Estimation of pollen dispersal distance in Job's tears (Coix lacryma-jobi L.) by using red leaf sheath as a morphological marker.

Job's tears (Coix lacryma-jobi L.) is grown widely in Asian countries and a crop that can fertilize with own pollen and pistils. The grains are used not only for food but also for medicinal purposes. The grain of many cultivars contains glutinous endosperm; only grains with this glutinous endosperm are suitable for use as medicine in Japan. Many wild types have non-glutinous endosperm and can easily cross with cultivar under natural environmental conditions. Because the non-glutinous endosperm trait is dominant to that of glutinous endosperm, F1 seeds produced by crosses between a cultivar and a wild type have non-glutinous endosperm. To reduce the rate of unwanted crosses, we investigated the pollen dispersal distance by using a red leaf sheath as a morphological marker. When plants were cultivated in rows 70 cm apart, the crossing rate was about 25%-35%. As the distance increased, the crossing rate decreased at a rate that could be fitted to a power approximation in fields without intervening plants and to an exponential equation in fields with intervening plants. Our data could be used as guidelines for preventing unwanted crossing with wild types when growing cultivars.

Genome sequencing reveals the genetic architecture of heterostyly and domestication history of common buckwheat.

Common buckwheat, Fagopyrum esculentum, is an orphan crop domesticated in southwest China that exhibits heterostylous self-incompatibility. Here we present chromosome-scale assemblies of a self-compatible F. esculentum accession and a self-compatible wild relative, Fagopyrum homotropicum, together with the resequencing of 104 wild and cultivated F. esculentum accessions. Using these genomic data, we report the roles of transposable elements and whole-genome duplications in the evolution of Fagopyrum. In addition, we show that (1) the breakdown of heterostyly occurs through the disruption of a hemizygous gene jointly regulating the style length and female compatibility and (2) southeast Tibet was involved in common buckwheat domestication. Moreover, we obtained mutants conferring the waxy phenotype for the first time in buckwheat. These findings demonstrate the utility of our F. esculentum assembly as a reference genome and promise to accelerate buckwheat research and breeding.

Development and chromosomal characterization of interspecific hybrids between common buckwheat (Fagopyrum esculentum) and a related perennial species (F. cymosum).

Common buckwheat (Fagopyrum esculentum) is an annual self-incompatible plant that is widely grown. The genus Fagopyrum comprises more than 20 species, including F. cymosum, a perennial that, unlike common buckwheat, is highly resistant to excess water. In this study, we developed interspecific hybrids between F. esculentum and F. cymosum via embryo rescue, to improve undesirable traits of common buckwheat, such as low tolerance to excess water. The interspecific hybrids were confirmed by genomic in situ hybridization (GISH). We also developed DNA markers to confirm the identity of the hybrids and if genes derived from each genome were inherited by the next generation. Observations of pollen indicated that the interspecific hybrids were essentially sterile. Unpaired chromosomes and abnormal segregation during meiosis were likely responsible for the pollen sterility of the hybrids. These findings could facilitate buckwheat breeding to produce lines that can withstand harsh environments with wild or related species in the genus Fagopyrum.

Genetic basis of maturity time is independent from that of flowering time and contributes to ecotype differentiation in common buckwheat (Fagopyrum esculentum Moench).

BACKGROUND: Common buckwheat is considered a quantitative short-day plant and is classified into the autumn (highly photoperiod sensitive), summer (weakly photoperiod sensitive), and intermediate ecotype. Understanding ecotype differentiation is essential for adaptive expansion and maximizing yield. The genetic analysis for ecotype has focused on photoperiod-dependent flowering time, whereas post-flowering traits such as seed set and maturity time might also regulate ecotype differentiation. RESULTS: A field experiment revealed that ecotype differentiation is mainly defined by the timing of seed set and maturation, whereas flowering time is less relevant. Thus, we focused on maturity time as a trait that defines the ecotype. To detect QTLs for maturity time, we developed two F2 populations derived from early × late-maturing accessions and intermediate × late-maturing accessions. Using genotyping by random amplicon sequencing-direct analysis, we generated a high-density linkage map. QTL analysis detected two major QTLs for maturity time, one in each F2 population. We also detected QTLs for flowering time at loci different from maturity time QTLs, which suggests that different genetic mechanisms regulate flowering and maturity. Association analysis showed that both QTLs for maturity time were significantly associated with variations in the trait across years. CONCLUSIONS: Maturity time appeared to be more suitable for explaining ecotype differentiation than flowering time, and different genetic mechanisms would regulate the timing of flowering and maturation. The QTLs and QTL-linked markers for maturity time detected here may be useful to extend the cultivation area and to fine-tune the growth period to maximize yield in buckwheat.

Determination of specific life changes on psychological distress during the COVID-19 pandemic.

The COVID-19 pandemic might affect many aspects of the community and a range of psychiatric risk factors due to life changes, including people's behaviors and perceptions. In this study, we aim to identify specific life changes that correlate with psychological distress within the social context of the COVID-19 pandemic in Japan. In July 2020, workers (company employees and civil servants) in Japan were recruited from local institutions that had not had any confirmed COVID-19 cases as well as neighborhoods that had only a few cases. Participants completed a COVID-19 mental health survey (N = 609; 66.9% male). Psychological distress was identified based on Kessler-6 scores (≥13). Life changes were assessed by an open-ended question about life changes in participants and their family, workplace, and community due to the COVID-19 pandemic. A convergent mixed-method approach was used to compare the context of perceived life changes in participants with psychological distress and those without. As a result, 8.9% of participants had psychological distress, and sex and age categories were different between those with psychological distress and those without. Among the participants who responded to the open-ended question, the biggest life change was "staying at home," and the next biggest life changes were "event cancellations" and "increased workload" in participants with psychological distress, and "no changes" and "mask-wearing" in those without psychological distress, respectively. Regarding emotional/perceptual changes, "stress," "fear," and "anger" were more frequently reported by participants with psychological distress than those without (P <0.001). By integrating these findings, we identified themes focusing on vulnerable characteristics related to psychological distress. This study may provide a source in society for mediating psychological distress during a pandemic.

Targeted amplicon sequencing + next-generation sequencing-based bulked segregant analysis identified genetic loci associated with preharvest sprouting tolerance in common buckwheat (Fagopyrum esculentum).

BACKGROUND: Common buckwheat (2n = 2x = 16) is an outcrossing pseudocereal whose seeds contain abundant nutrients and potential antioxidants. As these beneficial compounds are damaged by preharvest sprouting (PHS) and PHS is likely to increase with global warming, it is important to find efficient ways to develop new PHS-tolerant lines. However, genetic loci and selection markers associated with PHS in buckwheat have not been reported. RESULTS: By next-generation sequencing (NGS) of whole-genome of parental lines, we developed a genome-wide set of 300 markers. By NGS- based bulked segregant analysis (NGS-BSA), we developed 100 markers linked to PHS tolerance. To confirm the effectiveness of marker development from NGS-BSA data, we developed 100 markers linked to the self-compatibility (SC) trait from previous NGS-BSA data. Using these markers, we developed genetic maps with AmpliSeq technology, which can quickly detect polymorphisms by amplicon-based multiplex targeted NGS, and performed quantitative trait locus (QTL) analysis for PHS tolerance in combination with NGS-BSA. QTL analysis detected two major and two minor QTLs for PHS tolerance in a segregating population developed from a cross between the PHS-tolerant 'Kyukei 29' and the self-compatible susceptible 'Kyukei SC7'. We found different major and minor QTLs in other segregating populations developed from the PHS-tolerant lines 'Kyukei 28' and 'NARO-FE-1'. Candidate markers linked to PHS developed by NGS-BSA were located near these QTL regions. We also investigated the effectiveness of markers linked to these QTLs for selection of PHS-tolerant lines among other segregating populations. CONCLUSIONS: We efficiently developed genetic maps using a method combined with AmpliSeq technology and NGS-BSA, and detected QTLs associated with preharvest sprouting tolerance in common buckwheat. This is the first report to identify QTLs for PHS tolerance in buckwheat. Our marker development system will accelerate genetic research and breeding in common buckwheat.

Buckwheat heteromorphic self-incompatibility: genetics, genomics and application to breeding.

Common buckwheat (Fagopyrum esculentum Moench 2n = 2x = 16) is an outcrossing crop with heteromorphic self-incompatibility due to its distylous flowers, called pin and thrum. In pin plants, a long style is combined with short stamens and small pollen grains; in thrum plants, a short style is combined with long stamens and large pollen grains. Both the intra-morph self-incompatibility and flower morphology are controlled by a single genetic locus named the S locus; thrum plants are heterozygous (Ss) and pin plants are homozygous recessive (ss) at this locus. Self-incompatibility is an obstacle for establishing pure lines and fixation of agronomically useful genes. Elucidation of the molecular mechanism of heterostylous self-incompatibility of common buckwheat has continued for a quarter of a century. Recent advances in genomic and transcriptomic analyses using next-generation sequencing have made it possible to determine the genomic region harboring the buckwheat S locus and to identify novel genes at this locus. In this review, we summarize the current knowledge on buckwheat heterostyly gained from conventional and molecular genetics and genomics. We also discuss the application of these studies to breeding of common buckwheat.

Biosynthesis and regulation of flavonoids in buckwheat.

Buckwheat contains an abundance of antioxidants such as polyphenols and is considered a functional food. Among polyphenols, flavonoids have multiple functions in various aspects of plant growth and in flower and leaf colors. Flavonoids have antioxidant properties, and are thought to prevent cancer and cardiovascular disease. Here, we summarize the flavonoids present in various organs and their synthesis in buckwheat. We discuss the use of this information to breed highly functional and high value cultivars.

Development of co-dominant markers linked to a hemizygous region that is related to the self-compatibility locus (S) in buckwheat (Fagopyrum esculentum).

Common buckwheat (Fagopyrum esculentum) is a heterostylous self-incompatible (SI) species with two different flower morphologies, pin and thrum. The SI trait is controlled by a single gene complex locus, S. Self-compatible (SC) lines were developed by crossing F. esculentum and F. homotropicum; these lines have an SC gene, Sh , which is dominant over the s allele and recessive to the S allele. S-ELF3 has been identified as a candidate gene in the S locus and is present in the S and Sh but not s alleles. A single-nucleotide deletion in the S-ELF3 gene of the Sh allele results in a frame shift. To develop co-dominant markers to distinguish between ShSh and Shs plants, we performed a next-generation sequencing analysis in combination with bulked-segregant analysis. We developed four co-dominant markers linked to the S locus. We investigated the polymorphism frequency between a self-compatible line and leading Japanese buckwheat cultivars. Linkage between a developed sequence-tagged-site marker and flower morphology was confirmed using more than 1000 segregating plants and showed no recombination. The developed markers would be useful for buckwheat breeding and also to produce lines for genetic analysis such as recombinant inbred lines.

Efficient transient gene expression system using buckwheat hypocotyl protoplasts for large-scale experiments.

Buckwheat (Fagopyrum esculentum) is cultivated worldwide and its flour is used in a variety of food products. Although functional analyses of genes in buckwheat are highly desired, reliable methods to do it have yet to be developed. In this study we established a simple and efficient transient gene expression system using buckwheat protoplasts isolated from young hypocotyls using 96-well plates as a high-throughput platform. The transformation efficiency was comparable with that of similar systems, such as Arabidopsis mesophyll protoplasts. Stable results were obtained in a typical example of the experiment to examine transcription factor activity. This system shows potential for the large-scale analysis of gene function using protoplast isolated from fewer and younger plants than the conventional system and may provide novel information for efficient buckwheat breeding.

Genetic and genomic research for the development of an efficient breeding system in heterostylous self-incompatible common buckwheat (Fagopyrum esculentum).

Common buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is an annual crop that is cultivated widely around the world and contains an abundance of nutrients and bioactive compounds. However, the yield of buckwheat is low compared to that of other major crops, and it contains proteins that cause allergic reactions in some people. Much research has aimed to improve or eliminate these undesirable traits, and some major advances have recently been made. Here, we review recent advances in buckwheat breeding materials, tools, and methods, including the development of self-compatible lines, genetic maps, a buckwheat genome database, and an efficient breeding strategy. We also describe emerging breeding methods for high-value lines.

Identification of a gene encoding polygalacturonase expressed specifically in short styles in distylous common buckwheat (Fagopyrum esculentum).

Common buckwheat (Fagopyrum esculentum) is a heteromorphic self-incompatible (SI) species with two types of floral architecture: thrum (short style) and pin (long style). The floral morphology and intra-morph incompatibility are controlled by a single genetic locus, S. However, the molecular mechanisms underlying the heteromorphic self-incompatibility of common buckwheat remain unclear. To identify these mechanisms, we performed proteomic, quantitative reverse-transcription PCR, and linkage analyses. Comparison of protein profiles between the long and short styles revealed a protein unique to the short style. Amino-acid sequencing revealed that it was a truncated form of polygalacturonase (PG); we designated the gene encoding this protein FePG1. Phylogenetic analysis classified FePG1 into the same clade as PGs that function in pollen development and floral morphology. FePG1 expression was significantly higher in short styles than in long styles. It was expressed in flowers of a short-homostyle line but not in flowers of a long-homostyle line. Linkage analysis indicated that FePG1 was not linked to the S locus; it could be a factor downstream of this locus. Our finding of a gene putatively working under the regulation of the S locus provides useful information for elucidation of the mechanism of heteromorphic self-incompatibility.

Identification of a gene encoding glutathione S-transferase that is related to anthocyanin accumulation in buckwheat (Fagopyrum esculentum).

Anthocyanins are a group of flavonoids found in buckwheat (Fagopyrum esculentum) and many other plant species; however; little is known about their mechanisms of synthesis and regulation in buckwheat. We previously reported a spontaneous mutant buckwheat line that shows the green stem phenotype; this line does not accumulate anthocyanins but synthesizes flavonol and proanthocyanidin in the stem. Here, we used this line and lines developed by this line to search for genes related to anthocyanin accumulation in buckwheat. The lines with green stem showed flavonoid-3-O-glucosyltransferase activity against UDP-glucose, indicating that the flavonoid-3-O-glucosyltransferase gene was not controlling the green stem trait. We therefore searched the buckwheat genome database for a gene encoding glutathione S-transferase (GST), a flavonoid-binding protein that transports flavonoids to the vacuole, and identified a candidate gene, FeGST1. Expression analysis showed that FeGST1 was expressed in wild type buckwheat but not in the green stem lines. Linkage analysis with an F2 segregating population produced by crossing between the green stem line and a self-compatible line showed that FeGST1 segregated with stem color without any recombination. This indicates that the green stem trait could be caused by homozygous non-functional alleles of the FeGST1 locus.

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.

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

BACKGROUND: Dihydroflavonol 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. RESULTS: By 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. CONCLUSIONS: We 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.

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.

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 photoperiod sensitivity in common buckwheat by using markers for expressed sequence tags and photoperiod-sensitivity candidate genes.

Photoperiod sensitivity is an important trait related to crop adaptation and ecological breeding in common buckwheat (Fagopyrum esculentum Moench). Although photoperiod sensitivity in this species is thought to be controlled by quantitative trait loci (QTLs), no genes or regions related to photoperiod sensitivity had been identified until now. Here, we identified QTLs controlling photoperiod sensitivity by QTL analysis in a segregating F(4) population (n = 100) derived from a cross of two autogamous lines, 02AL113(Kyukei SC2)LH.self and C0408-0 RP. The F(4) progenies were genotyped with three markers for photoperiod-sensitivity candidate genes, which were identified based on homology to photoperiod-sensitivity genes in Arabidopsis and 76 expressed sequence tag markers. Among the three photoperiod-sensitivity candidate genes (FeCCA1, FeELF3 and FeCOL3) identified in common buckwheat, FeELF3 was associated with photoperiod sensitivity. Two EST regions, Fest_L0606_4 and Fest_L0337_6, were associated with photoperiod sensitivity and explained 20.0% and 14.2% of the phenotypic variation, respectively. For both EST regions, the allele from 02AL113(Kyukei SC2)LH.self led to early flowering. An epistatic interaction was also confirmed between Fest_L0606_4 and Fest_L0337_6. These results demonstrate that photoperiod sensitivity in common buckwheat is controlled by a pathway consisting of photoperiod-sensitivity candidate genes as well as multiple gene action.

Immunoassay using microfluid filters constructed by deep X-ray lithography.

Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (psi 40 microm) in 200 microm-thickness polymethylmethacrylate (PMMA) sheets (psi 3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunoglobulin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0-100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay.