Drought Tolerant QTL Mapping,genome Wide Identification And Functional Characterization Of Six Drought And/or Salt Stress Responsive Gene Families In Cotton

A backcross-inbred?BC₂F₂?population was screened under drought stress,genotyped through genotyping by sequencing?GBS?and quantitative trait loci?QTLs?mapped.Moreover,genes were mined within the stable QTL regions and their roles evaluated under drought conditions.Analyses were carried out on LEA,TH,CDK,CYP450,Alba and NLP gene families through genomewide identification in reference to the genomesequenced data of G.hirsutum [?AD?1],G.arboreum?A2?and G.raimondii?D5?.In each of the gene families,novel genes were identified and their functions were analyzed through forward and reverse genetics in Arabidopsis and upland cotton,respectively under drought,salt and nitrogen deficiency conditions.1.Drought QTL Mapping in A BC₂F₂ PopulationA genetic map of 10,888 SNP markers was developed from 188 BC₂F₂ lines derived from the cross of G.hirsutum and G.tomentosum.The genetic map spanned a distance of 4,191.3 c M,with an average marker distance of 0.1047 c M,covering an overall size of At and Dt subgenomes by 51% and 49%,respectively.The map was applied in determining the quantitative trait loci?QTLs?for drought tolerance with a total of 30 stable QTLs,of which three QTLs clustered Cluster 1,4 and 10 harbored more than one QTL per chromosome and thus declared as the major QTL clusters.The QTLs were skewed towards At subgenome,with 17 QTLs,while only 13 QTLs were mapped to the Dt subgenome.Eighty-nine?89?genes were mined within the GBS marker regions for physiological traits,which confers drought tolerance in cotton.The majority of the genes were interrupted by introns;only 15 genes were intronless accounting for 17%.The genes were involved in all the three GO functional annotations.Abiotic stress related mi RNAs were detected such as mi R164 known to be associated with NAC,MYB genes with a profound role in enhancing drought tolerance.Through RTq PCR,five genes were found to be the key genes involved in enhancing drought tolerance in cotton.2.Genomewide Identification of Six Gene FamiliesWe identified the following six gene families in three whole genomes sequenced species G.hirsutum,G.arboreum and G.raimondii,representing AD,A and D genomes in Gossypium,respectively:?1?LEA with 242,136 and 142 in number,which was classified into eight groups.LEA genes in the AD had fewer introns and were distributed in all the chromosomes.The majority of the duplicated LEA genes were segmental.The syntenic analysis showed that greater percentages of the LEA genes were conserved.Segmental gene duplication played a key role in the expansion of the LEA genes in cotton.Among all LEA genes,the LEA2 subfamily was the most abundant,showing hydrophobic in nature,and was further classified into six groups,designated as group 1 to 6.Phylogenetic tree analysis revealed orthologous gene pairs within the cotton genome.?2?CDK with 31,15,and 12 in number and distributed in 15,10,and 9 linkage groups of AD,D,and A genomes,respectively.All CDK genes were classified into six groups.?3?Trihelix transcription factors?TH?with 102,51 and 51 in number in AD,A,and D genomes,respectively.?4?CYP450 with 672,374,and 379 in number in AD,A,and D genomes.In tetraploid cotton?AD?,the CYPs genes were distributed in all 26 chromosomes,but clustered mainly in Ah05 and Ah12,and their homologs chromosomes in A and D genomes.?5?Alba)with 33,17 and 20 in number in AD,A and D genomes,which encoded protein lengths ranged from 62 to 312 aa with the molecular weight?MW?from 7.003 k Da to 34.55 k Da,grand average hydropathy?GRAVY?values of-1.012 to 0.609 and isoelectric?p I?values of-3 to 11.?6?NLP with 105,61 and 60 in number in AD,A and D genomes,respectively,which encoded protein lengths ranged from 63 aa to 1,403 aa with MW from 7 k Da to 152 k Da,p I ranged from 4 to 10,GRAVY ranged from-1to 0,showing hydrophilic in nature.3.Cis-Regulatory Elements,miRNA Prediction and GO Analysis in LEA and Alba Gene FamiliesSixty-three?63?miRNAs were found to target 89 LEA genes,such as miR164,ghr-miR394 among others.Gene ontology analysis revealed that LEA genes are involved in desiccation and defense responses.Similar mi RNAs were found to target other members of the gene families,for instance,among the Alba genes,such as gra-mi R8770 targeted four genes,gra-mi R8752 and gra-mi R8666 targeted three genes,each and gra-mi R8657 a,b,c,d,e targeted 10 genes each,while the rests targeted 1 to 2 genes each.Moreover,almost all the LEA genes in their promoter regions contained ABRE,MBS,W-Box,and TAC-elements,functionally known to be involved in drought and other stress responses.The same was observed in other functional gene families.Finally,GO revealed all the three gene annotations,molecular functions,biological processes,and cellular components,an indication that the proteins encoded by these gene families play an integral role in enhancing abiotic stress tolerance in plants4.RNA and RT-q PCR validation of protein encoded by the various gene familiesExpression profile analysis indicated that most of the LEA,LEA2,CDK,TH,CYP450,and NLP genes were highly expressed under drought and/or salt stress conditions,moreover,the LEA genes exhibited higher expression levels in wild species,G.tomentosum as opposed to drought susceptible cultivar,G.hirsutum.The tolerant genotypes have a greater ability to modulate genes under drought stress than the upland cotton cultivar.The finding provides comprehensive information on LEA genes in upland cotton,G.hirsutum and possible function in plants under drought stress.Across the various genes,some of the genes were uniquely highly upregulated,Cot_AD24498?LEA2?,Gh_D12G2017?CDK?,Gh_A05G2067?TH?,while for CYP450 and Alba,had two genes each,Gh_D07G1197 and Gh_A13G2057?CYP450?while Gh_D01G0884 and Gh_D01G0922?Alba?.These genes were further functionally characterized through forward and reverse genetics in Arabidopsis and cotton,respectively.5.Functional Characterization of Five Gene Families under Drought and/or Salt StressOverexpression of the transformed genes,Cot_AD24498?LEA2?,Gh_D12G2017?CDKF4?and Gh_A05G2067?GT-2?in Arabidopsis,revealed their significance in enhancing abiotic stress tolerance in plants.Overexpression of the LEA2 gene showed that the LEA2 genes were involved in promoting root growth and in turn conferring drought stress tolerance.The Gh_D12G2017?CDKF4?,Cot_AD24498?LEA2?and Gh_A05G2067?GT-2?overexpressed Arabidopsis lines exhibited higher concentration levels of the antioxidant enzymes,including peroxidase?POD?,superoxide dismutase?SOD?,and catalase?CAT?concentrations under drought and salt stress conditions with very low levels of oxidants.Moreover,cell membrane stability?CMS?,excised leaf water loss?ELWL?,saturated leaf weight?SLW?,and chlorophyll content measurements showed that the transgenic Arabidopsis lines were highly tolerant of either of the stress factors compared to their wild types?WT?.Similarly,stress-responsive genes RD29 A,SOS1,ABF4,and CBL1 were highly upregulated in the overexpressed lines but were significantly downregulated in wild types.Besides,the TH,CYP450,Alba and NLP-silenced cotton plants exhibited a high level of oxidative injury,due to high levels of oxidant enzymes,in addition to negative effects on CMS,ELWL,RLWC and chlorophyll content.Moreover,all the stress-responsive genes profiled were upregulated on the wild types,but significantly downregulated on the VIGS-plants.