Development Of Whole-genome Intron-length Polymorphic Functional Markers And Identification Of Genes Related To Salt Stress Tolerance In Cotton

Cotton is an important economic crop,affecting many aspects of human life,and playing an important role in the development of modern agriculture.While with the increasing influence of human activity in the world,human living and survival environment is deteriorating,exhibited by the expanded scope of the impact of extreme weather and terrific conditions.The changed environment also exerts great challenge to human agricultural production.In recent years,available land area is reducing,The state has adjusted the industrial structure of the planting industry.The changed environment also exerts great challenge to human agricultural production.As a result,cotton planting is gradually giving way to wheat,rice and other food crops,and transferred to the more arid,saline-alkali land.Therefore,enhancing cotton rsistance and tolerance to abiotic stresses become one of the important work for cotton breeding.Meanwhile,as a relative strong abiotic stress resistant crop,cotton has greater potential for abiotic stress resistant breeding.In a word,exploring the resistance gene,creating new materials for cotton resistance,and improving the ability of cotton abiotic stress resistance,is an important way to improve the land utilization and ensure the cotton production.The intron is a specific DNA sequence that is present in the noncoding genomic sequence of the gene.The intron exists in the precursor RNA when it is transcribed in the eukaryotic gene,and will be removed by splicing,finally it never exists in the mature RNA molecule.Introns are widely found in the eukaryotic genome and are an important part of the eukaryotic genome.In general,Compared with exons,introns contain more variations due to a lower selection pressure during the evolutionary process.Intron-spanning markers are which primers were designed in the flanking sequences of intron and amplified the region of intron.Intron length polymorphisms(ILPs)are the easiest identified molecular markers in introns.They can be conveniently detected by polymerase chain reaction(PCR),using primers designed for flanking exons.We developed a genome-wide cotton ILPs based on orthologs annotation from two sequenced diploid species,A-genome Gossypium arboreum and D-genome Gossypium raimondii.Also,these developed ILPs were used for association analysis of salt stress traits and potential function of candidate genes in cotton salt-tolerance.All the experimental results are as follows:1.Genome-wide development and identification of ILP markers in cotton:We identified 10180 putative ILP markers from 5021 orthologous genes.Among these,535 ILP markers from 9 gene families related to stress were selected for experimental verification.Polymorphic rates were 72.71%between G.arboreum and G.raimondii,and 36.45%between G.hirsutum acc.TM-1 and G.barbadense cv.Hai7124.2.Association analysis of salt stress traits in Upland cotton cultivars:Furthermore,we investigated the ILPs from 264 G.hirsutum accessions,14 polymorphic ILP markers were detected as a total.Coupled with previous simple sequence repeats(SSRs)evaluations and salt tolerance assays from the same individuals,we found a total of 25 marker-trait associations involved in nine ILPs.The nine genes,temporally named as Cl to C9.3.Potential function of candidate genes in cotton salt-tolerance:These nine genes showed the various expressions in different organs and tissues,and five genes(C3,C4,C5,C7 and C9)were significantly upregulated after salt treatment.We verified that the five genes play important roles in salt tolerance.Among them,C3 encodes WD40 repeat-like superfamily protein,C4 encodes WRKY DNA-binding protein,C5 encodes plasma membrane intrinsic protein,C7 encodes cytochrome P450 superfamily protein,and C9 encodes mitogen-activated protein kinase?Furthermore,the biological functions of these candidate gens in regulating cotton salt stress are verified by Virus Induced Gene Silencing(VIGS)technology.After the target gene was silenced,half of the plants per target gene-silenced plants were used for the salt stress treatment by irrigation 200 mM NaCl,and the others as water treatment control.we identified the candidate genes related to salt stress tolerance by observing the changes of physiological and biochemical indexes related to salt stress.A month later,there was no difference in growth between no-infiltration(CK),mock treated plants(TRV:00)and different target gene-silenced plant under tap water.However,cotton plants showed severe growth inhibition and lower true leaves defoliated under salt stress treatment,indicating that plant growth was impacted under salt stress.Especially,C3,C4,C7 and C9-silenced plants exhibited a serious true leaves defoliated than C5,no-infiltration(CK)and mock treated plants(TRV:00).Next,we measured seven traits related to salt tolerance for salt stress treatment and water treatment control plants.Compared salt stress treatment with water control plants,the plant height,shoot dry matter weight,and root dry matter weight was significantly decreased,and SOD and POD activity,and Pro and H2O2 content was drastically increased.We calculated the difference between salt stress treatment and water control of target gene-silenced plants(TRV:target-silenced gene NaCl,and TRV:target-silenced gene H2O),and the difference between salt stress treatment and water control of no-infiltration plants(CK NaCl and CK H2O),and compared the statistics significance between them.C4-silenced plants and C9-silenced plants were more distinct in five traits.Similarly,two traits in C7-silenced plants,one trait in C3-silenced plants and C5-silenced plants were also significantly changed,respectively.In a word,silencing of C3,C4,C5,C7 and C9 can significantly affect the salt tolerance in cotton.