Large-scale long terminal repeat insertions produced a significant set of novel transcripts in cotton.

Genomic analysis has revealed that the 1,637-Mb Gossypium arboreum genome contains approximately 81% transposable elements (TEs), while only 57% of the 735-Mb G. raimondii genome is occupied by TEs. In this study, we investigated whether there were unknown transcripts associated with TE or TE fragments and, if so, how these new transcripts were evolved and regulated. As sequence depths increased from 4 to 100 G, a total of 10,284 novel intergenic transcripts (intergenic genes) were discovered. On average, approximately 84% of these intergenic transcripts possibly overlapped with the long terminal repeat (LTR) insertions in the otherwise untranscribed intergenic regions and were expressed at relatively low levels. Most of these intergenic transcripts possessed no transcription activation markers, while the majority of the regular genic genes possessed at least one such marker. Genes without transcription activation markers formed their+1 and -1 nucleosomes more closely (only (117±1.4)bp apart), while twice as big spaces (approximately (403.5±46.0) bp apart) were detected for genes with the activation markers. The analysis of 183 previously assembled genomes across three different kingdoms demonstrated systematically that intergenic transcript numbers in a given genome correlated positively with its LTR content. Evolutionary analysis revealed that genic genes originated during one of the whole-genome duplication events around 137.7 million years ago (MYA) for all eudicot genomes or 13.7 MYA for the Gossypium family, respectively, while the intergenic transcripts evolved around 1.6 MYA, resultant of the last LTR insertion. The characterization of these low-transcribed intergenic transcripts can facilitate our understanding of the potential biological roles played by LTRs during speciation and diversifications.

A comprehensive overview of cotton genomics, biotechnology and molecular biological studies.

Cotton is an irreplaceable economic crop currently domesticated in the human world for its extremely elongated fiber cells specialized in seed epidermis, which makes it of high research and application value. To date, numerous research on cotton has navigated various aspects, from multi-genome assembly, genome editing, mechanism of fiber development, metabolite biosynthesis, and analysis to genetic breeding. Genomic and 3D genomic studies reveal the origin of cotton species and the spatiotemporal asymmetric chromatin structure in fibers. Mature multiple genome editing systems, such as CRISPR/Cas9, Cas12 (Cpf1) and cytidine base editing (CBE), have been widely used in the study of candidate genes affecting fiber development. Based on this, the cotton fiber cell development network has been preliminarily drawn. Among them, the MYB-bHLH-WDR (MBW) transcription factor complex and IAA and BR signaling pathway regulate the initiation; various plant hormones, including ethylene, mediated regulatory network and membrane protein overlap fine-regulate elongation. Multistage transcription factors targeting CesA 4, 7, and 8 specifically dominate the whole process of secondary cell wall thickening. And fluorescently labeled cytoskeletal proteins can observe real-time dynamic changes in fiber development. Furthermore, research on the synthesis of cotton secondary metabolite gossypol, resistance to diseases and insect pests, plant architecture regulation, and seed oil utilization are all conducive to finding more high-quality breeding-related genes and subsequently facilitating the cultivation of better cotton varieties. This review summarizes the paramount research achievements in cotton molecular biology over the last few decades from the above aspects, thereby enabling us to conduct a status review on the current studies of cotton and provide strong theoretical support for the future direction.

Molecular studies of cellulose synthase supercomplex from cotton fiber reveal its unique biochemical properties.

Cotton fiber is a highly elongated and thickened single cell that produces large quantities of cellulose, which is synthesized and assembled into cell wall microfibrils by the cellulose synthase complex (CSC). In this study, we report that in cotton (Gossypium hirsutum) fibers harvested during secondary cell wall (SCW) synthesis, GhCesA 4, 7, and 8 assembled into heteromers in a previously uncharacterized 36-mer-like cellulose synthase supercomplex (CSS). This super CSC was observed in samples prepared using cotton fiber cells harvested during the SCW synthesis period but not from cotton stem tissue or any samples obtained from Arabidopsis. Knock-out of any of GhCesA 4, 7, and 8 resulted in the disappearance of the CSS and the production of fiber cells with no SCW thickening. Cotton fiber CSS showed significantly higher enzyme activity than samples prepared from knock-out cotton lines. We found that the microfibrils from the SCW of wild-type cotton fibers may contain 72 glucan chains in a bundle, unlike other plant materials studied. GhCesA4, 7, and 8 restored both the dwarf and reduced vascular bundle phenotypes of their orthologous Arabidopsis mutants, potentially by reforming the CSC hexamers. Genetic complementation was not observed when non-orthologous CesA genes were used, indicating that each of the three subunits is indispensable for CSC formation and for full cellulose synthase function. Characterization of cotton CSS will increase our understanding of the regulation of SCW biosynthesis.

A Malvaceae-specific miRNA targeting the newly duplicated GaZIP1L to regulate Zn2+ ion transporter capacity in cotton ovules.

MicroRNAs (miRNAs) play critical roles in regulating gene expression in plants, yet their functions underlying cultivated diploid Gossypium arboreum cotton ovule development are largely unknown. Here, we acquired small RNA profiles from G. arboreum ovules and fibers collected at different growth stages, and identified 46 novel miRNAs that accounted for 23.7% of all miRNAs in G. arboreum reported in the latest plant sRNA database. Through analysis of 84 (including 38 conserved) differentially expressed G. arboreum miRNAs, we detected 215 putative protein-coding genes in 26 biological processes as their potential targets. A Malvaceae-specific novel miRNA named gar-miRN44 was found to likely regulate cotton ovule growth by targeting to a newly duplicated Zn2+ ion transporter gene GaZIP1L. During cotton ovule development, gar-miRN44 transcript level decreased sharply after 10 to 15 days post-anthesis (DPA), while that of the GaZIP1L increased significantly, with a concomitant increase of Zn2+ ion concentration in late ovule developmental stages. Molecular dynamics simulation and ion absorption analysis showed that GaZIP1L has stronger Zn2+ ion binding ability than the original GaZIP1, indicating that the newly evolved GaZIP1L may be more suitable for maintaining high Zn2+ ion transport capacity that is likely required for cotton ovule growth via enhanced cellulose synthase activities. Our systematic miRNA profiling in G. arboreum and characterization of gar-miRN44 not only contribute to the understanding of miRNA function in cotton, but also provide potential targets for plant breeding.

Multi-strategic RNA-seq analysis reveals a high-resolution transcriptional landscape in cotton.

Cotton is an important natural fiber crop, however, its comprehensive and high-resolution gene map is lacking. Here we integrate four complementary high-throughput techniques, including Pacbio long read Iso-seq, strand-specific RNA-seq, CAGE-seq, and PolyA-seq, to systematically explore the transcription landscape across 16 tissues or different organ types in Gossypium arboreum. We devise a computational pipeline, named IGIA, to reconstruct accurate gene structures from the integrated data. Our results reveal a dynamic and diverse transcriptional map in cotton: tissue-specific gene expression, alternative usage of TSSs and polyadenylation sites, hotspot of alternative splicing, and transcriptional read-through. These regulated events affect many genes in various aspects such as gain or loss of functional RNA motifs and protein domains, fine-tuning of DNA binding activity, and co-regulation for genes in the same complex or pathway. The methods and findings provide valuable resources for further functional genomic studies such as understanding natural SNP variations for plant community.