Genomic diversifications of five Gossypium allopolyploid species and their impact on cotton improvement.

Polyploidy is an evolutionary innovation for many animals and all flowering plants, but its impact on selection and domestication remains elusive. Here we analyze genome evolution and diversification for all five allopolyploid cotton species, including economically important Upland and Pima cottons. Although these polyploid genomes are conserved in gene content and synteny, they have diversified by subgenomic transposon exchanges that equilibrate genome size, evolutionary rate heterogeneities and positive selection between homoeologs within and among lineages. These differential evolutionary trajectories are accompanied by gene-family diversification and homoeolog expression divergence among polyploid lineages. Selection and domestication drive parallel gene expression similarities in fibers of two cultivated cottons, involving coexpression networks and N6-methyladenosine RNA modifications. Furthermore, polyploidy induces recombination suppression, which correlates with altered epigenetic landscapes and can be overcome by wild introgression. These genomic insights will empower efforts to manipulate genetic recombination and modify epigenetic landscapes and target genes for crop improvement.

The Gossypium longicalyx Genome as a Resource for Cotton Breeding and Evolution.

Cotton is an important crop that has made significant gains in production over the last century. Emerging pests such as the reniform nematode have threatened cotton production. The rare African diploid species Gossypium longicalyx is a wild species that has been used as an important source of reniform nematode immunity. While mapping and breeding efforts have made some strides in transferring this immunity to the cultivated polyploid species, the complexities of interploidal transfer combined with substantial linkage drag have inhibited progress in this area. Moreover, this species shares its most recent common ancestor with the cultivated A-genome diploid cottons, thereby providing insight into the evolution of long, spinnable fiber. Here we report a newly generated de novo genome assembly of G. longicalyx This high-quality genome leveraged a combination of PacBio long-read technology, Hi-C chromatin conformation capture, and BioNano optical mapping to achieve a chromosome level assembly. The utility of the G. longicalyx genome for understanding reniform immunity and fiber evolution is discussed.

Sub genome anchored physical frameworks of the allotetraploid Upland cotton (Gossypium hirsutum L.) genome, and an approach toward reference-grade assemblies of polyploids.

Like those of many agricultural crops, the cultivated cotton is an allotetraploid and has a large genome (~2.5 gigabase pairs). The two sub genomes, A and D, are highly similar but unequally sized and repeat-rich, which pose significant challenges for accurate genome reconstruction using standard approaches. Here we report the development of BAC libraries, sub genome specific physical maps, and a new-generation sequencing approach that will lead to a reference-grade genome assembly for Upland cotton. Three BAC libraries were constructed, fingerprinted, and integrated with BAC-end sequences (BES) to produce a de novo whole-genome physical map. The BAC map was partitioned by sub genomes through alignment to the diploid progenitor D-genome reference sequence with densely spaced BES anchor points and computational filtering. The physical maps were validated with FISH and genetic mapping of SNP markers derived from BES. Two pairs of homeologous chromosomes, A11/D11 and A12/D12, were used to assess multiplex sequencing approaches for completeness and scalability. The results represent the first sub genome anchored physical maps of Upland cotton, and a new-generation approach to the whole-genome sequencing, which will lead to the reference-grade assembly of allopolyploid cotton and serve as a general strategy for sequencing other polyploid species.

Epigenomic and functional analyses reveal roles of epialleles in the loss of photoperiod sensitivity during domestication of allotetraploid cottons.

BACKGROUND: Polyploidy is a pervasive evolutionary feature of all flowering plants and some animals, leading to genetic and epigenetic changes that affect gene expression and morphology. DNA methylation changes can produce meiotically stable epialleles, which are transmissible through selection and breeding. However, the relationship between DNA methylation and polyploid plant domestication remains elusive. RESULTS: We report comprehensive epigenomic and functional analyses, including ~12 million differentially methylated cytosines in domesticated allotetraploid cottons and their tetraploid and diploid relatives. Methylated genes evolve faster than unmethylated genes; DNA methylation changes between homoeologous loci are associated with homoeolog-expression bias in the allotetraploids. Significantly, methylation changes induced in the interspecific hybrids are largely maintained in the allotetraploids. Among 519 differentially methylated genes identified between wild and cultivated cottons, some contribute to domestication traits, including flowering time and seed dormancy. CONSTANS (CO) and CO-LIKE (COL) genes regulate photoperiodicity in Arabidopsis. COL2 is an epiallele in allotetraploid cottons. COL2A is hypermethylated and silenced, while COL2D is repressed in wild cottons but highly expressed due to methylation loss in all domesticated cottons tested. Inhibiting DNA methylation activates COL2 expression, and repressing COL2 in cultivated cotton delays flowering. CONCLUSIONS: We uncover epigenomic signatures of domestication traits during cotton evolution. Demethylation of COL2 increases its expression, inducing photoperiodic flowering, which could have contributed to the suitability of cotton for cultivation worldwide. These resources should facilitate epigenetic engineering, breeding, and improvement of polyploid crops.

DNA Sequence Evolution and Rare Homoeologous Conversion in Tetraploid Cotton.

Allotetraploid cotton species are a vital source of spinnable fiber for textiles. The polyploid nature of the cotton genome raises many evolutionary questions as to the relationships between duplicated genomes. We describe the evolution of the cotton genome (SNPs and structural variants) with the greatly improved resolution of 34 deeply re-sequenced genomes. We also explore the evolution of homoeologous regions in the AT- and DT-genomes and especially the phenomenon of conversion between genomes. We did not find any compelling evidence for homoeologous conversion between genomes. These findings are very different from other recent reports of frequent conversion events between genomes. We also identified several distinct regions of the genome that have been introgressed between G. hirsutum and G. barbadense, which presumably resulted from breeding efforts targeting associated beneficial alleles. Finally, the genotypic data resulting from this study provides access to a wealth of diversity sorely needed in the narrow germplasm of cotton cultivars.

Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement.

Upland cotton is a model for polyploid crop domestication and transgenic improvement. Here we sequenced the allotetraploid Gossypium hirsutum L. acc. TM-1 genome by integrating whole-genome shotgun reads, bacterial artificial chromosome (BAC)-end sequences and genotype-by-sequencing genetic maps. We assembled and annotated 32,032 A-subgenome genes and 34,402 D-subgenome genes. Structural rearrangements, gene loss, disrupted genes and sequence divergence were more common in the A subgenome than in the D subgenome, suggesting asymmetric evolution. However, no genome-wide expression dominance was found between the subgenomes. Genomic signatures of selection and domestication are associated with positively selected genes (PSGs) for fiber improvement in the A subgenome and for stress tolerance in the D subgenome. This draft genome sequence provides a resource for engineering superior cotton lines.

BAC-End Sequence-Based SNP Mining in Allotetraploid Cotton (Gossypium) Utilizing Resequencing Data, Phylogenetic Inferences, and Perspectives for Genetic Mapping.

A bacterial artificial chromosome library and BAC-end sequences for cultivated cotton (Gossypium hirsutum L.) have recently been developed. This report presents genome-wide single nucleotide polymorphism (SNP) mining utilizing resequencing data with BAC-end sequences as a reference by alignment of 12 G. hirsutum L. lines, one G. barbadense L. line, and one G. longicalyx Hutch and Lee line. A total of 132,262 intraspecific SNPs have been developed for G. hirsutum, whereas 223,138 and 470,631 interspecific SNPs have been developed for G. barbadense and G. longicalyx, respectively. Using a set of interspecific SNPs, 11 randomly selected and 77 SNPs that are putatively associated with the homeologous chromosome pair 12 and 26, we mapped 77 SNPs into two linkage groups representing these chromosomes, spanning a total of 236.2 cM in an interspecific F2 population (G. barbadense 3-79 × G. hirsutum TM-1). The mapping results validated the approach for reliably producing large numbers of both intraspecific and interspecific SNPs aligned to BAC-ends. This will allow for future construction of high-density integrated physical and genetic maps for cotton and other complex polyploid genomes. The methods developed will allow for future Gossypium resequencing data to be automatically genotyped for identified SNPs along the BAC-end sequence reference for anchoring sequence assemblies and comparative studies.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

Phylogenetic analysis of Gossypium L. using restriction fragment length polymorphism of repeated sequences.

Cotton is the world's leading textile fiber crop and is also grown as a bioenergy and food crop. Knowledge of the phylogeny of closely related species and the genome origin and evolution of polyploid species is significant for advanced genomics research and breeding. We have reconstructed the phylogeny of the cotton genus, Gossypium L., and deciphered the genome origin and evolution of its five polyploid species by restriction fragment analysis of repeated sequences. Nuclear DNA of 84 accessions representing 35 species and all eight genomes of the genus were analyzed. The phylogenetic tree of the genus was reconstructed using the parsimony method on 1033 polymorphic repeated sequence restriction fragments. The genome origin of its polyploids was determined by calculating the diploid-polyploid restriction fragment correspondence (RFC). The tree is consistent with the morphological classification, genome designation and geographic distribution of the species at subgenus, section and subsection levels. Gossypium lobatum (D7) was unambiguously shown to have the highest RFC with the D-subgenomes of all five polyploids of the genus, while the common ancestor of Gossypium herbaceum (A1) and Gossypium arboreum (A2) likely contributed to the A-subgenomes of the polyploids. These results provide a comprehensive phylogenetic tree of the cotton genus and new insights into the genome origin and evolution of its polyploid species. The results also further demonstrate a simple, rapid and inexpensive method suitable for phylogenetic analysis of closely related species, especially congeneric species, and the inference of genome origin of polyploids that constitute over 70 % of flowering plants.

Transcriptome analysis of extant cotton progenitors revealed tetraploidization and identified genome-specific single nucleotide polymorphism in diploid and allotetraploid cotton.

BACKGROUND: The most widely cultivated cotton (Gossypium hirsutum L., AD-genome) is derived from tetraploidization between A- and D-genome species. G. arboreum L. (A-genome) and G. raimondii Ulbr. (D-genome) are two of closely-related extant progenitors. Gene expression studies in allotetraploid cotton are complicated by the homoeologous loci of A- and D-genome origins. To develop genomic resources for gene expression and cotton breeding, we sequenced and assembled expressed sequence tags (ESTs) derived from G. arboreum and G. raimondii. RESULTS: Roche/454 FLX sequencing technology was employed to sequence normalized cDNA libraries prepared from leaves, roots, bolls, ovules, and fibers in G. arboreum and G. raimondii, respectively. Sequencing reads from two independent libraries in each species were combined to assemble high-quality EST contigs. The combined sequencing reads included 1,699,776 from A-genome and 1,464,815 from D-genome, which were clustered into 89,588 contigs in the A-genome and 65,542 contigs in the D-genome. These contigs represented ~80% of EST collections in Cotton Gene Index 11 (CGI11, March 2011). Compared to the D-genome transcript database, 27,537 and 10,452 contigs were unique transcripts in A and D genomes, respectively. Further analysis using self-blastn reduced the unigene contig number by 52% in A-genome and 57% in D-genome, suggesting that 50% or more of contigs are paralogs or isoforms within each species. The majority of EST contigs (73-81%) were conserved between A- and D-genomes, whereas 27% and 19% contigs were specific to A- and D-genomes, respectively. Using these ESTs, we generated a total of 75,754 genome-specific single nucleotide polymorphism (SNP) (gSNPs or GNPs) or homoeologous-specific SNPs (hSNPs) of 10,885 contigs or genes between A and D genomes, indicating a possibility of separating allelic expression for those genes in allotetraploid cotton. CONCLUSIONS: Expressed genes are highly redundant within each diploid progenitor and between A and D progenitor species, suggesting that diploid progenitors in cotton are likely ancient tetraploids. This large set of A- and D-genome ESTs and GNPs will be valuable resources for genome annotation, gene expression, and crop improvement in allotetraploid cotton.

miR828 and miR858 regulate homoeologous MYB2 gene functions in Arabidopsis trichome and cotton fibre development.

Although polyploidy is common in plants and some animals, mechanisms for functional divergence between homoeologous genes are poorly understood. MYB2 gene promotes cotton fibre development and is functionally homologous to Arabidopsis GLABROUS1 (GL1) in trichome formation. The most widely cultivated cotton is an allotetraploid (Gossypium hirsutum, AADD) that contains GhMYB2A and GhMYB2D homoeologs. Here we show that GhMYB2D mRNA accumulates more than GhMYB2A during fibre initiation and is targeted by miR828 and miR858, generating trans-acting siRNAs (ta-siRNAs) in the TAS4 family. Overexpressing GhMYB2A but not GhMYB2D complements the gl1 phenotype. Mutating the miR828-binding site or replacing its downstream sequence in GhMYB2D abolishes ta-siRNA production and restores trichome development in gl1 mutants. Moreover, disrupting Dicer-like protein 4 or RDR6, the biogenesis genes for ta-siRNAs, in the gl1 GhMYB2D overexpressors restores trichome development. These data support a unique role for microRNAs in functional divergence between target homoeologous genes that are important for evolution and selection of morphological traits.

Construction of a plant-transformation-competent BIBAC library and genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.).

BACKGROUND: Cotton, one of the world's leading crops, is important to the world's textile and energy industries, and is a model species for studies of plant polyploidization, cellulose biosynthesis and cell wall biogenesis. Here, we report the construction of a plant-transformation-competent binary bacterial artificial chromosome (BIBAC) library and comparative genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.) with one of its diploid putative progenitor species, G. raimondii Ulbr. RESULTS: We constructed the cotton BIBAC library in a vector competent for high-molecular-weight DNA transformation in different plant species through either Agrobacterium or particle bombardment. The library contains 76,800 clones with an average insert size of 135 kb, providing an approximate 99% probability of obtaining at least one positive clone from the library using a single-copy probe. The quality and utility of the library were verified by identifying BIBACs containing genes important for fiber development, fiber cellulose biosynthesis, seed fatty acid metabolism, cotton-nematode interaction, and bacterial blight resistance. In order to gain an insight into the Upland cotton genome and its relationship with G. raimondii, we sequenced nearly 10,000 BIBAC ends (BESs) randomly selected from the library, generating approximately one BES for every 250 kb along the Upland cotton genome. The retroelement Gypsy/DIRS1 family predominates in the Upland cotton genome, accounting for over 77% of all transposable elements. From the BESs, we identified 1,269 simple sequence repeats (SSRs), of which 1,006 were new, thus providing additional markers for cotton genome research. Surprisingly, comparative sequence analysis showed that Upland cotton is much more diverged from G. raimondii at the genomic sequence level than expected. There seems to be no significant difference between the relationships of the Upland cotton D- and A-subgenomes with the G. raimondii genome, even though G. raimondii contains a D genome (D5). CONCLUSIONS: The library represents the first BIBAC library in cotton and related species, thus providing tools useful for integrative physical mapping, large-scale genome sequencing and large-scale functional analysis of the Upland cotton genome. Comparative sequence analysis provides insights into the Upland cotton genome, and a possible mechanism underlying the divergence and evolution of polyploid Upland cotton from its diploid putative progenitor species, G. raimondii.

Genome physical mapping of polyploids: a BIBAC physical map of cultivated tetraploid cotton, Gossypium hirsutum L.

Polyploids account for approximately 70% of flowering plants, including many field, horticulture and forage crops. Cottons are a world-leading fiber and important oilseed crop, and a model species for study of plant polyploidization, cellulose biosynthesis and cell wall biogenesis. This study has addressed the concerns of physical mapping of polyploids with BACs and/or BIBACs by constructing a physical map of the tetraploid cotton, Gossypium hirsutum L. The physical map consists of 3,450 BIBAC contigs with an N50 contig size of 863 kb, collectively spanning 2,244 Mb. We sorted the map contigs according to their origin of subgenome, showing that we assembled physical maps for the A- and D-subgenomes of the tetraploid cotton, separately. We also identified the BIBACs in the map minimal tilling path, which consists of 15,277 clones. Moreover, we have marked the physical map with nearly 10,000 BIBAC ends (BESs), making one BES in approximately 250 kb. This physical map provides a line of evidence and a strategy for physical mapping of polyploids, and a platform for advanced research of the tetraploid cotton genome, particularly fine mapping and cloning the cotton agronomic genes and QTLs, and sequencing and assembling the cotton genome using the modern next-generation sequencing technology.

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.

Structure and size variations between 12A and 12D homoeologous chromosomes based on high-resolution cytogenetic map in allotetraploid cotton.

Cotton is a model system for studying polyploidization, genomic organization, and genome-size variation because the allotetraploid was formed 1-2 million years ago, which is old enough for sequence divergence but relatively recent to maintain genome stability. In spite of characterizing random genomic sequences in many polyploidy plants, the cytogenetic and sequence data that decipher homoeologous chromosomes are very limited in allopolyploid species. Here, we reported comprehensive analyses of integrated cytogenetic and linkage maps of homoeologous chromosomes 12A and 12D in allotetraploid cotton using fluorescence in situ hybridization and a large number of bacterial artificial chromosomes that were anchored by simple sequence repeat markers in the corresponding linkage maps. Integration of genetic loci into physical localizations showed considerable variation of genome organization, structure, and size between 12A and 12D homoeologous chromosomes. The distal regions of the chromosomes displayed relatively lower levels of structural and size variation than other regions of the chromosomes. The highest level of variation was found in the pericentric regions in the long arms of the two homoeologous chromosomes. The genome-size difference between A and D sub-genomes in allotetraploid cotton was mainly associated with uneven expansion or contraction between different regions of homoeologous chromosomes. As an attempt for studying on the polyploidy homoeologous chromosomes, these results are of general interest to the understanding and future sequencing of complex genomes in plant species.

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.

Transcriptome profiling, sequence characterization, and SNP-based chromosomal assignment of the EXPANSIN genes in cotton.

The knowledge of biological significance associated with DNA markers is very limited in cotton. SNPs are potential functional marker to tag genes of biological importance. Plant expansins are a group of extracellular proteins that directly modify the mechanical properties of cell walls, enable turgor-driven cell extension, and likely affect length and quality of cotton fibers. Here, we report the expression profiles of EXPANSIN transcripts during fiber elongation and the discovery of SNP markers, assess the SNP characteristics, and localize six EXPANSIN A genes to chromosomes. Transcriptome profiling of cotton fiber oligonucleotide microarrays revealed that seven EXPANSIN transcripts were differentially expressed when there was parallel polar elongation during morphogenesis at early stage of fiber development, suggesting that major and minor isoforms perform discrete functions during polar elongation and lateral expansion. Ancestral and homoeologous relationships of the six EXPANSIN A genes were revealed by phylogenetic grouping and comparison to extant A- and D-genome relatives of contemporary AD-genome cottons. The average rate of SNP per nucleotide was 2.35% (one SNP per 43 bp), with 1.74 and 3.99% occurring in coding and noncoding regions, respectively, in the selected genotypes. An unequal evolutionary rate of the EXPANSIN A genes at the subgenome level of tetraploid cotton was recorded. Chromosomal locations for each of the six EXPANSIN A genes were established by gene-specific SNP markers. Results revealed a strategy for discovering SNP markers in a polyploidy species like cotton. These markers could be useful to associate candidate genes with the complex fiber traits in MAS.

Accumulation of genome-specific transcripts, transcription factors and phytohormonal regulators during early stages of fiber cell development in allotetraploid cotton.

Gene expression during the early stages of fiber cell development and in allopolyploid crops is poorly understood. Here we report computational and expression analyses of 32 789 high-quality ESTs derived from Gossypium hirsutum L. Texas Marker-1 (TM-1) immature ovules (GH_TMO). The ESTs were assembled into 8540 unique sequences including 4036 tentative consensus sequences (TCs) and 4504 singletons, representing approximately 15% of the unique sequences in the cotton EST collection. Compared with approximately 178 000 existing ESTs derived from elongating fibers and non-fiber tissues, GH_TMO ESTs showed a significant increase in the percentage of genes encoding putative transcription factors such as MYB and WRKY and genes encoding predicted proteins involved in auxin, brassinosteroid (BR), gibberellic acid (GA), abscisic acid (ABA) and ethylene signaling pathways. Cotton homologs related to MIXTA, MYB5, GL2 and eight genes in the auxin, BR, GA and ethylene pathways were induced during fiber cell initiation but repressed in the naked seed mutant (N1N1) that is impaired in fiber formation. The data agree with the known roles of MYB and WRKY transcription factors in Arabidopsis leaf trichome development and the well-documented phytohormonal effects on fiber cell development in immature cotton ovules cultured in vitro. Moreover, the phytohormonal pathway-related genes were induced prior to the activation of MYB-like genes, suggesting an important role of phytohormones in cell fate determination. Significantly, AA sub-genome ESTs of all functional classifications including cell-cycle control and transcription factor activity were selectively enriched in G. hirsutum L., an allotetraploid derived from polyploidization between AA and DD genome species, a result consistent with the production of long lint fibers in AA genome species. These results suggest general roles for genome-specific, phytohormonal and transcriptional gene regulation during the early stages of fiber cell development in cotton allopolyploids.

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.

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.