Genome-wide identification,characterization,and expression analysis of aluminum-activated malate transporter genes(ALMTs)in Gossypium hirsutum L.

Aluminum-activated malate transporters(ALMT)are widely involved in plant growth and metabolic processes,including adaptation to acid soils,guard cell regulation,anion homeostasis,and seed development.Although ALMT genes have been identified in Arabidopsis,wheat,barley,and Lotus japonicus,little is known about its presence in Gossypium hirsutum L.In this study,ALMT gene recognition in diploid and tetraploid cotton were done using bioinformatics analysis that examined correlation between homology and evolution.Differentially regulated ALMT genetic profile in G.hirsutum was examined,using RNA sequencing and qRT-PCR,during six fiber developmental time-points,namely 5 d,7 d,10 d,15 d,20 d,and 25 d.We detected 36 ALMT genes in G.hirsutum,which were subsequently annotated and divided into seven sub-categories.Among these ALMT genes,34 had uneven distribution across 14/26 chromosomes.Conserved domains and gene structure analysis indicated that ALMT genes were highly conserved and composed of exons and introns.The GhALMT gene expression profile at different DPA(days post anthesis)in different varieties of G.hirsutum is indicative of a crucial role of ALMT genes in fiber development in G.hirsutum.This study provides basis for advancements in the cloning and functional enhancements of ALMT genes in enhancing fiber development and augmenting high quality crop production.

Transcriptional landscape of cotton roots in response to salt stress at single-cell resolution

Increasing soil salinization has led to severe reductions in plant yield and quality,and investigating the mo-lecular mechanism of salt stress response is therefore an urgent priority.In this study,we systematically analyzed the response of cotton roots to salt stress using single-cell transcriptomics technology;56281 high-quality cells were obtained from 5-day-old lateral root tips of Gossypium arboreum under natural growth conditions and different salt treatments.Ten cell types with an array of novel marker genes were identified and confirmed by in situ RNA hybridization,and pseudotime analysis of some specific cell types revealed their potential differentiation trajectories.Prominent changes in cell numbers under salt stress were observed for outer epidermal and inner endodermal cells,which were significantly enriched in response to stress,amide biosynthetic process,glutathione metabolism,and glycolysis/gluconeogenesis.Analysis of differentially expressed genes identified in multiple comparisons revealed other functional ag-gregations concentrated on plant-type primary cell wall biogenesis,defense response,phenylpropanoid biosynthesis,and metabolic pathways.Some candidate differentially expressed genes encoding transcrip-tion factors or associated with plant hormones also responsive to salt stress were identified,and the func-tion of Ga03G2153,annotated as auxin-responsive GH3.6,was confirmed by virus-induced gene silencing.The GaGH3.6-silenced plants showed a severe stress-susceptible phenotype,and physiological and biochemical measurements indicated that they suffered more significant oxidative damage.These results suggest that GaGH3.6 might participate in cotton salt tolerance by regulating redox processes.We thus construct a transcriptional atlas of salt-stressed cotton roots at single-cell resolution,enabling us to explore cellular heterogeneity and differentiation trajectories and providing valuable insights into the mo-lecular mechanisms that underlie plant stress tolerance.

Rapid Identification of a Candidate Gene Related to Fiber Strength Using a Superior Chromosome Segment Substitution Line from Gossypium hirsutum × Gossypium barbadense via Bulked Segregant RNA-Sequencing

Cotton is the most widely cultivated commercial crop producing natural fiber around the world.As a critical trait for fiber quality,fiber strength principally determined during the secondary wall thickening period.Based on the developed BC5F3:5 CSSLs(chromosome segment substitution lines)from Gossypium hirsutum CCRI36×G.barbadense Hai 1,the superior MBI9915 was chosen to construct the secondary segregated population BC7F2 with its recurrent parent CCRI36,which was subsequently subjected to Bulk segregant RNA-sequencing(BSR-seq)for rapid identification of candidate genes related to fiber strength.A total of 4 fiber-transcriptome libraries were separately constructed and sequenced,including two parents(CCRI36 and MBI9915)and two extreme pools at 20 DPA(days post anathesis).Through multiple comparisons,536 DEGs(differentially expressed genes)were overlapped at 20 DPA.Allelic-polymorphism comparison in mRNA sequences revealed 831 highly probable SNPs between two extreme pools related to fiber strength.Linkage analysis was performed between two extreme pools with SNP-index method.Eighteen correlated regions with 1981 annotation genes were obtained between two pools at 20 DPA,of which 12 common DEGs were similarly identified both between two parents and two pools.One gene(Gh_A07G0837)in the candidate region related to fiber strength was differentially expressed in both parents and extreme pools and involved in fiber strength development through reactive oxygen species(ROS)activity.Co-expression analysis of Gh_A07G0837 showed that Gh_A07G0837 may cooperate with other genes to regulate fiber strength.The reliability of BSR-seq results was validated by the quantitative real-time PCR(qRT-PCR)experiments on 5 common DGEs 20 DPA.Co-expressed analysis results indicated that there were some genes expressed especially low in MBI9915,resulting in good fiber strength.Focusing on bulked segregant analysis on the extreme pools derived from superior CSSL population,this study indicates that BSR-seq can be efficiently applied on rapid identification of candidate genes related to fiber strength,which make contributions to our understanding of fiber quality formation in cotton.

Dynamic Expression Analysis and Introgressive Gene Identification of Fiber Length Using Chromosome Segment Substitution Lines from G.hirsutum×G.barbadense

Fiber length is a critical trait that principally determines cotton spinning quality,while Upland cotton as the most widely cultivated Gossypium species around the world subjects to the relatively ordinary fiber performance.Chromosome segment substitution lines(CSSLs)have been introduced in cotton breeding to take full advantages of superior fiber quality and high yield from Sea Island and Upland cotton,respectively,which serve as ideal materials for elucidating the genetic mechanism of complex quantitative traits.Here,three CSSLs derived from CCRI45(G.hirsutum)×Hai1(G.barbadense),two superior(MBI7561 and MBI7747)and one(MBI7285)with ordinary fiber-quality,were subjected to transcriptome sequencing during fiber elongation together with their recurrent parent CCRI45,and 471.425 million clean reads were obtained with 91.47%average Q30 and 45.23%mean GC content.In total,5,673 differentially expressed genes(DEGs)were identified from multi-sample comparisons,which were mainly involved in the oxidation-reduction process,protein phosphorylation,regulation of transcription,DNA template,and carbohydrate metabolic process.Eight temporal expression patterns were monitored on the DEGs of different lines,of which the significantly enriched profile revealed higher similarities between two superior CSSLs or the ordinary CSSL and CCRI45 with respect to fiber performance.Based on the intersection between the predicted introgressive genes from RNAseq data and the published gene information from the G.barbadense genome,1,535 introgressive genes were identified in three CSSLs.Further analysis of the three common introgressive sections in superior CSSLs revealed eight candidate genes that were identified to be involved in fiber development,namely,O-fucosyltransferase family protein(GB_A02G0240),glutamine synthetase 2(GB_A02G0272),Ankyrin repeat family protein(GB_A02G0264),beta-6 tubulin(GB_D03G1742),WRKY DNA-binding protein 2(GB_D03G1655),quinolinate synthase(GB_D07G0623),nuclear factor Y,subunit B13(GB_D07G0631),and leucine-rich repeat transmembrane protein kinase(GB_D07G0797).Our results provide novel insights into the mechanism underlying fiber formation and lay a solid foundation for further high-efficiency determination of candidate genes by combining RNA-seq data and pivotal chromosome regions.