Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution.

Gossypium hirsutum has proven difficult to sequence owing to its complex allotetraploid (AtDt) genome. Here we produce a draft genome using 181-fold paired-end sequences assisted by fivefold BAC-to-BAC sequences and a high-resolution genetic map. In our assembly 88.5% of the 2,173-Mb scaffolds, which cover 89.6%∼96.7% of the AtDt genome, are anchored and oriented to 26 pseudochromosomes. Comparison of this G. hirsutum AtDt genome with the already sequenced diploid Gossypium arboreum (AA) and Gossypium raimondii (DD) genomes revealed conserved gene order. Repeated sequences account for 67.2% of the AtDt genome, and transposable elements (TEs) originating from Dt seem more active than from At. Reduction in the AtDt genome size occurred after allopolyploidization. The A or At genome may have undergone positive selection for fiber traits. Concerted evolution of different regulatory mechanisms for Cellulose synthase (CesA) and 1-Aminocyclopropane-1-carboxylic acid oxidase1 and 3 (ACO1,3) may be important for enhanced fiber production in G. hirsutum.

Distribution and evolution of cotton fiber development genes in the fibreless Gossypium raimondii genome.

Cotton fiber represents the largest single cell in plants and they serve as models to study cell development. This study investigated the distribution and evolution of fiber Unigenes anchored to recombination hotspots between tetraploid cotton (Gossypium hirsutum) At and Dt subgenomes, and within a parental diploid cotton (Gossypium raimondii) D genome. Comparative analysis of At vs D and Dt vs D showed that 1) the D genome provides many fiber genes after its merger with another parental diploid cotton (Gossypium arboreum) A genome although the D genome itself does not produce any spinnable fiber; 2) similarity of fiber genes is higher between At vs D than between Dt vs D genomic hotspots. This is the first report that fiber genes have higher similarity between At and D than between Dt and D. The finding provides new insights into cotton genomic regions that would facilitate genetic improvement of natural fiber properties.

Genome sequence of the cultivated cotton Gossypium arboreum.

The complex allotetraploid nature of the cotton genome (AADD; 2n = 52) makes genetic, genomic and functional analyses extremely challenging. Here we sequenced and assembled the Gossypium arboreum (AA; 2n = 26) genome, a putative contributor of the A subgenome. A total of 193.6 Gb of clean sequence covering the genome by 112.6-fold was obtained by paired-end sequencing. We further anchored and oriented 90.4% of the assembly on 13 pseudochromosomes and found that 68.5% of the genome is occupied by repetitive DNA sequences. We predicted 41,330 protein-coding genes in G. arboreum. Two whole-genome duplications were shared by G. arboreum and Gossypium raimondii before speciation. Insertions of long terminal repeats in the past 5 million years are responsible for the twofold difference in the sizes of these genomes. Comparative transcriptome studies showed the key role of the nucleotide binding site (NBS)-encoding gene family in resistance to Verticillium dahliae and the involvement of ethylene in the development of cotton fiber cells.

BAC-pool sequencing and analysis of large segments of A12 and D12 homoeologous chromosomes in upland cotton.

Although new and emerging next-generation sequencing (NGS) technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.). Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP) BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg). To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes.

A high-density simple sequence repeat and single nucleotide polymorphism genetic map of the tetraploid cotton genome.

Genetic linkage maps play fundamental roles in understanding genome structure, explaining genome formation events during evolution, and discovering the genetic bases of important traits. A high-density cotton (Gossypium spp.) genetic map was developed using representative sets of simple sequence repeat (SSR) and the first public set of single nucleotide polymorphism (SNP) markers to genotype 186 recombinant inbred lines (RILs) derived from an interspecific cross between Gossypium hirsutum L. (TM-1) and G. barbadense L. (3-79). The genetic map comprised 2072 loci (1825 SSRs and 247 SNPs) and covered 3380 centiMorgan (cM) of the cotton genome (AD) with an average marker interval of 1.63 cM. The allotetraploid cotton genome produced equivalent recombination frequencies in its two subgenomes (At and Dt). Of the 2072 loci, 1138 (54.9%) were mapped to 13 At-subgenome chromosomes, covering 1726.8 cM (51.1%), and 934 (45.1%) mapped to 13 Dt-subgenome chromosomes, covering 1653.1 cM (48.9%). The genetically smallest homeologous chromosome pair was Chr. 04 (A04) and 22 (D04), and the largest was Chr. 05 (A05) and 19 (D05). Duplicate loci between and within homeologous chromosomes were identified that facilitate investigations of chromosome translocations. The map augments evidence of reciprocal rearrangement between ancestral forms of Chr. 02 and 03 versus segmental homeologs 14 and 17 as centromeric regions show homeologous between Chr. 02 (A02) and 17 (D02), as well as between Chr. 03 (A03) and 14 (D03). This research represents an important foundation for studies on polyploid cottons, including germplasm characterization, gene discovery, and genome sequence assembly.

Polyploidization altered gene functions in cotton (Gossypium spp.).

Cotton (Gossypium spp.) is an important crop plant that is widely grown to produce both natural textile fibers and cottonseed oil. Cotton fibers, the economically more important product of the cotton plant, are seed trichomes derived from individual cells of the epidermal layer of the seed coat. It has been known for a long time that large numbers of genes determine the development of cotton fiber, and more recently it has been determined that these genes are distributed across At and Dt subgenomes of tetraploid AD cottons. In the present study, the organization and evolution of the fiber development genes were investigated through the construction of an integrated genetic and physical map of fiber development genes whose functions have been verified and confirmed. A total of 535 cotton fiber development genes, including 103 fiber transcription factors, 259 fiber development genes, and 173 SSR-contained fiber ESTs, were analyzed at the subgenome level. A total of 499 fiber related contigs were selected and assembled. Together these contigs covered about 151 Mb in physical length, or about 6.7% of the tetraploid cotton genome. Among the 499 contigs, 397 were anchored onto individual chromosomes. Results from our studies on the distribution patterns of the fiber development genes and transcription factors between the At and Dt subgenomes showed that more transcription factors were from Dt subgenome than At, whereas more fiber development genes were from At subgenome than Dt. Combining our mapping results with previous reports that more fiber QTLs were mapped in Dt subgenome than At subgenome, the results suggested a new functional hypothesis for tetraploid cotton. After the merging of the two diploid Gossypium genomes, the At subgenome has provided most of the genes for fiber development, because it continues to function similar to its fiber producing diploid A genome ancestor. On the other hand, the Dt subgenome, with its non-fiber producing D genome ancestor, provides more transcription factors that regulate the expression of the fiber genes in the At subgenome. This hypothesis would explain previously published mapping results. At the same time, this integrated map of fiber development genes would provide a framework to clone individual full-length fiber genes, to elucidate the physiological mechanisms of the fiber differentiation, elongation, and maturation, and to systematically study the functional network of these genes that interact during the process of fiber development in the tetraploid cottons.

The construction of a tetraploid cotton genome wide comprehensive reference map.

Integration of multiple genomic maps provides a higher density of markers and greater genome coverage, which not only facilitates the identification and positioning of QTLs and candidate genes, but it also provides a basic structure for the genome sequence assembly. However, the diversity in markers and populations used in individual mapping studies limits the ability to fully integrate the available data. By concentrating on marker orders rather than marker distances, published map data could be used to produce a comprehensive reference map (CRM) that includes a majority of known markers with optimally estimated order of those markers across the genome. In this study, a tetraploid cotton genome-wide CRM was constructed from 28 public cotton genetic maps. The initial CRM contained 7,424 markers and represented over 93% of the combined mapping information from the 28 individual maps. The current output is stored and displayed through CottonDB (http://www.cottondb.org), the public cotton genome database.

An integrated genetic and physical map of homoeologous chromosomes 12 and 26 in Upland cotton (G. hirsutum L.).

BACKGROUND: Upland cotton (G. hirsutum L.) is the leading fiber crop worldwide. Genetic improvement of fiber quality and yield is facilitated by a variety of genomics tools. An integrated genetic and physical map is needed to better characterize quantitative trait loci and to allow for the positional cloning of valuable genes. However, developing integrated genomic tools for complex allotetraploid genomes, like that of cotton, is highly experimental. In this report, we describe an effective approach for developing an integrated physical framework that allows for the distinguishing between subgenomes in cotton. RESULTS: A physical map has been developed with 220 and 115 BAC contigs for homeologous chromosomes 12 and 26, respectively, covering 73.49 Mb and 34.23 Mb in physical length. Approximately one half of the 220 contigs were anchored to the At subgenome only, while 48 of the 115 contigs were allocated to the Dt subgenome only. Between the two chromosomes, 67 contigs were shared with an estimated overall physical similarity between the two chromosomal homeologs at 40.0 %. A total of 401 fiber unigenes plus 214 non-fiber unigenes were located to chromosome 12 while 207 fiber unigenes plus 183 non-fiber unigenes were allocated to chromosome 26. Anchoring was done through an overgo hybridization approach and all anchored ESTs were functionally annotated via blast analysis. CONCLUSION: This integrated genomic map describes the first pair of homoeologous chromosomes of an allotetraploid genome in which BAC contigs were identified and partially separated through the use of chromosome-specific probes and locus-specific genetic markers. The approach used in this study should prove useful in the construction of genome-wide physical maps for polyploid plant genomes including Upland cotton. The identification of Gene-rich islands in the integrated map provides a platform for positional cloning of important genes and the targeted sequencing of specific genomic regions.

Cotton (Gossypium spp.) R2R3-MYB transcription factors SNP identification, phylogenomic characterization, chromosome localization, and linkage mapping.

R2R3-MYB transcription factors of plants are involved in the regulation of trichome length and density. Several of them are differentially expressed during initiation and elongation of cotton fibers. We report sequence phylogenomic characterization of the six MYB genes, their chromosomal localization, and linkage mapping via SNP marker in AD-genome cotton (2n = 52). Phylogenetic grouping and comparison to At- and Dt-genome putative ancestral diploid species of allotetraploid cotton facilitated differentiation between genome-specific polymorphisms (GSPs) and marker-suitable locus-specific polymorphisms (LSPs). The SNP frequency averaged one per 77 bases overall, and one per 106 and 30 bases in coding and non-coding regions, respectively. SNP-based multivariate relationships conformed to independent evolution of the six MYB homoeologous loci in the four tetraploid species. Nucleotide diversity analysis indicated that the six MYB loci evolved more quickly in the Dt- than At-genome. The greater variation in the Dt-D genome comparisons than that in At-A genome comparisons showed no significant bias among synonymous substitution, non-synonymous substitution, and nucleotide change in non-coding regions. SNPs were concordantly mapped by deletion analysis and linkage mapping, which confirmed their value as candidate gene markers and indicated the reliability of the SNP discovery strategy in tetraploid cotton species. We consider that these SNPs may be useful for genetic dissection of economically important fiber and yield traits because of the role of these genes in fiber development.

Complete assignment of the chromosomes of Gossypium hirsutum L. by translocation and fluorescence in situ hybridization mapping.

Significant progress has been made in the construction of genetic maps in the tetraploid cotton Gossypium hirsutum. However, six linkage groups (LGs) have still not been assigned to specific chromosomes, which is a hindrance for integrated genetic map construction. In the present research, specific bacterial artificial chromosome (BAC) clones constructed in G. hirsutum acc. TM-1 for these six LGs were identified by screening the BAC library using linkage group-specific simple-sequence repeats markers. These BAC clones were hybridized to ten translocation heterozygotes of G. hirsutum. L as BAC-fluorescence in situ hybridization probes, which allowed us to assign these six LGs A01, A02, A03, D02, D03, and D08 to chromosomes 13, 8, 11, 21, 24, and 19, respectively. Therefore, the 13 homeologous chromosome pairs have been established, and we have proposed a new chromosome nomenclature for tetraploid cotton.

Wide-cross whole-genome radiation hybrid mapping of the cotton (Gossypium barbadense L.) genome.

Whole-genome radiation hybrid mapping has been applied extensively to human and certain animal species, but little to plants. We recently demonstrated an alternative mapping approach in cotton (Gossypium hirsutum L.), based on segmentation by 5-krad gamma-irradiation and derivation of wide-cross whole-genome radiation hybrids (WWRHs). However, limitations observed at the 5-krad level suggested that higher doses might be advantageous. Here, we describe the development of an improved second-generation WWRH panel after higher dose irradiation and compare the resulting map to the 5-krad map. The genome of G. hirsutum (n = 26) was used to rescue the radiation-segmented genome of G. barbadense (n = 26) introduced via 8- and 12-krad gamma-irradiated pollen. Viable seedlings were not recovered after 12-krad irradiation, but 8-krad irradiation permitted plant recovery and construction of a 92-member WWRH mapping panel. Assessment of 31 SSR marker loci from four chromosomes revealed that the 8-krad panel has a marker retention frequency of ca. 76%, which is approximately equivalent to the rate of loss in a low-dose animal radiation hybrid panel. Retention frequencies of loci did not depart significantly from independence when compared between the A and D subgenomes, or according to positions along individual chromosomes. WWRH maps of chromosomes 10 and 17 were generated by the maximum likelihood RHMAP program and the general retention model. The resulting maps bolster evidence that WWRH mapping complements traditional linkage mapping and works in cotton, and that the 8-krad panel complements the 5-krad panel by offering higher rates of chromosome breakages, lower marker retention frequency, and more retention patterns.

Developmental and gene expression analyses of a cotton naked seed mutant.

Cotton fiber development is a fundamental biological phenomenon, yet the molecular basis of fiber cell initiation is poorly understood. We examined molecular and cellular events of fiber cell development in the naked seed mutant (N1N1) and its isogenic line of cotton (Gossypium hirsutum L. cv. Texas Marker-1, TM-1). The dominant mutation not only delayed the process of fiber cell formation and elongation but also reduced the total number of fiber cells, resulting in sparsely distributed short fibers. Gene expression changes in TM-1 and N1N1 mutant lines among four tissues were analyzed using spotted cotton oligo-gene microarrays. Using the Arabidopsis genes, we selected and designed approximately 1,334 70-mer oligos from a subset of cotton fiber ESTs. Statistical analysis of the microarray data indicates that the number of significantly differentially expressed genes was 856 in the leaves compared to the ovules (3 days post-anthesis, DPA), 632 in the petals relative to the ovules (3 DPA), and 91 in the ovules at 0 DPA compared to 3 DPA, all in TM-1. Moreover, 117 and 30 genes were expressed significantly different in the ovules at three and 0 DPA, respectively, between TM-1 and N1N1. Quantitative RT-PCR analysis of 23 fiber-associated genes in seven tissues including ovules, fiber-bearing ovules, fibers, and non-fiber tissues in TM-1 and N1N1 indicates a mode of temporal regulation of the genes involved in transcriptional and translational regulation, signal transduction, and cell differentiation during early stages of fiber development. Suppression of the fiber-associated genes in the mutant may suggest that the N1N1 mutation disrupts temporal regulation of gene expression, leading to a defective process of fiber cell elongation and development.

Cloning, characterization, and evolution of the NBS-LRR-encoding resistance gene analogue family in polyploid cotton (Gossypium hirsutum L.).

The nucleotide-binding site-leucine-rich repeat (NBS-LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. We cloned the NBS-LRR-encoding genes from polyploid cotton by a polymerase chain reaction-based approach. A sample of 150 clones was selected from the NBS-LRR gene sequence library and was sequenced, and 61 resistance gene analogs (RGA) were identified. Sequence analysis revealed that RGA are abundant and highly diverged in the cotton genome and could be categorized into 10 distinct subfamilies based on the similarities of their nucleotide sequences. The numbers of members vary many fold among different subfamilies, and gene index analysis showed that each of the subfamilies is at a different stage of RGA family evolution. Genetic mapping of a selection of RGA indicates that the RGA reside on a limited number of the cotton chromosomes, with those from a single subfamily tending to cluster and two of the RGA loci being colocalized with the cotton bacterial blight resistance genes. The distribution of RGA between the two subgenomes A and D of cotton is uneven, with RGA being more abundant in the A subgenome than in the D subgenome. The data provide new insights into the organization and evolution of the NBS-LRR-encoding RGA family in polyploid plants.

Wide-cross whole-genome radiation hybrid mapping of cotton (Gossypium hirsutum L.).

We report the development and characterization of a "wide-cross whole-genome radiation hybrid" (WWRH) panel from cotton (Gossypium hirsutum L.). Chromosomes were segmented by gamma-irradiation of G. hirsutum (n = 26) pollen, and segmented chromosomes were rescued after in vivo fertilization of G. barbadense egg cells (n = 26). A 5-krad gamma-ray WWRH mapping panel (N = 93) was constructed and genotyped at 102 SSR loci. SSR marker retention frequencies were higher than those for animal systems and marker retention patterns were informative. Using the program RHMAP, 52 of 102 SSR markers were mapped into 16 syntenic groups. Linkage group 9 (LG 9) SSR markers BNL0625 and BNL2805 had been colocalized by linkage analysis, but their order was resolved by differential retention among WWRH plants. Two linkage groups, LG 13 and LG 9, were combined into one syntenic group, and the chromosome 1 linkage group marker BNL4053 was reassigned to chromosome 9. Analyses of cytogenetic stocks supported synteny of LG 9 and LG 13 and localized them to the short arm of chromosome 17. They also supported reassignment of marker BNL4053 to the long arm of chromosome 9. A WWRH map of the syntenic group composed of linkage groups 9 and 13 was constructed by maximum-likelihood analysis under the general retention model. The results demonstrate not only the feasibility of WWRH panel construction and mapping, but also complementarity to traditional linkage mapping and cytogenetic methods.