# Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres.

Andrew H Paterson, Jonathan F Wendel, Heidrun Gundlach, Hui Guo, Jerry Jenkins, Dianchuan Jin, Danny Llewellyn, Kurtis C Showmaker, Shengqiang Shu, Joshua Udall, et al.

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► To cite this version:

Andrew H Paterson, Jonathan F Wendel, Heidrun Gundlach, Hui Guo, Jerry Jenkins, et al. Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres. Nature, 2012, 492 (7429), pp.423-427. 10.1038/nature11798. hal-01594869

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# Repeated polyploidization of *Gossypium* genomes and the evolution of spinnable cotton fibres

Andrew H. Paterson<fnref n="1" />, Jonathan F. Wendel², Heidrun Gundlach³, Hui Guo<fnref n="1" />, Jerry Jenkins⁴,⁵, Dianchuan Jin⁶, Danny Llewellyn⁷, Kurtis C. Showmaker⁸, Shengqiang Shu⁴, Joshua Udall⁹, Mi-jeong Yoo², Robert Byers⁹, Wei Chen⁶, Adi Doron-Faigenboim¹⁰, Mary V. Duke¹¹, Lei Gong², Jane Grimwood⁴,⁵, Corrinne Grover², Kara Grupp², Guanjing Hu², Tae-ho Lee<fnref n="1" />, Jingping Li<fnref n="1" />, Lifeng Lin<fnref n="1" />, Tao Liu⁶, Barry S. Marler<fnref n="1" />, Justin T. Page⁹, Alison W. Roberts¹², Elisson Romanel¹³, William S. Sanders⁸, Emmanuel Szadkowski², Xu Tan<fnref n="1" />, Haibao Tang<fnref n="1" />,¹⁴, Chunming Xu²,¹⁵, Jinpeng Wang⁶, Zining Wang<fnref n="1" />, Dong Zhang<fnref n="1" />, Lan Zhang⁶, Hamid Ashrafi¹⁶, Frank Bedon⁷, John E. Bowers<fnref n="1" />, Curt L. Brubaker⁷,¹⁷, Peng W. Chee¹⁸, Sayan Das<fnref n="1" />, Alan R. Gingle<fnref n="1" />, Candace H. Haigler¹⁹, David Harker⁹, Lucia V. Hoffmann²⁰, Ran Hovav¹⁰, Donald C. Jones²¹, Cornelia Lemke<fnref n="1" />, Shahid Mansoor<fnref n="1" />,²², Mehboob ur Rahman²², Lisa N. Rainville<fnref n="1" />, Aditi Rambani⁹, Umesh K. Reddy²³, Jun-kang Rong<fnref n="1" />, Yehoshua Saranga²⁴, Brian E. Scheffler¹¹, Jodi A. Scheffler¹¹, David M. Stelly²⁵, Barbara A. Triplett²⁶, Allen Van Deynze¹⁶, Maite F. S. Vaslin<fnref n="27" />, Vijay N. Waghmare²⁸, Sally A. Walford⁷, Robert J. Wright<fnref n="29" />, Essam A. Zaki³⁰, Tianzhen Zhang³¹, Elizabeth S. Dennis⁷, Klaus F. X. Mayer³, Daniel G. Peterson⁸, Daniel S. Rokhsar⁴, Xiyin Wang<fnref n="1" />,⁶ & Jeremy Schmutz⁴,⁵

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments<fnref n="1" />. Here we show that an abrupt five- to sixfold ploidy increase approximately 60 million years (Myr) ago, and allopolyploidy reuniting divergent *Gossypium* genomes approximately 1–2 Myr ago², conferred about 30–36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (*Gossypium hirsutum* and *Gossypium barbadense*), genetic complexity equalled only by *Brassica*³ among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred *Gossypium herbaceum* A and non-spinnable *Gossypium longicalyx* F genomes to one another and the outgroup D genome of non-spinnable *Gossypium raimondii*. The sequence of a *G. hirsutum* *A*<sub>t</sub>*D*<sub>t</sub> (in which ‘t’ indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in *G. hirsutum* recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.

The *Gossypium* genus is ideal for investigating emergent consequences of polyploidy. A-genome diploids native to Africa and Mexican D-genome diploids diverged ~5–10 Myr ago⁴. They were

reunited ~1–2 Myr ago by trans-oceanic dispersal of a maternal A-genome propagule resembling *G. herbaceum* to the New World², hybridization with a native D-genome species resembling *G. raimondii*, and chromosome doubling (Fig. 1). The nascent *A*<sub>t</sub>*D*<sub>t</sub> allopolyploid spread throughout the American tropics and subtropics, diverging into at least five species; two of these species (*G. hirsutum* and *G. barbadense*) were independently domesticated to spawn one of the world's largest industries (textiles) and become a major oilseed.

New insight into *Gossypium* biology is offered by a genome sequence of *G. raimondii* Ulbr. (chromosome number, 13) with ~8× longer scaffold N50 (18.8 versus 2.3 megabases (Mb)) compared with a draft⁵, and oriented to 98.3% (versus 52.4%)⁵ of the genome (Supplementary Table 1.3a). Across 13 pseudomolecules totalling 737.8 Mb, ~350 Mb (47%) of euchromatin span a gene-rich 2,059 centimorgan (cM), and ~390 Mb (53%) of heterochromatin span a repeat-rich 186 cM (Supplementary Discussion, sections 1.5 and 2.1). Despite having the least-repetitive DNA of the eight *Gossypium* genome types, *G. raimondii* is 61% transposable-element-derived (Supplementary Table 2.1). Long-terminal-repeat retrotransposons (LTRs) account for 53% of *G. raimondii*, but only 3% of LTR base pairs derive from 2,345 full-length elements. The 37,505 genes and 77,267 protein-coding transcripts annotated (Supplementary Table 2.3 and http://www.phytozome.com) comprise 44.9 Mb (6%) of the genome, largely in distal chromosomal regions (Supplementary Discussion, section 2.1).

Shortly after its divergence from an ancestor shared with *Theo-broma cacao* at least 60 Myr ago⁶, the cotton lineage experienced an abrupt five- to sixfold ploidy increase. Individual grape chromosome segments resembling ancestral eudicot genome structure, or corresponding cacao chromosome segments, generally have five

<footnote n="1">¹Plant Genome Mapping Laboratory, University of Georgia, Athens, Georgia 30602, USA. ²Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, Iowa 50011, USA. ³MIPS/IBIS Institute for Bioinformatics and System Biology, German Research Center for Environmental Health (GmbH), 85764 Neuherberg, Germany. ⁴Department of Energy Joint Genome Institute, Walnut Creek, California 94595, USA. ⁵HudsonAlpha Institute of Biotechnology, Huntsville, Alabama 35806, USA. ⁶Center for Genomics and Computational Biology, School of Life Sciences, and School of Sciences, Hebei United University, Tangshan, Hebei 063000, China. ⁷CSIRO Plant Industry, Canberra, ACT 2601, Australia. ⁸Institute for Genomics, Biocomputing & Biotechnology, Mississippi State University, Mississippi State, Mississippi 39762, USA. ⁹Plant and Wildlife Science Department, Brigham Young University, Provo, Utah 84602, USA. ¹⁰Department of Field Crops, Plant Sciences Institute, ARO, Bet-Dagan 50250, Israel. ¹¹Jamie Whitten Delta States Research Center, USDA-ARS, Stoneville, Mississippi 38776, USA. ¹²Department of Biological Sciences, University of Rhode Island, Kingston, Rhode Island 02881, USA. ¹³Departamento de Genética, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, 21941-901, Brazil. ¹⁴J. Craig Venter Institute, Rockville, Maryland 20850, USA. ¹⁵Key Laboratory of Molecular Epigenetics of MOE, Unit of Plant Epigenetics, Institute of Genetics & Cytology, Northeast Normal University, Renmin Street, 5268 Changchun, China. ¹⁶Plant Reproductive Biology Extension Center, University of California, Davis, California 95616, USA. ¹⁷Bayer CropScience, Technologiepark 38, 9052 Gent, Belgium. ¹⁸Coastal Plain Experiment Station, University of Georgia, Tifton, Georgia 31793, USA. ¹⁹Departments of Crop Science and Plant Biology, North Carolina State University, Raleigh, North Carolina 27695, USA. ²⁰Centro Nacional de Pesquisa em Algodão, EMBRAPA, Santo António de Góias, GO 75375-000, Brazil. ²¹Cotton Incorporated, Cary, North Carolina 27513, USA. ²²National Institute for Biotechnology & Genetic Engineering, Faisalabad 38000, Pakistan. ²³Department of Biology, West Virginia State University, Institute, West Virginia 25112, USA. ²⁴Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel. ²⁵Department of Soil and Crop Science, Texas A&M University, College Station, Texas 77843, USA. ²⁶Cotton Fiber Bioscience Research, USDA-ARS, New Orleans, Louisiana 70124, USA.</footnote>

<footnote n="27">²⁷Departamento de Microbiologia, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21941-971, Brazil. ²⁸Central Institute for Cotton Research, Nagpur, 440010 Maharashtra, India.</footnote>

<footnote n="29">²⁹Department of Plant Sciences, Texas Tech University, Lubbock, Texas 79415, USA. ³⁰Nucleic Acids Department, Genetic Engineering & Biotechnology Research Institute, 21934 Alexandria, Egypt. ³¹Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095 Jiangsu, China.</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1** | Evolution of spinnable cotton fibres. Paleohexaploidy in a eudicot ancestor (red, yellow and blue lines) formed a genome resembling that of grape (bottom right). Shortly after divergence from cacao (bottom left), the *Gossypium* lineage experienced a five- to sixfold ploidy increase. Spinnable fibre evolved in the A genome after its divergence from the F genome, and was further elaborated after the merger of A and D genomes ~1–2 Myr ago, forming the common ancestor of *G. hirsutum* (Upland) and *G. barbadense* (Egyptian, Sea Island and Pima) cottons.</figcaption></figure>

(infrequently six) best-matching *G. raimondii* regions and secondary matches resulting from pan-eudicot hexaploidy⁷,⁸ (Fig. 2 and Supplementary Table 3.1). Paralogous genes tracing to this five- to sixfold

ploidy increase show a single peak of synonymous nucleotide-substitution ($K_s$) values, suggesting either one, or multiple closely spaced, event(s) (Supplementary Fig. 3.5). Pairwise cytological similarity among A-genome chromosomes⁹ suggests the most recent event was a duplication.

Paleopolyploidy may have accelerated cotton mutation rates: for 7,021 co-linearity-supported gene triplets, $K_s$ rates and non-synonymous nucleotide-substitution ($K_a$) rates were, respectively, 19% and 15% larger for cotton-grape than cacao-grape comparisons (Supplementary Table 3.2). Adjusted for this acceleration (Supplementary Fig. 3.5), the cotton ploidy increase occurred about halfway between the pan-eudicot hexaploidy ($<125$ Myr ago¹⁰) and the present, near the low end of an estimated range of 57–70 Myr ago¹¹.

Paleopolyploidy increased the complexity of a Malvaceae-specific clade of Myb family transcription factors, perhaps contributing to the differentiation of epidermal cells into fibres rather than the mucilages of other Malvaceae. Among 204 R2R3, 8 R1R2R3 and 194 heterogeneous Myb transcription factors in *G. raimondii* (Supplementary Table 3.5), subgroup 9 has six members known only in Malvaceae (Fig. 3a), comprising a possible ‘fibre clade’ distinct from the *Arabidopsis thaliana* GL1-like subgroup 15 involved in trichome and root hair initiation and development¹². Expressed predominantly in early fibre development, elite cultivated tetraploid cottons have higher expression of five (50%) of ten subgroup 9 genes compared with wild (undomesticated) tetraploids (Fig. 3a and Supplementary Table 5.3). Some subgroup 9 genes are also active in leaves, hypocotyls and cotyledons (Supplementary Fig. 3.8), consistent with specialisation for different types of epidermal cell differentiation such as production of a ‘pulpy layer’ secreted from the teguments surrounding cacao seeds, and mucilages in other Malvaceae fruit (*Abelmoschus* (okra), *Cola* (kola)) and roots (*Althaea* (marshmallow)).

Cotton growers were early adopters of integrated pest management¹³ strategies to deploy intrinsic defences conferred by pest- and disease-resistance genes that evolved largely after the 5–6-fold ploidy increase.

<figure><img src="image_4.png" /><figcaption>**Figure 2** | Syntenic relationships among grape, cacao and cotton. **a**, Macro-synteny connecting blocks of >30 genes (grey lines). Highlighted regions (pink and red) trace to a common ancestor before the pan-eudicot hexaploidy⁷, with the *Gossypium* lineage five- to sixfold ploidy increase forming multiple derived</figcaption></figure>

regions. Inferred duplication depth in cotton varies (top). **b**, Micro-synteny of grape chromosome (Chr) 3, cacao chromosome 2 and five cotton chromosomes. Rectangles represent predicted genes, with connecting grey lines showing co-linear relationships. An example (1 grape, 1 cocoa, 5 cotton) is highlighted in red.
a

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b

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**Figure 3 |** **Paleo-evolution of cotton gene families.** *a*, Myb subgroup 9 (ref. 12) originated from a gene on the progenitor of cacao chromosome 2 that formed two adjacent copies after Malvales-Brassicales divergence and then triplicated in cotton, with subsequent loss of one chromosome 8 and two chromosome 12 paralogues. One extant paralogue traces to pan-eudicot hexaploidy, Tc04 g009420, and reduplicated in cotton (Gorai.012G052500.1 and Gorai.011G122800.1) and *Arabidopsis* (At3g01140 and At5g15310). The other, Tc01 g036330, has reduplicated in cotton (Gorai.004G157600.1 and Gorai.001G169700.1). Asterisk indicates increased gene expression in elite versus wild tetraploids (Supplementary Table 5.3). *b*, The most NBS-rich region of *T. cacao*, on chromosome 7, corresponds to regions of *G. raimondii* chromosome triplets 2/10/13 and 7/9/4. Cacao chromosome 7 NBSs form a single branch, indicating lineage-specific expansion. *G. raimondii* chromosome 7 and 13 NBSs form distinct branches, indicating cluster/tandem duplication (gene numbers also reflect physical proximity of genes to one another).

A total of 300 (0.8%) *G. raimondii* genes encode nucleotide-binding site (NBS) domains (Supplementary Table 3.6), largely of coiled-coil (CC)-NBS and CC-NBS-leucine rich repeat subgroups (165, 55%). Like cereals¹⁴, after paleopolyploidy *G. raimondii* evolved clusters of new

NBS-encoding genes. The most NBS-rich (21%) region of *T. cacao*, on chromosome 7, corresponds to parts of *G. raimondii* chromosome triplets 2/10/13 and 7/9/4. In total, 27% and 25% of 294 mapped *G. raimondii* NBS genes are on these parts of chromosomes 7 and 9, often clustered in otherwise gene-poor surroundings (Supplementary Fig. 2.2). Most NBS clusters are species and chromosome specific (Fig. 3b and Supplementary Table 3.7), indicating rapid turnover and/or concerted evolution after cotton paleopolyploidy. In total, 230 (76.7%) NBS-encoding genes have experienced striking mutations (as detailed below) in the A genome since A-F divergence, reflecting an ongoing plant-pathogen 'arms race' (Supplementary Table 3.8).

Changes in gene expression during domestication have contributed to the deposition of >90% cellulose in cotton fibres, single-celled models for studying cell wall and cellulose biogenesis¹⁵. *G. raimondii* has at least 15 cellulose synthase (CESA) sequences required for cellulose synthesis¹⁶ (Supplementary Table 3.3), with four single-gene *Arabidopsis* clades having three (CESA3, required in expanding primary walls) or two (CESA4, CESA7 and CESA8, each required in the thickening of secondary walls) clade members in *G. raimondii*¹⁶. *G. raimondii* has at least 35 cellulose-synthase-like (CSL) genes required for synthesis of cell wall matrix polysaccharides that surround cellulose microfibrils¹⁶ (Supplementary Table 3.4), including one family (CSLJ) absent in *Arabidopsis*¹⁶. Elite tetraploids have higher expression than wild cottons in 6 (40%) of 15 CESA genes and 12 (34%) of 35 CSL genes (Supplementary Table 5.3).

A total of 364 *G. raimondii* microRNA precursors from 28 conserved and 181 novel families (Supplementary Table 3.12), are predicted regulators of 859 genes enriched for molecule binding factors, catalytic enzymes, transporters and transcription factors (Supplementary Fig. 3.11, 12). Four conserved and 35 novel mRNAs were specifically expressed in *G. hirsutum* fibres, respectively targeting 53 and 318 genes, most with homology to proteins involved in fibre development (Supplementary Table 3.14, 15). Among 183,690 short interfering RNAs (siRNAs) found, 33,348 (18.15%) were on chromosome 13 (Supplementary Fig. 3.12), a vast enrichment. Small RNA¹⁷⁻¹⁹ biogenesis proteins include 13 argonaute, 6 dicer-like (DCL) and 5 RNA-dependent RNA polymerase orthologues (Supplementary Table 3.16). *G. raimondii* seems to be the first eudicot with two *DCL3* genes and two genes encoding RNA polymerase IVa (Supplementary Table 3.16), perhaps relating to control of its abundant retrotransposons.

From unremarkable hairs found on all *Gossypium* seeds, 'spinnable' fibres (fibres with a ribbon-like structure that allows for spinning into yarn) evolved in the A genome after divergence from the B, E and F genomes ~5–10 Myr ago⁴ (Fig. 1). To clarify the evolution of spinnable fibres, we sequenced the *G. herbaceum* A and *G. longicalyx* F genomes, which respectively differ from *G. raimondii* by 2,145,177 single-nucleotide variations (SNVs) and 477,309 indels, and 3,732,370 SNVs and 630,292 indels.

Specific genes are implicated in initial fibre evolution by both whole-gene and individual-nucleotide analyses. Across entire genes, 36 *G. herbaceum*–*G. raimondii* and 11 *G. herbaceum*–*G. longicalyx* orthologue pairs show evidence of diversifying selection (ω > 1, *P* < 0.05) (Supplementary Table 4.1). A notable example, with *G. herbaceum*–*G. raimondii* ω > 9, is Gorai.009G035800, a germin-like protein that is differentially expressed between normal and naked-seed cotton mutants during fibre expansion²⁰ and between wild and elite *G. barbadense* at 10 days post-anthesis (DPA; Supplementary Table 5.3).

Among 114,202 SNVs in 29,015 *G. herbaceum* genes after *G. herbaceum*–*G. longicalyx* A-F divergence (using D as outgroup, so F is the same as D, and A differs from both), we identified striking mutations including 1,090 non-synonymous mutations in 959 genes comprising the most severe 1% of functional impacts inferred using a modified entropy function²¹; 3,525 frameshift mutations (3,021 genes), 1,077 (987) premature stops, 527 (513) splice-site mutations, 102 (102) initiation alterations and 95 (94) extended reading frames (Supplementary Table 4.2, 3). These striking mutations have an average genomic
distribution (Supplementary Fig. 2.2) but are over-represented in genes coding for cell-wall-associated, kinase or nucleotide-binding proteins (Supplementary Table 4.5).

Striking mutations in the A-genome lineage are enriched ($P = 2.6 \times 10^{-18}$; Supplementary Discussion, section 4.4) within fibre-related quantitative trait locus (QTL) hotspots in $A_t D_t$ tetraploid cottons²², suggesting that post-allopolyploidy elaboration of fibre development¹ involved recursive changes in $A_t$ and new changes in $D_t$ genes. Striking A-genome mutations have orthologues in 1,051 $D_t$ and 951 $A_t$ fibre QTL hotspots. Likewise, sequencing of *G. hirsutum* cultivar Acala Maxxa revealed 495 striking mutations in 391 genes, with 83 (21.2%) in $D_t$ fibre QTL hotspots and 73 (18.7%) in $A_t$ hotspots (Supplementary Table 4.6).

QTL hotspots affecting multiple fibre traits²² may reflect coordinated changes in expression of functionally diverse cotton genes. A total of 671 (1.79%) genes with >100 reads per million reads were differentially expressed in fibres from wild versus domesticated *G. hirsutum* (mostly at 10 DPA) and/or *G. barbadense* (mostly at 20 DPA) (Supplementary Table 5.3). Among 48 genes upregulated in domesticated *G. hirsutum* at 10 DPA, 20 (42%) are among 1,582 (4.2%) genes within QTL hotspot $D_t$09.2 (ref. 22) affecting length, uniformity, and short-fibre content, with 13 (27%) out of 677 (1.8%) genes in homoeologous hotspot $A_t$09 affecting fibre elongation and fineness. Out of 45 genes downregulated in domesticated *G. barbadense* at 20 DPA, 16 (35.6%) map to $D_t$09.2, and 8 (17.7%) to $A_t$09. In 79% of cultivated *G. barbadense*, this $A_t$ region (which was then thought to be on chromosome 5, and is now known to be on chromosome 9) has been unconsciously introgressed by plant breeders with *G. hirsutum* DNA, suggesting an important contribution to productivity of *G. barbadense* cultivars²³.

A putative nuclear mitochondrial DNA (NUMT) sequence block²⁴ has an intriguing relationship with fibre improvement. A *G. raimondii* chromosome 1 region includes many genes closely resembling mitochondrial homologues ($K_s \sim 0.22$; Supplementary Table 4.7a). NUMT genes experienced a coordinated change in expression associated with *G. barbadense* domestication. The 105 (0.2%) genes upregulated in 10 DPA fibre of wild (versus elite) tetraploid *G. barbadense* (Supplementary Table 5.3) include 30 (37%; $P < 0.001$) of the 81 NUMT genes, including 8 NADH dehydrogenase and 4 cytochrome-c-related genes. All are within the QTL hotspot $D_t$01 that affects fibre fineness, length, and uniformity²², suggesting a fibre-specific change in electron transfer in *G. barbadense* domestication.

Emergent features of polyploids may be related to processes that render them no longer the sum of their progenitors and permit them to explore transgressive phenotypic innovations. Despite the A-genome origin of spinnable fibres, after 1–2 Myr of co-habitation in tetraploid nuclei most $A_t$ and $D_t$ homoeologues are now expressed in fibres at similar levels (Supplementary Table 5.4). Such convergence is not ubiquitous: gene families involved in the synthesis of seed oil show strong A bias in wild *G. hirsutum* and its sister *G. tomentosum*, but strong D bias in an improved *G. hirsutum* (Supplementary Table 5.6).

Recruitment of $D_t$-genome genes into tetraploid fibre development¹ may have involved non-reciprocal DNA exchanges from $A_t$ genes. In the ~40% of Acala Maxxa $A_t$ and $D_t$ genes that differ in sequence from their diploid progenitors (Fig. 4), most mutations are convergent, with $A_t$ genes converted to the $D_t$ state at more than twice the rate (25%) as the reciprocal (10.6%). Known to occur between cereal paralogues diverged by 70 Myr¹⁴, non-reciprocal DNA exchanges are more abundant between cotton $A_t$ and $D_t$ genes separated by only ~5–10 Myr⁴. Such non-reciprocal exchanges explain prior observations including incongruent gene tree topology for 10% (3 pairs) of *G. hirsutum* $A_t$ and $D_t$ homeologues in sequenced bacterial artificial chromosomes (BACs) (Supplementary Discussion, section 5.3); 13.2% of tetraploid DNA markers that showed different subgenomic affinities compared with the chromosomes to which they mapped, 9 of 13 being $D_t$ biased ($A_t$ to $D_t$)²⁵; and expressed-sequence-tag-based

<figure><img src="image_7.png" /><figcaption>**Figure 4** | Allelic changes between A- and D-genome diploid progenitors and the $A_t$ and $D_t$ subgenomes of *G. hirsutum* cultivar Acala Maxxa.</figcaption></figure>

evidence of phylogenetic incongruity for as many as 7% of homeologous genes²⁶.

Several factors may have favoured $D_t$-biased allele conversion in tetraploid cotton. The nascent polyploid may have gained fitness from D-genome alleles native to its New World habitat. Before fortifying its reproductive barriers, the nascent polyploid may have occasionally outcrossed to nearby D-genome diploids, increasing the likelihood of illegitimate recombination. Outcrossing may also have contributed to the origin of *Gossypium gossypioides*, sister to *G. raimondii* and the only D-genome cotton containing many otherwise A-genome-specific repetitive DNAs²⁷⁻²⁹. $D_t$-biased allele conversion may have contributed to slightly greater protein-coding nucleotide diversity in the $A_t$ compared with the $D_t$-genome (Supplementary Table 5.7).

Whereas the *G. raimondii* reference sequence and *G. hirsutum* short-read sequences reveal much about tetraploid cotton genome structure and polyploid evolution, high-contiguity sequencing of polyploids may elucidate still-cryptic features. Tetraploid cotton sequencing appears feasible: among six pairs of $A_t$ and $D_t$ BAC clones, the most similar pair shows 99.1% shared $D_t$-D and 97.6% $A_t$-D content (Supplementary Table 6.1), sufficient divergence to de-convolute shotgun sequence to the correct subgenome. Increased knowledge of molecular diversity is a foundation for integrating genomics with ecological and field-level knowledge of *Gossypium* species and their diverse adaptations to warm arid ecosystems on six continents.

## METHODS SUMMARY

**Sequencing.** Reads were collected from Applied Biosystems 3730xl, Roche 454 XLR and Illumina Genome Analyzer IIx machines at the Joint Genome Institute
(http://www.jgi.doe.gov/sequencing/protocols/prots_production.html) or Hudson-Alpha Institute and Beckman Coulter Genomics (BAC end sequence), and USDA-ARS Mid-South Area Genomics Laboratory (*G. longicalyx*, *G. arboreum* and *G. hirsutum*).

**Assembly.** Assembly of 80,765,952 sequence reads used a modification of Arachne v.20071016, integrating linear (15× genome coverage) and paired (3.1× genome coverage) Roche 454 libraries corrected using 41.9 Gb Illumina sequence, with 1.54× paired-end Sanger sequences from two subclone, six fosmid and two BAC libraries. Cotton genetic and physical maps, and *Vitis vinifera* and *T. cacao* synteny were used to identify 51 joins across 64 scaffolds to form the 13 chromosomes (Supplementary Discussion, section 1). The remaining scaffolds were screened for contamination to produce a final assembly of 1,033 scaffolds (19,735 contigs) and 761.4 Mb. Sequences are in NCBI for *G. raimondii* (BioProject accession PRJNA171262), *G. longicalyx* (accession F1-1, SRA061660), *G. herbaceum* (accession A1-97, SRA061243) and *G. hirsutum* (cultivar Acala Maxxa, SRS375727) genomes; *G. hirsutum* (SRA061240) and *G. barbadense* (SRA061309) fibre transcriptomes; *G. hirsutum* (SRA061456) seed transcriptomes; and *G. hirsutum* microRNAs (SRA061415).

**Annotation.** PERTRAN software was used to construct transcript assemblies from ~1.1 billion pairs of *G. raimondii* paired-end Illumina RNA-seq reads, 250 million *G. raimondii* single end reads, and 150 million *G. hirsutum* single end reads. PASA³⁰ was used to build transcript assemblies from 454 and Sanger resources (Supplementary Table 2.3). Loci were determined by transcript assembly and/or EXONERATE alignments of *A. thaliana*, *cacao*, rice, soybean, grape and poplar peptides to repeat-soft-masked *G. raimondii* genome using RepeatMasker. Gene models were predicted by three homology-based predictors (Supplementary Discussion, section 2.2). Best-scoring gene predictions were improved by PASA, then filtered on the basis of peptide homology or expressed-sequence-tag evidence to remove Pfam transposable element domain models. ClustalW alignments of amino acid sequences (Fig. 3) were used to guide coding sequence alignments. Phylogenetic trees were constructed by bootstrap neighbour-joining with a Kimura 2-parameter model using ClustalW2, assessing internal nodes with 1,000 replicates.

**Full Methods** and any associated references are available in the online version of the paper.

**Received 17 August; accepted 21 November 2012.**

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**Supplementary Information** is available in the online version of the paper.

**Acknowledgements** The work, conducted by the US Department of Energy Joint Genome Institute, is supported by the Office of Science of the US Department of Energy under Contract no. DE-AC02-05CH11231. The authors appreciate financial support from the US National Science Foundation (DBI 98-72630 to A.H.P., J.F.W., A.R.G.; DBI 02-11700 to J.F.W., A.H.P., J.U., A.R.G.; DBI 02-08311, IIP-0917856; IIP-1127755 to A.H.P.; IOS 1025947 to C.H.H.), USDA (ARS-58-6402-7-241, 58-6402-1-644 and 58-6402-1-645 to D.G.P.; ARS 6402-21310-003-00 to B.E.S.; NRI 00-52100-9685 and 02-35301-12045 to A.H.P.), Bayer CropScience and The Consortium for Plant Biotechnology Research (A.H.P.), Cotton, Inc. (P.W.C., D.C.J., A.H.P., D.M.S., A.V.-D., J.F.W.), Georgia State Support Committee (P.W.C., A.H.P.), Texas State Support Committee (R.J.W.), Pakistan–US Science and Technology Cooperation Program (P.W.C., S.M., A.H.P., M.u.R.), US–Egypt Science and Technology Cooperation Program (A.H.P., E.A.Z.), Fulbright Scholar Program (S.M., E.A.Z.), Conselho Nacional de Desenvolvimento Científico e Tecnológico PDJ150690/2012-6 (E.R.), Fundação de Amparo a Pesquisa Pensa Rio E-26/110.324/2010 (M.F.S.V.), Texas AgriLife (D.M.S.), and Brigham Young University (BYU) Mentored Environment Grants (J.U.). RNA-seq reads were mapped by students on Marylou at the Fulton Supercomputer Center at BYU. We thank L. S. Dure III, G. O. Myers, J. McD Stewart, T. A. Wilkins and J. Zhu for co-endorsing the sequencing of *G. raimondii* by the US Department of Energy.

**Author Contributions** A.H.P., D.S.R., J.S. and D.G.P. conceived the study. J.S., J.G., D.S.R., K.C.S., S.D., M.V.D., C.L., L.N.R., B.E.S. and J.A.S. performed sequencing and associated clone manipulations. A.H.P., J.F.W., D.L., E.S.D., J.U., E.R., Z.W., H.A., L.V.H., R.H., D.M.S., A.V.-D. and T.Z. contributed unpublished data. A.H.P., J.F.W., H.Guo, H.Gundlach, J.J., D.J., D.L., S.S., J.U., M.-J.Y., R.B., W.C., A.D.-F., L.G., C.G., K.G., G.H., T.-h.L., J.L., L.L., T.L., B.S.M., J.T.P., A.W.R., E.R., E.S., X.T., H.T., C.X., J.W., Z.W., D.Z., L.Z., F.B., C.H.H., D.H., L.V.H., R.H., S.M., M.F.S.V., S.A.W., T.Z., E.S.D., D.S.R., X.W. and J.S. analysed data. A.H.P., J.F.W., D.L., E.S.D., K.F.X.M., D.G.P. and J.S. wrote the manuscript. All authors discussed results and commented on the manuscript.

**Author Information** Sequences have been deposited in NCBI for *G. raimondii* (BioProject accession PRJNA171262), *G. longicalyx* (accession F1-1, SRA061660), *G. herbaceum* (accession A1-97, SRA061243) and *G. hirsutum* (cultivar Acala Maxxa, SRS375727) genomes; *G. hirsutum* (SRA061240) and *G. barbadense* (SRA061309) fibre transcriptomes; *G. hirsutum* (SRA061456) seed transcriptomes; and *G. hirsutum* microRNAs (SRA061415). Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to A.H.P. (paterson@plantbio.uga.edu) or J.S. (jschmutz@hudsonalpha.org).

<figure><img src="image_8.png" /></figure>

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METHODS

**Sequencing.** Reads were collected with standard protocols (http://www.jgi.doe.gov/sequencing/protocols/prots_production.html) on Applied Biosystems 3730xl, Roche 454 XLR and Illumina Genome Analyzer (GA)IIx machines at the US Department of Energy Joint Genome Institute. Linear 454 data included standard XLR (47 runs, 16.868 Gb) and pre-release FLX+ data (5 runs, 3.262 Gb). Eight paired 454, 3–4-kilobase (kb) average insert size and one paired 12-kb average insert size were sequenced on standard XLR (23 runs, 5.931 Gb). One standard 400-base pair (bp) fragment library was sequenced at 2 × 150 (7 channels, 41.9 Gb) on an Illumina GAIIx. One 2.5-kb average insert size (405,024 reads, 286.1 Mb), one 6.5-kb average insert size library (374,125 reads, 263.0 Mb), six fosmid libraries (1,222,643 reads, 702.1 Mb) of 34–39-kb insert size, and two BAC libraries (107,520 reads, 77.5 Mb) of 98-kb and 115-kb (73,728 reads, 48.8 Mb) average insert size were sequenced on both ends for a total of 2,183,240 Sanger reads of 1.38 Gb of high-quality bases. FLX+ data were collected at the Roche Service Center. BAC end sequence (BES) was collected using standard protocols at the HudsonAlpha Institute.

**Genome assembly and construction of pseudomolecule chromosomes.**
Organellar reads were removed by screening against mitochondria, chloroplast and ribosomal DNA. Any Roche 454 linear read <200 bp was discarded. Roche 454 paired reads in which either was shorter than 50 bp were discarded. An additional de-duplication step was applied to the 454 paired libraries that identifies and retains only one copy of each PCR duplicate. All remaining 454 reads were compared against a full Illumina GA2x run and any insertion/deletions in the 454 reads were corrected to match the Illumina alignments. The sequence reads were assembled using our modified version of Arachne v.20071016 (ref. 31) with parameters `maxcliq1` = 100, `correct1_passes` = 0 and `BINGE_AND_PURGE` = True, `bless` = False, `maxcliq1` = 200, `BINGE_AND_PURGE` = True, `lap_ratio` = 0.8, `max_bad_look` = 1000 (note Arachne error correction is on). This produced 1,263 scaffold sequences, with a scaffold L50 of 25.8 Mb, 58 scaffolds larger than 100 kb, and total genome size of 761.8 Mb. Scaffolds were screened against bacterial proteins, organelle sequences and non-redundant GenBank and removed if found to be a contaminant. Additional scaffolds were removed if they: (1) consisted of >95% 24-nucleotide sequences that occurred four other times in scaffolds larger than 50 kb; (2) contained only unanchored RNA sequences; or (3) were <1 kb in length.

The combination of BES/markers hybridized to fingerprint contigs³², 2,800 markers in a genetic map for the D genome in an A<sub>D</sub>1<sub>D</sub> plant³³ and 262 markers from the tetraploid genetic map³⁴, along with *Vitis vinifera* and *T. cacao* synteny was used to identify breaks in the initial assembly. Markers were aligned to the assembly using BLAT³⁵ (parameters: `-t` = dna -q = dna -minScore = 200 -extendThroughN). BES, physical map contigs, *V. vinifera* and *T. cacao* genes were aligned to the genome using BLAST³⁶. Scaffolds were broken if they contained linkage group/syntenic discontiguity coincident with an area of low BAC/ fosmid coverage. A total of 13 breaks were executed, and 64 of the broken scaffolds were oriented, ordered and joined using 51 joins to form the final assembly containing 13 pseudomolecule chromosomes. Each chromosome join is padded with 10,000 missing nucleotides. The final assembly contains 1,033 scaffolds (19,735 contigs) that cover 761.4 Mb of the genome with a contig L50 of 135.6 kb and a scaffold L50 of 62.2 Mb.

The assembly size is near the centre of genome-size estimates of 880 Mb from flow cytometry³⁷, 630 Mb from Feulgen cytophotometry³⁸, and 650 Mb³⁹ and 770 Mb⁴⁰ from re-naturation kinetics.

Completeness of the euchromatic portion of the genome assembly was assessed using 65,506 *G. raimondii* complementary DNAs obtained from GenBank, which were aligned to the assembly using BLAT³ (parameters: `-t` = dna -q = rna -extendThroughN). The aim of the completeness analysis was to obtain a measure of completeness of the assembly, rather than a comprehensive examination of gene space. cDNAs were aligned to the assembly using BLAT³⁵ (parameters: `-t` = dna -q = rna -extendThroughN) and alignments that comprised ≥90% base-pair identity and ≥85% EST coverage were retained. The screened alignments indicate that 57,170 out of 63,506 (90.3%) cDNAs aligned to the assembly. The cDNAs that failed to align were primarily composed of stretches of polynucleotide

sequences that failed to generate non-random alignments to any plant or other organism in the NCBI as of the release date.

**Annotation.** A total of 85,746 transcript assemblies were constructed from about 1.1 billion pairs of D5 paired-end Illumina RNA-seq reads, 55,294 transcript assemblies from 250 million D5 single-end Illumina RNA-seq reads and 62,526 transcript assemblies from 150 million *G. hirsutum* cotton single-end Illumina RNA-seq reads. All these transcript assemblies were constructed using PERTRAN software (in preparation). In total, 120,929 transcript assemblies were built using PASA³⁰ from 56,638 D5 Sanger ESTs, 2.5 million D5 Roche 454 RNA-seq reads and all of the RNA-seq transcript assemblies. An additional 133,073 transcript assemblies were constructed using PASA from 296,214 *G. hirsutum* cotton Sanger ESTs and about 2.9 million *G. hirsutum* cotton 454 reads. The larger number of transcript assemblies from fewer *G. hirsutum* sequences is due to the fragmented nature of the assemblies. Loci were determined by transcript assembly alignments and/or EXONERATE alignments of peptides from *A. thaliana*, cacao, rice, soybean, grape and poplar peptides to repeat-soft-masked D5 genome using RepeatMasker (http://www.repeatmasker.org) with up to 2,000-bp extensions on both ends, unless extending into another locus on the same strand. Gene models were predicted by homology-based predictors, FGENESH+⁴¹, FGENESH_EST (similar to FGENESH+, EST as splice site and intron input instead of peptide/ translated open-reading frames) and GenomeScan⁴². The best scored predictions for each locus are selected using multiple positive factors including EST and peptide support, and one negative factor: overlap with repeats. The selected gene predictions were improved by PASA. Improvement includes adding untranslated regions, splicing correction, and adding alternative transcripts. PASA-improved gene model peptides were subject to peptide-homology analysis to above-mentioned proteomes in order to obtain Cscore and peptide coverage. Cscore is a peptide BLASTP score ratio mutual best hit BLASTP score and peptide coverage is highest percentage of peptide aligned to the best of homologues. PASA-improved transcripts were selected on the basis of Cscore, peptide coverage, EST coverage and its coding sequence (CDS) overlapping with repeats. The transcripts were selected if their Cscore was larger than or equal to 0.5 and peptide coverage larger than or equal to 0.5, or if it had EST coverage, but its CDS overlapping with repeats was less than 20%. For gene models whose CDS overlaps with repeats for more than 20%, its Cscore needed to be at least 0.9 and homology coverage at least 70% to be selected. The selected gene models were subject to Pfam analysis and gene models whose peptide was more than 30% in Pfam transposable element domains were removed. The final gene set had 37,505 protein-coding genes and 77,267 protein-coding transcripts.

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