Mapping QTLs For Traits Related To Water Use Efficiency In Apple Under Drought Stress And Identification Of Candidate Genes

Apple?Malus domestica?cultivation area and production in China rank first in the world.Apple production is also an important industry to support the rural vitalization and poverty alleviation in China.However,water shortage is threatening the sustainable development of the apple industry in arid and semi-arid regions of China.Water-saving agriculture achieved by improving water use efficiency?WUE?,is the fundamental way for the sustainable development of apple industry in China's arid regions.In this study,the genetic population constructed using female parent cv.'Honeycrisp'with low WUE and male parent cv.'Qinguan'with high WUE as the materials,were used for RAD-Seq?Restriction-site Associated DNA Sequencing?,marker development and genetic linkage map construction.The quantitative trait loci?QTLs?for apple WUE and relative traits under well-watered and drought stress conditions were identified based on the genetic map.According to the stable QTLs for apple WUE,the effective molecular markers were validated.Additionally,the potential genes for apple WUE were screened out,and the functional characterizations of a candidate gene were detected.The aim of this study is to provide basic data for understanding molecular mechanisms and genetic improvement of apple WUE under drought stress.The main results of this study are as follows:1.Using RAD sequencing and a final mapping population of 350 F₁ seedlings derived from'Honeycrisp'x'Qinguan',we constructed an integrated map consisting of 10172 SNP?Single Nucleotide Polymorphisms?markers and spanning 2430.52 cM.All markers,including 4421 lm×ll,4688 nn×np,and 1603 hk×hk-type markers,were grouped into 17linkage groups?LGs?with the longest LG15 containing 574 markers and spanning 179.97 cM,and the shortest LG9 containing 590 markers and spanning 115.99 cM.Total 5351 markers?4421 lm×ll plus 930 hk×hk?constituted 17 LGs on the'Honeycrisp'map spanning 1837.61cM with 0.34 cM per marker,as well as 5623 markers?4688 nn×np plus 935 hk×hk?constituted 17 LGs on the'Qinguan'map spanning 1687.33 cM with 0.30 cM per marker.2.Under moderate drought and well-watered conditions in 2014 and 2015,11 indices involved in apple traits of stem growth,leaf area,leaf water status,leaf photosynthesis,and WUE were evaluated.And then,their relative QTLs were identified based on'Honeycrisp'x'Qinguan'genetic map.The results of population phenotypic evaluation showed that drought inhibited the growth,decreased leaf area?SLA?,net photosynthesis rate?Pn?,stomatal conductance?gs?and transpiration rate?Tr?,and reduced leaf water-holding capacity?WHC?,but increased the intrinsic water use efficiency?WUEi?and stable carbon isotope composition(?¹³C)of apple.WUEi and?¹³C showed obvious transgressive inheritance under the two watering conditions.Each phenotype value in the population showed continuous unimodal distribution,indicating their attributes of quantitative genetic traits.The results of Shapiro-Wilk tests for the population distributions of phenotypes in different years indicated that the environmental effects of relative water content?RWC?,WHC,Pn,and?¹³C were more significant than other indicators under drought stress.In two test years,the population WUEi under drought stress was significantly positively correlated with Pn,and had a significant negative correlation with gs and Tr.?¹³C was significantly and very significantly positively correlated with total leaf area?TLA?and Pn,respectively,and negatively correlated with specific leaf area?SLA?and Tr.The estimated values for the same phenotype of the population in different years showed significantly and very significant correlations,suggesting the repeatability of test phenotypes across years.The results of QTLs mapping showed that total 266 QTLs for these traits were identified,in which 22 QTLs related to intrinsic WUE?WUEi?,and 33 QTLs related to long-term WUE(?¹³C).Among them,co-located QTLs in 2014 and 2015,i.e.stable QTLs across years,for WUEi,WUEiDS14.LG17.2 and WUEiDS15.LG17 were detected.Also,stable QTLs for?¹³C,?13CDS14.LG8,?13CDS15.LG8.1,?13CDS14.LG15,?13CDS15.LG15.1,?13CDS14.LG16and?13CDS15.LG16.1,were found.In addition,22'QTL hot spots'were detected among QTLs for traits relative to apple WUE,one of which contained 2⁸ QTLs.Further genotyping by KASP confirmed the reliability of stable QTLs for?¹³C under drought stress on LG8,LG15 and LG16,as well as the effectiveness of SNP marker,lm1712?Chr8:12659110?,np2623?Chr15:36996403?and np2691?Chr16:4666838?involved in these QTLs.3.Based on QTLs for WUE in apple under drought stress,36 genes involved in biological processes of photoprotection,signalling,substance metabolism and transport,and transcriptional regulation were identified,which might relate to apple WUE.The identification of these genes laid the foundation for the study of regulatory mechanisms and genetic improvement of apple WUE.Among them,18 genes-MAPKKK,PP2A,GL1,ARF,ERF020,ATG4a,LAX,ATGP4,STN7,DnaJ,RLK,ARF17,GOLS1,MPL3,bZIP-1,VPS34,PIP5K1,and TAT7-showed different expression patterns or levels between'Qinguan'with high WUE and'Honeycrisp'with low WUE,so they could be prioritized for the future gene function researches.One of prioritized candidate genes,MDP0000217124,was further cloned because it was mined from the QTL for the long-term WUE(?¹³C)and induced significantly by drought stress.By analysing sequences and phylogenetics of multi-species,it was confirmed that this gene was MdDnaJ encoding apple DnaJ protein.The detection of cis-acting elements in promoter of MdDnaJ indicated 18 cis-acting elements involved in drought,heat shock,low temperature,hypoxia,defense and stress,and various hormones responsiveness.Apple MdDnaJ was transformed to Arabidopsis thaliana?Columbia‘??Col‘?.Three Arabidopsis lines over-expressing MdDnaJ were selected for abiotic stresses treatments,including natural drought stress combining re-watering and osmotic stress treatment using 200 mM mannitol.The results indicated that the lower levels of MDA and electrolyte leakage,and higher proline concentration were detected in plants of three transgenic Arabidopsis lines under natural drought stress for 20 d.Higher?¹³C levels were also detected in plants of three transgenic Arabidopsis lines than'Col'under drought stress.Three transgenic Arabidopsis lines exhibited significantly higher survival rates?64.0%,62.0%and 55.3%for OE-2,OE-4 and OE-5,respectively?than wild-type'Col'?17.9%?when being watered again.The growth inhibition of wild-type'Col'by osmotic stress was stronger than three transgenic Arabidopsis lines over-expressing MdDnaJ.Meanwhile,the fresh weights and root lengths of three transgenic Arabidopsis lines under osmotic stress were significantly higher and longer than wild-type'Col',respectively.MdDnaJ was also transformed to apple'Orin'callus.The osmotic stress treatment was performed using 150 mM mannitol for two transgenic apple callus lines.We detected the higher fresh weights and proline concentration,and lower MDA and electrolyte leakage levels in two transgenic'Orin'calluses under osmotic stress than in wild-type callus.These results demonstrated that apple MdDnaJ could confer tolerances to osmotic and drought stresses in plants,and also improve the WUE in plant under drought stress.