotting combined with Blue Native polyacrylamide gel electrophoresis. The signal intensity of the 250-kDa band decreased significantly, and 200- and 150-kDa bands appeared in bvORF20-expressing anthers. Transgenic callus expressing bvORF20 also generated the 200- and 150-kDa bands. The 200-kDa complex is likely to include both preSATP6 and bvORF20. Post-translational interaction between preSATP6 and bvORF20 appears to alter the higher order structure of preSATP6 that may lead to fertility restoration in sugar beet.

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
Yujiro Honma, Kazunori Taguchi, Hajime Hiyama, Rika Yui-Kurino, Tetsuo Mikami, Tomohiko Kubo
THEORETICAL AND APPLIED GENETICS, 127, 12, 2567, 2574, SPRINGER, 2014年12月, ［査読有り］
英語, 研究論文（学術雑誌）, By genetically eliminating the major restorer - of - fertility gene ( Rf ), a weak Rf gene was unveiled. It is an allele of Z , long known as an elusive Rf gene in sugar beet.
In the hybrid breeding of sugar beet, maintainer-genotype selection is a laborious process because of the dependence on test crossing, despite the very low occurrence of this genotype. Marker-assisted selection (MAS) of the maintainer genotype is highly desired by sugar beet breeders. The major restorer-of-fertility gene (Rf) was identified as Rf1, and its non-restoring allele (rf1) was discriminated at the DNA level; however, some of the rf1rf1 selections retained an as yet unidentified Rf, another target locus for MAS. The objective of this study was to identify this Rf. An rfrf1 plant was crossed to a cytoplasmic male-sterile sugar beet and then backcrossed to obtain progeny segregating the unidentified Rf. The progeny exhibited partial male-fertility restoration that was unstable in single plants. The segregation ratio of restored vs. non-restored plants suggested the involvement of a single Rf in this male-fertility restoration, designated as Rf2. We confirmed the feasibility of molecular tagging of Rf2 by identifying four shared amplified fragment length polymorphism (AFLP) fragments specific to 17 restored plants. Bulked segregant analysis also was performed to screen the Rf2-linked AFLP markers, which were subsequently converted into 17 sequence-tagged site markers. All the markers, as well two additional chromosome-IV-assigned markers, were linked to each other to form a single linkage map, on which Rf2 was located. Our data suggested that Rf2 is likely an allele of Z, long known as an elusive Rf gene in sugar beet. We also discuss the importance of Rf2 for sugar beet breeding.

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.)
Kazunori Taguchi, Hajime Hiyama, Rika Yui-Kurino, Aki Muramatsu, Tetsuo Mikami, Tomohiko Kubo
CROP SCIENCE, 54, 4, 1407, 1412, CROP SCIENCE SOC AMER, 2014年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Hybrid breeding of crops may involve the selection of reproductive traits, such as cytoplasmic male sterility (CMS), whose expression is controlled by cytoplasm and nuclear gene(s). Intense selection of a single cytoplasm and the consequent lack of cytoplasmic divergence is a potential danger, the so-called genetic vulnerability. However, little is known about the relationship between hybrid breeding and the diversity of nuclear genes that suppress the expression of CMS, termed restorer of fertility (Rf). Despite the multi-allelic nature of Rf at the molecular level, a common Rf variant was previously found to predominate in sugar beet maintainer lines that were selected for a specific genotype for propagating the CMS line. The question was raised as to the frequency of the common Rf variant before the hybrid breeding era. As the origin of Japanese maintainer lines can be genealogically traced back to seven nonhybrid cultivars, we investigated the allelic diversity of Rf in the seven cultivars using molecular markers. Our results indicated that Rf diversity differs among the cultivars but exceeds that of the maintainers in total, and the common variant in the maintainers is infrequent in all of the cultivars. Therefore, maintainer selection has involved selecting a small number of Rf variants in the founder population in Japan.

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
Mari Moritani, Kazunori Taguchi, Kazuyoshi Kitazaki, Hiroaki Matsuhira, Takaya Katsuyama, Tetsuo Mikami, Tomohiko Kubo
MOLECULAR BREEDING, 32, 1, 91, 100, SPRINGER, 2013年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Hybrid seed production in sugar beet relies on cytoplasmic male sterility (CMS). As time-consuming and laborious test crosses with a CMS tester are necessary to identify maintainer lines, development of a marker-assisted selection method for the rf gene (the nonrestoring allele of restorer-of-fertility locus) is highly desirable for sugar-beet breeding. To develop such a method, we investigated genetic variation at the Rf1 locus, one of two Rf loci known in sugar beet. After HindIII-digestion, genomic DNAs from beet plants known to have a restoring Rf1 allele yielded a range of hybridization patterns on agarose gels, indicating that Rf1 is a multi-allelic locus. However, the hybridization patterns of 22 of 23 maintainer lines were indistinguishable. The nucleotide sequences of the rf1 coding regions of these 22 maintainer lines were found to be identical, confirming that the lines had the same rf1 allele. Two PCR markers were developed that targeted a downstream intergenic sequence and an intron of Rf1. The electrophoretic patterns of both markers indicated multiple Rf1 alleles, one of which, named the dd(L) type, was associated with the maintainer genotype. To test the validity of marker-assisted selection, 147 sugar beet plants were genotyped using these markers. Additionally, the 147 sugar beet plants were crossed with CMS plants to determine whether they possessed the maintainer genotype. Analysis of 5038 F1 offspring showed that 53 % of the dd(L) plants, but none of the plants with other alleles, had the maintainer genotype. Thus, selection for the dd(L) type considerably enriched the proportion of plants with the maintainer genotype.

Evolutionary aspects of a unique internal mitochondrial targeting signal in nuclear-migrated rps19 of sugar beet (Beta vulgaris L.)
Muneyuki Matsunaga, Yoshiya Takahashi, Rika Yui-Kurino, Tetsuo Mikami, Tomohiko Kubo
GENE, 517, 1, 19, 26, ELSEVIER SCIENCE BV, 2013年03月, ［査読有り］
英語, 研究論文（学術雑誌）, The endosymbiotic theory postulates that many genes migrated from endosymbionts to the nuclear genomes of their hosts. Some migrated genes lack presequences directing proteins to mitochondria, and their mitochondrial targeting signals appear to be inscribed in the core coding regions as internal targeting signals (ITSs). ITSs may have evolved after sequence transfer to nuclei or ITSs may have pre-existed before sequence transfer. Here, we report the molecular cloning of a sugar beet gene for ribosomal protein S19 (Rps19; the first letter is capitalized when the gene is a nuclear gene). We show that sugar beet Rps19 (BvRps19) is an ITS-type gene. Based on amino-acid sequence comparison, dicotyledonous rps19s (the first letter is lower-cased when the gene is a mitochondrial gene), such as tobacco rps19 (Ntrps19), resemble an ancestral form of BvRps19. We investigated whether differences in amino-acid sequences between BvRps19 and Ntrps19 were involved in ITS evolution. Analyses of the intracellular localization of chimaeric GFP-fusion proteins that were transiently expressed in Welsh onion cells showed that Ntrps19-gfp was not localized in mitochondria. When several BvRps19-type amino acid substitutions, none of which was seen in any other angiosperm rps19, were introduced into Ntrps19-gfp, the modified Ntrps19-gfp became localized in mitochondria, supporting the notion that an ITS in BvRps19 evolved following sequence transfer to nuclei. Not all of these substitutions were seen in other ITS-type Rps19s, suggesting that the ITSs of Rps19 are diverse. (c) 2013 Elsevier B.V. All rights reserved.

Unusual and Typical Features of a Novel Restorer-of-Fertility Gene of Sugar Beet (Beta vulgaris L.)
Hiroaki Matsuhira, Hiroyo Kagami, Masayuki Kurata, Kazuyoshi Kitazaki, Muneyuki Matsunaga, Yuko Hamaguchi, Eiki Hagihara, Minoru Ueda, Michiyo Harada, Aki Muramatsu, Rika Yui-Kurino, Kazunori Taguchi, Hideto Tamagake, Tetsuo Mikami, Tomohiko Kubo
GENETICS, 192, 4, 1347, +, GENETICS SOC AM, 2012年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.

Mitochondrial minisatellite polymorphisms in fodder and sugar beets reveal genetic bottlenecks associated with domestication
Y. Yoshida, M. Matsunaga, D. Cheng, D. Xu, Y. Honma, T. Mikami, T. Kubo
BIOLOGIA PLANTARUM, 56, 2, 369, 372, SPRINGER, 2012年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Historically, sugar beets were selected from fodder beets. We used mitochondrial minisatellite loci to analyze cytoplasmic genetic diversity in fodder beet and sugar beet. Among the 8 sugar beet accessions examined we identified 3 multi-locus haplotypes. These 3 haplotypes were a subset of 5 haplotypes identified among the 29 fodder beet accessions examined. All but one haplotype in fodder beet comprised, in turn, a subset of 12 haplotypes identified previously in leaf beets. Such apparent decreases in cytoplasmic genetic diversity must result from genetic bottlenecks associated with domestication and the ensuing breeding processes. We also detected the haplotype associated with the male-sterile Owen cytoplasm of sugar beet in the fodder beet gene pool. Furthermore, the presence of a 39 kDa protein associated with the Owen cytoplasm was confirmed in two fodder beet plants by Western blot analysis. These results lead us to speculate that the Owen cytoplasm may have originated in fodder beet, from which sugar beet was derived.

Is RNA editing implicated in group II intron survival in the angiosperm mitochondrial genome?
Hiroyo Kagami, Hironori Nagano, Yoshiya Takahashi, Tetsuo Mikami, Tomohiko Kubo
GENOME, 55, 1, 75, 79, CANADIAN SCIENCE PUBLISHING, NRC RESEARCH PRESS, 2012年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Introns may be considered as optional because they are removed from mRNA molecules, but introns are fairly preserved for unknown reasons. Previously, the mitochondrial rps3 gene of sugar beet (Beta vulgaris L., Caryophyllales) was shown to represent a unique example of an intron loss. We have determined the distribution of the rps3 intron in 19 Caryophyllalean species. The intron was absent from the Amaranthaceae and the Achatocarpaceae. In the Caryophyllaceae, Dianthus japonicus rps3 was pseudogenized, but the intronic sequence was retained. Intact intron-bearing rps3 copies were cloned from Portulaca grandiflora and Myrtillocactus geometrizans, members of the sister clade of the Amaranthaceae Achatocarpaceae-Caryophyllaceae clade. Most of the C-to-U RNA-editing sites in P. grandiflora and M. geometrizans rps3 transcripts were homologous in the two species, as well as in the sugar beet rps3, which, unlike the other 12 rps3 transcripts, lacks editing in the exonic regions around the intron. Provided that the loss of editing preceded the loss of rps3 intron, it appears conceivable that a requirement for editing could have prevented the loss of group II introns retained in angiosperm mitochondrial genomes. This interpretation is an alternative to the conventional one that views the loss of editing as a mere trace of RNA-mediated gene conversion.

A horizontally transferred tRNA(Cys) gene in the sugar beet mitochondrial genome: evidence that the gene is present in diverse angiosperms and its transcript is aminoacylated
Kazuyoshi Kitazaki, Tomohiko Kubo, Hiroyo Kagami, Takuma Matsumoto, Asami Fujita, Hiroaki Matsuhira, Muneyuki Matsunaga, Tetsuo Mikami
PLANT JOURNAL, 68, 2, 262, 272, WILEY-BLACKWELL, 2011年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Of the two tRNA(Cys) (GCA) genes, trnC1-GCA and trnC2-GCA, previously identified in mitochondrial genome of sugar beet, the former is a native gene and probably a pseudo-copy, whereas the latter, of unknown origin, is transcribed into a tRNA [tRNA(Cys2)(GCA)]. In this study, the trnC2-GCA sequence was mined from various public databases. To evaluate whether or not the trnC2-GCA sequence is located in the mitochondrial genome, the relative copy number of its sequence to nuclear gene was assessed in a number of angiosperm species, using a quantitative real-time PCR assay. The trnC2-GCA sequence was found to exist sporadically in the mitochondrial genomes of a wide range of angiosperms. The mitochondrial tRNA(Cys2)(GCA) species from sugar beet (Beta vulgaris), spinach (Spinacea oleracea) and cucumber (Cucumis sativus) were found to be aminoacylated, indicating that they may participate in translation. We also identified a sugar beet nuclear gene that encodes cysteinyl-tRNA synthetase, which is dual-targeted to mitochondria and plastids, and may aminoacylate tRNA(Cys2)(GCA). What is of particular interest is that trnC1-GCA and trnC2-GCA co-exist in the mitochondrial genomes of eight diverse angiosperms, including spinach, and that the spinach tRNA(Cys1)(GCA) is also aminoacylated. Taken together, our observations lead us to surmise that trnC2-GCAmayhave been horizontally transferred to a common ancestor of eudicots, followed by co-existence and dual expression of trnC1-GCA and trnC2-GCA in mitochondria with occasional loss or inactivation of either trnC-GCA gene during evolution.

Identification and Precise Mapping of Resistant QTLs of Cercospora Leaf Spot Resistance in Sugar Beet (Beta vulgaris L.)
Kazunori Taguchi, Tomohiko Kubo, Hiroyuki Takahashi, Hideyuki Abe
G3-GENES GENOMES GENETICS, 1, 4, 283, 291, GENETICS SOC AM, 2011年09月, ［査読有り］
英語, 研究論文（学術雑誌）, The complex inheritance of resistance to Cercospora leaf spot (CLS), the most severe fungal foliar disease in sugar beet, was investigated by means of quantitative trait loci (QTL) analysis. Over a three year period, recombinant inbred lines (RILs) of sugar beet (Beta vulgaris L.), generated through a cross between lines resistant ('NK-310mm-O') and susceptible ('NK-184mm-O') to CLS, were field-tested for their resistance to the pathogen. Composite interval mapping (CIM) showed four QTL involved in CLS resistance to be consistently detected. Two resistant QTL (qcr1 on chromosome III, qcr4 on chromosome IX) bearing 'NK-310mm-O' derived alleles promoted resistance. Across 11 investigations, the qcr1 and qcr4 QTL explained approximately 10% and over 20%, respectively, of the variance in the resistance index. Two further QTL (qcr2 on chromosome IV, qcr3 on chromosome VI) bearing 'NK-184mm-O' derived alleles each explained about 10% of the variance. To identify the monogenic effect of the resistance, two QTL derived from 'NK-310mm-O' against the genetic background of 'NK-184mm-O', using molecular markers. The qcr1 and qcr4 were precisely mapped as single QTL, using progenies BC5F1 and BC2F1, respectively. The qcr1 that was located near e11m36-8 had CLS disease severity indices (DSI) about 15% lower than plants homozygous for the 'NK-184mm-O' genotype. As with qcr1, heterozygosis of the qcr4 that was located near e17m47-81 reduced DSI by about 45% compared to homozygosis. These two resistant QTL might be of particular value in marker-assisted selection (MAS) programs in CLS resistance progression.

Polymorphic minisatellites in the mitochondrial DNAs of Oryza and Brassica
Yujiro Honma, Yu Yoshida, Toru Terachi, Kinya Toriyama, Tetsuo Mikami, Tomohiko Kubo
CURRENT GENETICS, 57, 4, 261, 270, SPRINGER, 2011年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Polymorphic analyses of angiosperm mitochondrial DNA are rare in comparison with chloroplast DNA, because few target sequences in angiosperm mitochondrial DNA are known. Minisatellites, a tandem array of repeated sequences with a repeat unit of 10 to similar to 100 bp, are popular target sequences of animal mitochondria, but Beta vulgaris is the only known angiosperm species for which such an analysis has been conducted. From this lack of information, it was uncertain as to whether polymorphic minisatellites existed in other angiosperm species. Ten plant mitochondrial DNAs were found to contain minisatellite-like repeated sequences, most of which were located in intergenic regions but a few occurred in gene coding and intronic regions. Oryza and Brassica accessions were selected as models for the investigation of minisatellite polymorphism because substantial systematic information existed. PCR analysis of 42 Oryza accessions revealed length polymorphisms in four of the five minisatellites. The mitochondrial haplotypes of the 16 Oryza accessions with chromosomal complement (genome) types of CC, BBCC and CCDD were identical but were clearly distinguished from BB-genome accessions, a result consistent with the notion that the cytoplasmic donor parent of the amphidiploid species might be the CC-genome species. Twenty-nine accessions of six major cultivated species of Brassica were classified into five mitochondrial haplotypes based on two polymorphic minisatellites out of six loci. The haplotypes of Brassica juncea and Brassica carinata accessions were identical to Brassica rapa and Brassica nigra accessions, respectively. The haplotypes of Brassica napus accessions were heterogeneous and unique, results that were consistent with previous studies.

Mitochondrial genome diversity in Beta vulgaris L. ssp. vulgaris (Leaf and Garden Beet Groups) and its implications concerning the dissemination of the crop
Dayou Cheng, Yu Yoshida, Kazuyoshi Kitazaki, Shinya Negoro, Hiroyuki Takahashi, Dechang Xu, Tetsuo Mikami, Tomohiko Kubo
GENETIC RESOURCES AND CROP EVOLUTION, 58, 4, 553, 560, SPRINGER, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Four mitochondrial minisatellites were used to study cytoplasmic diversity in leaf and garden beet germplasm resources. Eleven multi-locus haplotypes were identified, of which one (named mitochondrial minisatellite haplotype 4, hereafter min04) was associated with male-sterile Owen cytoplasm and two others (min09 and min18), with a normal fertile cytoplasm. European leaf beet germplasm exhibited the greatest haplotype diversity, with min09 and min18 predominating. In North African leaf beet accessions, only these two haplotypes were observed, making it likely that North African accessions were descended from European genotypes. The prevalence of min18 was also noted in leaf beet from the Middle East and western Asia. Such a pattern contrasts with that found in east Asian leaf beet where the two haplotypes were extremely rare. The geographical structure of the mitochondrial haplotypes allowed us to infer possible dissemination pathways of leaf beet. Additionally, we showed that mitochondrial genome diversity was low in garden beet germplasm, with min18 being highly predominant. An explanation of this limited diversity may lie in the geographically restricted origin of as well as relatively short cultivation histories of garden beet.

Large 3' UTR of sugar beet rps3 is truncated in cytoplasmic male-sterile mitochondria
Muneyuki Matsunaga, Hironori Nagano, Tetsuo Mikami, Tomohiko Kubo
PLANT CELL REPORTS, 30, 2, 231, 238, SPRINGER, 2011年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Genomic alteration near or within mitochondrial gene is often associated with cytoplasmic male sterility (CMS). Its influence on the expression of the mitochondrial gene was proposed as one of the possible causes of CMS. In sugar beet mitochondrial rps3, whose downstream 1,056-bp region contains Norf246, an apparently non-functional open reading frame (ORF), was deleted in CMS mitochondria. In our previous study, normal rps3 (3.8 kb), CMS rps3 (2.7 kb), and Norf246 (3.8 and 0.9 kb) were shown to be transcribed. The present study was conducted to determine whether the deletion affected gene expression. Reverse transcription (RT)-PCR analysis revealed the co-transcription of rps3 and Norf246. By circularized RNA (CR) RT-PCR analysis, the 5' and 3' termini of the 3.8- and the 0.9-kb transcripts were determined. The results suggested that the 3.8-kb transcripts were the rps3 mRNA bearing similar to 464-base 5' untranslated region (UTR) and similar to 1,508-base 3' UTR, whereas no functional ORF was observed in the 0.9-kb transcripts. CR-RT-PCR revealed that the 3' UTR of the 2.7-kb transcripts was reduced to similar to 460 bases. However, no difference in the accumulation of RPS3 polypeptide and RNA editing was detected by protein gel blot analysis and cDNA sequencing. Although the deleted region encoded the truncated-atp9 that was edited, no influence on the pattern and frequency of RNA editing of genuine atp9 was evident. The results eliminated rps3 as a candidate for the CMS gene, making preSatp6, a unique ORF fused with CMS atp6, the sole CMS-associated region in sugar beet.

Increased accumulation of intron-containing transcripts in rice mitochondria caused by low temperature: is cold-sensitive RNA editing implicated?
Shiho Kurihara-Yonemoto, Tomohiko Kubo
CURRENT GENETICS, 56, 6, 529, 541, SPRINGER, 2010年12月, ［査読有り］
英語, 研究論文（学術雑誌）, An increase in the unspliced cox2 transcript and accompanying decrease in the frequency of RNA editing near the exon/intron junction (intron binding site 1, IBS1) have been reported in cold-treated wheat. Here, an attempt was made to clarify whether a similar phenomenon occurs in rice. Levels of unspliced cox2 transcript increased and its editing at the IBS was abolished after cold treatment. The accumulation of COXII protein remained unaffected. The accumulation of intron-containing transcripts of another eight mitochondrial genes, 23 introns in total, was analyzed by Northern blotting and semi-quantitative RT-PCR. An increase in 14 of the 23 intron-adjoining cDNA after cold treatment was observed. Six RNA editing sites in the IBS of four genes were tested as to their status by sequencing cDNA. One of these sites in the nad7 transcript showed a close association with splicing, with editing and splicing occurring simultaneously, irrespective of cold treatment. Two other sites in the intron-containing cox2 and rps3 transcripts were sensitive to cold, where editing frequency began to decrease 1 day after cold treatment, and finally exhibited a tight association with splicing 14 days later. The other sites were efficiently edited. The intron-spliced transcripts were fully edited at all six sites.

Molecular mapping of a gene conferring resistance to Aphanomyces root rot (black root) in sugar beet (Beta vulgaris L.)
Kazunori Taguchi, Kazuyuki Okazaki, Hiroyuki Takahashi, Tomohiko Kubo, Tetsuo Mikami
EUPHYTICA, 173, 3, 409, 418, SPRINGER, 2010年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Caused by Aphanomyces cochlioides Drechsler, Aphanomyces root rot is a serious disease of sugar beet (Beta vulgaris L.), for which sources of resistance are scarce. To identify the segregation pattern of the rare resistance trait found in Japanese sugar beet line 'NK-310mm-O', F(1) and BC(1)F(2) seedings, drawn from a cross between 'NK-310mm-O' and susceptible line 'NK-184mm-O', were inoculated with zoospores and their survival evaluated in the greenhouse. Resistance segregation followed was that of a single dominant gene, which was designated Acr1 (Aphanomyces cochlioides resistance 1). Molecular markers tightly linked to Acr1 were identified by bulked segregant analysis of two BC(1)F(2) populations. Fourteen AFLP markers linked to Acr1 were identified, the closest located within +/- 3.3 cM. Three F(5) lines and two BC(2)F(1) lines, selected on the basis of their Acr1-AFLP markers, were tested for their resistance to Aphanomyces root rot in a highly infested field. Results indicated that Acr1 conferred significant resistance to Aphanomyces root rot at the field level. Based on its linkage with CAPS marker tk, a representative marker for chromosome III, Acr1 was located on this chromosome. The clear linkage between tk and Rhizomania resistance trait Rz1, suggests the clustering of major disease resistance genes on chromosome III.

A new source of cytoplasmic male sterility found in wild beet and its relationship to other CMS types
Yuki Kawanishi, Hiroshi Shinada, Muneyuki Matsunaga, Yusuke Masaki, Tetsuo Mikami, Tomohiko Kubo
GENOME, 53, 4, 251, 256, NATL RESEARCH COUNCIL CANADA-N R C RESEARCH PRESS, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）, We found a number of male-sterile plants in a wild beet (Beta vulgaris L. subsp. maritima) accession line, FR4-31. The inheritance study of the male sterility indicated the trait to be of the cytoplasmic type. The mitochondrial genome of FR4-31 proved to lack the male-sterility-associated genes preSatp6 and orf129, which are characteristic of the Owen CMS and 1-12CMS(3) cytoplasms of beets, respectively. Instead, the truncated cox2 gene involved in G CMS originating from wild beets was present in the FR4-31 mitochondrial genome. In Southern hybridization using four mitochondrial gene probes, the FR4-31 cytoplasm showed patterns similar to those typical of the G cytoplasm. It is thus likely that the FR4-31 cytoplasm has a different CMS mechanism from both Owen CMS and I-12CMS(3), and that the FR4-31 and G cytoplasms resemble each other closely. A restriction map of the FR4-31 mitochondrial DNA was generated and aligned with those published for the Owen and normal fertile cytoplasms. The FR4-31 mitochondrial genome was revealed to differ extensively in arrangement from the Owen and normal genomes, and the male-sterile Owen and FR4-31 genomes seem to be derived independently from an ancestral genome.

The distribution of normal and male-sterile cytoplasms in Chinese sugar-beet germplasm
Dayou Cheng, Kazuyoshi Kitazaki, Dechang Xu, Tetsuo Mikami, Tomohiko Kubo
EUPHYTICA, 165, 2, 345, 351, SPRINGER, 2009年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Forty-two Chinese sugar-beet breeding lines were evaluated for the presence of normal and male-sterile (Owen) cytoplasms using polymorphisms in the chloroplast petG-psbE region as well as in the mitochondrial minisatellite loci. The polymorphisms detected allowed the distinction of three cytoplasm types over the whole sample, one being associated with Owen cytoplasm, a second with the maintainer inbred 'TK-81mm-O'-type cytoplasm (termed normal-1 cytoplasm) and a third with another maintainer inbred 'NK-310mm-O'-type cytoplasm (normal-2 cytoplasm). Western blot analysis was carried out to confirm that expression of the male-sterility-associated protein (preSATP6) occurred in plants with Owen cytoplasm but not in plants with either normal-1 or normal-2 cytoplasm. Of the 42 breeding lines examined, 14 had exclusively normal (normal-1 and/or normal- 2) cytoplasm and 11 had only Owen cytoplasm. The remaining 17 lines possessed both normal and Owen cytoplasms, and noticeably, some of these 17 lines have been expected to become the source of superior maintainer lines. The results thus show that molecular identification of the cytoplasm is required to avoid wasting resources on account of attempting to develop the maintainer genotype from plants with Owen cytoplasm.

Quantitative trait locus responsible for resistance to Aphanomyces root rot (black root) caused by Aphanomyces cochlioides Drechs. in sugar beet
Kazunori Taguchi, Naoki Ogata, Tomohiko Kubo, Shinji Kawasaki, Tetsuo Mikami
THEORETICAL AND APPLIED GENETICS, 118, 2, 227, 234, SPRINGER, 2009年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Aphanomyces root rot, caused by Aphanomyces cochlioides Drechs., is one of the most serious diseases of sugar beet ( Beta vulgaris L.). Identification and characterization of resistance genes is a major task in sugar beet breeding. To ensure the effectiveness of marker-assisted screening for Aphanomyces root rot resistance, genetic analysis of mature plants' phenotypic and molecular markers' segregation was carried out. At a highly infested Weld site, some 187 F(2) and 66 F(3) individuals, derived from a cross between lines 'NK-310mm-O' ( highly resistant) and 'NK-184mm-O' ( susceptible), were tested, over two seasons, for their level of resistance to Aphanomyces root rot. This resistance was classified into six categories according to the extent and intensity of whole plant symptoms. Simultaneously, two selected RAPD and 159 'NK-310mm-O' coupled AFLP were used in the construction of a linkage map of 695.7 cM. Each of nine resultant linkage groups was successfully anchored to one of nine sugar beet chromosomes by incorporating 16 STS markers. Combining data for phenotype and molecular marker segregation, a single QTL was identified on chromosome III. This QTL explained 20% of the variance in F2 population ( in the year 2002) and 65% in F3 lines ( 2003), indicating that this QTL plays a major role in the Aphanomyces root rot resistance. This is the first report of the genetic mapping of resistance to Aphanomyces-caused diseases in sugar beet.

A mitochondrial gene involved in cytochrome c maturation (ccmC) is expressed as a precursor with a long NH2-terminal extension in sugar beet
Kazuyoshi Kitazaki, Yuta Nomoto, Akihiro Aoshima, Tetsuo Mikami, Tomohiko Kubo
JOURNAL OF PLANT PHYSIOLOGY, 166, 7, 775, 780, ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2009年, ［査読有り］
英語, 研究論文（学術雑誌）, Extensive genome rearrangement is one of the major mechanisms of angiosperm mitochondrial evolution. As a by-product, some angiosperm mitochondrial genes exhibit divergent organization, but not all of these genes have been fully characterized. Sugar beet ccmC, which plays an important rote in cytochrome c maturation, harbors a unique extended NH2 terminal region of 277 amino acid residues (N-extension) instead of a conserved translational initiation codon. The 5' termini of two major RNA species were determined by primer extension analysis, which revealed that the larger transcript covered the entire N-extension. Nucleotide sequencing of the cDNA revealed that a total of 31 C-to-U RNA editing events occurred in the N-extension and the ccmC-homologous region (ccmC-core region), resulting in improvement of amino acid sequence conservation. Antiserum was raised against a synthetic peptide corresponding to the ccmC-core region and was used for protein get blot analysis of sugar beet and radish mitochondrial proteins. The detected 29.5-kDa signal band is shared by sugar beet and radish. Two additional larger signal bands are exclusively detected from sugar beet. The largest signal band is also detected by anti-N-extension antiserum. Our results indicate that sugar beet ccmC is translated as a long precursor with N-extension. (C) 2008 Elsevier GmbH. All rights reserved.

A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants
Masayuki P. Yamamoto, Hiroshi Shinada, Yasuyuki Onodera, Chihiro Komaki, Tetsuo Mikami, Tomohiko Kubo
PLANT JOURNAL, 54, 6, 1027, 1036, WILEY-BLACKWELL, 2008年06月, ［査読有り］
英語, 研究論文（学術雑誌）, In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST-ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.

Mitochondrial DNA phylogeny of cultivated and wild beets: Relationships among cytoplasmic male-sterility-inducing and nonsterilizing cytoplasms
Satsuki Nishizawa, Tetsuo Mikami, Tomohiko Kubo
GENETICS, 177, 3, 1703, 1712, GENETICS, 2007年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Cytoplasmic male sterility (CMS), the maternally inherited failure to produce functional pollen, has been used in the breeding of sugar beet (Beta vulgaris ssp. vulgaris). At least three different sources of CMS can be distinguished from one another as well as from normal fertile cytoplasm by polymorphisms in their mitochondrial genomes. Here we analyzed 50 accessions of cultivated and wild beets to investigate the phylogenetic relationships among male-sterility-inducing and normal cytoplasms. The haplotypes were characterized by the nucleotide sequence of the mitochondrial cox2-cox1 spacer region and mitochondrial minisatellite loci. The results indicated that (1) a normal cytoplasm line, cv. TK81-O, was situated at the major core node of the haplotype network, and (2) the three sterilizing cytoplasms in question derived independently from the core haplotype. The evolutionary pathway was investigated by physical mapping study of the mitochondrial genome of a wild beet (B. vulgaris ssp. orientalis) accession BGRC56777 which shared the same mitochondrial haplotype with TK81-O, but was not identical to TK81-O for the RFLP profiles of mitochondrial DNA. Interestingly, three sets of inverted repeated sequences appeared to have been involved in a series of recombination events during the course of evolution between the BGRC56777 and the TK81-O mitochondrial genomes.

An anther-specific lipid transfer protein gene in sugar beet: its expression is strongly reduced in male-sterile plants with Owen cytoplasm
Hiroaki Matsuhira, Hiroshi Shinada, Rika Yui-Kurino, Naonori Hamato, Mitsuhiro Umeda, Tetsuo Mikami, Tomohiko Kubo
PHYSIOLOGIA PLANTARUM, 129, 2, 407, 414, BLACKWELL PUBLISHING, 2007年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Differential screening of a sugar beet (normal cytoplasm line TK81-O) cDNA library made with anther tissues of various stages resulted in the isolation of a clone (#74-29) that hybridized to flower bud RNA but did not hybridize to RNA of vegetative organs. The clone contained an open reading frame (ORF) (designated bvLTP-1) that encoded a putative lipid transfer protein. We also identified a second copy (bvLTP-2) of the gene. In situ hybridization analysis demonstrated that expression of bvLTP-1 was confined to the tapetal cells of the anthers at the young microspore stage. Flower bud RNA was prepared from male-sterile sugar beet with Owen cytoplasm and fertility-restored plants and used for northern hybridization with the bvLTP-1 probe. Interestingly, bvLTP-1 was found to be expressed in the flower buds from the restored plants producing 30% or more stainable pollen, but not in the flower buds from completely sterile or poorly fertility-restored plants. These results lead us to suppose that the expression of bvLTP-1 is strongly reduced in the tapetum in response to mitochondrial dysfunction and subsequent physiological changes caused by the Owen cytoplasm.

The Owen mitochondrial genome in sugar beet (Beta vulgaris L.): possible mechanisms of extensive rearrangements and the origin of the mitotype-unique regions
Mizuho Satoh, Tomohiko Kubo, Tetsuo Mikami
THEORETICAL AND APPLIED GENETICS, 113, 3, 477, 484, SPRINGER, 2006年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The mitochondrial genomes of normal fertile and male-sterile (Owen CMS) cytoplasms of sugar beet are highly rearranged relative to each other and dozens of inversional recombinations and other reshuffling events must be postulated to interconvert the two genomes. In this paper, a comparative analysis of the entire nucleotide sequences of the two genomes revealed that most of the inversional recombinations involved short repeats present at their endpoints. Attention was also focused on the origin of the Owen CMS-unique mtDNA regions, which occupy 13.6% of the Owen genome and are absent from the normal mtDNA. BLAST search was performed to assign the sequences, and as a result, 7.6% of the unique regions showed significant homology to previously determined mitochondrial sequences, 17.9% to nuclear DNA, 4.6% to mitochondrial episomes, and 0.1% to plastid DNA. Southern blot analysis revealed that additional sequences of nuclear origin may be included within the unique regions. We also found that the copies of many short repeat families are scattered throughout the unique regions. This suggests that, in addition to the incorporation of foreign DNAs, extensive duplication of short repetitive sequences and continued scrambling of mtDNA sequences may be implicated in the generation of the Owen CMS-unique regions.

Sugar beet BAC library construction and assembly of a contig spanning Rf1, a restorer-of-fertility gene for Owen cytoplasmic male sterility
E Hagihara, H Matsuhira, M Ueda, T Mikami, T Kubo
MOLECULAR GENETICS AND GENOMICS, 274, 3, 316, 323, SPRINGER, 2005年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Rf1 is a nuclear gene that controls fertility restoration in cases of cytoplasmic male sterility caused by the Owen cytoplasm in sugar beet. In order to isolate the gene by positional cloning, a BAC library was constructed from a restorer line, NK198, with the genotype Rf1Rf1. The library contained 32,180 clones with an average insert size of 97.8 kb, providing 3.4 genome equivalents. Five AFLP markers closely linked to Rf1 were used to screen the library. As a result, we identified eight different BAC clones that were clustered into two contigs. The gap between the two contigs was filled by chromosome walking. To map the Rf1 region in more detail, we developed five cleaved amplified polymorphic sequence (CAPS) markers from the BAC DNAs identified, and carried out genotyping of 509 plants in the mapping population with the Rf1-flanking AFLP and CAPS markers. Thirteen plants in which recombination events had occurred in the vicinity of the Rf1 locus were identified and used to map the molecular markers relative to each other and to Rf1. In this way, we were able to restrict the possible location of the Rf1 gene to a minimum of six BAC clones spanning an interval of approximately 250 kb.

Molecular mapping of a fertility restorer gene for Owen cytoplasmic male sterility in sugar beet
E Hagihara, N Itchoda, Y Habu, S Iida, T Mikami, T Kubo
THEORETICAL AND APPLIED GENETICS, 111, 2, 250, 255, SPRINGER, 2005年07月, ［査読有り］
英語, 研究論文（学術雑誌）, We report here the molecular mapping of a fertility restorer gene (named Rf1) for Owen cytoplasmic male sterility in sugar beet. Eight AFLP and two RAPD markers, tightly linked to the Rf1 locus, were identified using bulked segregant analysis. Three AFLP markers, mAFEM972, mAFEM976 and mAFEM985, were found to co-segregate with the Rf1 allele in our mapping populations. With the help of RFLP markers, previously mapped on the sugar beet genome, we showed that Rf1 is positioned in the terminal region of linkage group Kiel III/Koeln IV. This map location agrees well with that found for the restorer gene X, which suggests that the Rf1 locus corresponds to the X locus. The availability of the molecular markers will facilitate the selection of maintainer-pollinator lines in breeding program and provide the foundation for map-based cloning of the Rf1 gene.

The 5'-leader sequence of sugar beet mitochondrial atp6 encodes a novel polypeptide that is characteristic of Owen cytoplasmic male sterility
MP Yamamoto, T Kubo, T Mikami
MOLECULAR GENETICS AND GENOMICS, 273, 4, 342, 349, SPRINGER, 2005年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Cytoplasmic male sterility (CMS) is a mitochondrially encoded trait, which is characterized by a failure of plants to produce viable pollen. We have investigated the protein profile of mitochondria from sugar beet plants with normal (fertile) or CMS cytoplasm, and observed that a 35-kDa polypeptlide is expressed in Owen CMS plants but not in normal plants. The variant 35-kDa polypeptide was found in CMS mitochondria placed in five different nuclear backgrounds. Interestingly, this polypeptide proved to be antigenically related to a 387-codon ORF (preSatp6) that is fused in-frame with the downstream atp6. T e presequence extension of the atp6 ORF is commonly found in higher plants, but whether or not it is normally expressed has hitherto remained unclear. Our study is thus the first to demonstrate that the atp6 presequence is actually translated in mitochondria. We also observed that preSATP6 is a mitochondrial membrane protein that assembles into a homogeneous 200-kDa protein complex. In organello translation experiments in the presence of protease inhibitors showed a reduction in the abundance of mature preSATP6 with time, suggesting that the mature preSATP6 may be derived by proteolytic processing of a translation product of the preSatp6/ Satp6 ORF.

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs
M Satoh, T Kubo, S Nishizawa, A Estiati, N Itchoda, T Mikami
MOLECULAR GENETICS AND GENOMICS, 272, 3, 247, 256, SPRINGER, 2004年10月, ［査読有り］
英語, 研究論文（学術雑誌）, The complete nucleotide sequence (501,020 bp) of the mitochondrial genome from cytoplasmic male-sterile (CMS) sugar beet was determined. This enabled us to compare the sequence with that previously published for the mitochondrial genome of normal, male-fertile sugar beet. The comparison revealed that the two genomes have the same complement of genes of known function. The rRNA and tRNA genes encoded in the CMS mitochondrial genome share 100% sequence identity with their respective counterparts in the normal genome. We found a total of 24 single nucleotide substitutions in 11 protein genes encoded by the CMS mitochondrial genome. However, none of these seems to be responsible for male sterility. In addition, several other ORFs were found to be actively transcribed in sugar beet mitochondria. Among these, Norf246 was observed to be present in the normal mitochondrial genome but absent from the CMS genome. However, it seems unlikely that the loss of Norf246 is causally related to the expression of CMS, because previous studies on mitochondrial translation products failed to detect the product of this ORF. Conversely, the CMS genome contains four transcribed ORFs (Satp6presequence, Scox2-2 , Sorf324 and Sorf119) which are missing from the normal genome. These ORFs, which are potential candidates for CMS genes, were shown to be generated by mitochondrial genome rearrangements.

Antisense inhibition of mitochondrial pyruvate dehydrogenase E1 alpha subunit in anther tapetum causes male sterility
R Yui, S Iketani, T Mikami, T Kubo
PLANT JOURNAL, 34, 1, 57, 66, WILEY-BLACKWELL, 2003年04月, ［査読有り］
英語, 研究論文（学術雑誌）, We hypothesized that cytoplasmic male sterility (CMS) in sugar beet may be the consequence of mitochondrial dysfunctions affecting normal anther development. To test the hypothesis, we attempted to mimic the sugar beet CMS phenotype by inhibiting the expression of mitochondrial pyruvate dehydrogenase (PDH), which is essential for the operation of the tricarboxylic acid (TCA) cycle. Screening with a cDNA library of sugar beet flower buds allowed the identification of two PDH E1alpha subunit genes (bvPDH_E1alpha-1 and bvPDH_E1alpha-2 ). bvPDH_E1alpha-1 was found to be highly expressed in tap roots, whereas bvPDH_E1alpha-2 was expressed most abundantly in flower buds. Green fluorescent protein (GFP) fusion of bvPDH_E1alpha revealed mitochondrial targeting properties. A 300-bp bvPDH_E1alpha-1 cDNA sequence (from +620 to +926) was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. Antisense expression of bvPDH_E1alpha-1 resulted in conspicuously decreased endogenous bvPDH_E1alpha-1 transcripts and male sterility. The tapetum in the male-sterile anthers showed swelling or abnormal vacuolation. It is also worth noting that in the sterile anthers, cell organelles, such as elaioplasts, tapetosomes and orbicules were poorly formed and microspores exhibited aberrant exine development. These features are shared by sugar beet CMS. The results thus clearly indicate that inhibition of PDH activity in anther tapetum is sufficient to cause male sterility, a phenocopy of the sugar beet CMS.

The rps4 gene in sugar beet mitochondria: insertion/deletion mutations occur within the gene but do not disrupt the reading frame
N Itchoda, T Kubo, A Estiati, MP Yamamoto, H Handa, T Mikami
JOURNAL OF PLANT PHYSIOLOGY, 159, 2, 211, 215, URBAN & FISCHER VERLAG, 2002年02月, ［査読有り］
英語, 研究論文（学術雑誌）, We have characterized a sugar beet mitochondrial gene, rps4, which is co-transcribed with the downstream gene nad6, The sugar beet RPS4 polypeptide has two deletions of 8 and 49 amino acid residues and a duplication of 14 residues in the middle of the reading frame, relative to the rapeseed and rice homologues. Nevertheless, transcripts of the rps4 were observed to be edited at 10 nucleotides, suggesting that the sugar beet rps4 is functional. Western blot analysis identified the translation product of 33 kDa. The extensive rearrangements in rps4 were also found in spinach but not in pea, green pepper and rose.

The sugar beet mitochondrial nad4 gene: an intron loss and its phylogenetic implication in the Caryophyllales
N Itchoda, S Nishizawa, H Nagano, T Kubo, T Mikami
THEORETICAL AND APPLIED GENETICS, 104, 2-3, 209, 213, SPRINGER-VERLAG, 2002年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The sugar beet mitochondrial gene for subunit IV of NADH dehydrogenase (nad4) has been characterized. Unlike the corresponding genes in wheat and turnip, sugar beet nad4 lacks the second intron (nad4-i2). Northern-blot analysis demonstrates transcription of the gene. A total of 19 RNA editing sites were identified in the sugar beet nad4 transcripts; interestingly, there is no editing in the region which flanks the lost intron. This observation is in favour of intron loss via homologous recombination of an edited RNA intermediate. We also found that the nad4-i2 intron is absent from the mitochondrial genomes of all examined members of the Caryophyllales, but present in the closely related orders, Polygonales and Plumbaginales, which suggests that the intron was lost in the common ancestor of the Caryophyllales.

The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel gene for tRNA(Cys)(GCA)
T Kubo, S Nishizawa, A Sugawara, N Itchoda, A Estiati, T Mikami
NUCLEIC ACIDS RESEARCH, 28, 13, 2571, 2576, OXFORD UNIV PRESS, 2000年07月, ［査読有り］
英語, 研究論文（学術雑誌）, We determined the complete nucleotide sequence of the mitochondrial genome of an angiosperm, sugar beet (Beta vulgaris cv TK81-O). The 368 799 bp genome contains 29 protein, five rRNA and 25 tRNA genes, most of which are also shared by the mitochondrial genome of Arabidopsis thaliana, the only other completely sequenced angiosperm mitochondrial genome. However, four genes identified here (namely rps13 trnF-GAA, ccb577 and trnC2-GCA) are missing in Arabidopsis mitochondria. In addition, four genes found in Arabidopsis (ccb228, rpl2, rpl16 and trnY2-GUA) are entirely absent in sugar beet or present only in severely truncated form. Introns, duplicated sequences, additional reading frames and inserted foreign sequences (chloroplast, nuclear and plasmid DNA sequences) contribute significantly to the overall size of the sugar beet mitochondrial genome. Nevertheless, 55.6% of the genome has no obvious features of information. We identified a novel tRNA(Cys) gene (trnC2-GCA) which shows no sequence homology with any tRNA(Cys) genes reported so far in higher plants. Intriguingly, this tRNA gene is actually transcribed into a mature tRNA, whereas the native tRNA(Cys) gene (trnC1-GCA) is most likely a pseudogene.

The nad4L-orf25 gene cluster is conserved and expressed in sugar beet mitochondria
T Kubo, MP Yamamoto, T Mikami
THEORETICAL AND APPLIED GENETICS, 100, 2, 214, 220, SPRINGER VERLAG, 2000年01月, ［査読有り］
英語, 研究論文（学術雑誌）, We have found that a gene coding for NADH dehydrogenase subunit 4L and a presumed gene, orf25, are Linked and co-transcribed with each other in sugar beet mitochondria. Ten and twelve C-to-U editing events were observed in the mRNAs of nad4L and orf25, respectively; the amino-acid sequence specified after editing is better-conserved in comparison with the homologues of other organisms. It is interesting to note that the translation initiation codon of nad4L is created by editing. The conservation of the nad4L-orf25 linkage was examined by PCR-amplification of the intergenic region. We obtained successful PCR products from five dicots (spinach, apple, snapdragon, petunia and tobacco) and two monocots (tulip and pineapple), but not in two poaceous plants, rice and maize. The intergenic region, when present, was found to be well-conserved in its sequence, suggesting a monophyletic origin of this linkage. Our result, together with previous reports of Arabidopsis and four poaceous species, favour the argument that the nad4L-orf25 linkage is conserved throughout angiosperms except in the Poaceae.

Variable number of tandem repeat loci in the mitochondrial genomes of beets
S Nishizawa, T Kubo, T Mikami
CURRENT GENETICS, 37, 1, 34, 38, SPRINGER VERLAG, 2000年01月, ［査読有り］
英語, 研究論文（学術雑誌）, We found four unrelated tandem repeat loci (TR1, TR2, TR3 and TR4) in the mitochondrial genomes of beets, with the TR1 locus embedded within a three-membered family of recombining repeat sequences (the rrn26-repeat). TR1 is composed of an array of 32-bp tandem repeats, the number of which varies from 2 to 13 among the seven beet genotypes examined. It is interesting to note that TR1 has 7-bp direct repeats flanking the array, which may be involved in the generation of the tandem repeat array. Such striking features are shared by the remaining TR loci, and this is thus the first description of minisatellite nucleotide sequences from a higher-plant mitochondrial genome.

Heterogeneity of the atp6 presequences in normal and different sources of male-sterile cytoplasms of sugar beet
Y Onodera, MP Yamamoto, T Kubo, T Mikami
JOURNAL OF PLANT PHYSIOLOGY, 155, 4-5, 656, 660, GUSTAV FISCHER VERLAG, 1999年10月, ［査読有り］
英語, 研究論文（学術雑誌）, We have characterized the mitochondrial atp6 loci from male-fertile (TK81-O), Owen CMS (TK81-MS), and wild beet-derived CMS (I-12CMS(3)) cytoplasms of sugar beet (Beta vulgaris L.). The TK81-O atp6 lacks a presequence characteristic of the atp6 genes from a variety of organisms. Interestingly, RNA editing was found to create an initiation codon in TK81-O atp6 mRNA, resulting in a 5 amino acid presequence. On the other hand, TK81-MS atp6 and I-12CMS(3) atp6 have presequences of 387 and 389 residues, respectively. Western blot analysis failed to reveal polymorphisms in size and abundance of the accumulated ATP6 polypeptide among the three genotypes. Our results thus favour the view that the presequence is proteolytically discarded upon maturation of the gene product and that atp6 itself is nor the cause of the sterility phenotype.

Alterations in organization and transcription of the mitochondrial genome of cytoplasmic male sterile sugar beet (Beta vulgaris L.)
T Kubo, S Nishizawa, T Mikami
MOLECULAR AND GENERAL GENETICS, 262, 2, 283, 290, SPRINGER VERLAG, 1999年09月, ［査読有り］
英語, 研究論文（学術雑誌）, We have constructed a physical map of the mitochondrial DNA of a cytoplasmic male sterile (CMS) sugar beet line, TK81-MS, and compared it with that published for normal fertile sugar beet (cv. TK81-O) to clarify the differences between the CMS and normal mitochondrial genomes. The TK81-MS genome is present as a single circular molecule of 481.8 kb, or as two molecules of 184.9 and 296.9 kb. The CMS genome was found to be highly rearranged relative to the normal mitochondrial genome, with at least fifteen rearrangement and/or inversion events being required to align the two DNAs. Analysis of transcription patterns of known mitochondrial genes and rearranged regions revealed six genes, coxI, coxII, atpA, atp6, rps3, and orf324, whose expression is altered in the CMS line relative to the normal line. Of these six, only the coxI transcript pattern differs between male-sterile and fertility-restored genotypes, making it likely that the coxI locus is involved in mediating CMS in sugar beet.

The ribosomal protein S7 gene is transcribed and edited in sugar beet mitochondria
A. Estiati, T. Kubo, Mikami
Physiologia Plantarum, 102, 2, 325, 327, 1998年02月, ［査読有り］
英語, 研究論文（学術雑誌）, We have isolated a mitochondrial rps7 gene from sugar beet (Beta vulgaris L. cv. TK81-O). It is present in a single genomic environment, with northern blot analysis showing transcription of this gene. Sequence analysis of RT-PCR-derived cDNA of rps7 showed that three RNA-editing events occurred on rps7 mRNAs. As far as we know, this is the first report of the complete identification of the RNA editing of a mitochondrial rps7 in the angiosperms.

A duplicated sequence in sugarbeet mitochondrial transcripts is differentially edited: Analysis of orfB and its derivative orf324 mRNAs
T Kubo, T Mikami
BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, 1307, 3, 259, 262, ELSEVIER SCIENCE BV, 1996年07月, ［査読有り］
英語, 研究論文（学術雑誌）, RNA editing of the duplicated sequences was investigated in the transcripts of orfB and orf324 genes from sugarbeet mitochondria. The olf324 shares the first 59 bp of the reading frame and 321 bp upstream sequence with orfB. Two cytidine-to-uridine editing sites were found in orfB, but the corresponding cytidine residues remained unchanged in the transcripts of orf324. In the vicinity of the non-edited cytidine residues within the shared sequence element of orf324 were identified three point mutations that may abolish orf324 editing. Our results also suggest that selection of editing sites depends on primary sequence.

PHYSICAL AND GENE ORGANIZATION OF MITOCHONDRIAL-DNA FROM THE FERTILE CYTOPLASM OF SUGAR-BEET (BETA-VULGARIS L)
T KUBO, YS TAKATO, T KINOSHITA, T MIKAMI
CURRENT GENETICS, 28, 3, 235, 241, SPRINGER VERLAG, 1995年08月, ［査読有り］
英語, 研究論文（学術雑誌）, We have constructed a complete physical map of the mitochondrial genome from the male-fertile cytoplasm of sugarbeet. The entire sequence complexity can be represented on a single circular master chromosome of 358 kb. This master chromosome contains three copies of one recombinationally active repeat sequence, with two copies in direct orientation and the other in inverted orientation. The positions of the rRNA genes and of 23 polypeptide genes, determined by filter hybridization, are scattered throughout the genome, with triplicate rrn26 genes located partially or entirely within the recombination-repeat elements. Three ribosomal-protein genes (rps1A, rps14 and rps19) were found to be absent from sugarbeet mtDNA. Our results also reveal that at least six regions homologous with cpDNA are dispersed in the mitochondrial genome.

THE CHLOROPLAST TRNP-TRNW-PETG GENE-CLUSTER IN THE MITOCHONDRIAL GENOMES OF BETA-VULGARIS, B-TRIGYNA AND B-WEBBIANA - EVOLUTIONARY ASPECTS
T KUBO, Y YANAI, T KINOSHITA, T MIKAMI
CURRENT GENETICS, 27, 3, 285, 289, SPRINGER VERLAG, 1995年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The chloroplast trnP-trnW-petG gene cluster has been identified in the mitochondrial DNA (mtDNA) of sugar beet (Beta vulgaris). The chloroplast-derived trnW gene is transcribed in the mitochondria; the other two genes, however, do not seem to be transcribed. This gene cluster is also present in the mitochondrial genomes of two wild Beta species, B. trigyna and B. webbiana. Sugar beet and the two wild relatives share 100% sequence identity in the coding regions of both the mitochondrial trnP and trnW genes. On the other hand, the petG genes from the wild Beta mtDNAs were found to be disrupted either by a 5-bp duplication (B. trigyna) or by a deletion of the 5' region (B. webbiana). A data-base search revealed that a conserved sequence of 60 bp is present in the trnP-trnW intergenic region of the mitochondrial genomes of the three Beta species as well as in other higher plants, including wheat and maize, and that the conserved sequence is absent from the chloroplast counterpart. Our results thus favour the hypothesis of a monophyletic origin of the trnP-trnW-petG cluster found in the plant mitochondrial genomes examined.

Physical maps of mitochondrial genomes from male-fertile and male-sterile sugar beets
T Kinoshita, T Mikami, T Kubo
CURRENT ISSUES IN PLANT MOLECULAR AND CELLULAR BIOLOGY, 22, 303, 308, KLUWER ACADEMIC PUBL, 1995年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）

THE SUGAR-BEET MITOCHONDRIAL GENOME CONTAINS AN ORF SHARING SEQUENCE HOMOLOGY WITH THE GENE FOR THE 30 KDA SUBUNIT OF BOVINE MITOCHONDRIAL COMPLEX-I
T KUBO, T MIKAMI, T KINOSHITA
MOLECULAR & GENERAL GENETICS, 241, 3-4, 479, 482, SPRINGER VERLAG, 1993年11月, ［査読有り］
英語, 研究論文（学術雑誌）, From a sugar beet mitochondrial DNA library, we have isolated an open reading frame (ORF192) showing extensive homology to the gene for the 30 kDa subunit of the bovine mitochondrial complex I (NADH: ubiquinone reductase). The ORF 192 was found to be actively transcribed to give an RNA of approximately 1.0 kb. We have designated this gene nad9. Transcripts from the nad9 locus are edited by five C to U transitions, increasing similarity with the amino acid sequence of the corresponding bovine and Neurospora crassa polypeptides. Southern blot hybridization also indicates that nad9 is present in the mitochondrial genomes of a variety of higher plant species.

未来のテンサイ遺伝育種学に必要なこと　　　　　　　　　　　　　　　
久保友彦, グリーンテクノ情報, 19, 1, 2023年06月, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］, ［国内誌］
日本語, その他

ガーデンビートはオリゴ糖原料作物になりうるか：てん菜との関係から考える
久保友彦, 早川諒, 森 春英, 月報 砂糖類・でん粉情報, 49, 54, 2022年03月, ［招待有り］, ［筆頭著者］, ［国内誌］
独立行政法人 農畜産業振興機構, 日本語, 記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）

応用展開が進みつつある植物ミトコンドリア基礎研究と今後の課題
Tomohiko Kubo, Takumi Arakawa, Kazuyoshi Kitazaki, Tomohiko Kazama, Mizuki Takenaka, Wataru Sakamoto, Naotada Ishihara, Takahiro Nakamura, Satoshi Niikura, Shin-ichi Arimura, Hirokazu Handa, Nobuya Koizuka, 育種学研究, 22, 1, 87, 94, 2020年06月01日, ［国内誌］
Japanese Society of Breeding, 日本語, 記事・総説・解説・論説等（学術雑誌）

What Does the Molecular Genetics of Different Types of Restorer-of-Fertility Genes Imply?
Tomohiko Kubo, Takumi Arakawa, Yujiro Honma, Kazuyoshi Kitazaki, Plants, 9, 3, 361, 361, 2020年03月13日, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］, ［国際誌］
Cytoplasmic male sterility (CMS) is a widely used trait for hybrid seed production. Although male sterility is caused by S cytoplasm (male-sterility inducing mitochondria), the action of S cytoplasm is suppressed by restorer-of-fertility (Rf), a nuclear gene. Hence, the genetics of Rf has attained particular interest among plant breeders. The genetic model posits Rf diversity in which an Rf specifically suppresses the cognate S cytoplasm. Molecular analysis of Rf loci in plants has identified various genes; however, pentatricopeptide repeat (PPR) protein (a specific type of RNA-binding protein) is so prominent as the Rf-gene product that Rfs have been categorized into two classes, PPR and non-PPR. In contrast, several shared features between PPR- and some non-PPR Rfs are apparent, suggesting the possibility of another grouping. Our present focus is to group Rfs by molecular genetic classes other than the presence of PPRs. We propose three categories that define partially overlapping groups of Rfs: association with post-transcriptional regulation of mitochondrial gene expression, resistance gene-like copy number variation at the locus, and lack of a direct link to S-orf (a mitochondrial ORF associated with CMS). These groups appear to reflect their own evolutionary background and their mechanism of conferring S cytoplasm specificity., MDPI AG, 英語, 記事・総説・解説・論説等（学術雑誌）

2018年第60回シンポジウム（シンポジウム・ワークショップ）報告について
久保 友彦, 育種学研究, 21, 1, 47, 48, 2019年, ［招待有り］, ［国内誌］
日本語, 会議報告等

ビーツはどこしゅっしんなんですか　　　　　　　　　　　　　　　
久保 友彦, 日本の学童ほいく, 515, 7月号, 39, 2018年06月, ［招待有り］, ［筆頭著者, 責任著者］, ［国内誌］
日本語, 記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）

農学校1年1組 てん菜の時間 ビートの種類と栽培の歴史　　　　　　　　　　　　　　　
久保 友彦, ニューカントリー, 65, 2月号, 48, 49, 2018年02月, ［招待有り］, ［筆頭著者, 責任著者］, ［国内誌］
日本語, 記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）

Sugar beet (Beta vulgaris L.)
Hiroyo Kagami, Masayuki Kurata, Hiroaki Matsuhira, Kazunori Taguchi, Tetsuo Mikami, Hideto Tamagake, Tomohiko Kubo, Agrobacterium Protocols: Third Edition, 1, 335, 347, 2014年10月09日, ［査読有り］
Creating transgenic plants is invaluable for the genetic analysis of sugar beet and will be increasingly important as sugar beet genomic technologies progress. A protocol for Agrobacterium-mediated transformation of sugar beet is described in this chapter. Our protocol is optimized for a sugar beet genotype that performs exceptionally well in tissue culture, including the steps of dedifferentiation, callus proliferation, and regeneration. Because of the infrequent occurrence of such a genotype in sugar beet populations, our protocol includes an in vitro propagation method for germplasm preservation. The starting materials for transgenic experiments are aseptic shoots grown from surface-sterilized seed balls. Callus is induced from leaf explants and subsequently infected with Agrobacterium. Plantlets are regenerated from transgenic callus and vernalized for flowering, if necessary. The efficiency of transformation was quite high
in our laboratory, the culture of only ten leaf explants, on average, generated one transgenic plant., Springer New York, 英語

ミトコンドリアは従順なオルガネラか–核・細胞質相互作用の背景–
久保 友彦, 寺地 徹, 育種学研究, 15, 2, 68, 71, 2013年, ［招待有り］
日本育種学会, 日本語, 記事・総説・解説・論説等（学術雑誌）

Erratum: Is RNA editing implicated in group II intron survival in the angiosperm mitochondrial genome? (Genome (2012) 55:1 (75-79))
Hiroyo Kagami, Hironori Nagano, Yoshiya Takahashi, Tetsuo Mikami, Tomohiko Kubo, Genome, 55, 6, 481, 2012年06月, ［査読有り］
英語

Molecular basis of cytoplasmic male sterility in beets: an overview
Tetsuo Mikami, Masayuki P. Yamamoto, Hiroaki Matsuhira, Kazuyoshi Kitazaki, Tomohiko Kubo, PLANT GENETIC RESOURCES-CHARACTERIZATION AND UTILIZATION, 9, 2, 284, 287, 2011年07月, ［査読有り］
Sugarbeet cultivars are almost exclusively hybrids, which are produced using the sole source of cytoplasmic male sterility (CMS), the so-called Owen CMS. Several alternative sources of CMS have been described. One of these, I-12CMS(3), was derived from wild beets collected in Pakistan, and another CMS source, GCMS, has a cytoplasmic origin in wild sea beets from France. During the past decade, male sterility-associated mitochondrial genes have been identified in these three CMS systems. Moreover, the recent development of a variety of DNA markers has permitted the genetic mapping of nuclear restorer-of-fertility genes for both Owen and GCMS. This review focuses on the mechanism of CMS in beets., CAMBRIDGE UNIV PRESS, 英語, 記事・総説・解説・論説等（学術雑誌）

Male Sterility-Inducing Mitochondrial Genomes: How Do They Differ?
Tomohiko Kubo, Kazuyoshi Kitazaki, Muneyuki Matsunaga, Hiroyo Kagami, Tetsuo Mikami, CRITICAL REVIEWS IN PLANT SCIENCES, 30, 4, 378, 400, 2011年, ［査読有り］
Twenty-nine mitochondrial genomes from 19 angiosperm species have been completely sequenced and have been found to vary in genome size and gene content. Seven of these mitochondrial genomes are known to induce cytoplasmic male sterility (CMS), and thus can be utilized for hybrid seed production or the prevention of pollen dispersal. Genome rearrangement frequently is observed in male sterility (MS)-inducing mitochondria, but it also occurs as part of the normal inter- or intraspecific variation in male fertile (MF) mitochondria. Sequence analyses have revealed that the repertoire of genuine genes is indistinguishable between MS-inducing and MF mitochondria. Deleterious mutations appear to be rare in MS-inducing mitochondria, which may be consistent with the lack of systemic manifestation of CMS. On the other hand, several nucleotide substitutions remain to be investigated for their potential mild effects. Various mitochondrial open reading frames (ORFs) are associated with CMS (CMS-ORFs). There are some common but not strict features shared by CMS-ORFs such as their uniqueness to the CMS mitochondrial genome, their association with genes for ATPase subunits, and the hydrophobic nature of their putative translation products. It should be noted that some CMS-ORFs do not satisfy all of these criteria, and ORFs that satisfy these criteria are not necessarily associated with CMS. Therefore, it is difficult to infer the capability of MS induction of mitochondrial genomes solely from their nucleotide sequences. Morphological, physiological, and molecular biological studies suggest that multiple mechanisms cause CMS. Nuclear genes that suppress CMS have been identified. Post-transcriptional suppression of CMS-ORFs mediated by a certain class of RNA binding proteins (pentatrico peptide repeat proteins) is the predominant mechanism of fertility restoration. On the other hand, CMS suppression that is not associated with post-transcriptional suppression of CMS-ORFs has also been reported, suggesting that various types of gene products are involved in fertility restoration., TAYLOR & FRANCIS INC, 英語, 書評論文,書評,文献紹介等

Cost of having the largest mitochondrial genome: evolutionary mechanism of plant mitochondrial genome　　　　　　　　　　　　　　　
Kitazaki K, Kubo T, Journal of Botany, 2010, 620137, 2010年, ［査読有り］
英語, 記事・総説・解説・論説等（学術雑誌）

Angiosperm mitochondrial genomes and mutations
Tomohiko Kubo, Kathleen J. Newton, MITOCHONDRION, 8, 1, 5, 14, 2008年01月, ［査読有り］
Flowering plants harbor the largest mitochondrial genomes reported so far. At present, the nucleotide sequences of 15 mitochondrial genomes from seven angiosperm species are available, making detailed comparative analysis feasible. The gene content is variable among the species, but the most striking feature is the fluidity of intergenic regions, where species-specific sequences predominate. Additionally, angiosperm mitochondrial genomes, even within a species, show a remarkable amount of rearrangement. We also review mitochondrial mutants in angiosperms from a genomic viewpoint, and discuss how they have arisen. The involvement of nuclear genes in mitochondrial genome stability and organization is currently being revealed through the analysis-of mutants. (C) 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved., ELSEVIER SCI LTD, 英語, 記事・総説・解説・論説等（学術雑誌）

Organization and variation of angiosperm mitochondrial genome
Tomohiko Kubo, Tetsuo Mikami, PHYSIOLOGIA PLANTARUM, 129, 1, 6, 13, 2007年01月, ［査読有り］
The mitochondrial genomes of angiosperms are the largest mitochondrial genomes so far reported and are highly variable in size among plant species. The comparative analysis of the angiosperm mitochondrial genomes at the nucleotide level has now become feasible for addressing long-standing questions, owing to the publication of five dicot and three monocot genomes. Whereas the identified genes and introns are rather well conserved, intergenic regions are highly variable in sequence, even between two close relatives. Promiscuous DNA and horizontally transferred sequence constitute part of the intergenic regions, but the origin of the majority of these regions is unknown. On the other hand, duplication and extensive rearrangement of preexisting sequences may be one of the explanations for the occurrence of unknown sequences. Functional aspects of the mitochondrial genome, such as RNA editing and expression of unique open reading frames (ORFs), can be changed under certain nuclear genotypes., BLACKWELL PUBLISHING, 英語, 書評論文,書評,文献紹介等

ミトコンドリアゲノムと細胞質雄性不稔性　　　　　　　　　　　　　　　
久保 友彦, 育種学研究, 8, 135, 141, 2006年, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

植物の性表現を変えるミトコンドリア遺伝子と核遺伝子の相互作用
久保友彦, 三上哲夫, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 50, 1803, 1807, 2005年11月, ［招待有り］
14 Suppl, 日本語, 記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）

アゼルバイジャンに自生するBeta lomatogonaについて
久保 友彦, 田口 和憲, てん菜研究会報 = Proceedings of the Sugar Beet Research Association, 45, 41, 43, 2004年05月
甘味資源振興会, 日本語

ロシアおよびアゼルバイジャンにおけるテンサイ遺伝資源の共同調査収集　　　　　　　　　　　　　　　
田口和憲, 久保友彦, Burenin VI, Burmistrov LA、Akparov Z、Alderov AA, 植物遺伝資源探索導入調査報告書, 20, 93, 101, 2004年
日本語, その他

アゼルバイジャンを知るための67章
久保, 友彦, 第3章 地理・気候・自然--アゼルバイジャンと植物遺伝資源
明石書店, 2018年08月, 9784750346724, 426p, 28-32, 日本語, 一般書・啓蒙書, ［分担執筆］

Frontiers of Agricultural Science　　　　　　　　　　　　　　　
久保 友彦, Sugar beet
Shoukadoh Book Sellers, 2015年, 9784879746856, ［分担執筆］

Agricultural Sciences for Human Sustainability (Graduate School of Agriculture, Hokkaido University Editorial Committee)　　　　　　　　　　　　　　　
Kubo T, Horizontal gene transfer to the plant mitochondrial genome
Kaiseisha Press, 2012年, ［分担執筆］

コーカサスを知るための60章（北川誠一，前田弘毅，廣瀬陽子，吉村貴之 編著）　　　　　　　　　　　　　　　
久保 友彦, 栽培植物起原地としてのコーカサス
2006年, 4750323012, 336, 23-26, 日本語, 一般書・啓蒙書, ［分担執筆］

植物育種学辞典（日本育種学会 編）　　　　　　　　　　　　　　　
久保 友彦, 「IPCR」，「RT-PCR」，「Rループ」，「塩基」，「相補性」，「DNA」，「DNA分解酵素」，「TAIL-PCR」，「Dループ」，「トポイソメラーゼ」，「ヌクレアーゼ」，「ヌクレオシド」，「ヌクレオチド」，「PCR法」，「ピリミジン塩基」，「プリン塩基」
培風館, 2005年, ［分担執筆］

Plant Mitochondria: From Gene to Function　　　　　　　　　　　　　　　
Kubo T, Inoue M, Nishizawa S, Mikami T, Structure of mitochondrial genomes of Beta vulgaris and B. maritima
Backhuys Publishers, 1998年, ［分担執筆］