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Average fiber quality of the two BILs tested in different environments based on Yu et al. [37]. (DOC 15Â kb)

Summary of RNA-Seq mapping reads to the TM-1 reference genome. (DOC 38Â kb)

Phylogenetic relationships, gene structure and motif compositions of the Gossypium barbadense SAUR genes. (A) The phylogenetic tree was constructed using MEGA 6.0 with the Neighbour-Joining (NJ) method with 1000 bootstrap replicates. (B) Exon/intron structures of SAUR genes from G. hirsutum. The introns, exons and UTRs are represented by black lines, green and blue boxes respectively. The scale bar represents 0.5Â kb. (C) Protein motif. Each motif is represented in the colored box. (PDF 677Â kb)

Sequence variations in the predicted LPAAT genes between the sequenced G. hirsutum (TM-1) and G. barbadense (3â 79 and Xinhai 21). The boxes indicate the variations of the A- and D- subgenomes and the ellipses indicate the SNPs between the two cultivated species; Gb1 and Gb2 indicate 3â 79 and Xinhai21 respectively. (PDF 22277 kb)

Phylogenetic relationships, gene structure and motif compositions of the Gossypium arboreum SAUR genes. (A) The phylogenetic tree was constructed using MEGA 6.0 with the Neighbour-Joining (NJ) method with 1000 bootstrap replicates. (B) Exon/intron structures of SAUR genes from G. arboreum. The introns, CDS are represented by black lines, green and blue boxes respectively. The scale bar represents 0.5Â kb. (C) Protein motif. Each motif is represented in the colored box. (PDF 2081Â kb)

A list of primers used for SSCP, HRM and qRT-PCR analyses. (DOC 43Â kb)

Summary of RNA-Seq data. (DOC 30Â kb)

Chromosome distribution of 239 DEGs with SNP/InDels between “Long” and “Short” in the Gossypium hirsutum genome from A01 to A13, and from D01 to D13. Genes with InDels are indicated in blue. (PDF 1348 kb)

Sequence alignment of the predicted SAUR genes between four sequenced Gossypium species. The red boxes indicate sequence variations of G. hirsutum (TM-1) and G. barbadense (3-79 and Xinhai 21). Gh1 indicate TM-1 sequenced by Zhang et al. (2015). Gb1 and Gb2 indicate 3-79 and Xinhai21 sequenced by Yuan et al. (2015) and Liu et al. (2015), respectively. Gr and Ga indicate two diploid genomes sequenced by Paterson et al. (2013) and Li et al. (2014), respectively. (PDF 335Â kb)

SQUAMOSA promoter binding protein-like (SPL) genes encode plant-specific transcription factors that are involved in many fundamental developmental processes. Certain SPL genes contain sequences complementary to miR156, a microRNA (miRNA) that plays a role in modulating plant gene expression. In this study, 30 SPL genes were identified in the reference genome of Gossypium raimondii and 24 GhSPLs were cloned from Gossypium hirsutum. G. raimondii is regarded as the putative contributor of the D-subgenome of G. hirsutum. Comparative analysis demonstrated sequence conservation between GhSPLs and other plant species. GhSPL genes could be classified into seven subclades based on phylogenetic analysis, diverse intron-exon structure, and motif prediction. Within each subclade, genes shared a similar structure. Sequence and experimental analysis predicted that 18 GhSPL genes are putative targets of GhmiR156. Additionally, tissue-specific expression analysis of GhSPL genes showed that their spatiotemporal expression patterns during development progressed differently, with most genes having high transcript levels in leaves, stems, and flowers. Finally, overexpression of GhSPL3 and GhSPL18 in Arabidopsis plants demonstrated that these two genes are involved in the development of leaves and second shoots and play an integral role in promoting flowering. The flowering integrator GhSOC1 may bind to the promoter of GhSPL3 but not GhSPL18 to regulate flowering. In conclusion, our analysis of GhSPL genes will provide some gene resources and a further understanding of GhSPL3 and GhSPL18 function in flowering promotion. Furthermore, the comparative genomics and functional analysis deepened our understanding of GhSPL genes during upland cotton vegetative and reproductive growth.

Six short season cotton cultivars (Gossypium hirsutum L.) were crossed in a dillalel crossing system to evaluate inheritance and interrelationship of phenotype and genotype of fiber quality traits (fiber length, uniformity index, fiber strength, micronaire and fiber elongation) and their correlation with earliness and yield traits. The study was carried out from year 2005 to 2008. The experiment design was a randomized complete block design with three replications. Additive, dominance and epistasis effects were analyzed with ADAA model. The results showed that fiber quality traits were mainly controlled by dominant genetic effects and also had a definitely proportion additive genetic effects. Micronaire, fiber length, fiber strength and fiber elongation had the highest broad sense heritability (HB) and by environment interaction (HBE) estimates, 0.618 (HB) and 0.267 (HBE) for micronaire, 0.510 (HB) and 0.287 (HBE) for fiber length, 0.452 (HB) and 0.316 (HBE) for fiber strength, 0.294 (HB) and 0.494(HBE) for fiber elongation, respectively. Narrow sense heritability (HN) was 0.258 for fiber elongation, 0.136 for fiber length, 0.127 for fiber strength and 0.110 for micronaire. Some F1 hybrids (A1×B2), (A2×A3) and (B1×A3) for fiber length, F1 (A2×B3) and (B1×B2) for fiber strength, F1 (B1×A2), (A2×B2), (A2×B3), (B1×A3), (B2×A3) and (B1×B2) for micronaire and F1 (A1×A2), (A1×A3), (A1×B1), (A1×B3) and (B2×A3) for fiber elongation had significant positive heterozygous dominance effects and affected by environment. Some F1 hybrids (A1×A2), (A1×B1), (A1×B3), (B1×A2) and (B2×A3) for fiber length, (A1×A3), (A1×B1) and (A1×B3) for fiber strength, (A1×A3), (A1×B1), (A1×B2) and (B2×B3) for micronaire and (A2×A3), (A2×B2), (A2×B3) and (B1×B2) for fiber elongation had significant positive epistatic effects. Among fiber quality traits, fiber length with fiber strength, uniformity index with fiber strength and micronaire, fiber strength with micronaire showed a positive and significant correlation. Only elongation had a significant negative correlation with fiber quality traits. Fiber quality traits had a negative correlation with earliness traits except micronaire. Fiber length and strength showed a significant negative correlation with lint yield, lint yield before frost, lint percentage and boll number. Uniformity index, micronaire and fiber elongation had a significant positive correlation with yield traits except boll weight and lint percentage. The results showed that the higher the yield and the earlier the maturing and the worse the fiber quality. Therefore, selections of fiber properties and integration of high yield, good fiber quality and early maturing traits should be performed in higher generations in short season cotton variety breeding program.

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.

Premature senescence is an important limited factor for the short season cotton (SSC), which decreases yield and declines fiber quality. The activity of antioxidant enzyme, methane dicarboxylic aldehyde (MDA) content and chlorophyll content is related with premature senescence. The present study is aimed to investigate the heredity and relationship of the antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), MDA content and chlorophyll content for breeding high yield and good quality of the SSC varieties. Six parents from two types of senescence varieties (type A, which matures early and displays premature senescence; type B, which matures early but does not display premature senescence) and their F1, F2 progenies in a diallel crossing scheme were planted in the experiment field. Phenotypic data on the traits were analyzed according to the Additive–Dominance–Epistasis and Additive–Dominance–Maternal–Paternal effects genetic model. CAT activity of dominant genetic effects were significant and large from 9 to 85 days after planting, the variance components were 34.69, 8.41, 31.93, 19.57 and 27.96 % at 9, 36, 50, 66 and 85 days, respectively. Additive-by-additive epistatic effects on CAT activity were significant and important from 9 to 85 days after planting. POD activity of additive genetic effects was significant and stable. There was not interaction by environment during 24 to 50 days after planting, the variance components were 9.82, 18.86 and 7.87 % at 24, 36 and 50 days, respectively. SOD activity of additive genetic effects was significant and stable. There was not interaction by environment during 66 to 100 days after planting, the variance components were 11.56, 11.80 and 13.84 % at 66, 85 and 100 days, respectively. MDA content of additive genetic effects was significant and stable. There was not interaction by environment during 50 to 100 days after planting, the variance components were 24.81, 13.81, 14.38 and 4.77 % at 50, 66, 85 and 100 days, respectively. Chlorophyll content was mainly controlled by significant dominance effects and main genetic effect from 9 to 66 days after planting, the dominant variance components were over 30 %. Maternal and paternal genetic effects on CAT and chlorophyll content were significant among 36–85 days after planting. Based on these results, it is suggested to selection of POD, SOD and MDA should be undertaken at early generation (F2, F3) and selection of chlorophyll and CAT should be undertaken at late generation (F4, F5) in SSC breeding programs.

The NAC domain genes encode a large family of plant-specific transcription factors that play diverse roles in plant development and stress regulation. In this study, a total of 60 full-length putative NAC genes were isolated from Gossypium hirsutum L. Based on their phylogeny, all GhNAC genes were clustered into seven distinct subfamilies, which exhibit functional similarity. Similarly, a phylogenetic tree for GhNAC genes and their motif showed close resemblance among the subfamilies. The isolated 60 full-length GhNAC genes were located on 13 different chromosomes of D sub-genome. Majority of the NACs showed specific temporal and spatial expression patterns in tissue-specific (fibers, cotyledon leaf, mature leaf, stem, root and flower) studies based on qRT-PCR analyses. Furthermore, the roles of GhNAC genes were monitored using qRT-PCR during leaf senescence and following treatment with ethylene, abscisic acid, gibberellic acid and drought or salinity. This first comprehensive study of GhNAC family elucidates the essential role of these genes in cotton development and in response to various stresses. This study lays fundamental foundations for future research and development in cotton genome.

The analysis of genetic correlations between fiber length (Len), strength (Str), micronaire, and 12 other traits was conducted using the additive (A)-dominance (D) genetic model, which considers genotype × environment interaction effects, in intraspecific upland cotton (Gossypium hirsutum L.) hybrids to effectively improve the quality of cotton cultivars in high planting density cases. Decision-making coefficients were computed based on the genetic correlation and path analysis of three fiber quality traits. The decision-making coefficient analysis of three fiber traits in cross breeding was beneficial for the improvement of Len by increasing the additive effects of Str and length of boll (LB) and decreasing lint percentage (LP), boll number of the top three fruit-bearing branches. The analysis was also beneficial for the improvement of Str and fiber fineness by increasing or decreasing the additive effects of other traits. Utilizing heterosis in hybrids was beneficial to the heterosis of Len by selecting the high dominance effects of number of nodes of the 1st fruit-bearing branch and LB and decreasing the dominance effects of diameter of boll (DB) and LP and for improving Str by increasing the dominance effects of DB and decreasing the dominance effects of number of fruit-bearing branches and number of nodes of the main stem (NNMS). Utilizing heterosis was also beneficial for improving fineness by increasing the dominance effects of LB, Str, and lint yield and decreasing the dominance effects of NNMS and Len.

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.

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.

The most widely grown tetraploid Gossypium hirsutum and G. barbadense differ greatly in yield potential and fiber quality and numerous quantitative trait loci (QTLs) have been reported. However, correspondence of QTLs between experiments and populations is poor due to limited number of markers, small population size and inaccurate phenotyping. The purpose of the present study was to map QTLs for yield, yield components and fiber quality traits using testcross progenies between a large interspecific F2 population and a commercial cotton cultivar as the tester. The results were compared to these from its F2 and F2:3 progenies. Of the 177 QTLs identified from the three populations, 65 fiber QTLs and 51 yield QTLs were unique with an average of 8–12 QTLs per traits. All the 26 chromosomes carried QTLs, but differed in the number of QTLs and the number of QTLs between fiber and yield QTLs. The congruence of QTLs identified across populations was higher (20–60 %) for traits with higher heritabilities including fiber quality, seed index and lint percentage, but lower (10–25 %) for lower heritability traits-seedcotton and lint yields. Major QTLs, QTL clusters for the same traits and QTL ‘hotspots’ for different traits were also identified. This research represents the first report using a testcross population in QTL mapping in interspecific cotton crosses and provides useful information for further comparative analysis and marker-assisted selection.

Background Lysophosphatidic acid acyltransferase (LPAAT) encoded by a multigene family is a rate-limiting enzyme in the Kennedy pathway in higher plants. Cotton is the most important natural fiber crop and one of the most important oilseed crops. However, little is known on genes coding for LPAATs involved in oil biosynthesis with regard to its genome organization, diversity, expression, natural genetic variation, and association with fiber development and oil content in cotton. Results In this study, a comprehensive genome-wide analysis in four Gossypium species with genome sequences, i.e., tetraploid G. hirsutum- AD1 and G. barbadense- AD2 and its possible ancestral diploids G. raimondii- D5 and G. arboreum- A2, identified 13, 10, 8, and 9 LPAAT genes, respectively, that were divided into four subfamilies. RNA-seq analyses of the LPAAT genes in the widely grown G. hirsutum suggest their differential expression at the transcriptional level in developing cottonseeds and fibers. Although 10 LPAAT genes were co-localised with quantitative trait loci (QTL) for cottonseed oil or protein content within a 25-cM region, only one single strand conformation polymorphic (SSCP) marker developed from a synonymous single nucleotide polymorphism (SNP) of the At-Gh13LPAAT5 gene was significantly correlated with cottonseed oil and protein contents in one of the three field tests. Moreover, transformed yeasts using the At-Gh13LPAAT5 gene with the two sequences for the SNP led to similar results, i.e., a 25–31% increase in palmitic acid and oleic acid, and a 16–29% increase in total triacylglycerol (TAG). Conclusions The results in this study demonstrated that the natural variation in the LPAAT genes to improving cottonseed oil content and fiber quality is limited; therefore, traditional cross breeding should not expect much progress in improving cottonseed oil content or fiber quality through a marker-assisted selection for the LPAAT genes. However, enhancing the expression of one of the LPAAT genes such as At-Gh13LPAAT5 can significantly increase the production of total TAG and other fatty acids, providing an incentive for further studies into the use of LPAAT genes to increase cottonseed oil content through biotechnology. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3594-9) contains supplementary material, which is available to authorized users.

DNA methylation is an important epigenetic modification regulating gene expression, genomic imprinting, transposon silencing and chromatin structure in plants and plays an important role in leaf senescence. However, the DNA methylation pattern during Gossypium hirsutum L. cotyledon senescence is poorly understood. In this study, global DNA methylation patterns were compared between two cotyledon development stages, young (J1) and senescence (J2), using methylated DNA immunoprecipitation (MeDIP-Seq). Methylated cytosine occurred mostly in repeat elements, especially LTR/Gypsy in both J1 and J2. When comparing J1 against J2, there were 1222 down-methylated genes and 623 up-methylated genes. Methylated genes were significantly enriched in carbohydrate metabolism, biosynthesis of other secondary metabolites and amino acid metabolism pathways. The global DNA methylation level decreased from J1 to J2, especially in gene promoters, transcriptional termination regions and regions around CpG islands. We further investigated the expression patterns of 9 DNA methyltransferase-associated genes and 2 DNA demethyltransferase-associated genes from young to senescent cotyledons, which were down-regulated during cotyledon development. In this paper, we first reported that senescent cotton cotyledons exhibited lower DNA methylation levels, primarily due to decreased DNA methyltransferase activity and which also play important role in regulating secondary metabolite process.

Fiber quality is one of the most important agronomic traits of cotton, and understanding the genetic basis of its target traits will accelerate improvements to cotton fiber quality. In this study, a panel comprising 355 upland cotton accessions was used to perform genome-wide association studies (GWASs) of five fiber quality traits in four environments. A total of 16, 10 and 7 SNPs were associated with fiber length (FL), fiber strength (FS) and fiber uniformity (FU), respectively, based on the mixed linear model (MLM). Most importantly, two major genomic regions (MGR1 and MGR2) on chromosome Dt7 and four potential candidate genes for FL were identified. Analyzing the geographical distribution of favorable haplotypes (FHs) among these lines revealed that two favorable haplotype frequencies (FHFs) were higher in accessions from low-latitude regions than in accessions from high-latitude regions. However, the genetic diversity of lines from the low-latitude regions was lower than the diversity of lines from the high-latitude regions in China. Furthermore, the FHFs differed among cultivars developed during different breeding periods. These results indicate that FHs have undergone artificial selection during upland cotton breeding in recent decades in China and provide a foundation for the further improvement of fiber quality traits.

To investigate the molecular mechanisms of fiber initiation in cotton (Gossypium spp.), an integrated approach combining transcriptome, iTRAQ-based proteome and genetic mapping was taken to compare the ovules of the Xuzhou 142 wild type (WT) with its fuzzless-lintless (fl) mutant at −3 and 0 day post-anthesis. A total of 1,953 mRNAs, 187 proteins and 131 phosphoproteins were differentially expressed (DE) between WT and fl and the levels of transcripts and their encoded proteins and phosphoproteins were highly congruent. A functional analysis suggested that the abundance of proteins were mainly involved in amino sugar, nucleotide sugar and fatty acid metabolism, one carbon pool for folate metabolism and flavonoid biosynthesis. qRT-PCR, Western blotting and enzymatic assays were performed to confirm the regulation of these transcripts and proteins. A molecular mapping located the lintless gene li3 in the fl mutant on chromosome 26 for the first time. A further in-silico physical mapping of DE genes with sequence variations between fl and WT identified one and four candidate genes in the li3 and n2 regions, respectively. Taken together, the transcript abundance, phosphorylation status of proteins at the fiber initiation stage and candidate genes have provided insights into regulatory processes underlying cotton fiber initiation.

Descriptive statistics for six traits related to early maturity in four different environments. Table S2. Correlations between the WGPs and related traits based on 185 upland cotton accessions in four environments. Table S3. Single nucleotide polymorphisms (SNPs) significantly associated with traits related to early maturity in upland cotton, as determined using the GLM and MLM. Table S4. Information on 185 upland cotton germplasms. Table S5. qRT-PCR primers. (DOCX 45 kb)

Heat maps of the 26 linkage groups. (PDF 4231Â kb)

Recombinants in the RIL population at locus qFT-D3-3 defining the core region between Marker25957 and Marker25965. CCRI36 is the maternal cultivar; G2005 is the paternal line; b is the homozygous allele conferred by CCRI36; a is the homozygous allele conferred by G2005; and h is heterozygous. (PDF 124Â kb)

As the longest known single-celled trichomes, cotton (Gossypium L.) fibers constitute a classic model system to investigate cell initiation and elongation. In this study, we used a high-throughput transcriptome sequencing technology to identify fiber-initiation-related single nucleotide polymorphism (SNP) markers and differentially expressed genes (DEGs) between the wild-type (WT) Upland cotton (G. hirsutum) Xuzhou 142 and its natural fuzzless-lintless mutant Xuzhou 142 fl. Approximately 700 million high-quality cDNA reads representing over 58 Gb of sequences were obtained, resulting in the identification of 28,610 SNPs--of which 17,479 were novel--from 13,960 expressed genes. Of these SNPs, 50% of SNPs in fl were identical to those of G. barbadense, which suggests the likely origin of the fl mutant from an interspecific hybridization between Xuzhou 142 and an unknown G. barbadense genotype. Of all detected SNPs, 15,555, 12,750, and 305 were classified as non-synonymous, synonymous, and pre-terminated ones, respectively. Moreover, 1,352 insertion/deletion polymorphisms (InDels) were also detected. A total of 865 DEGs were identified between the WT and fl in ovules at -3 and 0 days post-anthesis, with 302 candidate SNPs selected from these DEGs for validation by a high-resolution melting analysis and Sanger sequencing in seven cotton genotypes. The number of genotypic pairwise polymorphisms varied from 43 to 302, indicating that the identified SNPs are reliable. These SNPs should serve as good resources for breeding and genetic studies in cotton.

QTL and QTL coincidence region distributions on the genetic map. QTLs located in the coincidence regions are marked in blue. (PDF 343Â kb)

A promising way to uncover the genetic architectures underlying complex traits may lie in the ability to recognize the genetic variants and expression transcripts that are responsible for the traits’ inheritance. However, statistical methods capable of investigating the association between the inheritance of a quantitative trait and expression transcripts are still limited. In this study, we described a two-step approach that we developed to evaluate the contribution of expression transcripts to the inheritance of a complex trait. First, a mixed linear model approach was applied to detect significant trait-associated differentially expressed transcripts. Then, conditional analysis were used to predict the contribution of the differentially expressed genes to a target trait. Diallel cross data of cotton was used to test the application of the approach. We proposed that the detected differentially expressed transcripts with a strong impact on the target trait could be used as intermediates for screening lines to improve the traits in plant and animal breeding programs. It can benefit the discovery of the genetic mechanisms underlying complex traits.

It is well known that genetic mutation could be generated by physical treatment (for example, γ-irradiation) and chemical treatment (for example methylnitrosourea and ethyl methanesulfonate). Here we reported identification of a virescent mutation (vsp) after exposing the upland cotton (Gossypium hirsutum L.) CCRI58 seeds in space environments. vsp mutant was characterized at the morphological, agronomic, cellular and genetic levels. vsp mutant showed an earlier virescence and specific only to true leaves. Agronomic traits of vsp mutant, such as plant height, number of bolls, boll weight, yield and fiber quality were significantly lower than those of CCRI 58. Chlorophyll level, carotenoid level and photochemical efficiency of vsp mutant true leaves were significantly lower compared to CCRI 58 at young leave stage. Anatomical studies of chloroplasts showed that vsp mutant lacked grana in the thylakoids of the mesophyll cells at young leave stage, while CCRI 58 showed normal grana in the thylakoids of the mesophyll cells at young leave stage. This indicated that chlorophyll and carotenoid levels were related with chloroplast structure. Genetic analysis indicated that vsp was controlled by one recessive gene in nucleus. Allelic tests showed that vsp was nonallelic to 12 virescent genes currently available at Anyang, China. In summary, we identified a vsp mutant after exposing the upland cotton (Gossypium hirsutum L.) seeds in space environments. vsp could be a newly identified virescent gene. vsp may also be used as a marker in cotton breeding programs. Exposing seeds in space environments could cause new spectrum of genetic mutations and could be used for breeding programs.

Cotton is one of the most important oil-producing crops and the cottonseed meal provides important protein nutrients as animal feed. However, information on the genetic basis of cottonseed oil and protein contents is lacking. A backcross inbred line (BIL) population from a cross between Gossypium hirsutum as the recurrent parent and G. barbadense was used to identify quantitative trait loci (QTLs) for cottonseed oil, protein, and gossypol contents. The BIL population of 146 lines together with the two parental lines was tested in the same location for three years in China. Based on a genetic map of 392 SSR markers and a total genetic distance of 2,895.2 cM, 17 QTLs on 12 chromosomes for oil content, 22 QTLs on 12 chromosomes for protein content and three QTLs on two chromosomes for gossypol content were detected. Seed oil content was significantly and negatively correlated with seed protein content, which can be explained by eight QTLs for both oil and protein contents co-localized in the same regions but with opposite additive effects. This research represents the first report using a permanent advanced backcross inbred population of an interspecific hybrid population to identify QTLs for seed quality traits in cotton in three environments.

To better understand nitric oxide (NO) responsive proteins, we investigated the proteomic differences between untreated (control), sodium nitroprusside (SNP) treated, and carboxy-PTIO potassium salt (cPTIO, NO scavenger) followed by SNP treated cotton plants. This is the first study to examine the effect of different concentrations of NO on the leaf proteome in cotton using a label-free approach based on nanoscale ultraperformance liquid chromatography-electrospray ionization (ESI)-low/high-collision energy MS analysis (MS(E)). One-hundred and sixty-six differentially expressed proteins were identified. Forty-seven of these proteins were upregulated, 82 were downregulated, and 37 were expressed specifically under different conditions. The 166 proteins were functionally divided into 17 groups and localized to chloroplast, Golgi apparatus, cytoplasm, and so forth. The pathway analysis demonstrated that NO is involved in various physiological activities and has a distinct influence on carbon fixation in photosynthetic organisms and photosynthesis. In addition, this is the first time proteins involved in ethylene synthesis were identified to be regulated by NO. The characterization of these protein networks provides a better understanding of the possible regulation mechanisms of cellular activities occurring in the NO-treated cotton leaves and offers new insights into NO responses in plants.

Somatic embryogenesis (SE) is a critical step leading to plant regeneration in tissue culture of many plant species. The objective of the present study was to analyze the inheritance of SE in cotton (Gossypium hirsutum L.) using leaf petioles as explants. A high embryogenic callus (HEC)—producing line, W10, was selected by petiole callus culture from a commercial Chinese cotton cultivar CRI24 and crossed with a non embryogenic line, TM-1 and a low embryogenic (LEC) commercial Chinese cotton cultivar, CRI12, respectively. The parental lines, F1 and F2 were grown in field conditions for sources of leaf petioles as explants. The F1 plants were similar to the HEC parent in embryogenic callus (EC) induction, indicating that high EC ability is dominant. The classical Mendelian analysis showed that the high EC ability in the HEC line W10 is controlled by two independent dominant genes with complementary effect, designated Ec 1 and Ec 2 , while the LEC line CRI12 contains one dominant gene Ec 2 . A joint segregation analysis confirmed that SE ability in cotton is controlled by two major genes with epistatic effects along with other polygenes. A SSR marker analysis identified three quantitative trait loci (QTLs) on two linkage groups, one of which harbored a major QTL (qEc1) which is assigned to the major gene Ec 1 . This qualitative and quantitative genetic study has provided an incentive to fine map the genes responsible for SE towards the isolation of the SE genes in cotton.

The lack of genetic diversity within cultivated upland cotton (Gossypium hirsutum L.) has hindered the construction of genomewide linkage maps and their applications in genetics and breeding. The objective of this investigation was to develop candidate gene markers for fi ber quality and yield on the basis of approximately 90 genes implicated in fi ber development. Polymorphisms using sequence-tagged site (STS) and single nucleotide polymorphism (SNP) markers based on single strand conformation polymorphism (SSCP) and cleaved amplifi ed polymorphism (CAP) were evaluated among three upland and fi ve Pima cotton (G. barbadense L.) genotypes. Of the 90 primer pairs, 75 resulted in polymerase chain reaction amplifi cations, including 11 that yielded polymorphic STS markers. Of the 48 primer pairs that produced polymorphic SSCP markers, 27 yielded interspecifi c polymorphism, while 15 yielded both inter- and intraspecifi c polymorphisms. Six pairs yielded only intraspecifi c polymorphisms. A total of 18 SNPs, including four indels, were identifi ed in seven of the 15 fi ber gene fragments on the basis of direct DNA sequencing, and the average length was 350 bp, with a mean of 1.3 SNPs per fragment. The average rate of SNPs per nucleotide was 0.34%, and 0.31% and 0.41% in coding and noncoding regions, respectively. Eight of the 15 SNPs were interspecifi c and 78% were nucleotide substitutions, with the four indels contributing to interspecifi c polymorphism. Six selected SNPs were confi rmed by restriction enzyme digestion. The high level of SSCP polymorphism observed within a selected set of agronomically improved lines of upland cotton suggests that the use of SSCP will greatly facilitate genomewide mapping in upland cotton.

Molecular markers are playing an increasingly important role in map construction, QTL analysis, gene mapping and marker-assisted selection. Researchers hope the target gene and locus are as close as possible, one locus can present one gene, or linked with some important trait, then, individuals with useful trait can be selected through molecular markers se-lecting, and it’s the functional molecular marker. PCR-based molecular markers such as RAPD, SSR, AFLP amplified non-coding regions, or the whole genome randomly, the locus is far away from the gene of targeted trait, this limit the ap-plication of these molecular markers. This study established a kind of functional molecular markers based on intron of gene sequence, trying to link loci with gene sequence to achieve the purpose of its function. It used the conservative consistent sequence of intron splicing sites as its core sequence of amplification. ISAP is a PCR-based marker system, it has two kinds of primers: forward primer and reverse primer, both primers are 18 bases. Any of the primers can be used to construct a primer combination with the other kind of primers. Seventeen primers, 9 forward and 8 reverse, were used to construct 72 primer combinations, 67 of them showed polymorphism in a G. hirsutum cv. CCRI36 × G. barbadense cv. H7124 F2 popu-lation and a total of 212 loci were obtained. Together with 164 SRAP loci, these 212 loci were used to construct a genetic linkage map. ISAP markers distributed evenly in the entire linkage group, part of the region had a high saturation, might be the coding sequence-rich region. Sequencing results of 20 fragments showed that 85% of the sequences announced homol-ogy with published EST sequence stored in the NCBI which indicated that they were amplified adjacent to expressed se-quences. These results showed that ISAP marker system was simple, efficient, reliable, and had a relatively high polymor-phism, furthermore, it directly targeted gene sequence, was a functional molecular marker system. ISAP was also used to amplify other plants and good results were achieved.

Despite rapidly decreasing costs and innovative technologies, sequencing of angiosperm genomes is not yet undertaken lightly. Generating larger amounts of sequence data more quickly does not address the difficulties of sequencing and assembling complex genomes de novo. The cotton ( Gossypium spp.)

When alien DNA inserts into the cotton genome in a multicopy manner, several quantitative trait loci (QTLs) in the cotton genome are disrupted; these are called dQTL in this study. A transgenic mutant line is near-isogenic to its recipient, which is divergent for the dQTL from the remaining QTLs. Therefore, a set of data from a transgenic QTL line mutated by Agrobacterium-mediated transformation (30074), its recipient and their F1 hybrids, and three elite lines were analyzed under a modified additive-dominance model with genotype × environment interactions in three different environments to separate the genetic effects due to dQTL from whole-genome effects. Our result showed that dQTL had significant additive effects on lint percentage, boll weight, and boll number per square meter, while it had little genetic association with fiber traits, seed cotton yield, and lint yield. The dQTL in 30074 significantly increased lint percentage and boll number, while significantly decreasing boll weight, having little effect on fibre traits, while those from the recipient and three elite lines showed significant genetic effects on lint percentage. In addition, the remaining QTL other than dQTL had significant additive effects on seed cotton yield, fruiting branch number, uniformity index, micronaire, and short fibre index, and significant dominance effects on seed cotton yield, lint yield, and boll number per square meter. The additive and dominance effects under homozygous and heterozygous conditions for each line are also predicted in this study.

A high-density linkage map was constructed for an F2 population derived from an interspecific cross of cultivated allotetraploid species between Gossypium hirsutum L. and G. barbadense L. A total of 186 F2 individuals from the interspecific cross of "CRI 36 × Hai 7124" were genotyped at 1 252 polymorphic loci including a novel marker system, target region amplification polymorphism (TRAP). The map consists of 1 097 markers, including 697 simple se- quence repeats (SSRs), 171 TRAPs, 129 sequence-related amplified polymorphisms, 98 amplified fragment length polymorphisms, and two morphological markers, and spanned 4 536.7 cM with an average genetic distance of 4.1 cM per marker. Using 45 duplicated SSR loci among chromosomes, 11 of the 13 pairs of homologous chromosomes were identified in tetraploid cotton. This map will provide an essential resource for high resolution mapping of quantitative trait loci and molecular breeding in cotton.

Disease resistance (R) genes have been isolated from many plant species and R genes with domains of nucleotide binding sites (NBS) and leucine-rich repeats (LRR) represent the largest R gene family. The objective of this investigation was to test a resistance gene analog (RGA) anchored marker system, called amplified fragment length polymorphism (AFLP)-RGA in cotton (Gossypium spp.). The AFLP-RGA analysis uses one degenerate RGA primer designed from various NBS and LRR domains of R genes in combination with one selective AFLP primer in a PCR reaction. Out of a total of 446 AFLP-RGA bands amplified by 22 AFLP-RGA primer combinations, 76 (17.0%) and 37 (8.3%) were polymorphic within four G. hirsutum L. genotypes and four G. barbadense L. cotton genotypes, respectively. The number of polymorphic AFLP-RGA bands (256) between G. hirsutum and G. barbadense was much higher (57.4%). This level of polymorphism mirrors that of AFLP. The genetic similarity among the eight genotypes based on AFLP-RGA or AFLP lead to similar results in genotype grouping at the species and intraspecies level. However, RGA markers amplified by only degenerate RGA primers could not discriminate several genotypes. AFLP-RGA offers a great flexibility for numerous primer combinations in a genome-wide search for RGAs. Due to the distribution of RGAs or RGA clusters in the plant genome, genome-wide AFLP-RGA analysis provides a useful resource for candidate gene mapping of R genes for disease resistance in cotton.

Upland cotton (Gossypium hirsutum L.) is the most important fiber crop, and its lint-yield improvement is impeded due to its narrow genetic base and the lack of understanding of the genetic basis of yield. Backcross inbred lines (BILs) or near-isogenic lines (NILs) in the same genetic background differing in lint yield, developed through advanced backcrossing, provide an important genomic resource to study the molecular genetic basis of lint yield. In the present study, a high-yield (HY) group and a low-yield (LY) group each with three BILs were selected from a BIL population between G. hirsutum and G. barbadense. Using a microarray-based comparative transcriptome analysis on developing fibers at 10 days post-anthesis (DPA) between the two groups, 1486 differentially expressed genes (DEGs) were identified. A total of 212 DEGs were further mapped in the regions of 24 yield QTL and 11 yield trait QTL hotspots as reported previously, and 81 DEGs mapped with the 7 lint-yield QTL identified in the BIL population from which the two sets of BILs were selected. Gene Ontology annotations and Blast-Mapping-Annotation-KEGG analysis via Blast2GO revealed that more DEGs were associated with catalytic activity and binding, followed by transporters, nucleic acid binding transcription factors, structural molecules and molecular transducer activities. Six DEGs were chosen for a quantitative RT-PCR assay, and the results were consistent with the microarray analysis. The development of DEGs-based markers revealed that 7 single strand conformation polymorphism-based single nucleotide polymorphic (SSCP-SNP) markers were associated with yield traits, and 3 markers with lint yield. In the present study, we identified a number of yield and yield component QTL-co-localizing DEGs and developed several DEG-based SSCP-SNP markers for the traits, thereby providing a set of candidate genes for molecular breeding and genetic manipulation of lint yield in cotton.

Phytochrome system perceives the reduction in the ratio of red to far-red light when plants are grown under dense canopy. This signal, regarded as a warning of competition, will trigger a series of phenotypic changes to avoid shade. Progress has been made for several phytochrome signaling intermediates acting as positive regulators of accelerated elongation growth and promotion of flowering in shade-avoidance has been identified. Recently, a FPF1 homolog GhFPF1 was identified in upland cotton. Our data supported that transgenic Arabidopsis of over-expressing GhFPF1 displayed a constitutive shade-avoiding phenotype resembling phyB mutants in several respects such as accelerated elongation of hypocotyl and petioles, upward of leaf movement, and promoted flowering. In this addendum, by dissection of GhFPF1 acting as a component of shade-avoidance responses we suppose that GhFPF1 might influence the timing of the floral transition independently of shade-mediated early flowering. Furthermore, the opposite changes of IAA content in transgenic leaves and stems suggested that alteration of IAA storage and release took place during shade-avoidance responses.

In order to unravel the biochemical pathways and understand the molecular mechanisms involved in leaf senescence, suppression subtractive hybridization (SSH) was used to generate a cDNA library enriched for transcripts differentially expressed in developmental senescence cotyledons of upland cotton. After differential screening by membrane-based hybridization and subsequent confirmation by reverse Northern blot analysis, selected 678 clones were sequenced and analyzed. Sequencing of these cDNA fragments reveals that 216 of expressed sequence tags (ESTs) represented unique genes. Of these 216 cDNAs, 151 clones (69.9%) show significant homologies to previously known genes, while the remaining 65 do not match any known sequences. 151 unique ESTs are assigned to twelve different categories based on their putative functions generated by BLAST analysis. These SAG-encoded proteins are likely to participate in macromolecule degradation, nutrient recycling, detoxification of oxidative metabolites, and signaling and regulatory events. The expression pattern of selection of genes was confirmed using northern hybridization. Northern hybridization confirmed several distinct patterns, from expression at a very early stage to the terminal phase of the senescence syndrome. Clones encoding proteases and proteins involved in macromolecule degradation and gluconeogenesis, as well as stress-related genes, are up regulated in senescence cotyledons.

Premature leaf senescence occurs in the ultimate phase of the plant, and it occurs through a complex series of actions regulated by stress, hormones and genes. In this study, a proteomic analysis was performed to analyze the factors that could induce premature leaf senescence in two cotton cultivars. We successfully identified 443 differential abundant proteins (DAPs) from 7388 high-confidence proteins at four stages between non-premature senescence (NS) and premature senescence (PS), among which 158 proteins were over-accumulated, 238 proteins were down-accumulated at four stages, and 47 proteins displayed overlapped accumulation. All the DAPs were mapped onto 21 different categories on the basis of a Clusters of Orthologous Groups (COG) analysis, and 9 clusters were based on accumulation. Gene Ontology (GO) enrichment results show that processes related to stress responses, including responses to cold temperatures and responses to hormones, are significantly differentially accumulated. More importantly, the enriched proteins were mapped in The Arabidopsis Information Resource (TAIR), showing that 58 proteins play an active role in abiotic stress, hormone signaling and leaf senescence. Among these proteins, 26 cold-responsive proteins (CRPs) are significantly differentially accumulated. The meteorological data showed that the median temperatures declined at approximately 15 days before the onset of aging, suggesting that a decrease in temperature is tightly linked to an onset of cotton leaf senescence. Because accumulations of H₂O₂ and increased jasmonic acid (JA) were detected during PS, we speculate that two pathways associated with JA and H₂O₂ are closely related to premature leaf senescence in cotton.

Genetic architecture of branch traits has large influences on the morphological structure, photosynthetic capacity, planting density, and yield of Upland cotton (Gossypium hirsutum L.). This research aims to reveal the genetic effects of six branch traits, including bottom fruit branch node number (BFBNN), bottom fruit branch length (BFBL), middle fruit branch node number (MFBNN), middle fruit branch length (MFBL), upper fruit branch node number (UFBNN), and upper fruit branch length (UFBL). Association mapping was conducted for these traits of 39 lines and their 178 F1 hybrids in three environments. There were 20 highly significant Quantitative Trait SSRs (QTSs) detected by mixed linear model approach analyzing a full genetic model with genetic effects of additive, dominance, epistasis and their environment interaction. The phenotypic variation explained by genetic effects ranged from 32.64 ~ 91.61%, suggesting these branch traits largely influenced by genetic factors.

Background Male sterility is a common phenomenon in flowering plant species, and it has been successfully developed in several crops by taking advantage of heterosis. Using space mutation breeding of upland cotton, a novel photosensitive genetic male sterile (PGMS) mutant was isolated. To take advantage of the PGMS lines in cotton hybrid breeding, it is of great importance to study the molecular mechanisms of its male sterility. Results Delayed degradation of the PGMS anther tapetum occurred at different developmental stages as shown by analysis of anther cross-sections. To gain detailed insights into the cellular defects that occurred during PGMS pollen development, we used a differential proteomic approach to investigate the protein profiles of mutant and wild-type anthers at the tetrad, uninucleate and binucleate pollen stages. This approach identified 62 differentially expressed protein spots, including 19 associated with energy and metabolic pathways, 7 involved with pollen tube growth, 5 involved with protein metabolism, and 4 involved with pollen wall development. The remaining 27 protein spots were classified into other functional processes, such as protein folding and assembly (5 spots), and stress defense (4 spots). These differentially expressed proteins strikingly affected pollen development in the PGMS mutant anther and resulted in abnormal pollen grain formation, which may be the key reason for its male sterility. Conclusions This work represents the first study using comparative proteomics between fertile and PGMS cotton plants to identify PGMS-related proteins. The results demonstrate the presence of a complicated metabolic network in anther development and advance our understanding of the molecular mechanisms of microgamete formation, providing insights into the molecular mechanisms of male sterility. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0390-4) contains supplementary material, which is available to authorized users.

Salinization usually plays a primary role in soil degradation, which consequently reduces agricultural productivity. In this study, the effects of salinity on growth parameters, ion, chlorophyll, and proline content, photosynthesis, antioxidant enzyme activities, and lipid peroxidation of two cotton cultivars, [CCRI-79 (salt tolerant) and Simian 3 (salt sensitive)], were evaluated. Salinity was investigated at 0 mM, 80 mM, 160 mM, and 240 mM NaCl for 7 days. Salinity induced morphological and physiological changes, including a reduction in the dry weight of leaves and roots, root length, root volume, average root diameter, chlorophyll and proline contents, net photosynthesis and stomatal conductance. In addition, salinity caused ion imbalance in plants as shown by higher Na+ and Cl- contents and lower K+, Ca2+, and Mg2+ concentrations. Ion imbalance was more pronounced in CCRI-79 than in Simian3. In the leaves and roots of the salt-tolerant cultivar CCRI-79, increasing levels of salinity increased the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), but reduced catalase (CAT) activity. The activities of SOD, CAT, APX, and GR in the leaves and roots of CCRI-79 were higher than those in Simian 3. CAT and APX showed the greatest H2O2 scavenging activity in both leaves and roots. Moreover, CAT and APX activities in conjunction with SOD seem to play an essential protective role in the scavenging process. These results indicate that CCRI-79 has a more effective protection mechanism and mitigated oxidative stress and lipid peroxidation by maintaining higher antioxidant activities than those in Simian 3. Overall, the chlorophyll a, chlorophyll b, and Chl (a+b) contents, net photosynthetic rate and stomatal conductance, SOD, CAT, APX, and GR activities showed the most significant variation between the two cotton cultivars.

Background Leaf senescence is an important developmental programmed degeneration process that dramatically affects crop quality and yield. The regulation of senescence is highly complex. Although senescence regulatory genes have been well characterized in model species such as Arabidopsis and rice, there is little information on the control of this process in cotton. Here, the senescence process in cotton (Gossypium hirsutum L.) leaves was investigated over a time course including young leaf, mature leaf and leaf samples from different senescence stages using RNA-Seq. Results Of 24,846 genes detected by mapping the tags to Gossypium genomes, 3,624 genes were identified as differentially expressed during leaf senescence. There was some overlap between the genes identified here and senescence-associated genes previously identified in other species. Most of the genes related to photosynthesis, chlorophyll metabolism and carbon fixation were downregulated; whereas those for plant hormone signal transduction were upregulated. Quantitative real-time PCR was used to evaluate the results of RNA-Seq for gene expression profiles. Furthermore, 519 differentially expressed transcription factors were identified, notably WRKY, bHLH and C3H. In addition, 960 genes involved in the metabolism and regulation of eight hormones were identified, of which many genes involved in the abscisic acid, brassinosteroid, jasmonic acid, salicylic acid and ethylene pathways were upregulated, indicating that these hormone-related genes might play crucial roles in cotton leaf development and senescence. However, most auxin, cytokinin and gibberellin pathway-related genes were downregulated, suggesting that these three hormones may act as negative regulators of senescence. Conclusions This is the first high-resolution, multiple time-course, genome-wide comprehensive analysis of gene expression in cotton. These data are the most comprehensive dataset currently available for cotton leaf senescence, and will serve as a useful resource for unraveling the functions of many specific genes involved in cotton leaf development and senescence. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0433-5) contains supplementary material, which is available to authorized users.

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.

Knowledge of phosphorylation events and their regulation is crucial to understanding the functional biology of plant proteins, but very little is currently known about nitric oxide–responsive phosphorylation in plants. Here, we report the first large-scale, quantitative phosphoproteome analysis of cotton (Gossypium hirsutum) treated with sodium nitroprusside (nitric oxide donor) by utilizing the isobaric tag for relative and absolute quantitation (iTRAQ) method. A total of 1315 unique phosphopeptides, spanning 1528 non-redundant phosphorylation sites, were detected from 1020 cotton phosphoproteins. Among them, 183 phosphopeptides corresponding to 167 phosphoproteins were found to be differentially phosphorylated in response to sodium nitroprusside. Several of the phosphorylation sites that we identified, including RQxS, DSxE, TxxxxSP and SPxT, have not, to our knowledge, been reported to be protein kinase sites in other species. The phosphoproteins identified are involved in a wide range of cellular processes, including signal transduction, RNA metabolism, intracellular transport and so on. This study reveals unique features of the cotton phosphoproteome and provides new insight into the biochemical pathways that are regulated by nitric oxide.

WRKY proteins are major transcription factors involved in regulating plant growth and development. Although many studies have focused on the functional identification of WRKY genes, our knowledge concerning many areas of WRKY gene biology is limited. For example, in cotton, the phylogenetic characteristics, global expression patterns, molecular mechanisms regulating expression, and target genes/pathways of WRKY genes are poorly characterized. Therefore, in this study, we present a genome-wide analysis of the WRKY gene family in cotton (Gossypium raimondii and Gossypium hirsutum). We identified 116 WRKY genes in G. raimondii from the completed genome sequence, and we cloned 102 WRKY genes in G. hirsutum. Chromosomal location analysis indicated that WRKY genes in G. raimondii evolved mainly from segmental duplication followed by tandem amplifications. Phylogenetic analysis of alga, bryophyte, lycophyta, monocot and eudicot WRKY domains revealed family member expansion with increasing complexity of the plant body. Microarray, expression profiling and qRT-PCR data revealed that WRKY genes in G. hirsutum may regulate the development of fibers, anthers, tissues (roots, stems, leaves and embryos), and are involved in the response to stresses. Expression analysis showed that most group II and III GhWRKY genes are highly expressed under diverse stresses. Group I members, representing the ancestral form, seem to be insensitive to abiotic stress, with low expression divergence. Our results indicate that cotton WRKY genes might have evolved by adaptive duplication, leading to sensitivity to diverse stresses. This study provides fundamental information to inform further analysis and understanding of WRKY gene functions in cotton species.

Extensive studies on floral transition in model species have revealed a network of regulatory interactions between proteins that transduce and integrate developmental and environmental signals to promote or inhibit the transition to flowering. Previous studies indicated FLOWERING PROMOTING FACTOR 1 (FPF1) gene was involved in the promotion of flowering, but the molecular mechanism was still unclear. Here, FPF1 homologous sequences were screened from diploid Gossypium raimondii L. (D-genome, n = 13) and Gossypium arboreum L. genome (A-genome, n = 13) databases. Orthologous genes from the two species were compared, suggesting that distinctions at nucleic acid and amino acid levels were not equivalent because of codon degeneracy. Six FPF1 homologous genes were identified from the cultivated allotetraploid Gossypium hirsutum L. (AD-genome, n = 26). Analysis of relative transcripts of the six genes in different tissues revealed that this gene family displayed strong tissue-specific expression. GhFPF1, encoding a 12.0-kDa protein (Accession No: KC832319) exerted more transcripts in floral apices of short-season cotton, hinting that it could be involved in floral regulation. Significantly activated APETALA 1 and suppressed FLOWERING LOCUS C expression were induced by over-expression of GhFPF1 in the Arabidopsis Columbia-0 ecotype. In addition, transgenic Arabidopsis displayed a constitutive shade-avoiding phenotype that is characterized by long hypocotyls and petioles, reduced chlorophyll content, and early flowering. We propose that GhFPF1 may be involved in flowering time control and shade-avoidance responses.