QTL Mapping Of Flag Leaf Cuticular Wax And Functional Analysis Of Candidate Alkane Synthase Gene In Wheat

Cuticular wax,covering the outermost layer of the terrestrial plants,is an important barrier protecting tissue from environment stresses.It is mainly composed of a mixture of long chain of fatty acids and their derivatives.Alkane synthesis is the critical pathway for cuticular wax formation.Among them,CER1 holds the core component of the alkanes-forming pathway.In this study,several experiments were carried out to characterize the function of TaCER1 in wheat alkane synthesis.Firstly,a high-density genetic linkage map,consisted by SNP and SSR markers,was developed in a wheat HL population and used to identify QTLs for wheat flag leaf wax.Secondly,we analyzed the compositions and crystal structure of wax in different tissues or developmental stages of wheat.Thirdly,three genes related to alkane synthesis were selected based on a transcriptome analysis.Their function were characterized by subcellular localization,qRT-PCR and over-expression analysis.The mainly results were listed as follows:1.A multi-year statistics of the flag leaf epidermal waxiness were performed in a recombinant inbred line population(F6 generation),which was derived from the cross Heyne×Lakin by single-seed descent.After integrating the genetics map,two additive QTLs were identified for contributing to flag leaf glacousness.The major QTL(QFlg.hwwgr-3AL)was located on wheat chromosome 3AL,which was flanked by two markers IWA1831 and IWA8374 and could explain 17.5-37.8% of the phenotypic variation in different environments.The other QTL,flanked by marker IWA1939 and Xgwm261,could explain 11.3% phenotypic variation and is located on wheat chromosome 2DS(QFlg.hwwgr-2DS).Quantitative trait loci(QTL)analyses identified a major QTL region associated with the three major components of cuticular wax.This region located at the chromosomal region between the markers IWA8374 and IWA4298 at 7.8 cM apart on chromosome 3AL.Two additional SNPs,IWA8162 and IWA4296 were also mapped in the QTL region.The QTL for diketone(QDik.hwwgr-3AL)and primary alcohol(QAlc.hwwgr-3AL)exactly flanked markers IWA8162 and IWA4298 within a 6.3 cM interval.The QTL associating with alkane(QAlk.hwwgr-3AL)positioned marker interval of IWA8374-IWA8162 spanning only 1.5 cM with the favorable allele from ‘Heyne’ and explaining 26.8% of the phenotypic variation.2.Two cultivars(W87 and Chinese spring)with different wax content were selected and used to comparativly analyze the waxy components and crystal structures of wheat.Results showed that they were significantly different for wax contents and crystal structures at the different developmental stages of wheat,while a similar patter was observed for W87 and Chinese spring.At the seedling stage,wheat leaves mainly produced primary alcohols and displayed a relatively less wax coverage.As wheat growth,primary alcohols were gradually reduced,while the content of alkanes showed an acculmulation.Diketones and alkanes were predominating in the flowering spikes.3.Three wheat TaCER1 candidate genes were screened out based on the result of transcriptome sequence and bioinformatics analysis in wheat W87.Then,their full length cDNA were cloned from wheat spike.All of the three candidate TaCER1 genes displayed a typical CERl functional domain,including three conservative histidine-rich motifs(HX3HH+HX2HH+HX2HH)and a WAX2 structure in the C-terminal region.Further phylogenetic analysis demonstrated that the wheat TaCER1 fell into the same class with other homologs,such as AtCER1 and OsWDA1.Taken together,these results suggested that TaCER1 might be functional in wheat alkanes biosynthesis.4.The full sequences of TaCER1 were searched against wheat genome sequence to predict their chromosomal location.Results showed that TaCER1-6A,TaCER1-1A and TaCER1-2D were located on wheat chromosome 6AL,1AL and 2DL respectively.These results were further validated in a set of 11 wheat cv Chinese Spring(CS)nullisomic-tetrasomic substitution lines.In addition,the C-terminus of the TaCER1 coding sequence was fused with GFP and further transiently expressed in arabidopsis leaf protoplasts to investigate the subcellular location of TaCER1 s.The results demonstrated that a completely co-localized GFP signal was detected between TaCER1 s and ER marker,which was consistent with reported results that the majority of wax synthesis-related enzymes are located in the ER.5.qRT-PCR was carried out to analysis their expression patterns in different tissues and abiotic stresses.The results displayed that three TaCER1 genes were expressed in multiple tissues of wheat.A relatively low expression level was observed at wheat seedling stage,but they showed an increasing expression as plant growth.TaCER1 showed a higher expression level in mature leaves,leaf sheaths and glumes of wheat.In addition,three TaCER1 genes could be induced by ABA,drought,PEG and low temperature treatments.Their transcriptional level could increase after a few hours,but would gradually returns to the normal level.All the three TaCER1 s genes showed a similar expression pattern,but the magnitude of the change were different.6.For functional analysis of three TaCER1 s in alkanes biosynthesis,an over-expression construct,under the transcriptional control of rice OsGL1-6 gene promoter,was transformed into rice and arabidopsis,with empty vector as control.Compared to the empty vector,the total cuticular wax contents per leaf area were significantly higher in transgenic lines harboring TaCER1.The accumulation of total waxes was mainly attributed by the increasing of alkanes,while there were no significant changes for the other component contents,such as primary alcohols,aldehydes and fatty acids.Taken together,our data demonstrated that the three TaCER1 s were involved in the synthesis of wheat wax alkanes.The water loss rate and the chlorophyll extraction rate were reduced in the leaves of transgenic rice lines,the permeability of the cuticle was reduced and thus the drought resistance of the plants was improved.Taken together,we characterized the temporal and spatial distribution patterns for different wax composition in wheat,then QTL mapping of flag leaf cuticular wax using RILs population.Furthermore,three TaCER1 s were cloned and validated to play an important role in wheat wax alkane biosynthesis and involved in responding to drought and other environmental stresses.Ours results will provide foundation in wheat drought resistance improvement/breeding by regulation of genes related to alkanes biosynthesis.