Kyoko Yamane's research while affiliated with Gifu University and other places

In Japan, wasabi (Eutrema japonicum) is an important traditional condiment, and is recognized as an endemic species. In the present study, we generated a chromosome-level and haplotype-resolved reference genome for E. japonicum using PacBio CLR (continuous long reads), Illumina, and Hi-C sequencing data. The genome consists of 28 chromosomes that contain 1,512.1 Mb of sequence data, with a scaffold N50 length of 55.67 Mb. We also reported the subgenome and haplotype assignment of the 28 chromosomes by read-mapping and phylogenic analysis. Three validation methods (Benchmarking Universal Single-Copy Orthologs, Merqury, and Inspector) indicated that our obtained genome sequences were a high-quality and high-completeness genome assembly. Comparison of genome assemblies from previously published genomes showed that our obtained genome was of higher quality. Therefore, our genome will serve as a valuable genetic resource for both chemical ecology and evolution research of the genera Eutrema and Brassicaceae, as well as for wasabi breeding.

Wasabi (Japanese horseradish, Eutrema japonicum) is the only cultivated species in the genus Eutrema with functional components that provide a strong pungent flavor. To evaluate genetic resources for wasabi breeding, we surveyed variations in the two most abundant isothiocyanate (ITC) components in wasabi, allyl iso­thiocyanate (AITC) and 6-methylsulfinyl (hexyl) isothiocyanate (6-MSITC, hexaraphane). We also examined the phylogenetic relationships among 36 accessions of wild and cultivated wasabi in Japan using chloroplast DNA analysis. Our results showed that (i) the 6-MSITC content in currently cultivated wasabi accessions was significantly higher than in escaped cultivars, whereas the AITC content was not significantly different. (ii) Additionally, the 6-MSITC content in cultivated wasabi was significantly lower in the spring than during other seasons. This result suggested that the 6-MSITC content responds to environmental conditions. (iii) The phylogenetic position and the 6-MSITC content of accessions from Rebun, Hokkaido Prefecture had different profiles compared with those from southern Honshu, Japan, indicating heterogeneity of the Rebun populations from other Japanese wasabi accessions. (iv) The total content of AITC and 6-MSITC in cultivated wasabi was significantly higher than that of wild wasabi. In conclusion, old cultivars or landraces of wasabi, “zairai”, are the most suitable candidates for immediate use as genetic resources.

In Japan, two Eutrema species, wasabi (Eutrema japonicum, the important traditional Japanese condiment) and yuriwasabi (E. tenue), have been recognized as endemic species. We sequenced complete chloroplast (cp) genomes of seven wasabi and yuriwasabi accessions from Japan to study their phylogeny and evolution, using molecular dating of species divergence. Phylogenetic analyses of the complete cp DNA of these two Japanese species and five other Eurasian Eutrema species revealed that wasabi and yuriwasabi did not form a monophyletic group. One yuriwasabi accession (Gifu) formed a clade with E. yunnanense from China, indicating that this accession should be considered as a different species from the other yuriwasabi accessions. We reveal that Japanese Eutrema species diverged from the ‘E. yunnanense–yuriwasabi (Gifu)’ clade approximately 1.3 million years ago (Mya), suggesting that the connection between Japan and the Eurasian continent has existed more recently than the Quaternary period. The abundance of cp sequence data in this study also allowed the detection of genetic differentiation among wasabi cultivars. The two polymorphic sites detected between ‘Fujidaruma’ and ‘Shimane No.3’ were used to develop genotyping markers. The cp genome information provided here will thus inform the evolutionary histories of Japanese Eutrema species and help in genotyping wasabi cultivars.

Wasabi has been part of the Japanese diet for centuries, however, young people may be rejecting the traditional Japanese diet. In this study, we investigated whether the avoidance of wasabi in young people is an actual phenomenon using postal questionnaire surveys involving students in 15 agricultural high schools and residents in 3 senior care homes. Responses were obtained from 259 young students and 53 elderly people. A significantly higher rate of young students disliked wasabi compared with elderly subjects. A significantly higher rate of young female students disliked wasabi compared with young male students; the main reason was that female students experienced nasal irritation their noses stung after consumption. There was also a tendency for young students who liked wasabi to prefer fish to meat and to come from families that ate wasabi. The home environment was considered an important determinant of the preference. A previous study involving Korean and Japanese subjects, suggested a significant correlation between pungency preference and wasabi preference. The tendency to avoid wasabi and yet eat red pepper cannot be ignored if Japanese food culture is to survive. It is necessary to increase the opportunities for young Japanese people to eat raw wasabi

Chloroplast simple sequence repeats (cpSSRs) are widely distributed in the chloroplast genomes of all plant species, and are frequently employed for genotypic and phylogenetic analysis. However, information on intra- and interspecies variation in cpSSRs is lacking. In this study, we sequenced four intergenic (non-coding) chloroplast DNA regions in 57 accessions of 12 tetraploid, and 16 accessions of 4 hexaploid species of Triticum and Aegilops. These sequence data added to our previous data for diploid species in the same chloroplast regions. Intra- and interspecific genetic variation was analyzed for a total of 189 accessions of 13 diploid, 12 tetraploid, and 4 hexaploid species of Triticum and Aegilops, such that all species were represented by multiple accessions. The data were used to infer phylogenetic relationships within and among Triticum and Aegilops species. Based on this robust phylogenetic tree, seven of eight cpSSR loci clearly exhibited “size homoplasy,” referring to the fact that cpSSRs of identical size and DNA sequence can arise even if the alleles are not descended from a common ancestor. These data indicate that cpSSRs should be used with caution in phylogenetic analyzes. Interestingly, as observed from several previous studies, our data also suggest that observed mutation rates may increase significantly when mononucleotide (homopolymer) repeat numbers reach or exceed 9 bp. In the present report, using this sequence data set involving cpSSRs, 81 unique haplotypes among 189 accessions were detected, and five tetraploid Triticum and Aegilops species were successfully identified and genotyped. Our results indicate that combinations of nucleotide substitutions, indels and SSRs of chloroplast nucleotide sequences are available for genotyping at the species accession level.

We developed a multiplex PCR DNA marker for quick and easy identification of the AAGG-genome timopheevii lineage, including Triticum timopheevii , Triticum araraticum and hexaploid Triticum zhukovskyi (AAA m A m GG), and the AABB-genome emmer wheat lineage, including Triticum durum , Triticum dicoccum and Triticum dicoccoides . Distinguishing between tetraploid AAGG- and AABB-genome wheat species based on morphology is known to be difficult. This multiplex PCR system is based on the simultaneous PCR amplification of two chloroplast regions, matK and rbcL . The matK region molecularly distinguishes the two lineages, whereas the rbcL region is a positive control amplicon. We also examined whether the simple sequence repeat is a fixed mutation within species, using genetic resources in the collection of KOMUGI, Kyoto University, which comprises accessioned species collected across diverse geographical areas. The multiplex PCR marker distinguished AAGG from AABB species with complete accuracy.

This study examines the relationship between spike morphology and natural habitat for 84 accessions of four Aegilops species, belongs to section Sitopsis, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. sharonensis in genus Aegilops, section Sitopsis, wild relatives of Triticum aestivum L. These species are considered valuable genetic resources for future cultivation and breeding of domesticated wheat. The goals of the study were to: (1) document variation in spike morphology among these four species; (2) examine the relationship between spike morphology and native habitat; (3) document geographical distribution of distinct spike morphology; and (4) examine the relationship between spike morphology and heading time and value for these four species. The results reveal significant differences in spike morphology among species of section Sitopsis. The most noteworthy variation involved the absence/presence of lateral awn, such that species with lateral awn were restricted in coastal, though species without lateral awn were mainly distributed in inland. This suggests that local climate may be a determinant of variation in lateral awn, and that this trait may be subject to convergent evolution. Differences in heading time in sympatric area were also observed. The differences may enhance species divergence and could represent a lead speciation event. The results of this study will facilitate identification of populations or accessions of wild wheat with favorable traits and/or novel adaptive genes.

This paper reports the level of genetic differentiation between two Japanese and one Chinese species of Eutrema: E. japonicum, “wasabi”; its wild relative in Japan, E. tenue; and their wild relative in China, E. yunnanense. Phylogenetic analyses were based on the DNA sequence of the chloroplast trnK/matK region of 16 Brassicaceae and an outgroup species. Neighbor joining (NJ) and maximum parsimony (MP) trees were constructed, revealing that the three Eutrema species form a single clade clearly separated from other Brassicaceae species. The two Japanese Eutrema species are highly differentiated from Chinese E. yunnanense, and it is estimated that they diverged from E. yunnanense approximately 5 million years ago. An ethnobotanical survey was conducted among ethnic Chinese in Yunnan Province, and the results indicate that E. yunnanense is not perceived as “hot” in taste, while a pungent flavor is associated with wasabi; in addition, no evidence was found for the domestication of E. yunnanense. On the basis of the present molecular phylogenetic study and the ethnobotanical survey, we conclude that wasabi acquired its specific pungent flavor during its long botanical history in Japan, and that its subsequent domestication in Japan was because of this acquired pungent flavor. The culinary habit of using wasabi with raw fish has since become an important feature of Japanese cuisine and culture.