We assembled a total of 297,239 Gossypium hirsutum (Gh, a tetraploid cotton, AADD) expressed sequence tag (EST) sequences that were available in the National Center for Biotechnology Information database, with reference to the recently published G. raimondii (Gr, a diploid cotton, DD) genome, and obtained 49,125 UniGenes. The average lengths of the UniGenes were increased from 804 and 791 bp in two previous EST assemblies to 1,019 bp in the current analysis. The number of putative cotton UniGenes with lengths of 3 kb or more increased from 25 or 34 to 1,223. As a result, thousands of originally independent G. hirsutum ESTs were aligned to produce large contigs encoding transcripts with very long open reading frames, indicating that the G. raimondii genome sequence provided remarkable advantages to assemble the tetraploid cotton transcriptome. Significant different distribution patterns within several GO terms, including transcription factor activity, were observed between D- and A-derived assemblies. Transcriptome analysis showed that, in a tetraploid cotton cell, 29,547 UniGenes were possibly derived from the D subgenome while another 19,578 may come from the A subgenome. Finally, some of the in silico data were confirmed by reverse transcription polymerase chain reaction experiments to show the changes in transcript levels for several gene families known to play key role in cotton fiber development. We believe that our work provides a useful platform for functional and evolutionary genomic studies in cotton.

Virus-induced gene silencing (VIGS) offers a powerful approach for functional analysis of individual genes by knocking down their expression. We have adopted this approach to dissect gene functions in cotton resistant to Verticillium wilt, one of the most devastating diseases worldwide. We showed here that highly efficient VIGS was obtained in a cotton breeding line (CA4002) with partial resistance to Verticillium wilt, and GhMKK2 and GhVe1 are required for its resistance to Verticillium wilt. Arabidopsis AtBAK1/SERK3, a central regulator in plant disease resistance, belongs to a subfamily of somatic embryogenesis receptor kinases (SERKs) with five members, AtSERK1 to AtSERK5. Two BAK1 orthologs and one SERK1 ortholog were identified in the cotton genome. Importantly, GhBAK1 is required for CA4002 resistance to Verticillium wilt. Surprisingly, silencing of GhBAK1 is sufficient to trigger cell death accompanied with production of reactive oxygen species in cotton. This result is distinct from Arabidopsis in which AtBAK1 and AtSERK4 play redundant functions in cell death control. Apparently, cotton has only evolved SERK1 and BAK1 whereas AtSERK4/5 are newly evolved genes in Arabidopsis. Our studies indicate the functional importance of BAK1 in Verticillium wilt resistance and suggest the dynamic evolution of SERK family members in different plant species.

The MADS-box genes encode a large family of transcription factors having diverse roles in plant development. The SQUAMOSA (SQUA)/APETALA1 (AP1)/FRUITFULL (FUL) subfamily genes are essential regulators of floral transition and floral organ identity. Here we cloned two MADS-box genes, GhMADS22 and GhMADS23, belonging to the SQUA/AP1/FUL subgroup from Gossypium hirsutum L. Phylogenetic analysis and sequence alignment showed that GhMADS22 and GhMADS23 belonged to the euFUL and euAP1 subclades, respectively. The two genes both had eight exons and seven introns from the start codon to the stop codon according to the alignment between the obtained cDNA sequence and the Gossypium raimondii L. genome sequence. Expression profile analysis showed that GhMADS22 and GhMADS23 were highly expressed in developing shoot apices, bracts, and sepals. Gibberellic acid promoted GhMADS22 and GhMADS23 expression in the shoot apex. Transgenic Arabidopsis lines overexpressing 35S::GhMADS22 had abnormal flowers and bolted earlier than wild type under long-day conditions (16 h light/8 h dark). Moreover, GhMADS22 overexpression delayed floral organ senescence and abscission and it could also respond to abscisic acid. In summary, GhMADS22 may have functions in promoting flowering, improving resistance and delaying senescence for cotton and thus it may be a candidate target for promoting early-maturation in cotton breeding.

Upland cotton (Gossypium hirsutum L.) shows strong heterosis. However, heterosis is not widely utilized owing to the high cost of hybrid seed production. Creation of a photoperiod-sensitive genetic male sterile line could substantially reduce the cost of hybrid seed production in upland cotton. Such a mutant with virescent marker was found by space mutation in near-earth orbit and its traits had been stable after 4 years of selection in Anyang and Sanya, China. This mutant was fertile with an 11–12.5 h photoperiod when the temperature was higher than 21.5 °C and was sterile with a 13–14.5 h photoperiod. Genetic analysis indicated that both traits were controlled by a single recessive gene or two closely linked genes. Also, the cytological observations and transcriptome profiling analysis showed that the degradation of pollen grain cytoplasm should be the primary reason why the mutant line were male sterile under long-day conditions.

Cotton fuzz fibers, like Arabidopsis trichomes, are elongated unicells. It is postulated that a transcriptional complex of GLABRA1 (GL1), GLABRA3 (GL3), and TRANSPARENT TESTAGLABRA1 (TTG1) might be in existence in Arabidopsis as evidenced by their physical interaction in yeast, and the complex regulates expression of GLABRA2 (GL2) controlling trichome cell differentiation; it is also assumed that TRIPTYCHON (TRY) and CAPRICE (CPC) counteract the complex formation in neighboring cells. Here, the homologs GaMYB23 (a homolog of GL1), GaDEL65 (a homolog of GL3), GaTTG1, GaCPC and GaTRY were identified in Gossypium arboreum. We show that GaMYB23 can bind to and activate the promoters of GaCPC, GaGL2 and GaTRY, and that GaMYB23, GaTRY and GaTTG1 could interact with GaDEL65 in yeast and in planta. In situ analysis showed that GaMYB23, GaGL2, GaDEL65, and GaTRY were predominantly expressed in fuzz fiber, but GaTRY proteins were primarily found in undeveloped epidermal cells. A G. arboreum fuzzless mutant with consistently high level GaMYB23 transcript has lost the detectable GaMYB23-promoter of GaGL2 complex, corresponding to sharply reduced transcription of GaGL2. Our results support that cotton homologs to the genetic molecules regulating Arabidopsis trichome differentiation interacted in the epidermis of ovules and the redundant GaMYB23 serves as a negative regulator in fuzz-fiber patterning.

To get a broader view on the molecular mechanisms underlying somatic embryogenesis (SE) in cotton (Gossypium hirsutum L.), global analysis of cotton transcriptome dynamics during SE in different sister lines was performed using RNA-Seq. A total of 204 349 unigenes were detected by de novo assembly of the 214 977 462 Illumina reads. The quantitative reverse transcription–polymerase chain reaction (qRT-PCR) measurements were positively correlated with the RNA-Seq results for almost all the tested genes (R² = 0.841, correlation was significant at the 0.01 level). Different phytohormone (auxin and cytokinin) concentration ratios in medium and the endogenous content changes of these two phytohormones at two stages in different sister lines suggested the roles of auxin and cytokinin during cotton SE. On the basis of global gene regulation of phytohormone-related genes, numerous genes from all the differentially expressed transcripts were involved in auxin and cytokinin biosynthesis and signal transduction pathways. Analyses of differentially expressed genes that were involved in these pathways revealed the substantial changes in gene type and abundance between two sister lines. Isolation, cloning and silencing/overexpressing the genes that revealed remarkable up- or down-expression during cotton SE were important. Furthermore, auxin and cytokinin play a primary role in SE, but potential cross-talk with each other or other factors remains unclear.

Sucrose synthase (Sus) is a key enzyme in plant sucrose metabolism. In cotton, Sus (EC 2.4.1.13) is the main enzyme that degrades sucrose imported into cotton fibers from the phloem of the seed coat. This study demonstrated that the genomes of Gossypium arboreum L., G. raimondii Ulbr., and G. hirsutum L., contained 8, 8, and 15 Sus genes, respectively. Their structural organizations, phylogenetic relationships, and expression profiles were characterized. Comparisons of genomic and coding sequences identified multiple introns, the number and positions of which were highly conserved between diploid and allotetraploid cotton species. Most of the phylogenetic clades contained sequences from all three species, suggesting that the Sus genes of tetraploid G. hirsutum derived from those of its diploid ancestors. One Sus group (Sus I) underwent expansion during cotton evolution. Expression analyses indicated that most Sus genes were differentially expressed in various tissues and had development-dependent expression profiles in cotton fiber cells. Members of the same orthologous group had very similar expression patterns in all three species. These results provide new insights into the evolution of the cotton Sus gene family, and insight into its members' physiological functions during fiber growth and development.

NAC domain proteins are plant-specific transcription factors known to play diverse roles in various plant developmental processes. In the present study, we performed the first comprehensive study of the NAC gene family in Gossypium raimondii Ulbr., incorporating phylogenetic, chromosomal location, gene structure, conserved motif, and expression profiling analyses. We identified 145 NAC transcription factor (NAC-TF) genes that were phylogenetically clustered into 18 distinct subfamilies. Of these, 127 NAC-TF genes were distributed across the 13 chromosomes, 80 (55%) were preferentially retained duplicates located in both duplicated regions and six were located in triplicated chromosomal regions. The majority of NAC-TF genes showed temporal-, spatial-, and tissue-specific expression patterns based on transcriptomic and qRT-PCR analyses. However, the expression patterns of several duplicate genes were partially redundant, suggesting the occurrence of sub-functionalization during their evolution. Based on their genomic organization, we concluded that genomic duplications contributed significantly to the expansion of the NAC-TF gene family in G. raimondii. Comprehensive analysis of their expression profiles could provide novel insights into the functional divergence among members of the NAC gene family in G. raimondii.