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Cotton architecture is determined by the differentiation fate transition of axillary meristem (AM), and influences cotton yield and the efficiency of mechanized harvesting. We observed that the initiation of flowering primordium was earlier in early-maturing than that in late-maturing cultivars during the differentiation and development of AM. The RNA-Seq and expression level analyses showed that genes FLAVIN BINDING, KELCH REPEAT, F-BOX! (GhFKFl), and GIGANTEA (GhGI) were in response to circadian rhythms, and involved in the regulation of cotton flowering. The gene structure, predicted protein structure, and motif content analyses showed that in Arabidopsis, cotton, rapseed, and soybean, proteins GhFKF1 and GhGI were functionally conserved and share evolutionary origins. Compared to the wild type, in GhFKFl mutants that were created by the CRISPR/Cas9 system, the initiation of branch primordium was inhibited. Conversely, the knocking out of GhGI increased the number of AM differentiating into flower primordium, and there were much more lateral branch differentiation and development. Besides, we investigated that proteins GhFKF1 and GhGI can interact with each other. These results sug-gest that GhFKF1 and GhGI are key regulators of cotton architecture development, and may collaborate to regulate the differentiation fate transition of AM, ultimately influencing plant architecture. We describe a strategy for using the CRISPR/Cas9 system to increase cotton adaptation and productivity by optimizing plant architecture. [ABSTRACT FROM AUTHOR]

Cytochrome P450 proteins belong to one of the largest families of enzyme proteins in plants and play important roles in plant growth and development and the stress response. In our previous studies, a cytochrome P450 gene, GhCYP94C1 (cytochrome P450 94C1), was functionally characterized as a positive regulator of seed germination, main root elongation and early flowering. However, whether the gene has other potential functions remains to be further explored. In our study, expression analysis showed that GhCYP94C1 was highly expressed in roots and was suppressed by drought treatment. Endogenous silencing of GhCYP94C1 via virus-induced gene silencing (VIGS) increased drought resistance in cotton plants, which was accompanied by the upregulated expression of the abscisic acid (ABA) biosynthesis gene nine-cis-epoxycarotenoid dioxygenase 9 (GhNCED9) during drought stress. Our findings suggested that GhCYP94C1 may play an important role in drought resistance. Combined with previous research results, the present results provide a theoretical basis for future breeding of new cotton varieties with early maturation and drought resistance. [ABSTRACT FROM AUTHOR]

Seed vigor (SV) is a crucial trait determining the quality of crop seeds. Currently, over 80% of China's cotton-planting area is in Xinjiang Province, where a fully mechanized planting model is adopted, accounting for more than 90% of the total fiber production. Therefore, identifying SV-related loci and genes is crucial for improving cotton yield in Xinjiang. In this study, three seed vigor-related traits, including germination potential, germination rate, and germination index, were investigated across three environments in a panel of 355 diverse accessions based on 2,261,854 high-quality single-nucleotide polymorphisms (SNPs). A total of 26 significant SNPs were detected and divided into six quantitative trait locus regions, including 121 predicted candidate genes. By combining gene expression, gene annotation, and haplotype analysis, two novel candidate genes (Ghir_A09G002730 and Ghir_D03G009280) within qGR-A09-1 and qGI/GP/GR-D03-3 were associated with vigor-related traits, and Ghir_A09G002730 was found to be involved in artificial selection during cotton breeding by population genetic analysis. Thus, understanding the genetic mechanisms underlying seed vigor-related traits in cotton could help increase the efficiency of direct seeding by molecular marker-assisted selection breeding. [ABSTRACT FROM AUTHOR]

Calcium-Dependent Protein Kinases (CDPKs) involved in regulating downstream components of calcium signaling pathways play a role in tolerance to abiotic stresses and seed development in plants. However, functions of only a few cotton CDPKs have been clarified at present. In this study, 80 conserved CDPKs in Gossypium hirsutum L. were identified and characterized, which was divided into four subgroups. Among them, the transcript level of GhCDPK60 was significantly upregulated under drought and several hormone treatments. And we found that the expression levels of several stress-inducible genes down-regulated in GhCDPK60-silence cotton and up-regulated in GhCDPK60-overexpressing Arabidopsis. In addition, physiological analyses demonstrated that GhCDPK60 improved drought stress tolerance by improving the osmotic adjustment ability and reducing the accumulation of reactive oxygen species (ROS) in plants. These findings broaden our understanding of the biological roles of GhCDPK60 and mechanisms underlying drought stress tolerance in cotton. [ABSTRACT FROM AUTHOR]

Background: Small auxin-up RNA (SAUR) gene family is the largest family of early auxin response genes in higher plants, which have been implicated in the regulation of multiple biological processes. However, no comprehensive analysis of SAUR genes has been reported in cotton (Gossypium spp.). Results: In the study, we identified 145, 97, 214, and 176 SAUR homologous genes in the sequenced genomes of G. raimondii, G. arboreum, G. hirsutum, and G. barbadense, respectively. A phylogenetic analysis revealed that the SAUR genes can be classified into 10 groups. A further analysis of chromosomal locations and gene duplications showed that tandem duplication and segmental duplication events contributed to the expansion of the SAUR gene family in cotton. An exon-intron organization and motif analysis revealed the conservation of SAUR-specific domains, and the auxin responsive elements existed in most of the upstream sequences. The expression levels of 16 GhSAUR genes in response to an exogenous application of IAA were determined by a quantitative RT-PCR analysis. The genome-wide RNA-seq data and qRT-PCR analysis of selected SAUR genes in developing fibers revealed their differential expressions. The physical mapping showed that 20 SAUR genes were co-localized with fiber length quantitative trait locus (QTL) hotspots. Single nucleotide polymorphisms (SNPs) were detected for 12 of these 20 genes between G. hirsutum and G. barbadense, but no SNPs were identified between two backcross inbred lines with differing fiber lengths derived from a cross between the two cultivated tetraploids. Conclusions: This study provides an important piece of genomic information for the SAUR genes in cotton and lays a solid foundation for elucidating the functions of SAUR genes in auxin signaling pathways to regulate cotton growth. [ABSTRACT FROM AUTHOR]

Plant-specific NAC proteins comprise one of the largest transcription factor families in plants and play important roles in plant development and the stress response. Gossypium hirsutum L. is a major source of fiber, but its growth and productivity are limited by many biotic and abiotic stresses. In this study, the NAC domain gene GhNAC79 was functionally characterized in detail, and according to information about the cotton genome sequences, it was located on scaffold42.1, containing three exons and two introns. Promoter analysis indicated that the GhNAC79 promoter contained both basic and stress-related elements, and it was especially expressed in the cotyledon of Arabidopsis. A transactivation assay in yeast demonstrated that GhNAC79 was a transcription activator, and its activation domain was located at its C-terminus. The results of qRT-PCR proved that GhNAC79 was preferentially expressed at later stages of cotyledon and fiber development, and it showed high sensitivity to ethylene and meJA treatments. Overexpression of GhNAC79 resulted in an early flowering phenotype in Arabidopsis, and it also improved drought tolerance in both Arabidopsis and cotton. Furthermore, VIGS-induced silencing of GhNAC79 in cotton led to a drought-sensitive phenotype. In summary, GhNAC79 positively regulates drought stress, and it also responds to ethylene and meJA treatments, making it a candidate gene for stress studies in cotton. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Cotton (Gossypium spp.) fibers are single-celled elongated trichomes, the molecular aspects of genetic variation in fiber length (FL) among genotypes are currently unknown. In this study, two backcross inbred lines (BILs), i. e., NMGA-062 ("Long") and NMGA-105 ("Short") with 32.1 vs. 27.2 mm in FL, respectively, were chosen to perform RNA-Seq on developing fibers at 10 days post anthesis (DPA). The two BILs differed in 4 quantitative trait loci (QTL) for FL and were developed from backcrosses between G. hirsutum as the recurrent parent and G. barbadense. Results: In total, 51.7 and 54.3 million reads were obtained and assembled to 49,508 and 49,448 transcripts in the two genotypes, respectively. Of 1551 differentially expressed genes (DEGs) between the two BILs, 678 were up-regulated and 873 down-regulated in "Long"; and 703 SNPs were identified in 339 DEGs. Further physical mapping showed that 8 DEGs were co-localized with the 4 FL QTL identified in the BIL population containing the two BILs. Four SNP markers in 3 DEGs that showed significant correlations with FL were developed. Among the three candidate genes encoding for proline-rich protein, D-cysteine desulfhydrase, and thaumatin-like protein, a SNP of thaumatin-like protein gene showed consistent correlations with FL across all testing environments. Conclusions: This study represents one of the first investigations of positional candidate gene approach of QTL in cotton in integrating transcriptome and SNP identification based on RNA-Seq with linkage and physical mapping of QTL and genes, which will facilitate eventual cloning and identification of genes responsible for FL QTL. The candidate genes may serve as the foundation for further in-depth studies of the molecular mechanism of natural variation in fiber elongation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Gossypium hirsutum L., or upland cotton, is an important renewable resource for textile fiber. To enhance understanding of the genetic basis of cotton earliness, we constructed an intra-specific recombinant inbred line population (RIL) containing 137 lines, and performed linkage map construction and quantitative trait locus (QTL) mapping. Results: Using restriction-site associated DNA sequencing, a genetic map composed of 6,434 loci, including 6,295 single nucleotide polymorphisms and 139 simple sequence repeat loci, was developed from RIL population. This map spanned 4,071.98 cM, with an average distance of 0.63 cM between adjacent markers. A total of 247 QTLs for six earliness-related traits were detected in 6 consecutive years. In addition, 55 QTL coincidence regions representing more than 60 % of total QTLs were found on 22 chromosomes, which indicated that several earliness-related traits might be simultaneously improved. Fine-mapping of a 2-Mb region on chromosome D3 associated with five stable QTLs between Marker25958 and Marker25963 revealed that lines containing alleles derived from CCRI36 in this region exhibited smaller phenotypes and earlier maturity. One candidate gene (EMF2) was predicted and validated by quantitative real-time PCR in early-, medium- and late-maturing cultivars from 3- to 6-leaf stages, with highest expression level in early-maturing cultivar, CCRI74, lowest expression level in late-maturing cultivar, Bomian1. Conclusions: We developed an SNP-based genetic map, and this map is the first high-density genetic map for short-season cotton and has the potential to provide deeper insights into earliness. Cotton earliness-related QTLs and QTL coincidence regions will provide useful materials for QTL fine mapping, gene positional cloning and MAS. And the gene, EMF2, is promising for further study. [ABSTRACT FROM AUTHOR]

Background: Early maturity is one of the most important and complex agronomic traits in upland cotton (Gossypium hirsutum L). To dissect the genetic architecture of this agronomically important trait, a population consisting of 355 upland cotton germplasm accessions was genotyped using the specific-locus amplified fragment sequencing (SLAF-seq) approach, of which a subset of 185 lines representative of the diversity among the accessions was phenotypically characterized for six early maturity traits in four environments. A genome-wide association study (GWAS) was conducted using the generalized linear model (GLM) and mixed linear model (MLM). Results: A total of 81,675 SNPs in 355 upland cotton accessions were discovered using SLAF-seq and were subsequently used in GWAS. Thirteen significant associations between eight SNP loci and five early maturity traits were successfully identified using the GLM and MLM; two of the 13 associations were common between the models. By computing phenotypic effect values for the associations detected at each locus, 11 highly favorable SNP alleles were identified for five early maturity traits. Moreover, dosage pyramiding effects of the highly favorable SNP alleles and significant linear correlations between the numbers of highly favorable alleles and the phenotypic values of the target traits were identified. Most importantly, a major locus (rs13562854) on chromosome Dt3 and a potential candidate gene (CotAD_01947) for early maturity were detected. Conclusions: This study identified highly favorable SNP alleles and candidate genes associated with early maturity traits in upland cotton. The results demonstrate that GWAS is a powerful tool for dissecting complex traits and identifying candidate genes. The highly favorable SNP alleles and candidate genes for early maturity traits identified in this study should be show high potential for improvement of early maturity in future cotton breeding programs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 occur 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. [ABSTRACT FROM AUTHOR]

Background The cotton (Gossypium spp.) fiber cell is an important unicellular model for studying cell differentiation. There is evidence suggesting that phosphorylation is a critical posttranslational modification involved in regulation of a wide range of cell activities. Nevertheless, the sites of phosphorylation in G. hirsutum and their regulatory roles in fiber cell initiation are largely unknown. In this study, we employed a mass spectrometry-based phosphoproteomics to conduct a global and site-specific phosphoproteome profiling between ovules of a fuzzless-lintless (fl) Upland cotton (G. hirsutum) mutant and its isogenic parental wild type (WT) at -3 and 0 days post-anthesis (DPA). Results A total of 830 phosphopeptides and 1,592 phosphorylation sites from 619 phosphoproteins were identified by iTRAQ (isobaric tags for relative and absolute quantitation). Of these, 76 phosphoproteins and 1,100 phosphorylation sites were identified for the first time after searching the P3DB public database using the BLAST program. Among the detected phosphopeptides, 69 were differentially expressed between the fl mutant and its WT in ovules at -3 and 0 DPA. An analysis using the Motif-X program uncovered 19 phosphorylation motifs, 8 of which were unique to cotton. A further metabolic pathway analysis revealed that the differentially phosphorylated proteins were involved in signal transduction, protein modification, carbohydrate metabolic processes, and cell cycle and cell proliferation. Conclusions Our phosphoproteomics-based research provides the first global overview of phosphorylation during cotton fiber initiation, and also offers a helpful dataset for elucidation of signaling networks in fiber development of G. hirsutum. [ABSTRACT FROM AUTHOR]

Background: Genetic male sterility (GMS) in cotton (Gossypium hirsutum) plays an important role in the utilization of hybrid vigor. However, the molecular mechanism of the GMS is still unclear. While numerous studies have demonstrated that microRNAs (miRNA) regulate flower and anther development, whether different small RNA regulations exist in GMS and its wild type is unclear. A deep sequencing approach was used to investigate the global expression and complexity of small RNAs during cotton anther development in this study. Results: Three small RNA libraries were constructed from the anthers of three development stages each from fertile wild type (WT) and its GMS mutant cotton, resulting in nearly 80 million sequence reads. The total number of miRNAs and short interfering RNAs in the three WT libraries was significantly greater than that in the corresponding three mutant libraries. Sixteen conserved miRNA families were identified, four of which comprised the vast majority of the expressed miRNAs during anther development. In addition, six conserved miRNA families were significantly differentially expressed during anther development between the GMS mutant and its WT. Conclusions: The present study is the first to deep sequence the small RNA population in G. hirsutum GMS mutant and its WT anthers. Our results reveal that the small RNA regulations in cotton GMS mutant anther development are distinct from those of the WT. Further results indicated that the differently expressed miRNAs regulated transcripts that were distinctly involved in anther development. Identification of a different set of miRNAs between the cotton GMS mutant and its WT will facilitate our understanding of the molecular mechanisms for male sterility. [ABSTRACT FROM AUTHOR]

Background: Previous transcriptome profiling studies have investigated the molecular mechanisms of pollen and anther development, and identified many genes involved in these processes. However, only 51 anther ESTs of Upland cotton (Gossypium hirsutum) were found in NCBI and there have been no reports of transcriptome profiling analyzing anther development in Upland cotton, a major fiber crop in the word. Methodology/Principal Finding: Ninety-eight hundred and ninety-six high quality ESTs were sequenced from their 39-ends and assembled into 6,643 unigenes from a normalized, full-length anther cDNA library of Upland cotton. Combined with previous sequenced anther-related ESTs, 12,244 unigenes were generated as the reference genes for digital gene expression (DGE) analysis. The DGE was conducted on anthers that were isolated at tetrad pollen (TTP), uninucleate pollen (UNP), binucleate pollen (BNP) and mature pollen (MTP) periods along with four other tissues, i.e., roots (RO), stems (ST), leaves (LV) and embryos (EB). Through transcriptome profiling analysis, we identified 1,165 genes that were enriched at certain anther development periods, and many of them were involved in starch and sucrose metabolism, pentose and glucuronate interconversion, flavonoid biosynthesis, and ascorbate and aldarate metabolism. Conclusions/Significance: We first generated a normalized, full-length cDNA library from anthers and performed transcriptome profiling analysis of anther development in Upland cotton. From these results, 10,178 anther expressed genes were identified, among which 1,165 genes were stage-enriched in anthers. And many of these stage-enriched genes were involved in some important processes regulating anther development. [ABSTRACT FROM AUTHOR]

Background: Upland cotton, Gossypium hirsutum L., is one of the world's most important economic crops. In the absence of the entire genomic sequence, a large number of expressed sequence tag (EST) resources of upland cotton have been generated and used in several studies. However, information about the flower development of this species is rare. Methodology/Principal Findings: To clarify the molecular mechanism of flower development in upland cotton, 22,915 high-quality ESTs were generated and assembled into 14,373 unique sequences consisting of 4,563 contigs and 9,810 singletons from a normalized and full-length cDNA library constructed from pooled RNA isolated from shoot apexes, squares, and flowers. Comparative analysis indicated that 5,352 unique sequences had no high-degree matches to the cotton public database. Functional annotation showed that several upland cotton homologs with flowering-related genes were identified in our library. The majority of these genes were specifically expressed in flowering-related tissues. Three GhSEP (G. hirsutum L. SEPALLATA) genes determining floral organ development were cloned, and quantitative real-time PCR (qRT-PCR) revealed that these genes were expressed preferentially in squares or flowers. Furthermore, 670 new putative microsatellites with flanking sequences sufficient for primer design were identified from the 645 unigenes. Twenty-five EST- simple sequence repeats were randomly selected for validation and transferability testing in 17 Gossypium species. Of these, 23 were identified as true-to-type simple sequence repeat loci and were highly transferable among Gossypium species. Conclusions/Significance: A high-quality, normalized, full-length cDNA library with a total of 14,373 unique ESTs was generated to provide sequence information for gene discovery and marker development related to upland cotton flower development. These EST resources form a valuable foundation for gene expression profiling analysis, functional analysis of newly discovered genes, genetic linkage, and quantitative trait loci analysis. [ABSTRACT FROM AUTHOR]

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, F and F were grown in field conditions for sources of leaf petioles as explants. The F 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 and Ec, while the LEC line CRI12 contains one dominant gene Ec. 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. 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. [ABSTRACT FROM AUTHOR]

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 fiber quality and yield on the basis of approximately 90 genes implicated in fiber development. Polymorphisms using sequence-tagged site (STS) and single nucleotide polymorphism (SNP) markers based on single strand conformation polymorphism (SSCP) and cleaved amplified polymorphism (CAP) were evaluated among three upland and five Pima cotton (G. barbadense L.) genotypes. Of the 90 primer pairs, 75 resulted in polymerase chain reaction amplifications, including 11 that yielded polymorphic STS markers. Of the 48 primer pairs that produced polymorphic SSCP markers, 27 yielded interspecific polymorphism, while 15 yielded both inter- and intraspecific polymorphisms. Six pairs yielded only intraspecific polymorphisms. A total of 18 SNPs, including four indels, were identified in seven of the 15 fiber 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 interspecific and 78% were nucleotide substitutions, with the four indels contributing to interspecific polymorphism. Six selected SNPs were confirmed 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. [ABSTRACT FROM AUTHOR]

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

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 (RCA) anchored marker system, called amplified fragment length polymorphism (AFLP)-RGA in cotton (Gossypiun, 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 RCA 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-RCA analysis provides a useful resource for candidate gene mapping of R genes for disease resistance in cotton. [ABSTRACT FROM AUTHOR]

In certain plant species including cotton ( Gossypium hirsutum L. or Gossypium barbadense L.), the level of amplified fragment length polymorphism (AFLP) is relatively low, limiting its utilization in the development of genome-wide linkage maps. We propose the use of frequent restriction enzymes in combination with AFLP to cleave the AFLP fragments, called cleaved AFLP analysis (cAFLP). Using four Upland cotton genotypes ( G. hirsutum) and three Pima cotton ( G. barbadense), we demonstrated that cAFLP generated 67% and 132% more polymorphic markers than AFLP in Upland and Pima cotton, respectively. This resulted in 15.5 and 25.5 polymorphic cAFLP markers per AFLP primer combination, as compared to 9.1 and 11.0 polymorphic AFLP. The cAFLP-based genetic similarity (GS) is generally lower than the AFLP-based GS, even though both marker systems are overall congruent. In some cases, cAFLP can better resolve genetic relationships between genotypes, rendering a higher discriminatory power. Given the high-resolution power of capillary-based DNA sequencing system, we further propose that AFLP and cAFLP amplicons from the same primer combination can be pooled as one sample before electrophoresis. The combination produced an average of 18.5 and 31.0 polymorphic markers per primer pair in Upland and Pima cotton, respectively. Using several restriction enzyme combinations before pre-selective amplification in combination with various frequent 4 bp-cutters or 6 bp-cutters after selective amplification, the pooled AFLP and cAFLP will provide unlimited number of polymorphic markers for genome-wide mapping and fingerprinting. [ABSTRACT FROM AUTHOR]