Mining And Analyzing Genic Resources Of Salt Tolerance In Gossypium Darwinii

Salinity is a major threat for cotton seedling,yield and productivity.Owing to the continued genetic erosion of the upland cotton germplasm and due to intense selection and inbreeding,researchers' attentions have shifted towards wild cotton progenitors which unique traits can be introgressed into the cultivated cottons for their genetic performances to be improved.The present study was trying to mine and analysis the potential gneic resources for salt telorence in a wild allotetraploid cotton species,Gossypium darwinii.An accession,AD507,was used as target plant material representing G.darwinii and a cultivar,CCRI12,was used as reference plant material representing G.hirsutum.To determine the expression pattern and function of Fructose-1,6-bisphosphate Aldolase(FBA)gene family,whole genome sequened data of three cotton species,G.hirsutum(TM-1),G.arboreum(shixiya-1)and G.raimondii(D502)was introduced.A total of 37 FBA genes,19,9 and 9 in G.hirsutum,G.arboreum and G.raimondii,respectively,were found.Based on protein domains and phylogenetic analysis,FBA genes were categorized into two classes: class I and class II.Gene structure analysis and motif division of FBA genes in three Gossypium species showed that numerous genes of FBA have unequal distribution of introns.Combined syntenic blocks of FBA genes revealed 31 duplicated genes among the three-cotton species.Three kinds of duplications(segmental,tandem and dispersed)were observed but most of the FBA genes were found to be duplicated in segmental pattern and remain conserved,which could be a possible basis of expansion of FBA genes in cotton.Gene ontology results showed that FBA genes were involved in a couple of mechanisms such as vacuole compartmentalization,glycolytic process and zinc ion binding.The expression analysis under salt stress in the target material(AD507)and the reference material(CCRI12)depicted that most of class I genes were upregulated in salt tolerant species,G.darwinii;as compared to salt susceptible species,G.hirsutum.Similarly,the De Novo data were also used to characterize the Pkinase gene family.A total of 151 Pkinase genes was found in the three cotton species and,based on phylogenetic analysis,was categorized into 13 subfamilies.Syntenic analysis of gene blocks revealed 99 duplicated genes among G.hirsutum,G.arboreum and G.raimondii.Most of the genes were duplicated in segmental pattern.Expression pattern analysis in AD507 and CCRI12 showed that the Pkinase gene family possessed species-level variation in induction to salinity and G.darwinii had higher expression levels than G.hirsutum.Based on RNA sequence analysis and preliminary RT-q PCR verification,we hypothesized that the Pkinase gene family,regulated by transcription factors(TFs)and mi RNAs might play key roles in salt stress tolerance.These findings inferred comprehensive information on possible structure and function of FBA and Pkinase gene family in cotton under salt stress.This study provides a solid foundation for further in-depth evaluation of these genes in understanding the specific role of these genes in cotton in relation to salt stress tolerance and paves the way for breeding elite cotton cultivars with potential resistance genic resources to abiotic stresses.Further study was carried out to ues the BC2F2 population of cotton plants from salt tolerant wild G. darwinii,AD507,and the cultivated G.hirsutum CCRI12 salt susceptible parents for screening and identification of salt resistant candidate genes.Four significant genomic regions were determined,first genomic region on chromosome 19(1.86Mb),second and third genomic region on chromosome 26(1.06 Mb,1.94 Mb)and fourth on chromosome 08(1.41 Mb),respectively.The reads of Bulk01 and Bulk02 were aligned to the G.darwinii(AD507)and G.hirsutum(CCRI12)genomes,respectively,leading to the identification of 20,664,007 SNP/indel.After screening,6573 polymorphic markers were obtained.The SNP-index in resistant and susceptible bulks and ?(SNP-index)between resistant and susceptible bulks were measured.For further validation,the five candidate genes were passed through q RT-PCR analysis,the expression profile depicted that two highly upregulated genes in salt tolerant species G.darwinii,ID No.GOBAR_DD13798 and GOBAR_AA08552,showed the reverse in G.hirsutum,except one gene that showed only partial expression.The results indicated that these tow genes are possible salt tolerant.The validation under salt stress(200 mml Na CL)to G.darwinii(AD507)and G.hirsutum(CCRI12)comfirmed the two genes having possibly telorant to salt stress and much higher telorant level in AD507 than in CCRI12.Further study in this direction will help considerably in improving the crop yield and quality,and cultivating new varieties resistant against salt stress.