Yasuo Yasui

S-LOCUS EARLY FLOWERING 3 is exclusively present in the genomes of short-styled buckwheat plants that exhibit heteromorphic self-incompatibility.

The different forms of flowers in a species have attracted the attention of many evolutionary biologists, including Charles Darwin. In Fagopyrum esculentum (common buckwheat), the occurrence of dimorphic flowers, namely short-styled and long-styled flowers, is associated with a type of self-incompatibility (SI) called heteromorphic SI. The floral morphology and intra-morph incompatibility are both determined by a single genetic locus named the S-locus. Plants with short-styled flowers are heterozygous (S/s) and plants with long-styled flowers are homozygous recessive (s/s) at the S-locus. Despite recent progress in our understanding of the molecular basis of flower development and plant SI systems, the molecular mechanisms underlying heteromorphic SI remain unresolved. By examining differentially expressed genes from the styles of the two floral morphs, we identified a gene that is expressed only in short-styled plants. The novel gene identified was completely linked to the S-locus in a linkage analysis of 1,373 plants and had homology to EARLY FLOWERING 3. We named this gene S-LOCUS EARLY FLOWERING 3 (S-ELF3). In an ion-beam-induced mutant that harbored a deletion in the genomic region spanning S-ELF3, a phenotype shift from short-styled flowers to long-styled flowers was observed. Furthermore, S-ELF3 was present in the genome of short-styled plants and absent from that of long-styled plants both in world-wide landraces of buckwheat and in two distantly related Fagopyrum species that exhibit heteromorphic SI. Moreover, independent disruptions of S-ELF3 were detected in a recently emerged self-compatible Fagopyrum species and a self-compatible line of buckwheat. The nonessential role of S-ELF3 in the survival of individuals and the prolonged evolutionary presence only in the genomes of short-styled plants exhibiting heteromorphic SI suggests that S-ELF3 is a suitable candidate gene for the control of the short-styled phenotype of buckwheat plants.

Draft genome sequence of an inbred line of Chenopodium quinoa, an allotetraploid crop with great environmental adaptability and outstanding nutritional properties

Chenopodium quinoa Willd. (quinoa) originated from the Andean region of South America, and is a pseudocereal crop of the Amaranthaceae family. Quinoa is emerging as an important crop with the potential to contribute to food security worldwide and is considered to be an optimal food source for astronauts, due to its outstanding nutritional profile and ability to tolerate stressful environments. Furthermore, plant pathologists use quinoa as a representative diagnostic host to identify virus species. However, molecular analysis of quinoa is limited by its genetic heterogeneity due to outcrossing and its genome complexity derived from allotetraploidy. To overcome these obstacles, we established the inbred and standard quinoa accession Kd that enables rigorous molecular analysis, and presented the draft genome sequence of Kd, using an optimized combination of high-throughput next generation sequencing on the Illumina Hiseq 2500 and PacBio RS II sequencers. The de novo genome assembly contained 25 k scaffolds consisting of 1 Gbp with N50 length of 86 kbp. Based on these data, we constructed the free-access Quinoa Genome DataBase (QGDB). Thus, these findings provide insights into the mechanisms underlying agronomically important traits of quinoa and the effect of allotetraploidy on genome evolution.

Assembly of the draft genome of buckwheat and its applications in identifying agronomically useful genes

Buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is a nutritionally dense annual crop widely grown in temperate zones. To accelerate molecular breeding programmes of this important crop, we generated a draft assembly of the buckwheat genome using short reads obtained by next-generation sequencing (NGS), and constructed the Buckwheat Genome DataBase. After assembling short reads, we determined 387,594 scaffolds as the draft genome sequence (FES_r1.0). The total length of FES_r1.0 was 1,177,687,305 bp, and the N50 of the scaffolds was 25,109 bp. Gene prediction analysis revealed 286,768 coding sequences (CDSs; FES_r1.0_cds) including those related to transposable elements. The total length of FES_r1.0_cds was 212,917,911 bp, and the N50 was 1,101 bp. Of these, the functions of 35,816 CDSs excluding those for transposable elements were annotated by BLAST analysis. To demonstrate the utility of the database, we conducted several test analyses using BLAST and keyword searches. Furthermore, we used the draft genome as a reference sequence for NGS-based markers, and successfully identified novel candidate genes controlling heteromorphic self-incompatibility of buckwheat. The database and draft genome sequence provide a valuable resource that can be used in efforts to develop buckwheat cultivars with superior agronomic traits.

QTL analysis of heterostyly in Primula sieboldii and its application for morph identification in wild populations

BACKGROUND AND AIMS Primula sieboldii is a perennial clonal herb that is distributed around the Sea of Japan and is endangered in Japan. Its breeding system is characterized by heteromorphic self-incompatibility, and the morph ratio within a population is very important for reproductive success. The aims of this study were to construct a linkage map, map the S locus as a qualitative trait and quantitative trait loci (QTLs) for floral morphological traits related to heterostyly, and predict the morph type in wild populations by using molecular markers for devising a conservation strategy. METHODS A linkage map was constructed with 126 markers. The QTLs for four floral traits and the S locus were mapped. Using the genotypes of loci that were located near both the S locus and the QTLs with large effects, morphs of 59 wild genets were predicted. KEY RESULTS The linkage map consisted of 14 linkage groups (LGs). The S locus was mapped to LG 7. Major QTLs for stigma and anther heights were detected in the same region as the S locus. These QTLs exhibited high logarithm of the odds scores and explained a high percentage of the phenotypic variance (>85 %). By analysing these two traits within each morph, additional QTLs for each trait were detected. Using the four loci linked to the S locus, the morphs of 43 genets in three wild populations could be predicted. CONCLUSIONS This is the first report of a linkage map and QTL analysis for floral morphology related to heterostyly in P. sieboldii. Floral morphologies related to heterostyly are controlled by the S locus in LG 7 and by several QTLs in other LGs. Additionally, this study showed that molecular markers are effective tools for investigating morph ratios in a population containing the non-flowering individuals or during the non-flowering seasons.

Isolation and characterization of genes encoding leucoanthocyanidin reductase (FeLAR) and anthocyanidin reductase (FeANR) in buckwheat (Fagopyrum esculentum).

Proanthocyanidins (PAs) are a major group of flavonoids synthesized via the phenylpropanoid biosynthesis pathway, however the pathway has not been fully characterized in buckwheat. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are involved in the last steps of PA biosynthesis. To isolate the genes for these enzymes from buckwheat we performed PCR using degenerate primers and obtained cDNAs of ANR and LAR, which we designated FeANR and FeLAR1. A search for homologs in a buckwheat genome database with both sequences returned two more LAR sequences, designated FeLAR2 and FeLAR3. Linkage analysis with an F2 segregating population indicated that the three LAR loci were not genetically linked. We detected high levels of PAs in roots and cotyledons of buckwheat seedlings and in buds and flowers of mature plants. FeANR and FeLAR1-3 were expressed in most organs but had different expression patterns. Our findings would be useful for breeding and further analysis of PA synthesis and its regulation in buckwheat.

Diversification of 13S globulins, allergenic seed storage proteins, of common buckwheat.

The α polypeptide of the 13S globulin subunit of common buckwheat is the counterpart of the major allergenic β polypeptide. Trypsin digestibility varies between variants of the α polypeptide with and without a tandem repeat insert. To evaluate the intra-species diversity of 13S globulin, the comprehensive screening of a genomic DNA library was performed, resulting in the isolation of 14 and 3 genes for Met-poor and Met-rich subunits, respectively. Although most tandem repeat units were 45 bp in length, the two-repeat gene Glb2B and all one-repeat genes contained an additional 3 bp. Secondary structure predictions and polyacrylamide gel electrophoresis demonstrated that the sense strand of Glb2B-CCG, the additional 3 bp-deletion clone of Glb2B, formed a more rigid secondary structure than that of the wild-type. Thus, the large intra-species variation of 13S globulin revealed in this study and its diversification might be attributable to the unique nature of the tandem repeat sequences.

Phylogenetic analysis of AGAMOUS sequences reveals the origin of the diploid and tetraploid forms of self-pollinating wild buckwheat, Fagopyrum homotropicum Ohnishi

Fagopyrum homotropicum Ohnishi is a self-pollinating wild buckwheat species indigenous to eastern Tibet and the Yunnan and Sichuan Provinces of China. It is useful breeding material for shifting cultivated buckwheat (F. esculentum ssp. esculentum Moench) from out-crossing to self-pollinating. Despite its importance as a genetic resource in buckwheat breeding, the genetic variation of F. homotropicum is poorly understood. In this study, we investigated the genetic variation and phylogenetic relationships of the diploid and tetraploid forms of F. homotropicum based on the nucleotide sequences of a nuclear gene, AGAMOUS (AG). Neighbor-joining analysis revealed that representative individuals clustered into three large groups (Group I, II and III). Each group contained diploid and tetraploid forms of F. homotropicum. We identified tetraploid plants that had two diverged AG sequences; one belonging to Group I and the other belonging to Group II, or one belonging to Group II and the other belonging to Group III. These results suggest that the tetraploid form originated from at least two hybridization events between deeply differentiated diploids. The results also imply that the genetic diversity contributed by tetraploidization of differentiated diploids may have allowed the distribution range of F. homotropicum to expand to the northern areas of China.

Rapid genotyping with DNA micro-arrays for high-density linkage mapping and QTL mapping in common buckwheat (Fagopyrum esculentum Moench)

For genetic studies and genomics-assisted breeding, particularly of minor crops, a genotyping system that does not require a priori genomic information is preferable. Here, we demonstrated the potential of a novel array-based genotyping system for the rapid construction of high-density linkage map and quantitative trait loci (QTL) mapping. By using the system, we successfully constructed an accurate, high-density linkage map for common buckwheat (Fagopyrum esculentum Moench); the map was composed of 756 loci and included 8,884 markers. The number of linkage groups converged to eight, which is the basic number of chromosomes in common buckwheat. The sizes of the linkage groups of the P1 and P2 maps were 773.8 and 800.4 cM, respectively. The average interval between adjacent loci was 2.13 cM. The linkage map constructed here will be useful for the analysis of other common buckwheat populations. We also performed QTL mapping for main stem length and detected four QTL. It took 37 days to process 178 samples from DNA extraction to genotyping, indicating the system enables genotyping of genome-wide markers for a few hundred buckwheat plants before the plants mature. The novel system will be useful for genomics-assisted breeding in minor crops without a priori genomic information.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity

BackgroundDihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood.ResultsBy using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds.ConclusionsWe isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Buckwheat R2R3 MYB transcription factor FeMYBF1 regulates flavonol biosynthesis

Buckwheat (Fagopyrum esculentum) contains high amounts of flavonoids, especially flavonols (e.g., rutin), which are thought to be highly beneficial for human health. Little is known, however, about the regulation of flavonol synthesis in buckwheat. We identified a buckwheat gene encoding an R2R3 MYB transcription factor, and named this gene FeMYBF1. Analysis of the deduced amino acid sequence and phylogenetic analysis suggested that FeMYBF1 encodes an ortholog of the Arabidopsis flavonol regulators AtMYB11, AtMYB12 and AtMYB111. Expression of FeMYBF1 in a flavonol-deficient Arabidopsis triple mutant (myb11 myb12 myb111) restored flavonol synthesis. Constitutive expression of FeMYBF1 driven by the CaMV 35S promoter in Arabidopsis resulted in over-accumulation of flavonol glycosides and upregulation of the expression of AtFLS1. Transient expression assays showed that FeMYBF1 activated the promoter of the Arabidopsis gene encoding AtFLS1, and the promoters of buckwheat genes related to anthocyanin and proanthocyanidin synthesis such as dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX) in addition to genes encoding FLS. The results indicate that FeMYBF1 regulates flavonol synthesis and may have a role in synthesis of other flavonoid compounds, and also that buckwheat may have alternative pathway of flavonol synthesis through DFR and LDOX.