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Introduction: The simultaneous improvement of fiber quality and yield for cotton is strongly limited by the narrow genetic backgrounds of Gossypium hirsutum (Gh) and the negative genetic correlations among traits. An effective way to overcome the bottlenecks is to introgress the favorable alleles of Gossypium barbadense (Gb) for fiber quality into Gh with high yield., Objectives: This study was to identify superior loci for the improvement of fiber quality and yield., Methods: Two sets of chromosome segment substitution lines (CSSLs) were generated by crossing Hai1 (Gb, donor-parent) with cultivar CCRI36 (Gh) and CCRI45 (Gh) as genetic backgrounds, and cultivated in 6 and 8 environments, respectively. The kmer genotyping strategy was improved and applied to the population genetic analysis of 743 genomic sequencing data. A progeny segregating population was constructed to validate genetic effects of the candidate loci., Results: A total of 68,912 and 83,352 genome-wide introgressed kmers were identified in the CCRI36 and CCRI45 populations, respectively. Over 90 % introgressions were homologous exchanges and about 21 % were reverse insertions. In total, 291 major introgressed segments were identified with stable genetic effects, of which 66(22.98 %), 64(21.99 %), 35(12.03 %), 31(10.65 %) and 18(6.19 %) were beneficial for the improvement of fiber length (FL), strength (FS), micronaire, lint-percentage (LP) and boll-weight, respectively. Thirty-nine introgression segments were detected with stable favorable additive effects for simultaneous improvement of 2 or more traits in Gh genetic background, including 6 could increase FL/FS and LP. The pyramiding effects of 3 pleiotropic segments (A07:C45Clu-081, D06:C45Clu-218, D02:C45Clu-193) were further validated in the segregating population., Conclusion: The combining of genome-wide introgressions and kmer genotyping strategy showed significant advantages in exploring genetic resources. Through the genome-wide comprehensive mining, a total of 11 clusters (segments) were discovered for the stable simultaneous improvement of FL/FS and LP, which should be paid more attention in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

Cotton (Gossypium spp.) is the fifth largest oil crop in the world, and cottonseed provides abundant vegetable oil resources and industrial bioenergy fuels for people; therefore, it is of practical significance to increase the oil content of cotton seeds for improving the oil yield and economic benefits of planting cotton. Long-chain acyl-coenzyme A (CoA) synthetase ( LACS ) capable of catalyzing the formation of acyl-CoAs from free fatty acids has been proven to significantly participate in lipid metabolism, of which whole-genome identification and functional characterization of the gene family have not yet been comprehensively analyzed in cotton. In this study, a total of sixty-five LACS genes were confirmed in two diploid and two tetraploid Gossypium species, which were divided into six subgroups based on phylogenetic relationships with twenty-one other plants. An analysis of protein motif and genomic organizations displayed structural and functional conservation within the same group but diverged among the different group. Gene duplication relationship analysis illustrates the LACS gene family in large scale expansion through WGDs/segmental duplications. The overall Ka/Ks ratio indicated the intense purifying selection of LACS genes in four cotton species during evolution. The LACS genes promoter elements contain numerous light response cis-elements associated with fatty acids synthesis and catabolism. In addition, the expression of almost all GhLACS genes in high seed oil were higher compared to those in low seed oil. We proposed LACS gene models and shed light on their functional roles in lipid metabolism, demonstrating their engineering potential for modulating TAG synthesis in cotton, and the genetic engineering of cottonseed oil provides a theoretical basis.

Key Message: The fiber length-related qFL-A12-5 identified in CSSLs introgressed from Gossypium barbadense into Gossypium hirsutum was fine-mapped to an 18.8 kb region on chromosome A12, leading to the identification of the GhTPR gene as a potential regulator of cotton fiber length. Fiber length is a key determinant of fiber quality in cotton, and it is a key target of artificial selection for breeding and domestication. Although many fiber length-related quantitative trait loci have been identified, there are few reports on their fine mapping or candidate gene validation, thus hampering efforts to understand the mechanistic basis of cotton fiber development. Our previous study identified the qFL-A12-5 associated with superior fiber quality on chromosome A12 in the chromosome segment substitution line (CSSL) MBI7747 (BC 4 F 3:5 ). A single segment substitution line (CSSL-106) screened from BC 6 F 2 was backcrossed to construct a larger segregation population with its recurrent parent CCRI45, thus enabling the fine mapping of 2852 BC 7 F 2 individuals using denser simple sequence repeat markers to narrow the qFL-A12-5 to an 18.8 kb region of the genome, in which six annotated genes were identified in Gossypium hirsutum. Quantitative real-time PCR and comparative analyses led to the identification of GH_A12G2192 (GhTPR) encoding a tetratricopeptide repeat-like superfamily protein as a promising candidate gene for qFL-A12-5. A comparative analysis of the protein-coding regions of GhTPR among Hai1, MBI7747, and CCRI45 revealed two non-synonymous mutations. The overexpression of GhTPR resulted in longer roots in Arabidopsis, suggesting that GhTPR may regulate cotton fiber development. These results provide a foundation for future efforts to improve cotton fiber length., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cotton is the fifth-largest oil crop in the world. A high kernel oil content (KOC) and high stability are important cottonseed attributes for food security. In this study, the phenotype of KOC and the genotype-by-environment interaction factors were collectively dissected using 250 recombinant inbred lines, their parental cultivars sGK156 and 901-001, and CCRI70 across multi-environments. ANOVA and correlation analysis showed that both genotype and environment contributed significantly to KOC accumulation. Analyses of additive main effect multiplicative interaction and genotype-by-environment interaction biplot models presented the effects of genotype, environment, and genotype by environment on KOC performance and the stability of the experimental materials. Interaction network analysis revealed that meteorological and geographical factors explained 38% of the total KOC variance, with average daily rainfall contributing the largest positive impact and cumulative rainfall having the largest negative impact on KOC accumulation. This study provides insight into KOC accumulation and could direct selection strategies for improved KOC and field management of cottonseed in the future.

Key Message: Based on the integration of QTL-mapping and regulatory network analyses, five high-confidence stable QTL regions, six candidate genes and two microRNAs that potentially affect the cottonseed oil content were discovered. Cottonseed oil is increasingly becoming a promising target for edible oil with its high content of unsaturated fatty acids. In this study, a recombinant inbred line (RIL) cotton population was constructed to detect quantitative trait loci (QTLs) for the cottonseed oil content. A total of 39 QTLs were detected across eight different environments, of which five QTLs were stable. Forty-three candidate genes potentially involved in carbon metabolism, fatty acid synthesis and triacylglycerol biosynthesis processes were further obtained in the stable QTL regions. Transcriptome analysis showed that nineteen of these candidate genes expressed during  the developing cottonseed ovules and may affect the cottonseed oil content. Besides, transcription factor (TF) and microRNA (miRNA) co-regulatory network analyses based on the nineteen candidate genes suggested that six genes, two core miRNAs (ghr-miR2949b and ghr-miR2949c), and one TF GhHSL1 were considered to be closely associated with the cottonseed oil content. Moreover, four vital genes were validated by quantitative real-time PCR (qRT-PCR). These results provide insights into the oil accumulation mechanism in developing cottonseed ovules through the construction of a detailed oil accumulation model., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cotton seeds are typically covered by lint and fuzz fibres. Natural 'fuzzless' mutants are an ideal model system for identifying genes that regulate cell initiation and elongation. Here, using a genome-wide association study (GWAS), we identified a ~ 6.2 kb insertion, larINDEL FZ , located at the end of chromosome 8, composed of a ~ 5.0 kb repetitive sequence and a ~ 1.2 kb fragment translocated from chromosome 12 in fuzzless Gossypium arboreum. The presence of larINDEL FZ was associated with a fuzzless seed and reduced trichome phenotypes in G. arboreum. This distant insertion was predicted to be an enhancer, located ~ 18 kb upstream of the dominant-repressor GaFZ (Ga08G0121). Ectopic overexpression of GaFZ in Arabidopsis thaliana and G. hirsutum suggested that GaFZ negatively modulates fuzz and trichome development. Co-expression and interaction analyses demonstrated that GaFZ might impact fuzz fibre/trichome development by repressing the expression of genes in the very-long-chain fatty acid elongation pathway. Thus, we identified a novel regulator of fibre/trichome development while providing insights into the importance of noncoding sequences in cotton., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Large-scale genomic surveys of crop germplasm are important for understanding the genetic architecture of favorable traits. The genomic basis of geographic differentiation and fiber improvement in cultivated cotton is poorly understood. Here, we analyzed 3,248 tetraploid cotton genomes and confirmed that the extensive chromosome inversions on chromosomes A06 and A08 underlies the geographic differentiation in cultivated Gossypium hirsutum. We further revealed that the haplotypic diversity originated from landraces, which might be essential for understanding adaptative evolution in cultivated cotton. Introgression and association analyses identified new fiber quality-related loci and demonstrated that the introgressed alleles from two diploid cottons had a large effect on fiber quality improvement. These loci provided the potential power to overcome the bottleneck in fiber quality improvement. Our study uncovered several critical genomic signatures generated by historical breeding effects in cotton and a wealth of data that enrich genomic resources for the research community.

Improving yield is a primary mission for cotton (Gossypium hirsutum) breeders; development of cultivars with suitable architecture for high planting density (HPDA) can increase yield per unit area. We characterized a natural cotton mutant, AiSheng98 (AS98), which exhibits shorter height, shorter branch length, and more acute branch angle than wild-type. A copy number variant at the HPDA locus on Chromosome D12 (HPDA-D12), encoding a dehydration-responsive element-binding (DREB) transcription factor, GhDREB1B, strongly affects plant architecture in the AS98 mutant. We found an association between a tandem duplication of a c. 13.5 kb segment in HPDA-D12 and elevated GhDREB1B expression resulting in the AS98 mutant phenotype. GhDREB1B overexpression confers a significant decrease in plant height and branch length, and reduced branch angle. Our results suggest that fine-tuning GhDREB1B expression may be a viable engineering strategy for modification of plant architecture favorable to high planting density in cotton., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

Circular RNA is one of the endogenous non-coding RNAs with a covalently closed loop structure and largely involved in regulating gene expression. However, the abundance of circular RNAs and their regulatory functions during the early stages of fiber development are still not known. In this work, we conducted high-throughput sequencing of the Ligonlintless-1 and its wild-type at 0 DPA, 8 DPA and stem. A total of 2811 circular RNAs were identified and unevenly distributed across cotton chromosomes. We found 34, 142 and 27 circular RNAs were differentially expressed between Ligonlintless-1 and wild-type at 0 DPA, 8 DPA and stem, respectively. Both circular RNA-microRNA-mRNA network and MeJA treatment results in Ligonlintless-1 and wild-type might provide a strong indication of four circular RNAs and ghr_miR169b being important biological molecular associating with fiber development. The results provide new insight into the putative molecular function of circular RNAs in the regulation of fiber development., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Key Message: Background-independent (BI) and stably expressed (SE) quantitative trait loci (QTLs) were identified using two sets of introgression lines across multiple environments. Genetic background more greatly affected fiber quality traits than environmental factors. Sixty-one SE-QTLs, including two BI-QTLs, were novel and 48 SE-QTLs, including seven BI-QTLs, were previously reported. Cotton fiber quality traits are controlled by QTLs and are susceptible to environmental influence. Fiber quality improvement is an essential goal in cotton breeding but is hindered by limited knowledge of the genetic basis of fiber quality traits. In this study, two sets of introgression lines of Gossypium hirsutum × G. barbadense were used to dissect the QTL stability of three fiber quality traits (fiber length, strength and micronaire) across environments using 551 simple sequence repeat markers selected from our high-density genetic map. A total of 76 and 120 QTLs were detected in the CCRI36 and CCRI45 backgrounds, respectively. Nine BI-QTLs were found, and 78 (41.71%) of the detected QTLs were reported previously. Thirty-nine and 79 QTLs were SE-QTLs in at least two environments in the CCRI36 and CCRI45 backgrounds, respectively. Forty-eight SE-QTLs, including seven BI-QTLs, were confirmed in previous reports, and 61 SE-QTLs, including two BI-QTLs, were considered novel. These results indicate that genetic background more strongly impacts on fiber quality traits than environmental factors. Twenty-three clusters with BI- and/or SE-QTLs were identified, 19 of which harbored favorable alleles from G. barbadense for two or three fiber quality traits. This study is the first report using two sets of introgression lines to identify fiber quality QTLs across environments in cotton, providing insights into the effect of genetic backgrounds and environments on the QTL expression of fiber quality and important information for the genetic basis underlying fiber quality traits toward QTL cloning and molecular breeding.

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.

Cotton possesses certain physical features, including leaf and stem trichomes that help plants deter damage caused by insect pests, and to some extent, from abiotic factors as well. Among those features, trichomes (pubescence) hold a special place as a first line of defense and a managemental tool against sucking insect pests of cotton. Different insect pests of cotton (whiteflies, aphids, jassids, and boll weevil) severely damage the yield and quality of the crop. Likewise, whiteflies, aphids, jassids, and other insect pests are considered as potential carriers for cotton leaf curl viruses and other diseases. Genotyping by sequencing (GBS) study was conducted to understand and explore the genomic regions governing hairy (Pubescence) leaves and stem phenotypes. A total of 224 individuals developed from an intraspecific cross (densely haired cotton (Liaoyang duomao mian) × hairless cotton (Zong 128)) and characterized phenotypically for leaf and stem pubescence in different environments. Here we identify and report significant QTLs (quantitative trait loci) associated with leaf and stem pubescence, and the response of plant under pest (aphid) infestation. Further, we identified putative genes colocalized on chromosome A06 governing mechanism for trichome development and host-pest interaction. Our study provides a comprehensive insight into genetic architecture that can be employed to improve molecular marker-assisted breeding programs aimed at developing biotic (insect pests) resilient cotton cultivars.

Cotton is widely cultivated globally because it provides natural fibre for the textile industry and human use. To identify quantitative trait loci (QTLs)/genes associated with fibre quality and yield, a recombinant inbred line (RIL) population was developed in upland cotton. A consensus map covering the whole genome was constructed with three types of markers (8295 markers, 5197.17 centimorgans (cM)). Six fibre yield and quality traits were evaluated in 17 environments, and 983 QTLs were identified, 198 of which were stable and mainly distributed on chromosomes 4, 6, 7, 13, 21 and 25. Thirty-seven QTL clusters were identified, in which 92.8% of paired traits with significant medium or high positive correlations had the same QTL additive effect directions, and all of the paired traits with significant medium or high negative correlations had opposite additive effect directions. In total, 1297 genes were discovered in the QTL clusters, 414 of which were expressed in two RNA-Seq data sets. Many genes were discovered, 23 of which were promising candidates. Six important QTL clusters that included both fibre quality and yield traits were identified with opposite additive effect directions, and those on chromosome 13 (qClu-chr13-2) could increase fibre quality but reduce yield; this result was validated in a natural population using three markers. These data could provide information about the genetic basis of cotton fibre quality and yield and help cotton breeders to improve fibre quality and yield simultaneously., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Fiber quality and yield are important traits of cotton. Quantitative trait locus (QTL) mapping is a prerequisite for marker-assisted selection (MAS) in cotton breeding. To identify QTLs for fiber quality and yield traits, 4 backcross-generation populations (BC 1 F 1 , BC 1 S 1 , BC 2 F 1 , and BC 3 F 0 ) were developed from an interspecific cross between CCRI36 (Gossypium hirsutum L.) and Hai1 (G. barbadense L.). A total of 153 QTLs for fiber quality and yield traits were identified based on data from the BC 1 F 1 , BC 1 S 1 , BC 2 F 1 and BC 3 F 0 populations in the field and from the BC 2 F 1 population in an artificial disease nursery using a high-density genetic linkage map with 2292 marker loci covering 5115.16 centimorgans (cM) from the BC 1 F 1 population. These QTLs were located on 24 chromosomes, and each could explain 4.98-19.80% of the observed phenotypic variations. Among the 153 QTLs, 30 were consistent with those identified previously. Specifically, 23 QTLs were stably detected in 2 or 3 environments or generations, 6 of which were consistent with those identified previously and the other 17 of which were stable and novel. Ten QTL clusters for different traits were found and 9 of them were novel, which explained the significant correlations among some phenotypic traits in the populations. The results including these stable or consensus QTLs provide valuable information for marker-assisted selection (MAS) in cotton breeding and will help better understand the genetic basis of fiber quality and yield traits, which can then be used in QTL cloning.

Millions of hectares of land are too saline to produce economically valuable crop yields. Salt tolerance in cotton is an imperative approach for improvement in response to ever-increasing soil salinization. Little is known about the genetic basis of salt tolerance in cotton at the seedling stage. To address this issue, a genome-wide association study (GWAS) was conducted on a core collection of a genetically diverse population of upland cotton ( Gossypium hirsutum L.) comprising of 419 accessions, representing various geographic origins, including China, USA, Pakistan, the former Soviet Union, Chad, Australia, Brazil, Mexico, Sudan, and Uganda. Phenotypic evaluation of 7 traits under control (0 mM) and treatment (150 mM) NaCl conditions depicted the presence of broad natural variation in the studied population. The association study was carried out with the efficient mixed-model association eXpedited software package. A total of 17,264 single-nucleotide polymorphisms (SNPs) associated with different salinity stress tolerance related traits were found. Twenty-three candidate SNPs related to salinity stress-related traits were selected. Final key SNPs were selected based on the r 2 value with nearby SNPs in a linkage disequilibrium (LD) block. Twenty putative candidate genes surrounding SNPs, A10_95330133 and D10_61258588, associated with leaf relative water content, RWC_150, and leaf fresh weight, FW_150, were identified, respectively. We further validated the expression patterns of twelve candidate genes with qRT-PCR, which revealed different expression levels in salt-tolerant and salt-sensitive genotypes. The results of our GWAS provide useful knowledge about the genetic control of salt tolerance at the seedling stage, which could assist in elucidating the genetic and molecular mechanisms of salinity stress tolerance in cotton plants.

Background: C2H2-zinc finger protein family is commonly found in the plant, and it is known as the key actors in the regulation of transcription and vital component of chromatin structure. A large number of the C2H2-zinc finger gene members have not been well characterized based on their functions and structure in cotton. However, in other plants, only a few C2H2-zinc finger genes have been studied., Results: In this work, we performed a comprehensive analysis and identified 386, 196 and 195 C2H2-zinc finger genes in Gossypium hirsutum (upland cotton), Gossypium arboreum and Gossypium raimondii, respectively. Phylogenetic tree analysis of the C2H2-zinc finger proteins encoding the C2H2-zinc finger genes were classified into seven (7) subgroups. Moreover, the C2H2-zinc finger gene members were distributed in all cotton chromosomes though with asymmetrical distribution patterns. All the orthologous genes were detected between tetraploid and the diploid cotton, with 154 orthologous genes pair detected between upland cotton and Gossypium arboreum while 165 orthologous genes were found between upland cotton and Gossypium raimondii. Synonymous (Ks) and non-synonymous (Ka) nucleotide substitution rates (Ka/Ks) analysis indicated that the cotton C2H2-zinc finger genes were highly influenced mainly by negative selection, which maintained their protein levels after the duplication events. RNA-seq data and RT-qPCR validation of the RNA seq result revealed differential expression pattern of some the C2H2-zinc finger genes at different stages of cotton fiber development, an indication that the C2H2-zinc finger genes play an important role in initiating and regulating fiber development in cotton., Conclusions: This study provides a strong foundation for future practical genome research on C2H2-zinc finger genes in upland cotton. The expression levels of C2H2-zinc finger genes family is a pointer of their involvement in various biochemical and physiological functions which are directly related to cotton fiber development during initiation and elongation stages. This work not only provides a basis for determining the nominal role of the C2H2-zinc finger genes in fiber development but also provide valuable information for characterization of potential candidate genes involved in regulation of cotton fiber development.

Background: Long non-coding RNAs (LncRNAs) are part of genes, which are not translated into proteins and play a vital role in plant growth and development. Nevertheless, the presence of LncRNAs and how they functions in Ligon-lintless-1 mutant during the early cessation of cotton fiber development are still not well understood. In order to investigate the function of LncRNAs in cotton fiber development, it is necessary and important to identify LncRNAs and their potential roles in fiber cell development., Results: In this work, we identified 18,333 LncRNAs, with the proportion of long intergenic noncoding RNAs (LincRNAs) (91.5%) and anti-sense LncRNAs (8.5%), all transcribed from Ligon-lintless-1 (Li1) and wild-type (WT). Expression differences were detected between Ligon-lintless-1 and wild-type at 0 and 8 DPA (day post anthesis). Pathway analysis and Gene Ontology based on differentially expressed LncRNAs on target genes, indicated fatty acid biosynthesis and fatty acid elongation being integral to lack of fiber in mutant cotton. The result of RNA-seq and RT-qPCR clearly singles out two potential LncRNAs, LNC_001237 and LNC_017085, to be highly down-regulated in the mutant cotton. The two LncRNAs were found to be destabilized or repressed by ghr-miR2950. Both RNA-seq analysis and RT-qPCR results in Ligon-lintless-1 mutant and wild-type may provide strong evidence of LNC_001237, LNC_017085 and ghr-miR2950 being integral molecular elements participating in various pathways of cotton fiber development., Conclusion: The results of this study provide fundamental evidence for the better understanding of LncRNAs regulatory role in the molecular pathways governing cotton fiber development. Further research on designing and transforming LncRNAs will help not only in the understanding of their functions but will also in the improvement of fiber quality.

Metacaspases (MCs) are cysteine proteases that are important for programmed cell death (PCD) in plants. In this study, we identified 89 MC genes in the genomes of four Gossypium species ( Gossypium raimondii , Gossypium barbadense , Gossypium hirsutum , and Gossypium arboreum ), and classified them as type-I or type-II genes. All of the type-I and type-II MC genes contain a sequence encoding the peptidase C14 domain. During developmentally regulated PCD, type-II MC genes may play an important role related to fiber elongation, while type-I genes may affect the thickening of the secondary wall. Additionally, 13 genes were observed to be differentially expressed between two cotton lines with differing fiber strengths, and four genes ( GhMC02 , GhMC04 , GhMC07 , and GhMC08 ) were predominantly expressed in cotton fibers at 5-30 days post-anthesis (DPA). During environmentally induced PCD, the expression levels of four genes were affected in the root, stem, and leaf tissues within 6 h of an abiotic stress treatment. In general, the MC gene family affects the development of cotton fibers, including fiber elongation and fiber thickening while four prominent fiber- expressed genes were identified. The effects of the abiotic stress and hormone treatments imply that the cotton MC gene family may be important for fiber development. The data presented herein may form the foundation for future investigations of the MC gene family in Gossypium species.

Microtubules (MTs) are of importance to fiber development. The Xklp2 (TPX2) proteins as a class of microtubule-associated proteins (MAPs) play a key role in plant growth and development by regulating the dynamic changes of microtubules (MTs). However, the mechanism underlying this is unknown. The interactions between TPX2 proteins and tubulin protein, which are the main structural components, have not been studied in fiber development of upland cotton. Therefore, a genome-wide analysis of the TPX2 family was firstly performed in Gossypium hirsutum L. This study identified 41 GhTPX2 sequences in the assembled G . hirsutum genome by a series of bioinformatic methods. Generally, this gene family is phylogenetically grouped into six subfamilies, and 41 G . hirsutum TPX2 genes (GhTPX2s) are distributed across 21 chromosomes. A heatmap of the TPX2 gene family showed that homologous GhTPX2 genes, GhWDLA 2/7 and GhW DLA 4/9, have large differences in expression levels between two upland cotton recombinant inbred lines (69307 and 69362) that are different in fiber quality at 15 and 20 days post anthesis. The relative data indicate that these four genes are down-regulated under oryzalin, which causes microtubule depolymerization, as determined via qRT-PCR. A subcellular localization experiment suggested that GhWDLA 2 and GhWDLA 7 are localized to the microtubule cytoskeleton, and GhWDLA4 and GhWDLA9 are only localized to the nucleus. However, only GhWDLA7 between GhWDLA2 and GhWDLA7 interacted with GhTUA2 in the yeast two-hybrid assay. These results lay the foundation for further function study of the TPX2 gene family., Competing Interests: The authors declare no conflicts of interest.

Salinity is not only a major environmental factor which limits plant growth and productivity, but it has also become a worldwide problem. However, little is known about the genetic basis underlying salt tolerance in cotton. This study was carried out to identify marker-trait association signals of seven salt-tolerance-related traits and one salt tolerance index using association analysis for 215 accessions of Asiatic cotton. According to a comprehensive index of salt tolerance (CIST), 215 accessions were mainly categorized into four groups, and 11 accessions with high salinity tolerance were selected for breeding. Genome-wide association studies (GWAS) revealed nine SNP rich regions significantly associated with relative fresh weight (RFW), relative stem length (RSL), relative water content (RRWC) and CIST. The nine SNP rich regions analysis revealed 143 polymorphisms that distributed 40 candidate genes and significantly associated with salt tolerance. Notably, two SNP rich regions on chromosome 7 were found to be significantly associated with two salinity related traits, RFW and RSL, by the threshold of -log 10 P ≥ 6.0, and two candidate genes (Cotton_A_37775 and Cotton_A_35901) related to two key SNPs (Ca7_33607751 and Ca7_77004962) were possibly associated with salt tolerance in G. arboreum . These can provide fundamental information which will be useful for future molecular breeding of cotton, in order to release novel salt tolerant cultivars.

Upland cotton ( Gossypium hirsutum ) is grown for its elite fiber. Understanding differential gene expression patterns during fiber development will help to identify genes associated with fiber quality. In this study, we used two recombinant inbred lines (RILs) differing in fiber quality derived from an intra- hirsutum population to explore expression profiling differences and identify genes associated with high-quality fiber or specific fiber-development stages using RNA sequencing. Overall, 72/27, 1137/1584, 437/393, 1019/184, and 2555/1479 differentially expressed genes were up-/down-regulated in an elite fiber line (L1) relative to a poor-quality fiber line (L2) at 10, 15, 20, 25, and 30 days post-anthesis, respectively. Three-hundred sixty-three differentially expressed genes (DEGs) between two lines were colocalized in fiber strength (FS) quantitative trait loci (QTL). Short Time-series Expression Miner (STEM) analysis discriminated seven expression profiles; gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation were performed to identify difference in function between genes unique to L1 and L2. Co-expression network analysis detected five modules highly associated with specific fiber-development stages, especially for high-quality fiber tissues. The hub genes in each module were identified by weighted gene co-expression network analysis. Hub genes encoding actin 1, Rho GTPase-activating protein with PAK-box, TPX2 protein, bHLH transcription factor, and leucine-rich repeat receptor-like protein kinase were identified. Correlation networks revealed considerable interaction among the hub genes, transcription factors, and other genes.

Transcripts of uncertain coding potential (TUCP) are part of the LncRNAs, which encode some polypeptides. However, the abundance of TUCP transcripts and their roles in Ligon-linless-1 (Li-1) cotton mutant during the early termination of fiber development are still not documented. Li-1 mutant is one of the excellent modules for investigating fiber elongation processes due to its unique fiber developmental stages. To examine the function of TUCP in cotton fiber development, it is important to identify TUCPs and their involvement in fiber development. In this study, we found that 11104 TUCP transcripts were removed by coding potential criteria of Pfam domain scan. Additionally, differential expression levels of TUCP transcripts were detected between Li-1 mutant and the wild-type (WT), which imply their possible functions in cotton fiber development. These results further revealed that a great number of differentially expressed TUCP transcripts in cotton were identified at 8 DPA, followed by 0 DPA and stem. However, these might explain an undesirable function in cotton fiber development. The gene ontology and pathway analysis, based on differential expression patterns of TUCP transcripts on targeted genes, identified the transport process, cytoskeleton structure, membrane permeability and fatty acids. These give new insight into significant involvement in early cessation of cotton fiber development and abnormal stem. The RNA-seq and qRT-PCR expression analyses of TUCP transcripts evidently singled out three possible genes, TUCP_010675, TUCP_001475, TUCP_009444 and other targeted mRNAs. The expression pattern of TUCP transcripts and their mRNA targets provided valuable evidence for further investigations on the biological functions of TUCP in cotton fiber development. The study findings may serve as a useful tool for comparative analysis of TUCP transcripts in cotton species and assist in selection of the applicable candidate genes for further functional analyses, genetic improvement and genetic engineering of cotton fiber development.

GATA transcription factors (TFs), which bind to DNA in regulatory regions, are involved in cell differentiation and possess a type-IV zinc finger and a DNA-binding domain. GATA genes have been characterized in plant species such as Arabidopsis thaliana, Oryza sativa, and Glycine max, and their functions have been elucidated in A. thaliana. Although many Gossypium quantitative trait loci for fiber quality harbor GATA TFs, GATA genes have not yet been characterized in cotton. In this study, we identified 179 GATA genes from the genomes of three Gossypium species. We analyzed the phylogenetic relationships, chromosomal distribution, gene structure, expression pattern, and predicted promoters of all 179 Gossypium GATA genes (46 in G. raimondii, 46 in G. arboreum, and 87 in G. hirsutum). Phylogenetic analysis grouped the 179 GATA genes into four subfamilies. Domain analysis revealed that GATA domains in subfamilies I, II, and III were located near the C-terminal, whereas those in subfamily IV were adjacent to the N-terminal. RNA-seq and (Real-time PCR) qRT-PCR revealed that 39.1% (34/87) of GATA genes were expressed in growing plant tissues in G. hirsutum, but only 12.6% (11/87) were expressed during fiber development. In addition, 45.7% (21/46) and 26.1% (12/46) of GATA genes were expressed in G. arboreum and G. raimondii, respectively. Our results may be useful for elucidating the evolution, expression patterns, and functional divergence of GATA genes in Gossypium., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An allopolyploidization event formed allotetraploid Gossypium species from an A-genome diploid species and a D-genome diploid species. To explore the responses of transposable elements (TEs) to allopolyploidy, we assembled parallel TE datasets from G. hirsutum, G. arboreum and G. raimondii and analyzed the TE types and the effects of TEs on orthologous gene expression in the three Gossypium genomes. Gypsy was the most abundant TE type and most TEs were located ∼500 bp from genes in all three genomes. In G. hirsutum, 35.6% of genes harbored TE insertions, whereas insertions were more frequent in G. arboreum and G. raimondii. G. hirsutum had the highest proportion of uniquely matching 24-nt small interfering RNAs (siRNAs) that targeted TEs. TEs, particularly those targeted by 24-nt siRNAs, were associated with reduced gene expression, but the effect of TEs on orthologous gene expression varied substantially among species. Orthologous gene expression levels in G. hirsutum were intermediate between those of G. arboreum and G. raimondii, which did not experience TE expansion or reduction resulting from allopolyploidization. This study underscores the diversity of TEs co-opted by host genes and provides insights into the roles of TEs in regulating gene expression in Gossypium., (© 2018 Institute of Botany, Chinese Academy of Sciences.)

Background: Heterosis, a multigenic complex trait extrapolated as sum total of many phenotypic features, is widely utilized phenomenon in agricultural crops for about a century. It is mainly focused on establishing vigorous cultivars with the fact that its deployment in crops necessitates the perspective of genomic impressions on prior selection for metric traits. In spite of extensive investigations, the actual mysterious genetic basis of heterosis is yet to unravel. Contemporary crop breeding is aimed at enhanced crop production overcoming former achievements. Leading cotton improvement programs remained handicapped to attain significant accomplishments., Results: In mentioned context, a comprehensive project was designed involving a large collection of cotton accessions including 284 lines, 5 testers along with their respective F 1 hybrids derived from Line × Tester mating design were evaluated under 10 diverse environments. Heterosis, GCA and SCA were estimated from morphological and fiber quality traits by L × T analysis. For the exploration of elite marker alleles related to heterosis and to provide the material carrying such multiple alleles the mentioned three dependent variables along with trait phenotype values were executed for association study aided by microsatellites in mixed linear model based on population structure and linkage disequilibrium analysis. Highly significant 46 microsatellites were discovered in association with the fiber and yield related traits under study. It was observed that two-thirds of the highly significant associated microsatellites related to fiber quality were distributed on D sub-genome, including some with pleiotropic effect. Newly discovered 32 hQTLs related to fiber quality traits are one of prominent findings from current study. A set of 96 exclusively favorable alleles were discovered and C tester (A971Bt) posited a major contributor of these alleles primarily associated with fiber quality., Conclusions: Hence, to uncover hidden facts lying within heterosis phenomenon, discovery of additional hQTLs is required to improve fibre quality. To grab prominent improvement in influenced fiber quality and yield traits, we suggest the A971 Bt cotton cultivar as fundamental element in advance breeding programs as a parent of choice.

WD40 repeat proteins are largely distributed across the plant kingdom and play an important role in diverse biological activities. In this work, we performed genome-wide identification, characterization, and expression level analysis of WD40 genes in cotton. A total of 579, 318, and 313 WD40 genes were found in Gossypium hirsutum, G. arboreum, and G. raimondii, respectively. Based on phylogenetic tree analyses, WD40 genes were divided into 11 groups with high similarities in exon/intron features and protein domains within the group. Expression analysis of WD40 genes showed differential expression at different stages of cotton fiber development (0 and 8 DPA) and cotton stem. A number of miRNAs were identified to target WD40 genes that are significantly involved in cotton fiber development during the initiation and elongation stages. These include miR156, miR160, miR162, miR164, miR166, miR167, miR169, miR171, miR172, miR393, miR396, miR398, miR2950, and miR7505. The findings provide a stronger indication of WD40 gene function and their involvement in the regulation of cotton fiber development during the initiation and elongation stages.

Background: Salinity is a major abiotic stress that limits upland cotton growth and reduces fibre production worldwide. To reveal genetic regulation via transcript and protein levels after salt stress, we comprehensively analysed the global changes in mRNA, miRNA, and protein profiles in response to salt stress in two contrasting salt-tolerant cotton genotypes., Results: In the current study, proteomic and mRNA-seq data were combined to reveal that some genes are differentially expressed at both the proteomic and mRNA levels. However, we observed no significant change in mRNA corresponding to most of the strongly differentially abundant proteins. This finding may have resulted from global changes in alternative splicing events and miRNA levels under salt stress conditions. Evidence was provided indicating that several salt stress-responsive proteins can alter miRNAs and modulate alternative splicing events in upland cotton. The results of the stringent screening of the mRNA-seq and proteomic data between the salt-tolerant and salt-sensitive genotypes identified 63 and 85 candidate genes/proteins related to salt tolerance after 4 and 24 h of salt stress, respectively, between the tolerant and sensitive genotype. Finally, we predicted an interaction network comprising 158 genes/proteins and then discovered that two main clusters in the network were composed of ATP synthase (CotAD_74681) and cytochrome oxidase (CotAD_46197) in mitochondria. The results revealed that mitochondria, as important organelles involved in energy metabolism, play an essential role in the synthesis of resistance proteins during the process of salt exposure., Conclusion: We provided a plausible schematic for the systematic salt tolerance model; this schematic reveals multiple levels of gene regulation in response to salt stress in cotton and provides a list of salt tolerance-related genes/proteins. The information here will facilitate candidate gene discovery and molecular marker development for salt tolerance breeding in cotton.

Background: Cottonseed is one of the most important raw materials for plant protein, oil and alternative biofuel for diesel engines. Understanding the complex genetic basis of cottonseed traits is requisite for achieving efficient genetic improvement of the traits. However, it is not yet clear about their genetic architecture in genomic level. GWAS has been an effective way to explore genetic basis of quantitative traits in human and many crops. This study aims to dissect genetic mechanism seven cottonseed traits by a GWAS for genetic improvement., Results: A genome-wide association study (GWAS) based on a full gene model with gene effects as fixed and gene-environment interaction as random, was conducted for protein, oil and 5 fatty acids using 316 accessions and ~ 390 K SNPs. Totally, 124 significant quantitative trait SNPs (QTSs), consisting of 16, 21, 87 for protein, oil and fatty acids (palmitic, linoleic, oleic, myristic, stearic), respectively, were identified and the broad-sense heritability was estimated from 71.62 to 93.43%; no QTS-environment interaction was detected for the protein, the palmitic and the oleic contents; the protein content was predominantly controlled by epistatic effects accounting for 65.18% of the total variation, but the oil content and the fatty acids except the palmitic were mainly determined by gene main effects and no epistasis was detected for the myristic and the stearic. Prediction of superior pure line and hybrid revealed the potential of the QTSs in the improvement of cottonseed traits, and the hybrid could achieve higher or lower genetic values compared with pure lines., Conclusions: This study revealed complex genetic architecture of seven cottonseed traits at whole genome-wide by mixed linear model approach; the identified genetic variants and estimated genetic component effects of gene, gene-gene and gene-environment interaction provide cotton geneticist or breeders new knowledge on the genetic mechanism of the traits and the potential molecular breeding design strategy.

Upland cotton is the most important natural-fiber crop. The genomic variation of diverse germplasms and alleles underpinning fiber quality and yield should be extensively explored. Here, we resequenced a core collection comprising 419 accessions with 6.55-fold coverage depth and identified approximately 3.66 million SNPs for evaluating the genomic variation. We performed phenotyping across 12 environments and conducted genome-wide association study of 13 fiber-related traits. 7,383 unique SNPs were significantly associated with these traits and were located within or near 4,820 genes; more associated loci were detected for fiber quality than fiber yield, and more fiber genes were detected in the D than the A subgenome. Several previously undescribed causal genes for days to flowering, fiber length, and fiber strength were identified. Phenotypic selection for these traits increased the frequency of elite alleles during domestication and breeding. These results provide targets for molecular selection and genetic manipulation in cotton improvement.

The ancestors of Gossypium arboreum and Gossypium herbaceum provided the A subgenome for the modern cultivated allotetraploid cotton. Here, we upgraded the G. arboreum genome assembly by integrating different technologies. We resequenced 243 G. arboreum and G. herbaceum accessions to generate a map of genome variations and found that they are equally diverged from Gossypium raimondii. Independent analysis suggested that Chinese G. arboreum originated in South China and was subsequently introduced to the Yangtze and Yellow River regions. Most accessions with domestication-related traits experienced geographic isolation. Genome-wide association study (GWAS) identified 98 significant peak associations for 11 agronomically important traits in G. arboreum. A nonsynonymous substitution (cysteine-to-arginine substitution) of GaKASIII seems to confer substantial fatty acid composition (C16:0 and C16:1) changes in cotton seeds. Resistance to fusarium wilt disease is associated with activation of GaGSTF9 expression. Our work represents a major step toward understanding the evolution of the A genome of cotton.

The cellulose synthase gene superfamily, which includes the cellulose synthase (Ces) and cellulose synthase-like (Csl) families, is involved in the synthesis of cellulose and hemicellulose. This superfamily is critical for cotton fiber development in Gossypium species. Applying a series of bioinformatic methods, we identified 228 Ces/Csl genes from four Gossypium species (G. hirsutum, G. barbadense, G. arboreum, and G. raimondii). These genes were then grouped into 11 subfamilies based on phylogenetic relationships. A subsequent analysis of gene evolution revealed sites in CSLG and CSLJ genes that were under long-term positive selection pressure, with a posterior probability >0.95. Moreover, the d N :d S value for the CSLJ clade was 1.305, suggesting this subfamily was under positive selection pressure. Our data indicated that the d N :d S value ranged from 0.0084 to 0.9693 among the homologous Ces/Csl genes, implying they were under purifying selection pressure. Our transcriptome and qRT-PCR analyses revealed that CesA genes were more highly expressed in tetraploids than in diploids. However, the Csl expression levels exhibited the opposite trend. Furthermore, changes to promoter sequences may have influenced the expression of homologous Ces/Csl genes. Our findings may provide novel insights into the evolutionary relationships and expression patterns of the Ces/Csl genes in Gossypium species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Fiber quality and yield improvement are crucial for cotton domestication and breeding. With the transformation in spinning techniques and multiplicity needs, the development of cotton fiber quality and yield is of great importance. A genetic map of 5178 Single Nucleotide Polymorphism (SNP) markers were generated using 277 F 2:3 population, from an intra-specific cross between two upland cotton accessions, CCRI35 a high fiber quality as female and Nan Dan Ba Di Da Hua (NH), with good yield properties as male parent. The map spanned 4768.098 cM with an average distance of 0.92 cM. A total of 110 Quantitative Traits Loci (QTLs) were identified for 11 traits, but only 30 QTLs were consistent in at least two environments. The highest percentage of phenotypic variance explained by a single QTL was 15.45%. Two major cluster regions were found, cluster 1 (chromosome17-D03) and cluster 2 (chromosome26-D12). Five candidate genes were identified in the two QTL cluster regions. Based on GO functional annotation, all the genes were highly correlated with fiber development, with functions such as protein kinase and phosphorylation. The five genes were associated with various fiber traits as follows: Gh_D03G0889 linked to qFM-D03_cb, Gh_D12G0093 , Gh_D12G0410 , Gh_D12G0435 associated with qFS-D12_cb and Gh_D12G0969 linked to qFY-D12_cb. Further structural annotation and fine mapping is needed to determine the specific role played by the five identified genes in fiber quality and yield related pathway., Competing Interests: The authors declare no conflict of interest.

As high-strength cotton fibers are critical components of high quality cotton, developing cotton cultivars with high-strength fibers as well as high yield is a top priority for cotton development. Recently, chromosome segment substitution lines (CSSLs) have been developed from high-yield Upland cotton ( Gossypium hirsutum ) crossed with high-quality Sea Island cotton ( G. barbadense ). Here, we constructed a CSSL population by crossing CCRI45, a high-yield Upland cotton cultivar, with Hai1, a Sea Island cotton cultivar with superior fiber quality. We then selected two CSSLs with significantly higher fiber strength than CCRI45 (MBI7747 and MBI7561), and one CSSL with lower fiber strength than CCRI45 (MBI7285), for further analysis. We sequenced all four transcriptomes at four different time points postanthesis, and clustered the 44,678 identified genes by function. We identified 2200 common differentially-expressed genes (DEGs): those that were found in both high quality CSSLs (MBI7747 and MBI7561), but not in the low quality CSSL (MBI7285). Many of these genes were associated with various metabolic pathways that affect fiber strength. Upregulated DEGs were associated with polysaccharide metabolic regulation, single-organism localization, cell wall organization, and biogenesis, while the downregulated DEGs were associated with microtubule regulation, the cellular response to stress, and the cell cycle. Further analyses indicated that three genes, XLOC_036333 [mannosyl-oligosaccharide-α-mannosidase ( MNS1 )], XLOC_029945 ( FLA8 ), and XLOC_075372 ( snakin-1 ), were potentially important for the regulation of cotton fiber strength. Our results suggest that these genes may be good candidates for future investigation of the molecular mechanisms of fiber strength formation and for the improvement of cotton fiber quality through molecular breeding., (Copyright © 2017 Lu et al.)

MBI9915 is an introgression cotton line with excellent fiber quality. It was obtained by advanced backcrossing and continuous inbreeding from an interspecific cross between the upland cotton (Gossypium hirsutum) cultivar CCRI36 as the recurrent parent and the sea island cotton (G. barbadense) cultivar Hai1, as the donor parent. To study the genetic effects of the introgressed chromosome segments in G. hirsutum, an F2 secondary segregating population of 1537 individuals was created by crossing MBI9915 and CCRI36, and an F2:3 population was created by randomly selecting 347 individuals from the F2 generation. Quantitative trait locus (QTL) mapping and interaction for fiber length and strength were identified using IciMapping software. The genotype analysis showed that the recovery rate for MBI9915 was 97.9%, with a total 6 heterozygous segments and 13 homozygous segments. A total of 18 QTLs for fiber quality and 6 QTLs for yield related traits were detected using the two segregating generations. These QTLs were distributed across 7 chromosomes and collectively explained 0.81%-9.51% of the observed phenotypic variations. Six QTLs were consistently detected in two generations and 6 QTLs were identified in previous studies. A total of 13 pairs of interaction for fiber length and 13 pairs of interaction for fiber strength were identified in two generations. Among them, 3 pairs of interaction for fiber length and 3 pairs of interaction for fiber strength could be identified in all generations; 4 pairs of interactions affected fiber length and fiber strength simultaneously. The results clearly showed that 5 chromosome segments (Seg-5-1, Seg-7-1, Seg-8-1, Seg-20-2 and Seg-20-3) have important effects on fiber yield and quality. This study provides the useful information for gene cloning and marker-assisted breeding for excellent fiber related quality.

Upland cotton (Gossypium hirsutum) is the most important natural fiber crop in the world. The overall genetic diversity among cultivated species of cotton and the genetic changes that occurred during their improvement are poorly understood. Here we report a comprehensive genomic assessment of modern improved upland cotton based on the genome-wide resequencing of 318 landraces and modern improved cultivars or lines. We detected more associated loci for lint yield than for fiber quality, which suggests that lint yield has stronger selection signatures than other traits. We found that two ethylene-pathway-related genes were associated with increased lint yield in improved cultivars. We evaluated the population frequency of each elite allele in historically released cultivar groups and found that 54.8% of the elite genome-wide association study (GWAS) alleles detected were transferred from three founder landraces: Deltapine 15, Stoneville 2B and Uganda Mian. Our results provide a genomic basis for improving cotton cultivars and for further evolutionary analysis of polyploid crops.

Background: Pectin methylesterase (PME, EC 3.1.1.11) is a hydrolytic enzyme that utilizes pectin as substrates, and plays a significant role in regulating pectin reconstruction thereby regulating plant growth. Pectin is one of the important components of the plant cell wall, which forms the main structural material of cotton fiber. In this research, cotton genome information was used to identify PMEs., Results: We identified 80 (GaPME01-GaPME80) PME genes from diploid G. arboreum (A genome), 78 (GrPME01-GrPME78) PME genes from G. raimondii (D genome), and 135 (GhPME001-GhPME135) PME genes from tetraploid cotton G. hirsutum (AD genome). We further analyzed their gene structure, conserved domain, gene expression, and systematic evolution to lay the foundation for deeper research on the function of PMEs. Phylogenetic data indicated that members from the same species demonstrated relatively high sequence identities and genetic similarities. Analysis of gene structures showed that most of the PMEs genes had 2-3 exons, with a few having a variable number of exons from 4 to 6. There are nearly no differences in the gene structure of PMEs among the three (two diploid and one tetraploid) cotton species. Selective pressure analysis showed that the Ka/Ks value for each of the three cotton species PME families was less than one., Conclusion: Conserved domain analysis showed that PMEs members had a relatively conserved C-terminal pectinesterase domain (PME) while the N-terminus was less conserved. Moreover, some of the family members contained a pectin methylesterase inhibitor (PMEI) domain. The Ka/Ks ratios suggested that the duplicated PMEs underwent purifying selection after the duplication events. This study provided an important basis for further research on the functions of cotton PMEs. Results from qRT-PCR indicated that the expression level of different PMEs at various fiber developmental stages was different. Moreover, some of the PMEs showed fiber predominant expression in secondary wall thickening indicating tissue-specific expression patterns.

Background: Verticillium wilt (VW) caused by Verticillium dahliae (Kleb) is one of the most destructive diseases of cotton. The identification of highly resistant QTLs or genes in the whole cotton genome is quite important for developing a VW-resistant variety and for further molecular design breeding., Results: In the present study, BC 1 F 1 , BC 1 S 1 , and BC 2 F 1 populations derived from an interspecific backcross between the highly resistant line Hai1 (Gossypium barbadense L.) and the susceptible variety CCRI36 (G. hirsutum L.) as the recurrent parent were constructed. Quantitative trait loci (QTL) related to VW resistance were detected in the whole cotton genome using a high-density simple sequence repeat (SSR) genetic linkage map from the BC 1 F 1 population, with 2292 loci covering 5115.16 centiMorgan (cM) of the cotton (AD) genome, and the data concerning VW resistance that were obtained from four dates of BC 2 F 1 in the artificial disease nursery and one date of BC 1 S 1 and BC 2 F 1 in the field. A total of 48 QTLs for VW resistance were identified, and 37 of these QTLs had positive additive effects, which indicated that the G. barbadense alleles increased resistance to VW and decreased the disease index (DI) by about 2.2-10.7. These QTLs were located on 19 chromosomes, in which 33 in the A subgenome and 15 QTLs in the D subgenome. The 6 QTLs were found to be stable. The 6 QTLs were consistent with those identified previously, and another 42 were new, unreported QTLs, of which 31 QTLs were from G. barbadense. By meta-analysis, 17 QTL hotspot regions were identified and 10 of them were new, unreported hotspot regions. 29 QTLs in this paper were in 12 hotspot regions and were all from G. barbadense., Conclusions: These stable or consensus QTL regions warrant further investigation to better understand the genetics and molecular mechanisms underlying VW resistance. This study provides useful information for further comparative analysis and marker-assisted selection in the breeding of disease-resistant cotton. It may also lay an important foundation for gene cloning and further molecular design breeding for the entire cotton genome.

The capacity for ion compartmentalization among different tissues and cells is the key mechanism regulating salt tolerance in plants. In this study, we investigated the ion compartmentalization capacity of two upland cotton genotypes with different salt tolerances under salt shock at the tissue, cell and molecular levels. We found that the leaf glandular trichome could secrete more salt ions in the salt-tolerant genotype than in the sensitive genotype, demonstrating the excretion of ions from tissue may be a new mechanism to respond to short-term salt shock. Furthermore, an investigation of the ion distribution demonstrated that the ion content was significantly lower in critical tissues and cells of the salt-tolerant genotype, indicating the salt-tolerant genotype had a greater capacity for ion compartmentalization in the shoot. By comparing the membrane H + -ATPase activity and the expression of ion transportation-related genes, we found that the H + -ATPase activity and Na + /H + antiporter are the key factors determining the capacity for ion compartmentalization in leaves, which might further determine the salt tolerance of cotton. The novel function of the glandular trichome and the comparison of Na + compartmentalization between two cotton genotypes with contrasting salt tolerances provide a new understanding of the salt tolerance mechanism in cotton.

Background: MYB family proteins are one of the most abundant transcription factors in the cotton plant and play diverse roles in cotton growth and evolution. Previously, few studies have been conducted in upland cotton, Gossypium hirsutum. The recent release of the G. hirsutum genome sequence provides a great opportunity to identify and characterize the entire upland cotton MYB protein family., Results: In this study, we undertook a comprehensive genome-wide characterization and expression analysis of the MYB transcription factor family during cotton fiber development. A total of 524 non-redundant cotton MYB genes, among 1986 MYB and MYB-related putative proteins, were identified and classified into four subfamilies including 1R-MYB, 2R-MYB, 3R-MYB, and 4R-MYB. Based on phylogenetic tree analysis, MYB transcription factors were divided into 16 subgroups. The results showed that the majority (69.1 %) of GhMYBs genes belong to the 2R-MYB subfamily in upland cotton., Conclusion: Our comparative genomics analysis has provided novel insights into the roles of MYB transcription factors in cotton fiber development. These results provide the basis for a greater understanding of MYB regulatory networks and to develop new approaches to improve cotton fiber development.

Chromosome segment substitution lines MBI9804, MBI9855, MBI9752, and MBI9134, which were obtained by advanced backcrossing and continuously inbreeding from an interspecific cross between CCRI36, a cultivar of upland cotton (Gossypium hirsutum) as the recurrent parent, and Hai1, a cultivar of sea island cotton (G. barbadense) as the donor parent, were used to construct a multiple parent population of (MBI9804×MBI9855)×(MBI9752×MBI9134). The segregating generations of double-crossed F1 and F2 and F2:3 were used to map the quantitative trait locus (QTL) for fiber quality and yield-related traits. The recovery rate of the recurrent parent CCRI36 in the four parental lines was from 94.3%-96.9%. Each of the parental lines harbored 12-20 introgressed segments from Hai1across 21 chromosomes. The number of introgressed segments ranged from 1 to 27 for the individuals in the three generations, mostly from 9 to 18, which represented a genetic length of between 126 cM and 246 cM. A total of 24 QTLs controlling fiber quality and 11 QTLs controlling yield traits were detected using the three segregating generations. These QTLs were distributed across 11 chromosomes and could collectively explain 1.78%-20.27% of the observed phenotypic variations. Sixteen QTLs were consistently detected in two or more generations, four of them were for fiber yield traits and 12 were for fiber quality traits. One introgressed segment could significantly reduce both lint percentage and fiber micronaire. This study provides useful information for gene cloning and marker-assisted breeding for excellent fiber quality., Competing Interests: The authors have declared that no competing interests exist.

We hybridized 10 chromosome segment substitution lines (CSSLs) each from two CSSL populations and produced 50 F1 hybrids according to North Carolina Design II. We analyzed the genetic effects and heterosis of yield and yield components in the F1 hybrids and parents in four environments via the additive-dominance genetic model. Yield and yield components of the CSSLs were controlled by combined additive and dominance effects, and lint percentage was mainly controlled by additive effects, but boll weight, boll number, seedcotton yield and lint yield were mainly controlled by dominance effects. We detected significant interaction effects between genetics and the environment for all yields traits. Similar interactions were detected between two CSSL populations (Pop CCRI 36 and Pop CCRI 45). Significant positive mid-parent heterosis was detected for all yield traits in both populations, and significant positive better-parent heterosis was also detected for all yield traits except lint percentage. The differences among parents were relatively small, but significant heterosis was detected for yield and yield components. Therefore, the relationship between heterosis and genetic distance for yield traits is complicated and requires further study. These CSSLs represent useful tools for improving yield and yield components in cotton.

Background: Upland Cotton (Gossypium hirsutum) is one of the most important worldwide crops it provides natural high-quality fiber for the industrial production and everyday use. Next-generation sequencing is a powerful method to identify single nucleotide polymorphism markers on a large scale for the construction of a high-density genetic map for quantitative trait loci mapping., Results: In this research, a recombinant inbred lines population developed from two upland cotton cultivars 0-153 and sGK9708 was used to construct a high-density genetic map through the specific locus amplified fragment sequencing method. The high-density genetic map harbored 5521 single nucleotide polymorphism markers which covered a total distance of 3259.37 cM with an average marker interval of 0.78 cM without gaps larger than 10 cM. In total 18 quantitative trait loci of boll weight were identified as stable quantitative trait loci and were detected in at least three out of 11 environments and explained 4.15-16.70 % of the observed phenotypic variation. In total, 344 candidate genes were identified within the confidence intervals of these stable quantitative trait loci based on the cotton genome sequence. These genes were categorized based on their function through gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis and eukaryotic orthologous groups analysis., Conclusions: This research reported the first high-density genetic map for Upland Cotton (Gossypium hirsutum) with a recombinant inbred line population using single nucleotide polymorphism markers developed by specific locus amplified fragment sequencing. We also identified quantitative trait loci of boll weight across 11 environments and identified candidate genes within the quantitative trait loci confidence intervals. The results of this research would provide useful information for the next-step work including fine mapping, gene functional analysis, pyramiding breeding of functional genes as well as marker-assisted selection.

Background: The identification of quantitative trait loci (QTLs) that are stable and consistent across multiple environments and populations plays an essential role in marker-assisted selection (MAS). In the present study, we used 28,861 simple sequence repeat (SSR) markers, which included 12,560 Gossypium raimondii (D genome) sequence-based SSR markers to identify polymorphism between two upland cotton strains 0-153 and sGK9708. A total of 851 polymorphic primers were finally selected and used to genotype 196 recombinant inbred lines (RIL) derived from a cross between 0 and 153 and sGK9708 and used to construct a linkage map. The RIL population was evaluated for fiber quality traits in six locations in China for five years. Stable QTLs identified in this intraspecific cross could be used in future cotton breeding program and with fewer obstacles., Results: The map covered a distance of 4,110 cM, which represents about 93.2 % of the upland cotton genome, and with an average distance of 5.2 cM between adjacent markers. We identified 165 QTLs for fiber quality traits, of which 47 QTLs were determined to be stable across multiple environments. Most of these QTLs aggregated into clusters with two or more traits. A total of 30 QTL clusters were identified which consisted of 103 QTLs. Sixteen clusters in the At sub-genome comprised 44 QTLs, whereas 14 clusters in the Dt sub-genome that included 59 QTLs for fiber quality were identified. Four chromosomes, including chromosome 4 (c4), c7, c14, and c25 were rich in clusters harboring 5, 4, 5, and 6 clusters respectively. A meta-analysis was performed using Biomercator V4.2 to integrate QTLs from 11 environmental datasets on the RIL populations of the above mentioned parents and previous QTL reports. Among the 165 identified QTLs, 90 were identified as common QTLs, whereas the remaining 75 QTLs were determined to be novel QTLs. The broad sense heritability estimates of fiber quality traits were high for fiber length (0.93), fiber strength (0.92), fiber micronaire (0.85), and fiber uniformity (0.80), but low for fiber elongation (0.27). Meta-clusters on c4, c7, c14 and c25 were identified as stable QTL clusters and were considered more valuable in MAS for the improvement of fiber quality of upland cotton., Conclusion: Multiple environmental evaluations of an intraspecific RIL population were conducted to identify stable QTLs. Meta-QTL analyses identified a common chromosomal region that plays an important role in fiber development. Therefore, QTLs identified in the present study are an ideal candidate for MAS in cotton breeding programs to improve fiber quality.

Salinity is a major abiotic stress that affects plant growth and development. In this study, we performed a proteomic analysis of cotton roots and leaf tissue following exposure to saline stress. 611 and 1477 proteins were differentially expressed in the roots and leaves, respectively. In the roots, 259 (42%) proteins were up-regulated and 352 (58%) were down-regulated. In the leaves, 748 (51%) proteins were up-regulated and 729 (49%) were down-regulated. On the basis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, we concluded that the phenylalanine metabolism and starch and sucrose metabolism were active for energy homeostasis to cope with salt stress in cotton roots. Moreover, photosynthesis, pyruvate metabolism, glycolysis / gluconeogenesis, carbon fixation in photosynthetic organisms and phenylalanine metabolism were inhabited to reduce energy consumption. Characterization of the signaling pathways will help elucidate the mechanism activated by cotton in response to salt stress.

Determining how function evolves following gene duplication is necessary for understanding gene expansion. Transcription factors (TFs) are a class of proteins that regulate gene expression by binding to specific cis-acting elements in the promoters of target genes, subsequently activating or repressing their transcription. In the present study, we systematically examined the functional diversification of the NAC transcription factor (NAC-TFs) family by analyzing their chromosomal location, structure, phylogeny, and expression pattern in Gossypium raimondii (Gr) and G. arboreum (Ga). The 145 and 141 NAC genes identified in the Gr and Ga genomes, respectively, were annotated and divided into 18 subfamilies, which showed distinct divergence in gene structure and expression patterns during fiber development. In addition, when the functional parameters were examined, clear divergence was observed within tandem clusters, which suggested that subfunctionalization had occurred among duplicate genes. The expression patterns of homologous gene pairs also changed, suggestive of the diversification of gene function during the evolution of diploid cotton. These findings provide insights into the mechanisms underlying the functional differentiation of duplicated NAC-TFs genes in two diploid cotton species.

Background: Dwarf cottons are more resistant to damage from wind and rain and associated with stable, increased yields, and also desirable source for breeding the machine harvest varieties. In an effort to uncover the transcripts and miRNA networks involved in plant height, the transcriptome and small RNA sequencing were performed based on dwarf mutant Ari1327 (A1), tall-culm mutant Ari3697 (A3) and wild type Ari971 (A9) in Gossypium hirsutum., Methods: The stem apexes of wild-type upland cotton (Ari971) and its dwarf mutant (Ari1327) and tall-culm mutant (Ari3697) at the fifth true leaf stage were extracted for RNA, respectively. Transcriptome and small RNA libraries were constructed and subjected to next generation sequencing., Results: The transcriptome sequencing analysis showed that the enriched pathways of top 3 differentially expressed genes (DEGs) were categorized as carotenoid biosynthesis, plant-pathogen interaction and plant hormone signal transduction in both A1-A9 and A3-A9. The ABA and IAA related factors were differentially expressed in the mutants. Importantly, we found the lower expressed SAUR and elevated expressed GH3, and ABA related genes such as NCED and PP2C maybe relate to reduced growth of the plant height in Ari1327 which was consistent with the higher auxin and ABA content in this mutant. Furthermore, miRNA160 targeted to the auxin response factor (ARF) and miRNA166 (gma-miR166u and gma-miR166h-3p) targeted to ABA responsive element binding factor were related to the mutation in cotton. We have noticed that the cell growth related factors (smg7 targeted by gra-miR482 and 6 novel miRNAs and pectate-lyases targeted by osa-miR159f), the redox reactions related factors (Cytochrome P450 targeted by miR172) and MYB genes targeted by miR828, miR858 and miR159 were also involved in plant height of the cotton mutants. A total of 226 conserved miRNAs representing 32 known miRNA families were obtained, and 38 novel miRNAs corresponding to 23 unique RNA sequences were identified. Total 531 targets for 211 conserved miRNAs were obtained. Using PAREsnip, 27 and 29 miRNA/target conserved interactions were validated in A1-A9 and A3-A9, respectively. Furthermore, miRNA160, miRNA858 and miRNA172 were validated to be up-regulated in A1-A9 but down-regulated in A3-A9, whereas miRNA159 showed the opposite regulation., Conclusions: This comprehensive interaction of the transcriptome and miRNA at tall-culm and dwarf mutant led to the discovery of regulatory mechanisms in plant height. It also provides the basis for in depth analyses of dwarf mutant genes for further breeding of dwarf cotton.

Cotton (Gossypium hirsutum L.) is an important agricultural crop that provides renewable natural fiber resources for the global textile industry. Technological developments in the textile industry and improvements in human living standards have increased the requirement for supplies and better quality cotton. Upland cotton 0-153 is an elite cultivar harboring strong fiber strength genes. To conduct quantitative trait locus (QTL) mapping for fiber quality in 0-153, we developed a population of 196 recombinant inbred lines (RILs) from a cross between 0-153 and sGK9708. The fiber quality traits in 11 environments were measured and a genetic linkage map of chromosome 25 comprising 210 loci was constructed using this RIL population, mainly using simple sequence repeat markers and single nucleotide polymorphism markers. QTLs were identified across diverse environments using the composite interval mapping method. A total of 37 QTLs for fiber quality traits were identified on chromosome 25, of which 17 were stably expressed in at least in two environments. A stable fiber strength QTL, qFS-chr25-4, which was detected in seven environments and was located in the marker interval between CRI-SNP120491 and BNL2572, could explain 6.53%-11.83% of the observed phenotypic variations. Meta-analysis also confirmed the above QTLs with previous reports. Application of these QTLs could contribute to improving fiber quality and provide information for marker-assisted selection.

To introgress the good fiber quality and yield from Gossypium barbadense into a commercial Upland cotton variety, a high-density simple sequence repeat (SSR) genetic linkage map was developed from a BC1 F1 population of Gossypium hirsutum × Gossypium barbadense. The map comprised 2,292 loci and covered 5115.16 centiMorgan (cM) of the cotton AD genome, with an average marker interval of 2.23 cM. Of the marker order for 1,577 common loci on this new map, 90.36% agrees well with the marker order on the D genome sequence genetic map. Compared with five published high-density SSR genetic maps, 53.14% of marker loci were newly discovered in this map. Twenty-six quantitative trait loci (QTLs) for lint percentage (LP) were identified on nine chromosomes. Nine stable or common QTLs could be used for marker-assisted selection. Fifty percent of the QTLs were from G. barbadense and increased LP by 1.07%-2.41%. These results indicated that the map could be used for screening chromosome substitution segments from G. barbadense in the Upland cotton background, identifying QTLs or genes from G. barbadense, and further developing the gene pyramiding effect for improving fiber yield and quality., (© 2014 Institute of Botany, Chinese Academy of Sciences.)

To understand the mechanism of fiber development and pigmentation formation, the mRNAs of two cotton lines were sequenced: line Z128 (light brown fiber) was a selected mutant from line Z263 (dark brown fiber). The primary walls of the fiber cell in both Z263 and Z128 contain pigments; more pigments were laid in the lumen of the fiber cell in Z263 compared with that in Z128. However, Z263 contained less cellulose than Z128. A total of 71,895 unigenes were generated: 13,278 (20.26%) unigenes were defined as differentially expressed genes (DEGs) by comparing the library of Z128 with that of Z263; 5,345 (8.16%) unigenes were up-regulated and 7,933 (12.10%) unigenes were down-regulated. qRT-PCR and comparative transcriptional analysis demonstrated that the pigmentation formation in brown cotton fiber was possibly the consequence of an interaction between oxidized tannins and glycosylated anthocyanins. Furthermore, our results showed the pigmentation related genes not only regulated the fiber color but also influenced the fiber quality at the fiber elongation stage (10 DPA). The highly expressed flavonoid gene in the fiber elongation stage could be related to the fiber quality. DEGs analyses also revealed that transcript levels of some fiber development genes (Ca(2+)/CaM, reactive oxygen, ethylene and sucrose phosphate synthase) varied dramatically between these two cotton lines.

Background: Cotton (Gossypium spp.) is one of the major fibre crops of the world. Although it is classified as salt tolerant crop, cotton growth and productivity are adversely affected by high salinity, especially at germination and seedling stages. Identification of genes and miRNAs responsible for salt tolerance in upland cotton (Gossypium hirsutum L.) would help reveal the molecular mechanisms of salt tolerance. We performed physiological experiments and transcriptome sequencing (mRNA-seq and small RNA-seq) of cotton leaves under salt stress using Illumina sequencing technology., Results: We investigated two distinct salt stress phases--dehydration (4 h) and ionic stress (osmotic restoration; 24 h)--that were identified by physiological changes of 14-day-old seedlings of two cotton genotypes, one salt tolerant and the other salt sensitive, during a 72-h NaCl exposure. A comparative transcriptomics was used to monitor gene and miRNA differential expression at two time points (4 and 24 h) in leaves of the two cotton genotypes under salinity conditions. The expression patterns of differentially co-expressed unigenes were divided into six groups using short time-servies expression miner software. During a 24-h salt exposure, 819 transcription factor unigenes were differentially expressed in both genotypes, with 129 unigenes specifically expressed in the salt-tolerant genotype. Under salt stress, 108 conserved miRNAs from known families were differentially expressed at two time points in the salt-tolerant genotype. We further analyzed the predicted target genes of these miRNAs along with the transcriptome for each time point. Important expressed genes encoding membrane receptors, transporters, and pathways involved in biosynthesis and signal transduction of calcium-dependent protein kinase, mitogen-activated protein kinase, and hormones (abscisic acid and ethylene) were up-regulated. We also analyzed the salt stress response of some key miRNAs and their target genes and found that the expressions of five of nine target genes exhibited significant inverse correlations with their corresponding miRNAs. On the basis of these results, we constructed molecular regulatory pathways and a potential regulatory network for these salt-responsive miRNAs., Conclusions: Our comprehensive transcriptome analysis has provided new insights into salt-stress response of upland cotton. The results should contribute to the development of genetically modified cotton with salt tolerance.