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Alternative splicing (AS) is a crucial post-transcriptional regulatory mechanism that contributes to proteome complexity and versatility in different plant species. However, detailed AS exploration in diploid cotton during fiber development has not been reported. In this study, we comparatively analyzed G. arboreum and G. raimondii AS events during fiber development using transcriptome data and identified 9690 and 7617 AS events that were distributed in 6483 and 4859 genes, respectively. G. arboreum had more AS genes and AS events than G. raimondii, and most AS genes were distributed at both ends of all 13 chromosomes in both diploid cotton species. Four major AS types, including IR, SE, A3SS, and A5SS, were all experimentally validated through RT-PCR assays. G. arboreum and G. raimondii had only 1888 AS genes in common, accounting for one-third and one-half of the total number of AS genes, respectively. Furthermore, we found a lysine-specific demethylase coding gene with a different AS mechanism in G. arboreum and G. raimondii, in which AS isoforms lacked part of a key conserved domain. Our findings may provide new directions for the discovery of functional genes involved in cotton species differentiation. [ABSTRACT FROM AUTHOR]

Alternative splicing (AS) is a crucial post-transcriptional regulatory mechanism that contributes to proteome complexity and versatility in different plant species. However, detailed AS exploration in diploid cotton during fiber development has not been reported. In this study, we comparatively analyzed G. arboreum and G. raimondii AS events during fiber development using transcriptome data and identified 9690 and 7617 AS events that were distributed in 6483 and 4859 genes, respectively. G. arboreum had more AS genes and AS events than G. raimondii, and most AS genes were distributed at both ends of all 13 chromosomes in both diploid cotton species. Four major AS types, including IR, SE, A3SS, and A5SS, were all experimentally validated through RT-PCR assays. G. arboreum and G. raimondii had only 1888 AS genes in common, accounting for one-third and one-half of the total number of AS genes, respectively. Furthermore, we found a lysine-specific demethylase coding gene with a different AS mechanism in G. arboreum and G. raimondii, in which AS isoforms lacked part of a key conserved domain. Our findings may provide new directions for the discovery of functional genes involved in cotton species differentiation.

The diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function as opposed to the allotetraploid cotton species. As a prerequisite for this, a reliable and efficient method of high frequency plant regeneration in G. arboreum must be established. In this study, callus was induced from hypocotyl, root and cotyledon of G. arboreum seedlings on MSB medium (MS salts and B5 vitamins) with 0.09 µM 2, 4-D (2, 4-dichlorophenoxyacetic acid) and 2.32 µM KT (kinetin). During propagation, callus was effectively selected for subculture on different media based on cell morphology to induce embryogenic callus and somatic embryos. Embryogenic callus was induced on MS5 medium (MSB, 37.59 mM KNO3, 62.47 µM NH4NO3, 3% (w/v) glucose, 6.8 mM glutamine, 3.8 mM asparagine) from suspended cultures after several cycles of alternate solid MS3 medium—liquid MS4 medium culture, a key step in somatic embryogenesis. Solid MS3 medium was supplemented with 2.46 µM IBA (īndole-3-butyric acid), 0.93 µM KT, 6.8 mM Gln, 3.8 mM Asn; liquid MS4 medium was supplemented with 0.1 g/L NaCl, 0.1 g/L KCl and 0.1 g/L CuSO4. The solid–liquid alternate culture was effective for embryogenic callus induced in G. arboreum. MS5 medium with maltose (3% w/v) or glucose (1.5% w/v) + maltose (1.5% w/v) performed better than that added single glucose (3% w/v) for somatic embryo maturation and germination. The regenerated plants with well-developed roots were directly transferred to the soil or grafted onto germinated cotton plants. Plant regeneration via somatic embryogenesis in diploid cultivated species was established, but there is a need for improvement and optimization for gene functional analysis and gene editing in the diploid cotton species. Key message: Regenerated plants of G. arboreum, a cultivated diploid cotton, were obtained via somatic embryogenesis by adjusting the culture mode and medium compositions. [ABSTRACT FROM AUTHOR]

Abstract Background Root systems are critical for plant growth and development. The Casparian strip in root systems is involved in stress resistance and maintaining homeostasis. Casparian strip membrane domain proteins (CASPs) are responsible for the formation of Casparian strips. Results To investigate the function of CASPs in cotton, we identified and characterized 48, 54, 91 and 94 CASPs from Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively, at the genome-wide level. However, only 29 common homologous CASP genes were detected in the four Gossypium species. A collinearity analysis revealed that whole genome duplication (WGD) was the primary reason for the expansion of the genes of the CASP family in the four cotton species. However, dispersed duplication could also contribute to the expansion of the GaCASPs gene family in the ancestors of G. arboreum. Phylogenetic analysis was used to cluster a total of 85 CASP genes from G. arboreum and Arabidopsis into six distinct groups, while the genetic structure and motifs of CASPs were conserved in the same group. Most GaCASPs were expressed in diverse tissues, with the exception of that five GaCASPs (Ga08G0113, Ga08G0114, Ga08G0116, Ga08G0117 and Ga08G0118) that were highly expressed in root tissues. Analyses of the tissue and subcellular localization suggested that GaCASP27 genes (Ga08G0117) are membrane protein genes located in the root. In the GaCASP27 silenced plants and the Arabidopsis mutants, the lateral root number significantly increased. Furthermore, GaMYB36, which is related to root development was found to regulate lateral root growth by targeting GaCASP27. Conclusions This study provides a fundamental understanding of the CASP gene family in cotton and demonstrates the regulatory role of GaCASP27 on lateral root growth and development.

Hypocotyl phototropism is mediated by the phototropins and plays a critical role in seedling morphogenesis by optimizing growth orientation. However, the mechanisms by which phototropism influences morphogenesis require additional study, especially for polyploid crops such as cotton. Here, we found that hypocotyl phototropism was weaker in Gossypium arboreum than in G. raimondii (two diploid cotton species), and LC-MS analysis indicated that G. arboreum hypocotyls had a higher content of abscisic acid (ABA) and a lower content of indole-3-acetic acid (IAA) and bioactive gibberellins (GAs). Consistently, the expression of ABA2 , AAO3 , and GA2OX1 was higher in G. arboreum than in G. raimondii , and that of GA3OX was lower; these changes promoted ABA synthesis and the transformation of active GA to inactive GA. Higher concentrations of ABA inhibited the asymmetric distribution of IAA across the hypocotyl and blocked the phototropic curvature of G. raimondii. Application of IAA or GA3 to the shaded and illuminated sides of the hypocotyl enhanced and inhibited phototropic curvature, respectively, in G. arboreum. The application of IAA, but not GA, to one side of the hypocotyl caused hypocotyl curvature in the dark. These results indicate that the asymmetric distribution of IAA promotes phototropic growth, and the weakened phototropic curvature of G. arboreum may be attributed to its higher ABA concentrations that inhibit the action of auxin, which is regulated by GA signaling. [ABSTRACT FROM AUTHOR]

Key message: Transcription factors (TFs) are the key regulators in plant development and stress adaptation. Understanding interactions of TFs in cotton crop is important for enhancing stress tolerance and yield enhancement. Among plant TFs, bZIPs plays a major role in seed germination, flower development, biotic and abiotic stress response. Most of the bZIP proteins from cotton remains uncharacterized and can be utilized for crop improvement. Bioinformatics analysis of bZIPs of cultivated cotton species G. hirsutum along with two sub-genome species G. arboreum and G. raimondii at one platform will certainly help the researchers in the selection of specific cotton bZIP genes according to the close alignment with Arabidopsis orthologs or sub-genome homolog for functional characterization. Cotton is an important commodity in the world economy. Transcription factors (TFs) are the key regulators in plant development and stress adaptation. Understanding interactions of TFs in cotton crop is important for enhancing stress tolerance and yield enhancement. Among plant TFs, bZIPs plays a major role in seed germination, flower development, biotic and abiotic stress response. Most of the bZIP proteins from cotton remains uncharacterized and can be utilized for crop improvement. In this study, we have carried out genome-wide identification and bioinformatics characterization of basic leucine zipper domain proteins (bZIPs) from cultivated cotton species Gossypium hirsutum along with two sub-genome species of allotetraploid cotton, Gossypium arboreum and Gossypium raimondii. A total of 228 bZIP genes of G. hirsutum, 91 bZIP genes of G. arboreum and 86 bZIP genes of G. raimondii were identified from CottonGen database. Cotton bZIP genes were annotated in standard pattern according to their match with Arabidopsis bZIPs. Multiple genes with similar bZIP designations were observed in cotton, linked to the gene duplication. Cotton bZIPs are distributed across all 13 chromosomes with varied density. Phylogenetic characterization of all three cotton species bZIPs classified them into 12 subfamilies, namely A, B, C, D, E, F, G, H, I, J, K and S and further into eight subgroups according to their predicted functional similarities, viz., A1, A2, A3, C1, C2, S1, S2 and S3. Subfamilies A and S are having maximum number of bZIP genes, subfamilies B, H, J and K are single-member families. Cotton bZIP protein functions were predicted from identified motifs and orthologs from varied species. Basic region leucine zipper (BRLZ) domain analysis of G. raimondii bZIPs revealed the presence of conserved basic region motif N-X7-R/K in almost all subfamily members, variants are GrbZIP62 with N-X7-I motif and GrbZIP76 with K-X7-R motif. Leucine heptad repeats motif, are also present in variant numbers from two to nine with leucine or other hydrophobic amino acid at designated position among 12 subfamily members. STRING protein interaction network analysis of G. raimondii bZIPs observed strong interaction between A and D, B and K, and C and S subfamily members. [ABSTRACT FROM AUTHOR]

Background: Root systems are critical for plant growth and development. The Casparian strip in root systems is involved in stress resistance and maintaining homeostasis. Casparian strip membrane domain proteins (CASPs) are responsible for the formation of Casparian strips. Results: To investigate the function of CASPs in cotton, we identified and characterized 48, 54, 91 and 94 CASPs from Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively, at the genome-wide level. However, only 29 common homologous CASP genes were detected in the four Gossypium species. A collinearity analysis revealed that whole genome duplication (WGD) was the primary reason for the expansion of the genes of the CASP family in the four cotton species. However, dispersed duplication could also contribute to the expansion of the GaCASPs gene family in the ancestors of G. arboreum. Phylogenetic analysis was used to cluster a total of 85 CASP genes from G. arboreum and Arabidopsis into six distinct groups, while the genetic structure and motifs of CASPs were conserved in the same group. Most GaCASPs were expressed in diverse tissues, with the exception of that five GaCASPs (Ga08G0113, Ga08G0114, Ga08G0116, Ga08G0117 and Ga08G0118) that were highly expressed in root tissues. Analyses of the tissue and subcellular localization suggested that GaCASP27 genes (Ga08G0117) are membrane protein genes located in the root. In the GaCASP27 silenced plants and the Arabidopsis mutants, the lateral root number significantly increased. Furthermore, GaMYB36, which is related to root development was found to regulate lateral root growth by targeting GaCASP27. Conclusions: This study provides a fundamental understanding of the CASP gene family in cotton and demonstrates the regulatory role of GaCASP27 on lateral root growth and development. [ABSTRACT FROM AUTHOR]

Summary: Recombination breaks up ancestral linkage disequilibrium, creates combinations of alleles, affects the efficiency of natural selection, and plays a major role in crop domestication and improvement. However, there is little knowledge regarding the variation in the population‐scaled recombination rate in cotton. We constructed recombination maps and characterized the difference in the genomic landscape of the population‐scaled recombination rate between Gossypium hirsutum and G. arboreum and sub‐genomes based on the 381 sequenced G. hirsutum and 215 G. arboreum accessions. Comparative genomics identified large structural variations and syntenic genes in the recombination regions, suggesting that recombination was related to structural variation and occurred preferentially in the distal chromosomal regions. Correlation analysis indicated that recombination was only slightly affected by geographical distribution and breeding period. A genome‐wide association study (GWAS) was performed with 15 agronomic traits using 267 cotton accessions and identified 163 quantitative trait loci (QTL) and an important candidate gene (Ghir_COL2) for early maturity traits. Comparative analysis of recombination and a GWAS revealed that the QTL of fibre quality traits tended to be more common in high‐recombination regions than were those of yield and early maturity traits. These results provide insights into the population‐scaled recombination landscape, suggesting that recombination contributed to the domestication and improvement of cotton, which provides a useful reference for studying recombination in other species. Significance Statement: There is little knowledge regarding the population‐scaled recombination rate variation in cotton. Genome‐wide fine‐scaled recombination maps were constructed and characterized the population‐scaled recombination rate in Gossypium hirsutum and G. arboreum in the present study; and GWAS revealed that agronomic trait QTL tended to be co‐located in high‐recombination regions, which revealed that recombination contributed to the domestication and genetic improvement of cotton. [ABSTRACT FROM AUTHOR]

Abiotic stress is an important limiting factor in crop growth and yield around the world. Owing to the continued genetic erosion of the upland cotton germplasm due to intense selection and inbreeding, attention has shifted towards wild cotton progenitors which offer unique traits that can be introgressed into the cultivated cotton to improve their genetic performance. The purpose of this study was to characterize the Pkinase gene family in a previously developed genetic map of the F2 population derived from a cross between two cotton species: Gossypium hirsutum (CCRI 12-4) and Gossypium darwinii (5-7). Based on phylogenetic analysis, Pkinase (PF00069) was found to be the dominant domain with 151 genes in three cotton species, categorized into 13 subfamilies. Structure analysis of G. hirsutum genes showed that a greater percentage of genes and their exons were highly conserved within the group. Syntenic analysis of gene blocks revealed 99 duplicated genes among G. hirsutum, Gossypium arboreum and Gossypium raimondii. Most of the genes were duplicated in segmental pattern. Expression pattern analysis showed that the Pkinase gene family possessed species-level variation in induction to salinity and G. darwinii had higher expression levels as compared to G. hirsutum. Based on RNA sequence analysis and preliminary RT-qPCR verification, we hypothesized that the Pkinase gene family, regulated by transcription factors (TFs) and miRNAs, might play key roles in salt stress tolerance. These findings inferred comprehensive information on possible structure and function of Pkinase gene family in cotton under salt stress.

Crosses were attempted between accessions belonging to G. hirsutum and G. arboreum to develop interspecific hybrids through in-ovulo embryo culture. A mixture of growth regulators (GA[sub 3] @ 50 ppm + NAA @ 100 ppm) was applied at the base of pedicel of crossed buds for three successive days starting from day of pollination. The ovules were excised and cultured three days after pollination on various tissue culture media. Of the 26,300 ovules cultured, 33 germinated with germination percentage of 0.12. Germination was observed in seven crosses namely, LH 2108 x LD 327 (0.57%), F 2164 x LD 949 (0.33%), F 2164 x FDK 124 (0.30%), PIL 43 x LD 949 (0.083%), LH 2108 x LD 949 (0.066%), LH 2108 x FDK 124 (0.044%) and LH 1556 x LD 491 (0.083%). Two media namely MS4 (MS + Gamborg's B5 vitamins + 1.9 g/l KNO[sub 3]) and MS5 (MS + 1.0 mg/l IAA + 0.2 mg/l Kin) were found to promote more embryo germination. Hybridity of the interspecific hybrid between LH 2108 x LD 327 was confirmed using microsatellite markers. [ABSTRACT FROM AUTHOR]

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

The resurgence of cotton leaf curl disease (CLCuD) since 2001, a major threat to cotton production in Pakistan is associated with Cotton leaf curl Burewala virus (CLCuBuV). All the available cultivated genotypes from Gossypium hirsutum at this time are susceptible however; the native cotton G. arboreum is believed to be immune to CLCuD. To investigate its exact resistance/susceptibility levels, young seedlings of genotype of G. arboreum (Ravi) and G. hirsutum (CIM-496) were graft inoculated with CLCuBuV under glass-house condition. All the graft-inoculated plants of G. arboreum (Ravi) and G. hirsutum (CIM-496) initiated disease symptoms 25 and 10 days respectively after inoculation. However, disease symptoms remained mild in G. arboreum genotype Ravi (resistant), while severe in case of G. hirsutum genotype CIM-496 (highly susceptible) throughout the experiment (90 days after graft-inoculation). All the graft-inoculated plants were found to be positive for CLCuBuV and cotton leaf curl Multan beta Burewala (CLCuMBBur) through PCR. This is the first symptomatic evidence of G. arboreum to be infected with virus causing CLCuD through grafting. The information generated under the present investigations would be helpful to provide better under-standing regarding the molecular basis of resistance to resolve this issue. [ABSTRACT FROM AUTHOR]

Screenhouse experiments were conducted under free-choice conditions to determine the mechanism of resistance operating in cotton against whitefly, Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae). Twelve cotton genotypes belonging to Gossypium hirsutum and G. arboreum were assessed for oviposition preference by whitefly. The trichome density and length, distance from lower leaf surface to nearest vascular bundles, leaf lamina thickness and compactness of vascular bundles were estimated for each genotype and correlations with number of eggs laid were determined. The genotypes RS2098, CNH911, and PA183 were non-preferred for oviposition and exhibited an antixenosis mechanism of resistance. NHH44, LK861, Supriya, RS2013 and LD694 were categorized as moderately resistant while IS-376/4/1/20/72 and F846 were categorized as susceptible. Greater leaf lamina thickness and more compact vascular bundles were correlated with egg laying by whitefly. [ABSTRACT FROM AUTHOR]

A tri-specific hybrid with delayed pigment gland morphogenesis was obtained by crossing the amphidiploid of (G. arboreum × G. bickii) F1 and an upland cotton germplasm with pigment gland genotype of Gl2Gl2gl3gl3. The tri-specific hybrid was a typical interspecific hybrid with high sterile, and the chromosome configuration at meiosis MI of PMC was 2n = 52 = 41.04 I + 4.54 II + 0.57III + 0.04. The crossover value of bivalent was 1.19. Two fertile plants with objective character were obtained in BCs population by continuously backcrossing with Gl2Gl2gl3gl3 as recurrent parent to the tri-specific hybrid, and a new upland cotton germplasm, named ABH-0318, with delayed pigment gland morphogenesis trait was developed through selfing and screening. The pigment gland trait of ABH-0318 was stable, and there were almost no pigment glands observed in the dormant seeds, although there were a few pigment glands confined to cotyledon edges, and the gossypol content in the dormant seeds was 0.017% only, being a typical low gossypol cotton type. However, a large quantity of pigment glands emerged in cotyledons and other main organs of plant after seed germination, and the gossypol contents in the upper parts of the plant were similar to that of ordinary glanded cotton types. Genetic analysis demonstrated that the delayed pigment gland morphogenesis trait of this germplasm was controlled by the interaction of the genes located in two pigment gland loci, Gl2 and Gl3. Among them, the gene located in locus of Gl2, derived from Gbickii, was dominance to up-land cotton pigment gland alleles, Gl2 and gl2, but was recessive epistatic to another glanded gene Gl3, which was named Gl2b temporarily. While the gene located in the locus of Gl3 was a recessive gene come from upland cotton. [ABSTRACT FROM AUTHOR]

Fibers of three cotton cultivars ( Gossypium hirsutum L.) H-4, H-8 and ( G. arboreum) G. Cot-15, which shows variation in staple length were analyzed for growth in terms of fiber length and fresh and dry mass. From the growth analysis cotton fiber development is divided in four distinct phases i.e. (i) initiation (ii) elongation (iii) secondary thickening and (iv) maturation. Rate of fiber elongation and rate of water content shows close parallelism. Highly esterified and less esterified pectic fraction along with high and low molecular weight xyloglucan fractions were estimated from fiber walls of all the three cotton genotypes. Xyloglucans were fractioned in to high and low molecular weight by alkali treatment, 1 M and 4 M KOH respectively. Xyloglucan content shows inverse correlation with fiber elongation. Role of water content and wall components in determination of staple length in cotton genotypes is discussed. [ABSTRACT FROM AUTHOR]

Abiotic stress is an important limiting factor in crop growth and yield around the world. Owing to the continued genetic erosion of the upland cotton germplasm due to intense selection and inbreeding, attention has shifted towards wild cotton progenitors which offer unique traits that can be introgressed into the cultivated cotton to improve their genetic performance. The purpose of this study was to characterize the Pkinase gene family in a previously developed genetic map of the F2 population derived from a cross between two cotton species: Gossypium hirsutum (CCRI 12-4) and Gossypium darwinii (5-7). Based on phylogenetic analysis, Pkinase (PF00069) was found to be the dominant domain with 151 genes in three cotton species, categorized into 13 subfamilies. Structure analysis of G. hirsutum genes showed that a greater percentage of genes and their exons were highly conserved within the group. Syntenic analysis of gene blocks revealed 99 duplicated genes among G. hirsutum, Gossypium arboreum and Gossypium raimondii. Most of the genes were duplicated in segmental pattern. Expression pattern analysis showed that the Pkinase gene family possessed species-level variation in induction to salinity and G. darwinii had higher expression levels as compared to G. hirsutum. Based on RNA sequence analysis and preliminary RT-qPCR verification, we hypothesized that the Pkinase gene family, regulated by transcription factors (TFs) and miRNAs, might play key roles in salt stress tolerance. These findings inferred comprehensive information on possible structure and function of Pkinase gene family in cotton under salt stress. [ABSTRACT FROM AUTHOR]