Phenotypic And Genotypic Characterization Of Root System Architecture And Fiber Quality Traits In Cotton Using GWAS

Cotton(Gossypium spp.)is an important fiber crop,accounting for nearly half of the world’s textile production and having significant economic and social importance.Root systems play a crucial role in cotton plant development,providing anchorage to the ground and absorbing nutrients necessary for sustained productivity.In cotton,vigorous root growth during the early post-germination stages is essential for successful seedling establishment and productivity,making it a highly desirable trait for breeders and farmers.Despite its fundamental importance,the genetic control of cotton root growth remains poorly understood.Understanding the genetic basis of root system architecture(RSA)is essential for improving cotton productivity under stress conditions.In this study,we used 227 cotton accessions to characterize the phenotypic variation of RSA and the genetic architecture of primary root length with and without salt stress.Using clustering analysis,we identified 18 accessions with robust root phenotypes,indicating that these accessions have the potential for improving cotton productivity.Furthermore,the results of salt stress indicated that the emergence and early seedling phases in cotton were particularly vulnerable to salt stress,and root length showed significant plasticity under different concentrations of salt stress.After evaluating the response of diverse cotton accessions to salt stress,a wide range of variation in root length across the accessions was observed.We also observed that primary root length increased in response to salt stress in some genotypes at 250 m M of salt stress,while others showed no increase or decrease in primary root length.This suggests that some cotton accessions have the potential to adapt to salt stress,making them suitable for cultivation in saline soils.To identify the genetic basis of the observed phenotypic variation in primary root length,we conducted a genome-wide association study(GWAS)and identified two candidate genes,Gibberellin 20 oxidase 2(GA20ox2),which was identified under normal condition,and LONESOME HIGHWAY(LHW)was identified under salt stress condition.These genes are involved in hormonal pathways such as gibberellin and auxin signaling and contribute to differences in root length among cotton accessions.Our gene expression analysis revealed differential expression of these genes in short and long root accessions.We further confirmed the role of GA20ox2 in primary root growth by conducting virus-induced gene silencing,which significantly reduced primary root length.Similarly,population genetic analysis of 1-Mb genomic region containing the gene GA20ox2 revealed that GA20ox2 underwent artificial selection,leading to changes in the frequency of SNP variants.The frequency of the favorable C SNP was higher in wild accessions and landraces but reduced in modern improved cultivars,suggesting a loss of the favorable SNP among modern cultivars.Additionally,the level of genetic differentiation for GA20ox2 was highest between wild accessions and improved cultivars,indicating a possible selection pressure during cotton improvement.Moreover,positive values for some subpopulations,suggests a possible selection pressure on GA20ox2 during cotton improvement.Also,the frequency of T SNP was higher in improved accessions from China and increased in frequency in accessions from regions where tetraploid cotton was introduced later due to selection by humans.In addition to our findings of the LHW gene,belonging to the bHLH transcription factor family related to primary root development,observed that this gene exhibited high expression specifically in cotton fibers and ovules,indicating its involvement in fiber development.To further explore its genetic variations,we analyzed SNPs around this gene and identified significant associations of five SNPs with fiber length and six SNPs with fiber strength.Among the eight haplotypes identified for the LHW gene,related fiber quality traits,Hap4 was exclusively present in Gossypium hirsutum and was prominently represented.Notably,accessions carrying Hap4 demonstrated superior fiber length and strength,although they exhibited a lower fiber elongation rate.Interestingly,Hap4 was found predominantly in certain subpopulations,particularly in Chinese accessions,suggesting regional variations and a potential selective advantage or positive selection for this haplotype in specific cotton-growing regions of China.These observations indicate the influence of breeding practices targeting fiber quality traits on the prevalence of Hap4.Given these findings,we performed a comprehensive analysis of bHLH transcription factors in three different cotton species: Gossypium hirsutum,Gossypium barbadense,and Gossypium arboreum.Our investigation was mainly focused on the genetic variations of bHLH transcription factors in G.hirsutum and their association with four fiber quality traits.Notably,the highly expressed bHLH genes in cotton fibers showed significant associations with fiber length and strength.Specifically,we identified two genes,bHLH149 and bHLH96,which exhibited favorable haplotypes associated with improved fiber quality traits.Moreover,our population genetic analysis revealed that these favorable haplotypes have undergone selection during the improvement of cotton varieties,indicating their potential significance in breeding programs aimed at enhancing fiber quality traits.This study identified cotton accessions with superior root phenotype that have the potential to be used in breeding programs aimed at improving the root systems of cotton plants for abiotic stress tolerance.Moreover,our identification of specific bHLH genes and their favorable haplotypes for fiber quality traits suggests that they could be targeted in future cotton breeding programs for improving both fiber quality traits and root systems.Overall,the insights gained from this study can be utilized to develop improved cotton varieties that exhibit enhanced performance and productivity.