Quantitative Trait Locus Mapping for Plant Height and Branch Number in CCRI70 Recombinant Inbred Line Population of Upland Cotton (Gossypium hirsutum).

Upland cotton accounts for a high percentage (95%) of the world's cotton production. Plant height (PH) and branch number (BN) are two important agronomic traits that have an impact on improving the level of cotton mechanical harvesting and cotton yield. In this research, a recombinant inbred line (RIL) population with 250 lines developed from the variety CCRI70 was used for constructing a high-density genetic map and identification of quantitative trait locus (QTL). The results showed that the map harbored 8298 single nucleotide polymorphism (SNP) markers, spanning a total distance of 4876.70 centimorgans (cMs). A total of 69 QTLs for PH (9 stable) and 63 for BN (11 stable) were identified and only one for PH was reported in previous studies. The QTLs for PH and BN harbored 495 and 446 genes, respectively. Combining the annotation information, expression patterns and previous studies of these genes, six genes could be considered as potential candidate genes for PH and BN. The results could be helpful for cotton researchers to better understand the genetic mechanism of PH and BN development, as well as provide valuable genetic resources for cotton breeders to manipulate cotton plant architecture to meet future demands.

Comparative transcriptional and co-expression network analysis of two upland cotton accessions with extreme phenotypic differences reveals molecular mechanisms of fiber development.

Introduction: Upland cotton (Gossypium hirsutum) is the main source of natural fiber in the global textile industry, and thus its fiber quality and yield are important parameters. In this study, comparative transcriptomics was used to analyze differentially expressed genes (DEGs) due to its ability to effectively screen candidate genes during the developmental stages of cotton fiber. However, research using this method is limited, particularly on fiber development. The aim of this study was to uncover the molecular mechanisms underlying the whole period of fiber development and the differences in transcriptional levels. Methods: Comparative transcriptomes are used to analyze transcriptome data and to screen for differentially expressed genes. STEM and WGCNA were used to screen for key genes involved in fiber development. qRT-PCR was performed to verify gene expression of selected DEGs and hub genes. Results: Two accessions of upland cotton with extreme phenotypic differences, namely EZ60 and ZR014121, were used to carry out RNA sequencing (RNA-seq) on fiber samples from different fiber development stages. The results identified 704, 376, 141, 269, 761, and 586 genes that were upregulated, and 1,052, 476, 355, 259, 702, and 847 genes that were downregulated at 0, 5, 10, 15, 20, and 25 days post anthesis, respectively. Similar expression patterns of DEGs were monitored using short time-series expression miner (STEM) analysis, and associated pathways of DEGs within profiles were investigated. In addition, weighted gene co-expression network analysis (WGCNA) identified five key modules in fiber development and screened 20 hub genes involved in the development of fibers. Discussion: Through the annotation of the genes, it was found that the excessive expression of resistance-related genes in the early fiber development stages affects the fiber yield, whereas the sustained expression of cell elongation-related genes is critical for long fibers. This study provides new information that can be used to improve fibers in newly developed upland cotton genotypes.

UDP-glucose pyrophosphorylase: genome-wide identification, expression and functional analyses in Gossypium hirsutum.

In this study, a total of 66 UDP-glucose pyrophosphorylase (UGP) (EC 2.7.7.9) genes were identified from the genomes of four cotton species, which are the members of Pfam glycosyltransferase family (PF01702) and catalyze the reaction between glucose-1-phosphate and UTP to produce UDPG. The analysis of evolutionary relationship, gene structure, and expression provides the basis for studies on function of UGP genes in cotton. The evolutionary tree and gene structure analysis revealed that the UGP gene family is evolutionarily conserved. Collinearity and Ka/Ks analysis indicated that amplification of UGP genes is due to repetitive crosstalk generating between new family genes, while being under strong selection pressure. The analysis of cis-acting elements exhibited that UGP genes play important role in cotton growth, development, abiotic and hormonal stresses. Six UGP genes that were highly expressed in cotton fiber at 15 DPA were screened by transcriptome data and qRT-PCR analysis. The addition of low concentrations of IAA and GA3 to ovule cultures revealed that energy efficiency promoted the development of ovules and fiber clusters, and qRT-PCR showed that expression of these six UGP genes was differentially increased. These results suggest that the UGP gene may play an important role in fiber development, and provides the opportunity to plant researchers to explore the mechanisms involve in fiber development in cotton.

Linkage and association analyses reveal that hub genes in energy-flow and lipid biosynthesis pathways form a cluster in upland cotton.

Upland cotton is an important allotetraploid crop that provides both natural fiber for the textile industry and edible vegetable oil for the food or feed industry. To better understand the genetic mechanism that regulates the biosynthesis of storage oil in cottonseed, we identified the genes harbored in the major quantitative trait loci/nucleotides (QTLs/QTNs) of kernel oil content (KOC) in cottonseed via both multiple linkage analyses and genome-wide association studies (GWAS). In 'CCRI70' RILs, six stable QTLs were simultaneously identified by linkage analysis of CHIP and SLAF-seq strategies. In '0-153' RILs, eight stable QTLs were detected by consensus linkage analysis integrating multiple strategies. In the natural panel, thirteen and eight loci were associated across multiple environments with two algorithms of GWAS. Within the confidence interval of a major common QTL on chromosome 3, six genes were identified as participating in the interaction network highly correlated with cottonseed KOC. Further observations of gene differential expression showed that four of the genes, LtnD, PGK, LPLAT1, and PAH2, formed hub genes and two of them, FER and RAV1, formed the key genes in the interaction network. Sequence variations in the coding regions of LtnD, FER, PGK, LPLAT1, and PAH2 genes may support their regulatory effects on oil accumulation in mature cottonseed. Taken together, clustering of the hub genes in the lipid biosynthesis interaction network provides new insights to understanding the mechanism of fatty acid biosynthesis and TAG assembly and to further genetic improvement projects for the KOC in cottonseeds.

Multi-environment Evaluations Across Ecological Regions Reveal That the Kernel Oil Content of Cottonseed Is Equally Determined by Genotype and Environment.

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.

Identification and analysis of oil candidate genes reveals the molecular basis of cottonseed oil accumulation in Gossypium hirsutum L.

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.

Co-expression network and comparative transcriptome analysis for fiber initiation and elongation reveal genetic differences in two lines from upland cotton CCRI70 RIL population.

Upland cotton is the most widely planted for natural fiber around the world, and either lint percentage (LP) or fiber length (FL) is the crucial component tremendously affecting cotton yield and fiber quality, respectively. In this study, two lines MBZ70-053 and MBZ70-236 derived from G. hirsutum CCRI70 recombinant inbred line (RIL) population presenting different phenotypes in LP and FL traits were chosen to conduct RNA sequencing on ovule and fiber samples, aiming at exploring the differences of molecular and genetic mechanisms during cotton fiber initiation and elongation stages. As a result, 249/128, 369/206, 4296/1198 and 3547/2129 up-/down- regulated differentially expressed genes (DGEs) in L2 were obtained at -3, 0, 5 and 10 days post-anthesis (DPA), respectively. Seven gene expression profiles were discriminated using Short Time-series Expression Miner (STEM) analysis; seven modules and hub genes were identified using weighted gene co-expression network analysis. The DEGs were mainly enriched into energetic metabolism and accumulating as well as auxin signaling pathway in initiation and elongation stages, respectively. Meanwhile, 29 hub genes were identified as 14-3-3ω , TBL35, GhACS, PME3, GAMMA-TIP, PUM-7, etc., where the DEGs and hub genes revealed the genetic and molecular mechanisms and differences during cotton fiber development.

Genome-wide identification, evolution, expression, and alternative splicing profiles of peroxiredoxin genes in cotton.

Peroxiredoxin (PRX) is a ubiquitous thioredoxin-dependent peroxidase that can eliminate excessive free radicals produced by stress and protect cells from oxidative damage. PRXs are also involved in reactive oxygen species (ROS)- and redox-dependent signaling by performing redox interactions with other proteins and modify their redox status. At present, PRX family identification, evolution and regulation research has been conducted in some plants; however, systematic research about this family is lacking in cotton. In this study, a total of 44 PRXs were identified in the cotton genome. Phylogenetic and conserved active site analyses showed that the PRXs were divided into six subfamilies according to the conserved site (PxxxTxxC…S…W/F) and conserved cysteinyl residues positions. Segmental duplication and polyploid events were the main methods for PRX family expansion, and the PRXs of diploid G. arboreum were the donors of PRXs in the D subgenomes of allotetraploid G. hirsutum and G. barbadense during the evolution of the PRX family. qRT-PCR analysis confirmed that cis-acting elements play important roles in regulating the expression of PRXs. Alternative splicing events occurred in GhPRX14-D that can increased the complexity of transcripts in G. hirsutum. Subcellular localization showed that most PRX members were located in chloroplasts, the cytoplasmic membrane and the nucleus. Our results provide systematic support for a better understanding of PRXs in cotton and a starting point for further studies of the specific functions of PRXs in cotton.

Quantitative Trait Loci and Transcriptome Analysis Reveal Genetic Basis of Fiber Quality Traits in CCRI70 RIL Population of Gossypium hirsutum.

Upland cotton (Gossypium hirsutum) is widely planted around the world for its natural fiber, and producing high-quality fiber is essential for the textile industry. CCRI70 is a hybrid cotton plant harboring superior yield and fiber quality, whose recombinant inbred line (RIL) population was developed from two upland cotton varieties (sGK156 and 901-001) and were used here to investigate the source of high-quality related alleles. Based on the material of the whole population, a high-density genetic map was constructed using specific locus-amplified fragment sequencing (SLAF-seq). It contained 24,425 single nucleotide polymorphism (SNP) markers, spanning a distance of 4,850.47 centimorgans (cM) over 26 chromosomes with an average marker interval of 0.20 cM. In evaluating three fiber quality traits in nine environments to detect multiple environments stable quantitative trait loci (QTLs), we found 289 QTLs, of which 36 of them were stable QTLs and 18 were novel. Based on the transcriptome analysis for two parents and two RILs, 24,941 unique differentially expressed genes (DEGs) were identified, 473 of which were promising genes. For the fiber strength (FS) QTLs, 320 DEGs were identified, suggesting that pectin synthesis, phenylpropanoid biosynthesis, and plant hormone signaling pathways could influence FS, and several transcription factors may regulate fiber development, such as GAE6, C4H, OMT1, AFR18, EIN3, bZIP44, and GAI. Notably, the marker D13_56413025 in qFS-chr18-4 provides a potential basis for enhancing fiber quality of upland cotton via marker-assisted breeding and gene cloning of important fiber quality traits.

UDP-Glucose Dehydrogenases: Identification, Expression, and Function Analyses in Upland Cotton (Gossypium hirsutum).

UDP-glucose dehydrogenase (UGD; EC1.1.1.22) is a NAD+-dependent enzyme that catalyzes the two-fold oxidation of UDP-glucose (UDP-Glc) to produce UDP-glucuronic acid and plays an important role in plant cell wall synthesis. A total of 42 UGD genes from four Gossypium genomes including G. hirsutum, G. arboretum, G. barbadense, and G. raimondii were identified and found that the UGD gene family has conservative evolution patterns in gene structure and protein domain. The growth of fibers can be effectively promoted after adding the UDP-Glc to the medium, and the GhUGD gene expression enhanced. In addition, the transgenic Arabidopsis lines over-expressing GH_D12G1806 had longer root lengths and higher gene expression level than the wild-type plants of Columbia-0. These results indicated that UGD may play important roles in cotton fiber development and has a guiding significance for dissecting fiber development mechanism.

Insight into the relationship between S-lignin and fiber quality based on multiple research methods.

Cotton (Gossypium hirsutum) is an important cash crop, providing people with high quality natural fiber. Lignin is the main component of cotton fiber, second only to cellulose. As a main substance filled in the cellulose framework during the secondary wall thickening process, lignin plays a key role in the formation of cotton fiber quality. However, the mechanism behind it is still unclear. In this research, we screened candidate genes involved in lignin biosynthesis based on analysis of cotton genome and transcriptome sequence data. The authenticity of the transcriptome data was verified by qRT-PCR assay. Total 62 genes were identified from nine gene families. In the process, we found the key gene GhCAD7 that affects the biosynthesis of S-lignin and the ratio of syringyl/guaiacyl (S/G). In addition, in combination with the metabolites and transcriptome profiles of the line 0-153 with high fiber quality and the line sGK9708 with low fiber quality during cotton fiber development, we speculate that the ratio of syringyl/guaiacyl (S/G) is inseparable from the quality of cotton fiber. Finally, the S-type lignin synthesis branch may play a more important role in the formation of high-quality fiber. This work provides insights into the synthesis of lignin in cotton and lays the foundation for future research into improving fiber quality.

Genome-wide quantitative trait loci reveal the genetic basis of cotton fibre quality and yield-related traits in a Gossypium hirsutum recombinant inbred line population.

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.

Genome-Wide Identification and Expression Analysis of the Metacaspase Gene Family in Gossypium Species.

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.

Identification of TPX2 Gene Family in Upland Cotton and Its Functional Analysis in Cotton Fiber Development.

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 Gossypiumhirsutum 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, GhWDLA2/7 and GhWDLA4/9, 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 GhWDLA2 and GhWDLA7 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.

Co-Expression Network Analysis and Hub Gene Selection for High-Quality Fiber in Upland Cotton (Gossypium hirsutum) Using RNA Sequencing Analysis.

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.

Genome-wide identification and analysis of the evolution and expression patterns of the GATA transcription factors in three species of Gossypium genus.

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.

Genome-wide identification and analysis of the evolution and expression patterns of the cellulose synthase gene superfamily in Gossypium species.

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 dN:dS value for the CSLJ clade was 1.305, suggesting this subfamily was under positive selection pressure. Our data indicated that the dN:dS 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.

Genome-wide identification, phylogeny, and expression analysis of pectin methylesterases reveal their major role in cotton fiber development.

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.