【Objective】Long non-coding RNAs(lncRNAs) are a group of RNA molecules longer than 200 bp with no protein coding capacity, which are involved in various biological regulatory processes. In this study, we aim to analyze the RNA-sequencing data of two Gossypium arboreum isogenic lines, a fuzzless mutant (GA0149) and its wildtype (GA0146), to identify the lncRNA involved in early fuzz fiber development, providing a foundation for investigation the mechanism of fiber development. 【Method】We collected 0 DPA, 3 DPA and 5 DPA ovule and 8 DPA ovule and fiber from the G. arboreum fuzzless mutant GA0149 and its isogenic line GA0146 with normal fuzz and lint fibers, were used for RNA-seq to identify lncRNA and predict their target genes. Differentially expressed mRNA (DE-mRNA) and lncRNA(DE-lncRNAs) between the samples were identified. The KOBAS software was used to predict the KEGG enrichment pathways which DE-lncRNAs targets were involved in. To ensure the quality of high-through sequencing, 25 DE-lncRNAs were selected for RT-qPCR detection. 【Result】We identified 15 339 lncRNA-encoding transcripts that 11 595 lncRNAs were located to intergenic regions, 2 428 lncRNAs were classified as antisense lncRNAs, 350 were categorized as intronic lncRNAs and 966 belonged to sense lncRNAs. Compared to mRNAs, lncRNAs in Asian cotton showed shorter exons and lower GC content. Most of lncRNAs had cis-regulatory effects on their neighboring mRNAs. We identified 1 932 differentially expressed (DE) lncRNAs, with 8 134 predicted DE-lncRNA target genes. Further analysis showed that 788 genes (mRNA) were differentially expressed (DE-genes) during four fiber development stages. KEGG enrichment pathways analysis showed that DE-target-mRNAs were mainly enriched in plant hormone signal transduction and protein processing in endoplasmic reticulum. Co-expression network analysis revealed that lncRNA (MSTRG.454250.3) and its associated target genes showed identical expression trends during four fuzz fiber development stages, while lncRNAs (MSTRG.454261.4) and its associated target genes showed contrary expression tendency, exhibiting dramatic higher expression in fuzzless GA0149 compared to wildtype GA0146. The results of RT-qPCR analysis confirmed the authenticity of our RNA-seq data.【Conclusion】A total of 26 specifically expressed lncRNAs were identified which related to cotton fuzz fiber development process. We further confirmed that these lncRNAs affected the fuzz fiber development by regulating the expression of indole-3-acetic acid-amido synthetase (Ga03G2421) and Auxin-responsive protein (Ga05G1344) in the plant hormone signal transduction pathway.
【Objective】β-tubulin is the basic structural unit of cotton fiber, regulates fiber cell morphogenesis, and plays a vital role in fiber development. But there is less understood how β-tubulin gene influenced the distinct characteristic of fiber quality traits in cotton. In this study, members of the β-tubulin gene family were identified in cotton, their expression profiles were analyzed, and role of β-tubulin genes were explored for fiber quality. 【Method】BLAST method was used to identify members of the β-tubulin gene family in the genomes of four cotton species. ProtParam tool was utilized to analyze physicochemical properties, MEGA7.0 to construct phylogenetic tree, Mapchart2.2 to draw chromosomal localization map, MEME to analyze conserved motif, and PlantCARE to analyze promoter cis-acting elements. Expression levels of β-tubulin genes were characterized by using transcriptome data from 39 studies on fiber development. Spearman correlation analysis was used to identify candidate genes for fiber quality traits. 【Result】Importantly, 36, 37, 19 and 18 β-tubulin genes were identified in the genomes of Gossypium hirsutum (AD1), Gossypium barbadense (AD2), Gossypium arboretum (A2) and Gossypium raimondii (D5), respectively. The number of β-tubulin genes in tetraploid cotton species is almost double than that of diploid cotton species. Phylogenetic analysis classified these genes into 5 main clusters. Phylogenetic and collinearity analysis revealed that β-tubulin genes in Gossypium barbadense is closely related to Gossypium arboretum and Gossypium raimondii as compared to Gossypium hirsutum. Furthermore, all genes have typical conservative domains with Tubulin and Tubulin-C. The genes physicochemical properties showed amino acids range from 421 to 508 with isoelectric point of 4.68 to 5.09. The analysis of promoter cis-acting elements identified growth responsive, hormone responsive, and stress responsive elements which showed β-tubulin mediates various mechanisms of cell growth regulation. Interestingly, cluster analysis on 36 β-tubulin gene expression profiles showed 42% genes in cluster П had dominant expression in fiber. In particular, 1, 6, and 11 β-tubulin genes exhibited significant correlation with fiber micronaire value, fiber strength, and fiber length, respectively. Four genes were found to influence fiber length and fiber strength traits simultaneously. 【Conclusion】A total of 110 β-tubulin gene family members were identified in the four cotton species. Their physicochemical properties and sequences of amino acids were highly conserved and the promoter sequence had diverse regulatory elements. This study characterized the expression profiles as well as molecular function of β-tubulin gene family in cotton fiber. Further discovered the potential candidate genes that probably regulate fiber quality traits in cotton. Our results may have great potential for cotton fiber quality improvement by genetic engineering.
【Background】It is of great importance to improve the quality of cotton fiber to meet the improvement of cotton textile production and the pursue of people for high quality cotton. Fasciclin-like arabinogalactan proteins (FLAs) play an important role in the initial development of cotton fibers and secondary wall synthesis. 【Objective】Comprehensive identification and analysis of cotton FLA gene family members to reveal their common characteristics and specific expression patterns, provided a reference for the function study of FLAs in cotton fiber development.【Method】According to the whole genome data of cotton, members of FLA gene family were identified by HMMER3.0 and further verified by online softwares of Pfam and Smart. Physical and chemical properties and transmembrane domains of these proteins were analyzed by ExPASy and TMHMM. Phylogenetic tree construction, chromosome localization, collinearity analysis and protein conserved domain sequence alignment were conducted and displayed using GSDS, MCScanX, MEGA, MEME, TBtools and Jalview. Expression of FLA genes in different tissues were analyzed by cotton transcriptome data. Expression differences of GhFLAs in different developmental stages of ovules and fibers between different fiber quality materials was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Function of GhFLA05 was verified by virus induced gene silencing (VIGS). 【Result】A total of 41, 40, 20 and 21 FLA family members were identified in G.hirsutum, G. barbadense, G. arboreum and G. raimondii, respectively. The phylogenetic tree showed that cotton FLA proteins could be divided into four groups. Gene structure and motif composition were relatively conserved in each group. Further analysis of FLA proteins in Gossypium hirsutum showed that all 41 FLA members had 1-2 AGP-like glycosylation regions and 1-2 fasciclin-like domains (FAS), 37 of which contained signal peptide (SP) and 25 contained glycosylphosphatidylinositol anchored protein (GPI) anchoring signals. Subcellular localization showed that GhFLA05_D showing aggregated granules in the cytoplasm was probably localized in endoplasmic reticulum, and GhFLA18_A and GhFLA22 were expressed in cell membrane/wall, cytoplasm and nucleus. Transcriptome sequencing results showed that FLA proteins in Group A and B were mainly highly expressed in fibers, which may be involved in the process of cotton fiber elongation development and secondary wall thickening. In general, group A and B members had a similar expression pattern in two materials with significant differences in fiber quality and expressed mainly in the secondary wall development stage, especially in 20-25 DPA period. GhFLA05 exhibited specific expression at the secondary wall thickening stage with significant differences between two materials, which expressed with a high maximum value in earlier stage of secondary wall thickening stage in high specific strength material RIL229, suggesting GhFLA05 may take a part in the regulation of cotton fiber strength difference formation. The fiber strength and micronaire value decreased in GhFLA05 gene-silenced cotton plants by VIGS.【Conclusion】A sum of 122 FLA family members were identified in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii, which could be divided into four groups. Members of different groups had high structural and functional similarities, and the genes related to cotton fiber development were identified. It was clarified that GhFLA05 specifically expressed in the secondary wall synthesis stage, and closely related to the difference in fiber strength of different upland cotton materials.
【Objective】The two main types of allotetraploid cotton that are currently cultivated are upland cotton, known for its high yield and good adaptability, and island cotton, which boasts excellent fiber quality but lower yield. EXO70, a vital subunit of the exocyst complex, plays a significant role in plant growth, development, and stress response. By identifying and analyzing members of the EXO70 gene family in upland and island cotton at the whole-genome level, and studying their functions in fiber development and environmental adaptation, we can shed light on the molecular basis for the differences in traits between these two varieties. 【Method】The reference sequences of EXO70 protein in Arabidopsis were obtained from the TAIR database. HMMER, ExPASy, MEME, TBtools, and other analysis tools were used to identify and analyze the members of EXO70 gene family in the genomes of upland cotton TM-1 and island cotton Hai7124. The similarities and differences in gene expression patterns, correlations with crucial agronomic traits, and stress responses of this family were systematically compared. 【Result】Through genome-level analysis of upland cotton and sea-island cotton, 54 EXO70 family members were identified in both upland and sea-island cotton, which could be divided into eight subgroups based on phylogenetic analysis. Orthologous genes between upland cotton and sea-island cotton can be paired one-to-one and are distributed across the 20 chromosomes of both species. The majority of the members have single exons, while 12 pairs of homologous genes displayed significant differences in the reading frame sequences. Most orthologous genes in Gossypium hirsutum and Gossypium barbadense display similar expression patterns, but differences in expression levels are observed during the same fiber development stage, such as GH_A04G1208 and its orthologous gene GB_A04G1253 in subgroup A. Single gene association analysis revealed that more genes were associated with fiber quality traits in sea-island cotton, while more genes were associated with yield traits and environmental adversity sensitivity in upland cotton. Some trait association differences resulted from genetic structural differences between Gossypium hirsutum and Gossypium barbadense. Under different stresses, upland cotton showed a significantly higher number of induced genes compared to sea-island cotton. 【Conclusion】The sequence structure and gene expression patterns of the EXO70 family were found to be relatively conserved in both upland and island cotton during the formation, differentiation, and domestication of tetraploid cotton. However, in terms of EXO70 family members, island cotton had more genes related to fiber quality traits, while upland cotton had more genes related to yield traits and exhibited greater sensitivity to environmental stress.
Map-based cloning is a classical and effective method to identify candidate genes for specific phenotypic variants. Map-based cloning of functional genes plays important roles in the innovative utilization of germplasm resources, molecular design breeding and improving breeding efficiency. In recent years, the whole-genome sequencing of Gossypium raimondii, Gossypium arboreum, Gossypium hirsutum, and Gossypium barbadense has been completed and improved. Map-based cloning has entered into a crucial period. In 2016, the dominant glandless gene Gl2e (GoPGF) was the first map-based cloning gene in cotton. So far, 20 qualitative traits genes and 5 quantitative traits genes have been identified by map-based cloning technology. In this paper, research progress was systematically reviewed in fiber, gland, nectary, leaf type, plant architecture, plant color, and fertility in terms of gene symbols, names, chromosomal positioning, and candidate genes. Moreover, map-based cloning strategies were systematically reviewed in mapping populations and bulked segregate analysis-sequencing (BSA-seq). With the reduction of sequencing cost and utilization of BSA-seq, it is believed that more and more genes will be cloned by map-based cloning technology. In addition, transformation and genome editing have been successfully used to evaluate the function of the candidate gene in the target interval. It is believed that map-based cloning could provide a theoretical basis and genetic resources for molecular design breeding in cotton.
