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Date & time Oct 13
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cde228
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The genome of Chenopodium quinoa

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high quality, chromosome scale reference genome sequence for quinoa, which was produced using single molecule real time sequencing in combination with optical, chromosome contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub genomes in quinoa, and reduced coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

a, Seeds of C. suecicum, C. pallidicaule and quinoa. b, The proportion of gene pairs in each species binned according to Ks values. c, Maximum likelihood tree generated from 3,132 SNPs. Black branches, diploid species. Coloured branches, tetraploid species: red, quinoa; blue, C. berlandieri; yellow, C. hircinum. Branch values represent the percentage of 1,000 bootstrap replicates that support the topology. Scale bar represents substitutions per site. d, Evolutionary relationships of Chenopodium species, showing the hypothesized long range dispersal of an ancestral C. berlandieri to South America, and knock off van cleef and arpels emerald ring the eventual domestication of quinoa from C. hircinum, either from a single event that gave rise to highland and subsequently coastal quinoa (1), or in two events that gave rise to highland (2a) and coastal (2b) quinoa independently. Blue, red and yellow shading represents the geographic distribution of C. berlandieri, quinoa and C. hircinum, respectively.

a, Blue lines and green lines connect regions of the C. pallidicaule and C. suecicum genomes, respectively, with their orthologous regions in the quinoa genome based on BLASTN. Quinoa scaffolds are arranged based on their positions in linkage groups, with blue and green coloured bars indicating sub genome assignment based on mapped reads from the two diploid species. Scaffolds that could not be unambiguously assigned to a sub genome based on read mapping are shown in white. Grey bars separate neighbouring scaffolds. b, Homoeologous gene pairs in the A (blue chromosomes) and B (green chromosomes) sub genomes. c, Simplified representation of synteny between CqA12, CqB05, CqB03 and CqA10. Dotted lines connect large scale syntenic regions between the A (blue) and B (green) sub genomes. The scale bar indicates approximate positions defining the indicated syntenic blocks. For purposes van cleef and arpels engagement ring knock off of visualization, CqB05 and CqA10 were inverted. d, Syntenic relationships between B. vulgaris and the A and B sub genomes of quinoa. Colours distinguish quinoa regions syntenic to each B. vulgaris chromosome. Blue and green quinoa chromosomes indicate the A and B sub genomes, respectively.

a, The number of orthologous protein coding gene clusters shared between or unique to quinoa, C. pallidicaule, C. suecicum and B. vulgaris. b, The number of gene sets for which each gene has been retained as a single copy in each genome/sub genome (middle), or lost from the quinoa A (left) or B (right) sub genome. c, Maximum likelihood tree of flowering locus T (FT) sequences, indicating the presence of two sets of orthologues in quinoa and B. vulgaris. The tree is rooted on the branch containing FT from A. thaliana. Branch values represent the percentage of 1,000 bootstrap replicates that support the topology. Scale bar represents substitutions per site.

a, Imaging mass spectrometry visualization of selected masses, including saponins in the pericarp of a quinoa seed. Purple gradient bar, tentative phosphatidylcholine (34:1), ([M+Na]+ m/z=782.5610, calc. error); yellow gradient bar, tentative triacylglycerol (54:6), ([M+K]+ m/z=917.6971, calc. error); green gradient bar indicates a representative saponin phytolaccagenic acid with sugar chains hexose pentose hexose ([M+K]+ m/z=1173.5114, calc. error). Coloured bars represent the ion signal intensity scaled from 0% (bottom) to 50% (top) of maximum signal. Scale bars, 500m. b, Accumulation of saponins as measured by total acids during seed development. Illustrations represent fruit development at 12 and 24 days after anthesis. n=6, 5, 5 and 5, respectively, for 12, 16, 20 and 24 days after anthesis. c, The percentage difference in allele frequency of sweet progeny compared to bitter progeny in the Kurmi0654 (top) and AtlasCarina Red (bottom) populations. Alternating red and blue dots indicate positions of markers along alternating chromosomes, with unmapped markers in chromosome 0 shown in grey. Asterisk above the top panel indicates the approximate position of TSARL1. d, The saponin biosynthetic pathway, showing enzymes that catalyse each step of the pathway and the quinoa gene ID for genes encoding each enzyme. Boxes surrounding each gene ID are coloured according to their fold change in expression (log2) in sweet lines compared to bitter lines of Kurmi0654. Horizontal lines to the left of each gene ID represent the 2 kb region upstream of the start codon of each gene, with tick marks indicating the positions of motifs putatively recognized by TSARL1. e, Gene models of TSARL1 in bitter and sweet lines. Red asterisk, premature stop codon in sweet lines of Kurmi0654.

a, Representative gene model showing mapped RNA sequencing reads generated using Illumina or isoform sequencing technologies. The top and middle panels show isoform sequencing and RNA seq reads, respectively, that have been mapped to the chromosomal location containing the AUR62017258 gene model, which is shown on the bottom panel. Light grey lines in the top two panels indicate regions where reads were split to indicate introns positions. Full length isoform sequencing reads were able to span the 5 untranslated region, all exons, and the 3 untranslated region in a single read. b, Gene density and GC% in 100 kb windows in quinoa chromosomes. c, The frequency of annotation edit distance (AED) scores for the assemblies of quinoa (blue), C. pallidicaule (red) and C. suecicum (green).

Frequency of SNPs in the sequenced quinoa accessions, relative to the reference quinoa genome assembly, in a 1 Mb window size. y axis scale is from 0 to 10,000 SNPs. The innermost track shows scaffolds arranged according to their placement in the linkage groups, with scaffolds coloured according to sub genome assignment based on mapping sequencing reads from C. pallidicaule and C. suecicum, as in Fig. 2a. From inside to outside, the remaining tracks show SNPs in PI 634921, Atlas, CICA 17, Carina Red, Cherry Vanilla, Chucapaca, G 205 95DK, Ku 2, Kurmi, 0654, Ollague, Pasankalla, Real, Regalona and Salcedo INIA.

Twelve individual seeds of 17 bitter lines, and 12 pooled seeds from each of 16 sweet lines were analysed for derivatized saponins using gas chromatography/mass spectrometry. Data for sweet lines, including parent 0654, were consolidated into one box plot (Sweet). Box plots show median values (solid horizontal lines), 25th and 75th percentile values (box), 90th percentile values (whiskers) and outlier values (open circles). Quantification was performed using standards of oleanolic acid. Letters above each box plot represent statistically significant (P differences between groups based on Games Howell post hoc test.

a, Representative scanning electron microscopy image of a quinoa seed cross section, showing an example of a region (white box) from which measurements were taken. Scale bar, 1mm. b, Representative scanning electron microscopy image showing measurements of inner and outer seed coat layers. Scale bar, 30m c, Thickness of the internal seed coat layer in bitter and sweet lines. d, Thickness of the external seed coat layer in bitter and sweet lines. Letters above each box plot represent statistically significant (P differences between groups based on ANOVA. Box plots show median values (solid horizontal lines), 25th and 75th percentile values (box), 90th percentile values (whiskers) and outlier values (open circles). n=91, 160, 54, and 129 for internal bitter, internal sweet, external bitter, and external sweet, respectively.

a, Maximum likelihood tree of select bHLH peptide sequences from quinoa, Arabidopsis thaliana and Medicago truncatula, showing the close evolutionary relationship between the quinoa bHLH TSARL1 (AUR62017204) and the M. truncatula bHLHs TSAR1 and TSAR2 (MEDTR7G080780 and MEDTR4G066460, respectively). Branch values represent the percentage of 500 bootstrap replicates that support the topology. Subclades of the bHLH family, as defined in A. thaliana, are indicated on the right. b, Sequence alignment of TSARL bHLH sequences. Underlined blue, bHLH homology domain (solid line, helix; dashed line, loop). Underlined red, C terminal domain. Boxed, residues that confer specificity to E box DNA binding. Green boxed Arg, residue that selects for the central CG dinucleotide. AUR62017204_AS designates the alternatively spliced protein. c, Schematic drawing of full length TSARL1 (AUR62017204). Dashed lines indicate regions predicted to be flexible. Boxed region shows C terminal domain lost in alternative splice variant. d, Zoomed in view of the black dashed box in c, showing TSARL1 specifically binding the CACGHG motif.

a, Screenshot from Integrative Genomics Viewer (IGV) showing read alignment results in a 3 kb region (track 1, top) around TSARL1 (track 5, bottom). Shown are alignments for Atlas (track 2), Carina Red (track 3), and the merged F2 sweet lines (track 4). In tracks 2 4, the top portion shows coverage, and the bottom portion shows individual reads. The F2 merged data (track 4) shows evidence of three structural variants (labelled A, B and C) relative to the reference, whereas Atlas (track 2) only shows evidence of the G2078C SNP (labelled D). b, c, Screenshots from IGV showing before (a) and after (b) local re assembly of the reference sequence to include the B insertion site shown in panel a, illustrating the effect on read mapping from the merged F2 sweet lines. The dips in coverage and disconcordantly mapped reads (indicated by colours assigned to the reads) around this insertion site are resolved after re assembly.

Quinoa (Chenopodium quinoa Willd., 2n=4x=36) is a highly nutritious crop that is adapted to thrive in a wide range of agroecosystems. It was presumably first domesticated more than 7,000 years ago by pre Columbian cultures and was known as the 'mother grain' of the Incan Empire1. Quinoa has adapted to the high plains of the Andean Altiplano ( above sea level), where it has developed tolerance to several abiotic stresses2, 3, 4. Quinoa has gained international attention because of the nutritional value of its seeds, which are gluten free, have a low glycaemic index5, and contain an excellent balance of essential amino acids, fibre, lipids, carbohydrates, vitamins, and minerals6. Quinoa has the potential to provide a highly nutritious food source that can be grown on marginal lands not currently suitable for other major crops. This potential was recognized when the United Nations declared 2013 as the International Year of Quinoa, this being one of only three times a plant has received such a designation.

Despite its agronomic potential, quinoa is still an underutilized crop7, with relatively few active breeding programs8. Breeding efforts to improve the crop for important agronomic traits are needed to expand quinoa production worldwide. To accelerate the improvement of quinoa, we present here the allotetraploid quinoa genome. We demonstrate the utility of the genome sequence by identifying a gene that probably regulates the presence of seed triterpenoid saponin content. Moreover, we sequenced the genomes of additional diploid and tetraploid Chenopodium species to characterize genetic diversity within the primary germplasm pool for quinoa and to understand sub genome evolution in quinoa. Together, these resources provide the foundation for accelerating the genetic improvement of the crop, with the objective of enhancing global food security for a growing world population.

We sequenced and assembled the genome of the coastal Chilean quinoa accession PI 614886 (BioSample accession code SAMN04338310) using single molecule real time (SMRT) sequencing technology from Pacific Biosciences (PacBio) and optical and chromosome contact maps from BioNano Genomics9 and Dovetail Genomics10. The assembly contains 3,486 scaffolds, with a scaffold N50 of 3.84 Mb and 90% of the assembled genome contained in 439 scaffolds (Table 1). The total assembly size of 1.39 gigabases (Gb) is similar to the reported size estimates of the quinoa genome (1.45 1.50 Gb (refs 11,12)). To combine scaffolds into pseudomolecules, an existing linkage map from quinoa13 was integrated with two new linkage maps. The resulting map (Extended Data Fig. 1) of 6,403 unique markers spans a total length of 2,034 centimorgans (cM) and consists of 18 linkage groups (Supplementary Table 7), corresponding to the haploid chromosome number of quinoa. Pseudomolecules (hereafter referred to as chromosomes, which are numbered according to a previously published single nucleotide polymorphism (SNP) linkage map13) were created by anchoring 565 scaffolds to the linkage map, representing 1.18 Gb (85%) of the total assembly length (Table 1, Supplementary Data 1, Supplementary Data 2). This assembly represents a substantial improvement over the previously published quinoa draft genome sequence, which contained more than 24,000 scaffolds with 25% missing data14.

Predicted protein coding and microRNA genes (Supplementary Table 4) were annotated using a combination of ab initio prediction and transcript evidence gathered from RNA sequenced from multiple tissues using both RNA seq and PacBio isoform sequencing (Iso Seq) approaches (Extended Data Fig. 2a). The annotation contains 44,776 gene models (Supplementary Table 2, Extended Data Fig. 2b), which is in line with sequenced tetraploid species15, and includes 33,365 genes with annotation edit distance (AED)16, 17 values0.3 (Extended Data Fig. 2c). Of the genome, 64% was found to be repetitive, including a large proportion of long terminal repeat (LTR) transposable elements (Supplementary Table 1). A majority (97.3%) of the 956 genes in the Plantae BUSCO dataset18 were identified in the annotation (Supplementary Table 3), which is suggestive of a complete assembly and annotation. The utility of the assembly, linkage maps, and annotation was demonstrated by mapping the betalain locus and identifying candidate genes underlying stem pigmentation (Supplementary Information 7.1.6), which is often used as a morphological marker in breeding programs.

Quinoa resulted from the hybridization of ancestral A and B genome diploid species19. Single gene sequencing studies previously identified pools of North American and Eurasian diploids as candidate sources of the A and B sub genomes, respectively20, 21, 22, with hybridization occurring somewhere in North America. To understand genome structure and evolution in quinoa further, we sequenced, assembled, and annotated the A genome diploid C. pallidicaule (commonly called caahua or kaiwa) and the B genome diploid C. suecicum21 (Fig. 1a, Table 1). A high proportion of orthologous gene pairs in quinoa showed similar rates of synonymous substitutions per synonymous site (Ks), indicative of a whole genome duplication event (Fig. 1b). This probably represents the hybridization of ancestral diploid species, because a similar peak was not observed in C. pallidicaule or C. suecicum (Fig. 1b). Using mutation rates calculated for Arabidopsis thaliana23 and for core eukaryotes24, we estimate the tetraploidization to have occurred 3.3 6.3 million years ago.

a, Seeds of C. suecicum, C. pallidicaule and quinoa. b, The proportion of gene pairs in each species binned according to Ks values. c, Maximum likelihood tree generated from 3,132 SNPs. Black branches, diploid species. Coloured branches, tetraploid species: red, quinoa; blue, C. berlandieri; yellow, C. hircinum. Branch values represent the percentage of 1,000 bootstrap replicates that support the topology. Scale bar represents substitutions per site. d, Evolutionary relationships of Chenopodium species, showing the hypothesized long range dispersal of an ancestral C. berlandieri to South America, and the eventual domestication of quinoa from C. hircinum, either from a single event that gave rise to highland and subsequently coastal quinoa (1), or in two events that gave rise to highland (2a) and coastal (2b) quinoa independently. Blue, red and yellow shading represents the geographic distribution of C. berlandieri, quinoa and C. hircinum, respectively.

Multiple interfertile tetraploid species have arisen from the ancestral tetraploid following hybridization, including C. berlandieri and C. hircinum, although the evolutionary relationships among quinoa and its diploid and tetraploid relatives remain unclear25. To begin to resolve these issues, we re sequenced 15 additional quinoa samples representing the two major recognized groups of quinoa: highland and coastal (Supplementary Data 5). We also sequenced five accessions of C. berlandieri and one accession each of C. hircinum from the Pacific and Atlantic Andean watersheds (Supplementary Data 5). Phylogenetic analysis of these taxa indicates that North American C. berlandieri is the basal member of the species complex (Fig. 1c). knock off van cleef perlee ring Quinoa was thought to have been domesticated from C. hircinum in a single event, from which coastal quinoa was later derived (Fig. 1d, arrow 1); however, our sequencing data place a C. hircinum sample basal to coastal ecotypes (Fig. 1c), suggesting the possibility that quinoa was domesticated independently in highland and coastal environments (Fig. 1d, arrows 2a and 2b, respectively). Future analyses with deeper sampling of quinoa and C. hircinum will help clarify the relationship between C. hircinum and quinoa, as well as provide germplasm for breeding broadly adapted coastal quinoa cultivars for warm season production. The SNPs identified between these accessions and the reference quinoa genome a total of 7,809,381 (Extended Data Fig. 3, Supplementary Table 5), including 2,668,694 that are specific to quinoa will be useful in assessing genetic diversity and identifying genomic regions associated with desirable traits.

By mapping sequencing reads from C. pallidicaule and C. suecicum onto the quinoa scaffold assembly, and by performing BLASTN searches of each diploid against the quinoa assembly, 156 and 410 quinoa scaffolds (totalling 202.6 and 646.3 Mb) were assigned to the A and B sub genomes, respectively (Fig. 2a, Supplementary Data 6). A mini satellite repeat (18 24J) previously shown to be more abundant in the B sub genome26 is over represented in scaffolds assigned to the B sub genome (Supplementary Data 6). Nine chromosomes were assigned to each sub genome (chromosomes hereafter designated as CqA or CqB, followed by the chromosome number), with the B sub genome accounting for a larger percentage of both the genetic (1,087 cM) and physical (660 Mb) sizes than the A sub genome (946 cM, 524 Mb). This result was not unexpected, given the differences in the estimated genome sizes of C. suecicum (815 Mb) and C. pallidicaule (452 Mb) based on k mer analyses.

a, Blue lines and green lines connect regions of the C. pallidicaule and C. suecicum genomes, respectively, with their orthologous regions in the quinoa genome based on BLASTN. Quinoa scaffolds are arranged

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