Single-cell RNA sequencing opens a new era for cotton genomic research and gene functional analysis

Journal of Cotton Research, Journal Year: 2024, Volume and Issue: 7(1)

Published: June 4, 2024

Abstract Single-cell RNA sequencing (scRNA-seq) is one of the most advanced technologies for studying transcriptome landscape at single-cell revolution. It provides numerous advantages over traditional RNA-seq. Since it was first used to profile in plants 2019, has been extensively employed perform different research plants. Recently, scRNA-seq also quickly adopted by cotton community solve lots scientific questions which have never solved. In this comment, we highlighted significant progress employing genetic and genomic study its future potential applications.

Language: Английский

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Robust CRISPR/Mb2Cas12a genome editing tools in cotton plants

iMeta, Journal Year: 2024, Volume and Issue: 3(3)

Published: June 1, 2024

The efficiency and accuracy of the CRISPR/Mb2Cas12a system were demonstrated in cotton, achieving an over 90% at target sites. Notably, Mb2Cas12a exhibited significant tolerance under different temperatures ranging from 22°C to 32°C. Additionally, revealed effective editing more relaxed VTTV PAM sites cotton genome, which expanded genome range by approximately 2.6-fold than wide-type LbCas12a. Finally, a multiplex was also developed based on Mb2Cas12a, enabling simultaneous eight using single crRNA cassette. CRISPR/Cas9 has sparked biotechnological revolution various fields, serving as milestone for biotechnology mutagenesis with unparalleled simplicity, high efficiency, precision [1-4]. CRISPR/Cas12 possesses certain features that make it promising tool [5]. Recently, described perform plants. Importantly, can recognize not only TTTV (V = A, C, G) PAMs but identify expand whole [6]. However, this been tested plants yet. Upland Cotton (Gossypium hirsutum) is allotetraploid large complex (AADD, 2n 4x 52) 2.5 Gb, where most genes are duplicated. Although several CRISPR/Cas12a systems have plant species, limited number Cas12 proteins utilized editing. Most CRISPR/Cas studies used mixed dual promoter [7, 8]. Whether other types Cas RNA (sgRNA)/crRNA expression enhance remains be investigated. Furthermore, Cas9 Cas12a plants, effectiveness highly efficient multiple still needs [9, 10]. In light this, present study aims establish evaluate cassettes, finally assess feasibility applying First, we constructed vector termed Strategy 1. 1, cassette containing 21 nt direct repeat fused 23 guide driven Pol III (pOsUbi2) II (pGhU6.7), respectively. spaced tRNA (Figure 1A) [11]. To system, selected visual reporter GhPGF GhCLA1 genes. Transgenic generated through Agrobacterium-mediated transformation S1). Subsequent next-generation sequencing analysis up 94.62% GhPGF-crRNA1 site T0 (Figures 1B S3 Table main window ranged 13 26 downstream Only DNA deletions detected all edited deletion sizes mainly 7 18 bp 1C). These findings consistent our previous verification LbCas12a indicating similar pattern within family [7]. We observed decrease density gossypol glands both leaf stem tissues, leading considerable lacking 1D). Meanwhile, carrying GhCLA1-crRNA2 component partially bleached phenotype their leaves, reaching 71.34% 1B,D). chimeric because homozygous mutants unable survive soil. two sites, GhPGF-crRNA2 GhCLA1-crRNA1 S4−S7). Next, evaluated 2 3. 2, arrays pOsUbi2 individually. array processed Hammerhead ribozyme (HH) Hepatitis Delta Virus (HDV) ribozyme. 3, simultaneously pOsUbi2, named transcript unit (STU) [12]. same four targeted sequences 1 generate transgenic Like 3 achieved 94.33% 91.95% S1), respectively, showing patterns obvious phenotypes those events site, efficiencies 47.81% 78.18%, respectively 1B). Efficient well S3−S6). Our accurate It successfully combination three arrays: tRNA-crRNA-tRNA, HH-crRNA-HDV, STU. indicated remained essentially regardless whether or crRNA. specificity Sanger analyses no off-target mutation any predicted 1E S2). genotypes loci faithfully inherited T1 progeny, some new lines due presence protein besides existing 1F−H Remarkably, transgene-free (pgf-1 pgf-2) obtained genetic segregation mutations exhibiting glandless one major advantages technology traditional strategy (the phenotypes/traits relay transgene integration host cells) investigate activity temperature conditions, total 12 targeting adaptability test. subjected treatments 22°C, 25°C, 28°C, 32°C days 2A). Interestingly, found slightly affected lower temperatures, condition. For example, M71 (a 1) 90.26%, 94.62%, 91.18%, 92.29% 32°C, difference 2B). Analysis profiles these position Mb2Cas12 varied 2C S8). carried out differences S9). Previous reports nucleases sensitive [13, 14]. Differences among may primarily attributed (25–30°C) required tissue culture processes reducing nuclease. Nevertheless, research indicate displays excellent varying shows even conditions. Therefore, holds great promise friendly vast species prefer cooler growing scope conducted comprehensive genome-wide cotton. Results showed NGG recognized SpCas9 cover 21.52% Jin668 widely genotype transformation) reference genome. comparison, 23.21% whereas its TTV 59.59% represents 2.8-fold increase compared 2D). employed construct GhPGF, ~15.50% ATTV resulting 4-bp 2E). editable nor AsCas12a [15, 16]. capable effectively thereby further enhancing utility crRNAs designed five endogenous (GhPGF, GhCLA1, GhFAD, GhTAC1, GhMYB25-like) 2F). 79 calli plantlets, rates 1.45% 86.96%. Compared gene earlier crRNA1 39.7% system. Moreover, results middle units (loci 3–4) relatively lower, 1.82% 17.93%. contrast, higher ends unit, 39.7%, 71.47%, 58.62% crRNA1, crRNA7, crRNA8, 2G). suggest targets influenced crRNA-specific locations [17]. This observation could suitability during transcription small RNAs, while producing long transcripts Considering data calli/plantlets report, advisable conduct tests larger samples include regenerated addition calli, facilitate analysis. Taken together, suggested precise greatly expands wide adaptability. highlight potential valuable improvement summary, CRISPR/Mb2Cas12 significantly enhances capability extends established provides technical tools creation customized germplasm resources facilitates fundamental biological field. advancements hold advancing breeding studies. Shuangxia Jin, Xianlong Zhang, Yong Zhang project. Fengjiao Hui performed experiments, analyzed data, wrote manuscript. Xu Tang involved experiments construction. Bo Li, Muna Alariqi, Yongxue Hu, Guanying Wang extraction. Qingying Meng Zhongping whole-genome revised All authors read final manuscript approved publication. supported Science Technology Innovation 2030-Major Projects (2023ZD04074) Dr. 2030 (2022ZD0402001-04) Xu, National Fund China Distinguished Young Scholars (32325039 32272128) Hubei Hongshan Laboratory (2021hszd013) Jin. computations paper run bioinformatics computing platform Key Crop Genetic Improvement, Huazhong Agricultural University. declare conflict interest. No animals humans study. deposited NCBI submission SUB14445092 SUB14154255, BioProject accession PRJNA1067522 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1067522) PRJNA1111234 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1111234). scripts saved GitHub https://github.com/fengjiaohui/Mb2Cas12a. Supplementary materials (methods, figures, tables, graphical abstract, slides, videos, Chinese translated version) online DOI iMeta http://www.imeta.science/. Figure S1: application CRISPR/Mb2Cas12a-mediated S2: stability effects S3: Deep Sequencing result site. S4: S5: S6: S7: Frequency size induced S8: Editing strategies treatments. S9: targets. Primers positive sequences, Barcode primer amplicon gene. Summary off-targets predictions Cas-OFFinder GhPGF-crRNA1. Please note: publisher responsible content functionality supporting information supplied authors. Any queries (other missing content) should directed corresponding author article.

Language: Английский

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Time-Course Transcriptomics Analysis Reveals Molecular Mechanisms of Salt-Tolerant and Salt-Sensitive Cotton Cultivars in Response to Salt Stress

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(1), P. 329 - 329

Published: Jan. 2, 2025

Salt stress is an environmental factor that limits plant seed germination, growth, and survival. We performed a comparative RNA sequencing transcriptome analysis during germination of the seeds from two cultivars with contrasting salt tolerance responses. A transcriptomic comparison between salt-tolerant cotton cv Jin-mian 25 salt-sensitive Su-mian 3 revealed both similar differential expression patterns genotypes stress. The genes related to aquaporins, kinases, reactive oxygen species (ROS) scavenging, trehalose biosynthesis, phytohormone biosynthesis signaling include ethylene (ET), gibberellin (GA), abscisic acid (ABA), jasmonic (JA), brassinosteroid (BR) were systematically investigated cultivars. Despite involvement these in cotton’s response positive or negative ways, their levels mostly genotypes. Interestingly, PXC2 gene (Ghir_D08G025150) was identified, which encodes leucine-rich repeat receptor-like protein kinase (LRR-RLK). This showed induced pattern after treatment but not 3. Our multifaceted approach illustrated cotton.

Language: Английский

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Effects of Shade Stress on the Synthesis of Cellulose and Lignin in Maize Nodal Roots

Journal of Agronomy and Crop Science, Journal Year: 2025, Volume and Issue: 211(1)

Published: Jan. 1, 2025

ABSTRACT Low solar radiation is an important factor affecting maize root growth and development. Roots have anchoring function, their components are cellulose lignin. Here, shade experiments were conducted using nets with 50% light transmittance (L 50 ). The experiment was in 2021 2022 the ‘Xianyu 335’ variety under two nitrogen conditions (N 1 = 180 kg ha −1 N 2 240 ) to investigate effect of shading on structural carbohydrate content nodal roots. results showed that had a highly significant lignin contents. Compared normal 100 ), significantly decreased by 10.36%–13.87% 12.96%–18.68% Shading sucrose soluble sugar contents lignin‐related enzyme activities. positively correlated content. at silking (R1) stage synthase (SS) phosphate activities 15th leaf (V15) stage; R1 SS, acid invertase, tyrosine ammonia‐lyase, cinnamyl alcohol dehydrogenase, polyphenol oxidase peroxidase V15. Genes related synthesis, including ( SS CESA synthase‐interactive protein CSI1 Chitinase‐Like1 CTL1 STELLO2 STL2 downregulated shading, as synthesis‐related phenylalanine/tyrosine ammonia‐lyase PTAL 4‐coumarate‐CoA ligase 4CL POD genes. Auxin jasmonic affected thereby reducing synthesis. These findings provide theoretical support for development appropriate cultivation practices reduced radiation.

Language: Английский

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Reversing anther thermotolerance by manipulating the cis-elements in the promoter of a high-temperature upregulated gene Casein Kinase I in upland cotton

Science China Life Sciences, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 14, 2025

Language: Английский

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Applications of Virus-Induced Gene Silencing in Cotton

Plants, Journal Year: 2024, Volume and Issue: 13(2), P. 272 - 272

Published: Jan. 17, 2024

Virus-induced gene silencing (VIGS) is an RNA-mediated reverse genetics technique that has become effective tool to investigate function in plants. Cotton one of the most important economic crops globally. In past decade, VIGS been successfully applied cotton functional genomic studies, including those examining abiotic and biotic stress responses vegetative reproductive development. This article summarizes traditional vectors used system, visible markers for endogenous silencing, applications genomics, limitations how they can be addressed cotton.

Language: Английский

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High-quality genomes of Bombax ceiba and Ceiba pentandra provide insights into the evolution of Malvaceae species and differences in their natural fiber development

Plant Communications, Journal Year: 2024, Volume and Issue: 5(5), P. 100832 - 100832

Published: Feb. 5, 2024

Members of the Malvaceae family, including Corchorus spp., Gossypium Bombax and Ceiba are important sources natural fibers. In past decade, genomes several species have been assembled; however, evolutionary history differences in their fiber development remain to be clarified. Here, we report genome assembly annotation two plants from Malvaceae, ceiba pentandra, whose assembled sizes 783.56 Mb 1575.47 Mb, respectively. Comparative analysis revealed that whole-genome duplication Gypsy long terminal repeat retroelements major causes chromosome number (2n = 14 2n 96) size (234 2676 Mb) among species. We also used comparative genomic analyses reconstruct ancestral karyotype with 11 proto-chromosomes, providing new insights into trajectories MYB-MIXTA-like 3 is relatively conserved functions cell-fate determination epidermis. It appears perform this function any tissue where it expressed, i.e. fibers on endocarp B. ovule cotton. identified a structural variation cellulose synthase gene higher copy synthase-like genes as possible finer, less spinnable, weaker ceiba. Our study provides high-quality offers evolution formation through multi-omics analysis.

Language: Английский

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Non-B-form DNA is associated with centromere stability in newly-formed polyploid wheat

Science China Life Sciences, Journal Year: 2024, Volume and Issue: 67(7), P. 1479 - 1488

Published: April 16, 2024

Language: Английский

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A telomere-to-telomere cotton genome assembly reveals centromere evolution and a Mutator transposon-linked module regulating embryo development

Nature Genetics, Journal Year: 2024, Volume and Issue: 56(9), P. 1953 - 1963

Published: Aug. 15, 2024

Language: Английский

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GbPP2C80 Interacts with GbWAKL14 to Negatively Co‐Regulate Resistance to Fusarium and Verticillium wilt via MPK3 and ROS Signaling in Sea Island Cotton

Advanced Science, Journal Year: 2024, Volume and Issue: 11(30)

Published: June 18, 2024

Fusarium wilt (FW) is widespread in global cotton production, but the mechanism underlying FW resistance superior-fiber-quality Sea Island unclear. This study reveals that has been target of genetic improvement China since 2010s. The key nonsynonymous single nucleotide polymorphism (SNP, T/C) gene Gbar_D03G001670 encoding protein phosphatase 2C 80 (PP2C80) results an amino acid shift (L/S), which significantly associated with cotton. Silencing GbPP2C80 increases cotton, whereas overexpressing reduces Arabidopsis. and GbWAKL14 exist synergistically accessions haplotype forms "susceptible-susceptible" (TA) "resistant-resistant" (CC), interact each other. CRISPR/Cas9-mediated knockout enhances Verticillium (VW) upland overexpression weakens VW respond to by modulating reactive oxygen species (ROS) content via affecting MPK3 expression. In summary, two tandem genes on chromosome D03, GbPP2C80, GbWAKL14, functions as cooperative negative regulators disease defense, providing novel resources molecular markers for development resistant cultivars.

Language: Английский

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