DNA metabarcoding focused on difficult-to-culture protists: An effective approach to clarify biological interactions.

DNA metabarcoding on a single organism is a promising approach to clarify the biological interactions (e.g., predator-prey relationships and symbiosis, including parasitism) of difficult-to-culture protists. To evaluate the effectiveness of this method, Radiolaria and Phaeodaria, which are ecologically important protistan groups, were chosen as target taxa. DNA metabarcoding on a single organism focused on the V9 region of the 18S rRNA gene revealed potential symbionts, parasites and food sources of Radiolaria and Phaeodaria. Previously reported hosts and symbionts (parasites) were detected, and newly recognized combinations were also identified. The contained organisms largely differed between Radiolaria and Phaeodaria. In Radiolaria, members of the same order tended to contain similar organisms, and the taxonomic composition of possible symbionts, parasites, and food sources was fixed at the species level. Members of the same phaeodarian family, however, did not contain similar organisms, and body part (i.e., the central capsule or the phaeodium) was the most important factor that divided the taxonomic composition of detected organisms, implying that the selection of appropriate body part is important when trying to ascertain contained organisms, even for unicellular zooplankton. Our results show that DNA metabarcoding on a single organism is effective in revealing the biological interactions of difficult-to-culture protists.

Development of 12 sets of chromosome segment substitution lines that enhance allele mining in Asian cultivated rice.

Many agronomic traits that are important in rice breeding are controlled by multiple genes. The extensive time and effort devoted so far to identifying and selecting such genes are still not enough to target multiple agronomic traits in practical breeding in Japan because of a lack of suitable plant materials in which to efficiently detect and validate beneficial alleles from diverse genetic resources. To facilitate the comprehensive analysis of genetic variation in agronomic traits among Asian cultivated rice, we developed 12 sets of chromosome segment substitution lines (CSSLs) with the japonica background, 11 of them in the same genetic background, using donors representing the genetic diversity of Asian cultivated rice. Using these materials, we overviewed the chromosomal locations of 1079 putative QTLs for seven agronomic traits and their allelic distribution in Asian cultivated rice through multiple linear regression analysis. The CSSLs will allow the effects of putative QTLs in the highly homogeneous japonica background to be validated.

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.

IonBreeders: bioinformatics plugins toward genomics-assisted breeding.

Polymorphism information generated by next-generation sequencing (NGS) technologies has enabled applications of genome-wide markers assisted breeding. However, handling such large-scale data remains a challenge for experimental researchers and breeders, calling for the urgent development of a flexible and straightforward analysis tool for NGS data. We developed "IonBreeders" as bioinformatics plugins that implement general analysis steps from genotyping to genomic prediction. IonBreeders comprises three plugins, "ABH", "IMPUTATION", and "GENOMIC PREDICTION", for format conversion of genotyping data, preprocessing and imputation of genotyping data, and genomic prediction, respectively. "ABH" converts genotyping data derived from NGS into the ABH format, which is acceptable for our further plugins and with other breeding software tools, R/qtl, MapMaker, and AntMap. "IMPUTATION" filters out non-informative markers and imputes missing marker genotypes. In "GENOMIC PREDICTION", users can use four statistical methods based on their target trait, quantitative trait locus effect, and number of markers, and construct a prediction model for genomic selection. IonBreeders is operated in Torrent Suite, but can also handle genotype data in standard formats, e.g., Variant Call Format (VCF), by format conversion using free software or our provided scripts.

Detection of heading date QTLs in advanced-backcross populations of an elite indica rice cultivar, IR64.

IR64 is one of the world's most popular rice cultivars. To collect genetic factors involved in controlling its heading date, we developed 70 reciprocal advanced-backcross populations with a total of 6284 individuals at the BC4F2 generation from crosses between Koshihikari and IR64. We detected 29 QTLs associated with heading date on chromosomes 3, 5-8, 10, and 12. Twenty QTLs were located in the same chromosome regions as previously isolated heading date genes (Hd1, Hd6, Hd16, Ghd7, DTH8, Hd17, and Hd18). The rest were located in other chromosome regions. We found more number of QTLs than previous studies using mapping populations of IR64. Fine mapping in additional advanced-backcross populations clearly revealed that QTLs on the long arm of chromosome 7 are overlapping and seem to be a novel genetic factor for heading date because of their different locations from OsPRR37. Our results suggest that the difference in heading date between IR64 and Koshihikari is genetically controlled by many factors, and that a non-functional allele of Hd1 contributes to early heading of IR64 in the genetic background of functional alleles of other heading date QTLs and genes such as Hd6 and Hd16.

Detection of novel QTLs qDTH4.5 and qDTH6.3, which confer late heading under short-day conditions, by SSR marker-based and QTL-seq analysis.

Heading date is one of the most important traits in rice breeding. It is governed by multiple genes, including known quantitative trait loci (QTLs). In general, almost all japonica cultivars, including Nipponbare, head early under short-day (SD) conditions, but some indica cultivars, including Kasalath, head late. To explain this difference, we identified QTLs controlling heading date under SD conditions. We used NILs, CSSLs, and BILs from a cross between Nipponbare and Kasalath, and evaluated days to heading (DTH) under SD conditions. No NILs or CSSLs showed late heading, but two BILs (BIL-55 and BIL-78) had almost the same DTH as Kasalath. We developed an F2 population from a cross between BIL-55 and Nipponbare and performed QTL analysis using SSR markers. The late-heading phenotype was controlled by two known genes and at least two novel QTLs on chromosomes 4 and 6, named qDTH4.5 and qDTH6.3. These QTLs were confirmed by QTL-seq. The QTLs and polymorphisms detected here will provide useful information for further genetic studies and breeding under SD conditions at lower latitudes.

Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mungo).

Seed size is one of the most important traits in leguminous crops. We obtained a recessive mutant of blackgram that had greatly enlarged leaves, stems and seeds. The mutant produced 100% bigger leaves, 50% more biomass and 70% larger seeds though it produced 40% less number of seeds. We designated the mutant as multiple-organ-gigantism (mog) and found the mog phenotype was due to increase in cell numbers but not in cell size. We also found the mog mutant showed a rippled leaf (rl) phenotype, which was probably caused by a pleiotropic effect of the mutation. We performed a map-based cloning and successfully identified an 8 bp deletion in the coding sequence of VmPPD gene, an orthologue of Arabidopsis PEAPOD (PPD) that regulates arrest of cell divisions in meristematic cells. We found no other mutations in the neighboring genes between the mutant and the wild type. We also knocked down GmPPD genes and reproduced both the mog and rl phenotypes in soybean. Controlling PPD genes to produce the mog phenotype is highly valuable for breeding since larger seed size could directly increase the commercial values of grain legumes.

Hd18, Encoding Histone Acetylase Related to Arabidopsis FLOWERING LOCUS D, is Involved in the Control of Flowering Time in Rice.

Flowering time is one of the most important agronomic traits in rice (Oryza sativa L.), because it defines harvest seasons and cultivation areas, and affects yields. We used a map-based strategy to clone Heading date 18 (Hd18). The difference in flowering time between the Japanese rice cultivars Koshihikari and Hayamasari was due to a single nucleotide polymorphism within the Hd18 gene, which encodes an amine oxidase domain-containing protein and is homologous to Arabidopsis FLOWERING LOCUS D (FLD). The Hayamasari Hd18 allele and knockdown of Hd18 gene expression delayed the flowering time of rice plants regardless of the day-length condition. Structural modeling of the Hd18 protein suggested that the non-synonymous substitution changed protein stability and function due to differences in interdomain hydrogen bond formation. Compared with those in Koshihikari, the expression levels of the flowering-time genes Early heading date 1 (Ehd1), Heading date 3a (Hd3a) and Rice flowering locus T1 (RFT1) were lower in a near-isogenic line with the Hayamasari Hd18 allele in a Koshihikari genetic background. We revealed that Hd18 acts as an accelerator in the rice flowering pathway under both short- and long-day conditions by elevating transcription levels of Ehd1 Gene expression analysis also suggested the involvement of MADS-box genes such as OsMADS50, OsMADS51 and OsMADS56 in the Hd18-associated regulation of Ehd1 These results suggest that, like FLD, its rice homolog accelerates flowering time but is involved in rice flowering pathways that differ from the autonomous pathways in Arabidopsis.

WAG2 represses apical hook opening downstream from gibberellin and PHYTOCHROME INTERACTING FACTOR 5.

When penetrating the soil during germination, dicotyledonous plants protect their shoot apical meristem through the formation of an apical hook. Apical hook formation is a dynamic process that can be subdivided into hook formation, maintenance and opening. It has previously been established that these processes require the transport and signaling of the phytohormone auxin, as well as the biosynthesis and signaling of the phytohormones ethylene and gibberellin (GA). Here, we identify a molecular mechanism for an auxin-GA crosstalk by demonstrating that the auxin transport-regulatory protein kinase WAG2 is a crucial transcription target during apical hook opening downstream from GA signaling. We further show that WAG2 is directly activated by PHYTOCHROME INTERACTING FACTOR 5 (PIF5), a light-labile interactor of the DELLA repressors of the GA pathway. We find that wag2 mutants are impaired in the repression of apical hook opening in dark-grown seedlings and that this phenotype correlates with GA-regulated WAG2 expression in the concave (inner) side of the apical hook. Furthermore, wag2 mutants are also impaired in the maintenance or formation of a local auxin maximum at the site of WAG2 expression in the hook. WAG2 is a regulator of PIN auxin efflux facilitators and, in line with previous data, we show that this kinase can phosphorylate the central intracellular loop of all PIN-FORMED (PIN) proteins regulating apical hook opening. We therefore propose that apical hook opening is controlled by the differential GA-regulated accumulation of WAG2 and subsequent local changes in PIN-mediated auxin transport.

Genetic architecture of variation in heading date among Asian rice accessions.

BACKGROUND: Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions. RESULTS: To genetically dissect naturally occurring variation in rice heading date, we collected QTLs in advanced-backcross populations derived from multiple crosses of the japonica rice accession Koshihikari (as a common parental line) with 11 diverse rice accessions (5 indica, 3 aus, and 3 japonica) that originate from various regions of Asia. QTL analyses of over 14,000 backcrossed individuals revealed 255 QTLs distributed widely across the rice genome. Among the detected QTLs, 128 QTLs corresponded to genomic positions of heading date genes identified by previous studies, such as Hd1, Hd6, Hd3a, Ghd7, DTH8, and RFT1. The other 127 QTLs were detected in different chromosomal regions than heading date genes. CONCLUSIONS: Our results indicate that advanced-backcross progeny allowed us to detect and confirm QTLs with relatively small additive effects, and the natural variation in rice heading date could result from combinations of large- and small-effect QTLs. We also found differences in the genetic architecture of heading date (flowering time) among maize, Arabidopsis, and rice.

Advanced backcross QTL analysis reveals complicated genetic control of rice grain shape in a japonica × indica cross.

Grain shape is an important trait for improving rice yield. A number of quantitative trait loci (QTLs) for this trait have been identified by using primary F2 mapping populations and recombinant inbred lines, in which QTLs with a small effect are harder to detect than they would be in advanced generations. In this study, we developed two advanced mapping populations (chromosome segment substitution lines [CSSLs] and BC4F2 lines consisting of more than 2000 individuals) in the genetic backgrounds of two improved cultivars: a japonica cultivar (Koshihikari) with short, round grains, and an indica cultivar (IR64) with long, slender grains. We compared the ability of these materials to reveal QTLs for grain shape with that of an F2 population. Only 8 QTLs for grain length or grain width were detected in the F2 population, versus 47 in the CSSL population and 65 in the BC4F2 population. These results strongly suggest that advanced mapping populations can reveal QTLs for agronomic traits under complicated genetic control, and that DNA markers linked with the QTLs are useful for choosing superior allelic combinations to enhance grain shape in the Koshihikari and IR64 genetic backgrounds.

Hd16, a gene for casein kinase I, is involved in the control of rice flowering time by modulating the day-length response.

The alteration of photoperiod sensitivity has let breeders diversify flowering time in Oryza sativa (rice) and develop cultivars adjusted to a range of growing season periods. Map-based cloning revealed that the rice flowering-time quantitative trait locus (QTL) Heading date 16 (Hd16) encodes a casein kinase-I protein. One non-synonymous substitution in Hd16 resulted in decreased photoperiod sensitivity in rice, and this substitution occurred naturally in an old rice cultivar. By using near-isogenic lines with functional or deficient alleles of several rice flowering-time genes, we observed significant digenetic interactions between Hd16 and four other flowering-time genes (Ghd7, Hd1, DTH8 and Hd2). In a near-isogenic line with the weak-photoperiod-sensitivity allele of Hd16, transcription levels of Ehd1, Hd3a, and RFT1 increased under long-day conditions, and transcription levels of Hd3a and RFT1 decreased under short-day conditions. Expression analysis under continuous light and dark conditions showed that Hd16 was not likely to be associated with circadian clock regulation. Biochemical characterization indicated that the functional Hd16 recombinant protein specifically phosphorylated Ghd7. These results demonstrate that Hd16 acts as an inhibitor in the rice flowering pathway by enhancing the photoperiod response as a result of the phosphorylation of Ghd7.

QTL mapping for salt tolerance and domestication-related traits in Vigna marina subsp. oblonga, a halophytic species.

QTL mapping in F2 population [V. luteola × V. marina subsp. oblonga] revealed that the salt tolerance in V. marina subsp. oblonga is controlled by a single major QTL. The habitats of beach cowpea (Vigna marina) are sandy beaches in tropical and subtropical regions. As a species that grows closest to the sea, it has potential to be a gene source for breeding salt-tolerant crops. We reported here for the first time, quantitative trait loci (QTLs) mapping for salt tolerance in V. marina. A genetic linkage map was constructed from an F2 population of 120 plants derived from an interspecific cross between V. luteola and V. marina subsp. oblonga. The map comprised 150 SSR markers. The markers were clustered into 11 linkage groups spanning 777.6 cM in length with a mean distance between the adjacent markers of 5.59 cM. The F2:3 population was evaluated for salt tolerance under hydroponic conditions at the seedling and developmental stages. Segregation analysis indicated that salt tolerance in V. marina is controlled by a few genes. Multiple interval mapping consistently identified one major QTL which can explain about 50% of phenotypic variance. The flanking markers may facilitate transfer of the salt tolerance allele from V. marina subsp. oblonga into related Vigna crops. The QTL for domestication-related traits from V. marina are also discussed.

Complete Genome Sequence of Annamia dubia, filamentous colony-making Chroococcales with the analysis of FraC gene influencing filament integrity.

The complete genome of Annamia dubia was sequenced. The genome size is 4.02 Mbp, including 3886286 bp circular chromosome and four circular plasmids (31516, 42453, 38085 and 24903 bp). It included 3718 protein-coding sequences, 45 tRNA genes, three sets of rRNA genes, a microcystin biosynthesis gene cluster and six CRISPR (clustered regularly interspaced short palindromic repeat). Annamia is the only one genus in the Chroococcales that makes filamentous colonies. FraC and FraG were identified in the genome. These genes are required for the integrity of cell junctions and influencing filament integrity and are thought to be related to colony formation. These genes are first reported from Chroococcales, and may play a significant role in the colony formation of this species. In the phylogenetic tree of the FraC gene, A. dubia was located in the basal position of Oscillatoriales. The GC ratio of FraC gene of A. dubia is very low from the genome and the FraC gene of Microcoleaceae. The presence of these genes in the basal region and the low GC ratio suggests that the FraC gene in this species was introduced by horizontal gene transfer. Since the filamentous colony is a fundamental and important taxonomic feature for the classification of cyanobacteria, the possibility of horizontal transmission of genes involved in filamentous cyanobacterial colonies is an important discovery for the classification of cyanobacteria.

Complete chloroplast and mitochondrial genomes of Ditrichum rhynchostegium Kindb. (Ditrichaceae, Bryophyta).

The moss family Pottiaceae is one of the most diverse lineages of the subclass Dicranidae (haplolepideous mosses). Nevertheless, the phylogenetic relationships of Pottiaceae with other Dicranidae families remain unclear. To better understand the ancestral genomic structure and evolution of the Pottiaceae, herein, we present the chloroplast and mitochondrial genomes of Ditrichum rhynchostegium (Ditrichaceae, Bryophyta). The chloroplast genome is 124,628 bp long and displayed a circular structure composed of a large single-copy region, a small single-copy region, and a pair of inverted repeats. It has 118 genes, including 82 protein-coding genes, 32 tRNA genes, and four rRNA genes. The mitochondrial genome is 106,246 bp long and has a circular structure. It contains 67 genes, including 40 protein-coding genes, 24 tRNA genes, and three rRNA genes. Phylogenetic trees based on the coding sequences strongly support the sister relationship of D. rhynchostegium with all Pottiaceous accessions, and the dextrosely arranged operculum cells suggest its affinity for Pottiaceae. This study also demonstrates that long-read sequencing employing the Nanopore platform facilitates the repair of unassembled or misassembled organellar genomic regions.

Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds.

Genome sequencing is important for discovering critical genes in crops and improving crop breeding efficiency. Generally, fresh, young leaves are used for DNA extraction from plants. However, seeds, the storage form, are more efficient because they do not require cultivation and can be ground at room temperature. Yet, only a few DNA extraction kits or methods suitable for seeds have been developed to date. In this study, we introduced an improved (IMP) Boom method that is relatively low-cost, simple to operate, and yields high-quality DNA that can withstand long-read sequencing. The method successfully extracted approximately 8 µg of DNA per gram of seed weight from soybean seeds at an average concentration of 48.3 ng/µL, approximately 40-fold higher than that extracted from seeds using a common extraction method kit. The A260/280 and A260/230 values of the DNA were 1.90 and 2.43, respectively, which exceeded the respective quality thresholds of 1.8 and 2.0. The DNA also had a DNA integrity number value (indicating the degree of DNA degradation) of 8.1, higher than that obtained using the kit and cetyltrimethylammonium bromide methods. Furthermore, the DNA showed a read length N50 of 20.96 kbp and a maximum read length of 127.8 kbp upon long-read sequencing using the Oxford Nanopore sequencer, with both values being higher than those obtained using the other methods. DNA extracted from seeds using the IMP Boom method showed an increase in the percentage of the nuclear genome with a decrease in the relative ratio of chloroplast DNA. These results suggested that the proposed IMP Boom method can extract high-quality and high-concentration DNA that can be used for long-read sequencing, which cannot be achieved from plant seeds using other conventional DNA extraction methods. The IMP Boom method could also be adapted to crop seeds other than soybeans, such as pea, okra, maize, and sunflower. This improved method is expected to improve the efficiency of various crop-breeding operations, including seed variety determination, testing of genetically modified seeds, and marker-assisted selection.

DNA metabarcoding focusing on the plankton community: an effective approach to reconstruct the paleo-environment.

DNA metabarcoding (DNA-MB) targeting the whole plankton community is a promising approach in studies of sediment samples from water bodies, but its effectiveness in ancient material is not well demonstrated. We applied DNA-MB of plankton in a sediment core to reconstruct the paleo-environment of Lake Shinji, Japan, through a marine lagoon/freshwater lake transition during the past 2300 years. We interpreted core-sample plankton taxonomy and habitat by reference to the modern plankton community in water samples. OTUs (operational taxonomic units) belonging to Dictyochophyceae were 81.05% of the total reads in sediments. However, Ciliophora, Copepoda and Labyrinthulea formed the majority of plankton taxa in the water samples, suggesting that they are under-represented in sediment. A drastic change in plankton composition correlated with a large decrease in sediment sulfur concentration, implying the change of aquatic environment from marine lagoon to freshwater lake. This event took place ca. 1200 CE in Lake Shinji. A 250 year-long transitional period followed, during which the total DNA sequence reads were very low. This suggests that salinity fluctuations created a hostile environment for both marine and freshwater plankton species. Our results show that DNA-MB of the whole plankton community is effective in reconstructing paleo-environments.

Unique Salt-Tolerance-Related QTLs, Evolved in Vigna riukiuensis (Na+ Includer) and V. nakashimae (Na+ Excluder), Shed Light on the Development of Super-Salt-Tolerant Azuki Bean (V. angularis) Cultivars.

Wild relatives of crops have the potential to improve food crops, especially in terms of improving abiotic stress tolerance. Two closely related wild species of the traditional East Asian legume crops, Azuki bean (Vigna angularis), V. riukiuensis "Tojinbaka" and V. nakashimae "Ukushima" were shown to have much higher levels of salt tolerance than azuki beans. To identify the genomic regions responsible for salt tolerance in "Tojinbaka" and "Ukushima", three interspecific hybrids were developed: (A) azuki bean cultivar "Kyoto Dainagon" × "Tojinbaka", (B) "Kyoto Dainagon" × "Ukushima" and (C) "Ukushima" × "Tojinbaka". Linkage maps were developed using SSR or restriction-site-associated DNA markers. There were three QTLs for "percentage of wilt leaves" in populations A, B and C, while populations A and B had three QTLs and population C had two QTLs for "days to wilt". In population C, four QTLs were detected for Na+ concentration in the primary leaf. Among the F2 individuals in population C, 24% showed higher salt tolerance than both wild parents, suggesting that the salt tolerance of azuki beans can be further improved by combining the QTL alleles of the two wild relatives. The marker information would facilitate the transfer of salt tolerance alleles from "Tojinbaka" and "Ukushima" to azuki beans.

Phosphate starvation response precedes abscisic acid response under progressive mild drought in plants.

Drought severely damages crop production, even under conditions so mild that the leaves show no signs of wilting. However, it is unclear how field-grown plants respond to mild drought. Here, we show through six years of field trials that ridges are a useful experimental tool to mimic mild drought stress in the field. Mild drought reduces inorganic phosphate levels in the leaves to activate the phosphate starvation response (PSR) in soybean plants in the field. Using Arabidopsis thaliana and its mutant plants grown in pots under controlled environments, we demonstrate that PSR occurs before abscisic acid response under progressive mild drought and that PSR plays a crucial role in plant growth under mild drought. Our observations in the field and laboratory using model crop and experimental plants provide insight into the molecular response to mild drought in field-grown plants and the relationship between nutrition and drought stress response.

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.