# The genomic footprints of wild Saccharum species trace domestication, diversification, and modern breeding of sugarcane

Olivier Garsmeur, Simon Rio, Nicolas Pompidor, Anna Lipzen, Catherine

Hervouet, Théo Durand, Chris Daum, Yuko Yoshinaga, Mike Butterfield,

Alexander Sanchez, et al.

► To cite this version:

Olivier Garsmeur, Simon Rio, Nicolas Pompidor, Anna Lipzen, Catherine Hervouet, et al. The genomic footprints of wild Saccharum species trace domestication, diversification, and modern breeding of sugarcane. Cell, 2025, 188 (25), pp.7252-7266.e15. 10.1016/j.cell.2025.09.017. hal-05482917

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https://hal.science/hal-05482917v1

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# The genomic footprints of wild *Saccharum* species trace domestication, diversification, and modern breeding of sugarcane

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Olivier Garsmeur, Simon Rio, Nicolas Pompidor, ..., Kerrie Barry, Jeremy Schmutz, Angélique D'Hont

## Correspondence

garsmeur@cirad.fr (O.G.), dhont@cirad.fr (A.D.)

## In brief

Genomic analyses of 390 accessions reveal the domestication history of highly polyploid sugarcane and the wild contributors to its diversification and to modern cultivars.

## Highlights

* Repeated k-mers allow tracing of admixture in complex polyploids such as sugarcane

* Sugarcane domestication involved hybrids between distinct groups of wild *S. robustum*

* Diversification involved hybridization with wild *Saccharum* species and *Miscanthus*

* An unknown *Saccharum* contributor to modern cultivars was discovered

<figure><img src="image_3.png" /></figure>
## Article

# The genomic footprints of wild *Saccharum* species trace domestication, diversification, and modern breeding of sugarcane

Olivier Garsmeur,<sup>1,2,16,*</sup> Simon Rio,<sup>1,2,16</sup> Nicolas Pompidor,<sup>1,2</sup> Anna Lipzen,<sup>3</sup> Catherine Hervouet,<sup>1,2</sup> Théo Durand,<sup>1,2</sup>

Chris Daum,<sup>3</sup> Yuko Yoshinaga,<sup>3</sup> Mike Butterfield,<sup>4</sup> Alexander Sanchez,<sup>5</sup> George Piperidis,<sup>6</sup> Noa Lincoln,<sup>7</sup> Anna Hale,<sup>8</sup>

Jean Yves Hoarau,<sup>1,2,9</sup> Yoshifumi Terajima,<sup>10</sup> Prakash Lakshmanan,<sup>11</sup> Erik Sacks,<sup>12</sup> Shailendra Sharma,<sup>13</sup>

Marotea Vitrac,<sup>14</sup> Kerrie Barry,<sup>3</sup> Jeremy Schmutz,<sup>3,15</sup> and Angélique D'Hont<sup>1,2,17,*</sup>

<sup>1</sup>CIRAD, UMR Agap Institut, 34398 Montpellier, France

<sup>2</sup>UMR AGAP Institut, University Montpellier, CIRAD, INRAE, Institut Agro, Montpellier, France

<sup>3</sup>DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

<sup>4</sup>Centro de Tecnologia Canavieira, Piracicaba, SP, Brazil

<sup>5</sup>USDA-ARS National Clonal Germplasm Repository in Davis, Davis, CA, USA

<sup>6</sup>SRA, Sugar Research Australia, Brisbane, QLD 4000, Australia

<sup>7</sup>Tropical Plant and Soil Sciences, University of Hawai'i at Mānoa, Honolulu, HI 96822, USA

<sup>8</sup>USDA, Sugarcane Research Unit, Houma, LA 70360, USA

<sup>9</sup>eRcane, Sainte-Clotilde, La Réunion, France

<sup>10</sup>JIRCAS, International Research Center for Agricultural Sciences, Ishigaki, Japan

<sup>11</sup>Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China

<sup>12</sup>University of Illinois, Urbana-Champaign, IL, USA

<sup>13</sup>Chaudhary Charan Singh University, Meerut, India

<sup>14</sup>UMR 241 EIO, University of French Polynesia (UPF), Punaauia, French Polynesia

<sup>15</sup>Hudson Alpha Institute for Biotechnology, Huntsville, AL 35806, USA

<sup>16</sup>These authors contributed equally

<sup>17</sup>Lead contact

*Correspondence: garsmeur@cirad.fr (O.G.), dhont@cirad.fr (A.D.)

https://doi.org/10.1016/j.cell.2025.09.017

## SUMMARY

Sugarcane is a major crop of unclear origins due to its complex polyploid interspecific genome. We analyzed genome ancestries using whole-genome sequence data from 390 representative accessions based on repeated k-mers and chloroplast phylogeny. The results provided evidence that *Saccharum officinarum* was domesticated in the New Guinea region from the *S. robustum* wild species and revealed that its genome is a mosaic involving different *S. robustum* subgroups. We discovered a wild *Saccharum* contributor to most modern cultivars, likely originating from East Melanesia. We highlighted two early centers of sugarcane diversification associated with human transport, one in continental Asia through hybridization with different *S. spontaneum* subgroups and one in the Melanesian and Polynesian islands via hybridization with the discovered ancestor and *Miscanthus*. Finally, we revealed the genome ancestry of modern cultivars, highlighting untapped wild *Saccharum* diversity as a source of alleles for breeding programs.

## INTRODUCTION

Interspecific hybridization and polyploidization are major evolutionary forces in plants. They complicate the reconstruction of phylogenetic relationships between taxa, especially in higher-order polyploids. Reference genome assemblies and resequencing data have greatly helped unravel those relationships. The findings have revealed that inter(sub)specific hybridization is associated with the domestication and/or diversification of some major crops.<sup>1,2</sup> In vegetatively propagated crops in which recombination

events are rare, this has resulted in genomes that are mosaics of large ancestral contributor chromosome segments.<sup>3,4</sup>

Sugarcane is a vegetatively propagated cash crop that accounts for 80% of world sugar production, while being increasingly used for bioethanol production and the leading crop in terms of harvested biomass. Despite its considerable economic importance, sugarcane origin is not yet fully established and remains controversial.<sup>5-9</sup>

Sugarcane belongs to the *Saccharum* genus *sensus stricto* (s.s.) (Poaceae; Andropogoneae), which exclusively consists of higher-order polyploids (≥4×), featuring frequent interspecific
<table><caption>Table 1. Summary table of the 390 accessions analyzed</caption><thead><tr><th>Taxonomy</th><th>Number of accessions</th><th>Wild vs cultivated</th></tr></thead><tbody><tr><td><strong>Saccharum</strong></td><td></td><td></td></tr><tr><td>S. spontaneum</td><td>135</td><td>wild</td></tr><tr><td>S. robustum</td><td>43</td><td>wild</td></tr><tr><td>S. officinarum</td><td>72</td><td>formerly cultivated for sugar</td></tr><tr><td>S. barberi</td><td>15</td><td>formerly cultivated for sugar</td></tr><tr><td>S. sinense</td><td>6</td><td>formerly cultivated for sugar</td></tr><tr><td>S. edule</td><td>6</td><td>cultivated for edible inflorescence</td></tr><tr><td>S. sp</td><td>1</td><td>wild</td></tr><tr><td>Modern cultivars</td><td>96</td><td>cultivated for sugar or bioenergy</td></tr><tr><td><strong>Undetermined status</strong></td><td></td><td></td></tr><tr><td>S. maximum</td><td>8</td><td>wild</td></tr><tr><td><strong>Other genera</strong></td><td></td><td></td></tr><tr><td>Tripidium arundinaceum</td><td>2</td><td>wild</td></tr><tr><td>Miscanthus</td><td>3</td><td>wild</td></tr><tr><td>Erianthus fulvus</td><td>1</td><td>wild</td></tr><tr><td>Narenga</td><td>2</td><td>wild</td></tr></tbody></table>

hybridization. The subdivision of the *Saccharum* genus s.s. is a matter of debate,⁶,¹⁰ but a subdivision into six species is generally used by sugarcane technologists.¹¹,¹² Two of these are wild species: *S. robustum* (Brandes and Jeswiet ex Grassl.) and *S. spontaneum* (L.), which diverged around 1.3 mya.¹³ *S. spontaneum* (2n = 40–128) is morphologically diverse, with a distribution range extending from the Mediterranean Basin and Africa to Japan and Indonesia. This species generally has thin stalks with no or very-low sugar content. *S. robustum* (2n = 60, 80, and up to 200) is most likely native to Southeast Asia, i.e., southeast of Sulawesi, and has long thick stalks with generally little or no sugar content. The basic chromosome number in *S. robustum* is x = 10, similar to most *Andropogoneae* species, while in *S. spontaneum*, this number has evolved through chromosome rearrangements from x = 10 to x = 9 and x = 8, with this latter state being by far the most frequent.¹⁴,¹⁵ Four species exist only in cultivation (*S. officinarum*, *S. barberi*, *S. sinense*, and *S. edule*) and are considered by some authors to be horticultural groups, yet in practice, they are still referred to as species.

Many scenarios have been proposed regarding the origin of *S. officinarum* (L.) and the other horticultural groups.¹¹,¹² Several close genera (*Tripidium*, *Erianthus*, *Miscanthus*, *Narenga*, and *Sclerostachya*) may occasionally hybridize with *Saccharum*, and some authors have proposed that they are involved in the origin of sugarcane, referring to them as the “*Saccharum* complex.”¹¹,¹²,¹⁶,¹⁷ However, the most popular scenario is that sugarcane was domesticated from the *S. robustum* wild species in New Guinea around 8,000 years ago,¹⁸ giving rise to a series of clones that accumulated sugar in their stalks and were originally used for chewing. Botanists later identified them as *S. officinarum* (2n = 80) and referred to them as the “noble” canes. Some of these noble canes were subsequently trans-

ported by humans to the Asian continent, where they hybridized naturally with local forms of the *S. spontaneum* wild species, giving rise to a new series of cultivars (*S. barberi* [2n = 81–124] and *S. sinense* [2n = 116–120]) better adapted to subtropical environments and from which emerged sugar manufacturing.¹⁶ Nevertheless, some genomic studies have questioned this scenario.⁶,¹⁹ Two other small peculiar groups of uncertain origin have been reported: *S. edule* (Hassk.) (2n = 60–122), which is cultivated for its edible aborted inflorescence in Melanesia, and a group of canes that grows wild in South Pacific Islands while being referred to as *S. maximum* (Brongn.) Trin. (syn. *Erianthus maximus Brongn.*).¹¹,¹²,²⁰,²¹

The origin of modern sugarcane cultivars is well documented. They are all derived from a few interspecific hybridizations performed by breeders a century ago between the formerly cultivated groups *S. officinarum*, *S. barberi*, and the *S. spontaneum* wild species, followed by backcrossing with *S. officinarum*.¹¹,²² They are all highly polyploid and aneuploid, with around 120 chromosomes. Molecular cytogenetics studies have highlighted that 75%–85% of their chromosomes originated from *S. officinarum* and 15%–25% from *S. spontaneum*, including some chromosomes derived from interspecific recombinations.²³–²⁶

A few recent studies based on next-generation sequencing (NGS) have investigated *Saccharum* diversity based on dense single-nucleotide polymorphism (SNP) data,²⁷ while focusing mainly on the *S. spontaneum* wild species.¹⁵,²⁸

Another type of polymorphism based on transposable elements (TEs) is becoming accessible via whole-genome sequence (WGS) data. TEs account for a large part of plant genomes and are generally dispersed throughout the genome.²⁹ Even closely related species are often distinguished by the amplification of specific TEs that become durable markers of recent evolutionary history.³⁰ These characteristics have been exploited in the past to perform chromosome painting by genomic *in situ* hybridization, notably in sugarcane.²³–²⁶ TE diversity has recently been exploited, using repeated k-mers as a proxy, particularly to differentiate sub-genomes in allopolyploid assemblies.³⁰–³²

The objectives of this study were to (1) characterize the genetic diversity of wild *Saccharum* species, (2) determine the contribution of this diversity, along with that of closely related genera, to the origin of both ancient and modern sugarcane cultivars, and (3) to review the many sugarcane domestication and diversification scenarios. To this end, we exploited WGS data from 390 representative accessions as well as the recent chromosome-scale genome assembly of the R570 modern sugarcane cultivar.³³ We used chloroplast phylogeny and nuclear SNP diversity and developed methods based on repeated k-mers as proxies for TEs.

## RESULTS

A total of 300 accessions were sequenced in high-depth WGS (~15× per haplotype) and analyzed together with WGS data available for 90 *Saccharum* accessions (Tables 1 and S1).

### Chloroplast phylogenomics

Chloroplast genomes were assembled, and a phylogenetic tree was built based on their multiple alignments (Figures 1A
<figure><img src="image_4.png" /><figcaption>**Figure 1. Chloroplast phylogenomics and nuclear SNP-based PCoA**</figcaption></figure>

(A) Chloroplast phylogenomic tree of *Saccharum* and related genera

(B) Principal coordinate analysis (PCoA) based on nuclear SNP for *Saccharum* accessions.

In both panels, dots are colored according to taxonomy and for *S. officinarum* chlorotypes as indicated on the tree). Empty dots indicate wild hybrid accessions.
See also Figures S1, S2, and S4.

and S1). The chloroplast phylogenetic tree clearly separated *Saccharum* accessions from other genera with *Tripidium arundinaceum* (synonym *Erianthus arundinaceus*) being the furthest, followed by *Miscanthus*, *Erianthus fulvus*, and *Narenga* being the closest to *Saccharum*. The tree clearly differentiated the two wild species, i.e., *S. spontaneum* and *S. robustum*, and revealed diversity within both species. Two chlorotypes were shared by many accessions. Chlorotype I was shared by a small group of *S. robustum* accessions, part of the *S. officinarum* accessions and all *S. barberi*, *S. sinense*, and *S. maximum* accessions. Chlorotype II was found in another small group of *S. robustum* accessions, the remaining part of *S. officinarum* accessions, and all modern cultivars. *S. edule* accessions were located on *S. robustum* branches different from chlorotypes I and II.

## Relationship between *Saccharum* and close genera

A total of 18,074,258 SNPs were characterized by mapping of WGS reads on the R570 reference sequence masked for repetitive elements.³³ Nuclear SNP-based principal coordinate analysis (PCoA) revealed a clear differentiation between *Saccharum* and other genera (Figure S2A).

Within *Saccharum*, the PCoA opposed *S. spontaneum* to *S. robustum* and *S. officinarum* (Figures 1B and S2B). As expected, the *S. barberi* and *S. sinense* accessions and the modern cultivars had an intermediate position between *S. officinarum* and *S. spontaneum*. The *S. edule* accessions were grouped together with the *S. robustum* accessions.

Repeated k-mers (short repetitive sequences as a proxy for TEs) were generated from WGS data of the 390 accessions and characterized to identify genomic signatures of genera, species, or subgroups within these species. A total of 27 million

k-mers were selected (50 bp, minimum depth of 30× in at least one accession), and their distribution was analyzed in a graph displaying sets of k-mers (in columns) shared by accessions (in rows) (Figures 2A and S3).

Very large sets of k-mers were only shared between accessions representative of one of the studied genera: *Tripidium* (e.g., column 1, ~2.7 million k-mers), *Miscanthus* (column 2, ~1.8 million k-mers), *Narenga/Erianthus fulvus* (columns 4 and 5, ~0.7 and 0.6 million k-mers, respectively), and *Saccharum* (column 6, ~0.5 million k-mers) representing genomic signatures of these genera.

The signature k-mers from other genera were absent from all *Saccharum* accessions (wild and cultivated). The so-called *S. maximum* displayed signature k-mers from *Saccharum* (column 6) and from *Miscanthus* (column 2), indicating that they were intergeneric hybrids between these two genera.

## Genetic diversity within wild *Saccharum* species

Very large sets of repeated k-mers were shared between all *S. spontaneum* accessions but were absent in almost all *S. robustum* accessions (e.g., column 8: 169,824 k-mers) or, conversely, shared between all *S. robustum* accessions but absent in almost all *S. spontaneum* accessions (e.g., column 3: 742,387 k-mers) (Figures 2A and S3), thereby representing genomic signatures of these species. The few accessions that displayed repeated k-mers from both species also had an offset or intermediate position on the *Saccharum* nuclear SNP-based PCoA, supporting their interspecific origins (Figures 1B and S2B).

Within *S. spontaneum*, the nuclear SNP-based PCoA showed differentiation between accessions from the West to the East of Asia on the first axis, and from Northeast Asia (Japan, Taiwan,
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Figure 2. Repeated k-mer signatures and nuclear SNP-based PCoAs within *Saccharum*</figcaption></figure>

(A) Graph of repeated k-mers showing sets of k-mers (columns) shared between accessions (rows). Dot sizes indicate the average depth of k-mers in accessions. Dot colors indicate sets of k-mers representative of genera (black), species or subgroups (blue, *S. officinarum/S. robustum*; red, *S. spontaneum*; orange, *SEA S.*

(legend continued on next page)
Northeast, and China) to Southeast Asia (mainly Indonesia) on the second axis (Figures 2B and S2C). Accessions from the latter two origins formed two clear clusters and were also each located on different branches on the chloroplast phylogenetic tree (Figure S1). Accessions from Northeast Asia shared very large sets of k-mers (e.g., column 9: 129,707 k-mers) as well as accessions from Southeast Asia (e.g., column 11: 102,977 k-mers), thereby representing genomic signatures of these subgroups (Figure 2A). A more continuous distribution was observed on the PCoA for accessions from continental Asia, with accessions from the Northwest areas (mainly North India) located at one extreme and those from Southeast areas (mainly Thailand and China) at the other (Figure 2B). As for k-mers, many columns grouped together small numbers of accessions from continental Asia (e.g., column 32 and 68) that only partially correlated with their distribution on the PCoA and on the chloroplast phylogenetic tree (Figure S1).

Within *S. robustum*, the PCoA structuration was not linked to the geographical origin of the accessions (Figures 2C and S2D). Large sets of k-mers were shared only by part of the accessions (Figures 2A and S3) forming potential subgroups, with subgroup R1 (e.g., column 15: 56,404 k-mers), R2 (e.g., column 31: 19,548 k-mers), R3 (e.g., column 62: 7,775 k-mers), and R4 (e.g., column 196: 2,466 k-mers). Subgroups R1, R2, and R3 were also observed on the first plan of the *S. robustum* nuclear SNP-based PCoA, but accessions involved in subgroup R4 did not form a distinct cluster in the PCoA (Figures 2C and S2D), hence suggesting that we did not have pure representatives of this subgroup in our sample. Other accessions in the center of the PCoA may be hybrids between the identified subgroups and other *S. robustum* subgroups not represented in our sample. The subgroups based on repeated k-mers only partially correlated with the chloroplast phylogenetic tree, with in particular R4 accessions split between two branches shared with *S. officinarum*.

Finally, a few columns showed k-mers shared between all *S. robustum* accessions and either all *S. spontaneum* accessions from Northeast Asia (column 66: 7,102 k-mers) or SouthEast Asia (column 240: 2,080 k-mers).

## Contributions of wild ancestral groups to the genomes of *S. officinarum*

All large sets of repeated k-mers shared by all *S. officinarum* accessions were also shared by all *S. robustum* accessions (e.g., 742,387 in column 3); thus, no k-mer set was specific to all accessions of either species (Figures 2A and S3). These shared k-mers were thus considered as genomic signatures of both *S. robustum* and *S. officinarum*. A few accessions, referred to as *S. officinarum*, displayed signature k-mers from both *S. officinarum/S. robustum* and *S. spontaneum* and were also slightly offset from other *S. officinarum* accessions on the PCoA, thus indicating that they were hybrids (e.g., Brash and IM76-2444) (Figures 1B and S2B).

The *S. officinarum* nuclear SNP-based PCoA revealed no strong structuration, and no clear separation was observed between *S. officinarum* accessions with chlorotypes I and II (Figures 2D and S2E). According to the genetic similarity network built using SNP data, the few observed clusters of accessions likely corresponded to duplicates and/or somaclonal variants (Figure S4).

Finally, no large set of repeated k-mers was shared only between *S. officinarum* and other genera or shared only by some *S. officinarum* accessions, with the exception of four accessions from New Caledonia. These results fully support the hypothesis that *S. officinarum* was domesticated from *S. robustum*.

## Identification of an unknown ancestor

Four accessions (NC24, NC29, NC30, and NC93) previously classified as *S. officinarum* by botanists were particular since they shared large sets of repeated k-mers (e.g., Figure 2A, column 20: 40,619 k-mers) only among themselves and with two other peculiar accessions, i.e., NC100, an accession of uncertain taxonomic origin, and RaiateX, an accession described as *S. maximum*.³⁴ These four accessions were not particularly distant from the other *S. officinarum* accessions on the SNP-based PCoA (Figures 2D and S2E). To determine whether these sets of repeated k-mers represented polymorphisms within *S. officinarum* or an admixture with another contributor, we designed oligo probes based on signature k-mers of *S. officinarum/S. robustum* and hybridized them on chromosomes of the NC29 accession. The resulting *in situ* chromosome-painting pattern showed a majority of chromosomes labeled by the *S. officinarum/S. robustum* probes and a few entire chromosomes or chromosome segments not detected by the probes, which were thus inherited from another contributor (Figure 3).

The same chromosome-painting experiment revealed that ¹/₃ of the ~136 NC100 chromosomes originated from *S. officinarum/S. robustum* and for ²/₃ were from another contributor. For the RaiateX accession, the results showed that 19 chromosomes originated from *Miscanthus*, around 37 and 34 originated from *S. officinarum/S. robustum* and from another contributor respectively, while 8 chromosomes were the result of recombination between these two latter origins (Figure 3).

These particular k-mers were only shared by the six accessions (Figure 2A, column 20), all of which were hybrids with other known contributors. As no accession in our sample was a pure representative of this contributor, we qualified it as an unknown ancestor.

## Contributions of ancestral groups to the genomes of interspecific cultivars

The 96 modern cultivars and the 21 *S. barberi/S. sinense* traditional cultivars displayed the signature k-mers from *S. officinarum/S. robustum* and from distinct *S. spontaneum*.

*spontaneum*; light pink, NEA *S. spontaneum*; blue gradient, *S. robustum* R1 to R4 subgroups; purple, unknown ancestor). Other particular sets of shared k-mers (see Results) are indicated in gray.
(B-D) Nuclear SNP-based PCoA of (B) *S. spontaneum*, (C) *S. robustum*, and (D) *S. officinarum* with chlorotype I in dark green and chlorotype II in light green and accessions admixed with the unknown ancestor surrounded in pink.
See also Figures S2, S3, and S4.
<figure><img src="image_9.png" /><figcaption>**Figure 3. Chromosome in situ painting**</figcaption></figure>

(A–C) (A) NC29, (B) NC100, and (C) RaiateaX metaphase chromosomes hybridized with *S. officinarum/S. robustum* oligo probes detected in green, and for (C), total DNA of *Miscanthus* detected in red. Chromosomes were counterstained with DAPI (detected in gray).

subgroups. In addition, many modern cultivars displayed k-mers signatures from the unknown ancestral contributor. None of them displayed signature k-mers from other genera or large sets of k-mers shared only among themselves.

To quantify the contribution of each ancestral group to the genomes of these accessions, we developed an admixture inference approach based on the number of repeated k-mers occurrences. A model was first trained on all other accessions while considering seven ancestral groups, including *S. officinarum/S. robustum*, three *S. spontaneum* subgroups (continental Asia, Southeast Asia, and Northeast Asia), the unknown ancestral group, and to represent other genera: *Tripidium* and a group including *Miscanthus*, *Narenga*, and *Erianthus fulvus* (Figure S5; Table S2). This model was then applied to the 117 interspecific cultivars (Figure 4; Table S3).

### S. barberi and S. sinense

The estimated admixture revealed three main profiles that varied according to the proportions of the *S. officinarum/S. robustum* contribution (58% to 67%) and the *S. spontaneum* subgroups involved. A first group of three *S. barberi* accessions involved only *S. spontaneum* from continental Asia. The remaining *S. barberi* accessions involved *S. spontaneum* from continental Asia, Southeast Asia, and Northeast Asia. The six *S. sinense* accessions involved *S. spontaneum* from Southeast Asia and continental Asia. No other ancestral contributions were significantly detected. This grouping was consistent with the groups observed on the genetic similarity network, which reflected their previously suggested somaclonal origin (Figure S4).

### Modern hybrid cultivars

Estimated admixture proportions showed that modern cultivars inherited on average 74% of their genome from *S. officinarum/S. robustum* (range from 60% to 84%) and 25% from *S. spontaneum* (range from 15% to 40%) (Table S3). Most of the modern cultivars had *S. spontaneum* contributions from two origins, i.e., continental Asia (19% on average) and Southeast Asia (7% on average). A few of them had an additional *S. spontaneum* contribution from Northeast Asia. Finally, around ¾ of the modern cultivars had a significant contribution from the

unknown ancestral group (>0.1%), which represented around 1% of their genome.

### Contributions of ancestral groups to the genomes of S. edule and S. maximum

#### S. edule

The *S. edule* accessions grouped together with *S. robustum* on the nuclear SNP PCoA (Figures 1B, S2B, and S2D) and displayed the signature k-mers of *S. robustum/S. officinarum* but not of *S. spontaneum* or other genera (Figures 2A and S3). No set of repeated k-mers was specific to all of them, so they did not seem to represent or involve another taxon. These accessions were grouped with *S. robustum* on the chloroplast phylogenetic tree but not on the branches shared with *S. officinarum* (Figures 1A and S1). These results supported that these accessions are derived from *S. robustum*.

#### S. maximum

The *S. maximum* accessions displayed the signature k-mers from *S. officinarum/S. robustum* and *Miscanthus*, while the unknown ancestor was a third contributor for one of them (Figures 2A and S3). This showed that they were intergeneric hybrids that had inherited distinct proportions of *Saccharum* and *Miscanthus* genomes (Figure S5; Table S2).

### Distribution of signature k-mers on the R570 modern cultivar genome assembly

Our approach based on repeated k-mers was driven by the assumption that they would mainly correspond to repeated sequences dispersed along the genome. To verify this and to characterize ancestral contributions along chromosomes of a modern cultivar, we aligned signature k-mers of the ancestral groups that had contributed to modern cultivars onto the genome assembly of the R570 cultivar.³³ Although this cultivar has 2n = 114 chromosomes, this assembly comprises 67 pseudo-chromosomes since around 50% of the genome consists of segments present in 2 to 4 nearly identical copies collapsed in the assembly.

Signature k-mers corresponding to *S. officinarum*, *S. spontaneum*, and the unknown ancestor each aligned on distinct chromosomes or large chromosome segments and jointly covered the whole assembly (Figure 5A). This highlighted their dispersed nature and revealed a mosaic distribution with
<figure><img src="image_10.png" /><figcaption>**Figure 4.** AdmixKmer barplot of modern and traditional hybrid cultivars (*S. barberi* and *S. sinense*) considering seven ancestral groups
See also Figure S5.</figcaption></figure>

77%, 22%, and 1% of the assembly cumulatively corresponding to *S. officinarum*, *S. spontaneum*, and the unknown ancestor, respectively (Figures S6A–S6D).

Signature k-mers of the Southeast Asian *S. spontaneum* subgroup aligned with 1/3 of the chromosome segments contributed by *S. spontaneum*, corresponding to 7% of the R570 assembly (Figures 5A and S6E). Signature k-mers of the Northeast Asian subgroup were not shared with R570 (Figure 2A, column 9) and accordingly did not align significantly on the R570 assembly.

## Phylogenetic position of the unknown ancestor

To investigate the phylogenetic position of the unknown ancestor, we performed a phylogenetic analysis using 323 genes present on the chromosome 2C segment inherited from the unknown ancestor in R570, as well as on the six corresponding R570 hom(e)ologous chromosome 2 segments, and on the orthologous segments from *Erianthus fulvus* and *Sorghum* (Figure 5B). Based on the k-mer signatures, two of these R570 hom(e)ologous segments were identified as being inherited from *S. spontaneum* and four from *S. officinarum/S. robustum*. The resulting phylogenetic tree positioned the unknown ancestor within the monophyletic group of *Saccharum* haplotypes, with a branching distance closer to the *S. officinarum/S. robustum* haplotypes than to the *S. spontaneum* haplotypes.

## Contributions of the *S. robustum* subgroups to the *S. officinarum* genome

In a previous study based on the analysis of hom(e)ologous haplotypes from two genomic regions of the R570 modern cultivar, we suggested that chromosomes from two distinct contributors coexist in *S. officinarum* and that these contributions were shared with *S. robustum*.¹³ To further test this hypothesis, sets of k-mers shared by S. officinarum accessions, R570 and only part of the *S. robustum* accessions were aligned on the R570 assembly.

Signature k-mers of subgroup R4 (Figure 2A, column 196) aligned to several large segments of the R570 genome assembly producing a mosaic referred to as mosaic type 1 (Figure 5C). Signature k-mers of subgroups R1, R2, and R3 did not align significantly on the assembly, but k-mers shared between *S. robustum* subgroups R2 and R4 (e.g., column 169) aligned to several large segments of the R570 genome assembly producing a mosaic referred to as mosaic type 2 (Figure 5C). Similarly, k-mers shared between subgroups R1, R3, and R4 and subgroup R2 (with a low repetition level) (Figure 2A, column 53) produced a mosaic referred to as mosaic type 3 (Figure 5C). Intriguingly, type 2 and type 3 mosaics partially overlapped between themselves, and each completely overlapped with type 1, and jointly, the three mosaic types covered all *S. officinarum* chromosome segments of the R570 assembly (Figure 5D).

These results showed that chromosome segments from at least two ancestral contributors, and maybe more, coexist in *S. officinarum* and that these ancestral contributors correspond to different *S. robustum* sub-groups for some of which we do not have pure representatives in our study.

In the genomic region containing the ADH1 gene studied by Pompidor et al.,¹³ the *S. officinarum* haplotypes formed two phylogenetic groups referred to as genomes A and B. Comparison of this region with the R570 genome assembly revealed that haplotypes from genome B corresponded to a segment of chromosome 1A on which signature k-mers of the R4 *S. robustum* subgroup aligned (Figures 5D and S7). This suggests that genome B identified by Pompidor et al.¹³ corresponds to *S. robustum* subgroup R4.

## DISCUSSION

### Repeated k-mers as markers to detect admixture

Challenges in unraveling the origin of sugarcane and the relationships among *Saccharum* species are related to the highly complex polyploid nature of their genomes and their frequent
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

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<figure><img src="image_14.png" /></figure>

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<figure><img src="image_18.png" /><figcaption>**Figure 5. Genome in silico painting and phylogenomic analysis**</figcaption></figure>

(A) *In silico* painting of the R570 genome assembly using signature k-mers of *S. officinarum/S. robustum* (blue), the unknown contributor (purple), and *S. spontaneum* (red) with Southeast Asia *S. spontaneum* (orange).

(B) Phylogenetic tree based on 323 genes present on (1) the R570 chromosome 2c segment inherited from the unknown ancestor, (2) the six R570 hom(e)ologous segments (connected on (a) with gray-shaded areas), and (3) the *Erianthus fulvus* (Ef4) and *Sorghum* (Sb3) orthologous segments. Branches Sh-like support values are indicated.

*(legend continued on next page)*
hybridizations. To address this complexity, we have developed a multi-faceted approach that involves chloroplast phylogeny and nuclear SNP analysis and—to facilitate admixture detection— the analysis of repeated k-mers as a proxy for transposable elements (TEs).

We developed two methods, i.e., “KmerCity” and “AdmixKmer,” to analyze repeated k-mers from WGS data. In the first approach, we analyzed the distribution of the repeated k-mers throughout accessions using KmerCity. We were thus able to identify large sets of k-mers that were representative of the different genera, wild *Saccharum* species and some subgroups within these species. We showed that these k-mers mainly corresponded to LTR-retrotransposons and that they were dispersed throughout the genome (Document S1). The structure of the diversity observed based on these k-mers was globally found to be consistent with that observed with SNPs. One marked advantage of k-mers is their ability to detect an ancestral genome contribution, even if this contribution is small and even in the absence of pure representatives of that genome in the analyzed samples.

Furthermore, those admixtures can be validated and quantified through *in silico* mapping on an assembled genome or through *in situ* chromosome painting. We selected 50 bp k-mers to be able to design oligo-probes for fluorescent *in situ* hybridization (FISH). Specific oligo-probes were obtained for *S. officinarum*/*S. robustum* and *S. spontaneum* but not for subgroups within these species and the unknown contributor due to insufficient polymorphism.

In a second approach, we exploited the differences in the expected number of occurrences of k-mer counts according to the ancestral groups defined in the first approach, using AdmixKmer. This approach is an adaptation of the standard admixture modeling implemented in STRUCTURE³⁵ and ADMIXTURE,³⁶ where allele dosages are replaced by k-mer counts. This method allows quantification of ancestral contributions in hybrid genomes for which WGS data are available. A comparison of the results obtained using AdmixKmer and ADMIXTURE³⁶ highlighted the consistency between both approaches. However, ADMIXTURE was unable to detect the presence of the unknown ancestor and tended to create artificial ancestral groups among some of the hybrids (Document S2).

KmerCity and AdmixKmer are based on repeated k-mers, so they require limited sequencing depth, which is of particular interest for polyploid species. Both methods were validated using simulated data and data from well-characterized hybrids from another plant model,³ *Musa* (Document S2). In addition, the ability of our methods to detect admixture based on shared repeated k-mers was validated using the ABBA-BABA test (Document S2).

## Differentiation and diversity within the wild species:

### *S. spontaneum* and *S. robustum*

The geographical distribution of the two wild *Saccharum* species, i.e., *S. robustum* and *S. spontaneum*, now partially overlaps. However, it is probable that their original natural distribution was distinct to the east and west of the Wallace line.¹²

At the nuclear (k-mers and SNP) and chloroplast genome levels, a clear differentiation between the two wild species was observed, and both species displayed a large diversity. The very large genetic diversity within *S. spontaneum* has already been reported¹⁵,²⁸,³⁷ and correlated with their geographic distribution. This high genetic diversity is consistent with their large morphological diversity, variable chromosome numbers (2n = 40–128), their adaptability to various environments, and their wide geographical distribution. A substantial range of diversity was identified across the continental region of South Asia, extending from India to Thailand and China. While potential subgroups exist within continental South Asia region, the probable occurrence of gene flow between them complicated their clear delineation. Conversely, two clearly differentiated subgroups were observed in East Asia, one from Northeast Asia (including Japan and Taiwan) and one from Southeast Asia (including Indonesia).

Within *S. robustum*, whose genetic diversity has been little studied so far, a large genetic diversity was revealed with some structuration in potential subgroups, but without a clear link to geographical distribution. The k-mer analysis suggested that we may not have had pure representatives of all of these subgroups in our sample. Past changes in land connectivity due to the complex geological and climatic historical context of Southeast Asia could have cyclically put differentiated populations in contact in the past.³⁸ More recently, *S. robustum* accessions were also intensively used and thus likely widely disseminated by humans.³⁹ Accordingly, during the first experiments in the 1930s, various forms of *S. robustum* interbreeding with each other were observed and qualified as “hybrid swarm” and “a group of plants in an active state of evolution.”¹¹

We detected hybrids between both wild species, especially in Southeast Asia where their current geographic distributions overlap. Interestingly, we also found that the two groups of *S. spontaneum* accessions from Northeast and Southeast Asia each shared particular sets of repeated-k-mers with *S. robustum*, which may represent traces of very ancient genetic flow between these two species in Southeast Asia.

## Identification of a wild unknown contributor

Interestingly, the repeated k-mers approach has enabled the identification of a unique genomic contribution in six accessions from the South Pacific Islands. All of these accessions were

<footnote>(C) *In silico* painting of hom(e)ologous copies of chromosome 1 of the R570 genome assembly using *S. robustum* k-mers from columns 196, 169, and 53 (see Figure 2A). Colored boxes highlight segments included in mosaic type 1 (green), segments specific to mosaic type 2 or 3 (dark gray and black, respectively), and segments common to mosaic type 2 and 3 (light gray).</footnote>

<footnote>(D) Overlay of the three types of mosaics and the *in silico* painting obtained with signature k-mers of *S. robustum*/*S. officinarum* (blue) and *S. spontaneum* (red), including Southeast Asia *spontaneum* (orange). Location of *Adh1* haplotypes (from Pompidor et al.¹³) corresponding to genomes A and B are indicated. See also Figures S6 and S7.</footnote>
identified as hybrids with other species or genera; therefore, our sample lacked a pure representative of this unknown ancestor.

Phylogenetically, we showed that the unknown ancestor belongs to the *Saccharum* genus and is closer to *S. robustum* than to *S. spontaneum*. Pure representatives of this taxon need to be identified to determine whether it represents a new *Saccharum* species or a distant *S. robustum* subgroup. The geographical region in which these accessions were collected indicates that they are of East Melanesian/Polynesian origin, with New Caledonia as a potential geographical source of this unknown ancestor. New Caledonia is a biodiversity hotspot, globally renowned for the diversity and endemism of its flora, which are linked to its complex geological history.⁴⁰ Many groups are anomalously absent or depauperate in New Caledonia, although represented there by a sister group. In New Caledonia, *S. robustum* is not naturally present, the unknown ancestor could therefore have evolved there from a common ancestor with *S. robustum*.

## Refined domestication and diversification scenario

### S. officinarum

Our results clearly highlight that *S. officinarum* was domesticated from *S. robustum* and that no other genera or taxa were involved in the process (Figure 6). These results are in line with the scenario proposed by Brandes¹⁸ after the first expeditions to New Guinea in the 19th century during which *S. robustum* was discovered. In this region, very diverse forms of *S. robustum* and *S. officinarum* were discovered, with both groups being morphologically quite similar and differing mainly by their capacity to accumulate sucrose in their stalks.

In addition, our results suggest that this domestication involves distinct *S. robustum* subgroups. Explorers, during the first expeditions to New Guinea, observed that some of *S. robustum* forms were being used by man for building fences and houses, as well as for medicinal applications.³⁹ The initial steps of domestication thus likely involved non-food exploitation of *S. robustum*. These canes were thus intensively cultivated near habitation and widely disseminated, thereby offering many opportunities for hybridization between distinct forms of *S. robustum* and progressive selection of new canes with attractive characteristics, such as sugar in their stalks. After several steps of recombination, somaclonal variation and selection in subsistence gardens, clones with a high sucrose and low fiber could have gradually emerged and subsequently intensively vegetatively propagated.

Our *S. officinarum* sample was divided into two groups based on their chloroplast genomes. However, these two groups could not clearly be distinguished at the nuclear level. It is therefore conceivable that forms of *S. officinarum* that inherited similar nuclear ancestral contributions from different *S. robustum* subgroups, but distinct chloroplast genomes, have emerged in different regions or at different times. Evans and Joshi⁵ and Zhang et al.¹⁹ questioned the domestication of *S. officinarum* from *S. robustum*, based on estimated divergence times (0.65 and 0.39 mya, respectively) and distinct chloroplast genomes. Evans and Joshi⁶ additionally proposed *S. cultum* as a potential ancestor of modern cultivars. Our data do not support these conclusions, likely influenced by limited sampling. Notably, Pompi-

dor et al.¹³ reported similar divergence ranges (0.05 to 0.84 mya) within *S. officinarum* haplotypes from two genomic regions, which aligns with our interpretation that the observed divergence reflects differentiation among *S. robustum* subgroups, including those contributing to the origin of *S. officinarum*.

### S. barberi/S. sinense

*S. barberi* and *S. sinense* are canes that were previously cultivated in India and China, respectively. They are thinner than *S. officinarum* and have less sugar but are more adapted to sub-tropical environments. Our data indicate that they are interspecific hybrids between *S. officinarum* and *S. spontaneum* and that these hybrids constitute distinct somaclone groups. Our results are thus in line with Brandes¹⁸ hypothesis that *S. officinarum* clones were transported by humans to continental Asia where they naturally hybridized with *S. spontaneum* (Figure 6). We showed that during their long dissemination toward the West, *S. officinarum* and its derived hybrids have crossed with distinct *S. spontaneum* subgroups. Therefore, several generations were involved, as already suggested by their peculiar chromosome numbers²¹ as well as by the presence of interspecific recombinant chromosomes.⁴¹

### S. edule

*S. edule* is a traditional Melanesian vegetable cultivated in village gardens from New Guinea to Fiji for its edible aborted inflorescences. In our results, *S. edule* accessions could not be genetically differentiated from *S. robustum* and did not have a chloroplast genome in common with *S. officinarum*, supporting that they were directly derived from *S. robustum*. As *S. robustum* was grown intensively near habitations, humans likely identified mutants with edible aborted inflorescences and then multiplied them vegetatively.

### S. maximum

*S. maximum* (syn. *Erianthus maximus*) has been reported as wild populations in some South Pacific Islands (New Caledonia, Fiji, and the Cook, Society, Marquesas, and Austral islands).¹² Our results confirmed their intergeneric hybrid origin between *Saccharum* and *Miscanthus*, as previously suggested²⁰,²¹ (Figure 6). It is likely that *S. officinarum* is involved based on the presence of some sugar in their stalks and the absence of *S. robustum* in their distribution area.¹² One of the *S. maximum* accessions had a third genomic contribution from the unknown ancestor, thereby representing a distinct *S. maximus* type.

## Genomic constitution of modern cultivars

In the modern cultivars, the average *S. officinarum* (74%) and *S. spontaneum* (25%) contribution and the presence of interspecific chromosome recombinants are consistent with previous findings based on chromosome *in situ* painting.²³⁻²⁶ In most modern cultivars, we could differentiate *S. spontaneum* contributions from two origins: Southeast Asia and continental Asia. This was consistent with the historical records of pioneer interspecific breeding in the early 1900s, in Java and India, from which all modern cultivars derived. In the Java Sugar Experiment Station (Proefstation Oost Java), breeders exploited Glagah, a local *S. spontaneum* clone, from the Indonesian subgroup, through a natural hybrid (Kassoer). This led to the creation of successful cultivars, including POJ2878, the “wonder cane” that rapidly spread worldwide. In line with this pedigree, we showed that this cultivar
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>**Figure 6. Scenario for sugarcane domestication and diversification**</figcaption></figure>

(A) Geographical distribution of *S. robustum* (blue), *S. spontaneum* from continental Asia (red), from Southeast Asia (orange), and from Northeast Asia (light pink), the unknown ancestral contributor (purple), and *Miscanthus floridulus* (gray). Hypothesized centers of origin for *S. officinarum* (green), *S. barberi* and *S. sinense* (yellow), and *S. maximum* (dark gray). The hypothesized dispersal routes of *S. officinarum* are indicated by arrows.

(B) Scenario for sugarcane domestication and diversification with major hybridization and domestication events indicated by arrows. Wild species are circled in dotted lines.
had a *S. spontaneum* contribution only from the Southeast Asia subgroup. POJ2878 also had an exceptional breeding value, which is why it is involved in the pedigree of almost all modern cultivars.⁴² At the Sugarcane Breeding Institute in India, breeders exploited *S. barberi* and local *S. spontaneum* forms from the continental Asian subgroup.²² Consistently, the only cultivars we analyzed that had a *S. spontaneum* contribution only from continental Asia came from this Indian breeding program. The few modern cultivars, in which we identified a contribution from the Northeast Asia *S. spontaneum* subgroup, were logically mainly from Japan. In addition, a contribution from the unknown ancestor was identified in many cultivars, including R570. Historical records indicate that *S. officinarum* clones from the Pacific islands, including Polynesia and New Caledonia, were collected early, starting during the first expedition of Bougainville (1768) during which the famous Otahiti clone from Polynesia was supposedly collected. Subsequently, some of these clones were introduced, cultivated intensively, and used as breeding materials in numerous regions worldwide. Contributions from the unknown ancestor have been found in cultivar POJ2878, one of the early modern cultivars, suggesting that *S. officinarum* introgressed with this unknown ancestor were involved in the early phases of modern sugarcane breeding.

## Conclusion

Our results have refined the global scenario of sugarcane domestication proposed by Brandes¹⁸ supporting that *S. officinarum* and *S. edule* were domesticated from *S. robustum* in New Guinea and surrounding islands (Figure 6) and that *S. officinarum* domestication involved hybridization between distinct forms of *S. robustum*. They indicated one center of diversification in continental Asia where *S. officinarum* clones began being transported by humans 3,000 years ago¹⁶ and then naturally hybridized with distinct *S. spontaneum* forms, leading to the emergence of traditional *S. barberi* and *S. sinense* cultivars. They identified a second center of diversification in the Melanesian and Polynesian islands where *S. officinarum* was introduced during Melanesians migrations from 3,500 to 500 years ago⁴³ and naturally hybridized with an unknown *Saccharum* ancestor and with *Miscanthus*.

We showed that modern cultivars inherited contributions from Southeast Asia and continental Asia *S. spontaneum*, while *S. spontaneum* forms from Northeast Asia have seldom been used in breeding programs. They could thus serve as a valuable source of adaptive alleles, as demonstrated by the recent identification of smut resistance alleles.⁴⁴

Our discovery of an unknown *Saccharum* group is of particular significance, as it has contributed to most modern cultivars. No pure representative of this unknown contributor seems to be available in international collections. Therefore, it would be beneficial to organize prospections with the aim of collecting pure representatives of this group, which may be endangered in the current context of increasing anthropization, and which could represent a new source of adaptive alleles.

The methodologies based on repeated k-mers that we developed have been instrumental in detecting and characterizing admixed genomes and discovering an unknown genome. They

should be particularly useful for characterizing other genomes and populations combining admixture and polyploidy.

## Limitations of the study

We analyzed a large panel of 390 representatives of the *Saccharum* diversity with WGS data. This panel is particularly notable for *S. robustum*, as it was significantly underrepresented in previous analyses.

However, it is possible that our sample still does not contain all and enough pure representatives of the different *S. robustum* and *S. spontaneum* subgroups (particularly those from continental India), in order to fully define these subgroups and their contributions to the cultivars. This may have resulted in some degree of imprecision when identifying the exact contribution of *S. spontaneum* subgroups, especially to the genome of *S. barberi* and *S. sinense* cultivars that have emerged in continental Asia. It may also have hindered the clear identification of the contributions of *S. robustum* subgroups to the *S. officinarum* genome, by mapping of the *S. robustum* subgroup k-mers onto the R570 modern cultivar assembly. While we showed that at least two subgroups have contributed to the genome of *S. officinarum*, we were only able to suggest that additional subgroups could have contributed. In the future, access to high-quality haplotype-resolved genome assemblies of representative accessions of *S. robustum* and *S. officinarum* will also help refine those results.

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information should be directed to the lead contact, Angélique D'Hont (dhont@cirad.fr).

### Materials availability

This study did not generate new, unique reagents.

### Data and code availability

All the raw sequencing data generated in this study have been deposited in the NCBI database under BioProjects PRJNA1228676 and PRJNA1209834. WGS data of the 89 *S. spontaneum*, *S. officinarum*, LA Purple, and R570 cultivars are available from NCBI BioProject PRJNA721787 and the Read Archive Database SRX4627259 and SRX19712494, respectively.

The *Musa* WGS data used for the validation of methods is available from BioProjects PRJEB58004 and PRJEB28077.

Reference genome assemblies of R570 sugarcane cultivar (v2.1) and *Sorghum bicolor* (v 3.1.1) are available from Phytozome: https://phytozome-next.jgi.doe.gov/info/Sofficinarumxs spontaneum R570_v2_1 and Phytozome: https://phytozome-next.jgi.doe.gov/info/Sbicolor_v3_1_1, respectively. *Erianthus fulvus* genome assembly is available at http://sugarcane.zhangjisenlab.cn/SugarcaneDB/#/downloads.

The VCF file with 18 million nuclear SNPs, the chloroplast genome assemblies and the KmerCity database including 27 million repeated k-mers are available from the CIRAD Dataverse: https://doi.org/10.18167/DVN1/YOH50G.

KmerCity and AdmixKmer pipelines, as well as all the associated programs and original codes developed in this study are available at https://gitlab.cirad.fr/agap/seg/kmercity and https://gitlab.cirad.fr/agap/seg/admixkmer.

## ACKNOWLEDGMENTS

The work (proposal: 10.46936/10.25585/60001194) conducted by the US Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, was supported by the Office of Science
of the US Department of Energy operated under contract no. DE-AC02-05CH11231. The work conducted by CIRAD was supported by the International Consortium for Sugarcane Biotechnology. Data analyses were performed with the support of MESO@LR Bioinformatic Platform (University of Montpellier, France). We would like to thank all the people who have helped in sending plant materials, including Ivan Tonasso and Dong-Liang Huang and the CIRAD-INRAE Biological Resource Centre for Tropical Plants (CRB-PT) in the West Indies (Guadeloupe, France). We thank Clémence Giloupe Taillefer for her contribution to the cytogenetics experiments.

## AUTHOR CONTRIBUTIONS

O.G., S.R., and N.P. designed and developed the bioinformatics tools and performed the bioinformatics analyses; T.D. contributed to bioinformatics analysis; C.H. extracted DNA and performed cytogenetic analysis; A.L. performed resequencing data QA/QC; C.D. and Y.Y. oversaw library construction and sequencing; M.B., A.S., G.P., N.L., A.H., J.Y.H., Y.T., E.S., S.S., P.L., and M. V. provided plant materials; A.D., O.G., and S.R. interpreted the data and wrote the manuscript that was then improved by N.L., J.Y.H., Y.T., P.L., and J.S.; A. D. designed the project and coordinated the analyses; K.B. managed the JGI part of the project; A.D. and J.S. supervised the project; and all authors have read and approved the content of the manuscript.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* METHOD DETAILS

* DNA extraction

* Whole genome Sequencing

* *In situ* chromosome-painting

* QUANTIFICATION AND STATISTICAL ANALYSIS

* Read coverage and quality assessment

* Chloroplast genome assemblies and phylogeny

* SNP calling, similarity network and principal coordinate analyses

* Analyses based on repeated k-mers

* *In silico* painting of genome assembly

* *In situ* chromosome painting probe design

* Phylogenetic analyses of unknown ancestor

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cell.2025.09.017.

Received: February 13, 2025

Revised: July 11, 2025

Accepted: September 18, 2025

Published: October 14, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>Cyanine3 (Cy3) fluorescent dye</td><td>Thermo Fisher Scientific</td><td>N/A</td></tr><tr><td>ATTO-633 dye</td><td>Integrated DNA Technologies, Inc (IDT)</td><td>N/A</td></tr><tr><td>4'-6-diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher Scientific</td><td>N/A</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>All sequencing data generated in this study have been deposited in NCBI database; individual access numbers are provided in Table S1</td><td>This study</td><td>NCBI Bioproject: PRJNA1228676, PRJNA1209834; See Table S1</td></tr><tr><td>S. spontaneum accessions (89 accessions); individual access numbers are provided in Table S1.</td><td>Zhang et al.<sup>15</sup></td><td>NCBI Bioproject: PRJNA721787; See Table S1</td></tr><tr><td>S. officinarum LA purple (whole-genome resequencing data)</td><td>DOE Joint Genome Institute</td><td>SRA:SRX4627259</td></tr><tr><td>Sugarcane cultivar R570 (reference genome, version 2.1)</td><td>Healey et al.<sup>33</sup></td><td>https://phytozome-next.jgi.doe.gov/info/Sofficinarumxs spontaneum R570_v2_1</td></tr><tr><td>Sugarcane cultivar R570 (whole-genome resequencing data)</td><td>DOE Joint Genome Institute</td><td>SRA:SRX19712494</td></tr><tr><td>Sorghum bicolor (reference genome, version 3.1.1)</td><td>McCormick et al.<sup>61</sup></td><td>https://phytozome-next.jgi.doe.gov/info/Sbicolor_v3_1_1</td></tr><tr><td>Erianthus fulvus (reference genome, synonym E. rufipilus)</td><td>Wang et al.<sup>60</sup></td><td>http://sugarcane.zhangjisenlab.cn/SugarcaneDB/#/downloads</td></tr><tr><td>Musa whole genome resequencing data (used for the validation of methods)</td><td>Martin et al.<sup>3</sup></td><td>NCBI Bioproject: PRJEB58004, PRJEB28077</td></tr><tr><td>Chloroplast genome assemblies</td><td>This study</td><td>CIRAD Dataverse: https://doi.org/10.18167/DVN1/YOH50G</td></tr><tr><td>KmerCity database (27 M repeated k-mers selected)</td><td>This study</td><td>CIRAD Dataverse: https://doi.org/10.18167/DVN1/YOH50G</td></tr><tr><td>VCF file (18M nuclear SNPs)</td><td>This study</td><td>CIRAD Dataverse: https://doi.org/10.18167/DVN1/YOH50G</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>All the accessions analyzed in this study are described in Table S1</td><td>This study</td><td>See Table S1</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>BBDuk (BBmap version 38.90)</td><td>Join Genome Institute (JGI, USA), BBTools package</td><td>RRID:SCR_016969; https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/</td></tr><tr><td>MAFFT (version 7.475)</td><td>Katoh and Standley<sup>46</sup></td><td>RRID:SCR_011811; http://mafft.cbrc.jp/alignment/server/</td></tr><tr><td>Gblocks (version 0.91b)</td><td>Castresana et al.<sup>47</sup></td><td>RRID:SCR_015945</td></tr><tr><td>MrBayes (version 3.2)</td><td>Ronquist et al.<sup>48</sup></td><td>RRID:SCR_012067; http://mrbayes.sourceforge.net/</td></tr><tr><td>Treedataverse (R package library version 4.3.1)</td><td>Yu et al.<sup>49</sup></td><td>https://yulab-smu.top/treedata-book/</td></tr><tr><td>Mirabait (MIRA package version 4.0.2)</td><td>https://bioweb.pasteur.fr/packages/pack@mira@4.0.2</td><td>RRID:SCR_010731; http://sourceforge.net/p/mira-assembler/wiki/Home/</td></tr><tr><td>VcfHunter (process_reseq_1.0.py)</td><td>Garsmeur et al.<sup>50</sup></td><td>https://github.com/SouthGreenPlatform/VcfHunter</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>BWA-MEM2 (version 2.0)</td><td>Vasimuddin et al.<sup>51</sup></td><td>RRID:SCR_022192; https://github.com/bwa-mem2/bwa-mem2</td></tr><tr><td>GATK3 (version 3.6)</td><td>DePristo et al.<sup>52</sup></td><td>RRID:SCR_001876; https://gatk.broadinstitute.org/hc/en-us</td></tr><tr><td>bam-readcount (version 0.8.0)</td><td>Khanna et al.<sup>53</sup></td><td>RRID:SCR_023653; https://github.com/genome/bam-readcount</td></tr><tr><td>Polyimpute</td><td>This study</td><td>https://gitlab.cirad.fr/agap/seg/polyimpute</td></tr><tr><td>KmerCity</td><td>This study</td><td>https://gitlab.cirad.fr/agap/seg/kmercity</td></tr><tr><td>Kraken2 (version 2.1.2)</td><td>Wood et al.<sup>56</sup></td><td>RRID:SCR_026838; https://github.com/DerrickWood/kraken2</td></tr><tr><td>Kat (version 2.4.2)</td><td>Mapleson et al.<sup>57</sup></td><td>RRID:SCR_016741; https://github.com/TGAC/KAT</td></tr><tr><td>KCounts_2_intersections</td><td>This study</td><td>https://gitlab.cirad.fr/agap/seg/kmercity</td></tr><tr><td>Draw_KGraph</td><td>This study</td><td>https://gitlab.cirad.fr/agap/seg/kmercity</td></tr><tr><td>AdmixKmer</td><td>This study</td><td>https://gitlab.cirad.fr/agap/seg/admixkmer</td></tr><tr><td>ADMIXTURE</td><td>Alexander et al.<sup>36</sup></td><td>RRID:SCR_001263</td></tr><tr><td>Build_KBlocks</td><td>This study</td><td>https://gitlab.cirad.fr/agap/seg/kmercity</td></tr><tr><td>InSilicoPainting</td><td>This study</td><td>https://gitlab.cirad.fr/agap/seg/kmercity</td></tr><tr><td>MCScanX (version 1.0.0)</td><td>Wang et al.<sup>59</sup></td><td>RRID:SCR_022067; https://github.com/wyp1125/MCScanX</td></tr><tr><td>FastTree2</td><td>Price et al.<sup>62</sup></td><td>RRID:SCR_015501; http://www.microbesonline.org/fasttree/</td></tr><tr><td>Figtree (version 1.4.4)</td><td>https://github.com/rambaut/figtree/releases/tag/v1.4.4</td><td>RRID:SCR_008515; http://tree.bio.ed.ac.uk/software/figtree</td></tr><tr><td>BLASTN (version 2.12.0+)</td><td>https://ncbiinsights.ncbi.nlm.nih.gov/2021/07/09/blast-2-12-0/</td><td>RRID:SCR_004870; http://blast.ncbi.nlm.nih.gov/Blast.cgi</td></tr><tr><td>GetOrganelle (version 1.7.3.5)</td><td>Jin et al.<sup>45</sup></td><td>RRID:SCR_022963; https://github.com/Kinggerm/GetOrganelle</td></tr><tr><td>FastTQC (version 0.11.7)</td><td>http://www.bioinformatics.babraham.ac.uk/projects/fastqc/</td><td>RRID:SCR_014583</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Leaves were obtained from different institute collections for 300 accessions representative of the *Saccharum* genus including *S. spontaneum*, *S. robustum*, *S. edule*, *S. officinarum*, *S. barberi*, *S. sinense*, *S. maximum*, and modern cultivars, as well as a few representatives of close genera (*Tripidium arundinaceum*, *Miscanthus*, *Narenga* and *Erianthus fulvus*). Additional 150bp paired-end WGS data were available in public repositories representing 89 *S. spontaneum* accessions (Zhang et al.,¹⁵,²⁸ https://www.ncbi.nlm.nih.gov/bioproject/PRJNA721787), and one *S. officinarum* accession (LA-Purple, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA333303). See Table S1 for complete description of the 390 accessions analyzed.

## METHOD DETAILS

### DNA extraction

Total genomic DNA was extracted from 500 mg of fresh leaves using MATAB reagent and the quality of DNA was verified by electrophoresis on a 1% gel agarose with ethidium bromide staining. DNA quantification was performed using Thermo Scientific NanoDrop 2000c spectrophotometer.

### Whole genome Sequencing

Library construction and 150 bp paired-end sequencing were performed at the Joint Genome Institute (290 accessions) or, for a few of them, at GeneWiz (Azenta life Sciences, 10 accessions). Genomic DNA (200 ng) was sheared to 600 bp using a Covaris LE220, size-selected via double-SPRI (TotalPure NGS beads, Omega Bio-tek), end-repaired, A-tailed, and ligated with dual-index Illumina adaptors (IDT, Inc). Libraries were quantified using KAPA Biosystems' next-generation sequencing library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument. Paired-end (2x150bp) sequencing was performed on Illumina NovaSeq sequencer.
## In situ chromosome-painting

Chromosome preparations and hybridizations were performed as described in D'Hont et al.²⁴ Genomic DNA from Miscanthus (NG77-22) was labelled by random priming with Cy3, and the S. officinarum oligo-probes were labelled with ATTO-633. After counterstaining with 4'-6-diamidino-2-phenylindole (DAPI, Vectashield Mounting Media with DAPI), images were captured with a Hamamatsu CCD camera.

## QUANTIFICATION AND STATISTICAL ANALYSIS

### Read coverage and quality assessment

Genomic DNA was sequenced with an intended depth of 15x per haplotype. In practice, an average depth of 21x was achieved yielding from 298M to 2295M reads per sample. Saccharum accessions have variable ploidy levels and chromosome numbers are not available for all of them. Therefore, when available in the literature, reported chromosome numbers were used to determine the ploidy levels. In cases where chromosome numbers were not available, we relied on the most frequent chromosome numbers for the species or subgroup considered (Table S1). Total genome size was estimated by multiplying the monoploid genome size (~800 Mbp for S. spontaneum and ~900 Mbp for S. officinarum) by the inferred ploidy level. Sequencing genome coverage was then calculated by dividing the total number of 150 bp reads generated by the estimated total genome size. Read quality was assessed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and an average of 95.4% of reads had a quality score of Q>=30 (99.9% accuracy). BBDuk from the BBTools package (https://jgi.doe.gov/data-and-tools/bbtools/) was used to trim reads for Illumina adapters and base quality (ktrim=r, k=23, mink=11, hdist=11, qtrim=r, trimq=20, maq=10, minlen=30, tpe, tbo).

### Chloroplast genome assemblies and phylogeny

The chloroplast genome of all accessions was assembled using GetOrganelle⁴⁵ into a single circular sequence, with the exception of six accessions (E2, NG57-40, GT11, ROC22, YC75-284, YC96-69) for which the assembly was fragmented and which were excluded from subsequent analyses. Multiple alignment of chloroplast genomes was done using MAFFT⁴⁶ and filtered for poorly aligned positions and divergent DNA regions using Gblocks,⁴⁷ resulting in 848 SNPs. The inference of the phylogenetic tree was performed using MrBayes⁴⁸ considering the “GTR+I+Γ” model and 20M iterations. The phylogenetic tree was represented using the “treedataverse” meta R-package.⁴⁹

### SNP calling, similarity network and principal coordinate analyses

The SNP calling was performed using a monoploid reference sequence with masked repetitive elements derived from the polyploid genome assembly of R570³³ by choosing the longest chromosome from each hom(e)ology group, and excluding chromosomes rearranged in S. spontaneum compared to S. officinarum (i.e. Chr1A, Chr2A, Chr3A, Chr4A, Chr5A, Chr6A, Chr7A, Chr8A, Chr9A, and Chr10A). The VcfHunter SNP calling workflow was applied (https://github.com/SouthGreenPlatform/VcfHunter)⁵⁰ with the following steps: (i) mapping of the selected sequencing reads using BWA MEM2,⁵¹ (ii) local realignment of reads around indels using GATK3 IndelRealigner,⁵² (iii) allele count reporting using bam-readcount⁵³ and (iv) VCF generation with diploid-like genotype classes: 0/0, 0/1 and 1/1, as described in Garsmeur et al.⁵⁰ Only high-confidence SNPs with less than 5% missing values across all 390 accessions were retained, leading to a total of 18,074,258 bi-allelic SNPs. In addition to this diploid-like genotype coding, an alternative coding based on allele read depth ratios (i.e. allele read depth to the total read depth) was considered as a proxy of allele dosages.

To identify duplicates or accessions with very similar genetic profiles, the percentage of identical diploid-like genotyping data-points between each pair of accessions was calculated on their shared set of non-missing SNPs. Accessions with at least 99% similarity were represented as connected on a genetic similarity network graph.

Principal coordinate analyses (PCAs) were performed on the whole population or on subsets of the whole population using a Euclidean distance matrix obtained from imputed SNP data coded as read depth ratios. The imputation was performed using the R-package 'PolyImpute' developed in this study (https://gitlab.cirad.fr/agap/seg/polyimpute), see detailed description of the method in Document S3, adapted from the expectation-maximization (EM) algorithm presented in Poland et al.⁵⁴ In summary, genotype data-points with a sequencing depth of less than 30 were considered as ambiguous and updated based on their overall genomic resemblance with other accessions.

### Analyses based on repeated k-mers

The workflow 'KmerCity' (https://gitlab.cirad.fr/agap/seg/kmercity) was developed to identify k-mer sequences occurring abundantly in genomes, and to report their comparative frequencies and distribution in the panel of accessions analyzed.

Before running the pipeline, WGS data were filtered out for chloroplast and mitochondrial sequences with Mirabait (https://mirassembler.sourceforge.net/docs/DefinitiveGuideToMIRA.html) using the R570 chloroplast genome assembled in this study and the mitochondrial genome assembly of Khon Kaen 3.⁵⁵ In addition, WGS data were filtered out for potential contamination with Kraken 2⁵⁶ using a standard database (i.e. archaea, bacteria, viral, plasmid, human and UniVec_Core).

The KmerCity pipeline was run on all accessions and included the following steps: i) for each accession, 20M sequencing reads were randomly subsampled from WGS data, ii) 50-mers were generated from read subsamples and for each accession k-mer counts were reported into individual tables using kat,⁵⁷ iii) repeated k-mers were selected by retaining only those observed at a depth
superior or equal to 30x in at least one accession and when a k-mer was selected based on this criterion, its depth was reported in all accessions, including those where the depth was below 30x, iv) a repeated k-mer count database aggregating individual k-mer count tables was created and included 27,015,331 k-mers.

The repeated k-mer count database was then processed with the in-house script 'KCounts_2_intersection.pl' to identify intersections that correspond to sets of k-mer sequences shared by at least two accessions and absent or with a low depth (<10x) in all others. A graph of repeated k-mer was built based on a method adapted from the UpSet plot visualization with the in-house script 'Draw_KGraph.pl' in which sets of shared k-mers are represented as columns and accessions as rows. A total of ~7.3 million columns corresponding to sets of kmers shared by at least two accessions were obtained, each including from 10 to ~2.7 million k-mers.

Genome-wide admixture inference was performed using the same repeated k-mer count database and the R-package 'AdmixKmer' developed in this study (https://gitlab.cirad.fr/agap/seg/admixkmer), see detailed description of the method in Document S4, consisting of an EM algorithm adapted from van Oosten.⁵⁸ In the first step, model parameters were estimated from ancestral accessions, excluding hybrids (i.e. *S. barberi*, *S. sinense* and modern cultivars), by considering six ancestral groups. Given the limited contribution of the unknown ancestor to the population of ancestral accessions, with no pure representatives, it is very difficult to detect it automatically. To solve this issue, in the second step, admixture proportions of accessions with substantial contribution from this unknown contributor (i.e. NC100, NC24, NC29, NC30, and Raiatea) were set manually based on cytogenetics observations (Table S2). In the third step, expected k-mer counts were re-estimated considering fixed admixture proportions, including those corresponding to the unknown ancestral group. In the last step, these estimates of expected k-mer counts were treated as fixed in a new model used to estimate the admixture proportions of hybrids.

## In silico painting of genome assembly

Sets of k-mer signatures representative of species or sub-groups were aligned onto the R570 genome assembly using BLASTN and perfect hits were retained (BLAST options: -task blastn-short, -dust no, -perc_identity 100, -word_size 50, -qcov_hsp_percent 100, -max_target_seqs 100000). For each BLAST alignment, the in-house program 'Build_KBlocks.pl' was used to calculate the k-mer density within 100 kb bins along the genome assembly and to distinguish bins with high k-mer density from those with a lower k-mer density. A bin was considered to have a high k-mer density if the number of k-mers in that bin was greater than or equal to half the average number of k-mers in all bins.

Adjacent 100Kbp bins with high-kmer density were merged into blocks if the distance separating them did not exceed 2Mbp. The in-house script 'InSilicoPainting.pl' was then used to produce ideograms displaying the location of kmers that fall into these blocks along the R570 genome assembly (Figures 4 and S5). Buid_Kblocks.pl and InSilicoPainting.pl programs are available at https://gitlab.cirad.fr/agap/seg/kmercity.

## In situ chromosome painting probe design

The design of *S. officinarum* specific oligo probes was based on the sequences of *S. officinarum/S. robustum* signature k-mers (Figure 2A, column 3) and subcontracted to Arbor Biosciences (Ann Arbor, MI, USA). K-mers were cut into overlapping probe candidates (43 to 47 bp) to account for a narrow Tm distribution. The genome assembly of R570³³ was used to verify the distribution of candidate probes and reject those with potential cross-hybridization with chromosome segments other than *S. officinarum*. A total of 4,000 highly repeated oligo probes distributed exclusively in *S. officinarum* chromosome segments were selected.

## Phylogenetic analyses of unknown ancestor

The toolkit MCScanX⁵⁹ was used to identify the collinear genes models conserved between the chromosome segment inherited from the unknown ancestor in R570 (Chr2C from position 55,409,515 to 89,674,989), the six corresponding R570 hom(e)ologous chromosome segments (Chr2A, Chr2B, Chr2D, Chr2E, Chr2F, Chr2G) and the orthologous segments from *Erianthus fulvus*⁶⁰ and *Sorghum*.⁶¹ A total of 323 groups of syntenic genes were identified and for each of them a multiple sequence alignment was performed with MAFFT⁴⁶ followed by a cleanup of misaligned and ambiguous positions with Gblocks.⁴⁷ A phylogenetic tree was built with FastTree2⁵² using the general time reversible model (GTR) of nucleotide substitution, and represented with FigTree (v1.4.4, https://github.com/rambaut/figtree/releases).
Supplemental figures

<figure><img src="image_21.png" /></figure>

* Tripidium / Miscanthus / E. fulvus / Narenga

* S. maximum

* S. spontaneum

* Northeast S. spontaneum

* Southeast S. spontaneum

* S. robustum

* R1 S. robustum

* R2 S. robustum

* R3 S. robustum

* R4 S. robustum

* S. edule

* S. officinarum (chlorotype II)

* S. officinarum (chlorotype I)

* S. barberi / S. sinense

* Modern cultivars

(legend on next page)
<figure><img src="image_22.png" /></figure>

Figure S1. Chloroplast phylogenomic tree of *Saccharum* accessions and related genera, related to Figure 1A

Colored dots represent accessions with *Tripidium*, *Miscanthus*, *E. fulvus*, *Narenga* (black), *S. spontaneum* (red), *S. robustum* (dark blue), *S. edule* (light blue), *S. officinarum* with chlorotype I (dark green), *S. officinarum* with chlorotype II chloroplast (light green), *S. barberi* and *S. sinense* (yellow), *S. maximum* (gray), and modern cultivars (brown). Empty dots represent wild hybrid accessions. The dot outlines are colored according to the *S. robustum* or *S. spontaneum* nuclear subgroups to which the accessions belong.
<figure><img src="image_23.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_27.png" /></figure>
<figure><img src="image_28.png" /></figure>

Figure S2. Nuclear SNP-based PCoAs of *Saccharum* and related genera, related to Figures 1B and 2B–2D

(A–E) (A) *Saccharum* and related genera, (B) *Saccharum*, (C) *spontaneum*, (D) *robustum*, and (E) *S. officinarum*. Colored dots represent accessions with *Tripidium*, *Miscanthus*, *E. fulvus*, *Narenga* (black), *S. spontaneum* (red), *S. robustum* (dark blue), *S. edule* (light blue), *S. officinarum* with chlorotype I (dark green), *S. officinarum* with chlorotype II chloroplast (light green), *S. barberi* and *S. sinense* (yellow), *S. maximum* (gray), and modern cultivars (brown). Empty dots represent wild hybrid accessions.
<figure><img src="image_29.png" /></figure>

(legend on next page)
<figure><img src="image_30.png" /></figure>

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<caption>Figure S3. Shared repeated k-mers among the 390 *Saccharum* accessions and related taxa, related to Figure 2A</caption>

The graph includes the 390 accessions (rows) and the 300 largest repeated k-mers sets (columns) shared between some of these accessions. A dot indicates that all k-mers from a given set are present in the accession with a size proportional to the average k-mer count in the accessions (see scale). Accession names and dots are colored according to the taxonomy: related genera in black, *S. maximum* in gray, *S. robustum* and *S. edule* in blue, *S. officinarum* in green, *S. spontaneum* in red, *S. barberi* and *S. sinense* in yellow, and modern cultivars in brown.
<figure><img src="image_31.png" /><figcaption>Figure S4. Network of genotype similarity, related to Figures 1B and 2B-2D</figcaption></figure>

Accessions are represented as nodes and linked by edges when a similarity in SNP diploid-like genotyping datapoints is superior or equal to 99%. Nodes are colored according to the taxonomy of accessions with related genera in black (*Tripidium*, *Miscanthus*, *E. fulvus*, and *Narenga*), *S. spontaneum* in red, *S. robustum* in dark blue, *S. edule* in light blue, *S. officinarum* with group II chloroplast in dark green, *S. officinarum* with group I chloroplast in light green, *S. barberi* and *S. sinense* in yellow, *S. maximum* in gray, and modern cultivars in brown.
<figure><img src="image_32.png" /><figcaption>Figure S5. K-mer-based admixture barplot for *Saccharum* and related genera excluding hybrids, related to Figure 4</figcaption></figure>

Admixture proportions were estimated excluding hybrids (*S. barberi*, *S. sinense*, and modern cultivars) considering seven ancestral groups: (1) *Tripidium* in dark gray; (2) a group formed of *Miscanthus*, *E. fulvus*, and *Narenga* in light gray; (3) the unknown ancestral contributor in purple; (4) a group formed of *S. robustum* and *S. officinarum* in blue; (5) continental *S. spontaneum* in red; (6) Southeast Asia (SEA) *S. spontaneum* in orange; and (7) Northeast Asia (NEA) *S. spontaneum* in pink.
A

<figure><img src="image_33.png" /></figure>

B

<figure><img src="image_34.png" /></figure>

C

<figure><img src="image_35.png" /></figure>

E

<figure><img src="image_36.png" /></figure>

D

<figure><img src="image_37.png" /></figure>
Figure S6. *In silico* painting of R570 genome assembly, related to Figure 5A

(A) Using signature k-mer of *S. officinarum/robustum* species (column 3).

(B) Using signature k-mer of *S. spontaneum* species (column 8).

(C) Using signature k-mer of the unknown ancestral contributor (column 20).

(D) Using the three sets of signature k-mers *S. offinarum/robustum* (column 3), *S. spontaneum* (column 8) and the unknown ancestral contributor (column 20).

(E) Using signature k-mer of Southeast Asia *S. spontaneum* (column 11).
<figure><img src="image_38.png" /><figcaption>**Figure S7. Correspondence between the Adh1 haplotypes identified by Pompidor et al. and the R570 genome assembly, related to Figure 5D**
On the left is a phylogenetic tree showing the 12 haplotypes and their origins. These are either *S. officinarum/robustum* (A and B) or *S. spontaneum* (from Pompidor et al.¹⁵). On the right is a dot plot showing the six copies of chromosome 1 present in the R570 assembly,³³ with a colored bar at the top indicating their ancestral mosaic origin as defined in Figure 5D. At the bottom are gray bars showing the estimated number of copies of chromosome 1 segments in the R570 genome (from Healey et al.³³ The two B haplotypes from Pompidor et al.¹³ correspond to a single chromosome segment (on chromosome 1a) due to the collapse of highly similar haplotypes during the genome assembly process.³³</figcaption></figure>