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Cotton fibers are the main raw materials of the textile industry. Exogenous superior fiber genes have been introduced into upland cotton to develop high-yield cultivars with excellent fiber quality. We used a single chromosomal segment on the chromosome A07 substitution line SL7, with high fiber strength, to investigate the molecular mechanism underlying its fiber quality. RNA-seq and KEGG analysis showed that 70 differentially expressed genes were enriched in plant hormone transduction pathways, including auxin, ethylene and abscisic acid, in fibers at 10 days post-anthesis (DPA). Among these, fiber-development related transcription factors MYB and NAC, including Gh_A11G0981 (MYB108), Gh_A03G0887 (NAC029), and Gh_A08G1691 (NAC021), were significantly upregulated in SL7, as were numerous cellulose synthase-like (CSL) genes involved in non-cellulose polysaccharide and cell wall synthesis. The hemicellulose content of SL7 was significantly higher than that of L22, an upland cotton cultivar. These results suggest that key genes in the introgressed chromosomal segment of SL7 regulate the expression of transcription-factor genes via hormone-transduction pathways, thereby inducing the expression of genes involved in secondary wall synthesis and ultimately improving fiber quality. This study has shed light on the molecular mechanism of fiber development and will contribute to the improvement of fiber quality of upland cotton by molecular breeding. Keywords: Cotton fiber quality, Single-fragment substitution line, RNA-seq, Hormone transduction pathway, Transcription factor

Summary Fine mapping QTLs and identifying candidate genes for cotton fibre‐quality and yield traits would be beneficial to cotton breeding. Here, we constructed a high‐density genetic map by specific‐locus amplified fragment sequencing (SLAF‐seq) to identify QTLs associated with fibre‐quality and yield traits using 239 recombinant inbred lines (RILs), which was developed from LMY22 (a high‐yield Gossypium hirsutumL. cultivar) × LY343 (a superior fibre‐quality germplasm with G. barbadenseL. introgressions). The genetic map spanned 3426.57 cM, including 3556 SLAF‐based SNPs and 199 SSR marker loci. A total of 104 QTLs, including 67 QTLs for fibre quality and 37 QTLs for yield traits, were identified with phenotypic data collected from 7 environments. Among these, 66 QTLs were co‐located in 19 QTL clusters on 12 chromosomes, and 24 QTLs were detected in three or more environments and determined to be stable. We also investigated the genomic components of LY343 and their contributions to fibre‐related traits by deep sequencing the whole genome of LY343, and we found that genomic components from G. hirsutum races (which entered LY343 via its G. barbadense parent) contributed more favourable alleles than those from G. barbadense. We further identified six putative candidate genes for stable QTLs, including Gh_A03G1147 (GhPEL6), Gh_D07G1598 (GhCSLC6) and Gh_D13G1921 (GhTBL5) for fibre‐length QTLs and Gh_D03G0919 (GhCOBL4), Gh_D09G1659 (GhMYB4) and Gh_D09G1690 (GhMYB85) for lint‐percentage QTLs. Our results provide comprehensive insight into the genetic basis of the formation of fibre‐related traits and would be helpful for cloning fibre‐development‐related genes as well as for marker‐assisted genetic improvement in cotton.

Background The circadian clock not only participates in regulating various stages of plant growth, development and metabolism, but confers plant environmental adaptability to stress such as drought. Pseudo-Response Regulators (PRRs) are important component of the central oscillator (the core of circadian clock) and play a significant role in plant photoperiod pathway. However, no systematical study about this gene family has been performed in cotton. Methods PRR genes were identified in diploid and tetraploid cotton using bioinformatics methods to investigate their homology, duplication and evolution relationship. Differential gene expression, KEGG enrichment analysis and qRT-PCR were conducted to analyze PRR gene expression patterns under diurnal changes and their response to drought stress. Results A total of 44 PRR family members were identified in four Gossypium species, with 16 in G. hirsutum, 10 in G. raimondii, and nine in G. barbadense as well as in G. arboreum. Phylogenetic analysis indicated that PRR proteins were divided into five subfamilies and whole genome duplication or segmental duplication contributed to the expansion of Gossypium PRR gene family. Gene structure analysis revealed that members in the same clade are similar, and multiple cis-elements related to light and drought stress response were enriched in the promoters of GhPRR genes. qRT-PCR results showed that GhPRR genes transcripts presented four expression peaks (6 h, 9 h, 12 h, 15 h) during 24 h and form obvious rhythmic expression trend. Transcriptome data with PEG treatment, along with qRT-PCR verification suggested that members of clade III (GhPRR5a, b, d) and clade V (GhPRR3a and GhPRR3c) may be involved in drought response. This study provides an insight into understanding the function of PRR genes in circadian rhythm and in response to drought stress in cotton.

Leaves are the most important organs for photosynthesis. Many cotton leaf mutants (Gossypium spp.) have been reported and broadened the insights of leaf development. A natural occurring small, crinkled leaf (scl) mutant of upland cotton (Gossypium hirsutum L.) occurs in SY321 (wild type, WT), a high-yield upland cotton cultivar that is widely planted in China. The scl has a small and crinkled leaf phenotype. The shoot apical meristem (SAM) of scl did not form the essential typical layered structure, and the cell layers were arranged in a disordered manner. The free IAA concentration of shoot tips was significantly reduced in the scl according to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Gene expression profiles showed that 432 genes were differentially expressed between scl and WT. The top 20 enriched pathways were analyzed using the Kyoto encyclopedia of genes and genomes (KEGG) database and four pathways, including flavonoid biosynthesis, photosynthesis, RNA transport, and carbon fixation in photosynthetic organisms, were considered as significantly enriched pathways. Furthermore, a number of differentially expressed genes related to auxin activity, including flavonoids biosynthesis, auxin perception and signal transduction pathway, auxin transport, and ubiquitin-mediated proteolysis pathways, were identified, and the altered expressions of these genes might be responsible for the aberrant phenotype of the scl.

A VIGS method by agroinoculation of cotton seeds was developed for gene silencing in young seedlings and roots, and applied in functional analysis of GhBI-1 in response to salt stress. Virus-induced gene silencing (VIGS) has been widely used to investigate the functions of genes expressed in mature leaves, but not yet in young seedlings or roots of cotton (Gossypium hirsutum L.). Here, we developed a simple and effective VIGS method for silencing genes in young cotton seedlings and roots by soaking naked seeds in Agrobacterium cultures carrying tobacco rattle virus (TRV)-VIGS vectors. When the naked seeds were soaked in Agrobacterium cultures with an OD600 of 1.5 for 90 min, it was optimal for silencing genes effectively in young seedlings as clear photo-bleaching phenotype in the newly emerging leaves of pTRV:GhCLA1 seedlings were observed at 12-14 days post inoculation. Silencing of GhPGF (cotton pigment gland formation) by this method resulted in a 90% decrease in transcript abundances of the gene in roots at the early development stage. We further used the tool to investigate function of GhBI-1 (cotton Bax inhibitor-1) gene in response to salt stress and demonstrated that GhBI-1 might play a protective role under salt stress by suppressing stress-induced cell death in cotton. Our results showed that the newly established VIGS method is a powerful tool for elucidating functions of genes in cotton, especially the genes expressed in young seedlings and roots.

Plant roots and soil microorganisms interact with each other mainly in the rhizosphere. Changes in the community structure of the rhizosphere microbiome are influenced by many factors. In this study, we determined the community structure of rhizosphere bacteria in cotton, and studied the variation of rhizosphere bacterial community structure in different soil types and developmental stages using TM-1, an upland cotton cultivar (Gossypium hirsutum L.) and Hai 7124, a sea island cotton cultivar (G. barbadense L.) by high-throughput sequencing technology. Six bacterial phyla were found dominantly in cotton rhizosphere bacterial community including Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria, and Verrucomicrobia. The abundance of Acidobacteria, Cyanobacteria, Firmicutes, Planctomycetes and Proteobacteria were largely influenced by cotton root. Bacterial α-diversity in rhizosphere was lower than that of bulk soil in nutrient-rich soil, but higher in cotton continuous cropping field soil. The β-diversity in nutrient-rich soil was greater than that in continuous cropping field soil. The community structure of the rhizosphere bacteria varied significantly during different developmental stages. Our results provided insights into the dynamics of cotton rhizosphere bacterial community and would facilitate to improve cotton growth and development through adjusting soil bacterial community structure artificially.

Rhizosphere fungal communities exert important influential forces on plant growth and health. However, information on the dynamics of the rhizosphere fungal community structure of the worldwide economic crop, cotton (Gossypium spp.), is limited. Next-generation sequencing of nuclear ribosomal internal transcribed spacer-1 (ITS1) was used to characterize the rhizosphere fungal communities of worldwide cultivated tetraploid cotton using G. hirsutum cv. TM-1 (upland cotton) and G. barbadense cv. Hai 7124 (island cotton). Plants were grown in field soil (FS) that had been continuously cropped with cotton and nutrient-rich soil (NS) that had not been cropped. Fungal species richness, diversity, and community composition were analyzed and compared among soil resources, cotton genotypes, and developmental stages. We found that the fungal community structure between the rhizosphere and bulk soil of cotton were different and the rhizosphere fungal communities were significantly varied between FS and NS. These results suggest that cotton rhizosphere fungal community structure variation was primarily determined by the interaction of cotton roots with different soil resources. We also found that the community composition of cotton rhizosphere fungi varied significantly during different developmental stages, suggesting that developmental stages were also important factors in the dynamics of rhizosphere fungal communities for the varying dominant fungal genera of the rhizosphere. In addition, we also observed that fungal pathogens were clearly increased at certain developmental stages, suggesting a higher infection rate and a high incidence of corresponding soil-borne disease in each stage. This research illustrates the characteristics of cotton rhizosphere fungal communities and provides important information for understanding the potential influences of rhizosphere fungal communities on cotton growth and health.