Genome-wide association study for boll weight in Gossypium hirsutum races.

High yield has always been an essential target in almost all of the cotton breeding programs. Boll weight (BW) is a key component of cotton yield. Numerous linkage mapping and genome-wide association studies (GWAS) have been performed to understand the genetic mechanism of BW, but information on the markers/genes controlling BW remains limited. In this study, we conducted a GWAS for BW using 51,268 high-quality single-nucleotide polymorphisms (SNPs) and 189 Gossypium hirsutum accessions across five different environments. A total of 55 SNPs significantly associated with BW were detected, of which 29 and 26 were distributed in the A and D subgenomes, respectively. Five SNPs were simultaneously detected in two environments. For TM5655, TM8662, TM36371, and TM50258, the BW grouped by alleles of each SNP was significantly different. The ± 550 kb regions around these four key SNPs contained 262 genes. Of them, Gh_A02G1473, Gh_A10G1765, and Gh_A02G1442 were expressed highly at 0 to 1 days post-anthesis (dpa), - 3 to 0 dpa, and - 3 to 0 dpa in ovule of TM-1, respectively. They were presumed as the candidate genes for fiber cell differentiation, initiation, or elongation based on gene annotation of their homologs. Overall, these results supplemented valuable information for dissecting the genetic architecture of BW and might help to improve cotton yield through molecular marker-assisted selection breeding and molecular design breeding.

Genetic dissection of lint percentage in short-season cotton using combined QTL mapping and RNA-seq.

KEY MESSAGE: In total, 17 QTLs for lint percentage in short-season cotton, including three stable QTLs, were detected. Twenty-eight differentially expressed genes located within the stable QTLs were identified, and two genes were validated by qRT-PCR. The breeding and use of short-season cotton have significant values in addressing the question of occupying farmlands with either cotton or cereals. However, the fiber yields of short-season cotton varieties are significantly lower than those of middle- and late-maturing varieties. How to effectively improve the fiber yield of short-season cotton has become a focus of cotton research. Here, a high-density genetic map was constructed using genome resequencing and an RIL population generated from the hybridization of two short-season cotton accessions, Dong3 and Dong4. The map contained 4960 bin markers across the 26 cotton chromosomes and spanned 3971.08 cM, with an average distance of 0.80 cM between adjacent markers. Based on the genetic map, quantitative trait locus (QTL) mapping for lint percentage (LP, %), an important yield component trait, was performed. In total, 17 QTLs for LP, including three stable QTLs, qLP-A02, qLP-D04, and qLP-D12, were detected. Three out of 11 non-redundant QTLs overlapped with previously reported QTLs, whereas the other eight were novel QTLs. A total of 28 differentially expressed genes associated with the three stable QTLs were identified using RNA-seq of ovules and fibers at different seed developmental stages from the parental materials. The two genes, Ghir_A02G017640 and Ghir_A02G018500, may be related to LP as determined by further qRT-PCR validation. This study provides useful information for the genetic dissection of LP and promotes the molecular breeding of short-season cotton.

Genome-Wide Association Study of Lint Percentage in Gossypium hirsutum L. Races.

Lint percentage is one of the most essential yield components and an important economic index for cotton planting. Improving lint percentage is an effective way to achieve high-yield in cotton breeding worldwide, especially upland cotton (Gossypium hirsutum L.). However, the genetic basis controlling lint percentage has not yet been systematically understood. Here, we performed a genome-wide association mapping for lint percentage using a natural population consisting of 189 G. hirsutum accessions (188 accessions of G. hirsutum races and one cultivar TM-1). The results showed that 274 single-nucleotide polymorphisms (SNPs) significantly associated with lint percentage were detected, and they were distributed on 24 chromosomes. Forty-five SNPs were detected at least by two models or at least in two environments, and their 5 Mb up- and downstream regions included 584 makers related to lint percentage identified in previous studies. In total, 11 out of 45 SNPs were detected at least in two environments, and their 550 Kb up- and downstream region contained 335 genes. Through RNA sequencing, gene annotation, qRT-PCR, protein-protein interaction analysis, the cis-elements of the promotor region, and related miRNA prediction, Gh_D12G0934 and Gh_A08G0526 were selected as key candidate genes for fiber initiation and elongation, respectively. These excavated SNPs and candidate genes could supplement marker and gene information for deciphering the genetic basis of lint percentage and facilitate high-yield breeding programs of G. hirsutum ultimately.

Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection.

Investigation of cotton response to nematode infection will allow us to better understand the cotton immune defense mechanism and design a better biotechnological approach for efficiently managing pest nematodes in cotton. In this study, we firstly treated cotton by root knot nematode (RKN, Meloidogyne incognita) infections, then we employed the high throughput deep sequencing technology to sequence and genome-widely identify all miRNAs in cotton; finally, we analyzed the functions of these miRNAs in cotton response to RKN infections. A total of 266 miRNAs, including 193 known and 73 novel miRNAs, were identified by deep sequencing technology, which belong to 67 conserved and 66 novel miRNA families, respectively. A majority of identified miRNA families only contain one miRNA; however, miR482 family contains 14 members and some others contain 2-13 members. Certain miRNAs were specifically expressed in RKN-infected cotton roots and others were completely inhibited by RKN infection. A total of 50 miRNAs were differentially expressed after RKN infection, in which 28 miRNAs were up-regulated and 22 were inhibited by RKN treatment. Based on degradome sequencing, 87 gene targets were identified to be targeted by 57 miRNAs. These miRNA-targeted genes are involved in the interaction of cotton plants and nematode infection. Based on GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, 466 genes from all 636 miRNA targets were mapped to 6340 GO terms, 181 genes from 228 targets of differentially expressed miRNAs were mapped to 1588 GO terms. The GO terms were then categorized into the three main GO classes: biological processes, cellular components, and molecular functions. The targets of differentially expressed miRNAs were enriched in 43 GO terms, including 22 biological processes, 10 cellular components, and 11 molecular functions (p < 0.05). Many identified processes were associated with organism responses to the environmental stresses, including regulation of nematode larval development, response to nematode, and response to flooding. Our results will enhance the study and application of developing new cotton cultivars for nematode resistance.

Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity.

Verticillium dahliae (V. dahliae) infects roots and colonizes the vascular vessels of host plants, significantly reducing the economic yield of cotton and other crops. In this study, the protein VdTHI20, which is involved in the thiamine biosynthesis pathway, was characterized by knocking out the corresponding VdTHI20 gene in V. dahliae via Agrobacterium tumefaciens-mediated transformation (ATMT). The deletion of VdTHI20 resulted in several phenotypic defects in vegetative growth and conidiation and in impaired virulence in tobacco seedlings. We show that VdTHI20 increases the tolerance of V. dahliae to UV damage. The impaired vegetative growth of ΔVdTHI20 mutant strains was restored by complementation with a functional copy of the VdTHI20 gene or by supplementation with additional thiamine. Furthermore, the root infection and colonization of the ΔVdTHI20 mutant strains were suppressed, as indicated by green fluorescent protein (GFP)-labelling under microscope observation. When the RNAi constructs of VdTHI20 were used to transform Nicotiana benthamiana, the transgenic lines expressing dsVdTHI20 showed elevated resistance to V. dahliae. Together, these results suggest that VdTHI20 plays a significant role in the pathogenicity of V. dahliae. In addition, the pathogenesis-related gene VdTHI20 exhibits potential for controlling V. dahliae in important crops.

Dissecting the genetic architecture of seed-cotton and lint yields in Upland cotton using genome-wide association mapping.

Seed-cotton yield (SY) and lint yield (LY) are the most important yield traits of cotton. Thus, it is critical to dissect their genetic architecture. Upland cotton (Gossypium hirsutum) is widely grown worldwide. In this study, a genome-wide association mapping was performed based on the CottonSNP80K array to dissect the genetic architecture of SY and LY in Upland cotton. Twenty-three significant associations were detected within four environments, including 11 associated with SY and 12 associated with LY. Seven single nucleotide polymorphisms (SNPs), TM234, TM237, TM247, TM255, TM256, TM263, and TM264, were co-associated with the two traits, which may indicate pleiotropy or intergenic tight linkages. Five SNPs, TM13332, TM39771, TM57119, TM81653, and TM81660, were coincided with those of previous reports and could be used in marker-assisted selection. Combining functional annotations with expression analyses of the genes identified within 400 kb of the significantly associated SNPs, we hypothesize that the three genes, Gh_D05G1077 and Gh_D13G1571 for SY, and Gh_A11G0775 for LY, may have the potential to increase cotton yield. The results would provide useful information for understanding the genetic basis of yield traits in Upland cotton and for facilitating its high-yield breeding through molecular design.

Small RNA Sequencing Reveals Regulatory Roles of MicroRNAs in the Development of Meloidogyne incognita.

MicroRNAs (miRNAs) are an extensive class of small regulatory RNAs. Knowing the specific expression and functions of miRNAs during root-knot nematode (RKN) (Meloidogyne incognita) development could provide fundamental information about RKN development as well as a means to design new strategies to control RKN infection, a major problem of many important crops. Employing high throughput deep sequencing, we identified a total of 45 conserved and novel miRNAs from two developmental stages of RKN, eggs and J2 juveniles, during their infection of cotton (Gossypium hirsutum L.). Twenty-one of the miRNAs were differentially expressed between the two stages. Compared with their expression in eggs, two miRNAs were upregulated (miR252 and miRN19), whereas 19 miRNAs were downregulated in J2 juveniles. Nine miRNAs were expressed at high levels, with >1000 reads per mapped million (RPM) sequenced reads in both eggs and J2 juveniles (miR1, miR124, miR2-3p, miR252, miR279, miR57-5p, miR7904, miR87, and miR92). Three miRNAs were only expressed in eggs (miR4738, miRN3, and miRN5). These differentially expressed miRNAs may control RKN development by regulating specific protein-coding genes in pathways associated with RKN growth and development.

Xylem sap in cotton contains proteins that contribute to environmental stress response and cell wall development.

The xylem sap of a plant is primarily responsible for transporting molecules from the underground root system to the aboveground parts of the plant body. In order to understand the role that roots play in cotton growth and development, the components present in xylem sap must be elucidated. In this study, we used a shotgun HPLC-ESI-MS/MS proteomics approach to identify 455 peptides from the xylem sap of field-grown cotton plants at peak blooming stage. Of these peptides, 384 (84.4%) were found to be secreted proteins and 320 (70.3%) had special molecular functions. Based on Gene Ontology (GO) analysis, 348 peptides were annotated in terms of molecular function, biological process, and cellular localization, with 46.9 and 45.1% being related to catalytic activity and binding activity, respectively. Many xylem sap-containing proteins were predicted to be involved in different phases of xylem differentiation including cell wall metabolism, secondary cell wall development and patterning, and programmed cell death. The identification of starch and sucrose hydrolyzing enzymes implicated the interaction between roots and aboveground parts on the aspect of carbohydrate metabolism. Many of the proteins identified in this study are involved in defense mechanisms including pathogen-related proteins, such as peroxidases, chitinases, and germin-like proteins, proteases involved in disease resistance, and phytoalexin phenylpropanoid synthesis-related proteins. The majority of identified signaling proteins were fasciclin-like arabinogalactan proteins and kinases. The results of this study provide useful insight into the communication mechanisms between cotton roots and the rest of the cotton plant.

Genome-wide identification of auxin response factor (ARF) genes and its tissue-specific prominent expression in Gossypium raimondii.

Auxin response factors (ARFs) are recently discovered transcription factors that bind with auxin response elements (AuxRE, TGTCTC) to regulate the expression of early auxin-responsive genes. To our knowledge, the ARF gene family has never been characterized in cotton, the most important fiber crop in the world. In this study, a total of 35 ARF genes, named as GrARFs, were identified in a diploid cotton species Gossypium raimondii. The 35 ARF genes were located in 12 of the 13 cotton chromosomes; the intron/exon distribution of the GrARF genes was similar among sister pairs, whereas the divergence of some GrARF genes suggests the possibility of functional diversification. Our results show that the middle domains of nine GrARF proteins rich in glutamine (Q) are activators, while 26 other GrARF proteins rich in proline (P), serine (S), and threonine (T) are repressors. Our results also show that the expression of GrARF genes is diverse in different tissues. The expression of GrARF1 was significantly higher in leaves, whereas GrARF2a had higher expression level in shoots, which implicates different roles in the tested tissues. The GrARF11 has a higher expression level in buds than that in leaves, while GrARF19.2 shows contrasting expression patterns, having higher expression in leaves than that in buds. This suggests that they play different roles in leaves and buds. During long-term evolution of G. raimondii, some ARF genes were lost and some arose. The identification and characterization of the ARF genes in G. raimondii elucidate its important role in cotton that ARF genes regulate the development of flower buds, sepals, shoots, and leaves.

Small RNA sequencing identifies miRNA roles in ovule and fibre development.

MicroRNAs (miRNAs) have been found to be differentially expressed during cotton fibre development. However, which specific miRNAs and how they are involved in fibre development is unclear. Here, using deep sequencing, 65 conserved miRNA families were identified and 32 families were differentially expressed between leaf and ovule. At least 40 miRNAs were either leaf or ovule specific, whereas 62 miRNAs were shared in both leaf and ovule. qRT-PCR confirmed these miRNAs were differentially expressed during fibre early development. A total of 820 genes were potentially targeted by the identified miRNAs, whose functions are involved in a series of biological processes including fibre development, metabolism and signal transduction. Many predicted miRNA-target pairs were subsequently validated by degradome sequencing analysis. GO and KEGG analyses showed that the identified miRNAs and their targets were classified to 1027 GO terms including 568 biological processes, 324 molecular functions and 135 cellular components and were enriched to 78 KEGG pathways. At least seven unique miRNAs participate in trichome regulatory interaction network. Eleven trans-acting siRNA (tasiRNA) candidate genes were also identified in cotton. One has never been found in other plant species and two of them were derived from MYB and ARF, both of which play important roles in cotton fibre development. Sixteen genes were predicted to be tasiRNA targets, including sucrose synthase and MYB2. Together, this study discovered new miRNAs in cotton and offered evidences that miRNAs play important roles in cotton ovule/fibre development. The identification of tasiRNA genes and their targets broadens our understanding of the complicated regulatory mechanism of miRNAs in cotton.

Deep sequencing reveals important roles of microRNAs in response to drought and salinity stress in cotton.

Drought and salinity are two major environmental factors adversely affecting plant growth and productivity. However, the regulatory mechanism is unknown. In this study, the potential roles of small regulatory microRNAs (miRNAs) in cotton response to those stresses were investigated. Using next-generation deep sequencing, a total of 337 miRNAs with precursors were identified, comprising 289 known miRNAs and 48 novel miRNAs. Of these miRNAs, 155 miRNAs were expressed differentially. Target prediction, Gene Ontology (GO)-based functional classification, and Kyoto Encyclopedia of Genes and Genomes (KEGG)-based functional enrichment show that these miRNAs might play roles in response to salinity and drought stresses through targeting a series of stress-related genes. Degradome sequencing analysis showed that at least 55 predicted target genes were further validated to be regulated by 60 miRNAs. CitationRank-based literature mining was employed to determinhe the importance of genes related to drought and salinity stress. The NAC, MYB, and MAPK families were ranked top under the context of drought and salinity, indicating their important roles for the plant to combat drought and salinity stress. According to target prediction, a series of cotton miRNAs are associated with these top-ranked genes, including miR164, miR172, miR396, miR1520, miR6158, ghr-n24, ghr-n56, and ghr-n59. Interestingly, 163 cotton miRNAs were also identified to target 210 genes that are important in fibre development. These results will contribute to cotton stress-resistant breeding as well as understanding fibre development.

Selection and validation of reliable reference genes in Gossypium raimondii.

OBJECTIVE: To identify reliable reference genes for gene expression analysis in Gossypium raimondii. RESULTS: Five different software tools, geNorm, NormFinder, BestKeeper, ReFinder and ∆Ct method were employed to analyze the qRT-PCR data systematically of 12 housekeeping genes. SAD and TUA11 showed relatively stable expression levels in all tissues (i.e. leaves, shoots, buds, and sepals). We then limited our analysis to each plant part and identified tissue-specific reference genes. Our results showed TUA11, TUB6 and EF1a, EF1a, MZA and GAPC2, MZA, GAPC2, SAD and TUA11, and UBQ and MZA were reliable reference genes in leaves, shoots, buds, and sepals, respectively. CONCLUSION: Some genes were commonly identified as candidate reference genes in more than two tissue, while others were tissue-specific. Thus, our study allows choosing an appropriate control gene based on sampling for gene expression analysis.

Genome-wide identification of KNOX transcription factors in cotton and the role of GhKNOX4-A and GhKNOX22-D in response to salt and drought stress.

Cotton is one of the most important economic and fiber crops in the world. KNOX is one class of universal transcription factors, which plays important roles in plant growth and development as well as response to different stresses. Although there are many researches on KNOXs in other plant species, there are few reports on cotton. In this study, we systematically and comprehensively identified all KNOX genes in upland cotton and its two ancestral species; we also studied their functions by employing RNA-seq analysis and virus-induced gene silence (VIGS). A total of 89 KNOX genes were identified from three cotton species. Among them, 44 were from upland cotton, 22 and 23 were found in its ancestral species G. raimondii and G. arboreum, respectively. Plant polyploidization and domestication play a selective force driving KNOX gene evolution. Phylogenetic analysis displayed that KNOX genes were evolved into three Classes. The intron length and exon number differed in each Class. Transcriptome data showed that KNOX genes of Class II were widely expressed in multiple tissues, including fiber. The majority of KNOX genes were induced by different abiotic stresses. Additionally, we found multiple cis-elements related to stress in the promoter region of KNOX genes. VIGS silence of GhKNOX4-A and GhKNOX22-D genes showed significant growth and development effect in cotton seedlings under salt and drought treatments. Both GhKNOX4-A and GhKNOX22-D regulated plant tolerance; silencing both genes induced oxidative stresses, evidenced by reduced SOD activity and induced leave cell death, and also enhanced stomatal open and water loss. Thus, GhKNOX4-A and GhKNOX22-D may contribute to drought response by regulating stomata opening and oxidative stresses.

Genome-Wide Dissection of the Genetic Basis for Drought Tolerance in Gossypium hirsutum L. Races.

Drought seriously threats the growth and development of Gossypium hirsutum L. To dissect the genetic basis for drought tolerance in the G. hirsutum L. germplasm, a population, consisting of 188 accessions of G. hirsutum races and a cultivar (TM-1), was genotyped using the Cotton80KSNP biochip, and 51,268 high-quality single-nucleotide polymorphisms (SNPs) were obtained. Based on the phenotypic data of eight drought relative traits from four environments, we carried out association mapping with five models using GAPIT software. In total, thirty-six SNPs were detected significantly associated at least in two environments or two models. Among these SNPs, 8 and 28 (including 24 SNPs in 5 peak regions) were distributed in the A and D subgenome, respectively; eight SNPs were found to be distributed within separate genes. An SNP, TM73079, located on chromosome D10, was simultaneously associated with leaf fresh weight, leaf wilted weight, and leaf dry weight. Another nine SNPs, TM47696, TM33865, TM40383, TM10267, TM59672, TM59675, TM59677, TM72359, and TM72361, on chromosomes A13, A10, A12, A5, D6, and D9, were localized within or near previously reported quantitative trait loci for drought tolerance. Moreover, 520 genes located 200 kb up- and down-stream of 36 SNPs were obtained and analyzed based on gene annotation and transcriptome sequencing. The results showed that three candidate genes, Gh_D08G2462, Gh_A03G0043, and Gh_A12G0369, may play important roles in drought tolerance. The current GWAS represents the first investigation into mapping QTL for drought tolerance in G. hirsutum races and provides important information for improving cotton cultivars.

Impact of potassium deficiency on cotton growth, development and potential microRNA-mediated mechanism.

The goal of this study was to investigate the impact of potassium deficiency on cotton seedling growth and development at the individual, physiological, biochemical, and molecular levels. Potassium is an important plant nutrient; our results show that potassium deficiency significantly affected cotton seedling growth and development, evidenced by reduced plant height, and total areas of the leaves and roots as well as further reduced both fresh and dry biomass of the entire plants. Potassium deficiency also significantly inhibited root and leaf respiration and leaf photosynthesis. Compared with the controls, potassium deficiency significantly inhibited root elongation and total root surface areas that further inhibited cotton seedlings to uptake nutrients from the medium. Potassium deficiency induced aberrant expression of both microRNAs (miRNAs) and their protein-coding targets. These miRNAs regulate plant root development as well as response to abiotic stresses. Potassium deficiency altered the expression of miRNAs that regulate the expression of protein-coding genes controlling root development and response to potassium deficiency. miRNAs regulate root development and further control plant development in cotton seedlings under potassium deficiency. In summary, potassium deficiency significantly affected the cotton seedling photosynthesis and respiration that resulted in inhibition of cotton seedling growth and development potentially due to the miRNA-mediated mechanism.

Pollen Tube Pathway-Mediated Cotton Transformation.

Although many gene transfer methods have been employed for successfully obtaining transgenic cotton, the major constraint in cotton improvement is the limitation of genotype because the majority of transgenic methods require plant regeneration from a single transformed cell which is limited by cotton tissue culture. Comparing with other plant species, it is difficult to induce plant regeneration from cotton; currently, only a limited number of cotton cultivars can be cultured for obtaining regenerated plants. Thus, developing a simple and genotype-independent genetic transformation method is particularly interested for cotton. In this chapter, we present a simple, cost-efficient, and genotype-independent cotton transformation method - pollen tube pathway-mediated transformation. This method uses pollen tube pathway to deliver transgene into cotton embryo sacs and then insert foreign genes into cotton genome. There are three major steps for pollen tube pathway-mediated genetic transformation, which include injection of foreign genes into pollen tube, integration of foreign genes into plant genome, and selection of transgenic plants.

Overexpression of miRNA in Cotton via Agrobacterium-Mediated Transformation.

microRNAs (miRNAs) are an extensive class of newly identified endogenous small regulatory molecules. Many studies show that miRNAs play a critical role in almost all biological and metabolic progress through targeting protein-coding genes for mRNA cleavage or translation inhibition. Many miRNAs are also identified from cotton using computational and/or experimental approaches, including the next-generation deep sequencing technology. However, their functions are unclear. In this chapter, we describe a detailed method for overexpressing miRNA, miR156 as an example, in cotton using Agrobacterium-mediated genetic transformation. This provides an approach to investigate the function and regulatory mechanism of miRNAs in cotton.

Integrative Analysis of the lncRNA and mRNA Transcriptome Revealed Genes and Pathways Potentially Involved in the Anther Abortion of Cotton (Gossypium hirsutum L.).

Cotton plays an important role in the economy of many countries. Many studies have revealed that numerous genes and various metabolic pathways are involved in anther development. In this research, we studied the differently expressed mRNA and lncRNA during the anther development of cotton between the cytoplasmic male sterility (CMS) line, C2P5A, and the maintainer line, C2P5B, using RNA-seq analysis. We identified 17,897 known differentially expressed (DE) mRNAs, and 865 DE long noncoding RNAs (lncRNAs) that corresponded to 1172 cis-target genes at three stages of anther development using gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of DE mRNAs; and cis-target genes of DE lncRNAs probably involved in the degradation of tapetum cells, microspore development, pollen development, and in the differentiation, proliferation, and apoptosis of the anther cell wall in cotton. Of these DE genes, LTCONS_00105434, LTCONS_00004262, LTCONS_00126105, LTCONS_00085561, and LTCONS_00085561, correspond to cis-target genes Ghir_A09G011050.1, Ghir_A01G005150.1, Ghir_D05G003710.2, Ghir_A03G016640.1, and Ghir_A12G005100.1, respectively. They participate in oxidative phosphorylation, flavonoid biosynthesis, pentose and glucuronate interconversions, fatty acid biosynthesis, and MAPK signaling pathway in plants, respectively. In summary, the transcriptomic data indicated that DE lncRNAs and DE mRNAs were related to the anther development of cotton at the pollen mother cell stage, tetrad stage, and microspore stage, and abnormal expression could lead to anther abortion, resulting in male sterility of cotton.

Single-Locus and Multi-Locus Genome-Wide Association Studies in the Genetic Dissection of Fiber Quality Traits in Upland Cotton (Gossypium hirsutum L.).

A major breeding target in Upland cotton (Gossypium hirsutum L.) is to improve the fiber quality. To address this issue, 169 diverse accessions, genotyped by 53,848 high-quality single-nucleotide polymorphisms (SNPs) and phenotyped in four environments, were used to conduct genome-wide association studies (GWASs) for fiber quality traits using three single-locus and three multi-locus models. As a result, 342 quantitative trait nucleotides (QTNs) controlling fiber quality traits were detected. Of the 342 QTNs, 84 were simultaneously detected in at least two environments or by at least two models, which include 29 for fiber length, 22 for fiber strength, 11 for fiber micronaire, 12 for fiber uniformity, and 10 for fiber elongation. Meanwhile, nine QTNs with 10% greater sizes (R2) were simultaneously detected in at least two environments and between single- and multi-locus models, which include TM80185 (D13) for fiber length, TM1386 (A1) and TM14462 (A6) for fiber strength, TM18616 (A7), TM54735 (D3), and TM79518 (D12) for fiber micronaire, TM77489 (D12) and TM81448 (D13) for fiber uniformity, and TM47772 (D1) for fiber elongation. This indicates the possibility of marker-assisted selection in future breeding programs. Among 455 genes within the linkage disequilibrium regions of the nine QTNs, 113 are potential candidate genes and four are promising candidate genes. These findings reveal the genetic control underlying fiber quality traits and provide insights into possible genetic improvements in Upland cotton fiber quality.

MicroRNA expression profiles during cotton (Gossypium hirsutum L) fiber early development.

The role of microRNAs (miRNAs) during cotton fiber development remains unclear. Here, a total of 54 miRNAs belonging to 39 families were selected to characterize miRNA regulatory mechanism in eight different fiber development stages in upland cotton cv BM-1. Among 54 miRNAs, 18 miRNAs were involved in cotton fiber initiation and eight miRNAs were related to fiber elongation and secondary wall biosynthesis. Additionally, 3,576 protein-coding genes were candidate target genes of these miRNAs, which are potentially involved in cotton fiber development. We also investigated the regulatory network of miRNAs and corresponding targets in fiber initiation and elongation, and secondary wall formation. Our Gene Ontology-based term classification and KEGG-based pathway enrichment analyses showed that the miRNA targets covered 220 biological processes, 67 molecular functions, 45 cellular components, and 10 KEGG pathways. Three of ten KEGG pathways were involved in lignan synthesis, cell elongation, and fatty acid biosynthesis, all of which have important roles in fiber development. Overall, our study shows the potential regulatory roles of miRNAs in cotton fiber development and the importance of miRNAs in regulating different cell types. This is helpful to design miRNA-based biotechnology for improving fiber quality and yield.