Identification And Mapping Of QTL/Genes For Salinity Tolerance In Barley(Hordeum Vulgare L.)

Salinity stress is one of the major abiotic stresses which affect grain yield and quality in barley.The use of salinity tolerant varieties is the most effective and economical way of reducing salinity damage.Barley(Hordeum vulgare L.)is the fourth largest cereal crop grown worldwide,which is extensively utilised in livestock feed,food,brewing or even medicine.As the most salinity tolerant cereal crop,barley is one of the pioneer crops grown popularly for improving saline or alkali farming land.In this project,a natural population and three DH populations were used to screen for salinity tolerance QTL.Physiology and biochemistry,transcriptomics and proteomics analysis were employed to investigate physiological and molecular mechanisms for the tolerance using two pairs of near isogenic lines(NILs)differing in a major salinity tolerance QTL QSl.TxNn.2H on 2H.The main findings are listed below:(1).A total of 215 barley accessions collected worldwide were used to identify salinity tolerance QTL through genome wide association studies(GWAS)with three models GLM,MLM and FarmCPU.A significant association between marker SNP6867 on 2H 10216375bp and salinity tolerance was identified using all three models,with the-logio(P)value of 3.46E-11 from GLM,9.47E-07 from MLM and 1.18E-06 from FarmCPU.The marker(SNP6867)showed a very significant correlation with salinity tolerance(r=0.61)and was located within a gene(HORVU2Hr1G0004550)encoding cytochrome p450 superfamily protein.A significant marker(SNP1 7403)trait association was detected on 3H 37272409bp from both FarmCPU and GLM models,with-log10(P)values of 2.40E-06 and 2.68E-09,respectively.The marker(SNP17403)showed a very significant correlation with salinity tolerance(r=0.62)and was located within a gene(HORVU3Hr1G015 730)encoding MATE efflux family protein.(2)QTL mapping for salinity tolerance was conducted in two DH populations.The first population(CG)was constructed from a six-row feed barley,CM72,which is salinity tolerant,and a two-row malting barley variety,Gairdner,which is salinity sensitive.The second population(SG)is originated from a cross between a two-row wild barley,SRY01,which shows high salinity tolerance,and Gairdner.A major salinity tolerant QTL(qSCG2.1)with the tolerant parent CM72 contributing tolerance was detected in CG population.qSCG2.1 was mapped to 2.27-5.38 cM genetic interval,with the nearest marker located at 5.38 cM(128349163bp)on 2H,determining 82.7%of the phenotypic variation.Two salinity tolerance QTL were detected in SG population.The QTL qSSG2.1 was mapped to 0.00-2.52 cM genetic interval,with the nearest marker located at 2.18cM(10898658bp)on 2H.qSSG2.1 was located at a similar position to qSCG2.1,the 2H major locus detected by GWAS analysis and QSI.TxNn.2H but determined a relatively less phenotypic variation(26.1%).QSl.TxNn.2H was detected before in another DH population constructed by TX9425 and Naso Nijo(NT population).The other QTL(qSSG3.1)was mapped to 118.18-122.78 cM genetic interval,with the nearest marker located at 122,17cM(610833591bp)on 3H,determining 14.40%phenotypic variation.Gairdner contributed the tolerance in this QTL.(3).Two pairs of NILs(N33 and N53 are salinity sensitive lines,T46 and T66 are salinity tolerance lines)differing in QSl.TxNn.2H were used to analyze the differences of ion homeostasis,proline content and scavenging capacity of reactive oxygen species(ROS)in roots and leaves after 0,2,4 and 6 days salinity application.The roots and leaves of one pair of NILs(N33 and T46)were used for proteomic analysis under normal conditions and after exposure to 300mM NaCl for 96 hours.Tolerant NILs(T46 and T66)showed salinity tolerance with little symptoms of leaf chlorosis or wilting,while sensitive ones,N33 and N53,showed salinity sensitivity with severe chlorosis and a low survival rate.Both tolerant NILs showed a significantly lower Na+/K+ratio,H2O2,MDA,and proline but a greater enhancement in antioxidant enzymatic activities than both sensitive NILs.A total of 53 and 51 differentially expressed protein spots were identified through tandem mass spectrometry analysis in the leaves and roots,respectively.Proteins that are associated with photosynthesis,ROS scavenging,and ATP synthase were found to be specifically upregulated in the tolerant NILs.It is suggested that the tolerance allele of the QTL QSl.TxNn.2H improve salinity tolerance by controlling Na+loading into xylem through Ca2+signal.This signaling network involves reductions of Na+toxicity in leaves,upregulations of proteins related to photosynthesis,ROS scavenging,and ATP synthase to protect the photosynthetic apparatus,thus alleviating oxidative damage,providing additional energy which contributes to the salinity tolerance.(4).Transcriptome analysis was performed using the roots of N33 and T46.Compared to control,1173 genes were significantly down-regulated,and 880 genes were up-regulated in salinity sensitive line N33,while 1204 genes were highly down-regulated and 1196 genes up-regulated in tolerant line T46.Genes related to nitrogen transportation,Cl-,Na+,K+and Ca2+transportation,Ca2+signaling,hormones,ATPase,LEA were highly clustered in tolerant line T46.(5).The F2 population developed from N33 and T46 was used for fine mapping of QSI.TxNn.2H.This QTL has been mapped to a 1.9 Mb physical distance,containing a total of 58 predicted genes.Three new recombinants in F3 were found and will be used for further fine mapping.In the fine mapped region,6 significant SNPs were located in 3 genes,which encodes cytochrome P450 family protein,zinc finger family protein,and glyoxylate/hydroxypyruvate reductase B protein,respectively.Among the 58 genes in the fine-mapping region,genes encoding heat shock protein 90,chloramphenicol acetyltransferase-like domain superfamily and subtilisin-like protease were significantly up-regulated in the salinity tolerant line.The genes encoding cytochrome P450 family,zinc finger family proteins and heat shock protein 90 genes were three possible candidate genes for QSl.TxNn.2H.(6).A DH population(TamF population)constructed from a wild barley Tam407227 and Australian cultivar Franklin was used to map salinity tolerance QTL by scoring the plant damage under either single salinity stress or combined stress of salinity and waterlogging.Na+content was measured under salinity only stress.A significant correlation was found between the damage scores under salinity stress and those under combined stress(R2=0.475).The results showed that the performance of the population under combined stress was mainly determined by salinity tolerance.Damage scores under salinity conditions showed a weak positive correlation with Na+content(R2=0.146),suggesting that Na+ content in leaves was not the major determinant of salinity tolerance in this population.A total of four QTL were identified under salinity stress and six QTL were identified under the combined stress.Three QTL(qS5.1/qSW5.1,qS5.2/qSW5.2 and qS7.1/qSW7.1)were identified under both salinity and combined salinity and waterlogging stresses,contributing to a proportion of phenotypic variation from 5.7%to 28.8%.CAPS markers were designed for use in breeding programs using the molecular markers closely linked to these three QTL.Two QTL controlling Na+content in leaves under salinity stress were detected.One QTL(qNra7.1)was located on 7H,accounting for 14.1%of the phenotypic variation.The other QTL(qNra1.1)was located on 1H,determining 11.4%of the phenotypic variation.qNa1.1 was located at the same position as previously reported Na+exclusion gene HvNax4.Sequence analysis of the candidate gene HvCBL4 revealed that one SNP difference between the two parents leads to the substitution of alanine to threonine,which potentially affects the structure and function of the HvCBL4 protein.Covariate QTL analysis showed that qS7.1 may be closely related to qNa7.1 and HvNax3,which controls Na+content in leaves.However,the relationship between qS7.1,qNa 71.1and HvNax3 needs to be further investigated.