Hiroyuki Kakui

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.

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.

Sugi (Cryptomeria japonica D. Don) is an important forestry species in Japan. C. japonica ‘Spiralis’ is a mutant with twisted needles. The mutant is called Yore-sugi in Japan and is known as ‘Rasen’ in other countries. The twisted trait is regulated by a dominant gene called TWISTED NEEDLES, and it can be identified by observing the needles within 1 to 2 months after germination. The TWISTED NEEDLES gene may be useful for improving the efficiency of genome editing technology in C. japonica. In this study, we attempted to identify the linkage group of the TWISTED NEEDLES locus and investigate markers that sandwich this locus using the mapping family. First, we identified the linkage group containing the TWISTED NEEDLES locus based on the distortion from the expected segregation ratio using 32 mutant individuals of the mapping family. The segregation distortion showed that the TWISTED NEEDLES locus was located on the 11th linkage group (LG11). Next, a linkage map of LG11 was constructed based on genotype data from the single-nucleotide polymorphism (SNP) markers and double digested restriction site-associated DNA sequencing (ddRAD-seq) using 123 individuals of the MMY-1 family. On this map, six markers were located at the same position as the TWISTED NEEDLES locus. To investigate markers sandwiching the TWISTED NEEDLES locus, a partial linkage map around the TWISTED NEEDLES locus was constructed using 643 individuals of the MMY-1 family. The TWISTED NEEDLES locus was located in the 0.6 cM region between gSNP01822 and the other five markers (Contig_4705-179, Contig_4518-93, Contig_4398-118, gSNP04056, and Contig_4970-113).

Pollinosis, also known as pollen allergy or hay fever, is a global problem caused by pollen produced by various plant species ^1–6 . 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 ⁷ . The MS4 ( MALE STERILITY 4 ) 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 TKPR1 ( TETRAKETIDE α-PYRONE REDUCTASE ) gene in this region. Transformation experiments using Arabidopsis thaliana showed that single-nucleotide substitution of CjTKPR1 determines pollen production. Broad conservation of TKPR1 beyond plant division could lead to the creation of pollen-free plant not only for Japanese cedar but also for broader plant species.

Japanese cedar (Cryptomeria japonica) is the most important timber species in Japan; however, its pollen is the primary cause of pollinosis in Japan. The total number of pollen grains produced by a single tree is determined by the number of male strobili (male flowers) and the number of pollen grains per male strobilus. While the number of male strobili is a visible and well-investigated trait, little is known about the number of pollen grains per male strobilus. We hypothesized that genetic and environmental factors affect the pollen number per male strobilus and explored the factors that affect pollen production and genetic variation among clones. We counted pollen numbers of 523 male strobili from 26 clones using a cell counter method that we recently developed. Piecewise Structural Equation Modeling (pSEM) revealed that the pollen number is mostly affected by genetic variation, male strobilus weight, and pollen size. Although we collected samples from locations with different environmental conditions, statistical modeling succeeded in predicting pollen numbers for different clones sampled from branches facing different directions. Comparison of predicted pollen numbers revealed that they varied >3-fold among the 26 clones. The determination of the factors affecting pollen number and a precise evaluation of genetic variation will contribute to breeding strategies to counter pollinosis. Furthermore, the combination of our efficient counting method and statistical modeling will provide a powerful tool not only for Japanese cedar but also for other plant species.

Background: The determination of pollen number is important in evolutionary, agricultural, and medical studies. Tree species of the Cupressaceae family cause serious pollinosis worldwide. Although Japanese cedar (Cryptomeria japonica) is the most important forestry species in Japan, it is also the biggest cause of pollinosis in the country. Japanese cedar trees have been selected for growth speed and superior morphological traits and then cloned. These clones may vary in their pollen production, but there has been little research on how many pollen grains are produced by a single male strobilus (flower). A recently reported method for counting pollen number with a cell counter was applicable to Arabidopsis species and wheat, but was not suitable for Japanese cedar because the strobilus does not open with heating (e.g. 60 °C, overnight). Results: Here, we report an improved pollen counting method for Japanese cedar using a precise and rapid cell counter in combination with home-made mesh columns. The male strobilus was gently crushed using a pestle. Large and small debris were then removed using 100- and 20-μm mesh columns, respectively. We successfully detected pollen sizes and numbers that differed between two clones using this method. Conclusions: This improved method is not only suitable for counting pollen from Japanese cedar, but could also be applied to other species of the Cupressaceae family with hard scale tissue covering the pollen. Moreover, this method could be applied to a broader range of plant species, such as wheat, because there is no need to wait for anthesis and debris can be removed efficiently.

BackgroundThe determination of pollen number is important in evolutionary, agricultural, and medical studies. Tree species of the Cupressaceae family cause serious pollinosis worldwide. Although Japanese cedar (Cryptomeria japonica) is the most important forestry species in Japan, it is also the biggest cause of pollinosis in the country. Japanese cedar trees have been selected for growth speed and superior morphological traits and then cloned. These clones may vary in their pollen production, but there has been little research on how many pollen grains are produced by a single male strobilus (flower). A recently reported method for counting pollen number with a cell counter was applicable to Arabidopsis species and wheat, but was not suitable for Japanese cedar because the strobilus does not open with heating (e.g. 60°C, overnight). ResultsHere, we report an improved pollen counting method for Japanese cedar using a precise and rapid cell counter in combination with home-made mesh columns. The male strobilus was gently crushed using a pestle. Large and small debris were then removed using 100- and 20-μm mesh columns. We successfully detected pollen sizes and numbers that differed between two clones using this method. ConclusionsThis improved method is not only suitable for counting pollen from Japanese cedar, but could also be applied to other species of the Cupressaceae family with hard scale tissue covering the pollen. Moreover, this method could be applied to a broader range of plant species, such as wheat, because there is no need to wait for anthesis and debris can be removed efficiently.

The number of pollen grains is a critical part of the reproductive strategies in plants and varies greatly between and within species. In agriculture, pollen viability is important for crop breeding. It is a laborious work to count pollen tubes using a counting chamber under a microscope. Here, we present a method of counting the number of pollen grains using a cell counter. In this method, the counting step is shortened to 3 min per flower, which, in our setting, is more than five times faster than the counting chamber method. This technique is applicable to species with a lower and higher number of pollen grains, as it can count particles in a wide range, from 0 to 20,000 particles, in one measurement. The cell counter also estimates the size of the particles together with the number. Because aborted pollen shows abnormal membrane characteristics and/or a distorted or smaller shape, a cell counter can quantify the number of normal and aborted pollen separately. We explain how to count the number of pollen grains and measure pollen size in Arabidopsis thaliana, Arabidopsis kamchatica, and wheat (Triticum aestivum).

The number of male gametes is critical for reproductive success and varies between and within species. The evolutionary reduction of the number of pollen grains encompassing the male gametes is widespread in selfing plants. Here, we employ genome-wide association study (GWAS) to identify underlying loci and to assess the molecular signatures of selection on pollen number-associated loci in the predominantly selfing plant Arabidopsis thaliana. Regions of strong association with pollen number are enriched for signatures of selection, indicating polygenic selection. We isolate the gene REDUCED POLLEN NUMBER1 (RDP1) at the locus with the strongest association. We validate its effect using a quantitative complementation test with CRISPR/Cas9-generated null mutants in nonstandard wild accessions. In contrast to pleiotropic null mutants, only pollen numbers are significantly affected by natural allelic variants. These data support theoretical predictions that reduced investment in male gametes is advantageous in predominantly selfing species.

Polyploidization has played an important role in the speciation and diversification of plant species. However, genetic analyses of polyploids are challenging because the vast majority of the model species are diploids. The allotetraploid Arabidopsiskamchatica, which originated through the hybridization of the diploid Arabidopsis halleri and Arabidopsis lyrata, is an emerging model system for studying various aspects of polyploidy. However, a transgenic method that allows the insertion of a gene of interest into A. kamchatica is still lacking. In this study, we investigated the early development of pistils in A. kamchatica and confirmed the formation of open pistils in young flower buds (stages 8–9), which is important for allowing Agrobacterium to access female reproductive tissues. We established a simple Agrobacterium-mediated floral dip transformation method to transform a gene of interest into A. kamchatica by dipping A. kamchatica inflorescences bearing many young flower buds into a 5% sucrose solution containing 0.05% Silwet L-77 and Agrobacterium harboring the gene of interest. We showed that a screenable marker comprising fluorescence-accumulating seed technology with green fluorescent protein was useful for screening the transgenic seeds of two accessions of A. kamchatica subsp. kamchatica and an accession of A. kamchatica subsp. kawasakiana.