Chiaki Muto

The power of single molecule real-time sequencing technology in the de novo assembly of a eukaryotic genome

Second-generation sequencers (SGS) have been game-changing, achieving cost-effective whole genome sequencing in many non-model organisms. However, a large portion of the genomes still remains unassembled. We reconstructed azuki bean (Vigna angularis) genome using single molecule real-time (SMRT) sequencing technology and achieved the best contiguity and coverage among currently assembled legume crops. The SMRT-based assembly produced 100 times longer contigs with 100 times smaller amount of gaps compared to the SGS-based assemblies. A detailed comparison between the assemblies revealed that the SMRT-based assembly enabled a more comprehensive gene annotation than the SGS-based assemblies where thousands of genes were missing or fragmented. A chromosome-scale assembly was generated based on the high-density genetic map, covering 86% of the azuki bean genome. We demonstrated that SMRT technology, though still needed support of SGS data, achieved a near-complete assembly of a eukaryotic genome.

Novel Genetic Resources in the Genus Vigna Unveiled from Gene Bank Accessions.

The genus Vigna (Fabaceae) consists of five subgenera, and includes more than 100 wild species. In Vigna, 10 crops have been domesticated from three subgenera, Vigna, Plectrotropis, and Ceratotropis. The habitats of wild Vigna species are so diverse that their genomes could harbor various genes responsible for environmental stress adaptation, which could lead to innovations in agriculture. Since some of the gene bank Vigna accessions were unidentified and they seemed to be novel genetic resources, these accessions were identified based on morphological traits. The phylogenetic positions were estimated based on the DNA sequences of nuclear rDNA-ITS and chloroplast atpB-rbcL spacer regions. Based on the results, the potential usefulness of the recently described species V. indica and V. sahyadriana, and some wild Vigna species, i.e., V. aconitifolia, V. dalzelliana, V. khandalensis, V. marina var. oblonga, and V. vexillata, was discussed.

Reciprocal translocation identified in Vigna angularis dominates the wild population in East Japan

Using an F2 population derived from cultivated and wild azuki bean, we previously detected a reciprocal translocation and a seed size QTL near the translocation site. To test the hypothesis that the translocation in the cultivated variety contributed to the larger seed size, we performed further linkage analyses with several cross combinations between cultivated and wild azuki beans. In addition, we visually confirmed the translocation by cytogenetic approach using 25 wild and cultivated accessions. As a result, we found the translocation-type chromosomes in none of the cultivated accessions, but in a number of the wild accessions. Interestingly, all the wild accessions with the translocation were originally collected from East Japan, while all the accessions with normal chromosomes were from West Japan or the Sea of Japan-side region. Such biased geographical distribution could be explained by the glacial refugium hypothesis, and supported narrowing down the domestication origin of cultivated azuki bean.

Salt tolerance in wild relatives of adzuki bean, Vigna angularis (Willd.) Ohwi et Ohashi

Salt stress is becoming a serious problem in food production field. To find sources of salt tolerance, we screened 74 accessions of adzuki bean (Vigna angularis) and 145 accessions of cross-compatible wild relatives (seven species). We performed the primary screening in soil culture and the secondary screening in hydroponic culture, and identified JP205833 of V. riukiuensis (strain ‘Tojinbaka’) and JP107879 of V. nakashimae (strain ‘Ukushima’) as the valuable source of salt tolerance. We found these two strains had different salt tolerance mechanism, where ‘Ukushima’ prevented Na+ accumulation in leaves by filtering Na+ in roots and stems, while ‘Tojinbaka’ accumulated Na+ throughout the whole plant body. We also found ‘Tojinbaka’ and ‘Ukushima’ could retain photosynthesis even under salt stress. In addition, ‘Ukushima’ and especially ‘Tojinbaka’ showed even better growth in a salt-damaged field in Fukushima, Japan where soybean cultivar ‘Tachinagaha’ could not survive. Since both salt tolerant strains are cross-compatible with adzuki bean, our results will facilitate developing salt tolerant cultivar by introducing two different mechanisms of salt tolerance.

Diversity and Evolution of Salt Tolerance in the Genus Vigna

Breeding salt tolerant plants is difficult without utilizing a diversity of wild crop relatives. Since the genus Vigna (family Fabaceae) is comprised of many wild relatives adapted to various environmental conditions, we evaluated the salt tolerance of 69 accessions of this genus, including that of wild and domesticated accessions originating from Asia, Africa, Oceania, and South America. We grew plants under 50 mM and 200 mM NaCl for two weeks and then measured the biomass, relative quantum yield of photosystem II, leaf Na+ concentrations, and leaf K+ concentrations. The accessions were clustered into four groups: the most tolerant, tolerant, moderately susceptible, and susceptible. From the most tolerant group, we selected six accessions, all of which were wild accessions adapted to coastal environments, as promising sources of salt tolerance because of their consistently high relative shoot biomass and relative quantum yield. Interestingly, variations in leaf Na+ concentration were observed between the accessions in the most tolerant group, suggesting different mechanisms were responsible for their salt tolerance. Phylogenetic analysis with nuclear DNA sequences revealed that salt tolerance had evolved independently at least four times in the genus Vigna, within a relatively short period. The findings suggested that simple genetic changes in a few genes might have greatly affected salt tolerances. The elucidation of genetic mechanisms of salt tolerances in the selected accessions may contribute to improving the poor salt tolerance in legume crops.

A Homoploid Hybrid Between Wild Vigna Species Found in a Limestone Karst.

Genus Vigna comprise several domesticated species including cowpea and mungbean, and diverse wild species. We found an introgressive hybrid population derived from two wild species, Vigna umbellata and Vigna exilis, in Ratchaburi district, Thailand. The hybrid was morphologically similar to V. umbellata but habituated in a limestone rock mountain, which is usually dominated by V. exilis. Analyzing simple sequence repeat loci indicated the hybrid has undergone at least one round of backcross by V. umbellata. We found the hybrid acquired vigorous growth from V. umbellata and drought tolerance plus early flowering from V. exilis, and thus has taken over some habitats of V. exilis in limestone karsts. Given the wide crossability of V. umbellata, the hybrid can be a valuable genetic resource to improve drought tolerance of some domesticated species.

Synthesis of Sulfo-Sialic Acid Analogues: Potent Neuraminidase Inhibitors in Regards to Anomeric Functionality

The design, synthesis and application of N-acetylneuraminic acid-derived compounds bearing anomeric sulfo functional groups are described. These novel compounds, which we refer to as sulfo-sialic acid analogues, include 2-decarboxy-2-deoxy-2-sulfo-N-acetylneuraminic acid and its 4-deoxy-3,4-dehydrogenated pseudoglycal. While 2-decarboxy-2-deoxy-2-sulfo-N-acetylneuraminic acid contains no further modifications of the 2-deoxy-pyranose ring, it is still a more potent inhibitor of avian-origin H5N1 neuraminidase (NA) and drug-resistant His275Tyr NA as compared to the oxocarbenium ion transition state analogue 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The sulfo-sialic acid analogues described in this report are also more potent inhibitors of influenza NA (up to 40-fold) and bacterial NA (up to 8.5-fold) relative to the corresponding anomeric phosphonic acids. These results confirm that this novel anomeric sulfo modification offers great potential to improve the potency of next-generation NA inhibitors including covalent inhibitors.

A new taxonomic treatment for some wild relatives of mungbean (Vigna radiata (L.) Wilcz.) based on their molecular phylogenetic relationships and morphological variations

Mungbean (Vigna radiata (L.) Wilcz.) is an important leguminous crop cultivated mainly in Asia. Its wild relatives are considered useful genetic resources for mungbean breeding. However, the taxonomic history of mungbean and its wild relatives is complicated and some confusion is still present in recent publications. In this study, we examined the rDNA-ITS sequences and morphological characteristics of 83 gene bank accessions closely related to mungbean. As a result, we classified the 83 accessions into five species and one unclassified accession. The proper taxon name for each species was determined (Vigna grandiflora (Prain) Tateishi et Maxted, Vigna mungo (L.) Hepper, V. radiata, Vigna subramaniana (Babu ex Raizada) Raizada, and Vigna trinervia (Heyne ex Wight et Arn.) Tateishi et Maxted) based on a review of their taxonomic history and morphological comparisons between gene bank accessions and type specimens. A new taxonomic treatment is proposed and a morphological key has been prepared. In this treatment, Vigna radiata (L.) Wilcz. var. setulosa (Dalz.) Ohwi et Ohashi is treated as a synonym of Vigna radiata (L.) Wilcz. var. sublobata (Roxb.) Verdc., and Vigna hainiana Babu, Gopinathan et Sharma is a synonym of V. subramaniana. Accession ‘NI1135’ was revealed to be most closely related to, but is considerably differentiated from, mungbean (V. radiata) based on its rDNA-ITS sequences. It also has distinguishing morphological characters. Plants with similar morphologies and DNA profiles might be distributed in the Indian Himalaya. However, since only one accession is available at present, the taxonomic classification of ‘NI1135’ needs to be reconsidered in the future.

Diversity of drought tolerance in the genus Vigna

Wild relatives of crop plants are thought as reservoir of prominent genetic resources for abiotic stress tolerance. However, insufficient information on genetic variation and phenotypic traits restricts their use for crop breeding. This study focused on wild species of genus Vigna (family Fabaceae) originated from highly humid to arid regions. To clarify the diversity of drought tolerance during the vegetative stage, 69 accessions, including 15 domesticated, and 54 wild accessions, were evaluated under two drought conditions of non-terminal and terminal stresses. In the non-terminal drought condition, the plants were grown in pipes of different heights where surface soil water content decreased faster in pipes with greater height. Relative shoot biomass was used for tolerance evaluation and we identified 19 drought tolerant accessions. Almost of them were wild accessions showing higher relative shoot biomass than that in the domesticated accessions. Domesticated species were mostly classified as drought susceptible but could be improved using tolerant conspecific wild ancestors with cross-compatibility. The tolerance was related with higher plant water status presumably due to small water consumption. However, the variation of drought tolerance could not be explained by simple tolerance factor alone, and other tolerance mechanisms such as deep rooting and increasing in root biomass were found in the tolerant accessions. In the terminal drought condition, the plants were grown in small pots, and the watering was stopped to expose them extreme and rapid soil water scarcity. The tolerance was evaluated as the number of days until wilting. However, the accessions found to be tolerant in the pot experiment were not the same as those in the pipe experiment. In this condition, plant water status was not related with the length of days to wilting. This indicates that different mechanisms are necessary for adaptation to each of the non-terminal and terminal drought conditions. Many accessions were tolerant to one of the conditions, although we identified that some accessions showed tolerance in both experiments. The great diversity in drought tolerance in the genus Vigna might serve to both improve crop drought tolerance and understand the mechanisms of adaptation in drought-prone environments.