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

Abstract

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.

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Fig. 1: Sequencing and assembly of F. esculentum PL4.
Fig. 2: Genome structure of F. esculentum PL4.
Fig. 3: Development of a waxy buckwheat.
Fig. 4: Genetic architecture of the F. esculentum mating system.
Fig. 5: Population structure of cultivated and wild common buckwheat.

Data availability

The final genome assemblies, annotations, RNA sequences and raw genome sequence data generated in this study have all been deposited in the DNA Data Bank of Japan (DDBJ) database under BioProjects PRJDB15031 (assembly of F. esculentum PL4: BSUD01000001–BSUD01003041, assembly of F. esculentum GS1: BSUE01000001–BSUE01042256, raw reads of various F. esculentum accessions: DRR438014DRR438137 and DRR477348477353) and PRJDB15175 (assembly of F. homotropicum: BSWB01000001–BSWB01000436, raw reads of F. homotropicum: DRR438312). The final genome assemblies and annotations are also available from the Buckwheat Genome DataBase (BGDB) (http://buckwheat.kazusa.or.jp), as well as the initial scaffolds and contigs used to construct the final assemblies. Publicly available datasets from the following databases and websites were used in this study: NR database of NCBI, UniProtKB (https://www.uniprot.org), PFAM, Phytozome (https://phytozome-next.jgi.doe.gov), TAIR (https://www.arabidopsis.org), MBKbase (https://www.mbkbase.org) and CARNIVOROM. Source data are provided with this paper.

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Acknowledgements

This work was supported by KAKEN-HI (grants 20K06761 and 21H00356 to J.A.F., 22H05172 and 22H05181 to K.S., and 18KK0172 to Y.Y.); ACT-X ‘Environments and Biotechnology’ from the Japan Science and Technology Agency (JST) (grant JPMJAX20BA to R.T.); Cabinet Office, Government of Japan, Moonshot R&D Program for Agriculture, Forestry and Fisheries to Y.Y.; the research programme on development of innovative technology from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN) (grant JPJ007097 to T.H.); and the Leverhume Trust (grant RPG-2017-196 to M.K.J.). We thank S. Wright for helpful discussions and providing genomic data of R. hastatulus, K. L. Farquharson for language-editing support of the manuscript and RIKEN HOKUSAI and the National Institute of Genetics for providing computational resources.

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J.A.F., R.T., S.K., T.K.-T., M.K.J., H.H., T. Ota and Y.Y. wrote the paper. R.T., K.M., E.O.-T., T.H. and Y.Y. prepared materials for genome assembly. J.A.F., Y.D., C.L., M.L., H.V.H., M.K.J., D.L.L., T. Ohsako and Y.Y. prepared materials for whole-genome sequencing used for population genetic analyses. N.M., K.N., T.N., H.S., M.U. and Y.Y. prepared materials for mutagenesis. J.A.F., E.Y., N.T., K.S., H.H., T. Ota and Y.Y. performed computational data analyses. S.K., E.O.-T., K.F., T.H., K.M., N.M., H.S., M.U., D.M., M.N., K.S. and Y.Y. performed experiments. All authors read and approved the final manuscript.

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Correspondence to Jeffrey A. Fawcett, Chengyun Li, Hideki Hirakawa, Tatsuya Ota or Yasuo Yasui.

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Extended data

Extended Data Fig. 1 Comparison of F. esculentum PL4 pseudomolecules with linkage map.

Left grey bars indicate the PL4 pseudomolecules and right grey bars indicate linkage maps from Yabe et al.16. Positions of the array markers developed by the same previous study are shown by black lines. Markers where the order matches between the pseudomolecules and the linkage map are connected by blue lines, and those that do not match are connected by red lines. The thickness of the lines is proportional to the number of markers. The marker on P1_3 (FE140468) that is anchored to Chr1 is connected by a red dotted line. The map position of S locus, which contains the S-ELF3 gene, is 84.2 cM on the linkage map of Yabe et al.16. Sh denotes the locus containing the homologue of S-ELF3 which has a nonsense mutation in F. esculentum PL4 (see also Supplementary Fig. 30). We note that the notations of P1_8.1 and P1_8.2 are incorrectly interchanged in the Figure 2 of Yabe et al.16.

Extended Data Fig. 2 Nucleotide divergence between F. esculentum PL4 and F. homotropicum across the genome.

Nucleotide divergence, that is, the average number of differences per site, was calculated across a sliding window of 2 Mb with a step of 400 kb based on results of MUMMER. Regions in F. esculentum PL4 that were masked by RepeatMasker using the TE library constructed for F. esculentum PL4 were excluded. Windows with < 10,000 aligned sites were not plotted. Regions with a divergence of < 0.001, which are to be likely regions in F. esculentum PL4 that are derived from F. homotropicum, are shown in grey and correspond to regions indicated in Fig. 2a.

Source data

Extended Data Fig. 3 Nucleotide divergence between the flanking LTRs of full-length LTR retrotransposons of the three Fagopyrum species.

Nucleotide divergence was calculated between each flanking LTR whose alignment length was ≥100 nucleotides. a,Gypsy (F. esculentum: n = 24,585, F. homotropicum: n = 24,342, F. tataricum: n = 4,359) and Copia-type LTR retrotransposons (F. esculentum: n = 2,750, F. homotropicum: n = 2,700, F. tataricum: n = 1,028). b,Athila (F. esculentum: n = 9,830, F. homotropicum: n = 10,389, F. tataricum: n = 425), CRM (F. esculentum: n = 1,154, F. homotropicum: n = 1,073, F. tataricum: n = 166), and Tekay family (F. esculentum: n = 1,174, F. homotropicum: n = 1,197, F. tataricum: n = 1,427) of the Gypsy-type LTR retrotransposons.

Source data

Extended Data Fig. 4 Distribution of various types of Transposable Elements across the F. esculentum genome.

For LTR retrotransposons, only full-length LTR retrotransposons whose nucleotide divergence could be calculated with an alignment length of ≥100 nucleotides are shown and the colour represents the nucleotide divergence between the flanking LTRs. The divergence corresponds to those shown in Extended Data Fig. 3 and Supplementary Fig. 11. Those with divergence > 0.1 are shown as 0.1. Athila_11 and Athila_18 are the two largest Gypsy-type subfamilies. CRM_44 and CRM_88 are Gypsy-type subfamilies of the CRM clade not associated with centromeric regions in F. esculentum and F. homotropicum, whereas the remaining CRM subfamilies (that is, other) are associated with centromeric regions in F. esculentum and F. homotropicum (FeCEN). The numbers 11, 18, 44, and 88 correspond to the ClusterIDs of Supplementary Table 17 (see also Supplementary Fig. 12). Number of elements plotted are as follows - Copia: n = 2,750, Athila_18: n = 2,712, Athila_11: n = 2,603, Athila_other: n = 4,515, Tekay: n = 1,174, CRM_44: n = 201, CRM_88: n = 321, CRM_other: n = 632, LINE/SINE: n = 6,818, Helitron: n = 1,098.

Source data

Extended Data Fig. 5 Number and timing of whole-genome duplications (WGDs) in the ancestor of Fagopyrum.

a, Phylogenetic relationship of Caryophyllales species relevant to determining the timing of WGDs. b, Number of gene families (n = 159) where 0, 1, 2, 3, or 4 gene duplications likely corresponding to WGDs were placed at each branch shown in a by phylogenetic analysis. Filled and unfilled bars indicate the number of gene families with and without ≥70% bootstrap support, respectively. c, Age estimates of the two WGDs based on phylogenetic dating analysis of gene families with two WGDs in branch C. The bar plots indicate the median age estimates of each gene family (younger WGD: n = 80, older WGD: n = 77) which correspond to the estimates of Prior Setting 1 in Supplementary Table 21. The line plots are based on all ages of the MCMC analyses combined (F. esculentum-F. tataricum: n = 2,367,263, Fagopyrum-Rumex: n = 1,368,152, younger WGD: n = 720,080, older WGD: n = 693,077). Note that the age distribution of F. esculentum-F. tataricum and Fagopyrum-Rumex follow the prior age constraints assigned to both nodes (Supplementary Table 20). See also Supplementary Fig. 19 for age estimates without various prior age constraints. d,Ks distributions of orthologous gene pairs of F. esculentum and F. homotropicum (n = 21,192), F. esculentum and F. tataricum (n = 17,682) identified by OrthoFinder. e,Ks distributions of orthologous gene pairs of F. esculentum and R. hastatulus (n = 10,397), F. esculentum and A. vesiculosa (n = 9,215), F. esculentum and B. vulgaris (n = 9,466) identified by OrthoFinder, and collinear gene duplicates (n = 3,620) of F. esculentum identified by MCScanX.

Source data

Extended Data Fig. 6 Molecular evolution of Fag e 2 genes and sequence of Fag e 2 knockout mutant.

a, Amino acid alignment of Fag e 2 genes in the three Fagopyrum species. The epitope sequence25 is indicated by a red square. Conserved cysteine residues of Fag e 2 homologues are indicated by red arrowheads. The background colour is proportional to the degree of similarity of each residue compared to its aligned column b, Maximum likelihood phylogenetic tree of Fag e 2 genes constructed with IQ-TREE based on the amino acid alignment of the gene family including Caryophyllales species identified by OrthoFinder. Tree is unrooted and bootstrap values of ≥80% are indicated by each node. Scale bar indicates branch length. Nodes corresponding to tandem duplications are indicated by blue diamonds. c, Amino acid alignment of the epitope sequence indicated by a red square in a. The position conserved across all sequences is indicated by asterisk. The three groups correspond to those in b. d, Upper panel shows the amino acid sequence encoded by the EMS-induced Fag e 2 gene. Red letters indicate epitope amino acids. Green letters indicate the eight Cys residues conserved within plant 2S albumins. Lower panel indicates the results of Sanger sequencing of the mutant/wild type heterozygote (left) and wild type homozygote (right).

Extended Data Fig. 7 Characterization of S- and s-haplotypes in F. esculentum GS1 scaffolds.

a, Dotplot based on minimap2 between scaffold 2156 which contains S-ELF3 (S-haplotype) and scaffold 21180 (s-haplotype). b, Gene-based collinearity between scaffold 2156 and scaffold 21180 and the collinear regions in F. esculentum PL4 Chr 1 and Chr 6. Orange and light blue horizontal bars indicate genes on Chr 1 and Chr 6, respectively. Genes in squares of the same colours are homologous and thus most probably allelic in F. esculentum GS1. The hemizygous region containing the S locus, indicated by thick black lines, can therefore be restricted to between the two genes, FesPL4_r1.1_Chr1.g191810.1 and FesPL4_r1.1_Chr1.g199860.1. Genomic structure and RNA-Seq analysis of the scaffold 21180 region between these two genes are shown in Supplementary Fig. 27. c, Diagram describing the proposed origin of Sh-haplotype in F. homotropicum. The hemizygous region of the S-haplotype flanked by two genes was translocated from Chr 6 to Chr 1. This translocation is consistent with comparison with a previously developed linkage map (Extended Data Fig. 1) and results of allelism test crosses (Supplementary Fig. 31). A frameshift mutation resulted in a loss-of-function S-ELF3 (s-elf3-ψ1) before or after the translocation, whereas the putative genes encoding for the stamen phenotype, IPPA has remained functional, where IP, P, and A encode for pollen incompatibility, pollen size, and anther height, respectively.

Extended Data Fig. 8 Change of thrum to long homostyle flowers caused by loss-of-function mutation in S-ELF3.

a, Illustration of phenotype changes by loss-of-function mutation in S-ELF3 of the EMS mutant. b, Pollen tube growth after three hours of crossing the F. esculentum EMS mutant (long homostyle) pistil with the wild type thrum pollen (left panel) and the wild type pin pollen (right panel). Thrum pollen tubes reached the ovule (left panel, yellow arrowhead), whereas pin pollen tubes were arrested in the long styles of the EMS mutant (right panel, red arrowheads). Scale bar = 0.2mm. c, Style length of 10 wild type thrum, wild type pin, F. esculentum PL4 (long homostyle), and F. esculentum EMS mutant (long homostyle) flowers. d, Anther filament length of 10 wild type thrum, wild type pin, F. esculentum PL4 (long homostyle), and F. esculentum EMS mutant (long homostyle) flowers. The box plots show the median and first and third quartiles with the whiskers extending to 1.5 times the inter-quartile range. All wild types used here are F. esculentum cv. Harunoibuki.

Extended Data Fig. 9 Molecular evolutionary analyses of S-ELF3.

a, Maximum likelihood phylogenetic tree based on the amino acid sequences of the homologues of S-ELF3 and putative evolutionary scenario of S-ELF3. Sequences of S-ELF3 are from a previously study11 under the following GenBank accessions numbers - AB641416 (F. esculentum S-ELF3), AB641417 (F. cymosum S-ELF3), and AB641418 (F. urophyllum S-ELF3). Sources of the remaining sequences are as described in Supplementary Table 19. Alignment was performed using MAFFT with the option -linsi and filtered with trimAl with the options ‘-automated1 -resoverlap 0.7 -seqoverlap 50’. Phylogenetic tree was constructed using the resulting alignments as input with IQ-TREE with 1,000 bootstrap replicates. The tree was rooted using the two Arabidopsis genes and FesPL4_sc0096.1.g000180.1 as outgroup. The F. tataricum ortholog of FesPL4_r1.1_Chr5.g038430.1 (FtPinG0006101400.01.T01) was filtered out as the result of trimAl. Bootstrap values of ≥80% are indicated next to each node. The magenta dot corresponds to a whole-genome duplication. G and IS encode for style length and style incompatibility, respectively. b,Ks between the S-ELF3 genes in F. esculentum, F. cymosum, F. urophyllum, and the paralogs in F. esculentum. Ks was calculated using codeml of the PAML package. The Ks estimates between FesPL4_sc0096.1.g000180.1 and the other FagopyrumS-ELF3 homologues are > 3 and not shown.

Extended Data Fig. 10 Candidate region of artificial selection.

a, Nucleotide diversity π of cultivated, Wild Tib, and other wild accessions excluding Wild Tib of 50-60 Mb region of Chr1 containing the peak of PBS. π was calculated for each 1 Mb sliding window with a step of 200 kb. b, Lengths of haplotype identical to the reference genome in each phased genome are shown. Each haplotype was extended up- and downstream from a core SNP (Chr1 52,562,436) within the low diversity region until a mismatch to the reference genome is encountered. The region depicted corresponds to Chr1 52,325,106 to 52,906,013. Black rectangles indicate predicted protein-coding genes in the region.

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Source Data Extended Data Fig. 4

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Fawcett, J.A., Takeshima, R., Kikuchi, S. et al. Genome sequencing reveals the genetic architecture of heterostyly and domestication history of common buckwheat. Nat. Plants 9, 1236–1251 (2023). https://doi.org/10.1038/s41477-023-01474-1

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