Screening Barley Germplasm For Salt Tolerance And Its Associated Genetic Analyses

About 20% of agricultural land is affected by salinization globally,but most of crop cultivars are sensitive to salinity.Salt tolerance for plant has been defined as the ability to grow and accomplish its life cycle under saline conditions.Due to the long developmental period in crops,their salt tolerances were focused on one or several specific stages,and therefore,could be divided into short-term and long-term salt tolerance.Barley is the most tolerant crop to salt stress,therefore,it is considered as a model species for studies of salt tolerance.Barley genotypes are frequently tested by short-term or long-term salt tolerance for its mechanisms in physiological and genetic regulation.Seed germination is the initial stage for crop,which is largely affected seedling emergence and further crop growth.However,relevant salt-stress studies in germination are limited,not only investigated by poor germplasm,but also lack of comprehensive methodology to describe its dynamic salt-induced responses,which will subsequently affect the genotype screening and its genetic achievements.Salinity is limiting seed imbibition of germination by its lower water potential,which is also regarded as the component of osmotic stress.However,whether the salt tolerance of germinating barley depends on the germination or osmotic responsive loci are still under discussion.Therefore:1.In this study,total 315 barley genotypes were tested their germination responses under Na Cl simulated multiple saline conditions（0,80,160,240,320 and 400 m M）,and PEG（Polyethylene glycol）mediated osmotic treatments（0,5,10,15,20 and 25%）by using a novel vertical germination systems.The final seedling root and shoot length were simulated by two functions of Sigmoid model for their dynamic responsive curves.The results showed that osmotic root length curves were distinct from its shoot length curves and salt induced seedling curves.Different barley populations also displayed various salt responses with wild barley and Guizhou landrace which are much sensitive to salinities rather than Chinese and foreign cultivars.Key parameters are extracted from dynamic curves and named as response parameter（REPs）,including Lmax,AUC,Salt80-20 and PEG80-20.Combining with primary seedling length,there are 56 traits in total.Four key REPs,e.g.RL-Salt70,RL-AUC,SL-Salt60 and SL-Salt50,were exhibited the largest statistic differences among four barley populations.Considering the both results of PCA on 56 traits and variation in four key REPs,most tolerance genotypes are Vertige,Nudinka,Supi-4,Yangnongpi-2,Orchidea and Harrington,and most susceptible genotypes are Nah34,ZY50,ZY41,Nah22,Nah23 and Nah28.2.Combining the phenotypic data of previous European winter barley panel with 4884 SNPs for genome wide association mapping on 56 phenotypes,28 independent QTLs were identified by traits derived from non-stress,salt stress and osmotic stress conditions.Compared with 20 QTLs mapped from germination under osmotic stress,only 10 QTLs are responsive in salt stress conditions.Only one non-specific stress QTL（QTL5）inherited pleiotropic effects for seedling growth under three conditions and targeted as Hv5 PTase a candidate,another locus（QTL27）overlapped between salt and PEG stress associated with Hv DHN5,a co-localized QTL（QTL25）for both root and shoot growth was also identified with two candidates of Hv CNGC and Hv CML.To conclude,very few genetic locus of germination are able to responding to both salt and PEG stress,these two stresses are also possibly to utilize the same QTL.In general,the genetic background for salt tolerance of germinating barley is quite distanced from no-stress and PEG stress.3.Multiple saline and PEG treatments were applied to characterize the response of seedlings to salt stress in two barley cultivars（Nure and Tremois）and their double-haploid population for linkage mapping,and the results did not contained any overlapped QTLs among three growth conditions and two stresses.However,key REPs Salt80-40 and existing genetic map led to the identification of a new major QTL for root elongation under salt stress on chromosome 7HS,with 14.5 of LOD and 45% of phenotypic explanation.Gene-based markers were developed from the rice syntenic genomic region to restrict the QTL interval to Bin2.1 of barley chromosome 7HS,which is also mapped by two flanking markers Morex_Contig₄50119 and Morex_Contig₂179585.Considering the largest sequence variation existed in Hv SLR1 encoding DELLA protein between Nure and Tremois,we suggested it as a suitable candidate gene within this QTL region.No responses of further QTL test under PEG stress and no morphological variations between extreme genotypes under no-stress conditions demonstrated it as a salt-stress specific locus.Moreover,results in SIMR of extreme genotypes indicated root growth is not only inhibited but also reoriented by the increasing root tips,diameters and root hair density.Sixty barley recombinant inbred lines derived from Lewis × Karl cross were grown in four different environments,and the seed contents of starch,total soluble protein,phytate,total phenolics,total flavonoids and total inorganic phosphorus were determined in the harvested grains,in parallel with measurements of seedling growth under saline treatments from 0 to 400 m M Na Cl.ANOVA showed a significant environment/source effect for both seed components and seedling growth,although the previous was much affected by the seed-production environment.Modeling seedling length across multiple salinities for each seed source showed that the environment with the most saline-tolerant root-growth curve was associated with highest seed phosphorus content,which was determined by soil available phosphorus content.Correlations between seed components and seedling growth traits highlighted phytate and Pi as key components for seedling growth under moderate salinities,and their correlation dynamics indicated the unique roles of phytate for germinating barley under high salinity.Further GWAS mapping on P content,phytase acid phosphatase and fast germination in Europe winter barley panel revealed that,not only Pi locus overlapped RL-S4（QTL88）,but also QTLs controlling Pa content overlapped with loci mapped from non-stressed,salt stress and osmotic stress traits（QTL73 and QTL74）,confirming phytate degradation benefits its further germination under stress.5.In the aim of verifying whether salt tolerance of germinating barley is still different from non-stressed seedling and short-term salt tolerance of 21-day,7 days’ salt stress on seed germination was extended to 21 days.Therefore,European winter barley panel was applied for salt stresses by using soil substrate as medium within seedling dishes.The substrate of Method1（M1）was incubated by salt solutions of 160 m M and 240 m M Na Cl before the seed sowing,comparing to the Method2（M2）and Method3（M3）with substrate absorbing fresh water first and salt solutions（160 m M and 240 m M Na Cl）later at 7 and 14 days.Meanwhile,three seedling stages were harvested at 7,14 and 21 days from no-stress condition.Correlation network suggested traits of M1 are highly linked with no-stress seedling but quite isolated from M2 and M3.The comparison of their GWAS results confirmed only 1 QTL overlapped between M1 and M3,and 2 QTLs co-mapped by M1 and control conditions,indicating the same unique genetic control for salt tolerance of 21-day salt-induced seedling originated from germination.Moreover,the Hv Nax3 locus which was associated by M2 traits is not mapped any of M3 traits,plus less correlation between traits of M2 and M3 were obtained,suggesting the stress timepoint is also crucial for short-term salt tolerance.6.Most of short-term or long-term salt tolerance focused on aboveground plant for phenotyping,but root system is less mapped in genetic even though it served as the first organ sensing soil salt stress,thus,understanding genetic loci of developmental root system and its interactions with salt stress is of great significance to improve crops salt tolerance.Therefore,European winter barley panel with 143 varieties was profiled comprehensively for 13 morphological traits at 4-leaf-stage,including root biomass,seminal root length,lateral root length and other parameters.9 distinguished QTLs from 3H,4H,5H and 6H were detected from 2 distinct trait subgroups（Sub1 and Sub2）which confirmed by correlation-based network analysis.Genetic structure of this barley panel was strongly affected by row-type factor resulting in significantly association between row-type and gene Sdw1,with the consequences of Sub1 traits largely influenced by row-type and their associated QTL92 targeted by Sdw1.LRL and RT traits are associated with LRR-RLK and PIN genes on 3H.The validations on 20 extreme genotypes provided a dramatic shift of lateral root density from 2-leaf-to 4-leaf-stage,suggesting an important transient stage of 4-leaf-stage for barley lateral root development.Moreover,locus of RV（QTL93）is overlapped with QTLs mapped from seedling root and targeted b HLH transcriptal factor,root related QTL98 is co-localized with QTL65 salt-stress M1 traits at a region containing two ERF genes.The results indicated that the one locus could not only determine the roots in different stages,but also regulate root growth under non-stress and salt stress conditions.To conclude,salt tolerance of barley germination may not depend on germinating superiorities,not mainly associated with homeostasis regulators,whereas dominant by transcriptal factors and hormonal regulation in response to salt stress,and may correlated to seed phosphorus and the ability of degradating seed organic phosphorus in germination,but diverse with salt tolerance of seedling stage genetically,and linked with genetic control the root development.These studies above provide an important basis for genetic procedure of barley salt tolerance at germination stage.