Nuclear DNA segments homologous to mitochondrial DNA are obstacles for detecting heteroplasmy in sugar beet (Beta vulgaris L.).

Heteroplasmy, the coexistence of multiple mitochondrial DNA (mtDNA) sequences in a cell, is well documented in plants. Next-generation sequencing technology (NGS) has made it feasible to sequence entire genomes. Thus, NGS has the potential to detect heteroplasmy; however, the methods and pitfalls in heteroplasmy detection have not been fully investigated and identified. One obstacle for heteroplasmy detection is the sequence homology between mitochondrial-, plastid-, and nuclear DNA, of which the influence of nuclear DNA segments homologous to mtDNA (numt) need to be minimized. To detect heteroplasmy, we first excluded nuclear DNA sequences of sugar beet (Beta vulgaris) line EL10 from the sugar beet mtDNA sequence. NGS reads were obtained from single plants of sugar beet lines NK-195BRmm-O and NK-291BRmm-O and mapped to the unexcluded mtDNA regions. More than 1000 sites exhibited intra-individual polymorphism as detected by genome browsing analysis. We focused on a 309-bp region where 12 intra-individual polymorphic sites were closely linked to each other. Although the existence of DNA molecules having variant alleles at the 12 sites was confirmed by PCR amplification from NK-195BRmm-O and NK-291BRmm-O, these variants were not always called by six variant-calling programs, suggesting that these programs are inappropriate for intra-individual polymorphism detection. When we changed the nuclear DNA reference, a numt absent from EL10 was found to include the 309-bp region. Genetic segregation of an F2 population from NK-195BRmm-O x NK-291BRmm-O supported the numt origin of the variant alleles. Using four references, we found that numt detection exhibited reference dependency, and extreme polymorphism of numts exists among sugar beet lines. One of the identified numts absent from EL10 is also associated with another intra-individual polymorphic site in NK-195mm-O. Our data suggest that polymorphism among numts is unexpectedly high within sugar beets, leading to confusion about the true degree of heteroplasmy.

Identification and characterization of a semi-dominant restorer-of-fertility 1 allele in sugar beet (Beta vulgaris).

KEY MESSAGE: 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.

Identification of molecular variants of the nonrestoring restorer-of-fertility 1 allele in sugar beet (Beta vulgaris L.).

KEY MESSAGE: 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.