Masahiro Kasahara

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.

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.

Body shape and size diversity and their evolutionary rates correlate with species richness at the macroevolutionary scale. However, the molecular genetic mechanisms underlying the morphological diversification across related species are poorly understood. In beetles, which account for one-fourth of the known species, adaptation to different trophic niches through morphological diversification appears to have contributed to species radiation. Here, we explored the key genes for the morphological divergence of the slender to stout body shape related to divergent feeding methods on large to small snails within the genus Carabus. We show that the zinc-finger transcription factor encoded by odd-paired (opa) controls morphological variation in the snail-feeding ground beetle Carabus blaptoides. Specifically, opa was identified as the gene underlying the slender to stout morphological difference between subspecies through genetic mapping and functional analysis via gene knockdown. Further analyses revealed that changes in opa cis-regulatory sequences likely contributed to the differences in body shape and size between C. blaptoides subspecies. Among opa cis-regulatory sequences, single nucleotide polymorphisms on the transcription factor binding sites may be associated with the morphological differences between C. blaptoides subspecies. opa was highly conserved in a wide range of taxa, especially in beetles. Therefore, opa may play an important role in adaptive morphological divergence in beetles.

The rates of appearance of new mutations play a central role in evolution. However, mutational processes in natural environments and their relationship with growth rates are largely unknown, particular in tropical ecosystems with high biodiversity. Here, we examined the somatic mutation landscapes of two tropical trees, Shorea laevis (slow-growing) and S. leprosula (fast-growing), in central Borneo, Indonesia. Using newly-constructed genomes, we identified a greater number of somatic mutations in tropical trees than in temperate trees. In both species, we observed a linear increase in the number of somatic mutations with physical distance between branches. However, we found that the rate of somatic mutation accumulation per meter of growth was 3.7-fold higher in S. laevis than in S. leprosula. This difference in the somatic mutation rate was scaled with the slower growth rate of S. laevis compared to S. leprosula, resulting in a constant somatic mutation rate per year between the two species. We also found that somatic mutations are neutral within an individual, but those mutations transmitted to the next generation are subject to purifying selection. These findings suggest that somatic mutations accumulate with absolute time and older trees have a greater contribution towards generating genetic variation.

Pollinosis, also known as pollen allergy or hay fever, is a global problem caused by pollen produced by various plant species. The wind-pollinated Japanese cedar (Cryptomeria japonica) is the largest contributor to severe pollinosis in Japan, where increasing proportions of people have been affected in recent decades. The MALE STERILITY 4 (MS4) locus of Japanese cedar controls pollen production, and its homozygous mutants (ms4/ms4) show abnormal pollen development after the tetrad stage and produce no mature pollen. In this study, we narrowed down the MS4 locus by fine mapping in Japanese cedar and found TETRAKETIDE α-PYRONE REDUCTASE 1 (TKPR1) gene in this region. Transformation experiments using Arabidopsis thaliana showed that single-nucleotide substitution (“T” to “C” at 244-nt position) of CjTKPR1 determines pollen production. Broad conservation of TKPR1 beyond plant division could lead to the creation of pollen-free plants not only for Japanese cedar but also for broader plant species.

Japanese cedar ( Cryptomeria japonica D. Don) is the most important Japanese forest tree, occupying about 44% of artificial forests in Japan, and planted in East Asia, Azores Archipelago, and some islands in the Indian Ocean. Although the huge genome of the species (ca. 11 Gb) with abundant repeat elements might have been an obstacle for genetic analysis, the species is easily propagated by cutting, flowered by plant hormones like gibberellic acid, transformed by agrobacterium, and edited by CRISPR/Cas9. These characteristics of C. japonica are preferable to make the species a model conifer for which reference genome sequences are necessary. In this study, we report the first chromosome-level assembly for C. japonica (2n = 22) using a third generation selfed progeny with an estimated homozygosity of 0.96. Young leaf tissue was used to extract high-molecular-weight DNA (>50 kb) for HiFi PacBio long read sequencing and to construct Hi-C/Omni-C library for Illumina short read sequencing. Using the 29× and 26× genome coverage of HiFi and Illumina reads, respectively, de novo assembly resulted in 2,650 contigs (9.1 Gb in total) with N50 contig size of 12.0 Mb. The Hi-C analysis mapped 97% of the nucleotides on the 11 chromosomes. The assembly was verified by comparing with a consensus linkage map of 7,785 markers. The BUSCO analysis confirmed ~91% of conserved genes. Annotations of genes, repeat elements and synteny with other Cupressaceae and Pinaceae species were performed, providing fundamental resources for genomic research of conifers.

The rates of appearance of new mutations play a central role in evolution. However, mutational processes in natural environments and their relationship with growth rates are largely unknown, particular in tropical ecosystems with high biodiversity. Here, we examined the somatic mutation landscapes of two tropical trees, Shorea laevis (slow-growing) and S. leprosula (fast-growing), in central Borneo, Indonesia. Using newly-constructed genomes, we identified a greater number of somatic mutations in tropical trees than in temperate trees. In both species, we observed a linear increase in the number of somatic mutations with physical distance between branches. However, we found that the rate of somatic mutation accumulation per meter of growth was 3.7-fold higher in S. laevis than in S. leprosula . This difference in the somatic mutation rate was scaled with the slower growth rate of S. laevis compared to S. leprosula, resulting in a constant somatic mutation rate per year between the two species. We also found that somatic mutations are neutral within an individual, but those mutations transmitted to the next generation are subject to purifying selection. These findings suggest that somatic mutations accumulate with absolute time and older trees have a greater contribution towards generating genetic variation. Significance Statement: The significance of our study lies in the discovery of an absolute time-dependent accumulation of somatic mutations in long-lived tropical trees, independent of growth rate. Through a comparative analysis of somatic mutation landscapes in slow- and fast-growing species, we observed a clock-like accumulation of somatic mutations in both species, regardless of their growth rates. Although the majority of somatic mutations were restricted to a single branch, we also identified mutations present in multiple branches, likely transmitted during growth. Our findings suggest that older trees make a greater contribution towards generating genetic variation.

Summary: The Closterium peracerosum–strigosum–littorale complex (Closterium, Zygnematophyceae) has an isogamous mating system. Members of the Zygnematophyceae are the closest relatives to extant land plants and are distantly related to chlorophytic models, for which a genetic basis of mating type (MT) determination has been reported. We thus investigated MT determination in Closterium. We sequenced genomes representing the two MTs, mt+ and mt−, in Closterium and identified CpMinus1, a gene linked to the mt− phenotype. We analyzed its function using reverse genetics methods. CpMinus1 encodes a divergent RWP‐RK domain‐containing‐like transcription factor and is specifically expressed during gamete differentiation. Introduction of CpMinus1 into an mt+ strain was sufficient to convert it to a phenotypically mt− strain, while CpMinus1‐knockout mt− strains were phenotypically mt+. We propose that CpMinus1 is the major MT determinant that acts by evoking the mt− phenotype and suppressing the mt+ phenotype in heterothallic Closterium. CpMinus1 likely evolved independently in the Zygnematophyceae lineage, which lost an egg–sperm anisogamous mating system. mt− specific regions possibly constitute an MT locus flanked by common sequences that undergo some recombination.

Background: Delivering tools for genome analysis to users is often difficult given the complex dependencies and conflicts of such tools. Container virtualization systems (such as Singularity) isolate environments, thereby helping developers package tools. However, these systems lack mutual composability, i.e., an easy way to integrate multiple tools in different containers and/or on the host. Another issue is that one may be unable to use a single container system of the same version at all the sites being used, thus discouraging the use of container systems. Results: We developed LPMX, an open-source pure rootless composable container system that provides composability; i.e., the system allows users to easily integrate tools from different containers or even from the host. LPMX accelerates science by letting researchers compose existing containers and containerize tools/pipelines that are difficult to package/containerize using Conda or Singularity, thereby saving researchers’ precious time. The technique used in LPMX allows LPMX to run purely in userspace without root privileges even during installation, thus ensuring that we can use LPMX at any Linux clusters with major distributions. The lowest overhead for launching containers with LPMX gives us courage to isolate tools as much as possible into small containers, thereby minimizing the chance of conflicts. The support for the layered file system keeps the total size of container images for a single genomic pipeline modest, as opposed to Singularity, which uses mostly a flat single-layer image. Conclusions: LPMX is pure rootless container engine with mutual composability, thus saving researchers’ time, and accelerating science.

Summary: Delivering tools for genome analysis to users is often difficult given their complex dependencies and conflicts. Container virtualization systems such as Singularity isolate environments, helping developers avoid conflicts between tools. However, they lack composability , an easy way to integrate multiple tools in different containers or multiple tools both in a container and a host, which compromises the use of container systems in genome research. Another issue is that one may not be able to use a single container system of the same version at all sites they use, which discourages the use of container systems. To this end, we present a pure rootless composable container system, LPMX, that provides composability for letting developers easily integrate tools in different existing containers or on host, allowing researchers to compose existing containers. LPMX is pure rootless, so it does not require root privilege neither during installation nor at runtime, allowing researchers to use LPMX across sites without asking permissions from administrators. LPMX provides a pure userspace layered filesystem with at least an order of magnitude lower overhead for launching a new process than existing container systems. LPMX can import Docker and Singularity images. Availability and Implementation: The source code of LPMX is available at https://github.com/jasonyangshadow/lpmx under Apache 2.0 License. Contact: mkasa@k.u-tokyo.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online.