Cotton (Gossypium hirsutum) VIRMA as an N6-Methyladenosine RNA Methylation Regulator Participates in Controlling Chloroplast-Dependent and Independent Leaf Development.

N6-methyladenosine (m6A) is one of the most abundant internal modifications of mRNA, which plays important roles in gene expression regulation, and plant growth and development. Vir-like m6A methyltransferase associated (VIRMA) serves as a scaffold for bridging the catalytic core components of the m6A methyltransferase complex. The role of VIRMA in regulating leaf development and its related mechanisms have not been reported. Here, we identified and characterized two upland cotton (Gossypium hirsutum) VIRMA genes, named as GhVIR-A and GhVIR-D, which share 98.5% identity with each other. GhVIR-A and GhVIR-D were ubiquitously expressed in different tissues and relatively higher expressed in leaves and main stem apexes (MSA). Knocking down the expression of GhVIR genes by the virus-induced gene silencing (VIGS) system influences leaf cell size, cell shape, and total cell numbers, thereby determining cotton leaf morphogenesis. The dot-blot assay and colorimetric experiment showed the ratio of m6A to A in mRNA is lower in leaves of GhVIR-VIGS plants compared with control plants. Messenger RNA (mRNA) high-throughput sequencing (RNA-seq) and a qRT-PCR experiment showed that GhVIRs regulate leaf development through influencing expression of some transcription factor genes, tubulin genes, and chloroplast genes including photosystem, carbon fixation, and ribosome assembly. Chloroplast structure, chlorophyll content, and photosynthetic efficiency were changed and unsuitable for leaf growth and development in GhVIR-VIGS plants compared with control plants. Taken together, our results demonstrate GhVIRs function in cotton leaf development by chloroplast dependent and independent pathways.

GhTCE1-GhTCEE1 dimers regulate transcriptional reprogramming during wound-induced callus formation in cotton.

Wounded plant cells can form callus to seal the wound site. Alternatively, wounding can cause adventitious organogenesis or somatic embryogenesis. These distinct developmental pathways require specific cell fate decisions. Here, we identify GhTCE1, a basic helix-loop-helix family transcription factor, and its interacting partners as a central regulatory module of early cell fate transition during in vitro dedifferentiation of cotton (Gossypium hirsutum). RNAi- or CRISPR/Cas9-mediated loss of GhTCE1 function resulted in excessive accumulation of reactive oxygen species (ROS), arrested callus cell elongation, and increased adventitious organogenesis. In contrast, GhTCE1-overexpressing tissues underwent callus cell growth, but organogenesis was repressed. Transcriptome analysis revealed that several pathways depend on proper regulation of GhTCE1 expression, including lipid transfer pathway components, ROS homeostasis, and cell expansion. GhTCE1 bound to the promoters of the target genes GhLTP2 and GhLTP3, activating their expression synergistically, and the heterodimer TCE1-TCEE1 enhances this activity. GhLTP2- and GhLTP3-deficient tissues accumulated ROS and had arrested callus cell elongation, which was restored by ROS scavengers. These results reveal a unique regulatory network involving ROS and lipid transfer proteins, which act as potential ROS scavengers. This network acts as a switch between unorganized callus growth and organized development during in vitro dedifferentiation of cotton cells.

Founder transformants of cotton (Gossypium hirsutum L.) obtained through the introduction of DS-Red, Rec, Rep and CRISPR/Cas9 expressing constructs for developing base lines of recombinase mediated gene stacking.

Cotton being the major fiber crop across the world is exposed to numerous biotic and abiotic stresses. Genetic transformation of cotton is vital to meet the world's food, feed and fiber demands. Genetic manipulation by randomly transferring the genes emanate variable gene expression. Targeted gene insertion by latest genome editing tools results in predictable expression of genes at a specified location. Gene stacking technology emerged as an adaptive strategy to combat biotic and abiotic stresses by integrating 2-3 genes simultaneously and at a specific site to avoid variable gene expression at diverse locations. This study explains the development of cotton's founder transformants to be used as a base line for multiple gene stacking projects. We introduced Cre and PhiC31 mediated recombination sites to specify the locus of incoming genes. CRISPR-Cas9 gene was integrated for developing CRISPR based founder lines of cotton. Cas9 gene along with gRNA was integrated to target Rep (replication) region of cotton leaf curl virus. Replication region of virus was specifically targeted to diminish further proliferation and preventing the virus to develop new strains. To successfully develop these primary transformants, a model transformation system has been optimized with the red color visualization (DS-Red). Following red color transformation system, three baselines with recombination specified site (Rec), targeted replication region (Rep) and Cas9 founder lines have been developed. These founder transformants are useful for developing recombinase mediated and CRISPR/Cas9 based originator lines of cotton. Moreover, these transformants will set up a base system for all the recombinase mediated gene stacking projects.

Germination and seed persistence of Amaranthus retroflexus and Amaranthus viridis: Two emerging weeds in Australian cotton and other summer crops.

Redroot pigweed (Amaranthus retroflexus L.) and slender amaranth (Amaranthus viridis L.) are becoming problematic weeds in summer crops, including cotton in Australia. A series of laboratory and field experiments were performed to examine the germination ecology, and seed persistence of two populations of A. retroflexus and A. viridis collected from the Goondiwindi and Gatton regions of Australia. Both populations of A. retroflexus and A. viridis behaved similarly to different environmental conditions. Initial dormancy was observed in fresh seeds of both species; however, germination reached maximum after an after-ripening period of two months at room temperature. Light was not a mandatory prerequisite for germination of both species as they could germinate under complete darkness. Although both species showed very low germination at the alternating day/night temperature of 15/5 C, these species germinated more than 40% between ranges of 25/15 C to 35/25 C. Maximum germination of A. retroflexus (93%) and A. viridis (86%) was observed at 35/25 C and 30/20, respectively. Germination of A. retroflexus and A. viridis was completely inhibited at osmotic potentials of -1.0 and -0.6 MPa, respectively. No germination was observed in both species at the sodium chloride concentration of 200 mM. A. retroflexus seedling emergence (87%) was maximum from the seeds buried at 1 cm while the maximum germination of A. viridis (72%) was observed at the soil surface. No seedling emergence was observed from a burial depth of 8 cm for both species. In both species, seed persistence increased with increasing burial depth. At 24 months after seed placement, seed depletion ranged from 75% (10 cm depth) to 94% (soil surface) for A. retroflexus, and ranged from 79% to 94% for A. viridis, respectively. Information gained from this study will contribute to an integrated control programs for A. retroflexus and A. viridis.

An Unexpected Regulatory Sequence from Rho-Related GTPase6 Confers Fiber-Specific Expression in Upland Cotton.

Cotton fibers, single seed trichomes derived from ovule epidermal cells, are the major source of global textile fibers. Fiber-specific promoters are desirable to study gene function and to modify fiber properties during fiber development. Here, we revealed that Rho-related GTPase6 (GhROP6) was expressed preferentially in developing fibers. A 1240 bp regulatory region of GhROP6, which contains a short upstream regulatory sequence, the first exon, and the partial first intron, was unexpectedly isolated and introduced into transgenic cotton for analyzing promoter activity. The promoter of GhROP6 (proChROP6) conferred a specific expression in ovule surface, but not in the other floral organs and vegetative tissues. Reverse transcription PCR analysis indicated that proGhROP6 directed full-length transcription of the fused ß-glucuronidase (GUS) gene. Further investigation of GUS staining showed that proChROP6 regulated gene expression in fibers and ovule epidermis from fiber initiation to cell elongation stages. The preferential activity was enriched in fiber cells after anthesis and reached to peak on flowering days. By comparison, proGhROP6 was a mild promoter with approximately one-twenty-fifth of the strength of the constitutive promoter CaMV35S. The promoter responded to high-dosage treatments of auxin, gibberellin and salicylic acid and slightly reduced GUS activity under the in vitro treatment. Collectively, our data suggest that the GhROP6 promoter has excellent activity in initiating fibers and has potential for bioengineering of cotton fibers.

RNA-Seq with a novel glabrous-ZM24fl reveals some key lncRNAs and the associated targets in fiber initiation of cotton.

BACKGROUND: Cotton fiber is an important natural resource for textile industry and an excellent model for cell biology study. Application of glabrous mutant cotton and high-throughput sequencing facilitates the identification of key genes and pathways for fiber development and cell differentiation and elongation. LncRNA is a type of ncRNA with more than 200 nt in length and functions in the ways of chromatin modification, transcriptional and post-transcriptional modification, and so on. However, the detailed lncRNA and associated mechanisms for fiber initiation are still unclear in cotton. RESULTS: In this study, we used a novel glabrous mutant ZM24fl, which is endowed with higher somatic embryogenesis, and functions as an ideal receptor for cotton genetic transformation. Combined with the high-throughput sequencing, fatty acid pathway and some transcription factors such as MYB, ERF and bHLH families were identified the important roles in fiber initiation; furthermore, 3,288 lncRNAs were identified, and some differentially expressed lncRNAs were also analyzed. From the comparisons of ZM24_0 DPA vs ZM24_-2 DPA and fl_0 DPA vs ZM24_0 DPA, one common lncRNA MSTRG 2723.1 was found that function upstream of fatty acid metabolism, MBY25-mediating pathway, and pectin metabolism to regulate fiber initiation. In addition, other lncRNAs MSTRG 3390.1, MSTRG 48719.1, and MSTRG 31176.1 were also showed potential important roles in fiber development; and the co-expression analysis between lncRNAs and targets showed the distinct models of different lncRNAs and complicated interaction between lncRNAs in fiber development of cotton. CONCLUSIONS: From the above results, a key lncRNA MSTRG 2723.1 was identified that might mediate some key genes transcription of fatty acid metabolism, MYB25-mediating pathway, and pectin metabolism to regulate fiber initiation of ZM24 cultivar. Co-expression analysis implied that some other important lncRNAs (e.g., MSTRG 3390.1, MSTRG 48719.1, and MSTRG 31176.1) were also showed the different regulatory model and interaction between them, which proposes some valuable clues for the lncRNAs associated mechanisms in fiber development.

Identification and expression analysis of arabinogalactan protein genes in cotton reveal the function of GhAGP15 in Verticillium dahliae resistance.

Verticillium wilt, primarily caused by the fungal pathogen Verticillium dahliae, is a serious disease in cotton. Arabinogalactan proteins (AGPs), a class of hydroxyproline-rich glycoproteins, have been widely implicated in plant growth and environmental adaptation. The purpose of this study is to identify and characterize AGP members in cotton plants and explore their roles in responding to environmental stressors. In total, 65 GhAGP members were identified in upland cotton (Gossypium hirsutum), along with 43, 35, and 37 AGP members that were also identified in G. barbadense, G. arboreum, and G. raimondii, respectively. According to gene structure and protein domains analysis, GhAGP genes in upland cotton are highly conserved. Meanwhile, tandem duplication events have occurred frequently throughout cotton's evolutionary history. Expression analysis showed that GhAGP genes were widely expressed during growth and development and in response to abiotic stressors. Many cis-elements related to hormonal responses and environmental stressors were detected in GhAGP promoter regions. GhAGP genes participate in responding to cold, drought, and salt stress, and were sensitive to ET signaling. Furthermore, the expression level of GhAGP15 was elevated during V. dahliae infection and resistance against V. dahliae in upland cotton was significantly weakened by silencing GhAGP15 using a virus-induced gene silencing (VIGS) approach. Our results further suggest that the function of GhAGP15 in V. dahliae resistance might be involved in regulation of the JA, SA, and reactive oxygen species (ROS) pathways. The comprehensive analysis of AGP genes in cotton performed in this study provides a basic framework for further functional research of these genes.

Novel insights into water-deficit-responsive mRNAs and lncRNAs during fiber development in Gossypium hirsutum.

BACKGROUND: The fiber yield and quality of cotton are greatly and periodically affected by water deficit. However, the molecular mechanism of the water deficit response in cotton fiber cells has not been fully elucidated. RESULTS: In this study, water deficit caused a significant reduction in fiber length, strength, and elongation rate but a dramatic increase in micronaire value. To explore genome-wide transcriptional changes, fibers from cotton plants subjected to water deficit (WD) and normal irrigation (NI) during fiber development were analyzed by transcriptome sequencing. Analysis showed that 3427 mRNAs and 1021 long noncoding RNAs (lncRNAs) from fibers were differentially expressed between WD and NI plants. The maximum number of differentially expressed genes (DEGs) and lncRNAs (DERs) was identified in fibers at the secondary cell wall biosynthesis stage, suggesting that this is a critical period in response to water deficit. Twelve genes in cotton fiber were differentially and persistently expressed at ≥ five time points, suggesting that these genes are involved in both fiber development and the water-deficit response and could potentially be used in breeding to improve cotton resistance to drought stress. A total of 540 DEGs were predicted to be potentially regulated by DERs by analysis of coexpression and genomic colocation, accounting for approximately 15.76% of all DEGs. Four DERs, potentially acting as target mimics for microRNAs (miRNAs), indirectly regulated their corresponding DEGs in response to water deficit. CONCLUSIONS: This work provides a comprehensive transcriptome analysis of fiber cells and a set of protein-coding genes and lncRNAs implicated in the cotton response to water deficit, significantly affecting fiber quality during the fiber development stage.

Biochemical and Expression Analyses Revealed the Involvement of Proanthocyanidins and/or Their Derivatives in Fiber Pigmentation of Gossypium stocksii.

The wild cotton species Gossypium stocksii produces a brown fiber that provides a valuable resource for the color improvement of naturally colored cotton (NCC) fiber. However, the biochemical basis and molecular mechanism of its fiber pigmentation remain unclear. Herein, we analyzed the dynamics of proanthocyanidins (PAs) accumulation in developing the fiber of G. stocksii, which suggested a similar role of PAs and/or their derivatives in the fiber coloration of G. stocksii. In addition, comparative transcriptomics analyses revealed that the PA biosynthetic genes were expressed at higher levels and for a longer period in developing fibers of G. stocksii than G. arboreum (white fiber), and the transcription factors, such as TT8, possibly played crucial regulatory roles in regulating the PA branch genes. Moreover, we found that the anthocyanidin reductase (ANR) was expressed at a higher level than the leucoanthocyanidin reductases (LARs) and significantly upregulated during fiber elongation, suggesting a major role of ANR in PA synthesis in G. stocksii fiber. In summary, this work revealed the accumulation of PAs and the expression enhancement of PA biosynthetic genes in developing fibers of G. stocksii. We believe this work will help our understanding of the molecular mechanisms of cotton fiber coloration and further promote the future breeding of novel NCCs.

Genomic interrogation of a MAGIC population highlights genetic factors controlling fiber quality traits in cotton.

Cotton (Gossypium hirsutum L.) fiber is the most important resource of natural and renewable fiber for the textile industry. However, the understanding of genetic components and their genome-wide interactions controlling fiber quality remains fragmentary. Here, we sequenced a multiple-parent advanced-generation inter-cross (MAGIC) population, consisting of 550 individuals created by inter-crossing 11 founders, and established a mosaic genome map through tracing the origin of haplotypes that share identity-by-descent (IBD). We performed two complementary GWAS methods-SNP-based GWAS (sGWAS) and IBD-based haplotype GWAS (hGWAS). A total of 25 sQTLs and 14 hQTLs related to cotton fiber quality were identified, of which 26 were novel QTLs. Two major QTLs detected by both GWAS methods were responsible for fiber strength and length. The gene Ghir_D11G020400 (GhZF14) encoding the MATE efflux family protein was identified as a novel candidate gene for fiber length. Beyond the additive QTLs, we detected prevalent epistatic interactions that contributed to the genetics of fiber quality, pinpointing another layer for trait variance. This study provides new targets for future molecular design breeding of superior fiber quality.

Conformance of sowing dates for maximizing heat use efficiency and seed cotton yield in arid to semi-arid cotton zone of Pakistan.

Pakistan is placed among the most vulnerable countries with relation to climate change and its impacts on agricultural productivity. Cotton is staged as the cash crop of the country and the main source of raw material for textile, oil, and feed industry. Varying environmental attributes have significant effects on the duration of vegetative and reproductive stages of cotton crop. To evaluate the potential impacts of varied temperatures regimes in different sowing times, field experiments were carried out throughout the cotton growing areas of Pakistan from Faisalabad in Central Punjab to RYK in Southern Punjab and Sakrand in Sindh to Dera Ismail Khan in Khyber Pakhtunkhwa (KPK) Province. Crop was sown on six different sowing dates starting from 1st March towards 15th May with 2-week intervals for two crop seasons (2016 and 2017). The timing of phenological events like emergence, squaring, flowering, and boll opening was recorded on calendar days and cumulative heat units (GDDs) were calculated for flowering and boll opening stages. Heat use efficiency for these sowing times was estimated. Data regarding yield-related parameters like opened bolls per plant, average boll weight, and seed cotton yield were also recorded during the study. Results revealed that duration of the growth stages was significantly affected by variation in mean thermal kinetics in varied sowing times in all four different environments. Seed cotton yield and heat use efficiency were also varied among the locations and sowing dates. The maximum seed cotton yield was recorded in Sakrand location at 15th April sowing date. The dependence of the phenological advancement on temperature and negative impacts of higher thermal stress on cotton productivity were also confirmed throughout the cotton growing zone of Pakistan.

Exogenous application of bio-stimulant and growth retardant improved the productivity of cotton cultivars under different planting arrangement / A aplicação exógena de bioestimulante e regulador de crescimento na melhoria da produtividade de cultivares de algodão sob diferentes configurações de plantio

Optimum planting arrangement is an important attribute for efficient utilization of available resources and to obtain high yield of cotton. Application of plant growth promoter and retardant on cotton in improved planting density are the innovative techniques in the establishment of more productive cotton crop. Therefore, we planned a field study to assess the role of bio-stimulant and growth retardant in the resource utilization efficiency of cotton cultivars planted under variable row spacing at Agronomic Research Area Bahauddin Zakariya University and Usmania Agricultural Farm Shujabad during Kharif 2012. Experimental treatments consisted of cotton genotypes viz. CIM-573 and CIM-598, cultivated under conventional (75 cm), medium (50 cm) and ultra-narrow row spacing (25 cm) with foliar spray of bio-stimulant (moringa leaf extract) and growth retardant (mepiquate chloride), either sole or in combination, keeping distilled water as a control. Exogenously applied MLE alone and MLE + MC significantly enhanced the number of squares, flowers and green bolls per plant leading to higher cotton seed and lint yield of CIM 598 cultivar cultivated under conventional row spacing. While application of MC alone and MLE + MC produced maximum micronaire value, fiber strength and fiber uniformity ratio of CIM 573 cultivar cultivated under conventional row spacing. The results suggested that application of MLE is a possible approach to enhance the cotton productivity and the use of MC to enhance the fiber quality attributes under conventional row spacing.

A configuração ideal de plantio é um atributo importante para a utilização eficiente dos recursos disponíveis e para obter alto rendimento de algodão. A aplicação de promotores de crescimento de plantas e reguladores de crescimento no algodão em uma densidade de plantio adequada são técnicas inovadoras na obtenção de safras de algodão mais produtivas. Portanto, foi planejado um estudo de campo para avaliar o papel de um bioestimulante e um regulador de crescimento na eficiência da utilização de recursos de cultivares de algodão plantadas com espaçamento variável entre linhas na Área de Pesquisa Agronômica Universidade Bahauddin Zakariya e Usmania Agricultural Farm Shujabad durante Kharif 2012. Os tratamentos experimentais consistiram em genótipos de algodão viz. CIM-573 e CIM-598, cultivadas sob espaçamento convencional (75 cm), médio (50 cm) e ultraestreito (25 cm) e pulverização foliar de bioestimulante (extrato de folha de moringa) e regulador de crescimento (cloreto de mepiquato)), sozinho ou combinado, mantendo a água destilada como controle. O MLE aplicado exogenamente sozinho e o MLE + MC aumentaram significativamente o número de quadrados, flores e cápsulas verdes por planta, levando a um maior rendimento de sementes e fibra de algodão da cultivar CIM 598 cultivada sob espaçamento convencional entre fileiras. Enquanto a aplicação de MC sozinho e MLE + MC produziu valor máximo de micronaire, resistência da fibra e razão de uniformidade da fibra da cultivar CIM 573 cultivada sob espaçamento convencional entre linhas. Os resultados sugeriram que a aplicação do MLE é uma abordagem possível para aumentar a produtividade do algodão e o uso de MC para aprimorar os atributos de qualidade da fibra no espaçamento convencional entre linhas.

The application of ammonium sulphate and amino acid on cotton: effects on can improve growth, yield, quality and nitrogen absorption / Aplicação de sulfato de amônio e aminoácido no algodão: efeitos sobre o crescimento, o rendimento, a qualidade e a absorção de nitrogênio

A field study was carried out to determine the influence of foliage applied plant growth promoter and retardant in improving soil applied sulphur fertilizer use efficiency in cotton during two consecutive summers 2014 and 2015. Experimental trial comprised of three different sources of sulphur (ammonium sulphate, potassium sulphate and elemental sulphur) and foliar spray of plant growth promoter and growth retardant including tap water was taken as control. Among treatments soil applied ammonium sulphate with foliage applied amino acid produced maximum plant height, sympodial branches, pods per plant, seed cotton yield, fiber yield, biological yield, protein contents, oil contents and leaf nitrogen uptake as compared to the other treatments. Whereas, soil applied potassium sulphate with foliar spray of mepiquat chloride on cotton significantly improved the boll weight and leaf potassium uptake. We conclude that soil applied ammonium sulphate and foliage spray of amino acid was more effective in improving the productivity and quality attributes of cotton.

Foi realizado um estudo de campo para determinar a influência do promotor de crescimento das plantas e retardador da folhagem em algodão, para melhora da eficiência do uso de fertilizantes à base de enxofre aplicados no solo durante dois verões consecutivos (2014 e 2015). O ensaio experimental foi composto de três fontes diferentes de enxofre (sulfato de amônio, sulfato de potássio e enxofre elementar) e pulverização foliar do promotor de crescimento de plantas e retardador de crescimento, incluindo água da torneira que foi tomada como controle. Entre os tratamentos, o sulfato de amônio aplicado no solo com aminoácido aplicado na folhagem produziu o máximo na altura da planta, ramos simodiais, capulhos por planta, rendimento de algodão em caroço, rendimento de fibra, rendimento biológico, conteúdo de proteínas, conteúdo de óleo e absorção de nitrogênio nas folhas quando comparado a outros tratamentos. Enquanto o solo fertilizado com sulfato de potássio e aplicação foliar de cloreto de mepiquat no algodão melhorou, significativamente, o peso do capulho e a absorção de potássio nas folhas. Sulfato de amônio aplicado no solo e a aplicação foliar de aminoácidos foram mais eficazes na melhora dos atributos de produtividade e qualidade do algodão.

Identification of Raf-Like Kinases B Subfamily Genes in Gossypium Species Revealed GhRAF42 Enhanced Salt Tolerance in Cotton.

Salinity is a critical abiotic factor that significantly reduces agricultural production. Cotton is an important fiber crop and a pioneer on saline soil, hence genetic architecture that underpins salt tolerance should be thoroughly investigated. The Raf-like kinase B-subfamily (RAF) genes were discovered to regulate the salt stress response in cotton plants. However, understanding the RAFs in cotton, such as Enhanced Disease Resistance 1 and Constitutive Triple Response 1 kinase, remains a mystery. This study obtained 29, 28, 56, and 54 RAF genes from G. arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively. The RAF gene family described allopolyploidy and hybridization events in allotetraploid cotton evolutionary connections. Ka/Ks analysis advocates that cotton evolution was subjected to an intense purifying selection of the RAF gene family. Interestingly, integrated analysis of synteny and gene collinearity suggested dispersed and segmental duplication events involved in the extension of RAFs in cotton. Transcriptome studies, functional validation, and virus-induced gene silencing on salt treatments revealed that GhRAF42 is engaged in salt tolerance in upland cotton. This research might lead to a better understanding of the role of RAFs in plants and the identification of suitable candidate salt-tolerant genes for cotton breeding.

Path analysis based on genetic association of yield components and insects pest in upland cotton varieties.

Gossypium hirsutum L. is also called upland cotton or Mexican cotton. It is the most widely cultivated species of cotton in the whole world. Globally, about 90% of all cotton production comes from the cultivars derived from this species. Some genetic parameters like monopodial branches per plant, sympodial branches per plant, sympodial branch length, bolls per plant, boll weight, sympo-boll distance, Ginning Out Turn%, staple length (rg = 0.9199**), and fiber strength along with seed cotton yield were evaluated for their potential utilization via selection in seed cotton yield improvement. Significant positive genetic correlations were estimated for monopodial branches per plant (rg = 0.9722**), sympodial branches per plant (rg = 0.7098**), sympodial branch length (rg = 0.617**), bolls per plant (rg = 0.8271**), boll weight (rg = 0.8065**), sympo-boll distance (rg = 0.6507**), Ginning Out Turn (GOT)% (rg = 0.7541**), staple length (rg = 0.9199**), and fiber strength (rg = 0.7534**) with seed cotton yield. A path analysis of all the yield traits under study revealed strong positive direct effects of monopodial branch length (1.1556), sympo-boll distance (0.8173) and staple length (0.7633), while plant height exerted a highly strong direct negative effect (-1.2096) on yield. It is concluded that a direct selection based on monopodial branch length and sympo-boll distance, and staple length is effective, whereas, monopodial branch length, and sympodial branch length are good selection indicators via bolls per plant for yield improvement in cotton.

Silencing GhJUB1L1 (JUB1-like 1) reduces cotton (Gossypium hirsutum) drought tolerance.

Drought stress massively restricts plant growth and the yield of crops. Reducing the deleterious effects of drought is necessary for agricultural industry. The plant-specific NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) are widely involved in the regulation of plant development and stress response. One of the NAC TF, JUNGBRUNNEN1 (JUB1), has been reported to involve in drought resistance in Arabidopsis. However, little is known of how the JUB1 gene respond to drought stress in cotton. In the present study, we cloned GhJUB1L1, a homologous gene of JUB1 in upland cotton. GhJUB1L1 is preferentially expressed in stem and leaf and could be induced by drought stress. GhJUB1L1 protein localizes to the cell nucleus, and the transcription activation region of which is located in the C-terminal region. Silencing GhJUB1L1 gene via VIGS () reduced cotton drought tolerance, and retarded secondary cell wall (SCW) development. Additionally, the expression of some drought stress-related genes and SCW synthesis-related genes were altered in the GhJUB1L1 silencing plants. Collectively, our findings indicate that GhJUB1L1 may act as a positive regulator in response to drought stress and SCW development in cotton. Our results enriched the roles of NAC TFs in cotton drought tolerance and laid a foundation for the cultivation of transgenic cotton with higher drought tolerance.

Comparative transcriptome analysis of fiber and nonfiber tissues to identify the genes preferentially expressed in fiber development in Gossypium hirsutum.

Cotton is an important natural fiber crop and economic crop worldwide. The quality of cotton fiber directly determines the quality of cotton textiles. Identifying cotton fiber development-related genes and exploring their biological functions will not only help to better understand the elongation and development mechanisms of cotton fibers but also provide a theoretical basis for the cultivation of new cotton varieties with excellent fiber quality. In this study, RNA sequencing technology was used to construct transcriptome databases for different nonfiber tissues (root, leaf, anther and stigma) and fiber developmental stages (7 days post-anthesis (DPA), 14 DPA, and 26 DPA) of upland cotton Coker 312. The sizes of the seven transcriptome databases constructed ranged from 4.43 to 5.20 Gb, corresponding to approximately twice the genome size of Gossypium hirsutum (2.5 Gb). Among the obtained clean reads, 83.32% to 88.22% could be compared to the upland cotton TM-1 reference genome. By analyzing the differential gene expression profiles of the transcriptome libraries of fiber and nonfiber tissues, we obtained 1205, 1135 and 937 genes with significantly upregulated expression at 7 DPA, 14 DPA and 26 DPA, respectively, and 124, 179 and 213 genes with significantly downregulated expression. Subsequently, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analyses were performed, which revealed that these genes were mainly involved in catalytic activity, carbohydrate metabolism, the cell membrane and organelles, signal transduction and other functions and metabolic pathways. Through gene annotation analysis, many transcription factors and genes related to fiber development were screened. Thirty-six genes were randomly selected from the significantly upregulated genes in fiber, and expression profile analysis was performed using qRT-PCR. The results were highly consistent with the gene expression profile analyzed by RNA-seq, and all of the genes were specifically or predominantly expressed in fiber. Therefore, our RNA sequencing-based comparative transcriptome analysis will lay a foundation for future research to provide new genetic resources for the genetic engineering of improved cotton fiber quality and for cultivating new transgenic cotton germplasms for fiber quality improvement.

Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus.

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3-5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20-40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60-70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.

Assessing the impacts of irrigation termination periods on cotton productivity under strategic deficit irrigation regimes.

Determining optimum irrigation termination periods for cotton (Gossypium hirsutum L.) is crucial for efficient utilization and conservation of finite groundwater resources of the Ogallala Aquifer in the Texas High Plains (THP) region. The goal of this study was to suggest optimum irrigation termination periods for different Evapotranspiration (ET) replacement-based irrigation strategies to optimize cotton yield and irrigation water use efficiency (IWUE) using the CROPGRO-Cotton model. We re-evaluated a previously evaluated CROPGRO-Cotton model using updated yield and in-season physiological data from 2017 to 2019 growing seasons from an IWUE experiment at Halfway, TX. The re-evaluated model was then used to study the effects of combinations of irrigation termination periods (between August 15 and September 30) and deficit/excess irrigation strategies (55%-115% ET-replacement) under dry, normal and wet years using weather data from 1978 to 2019. The 85% ET-replacement strategy was found ideal for optimizing irrigation water use and cotton yield, and the optimum irrigation termination period for this strategy was found to be the first week of September during dry and normal years, and the last week of August during wet years. Irrigation termination periods suggested in this study are useful for optimizing cotton production and IWUE under different levels of irrigation water availability.

GhGASA10-1 promotes the cell elongation in fiber development through the phytohormones IAA-induced.

BACKGROUND: Cotton is an important cash crop. The fiber length has always been a hot spot, but multi-factor control of fiber quality makes it complex to understand its genetic basis. Previous reports suggested that OsGASR9 promotes germination, width, and thickness by GAs in rice, while the overexpression of AtGASA10 leads to reduced silique length, which is likely to reduce cell wall expansion. Therefore, this study aimed to explore the function of GhGASA10 in cotton fibers development. RESULTS: To explore the molecular mechanisms underlying fiber elongation regulation concerning GhGASA10-1, we revealed an evolutionary basis, gene structure, and expression. Our results emphasized the conservative nature of GASA family with its origin in lower fern plants S. moellendorffii. GhGASA10-1 was localized in the cell membrane, which may synthesize and transport secreted proteins to the cell wall. Besides, GhGASA10-1 promoted seedling germination and root extension in transgenic Arabidopsis, indicating that GhGASA10-1 promotes cell elongation. Interestingly, GhGASA10-1 was upregulated by IAA at fiber elongation stages. CONCLUSION: We propose that GhGASA10-1 may promote fiber elongation by regulating the synthesis of cellulose induced by IAA, to lay the foundation for future research on the regulation networks of GASA10-1 in cotton fiber development.