High-density genetic map construction and QTL mapping of a zigzag-shaped stem trait in tea plant (Camellia sinensis).

The highly unique zigzag-shaped stem phenotype in tea plants boasts significant ornamental value and is exceptionally rare. To investigate the genetic mechanism behind this trait, we developed BC1 artificial hybrid populations. Our genetic analysis revealed the zigzag-shaped trait as a qualitative trait. Utilizing whole-genome resequencing, we constructed a high-density genetic map from the BC1 population, incorporating 5,250 SNP markers across 15 linkage groups, covering 3,328.51 cM with an average marker interval distance of 0.68 cM. A quantitative trait locus (QTL) for the zigzag-shaped trait was identified on chromosome 4, within a 61.2 to 97.2 Mb range, accounting for a phenotypic variation explained (PVE) value of 13.62%. Within this QTL, six candidate genes were pinpointed. To better understand their roles, we analyzed gene expression in various tissues and individuals with erect and zigzag-shaped stems. The results implicated CsXTH (CSS0035625) and CsCIPK14 (CSS0044366) as potential key contributors to the zigzag-shaped stem formation. These discoveries lay a robust foundation for future functional genetic mapping and tea plant genetic enhancement.

CsEXL3 regulate mechanical harvest-related droopy leaves under the transcriptional activation of CsBES1.2 in tea plant.

Due to a labor shortage, the mechanical harvesting of tea plantations has become a focal point. However, mechanical harvest efficiency was hampered by droopy leaves, leading to a high rate of broken tea shoots and leaves. Here, we dissected the genetic structure of leaf droopiness in tea plants using genome-wide association studies (GWAS) on 146 accessions, combined with transcriptome from two accessions with contrasting droopy leaf phenotypes. A set of 16 quantitative trait loci (QTLs) containing 54 SNPs and 34 corresponding candidate genes associated with droopiness were then identified. Among these, CsEXL3 (EXORDIUM-LIKE 3) from Chromosome 1 emerged as a candidate gene. Further investigations revealed that silencing CsEXL3 in tea plants resulted in weaker vascular cell malformation and brassinosteroid-induced leaf droopiness. Additionally, brassinosteroid signal factor CsBES1.2 was proved to participate in CsEXL3-induced droopiness and vascular cell malformation via using the CsBES1.2-silencing tea plant. Notably, CsBES1.2 bound on the E-box of CsEXL3 promoter to transcriptionally activate CsEXL3 expression as CUT&TAG based ChIP-qPCR and ChIP-seq suggested in vivo as well as EMSA and Y1H indicated in vitro. Furthermore, CsEXL3 instead of CsBES1.2 decreased lignin content and the expressing levels of lignin biosynthesis genes. Overall, our findings suggest that CsEXL3 regulates droopy leaves, partially through the transcriptional activation of CsBES1.2, with the potential to improve mechanical harvest efficiency in tea plantations.

Bulked Segregant RNA-Seq Reveals Different Gene Expression Patterns and Mutant Genes Associated with the Zigzag Pattern of Tea Plants (Camellia sinensis).

The unique zigzag-patterned tea plant is a rare germplasm resource. However, the molecular mechanism behind the formation of zigzag stems remains unclear. To address this, a BC1 genetic population of tea plants with zigzag stems was studied using histological observation and bulked segregant RNA-seq. The analysis revealed 1494 differentially expressed genes (DEGs) between the upright and zigzag stem groups. These DEGs may regulate the transduction and biosynthesis of plant hormones, and the effects on the phenylpropane biosynthesis pathways may cause the accumulation of lignin. Tissue sections further supported this finding, showing differences in cell wall thickness between upright and curved stems, potentially due to lignin accumulation. Additionally, 262 single-nucleotide polymorphisms (SNPs) across 38 genes were identified as key SNPs, and 5 genes related to zigzag stems were identified through homologous gene function annotation. Mutations in these genes may impact auxin distribution and content, resulting in the asymmetric development of vascular bundles in curved stems. In summary, we identified the key genes associated with the tortuous phenotype by using BSR-seq on a BC1 population to minimize genetic background noise.

A key mutation in magnesium chelatase I subunit leads to a chlorophyll-deficient mutant of tea (Camellia sinensis).

Tea (Camellia sinensis) is a highly important beverage crop renowned for its unique flavour and health benefits. Chlorotic mutants of tea, known worldwide for their umami taste and economic value, have gained global popularity. However, the genetic basis of this chlorosis trait remains unclear. In this study, we identified a major-effect quantitative trait locus (QTL), qChl-3, responsible for the chlorosis trait in tea leaves, linked to a non-synonymous polymorphism (G1199A) in the magnesium chelatase I subunit (CsCHLI). Homozygous CsCHLIA plants exhibited an albino phenotype due to defects in magnesium protoporphyrin IX and chlorophylls in the leaves. Biochemical assays revealed that CsCHLI mutations did not affect subcellular localization or interactions with CsCHLIG and CsCHLD. However, combining CsCHLIA with CsCHLIG significantly reduced ATPase activity. RNA-seq analysis tentatively indicated that CsCHLI inhibited photosynthesis and enhanced photoinhibition, which in turn promoted protein degradation and increased the amino acid levels in chlorotic leaves. RT-qPCR and enzyme activity assays confirmed the crucial role of asparagine synthetase and arginase in asparagine and arginine accumulation, with levels increasing over 90-fold in chlorotic leaves. Therefore, this study provides insights into the genetic mechanism underlying tea chlorosis and the relationship between chlorophyll biosynthesis and amino acid metabolism.

Characterization of two O-methyltransferases involved in the biosynthesis of O-methylated catechins in tea plant.

Tea is known for having a high catechin content, with the main component being (-)-epigallocatechin gallate (EGCG), which has significant bioactivities, including potential anti-cancer and anti-inflammatory activity. The poor intestinal stability and permeability of EGCG, however, undermine these health-improving benefits. O-methylated EGCG derivatives, found in a few tea cultivars in low levels, have attracted considerable interest due to their increased bioavailability. Here, we identify two O-methyltransferases from tea plant: CsFAOMT1 that has a specific O-methyltransferase activity on the 3''-position of EGCG to generate EGCG3''Me, and CsFAOMT2 that predominantly catalyzes the formation of EGCG4″Me. In different tea tissues and germplasms, the transcript levels of CsFAOMT1 and CsFAOMT2 are strongly correlated with the amounts of EGCG3''Me and EGCG4''Me, respectively. Furthermore, the crystal structures of CsFAOMT1 and CsFAOMT2 reveal the key residues necessary for 3''- and 4''-O-methylation. These findings may provide guidance for the future development of tea cultivars with high O-methylated catechin content.

Identification of QTL controlling volatile terpene contents in tea plant (Camellia sinensis) using a high-aroma 'Huangdan' x 'Jinxuan' F1 population.

Aroma is an important factor affecting the character and quality of tea. The improvement of aroma trait is a crucial research direction of tea plant breeding. Volatile terpenes, as the major contributors to the floral odors of tea products, also play critical roles in the defense responses of plants to multiple stresses. However, previous studies have largely focused on the aroma formation during the manufacture of tea or the comparison of raw tea samples. The mechanisms causing different aroma profiles between tea cultivars have remained underexplored. In the current study, a high-density genetic linkage map of tea plant was constructed based on an F1 population of 'Huangdan' × 'Jinxuan' using genotyping by sequencing. This linkage map covered 1754.57 cM and contained 15 linkage groups with a low inter-marker distance of 0.47 cM. A total of 42 QTLs associated with eight monoterpene contents and 12 QTLs associated with four sesquiterpenes contents were identified with the average PVE of 12.6% and 11.7% respectively. Furthermore, six candidate genes related to volatile terpene contents were found in QTL cluster on chromosome 5 by RNA-seq analysis. This work will enrich our understanding of the molecular mechanism of volatile terpene biosynthesis and provide a theoretical basis for tea plant breeding programs for aroma quality improvement.

Comprehensive analysis and expression profiles of the AP2/ERF gene family during spring bud break in tea plant (Camellia sinensis).

BACKGROUND: AP2/ERF transcription factors (AP2/ERFs) are important regulators of plant physiological and biochemical metabolism. Evidence suggests that AP2/ERFs may be involved in the regulation of bud break in woody perennials. Green tea is economically vital in China, and its production value is significantly affected by the time of spring bud break of tea plant. However, the relationship between AP2/ERFs in tea plant and spring bud break remains largely unknown. RESULTS: A total of 178 AP2/ERF genes (CsAP2/ERFs) were identified in the genome of tea plant. Based on the phylogenetic analysis, these genes could be classified into five subfamilies. The analysis of gene duplication events demonstrated that whole genome duplication (WGD) or segmental duplication was the primary way of CsAP2/ERFs amplification. According to the result of the Ka/Ks value calculation, purification selection dominated the evolution of CsAP2/ERFs. Furthermore, gene composition and structure analyses of CsAP2/ERFs indicated that different subfamilies contained a variety of gene structures and conserved motifs, potentially resulting in functional differences among five subfamilies. The promoters of CsAP2/ERFs also contained various signal-sensing elements, such as abscisic acid responsive elements, light responsive elements and low temperature responsive elements. The evidence presented here offers a theoretical foundation for the diverse functions of CsAP2/ERFs. Additionally, the expressions of CsAP2/ERFs during spring bud break of tea plant were analyzed by RNA-seq and grouped into clusters A-F according to their expression patterns. The gene expression changes in clusters A and B were more synchronized with the spring bud break of tea plant. Moreover, several potential correlation genes, such as D-type cyclin genes, were screened out through weighted correlation network analysis (WGCNA). Temperature and light treatment experiments individually identified nine candidate CsAP2/ERFs that may be related to the spring bud break of tea plant. CONCLUSIONS: This study provides new evidence for role of the CsAP2/ERFs in the spring bud break of tea plant, establishes a theoretical foundation for analyzing the molecular mechanism of the spring bud break of tea plant, and contributes to the improvement of tea cultivars.

Deeply functional identification of TCS1 alleles provides efficient technical paths for low-caffeine breeding of tea plants.

Caffeine is an important functional component in tea, which has the effect of excitement and nerve stimulation, but excessive intake can cause insomnia and dysphoria. Therefore, the production of tea with low-caffeine content can meet the consumption needs of certain people. Here, in addition to the previous alleles of the tea caffeine synthase (TCS1) gene, a new allele (TCS1h) from tea germplasms was identified. Results of in vitro activity analysis showed that TCS1h had both theobromine synthase (TS) and caffeine synthase (CS) activities. Site-directed mutagenesis experiments of TCS1a, TCS1c, and TCS1h demonstrated that apart from the 225th amino acid residue, the 269th amino acid also determined the CS activity. GUS histochemical analysis and dual-luciferase assay indicated the low promoter activity of TCS1e and TCS1f. In parallel, insertion and deletion mutations in large fragments of alleles and experiments of site-directed mutagenesis identified a key cis-acting element (G-box). Furthermore, it was found that the contents of purine alkaloids were related to the expression of corresponding functional genes and alleles, and the absence or presence and level of gene expression determined the content of purine alkaloids in tea plants to a certain extent. In summary, we concluded TCS1 alleles into three types with different functions and proposed a strategy to effectively enhance low-caffeine tea germplasms in breeding practices. This research provided an applicable technical avenue for accelerating the cultivation of specific low-caffeine tea plants.

The potential effects of chlorophyll-deficient mutation and tree_age on the accumulation of amino acid components in tea plants.

Albino tea has been receiving growing attention on the tea market due to its attractive appearance and fresh taste, mainly caused by high amino acid contents. Here, variations in the contents of five free amino acids in relation to pigment contents and tree age in two hybrid populations'Longjin 43'(♀) × 'Baijiguan'(♂) and 'Longjin 43'(♀) ×'Huangjinya'(♂) with 334 first filial generation individuals including chlorophyll-deficient and normal tea plants were investigated. The data showed that the contents of main amino acids in all filial generation gradually decreased as plant age increased. Principal component analysis indicated that the amino acid content of individual plant tended to be stable with the growth of plants. Correlation analysis clarified that several main amino acids were significantly negatively correlated with chlorophyll a, chlorophyll b and carotenoid contents. Our results showed that the accumulation of amino acids in tea plant was closely related to leaf color variation and the tree age during growing period.

COTTONOMICS: a comprehensive cotton multi-omics database.

The rapid advancement of sequencing technology, including next-generation sequencing (NGS), has greatly improved sequencing efficiency and decreased cost. Consequently, huge amounts of genomic, transcriptomic and epigenetic data concerning cotton species have been generated and released. These large-scale data provide immense opportunities for the study of cotton genomic structure and evolution, population genetic diversity and genome-wide mining of excellent genes for important traits. However, the complexity of NGS data also causes distress, as it cannot be utilized easily. Here, we presented the cotton omics data platform COTTONOMICS (http://cotton.zju.edu.cn/), an easily accessible web database that integrates 32.5 TB of omics data including seven assembled genomes, resequencing data from 1180 allotetraploid cotton accessions and RNA-sequencing (RNA-seq), small RNA-sequencing (smRNA-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), DNase hypersensitive sites sequencing (DNase-seq) and Bisulfite sequencing (BS-seq). COTTONOMICS allows users to employ various search scenarios and retrieve information concerning the cotton genomes, genomic variation (Single nucleotide polymorphisms (SNPs) and Insertion and Deletion (InDels)), gene expression, smRNA expression, epigenetic regulation and quantitative trait locus (QTLs). The user-friendly web interface offers a variety of modules for storing, retrieving, analyzing and visualizing cotton multi-omics data to diverse ends, thereby enabling users to decipher cotton population genetics and identify potential novel genes that influence agronomically beneficial traits. Database URL: http://cotton.zju.edu.cn.

Genome sequence of Gossypium anomalum facilitates interspecific introgression breeding.

Crop wild relatives are an important reservoir of natural biodiversity. However, incorporating wild genetic diversity into breeding programs is often hampered by reproductive barriers and a lack of accurate genomic information. We assembled a high-quality, accurately centromere-anchored genome of Gossypium anomalum, a stress-tolerant wild cotton species. We provided a strategy to discover and transfer agronomically valuable genes from wild diploid species to tetraploid cotton cultivars. With a (Gossypium hirsutum × G. anomalum)2 hexaploid as a bridge parent, we developed a set of 74 diploid chromosome segment substitution lines (CSSLs) of the wild cotton species G. anomalum in the G. hirsutum background. This set of CSSLs included 70 homozygous substitutions and four heterozygous substitutions, and it collectively contained about 72.22% of the G. anomalum genome. Twenty-four quantitative trait loci associated with plant height, yield, and fiber qualities were detected on 15 substitution segments. Integrating the reference genome with agronomic trait evaluation of the CSSLs enabled location and cloning of two G. anomalum genes that encode peroxiredoxin and putative callose synthase 8, respectively, conferring drought tolerance and improving fiber strength. We have demonstrated the power of a high-quality wild-species reference genome for identifying agronomically valuable alleles to facilitate interspecific introgression breeding in crops.

Construction of a high-density genetic map and identification of QTLs related to agronomic and physiological traits in an interspecific (Gossypium hirsutum × Gossypium barbadense) F2 population.

BACKGROUND: Advances in genome sequencing technology, particularly restriction-site associated DNA sequence (RAD-seq) and whole-genome resequencing, have greatly aided the construction of cotton interspecific genetic maps based on single nucleotide polymorphism (SNPs), Indels, and other types of markers. High-density genetic maps can improve accuracy of quantitative trait locus (QTL) mapping, narrow down location intervals, and facilitate identification of the candidate genes. RESULT: In this study, 249 individuals from an interspecific F2 population (TM-1 and Hai7124) were re-sequenced, yielding 6303 high-confidence bin markers spanning 5057.13 cM across 26 cotton chromosomes. A total of 3380 recombination hot regions RHRs were identified which unevenly distributed on the 26 chromosomes. Based on this map, 112 QTLs relating to agronomic and physiological traits from seedling to boll opening stage were identified, including 15 loci associated with 14 traits that contained genes harboring nonsynonymous SNPs. We analyzed the sequence and expression of these ten candidate genes and discovered that GhRHD3 (GH_D10G0500) may affect fiber yield while GhGPAT6 (GH_D04G1426) may affect photosynthesis efficiency. CONCLUSION: Our research illustrates the efficiency of constructing a genetic map using binmap and QTL mapping on the basis of a certain size of the early-generation population. High-density genetic map features high recombination exchanges in number and distribution. The QTLs and the candidate genes identified based on this high-density genetic map may provide important gene resources for the genetic improvement of cotton.

Cotton genes GhMML1 and GhMML2 control trichome branching when ectopically expressed in tobacco.

Trichomes exhibit extraordinary diversity in shape, ultrastructure, distribution, secretion capability, biological functions, and morphological differences, which are strongly associated with their multifunction. Previous researches showed MIXTA-like transcription factors involved in regulating trichome initiation and patterning via forming MYB-bHLH-WD40 transcriptional activator complex to induce the expression of downstream genes. Here, we report the characteristics and role of GhMML1 and GhMML2, members of subgroup 9 of the R2R3-type MYB TFs. GhMML1 and GhMML2 were preferentially targeted to the nucleus and prominently expressed in the early stage during fiber development. Ectopic expression of GhMML1 and GhMML2 respectively in the transgenic tobacco plants changed the morphological characteristics of leaf trichomes; that is, the unbranched trichomes turned into multiple branched, and in the meantime, the density of trichomes was reduced on the surface of the leaf. Y2H and LCI assay revealed that both GhMML1 and GhMML2 could physically interact with a bZIP transcription factor family protein (GhbZIP) in vivo and in vitro. It has been reported that GhbZIP's homolog TAG3 in Arabidopsis is involved in the asymmetric growth of leaves and flowers via direct interaction with BOP1. Taken together, our results demonstrated that two MYB MIXTA-like proteins, GhMML1 and GhMML2, together with GhbZIP might form a multimeric complex to involve in trichome development. This study highlights the importance of MIXTA-like genes from TF subgroup 9 and will help to uncover the molecular mechanism underlying differential trichomes and their development.

Transcriptomic and Metabolomic Analyses Provide Insights Into an Aberrant Tissue of Tea Plant (Camellia sinensis).

Tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most important economic crops with multiple mutants. Recently, we found a special tea germplasm that has an aberrant tissue on its branches. To figure out whether this aberrant tissue is associated with floral bud (FB) or dormant bud (DB), we performed tissue section, transcriptome sequencing, and metabolomic analysis of these tissues. Longitudinal sections indicated the aberrant tissue internal structure was more like a special bud (SB), but was similar to that of DB. Transcriptome data analysis showed that the number of heterozygous and homozygous SNPs was significantly different in the aberrant tissue compared with FB and DB. Further, by aligning the unmapped sequences of the aberrant tissue to the Non-Redundant Protein Sequences (NR) database, we observed that 36.13% of unmapped sequences were insect sequences, which suggested that the aberrant tissue might be a variation of dormant bud tissue influenced by the interaction of tea plants and insects or pathogens. Metabolomic analysis showed that the differentially expressed metabolites (DEMs) between the aberrant tissue and DB were significantly enriched in the metabolic pathways of biosynthesis of plant hormones and biosynthesis of phenylpropanoids. Subsequently, we analyzed the differentially expressed genes (DEGs) in the above mentioned two tissues, and the results indicated that photosynthetic capacity in the aberrant tissue was reduced, whereas the ethylene, salicylic acid and jasmonic acid signaling pathways were activated. We speculated that exogenous infection induced programmed cell death (PCD) and increased the lignin content in dormant buds of tea plants, leading to the formation of this aberrant tissue. This study advanced our understanding of the interaction between plants and insects or pathogens, providing important clues about biotic stress factors and key genes that lead to mutations and formation of the aberrant tissue.

GoNe encoding a class VIIIb AP2/ERF is required for both extrafloral and floral nectary development in Gossypium.

The floral nectary, first recognized and described by Carl Linnaeus, is a remarkable organ that serves to provide carbohydrate-rich nectar to visiting pollinators in return for gamete transfer between flowers. Therefore, the nectary has indispensable biological significance in plant reproduction and even in evolution. Only two genes, CRC and STY, have been reported to regulate floral nectary development. However, it is still unknown what genes contribute to extrafloral nectary development. Here, we report that a nectary development gene in Gossypium (GoNe), annotated as an APETALA 2/ethylene-responsive factor (AP2/ERF), is responsible for the formation of both floral and extrafloral nectaries. GoNe plants that are silenced via virus-induced gene silencing technology and/or knocked out by Cas9 produce a nectariless phenotype. Point mutation and gene truncation simultaneously in duplicated genes Ne1 Ne2 lead to impaired nectary development in tetraploid cotton. There is no difference in the expression of the CRC and STY genes between the nectary TM-1 and the nectariless MD90ne in cotton. Therefore, the GoNe gene responsible for the formation of floral and extrafloral nectaries may be independent of CRC and STY. A complex mechanism might exist that restricts the nectary to a specific position with different genetic factors. Characterization of these target genes regulating nectary production has provided insights into the development, evolution, and function of nectaries and insect-resistant breeding.

Identifying Conserved Functional Gene Modules Underlying the Dynamic Regulation of Tea Plant Development and Secondary Metabolism.

Tea plants adjust development and metabolism by integrating environmental and endogenous signals in complex but poorly defined gene networks. Here, we present an integrative analysis framework for the identification of conserved modules controlling important agronomic traits using a comprehensive collection of RNA-seq datasets in Camellia plants including 189 samples. In total, 212 secondary metabolism-, 182 stress response-, and 182 tissue development-related coexpressed modules were revealed. Functional modules (e.g., drought response, theobromine biosynthesis, and new shoot development-related modules) and potential regulators that were highly conserved across diverse genetic backgrounds and/or environmental conditions were then identified by cross-experiment comparisons and consensus clustering. Moreover, we investigate the preservation of gene networks between Camellia sinensis and other Camellia species. This revealed that the coexpression patterns of several recently evolved modules related to secondary metabolism and environmental adaptation were rewired and showed higher connectivity in tea plants. These conserved modules are excellent candidates for modeling the core mechanism of tea plant development and secondary metabolism and should serve as a great resource for hypothesis generation and tea quality improvement.

Repressed Gene Expression of Photosynthetic Antenna Proteins Associated with Yellow Leaf Variation as Revealed by Bulked Segregant RNA-seq in Tea Plant Camellia sinensis.

The young leaves and shoots of albino tea cultivars are usually characterized as having a yellow or pale color, high amino acid, and low catechin. Increasing attention has been paid to albino tea cultivars in recent years because their tea generally shows high umami and reduced astringency. However, the genetic mechanism of yellow-leaf variation in albino tea cultivar has not been elucidated clearly. In this study, bulked segregant RNA-seq (BSR-seq) was performed on bulked yellow- and green-leaf hybrid progenies from a leaf color variation population. A total of 359 and 1134 differentially expressed genes (DEGs) were identified in the yellow and green hybrid bulked groups (Yf vs Gf) and parent plants (Yp vs Gp), respectively. The significantly smaller number of DEGs in Yf versus Gf than in Yp versus Gp indicated that individual differences could be reduced within the same hybrid progeny. Analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes revealed that the photosynthetic antenna protein was most significantly enriched in either the bulked groups or their parents. Interaction was found among light-harvesting chlorophyll a/b -binding proteins (LHC), heat shock proteins (HSPs), and enzymes involved in cuticle formation. Combined with the transcriptomic expression profile, results showed that the repressed genes encoding LHC were closely linked to aberrant chloroplast development in yellow-leaf tea plants. Furthermore, the photoprotection and light stress response possessed by genes involved in HSP protein interaction and cuticle formation were discussed. The expression profile of DEGs was verified via quantitative real-time PCR analysis of the bulked samples and other F1 individuals. In summary, using BSR-seq on a hybrid population eliminated certain disturbing effects of genetic background and individual discrepancy, thereby helping this study to intensively focus on the key genes controlling leaf color variation in yellow-leaf tea plants.

Role of phasiRNAs from two distinct phasing frames of GhMYB2 loci in cis- gene regulation in the cotton genome.

BACKGROUND: Phased small interfering RNA (phasiRNA) is primarily derived from the 22-nt miRNA targeting loci. GhMYB2, a gene with potential roles in cotton fiber cell fate determination, is a target gene of miR828 and miR858 in the generation of phasiRNAs. RESULTS: In the presented work, through the evaluation of phasing scores and phasiRNA distribution pattern, we found that phasiRNAs from GhMYB2 were derived from the 3' cleavage fragments of 22-nt miR828 and 21-nt miR858 respectively. These two miRNA targeting sites initiated two phasing frames on transcripts of one locus. By means of RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we further demonstrated that phasiRNAs derived from the two phasing frames played a role in cis-regulation of GhMYB2. The phasiRNAs derived from GhMYB2 were expressed in the somatic tissues, especially in anther and hypocotyl. We further employed our previous small RNA sequencing data as well as the degradome data of cotton fiber bearing ovules, anthers, hypocotyls and embryogenic calli tissues published in public databases, to validate the expression, phasing pattern and functions of phasiRNAs. CONCLUSIONS: The presenting research provide insights of the molecular mechanism of phasiRNAs in regulation of GhMYB2 loci.