Yoshinari Moriguchi

Background: Japanese cedar ( Cryptomeria japonica D. Don) is a major plantation species in Japan, but its abundant pollen production is a primary cause of seasonal allergic rhinitis (pollinosis). To mitigate this public health issue, the use of male-sterile cultivars has been promoted. Five types of recessive male-sterile mutations ( ms1 – ms5 ) have been identified, and the causal genes and mutations for MS1 and MS4 have been elucidated. However, the gene responsible for MS2-type male sterility remains unknown. Results: We aimed to identify the candidate gene responsible for MS2-type male sterility using a map-based cloning strategy. High-resolution linkage mapping localized the MS2 locus to a 1.56 cM interval on linkage group 5, corresponding to an 8.64 Mb region of the reference genome. Ninety-one genes in this region were subjected to functional annotation, gene expression analysis, and mutation screening. Among these, a single gene, SUGI_0493010, encoding a GDSL-type esterase/lipase protein (GELP), fulfilled all three criteria: it showed homology to pollen development genes in Arabidopsis thaliana , was specifically expressed in male strobili, and carried a deleterious amino acid substitution (S40F) within the predicted catalytic domain in ms2 mutant. The same mutation was also detected in a heterozygous individual ( Ms2/ms2 ) from a separate breeding population, whose genotype was confirmed through progeny testing. Structural annotation revealed that the affected serine residue lies within the conserved GDSL motif, suggesting a functional disruption of enzymatic activity. Conclusions: Our results strongly suggest that SUGI_0493010 ( GELP ) is the candidate gene for MS2-type male sterility in C. japonica . This finding enhances our understanding of male sterility mechanisms in conifers and provides a valuable genetic resource for breeding pollen-free trees. The study also demonstrates the effectiveness of combining genetic mapping with transcriptomic and mutational data in forest tree genomics.

近年，スギ雄性不稔遺伝子MS1の候補遺伝子としてCJt020762が同定され，これまでに2つの雄性不稔対立遺伝子（ms1-1およびms1-2）が発見されている。本研究では，CJt020762のイントロンの一部を含めた全コード配列をPCR増幅して増幅断片の長さの違いを識別するPCRプライマーセット（DNAマーカー）を開発し，日本各地の育種素材1,500個体を対象に，挿入または欠失変異を持つ個体をマーカー選抜した。その結果，岩手県の育種素材で新たにコード配列に4塩基の欠失変異をヘテロ接合型で持つ2個体を選抜し，この対立遺伝子をms1-3と命名した。交配試験の結果，ms1-1およびms1-3をともに保有する個体（ms1-1/ms1-3）は雄性不稔であり，ms1-3が無花粉スギの育種に利用できることが示された。また本研究では，イントロン内に重複変異を生じた個体も認められたが，その個体は雄性不稔とならないことも示された。

Background: Japanese cedar ( Cryptomeria japonica D. Don) is a major plantation species in Japan, but its abundant pollen production is a primary cause of seasonal allergic rhinitis (pollinosis). To mitigate this public health issue, the use of male-sterile cultivars has been promoted. Five types of recessive male-sterile mutations ( ms1 – ms5 ) have been identified, and the causal genes and mutations for MS1 and MS4 have been elucidated. However, the gene responsible for MS2-type male sterility remains unknown. Results: We aimed to identify the candidate gene responsible for MS2-type male sterility using a map-based cloning strategy. High-resolution linkage mapping localized the MS2 locus to a 1.56 cM interval on linkage group 5, corresponding to an 8.64 Mb region of the reference genome. Ninety-one genes in this region were subjected to functional annotation, gene expression analysis, and mutation screening. Among these, a single gene, SUGI_0493010 , encoding a GDSL-type esterase/lipase protein (GELP), fulfilled all three criteria: it showed homology to pollen development genes in Arabidopsis thaliana , was specifically expressed in male strobili, and carried a deleterious amino acid substitution (S40F) within the predicted catalytic domain in ms2 mutant. The same mutation was also detected in a heterozygous individual ( Ms2/ms2 ) from a separate breeding population, whose genotype was confirmed through progeny testing. Structural annotation revealed that the affected serine residue lies within the conserved GDSL motif, suggesting a functional disruption of enzymatic activity. Conclusions: Our results strongly suggest that SUGI_0493010 ( GELP ) is the causal gene for MS2-type male sterility in C. japonica . This finding enhances our understanding of male sterility mechanisms in conifers and provides a valuable genetic resource for breeding pollen-free trees. The study also demonstrates the effectiveness of combining genetic mapping with transcriptomic and mutational data in forest tree genomics.

Sugi-pollinosis poses a significant socioeconomic and public health concern in Japanese society. Consequently, the use of male-sterile plants (pollen-free plants or PFPs) is anticipated in reforestation efforts. In this context, we developed an improved, simplified method for efficiently propagating sugi PFPs. In the present study, we compared the efficiency of different embryogenic cell lines (ECLs) in producing somatic embryos and examined how effectively these embryos germinate and convert into plantlets. We also evaluated the germination potential of somatic embryos stored for various durations at temperatures above freezing and room temperature. The production efficiency of somatic embryos ranged from 129.6 to 504.1 per plate, with an average of 349.8 across the ECLs tested. The overall average germination and conversion rates of somatic embryos were found to be 93.9% and 92.4%, respectively. Furthermore, although differences were observed among the evaluated genotypes, our five-year study demonstrated that sugi somatic embryos could be stored at 25 °C, 15 °C, or 5 °C for 6, 12, or 24 months, respectively, without a notable decline in germination capacity. The developed method enhances flexibility in plant production scheduling and facilitates the optimal timing for transferring somatic seedlings to the field.

Coastal dune plants play an important role in maintaining the stability of coastal dune areas. Artemisia stelleriana Besser. is a coastal dune plant with a northern distribution, and populations are scattered and isolated in Japan due to habitat loss, leading to its classification as an endangered species in several prefectures. However, very few ecological and genetic studies of A . stelleriana have been conducted worldwide. This study was performed to investigate the self‐compatibility, flower structure, and flowering phenology, seed thalassochory potential, and clonal propagation ability of A . stelleriana, along with the genetic diversity of four populations in Niigata Prefecture. Our results showed that A . stelleriana is self‐compatible, with a flower head composed of female and hermaphrodite florets that have overlapping flowering periods. The species also exhibits a relatively short seed floating period, which leads to limited seed dispersal by sea currents, and possesses a weak clonal structure due to its low ability for clonal propagation. In four populations in Niigata Prefecture, most individuals were fixed for a single homozygous genotype at all loci. Artemisia stelleriana is considered to have a flower structure suitable for self‐pollination, and it is assumed that this extremely low genetic diversity has been established through repeated self‐pollination within the population, with little gene flow between populations. Investigation of the genetic diversity and structure is necessary in the other four prefectures, where A. stelleriana is classified as an endangered species, in the future.

Background: The Japanese cedar (Cryptomeria japonica D. Don) is one of the most important Japanese forest trees, occupying approximately 44% of artificial forests and planted in East Asia, the Azores Archipelago, and certain islands in the Indian Ocean. Although the huge genome of the species (ca. 9 Gbp) with abundant repeat elements may have represented an obstacle for genetic analysis, this species is easily propagated by cutting, flowered by gibberellic acid, transformed by Agrobacterium, and edited by CRISPR/Cas9. These characteristics of C. japonica recommend it as a model conifer species for which reference genome sequences are necessary. Results: Herein, we report the first chromosome-level assembly of C. japonica (2n = 22) using third-generation selfed progeny (estimated homozygosity rate = 0.96). Young leaf tissue was used to extract high molecular weight DNA (> 50 kb) for HiFi PacBio long-read sequencing and to construct an Hi-C/Omni-C library for Illumina short-read sequencing. The 29× and 26× genome coverage of HiFi and Illumina reads, respectively, for de novo assembly yielded 2,651 contigs (9.1 Gbp, N50 contig size 12.0 Mbp). Hi-C analysis mapped 97% of the nucleotides on 11 chromosomes. The assembly was verified through comparison with a consensus linkage map comprising 7,781 markers. BUSCO analysis identified ∼ 91% conserved genes. Conclusions: Annotations of genes and comparisons of repeat elements with other Cupressaceae and Pinaceae species provide a fundamental resource for conifer research.

This study presents the development of a simple DNA extraction method and a novel PCR-RFLP protocol for genotyping the MALE STERILITY 4 ( MS4 ) gene in Cryptomeria japonica as a proof of concept. Traditional CTAB-based DNA extraction methods, while effective, involve hazardous chemicals and require high-speed centrifugation, which are impractical in many field settings. Our approach utilizes a household dish detergent-based buffer, sodium chloride, and polyvinylpyrrolidone K-30 to extract DNA from C. japonica needle leaf tissues—without the need for liquid nitrogen. The simplicity of this method makes it more accessible and environmentally friendly. The extracted DNA was successfully used in PCR-RFLP analysis, targeting a single nucleotide polymorphism in the MS4 gene, demonstrating its efficacy for genotyping. The PCR-RFLP markers reliably discriminated between individual genotypes, confirming the practical application of our simple extraction method, even for conifers containing inhibitory substances. This technique is particularly advantageous for use in arboretums and field stations, where the use of hazardous chemicals, specialized equipment, and a draft chamber is limited. Our study contributes to genetic resource management by providing an easy, reliable, and safer method for DNA extraction and genetic analysis.