Finemapping And Functional Analysis Of The Major QTL Affecting Chlorophyll Content Of Flag Leaf In Rice

Chlorophyll is the key pigment in plants that takes part in photosynthesis by capturing,transmitting and transferring light energy to reaction center,closely related to photosynthetic rate and plant nutrition,and plays an important role in regulating photosynthetic physiology,yield and quality of crops.Rice is one of the most important food crops.The identification and cloning of rice chlorophyll content related genes will help breeders use biotechnology to design and utilize chlorophyll content,improve rice light energy utilization efficiency,and thereby increase rice biological yield.In our study,a series of QTL related to flag leaf chlorophyll content and other agronomic traits was identified from a set of chromosome segment substitution lines(CSSLs)constructed by Malaysian common wild rice(IRGC105491,ACC10)and indica rice ZS97B(ZS97).A major locus qSPAD1.2 affecting the chlorophyll content of flag leaves after heading was finely mapped,cloned and performed a functional analysis.The main results were as follows:1.One hundred eleven lines from wild rice CSSLs population were developed andgenotyped by using rice genome-wide SNP microarray and phenotyped.A total of49 QTLs was detected for a number of important agronomic traits,includingeffective panicle number,panicle length,primary branch number,grain numberper panicle,1000-grain weight,yield per plant,heading date,plant height,andchlorophyll content.A major QTL of them,qSPAD1.2,affecting the chlorophyllcontent of flag leaves,was found explaining 26.9%of phenotypic variation in theCSSLs population.2.A HIF(Heterogeneous inbred family)population was constructed derived fromone introgressive line HBZ7 containing the heterozygous genomic region atqSPAD1.2.The QTL analysis indicated qSPAD1.2 has a major effect on the flagleaf chlorophyll content,and the ACC10 allele was incompletely dominant toZS97 alleles.By progeny investigating the phenotype of the recombinant plants,qSPAD1.2 was finely mapped in a 10.3-kb region between SPRD19 and RM11962,which contained two genes,LOC?Os01g65770 and LOC?Os01g65780.Theformer is predicted an expression protein gene,and the latter is aglycosyltransferase gene.3.Real-time quantitative PCR test showed that Os01g65780 was expressed in a highlevel at the seedling stage and the late stage of panicle development,moreover,there was a significant difference in a pair of QTL near-isogenic lines,and theexpression level in NILAC containing the wild rice allele was significantly higherthan that in NILZS.The alignment of nucleotide sequence between ACC10 andZS97 revealed that there were 18 SNPs and 8 In Dels in the promoter region,8SNPs and 3 In Dels in the coding region.A non-synonymous mutation in the thirdexon resulted in a change of the amino acid polarity(V267M).4.Transgenic experiments showed that both overexpression Os01g65780^AC andOs01g65780^ZS significantly reduced SPAD(Soil Plant Analysis Development)value,chlorophyll b and nitrogen content of flag leaves.Compared with thenegative controls,the RNAi line R780^ZSsignificantly increased the SPAD value,chlorophyll b and nitrogen content of flag leaf.Differences of leaf phenotype wereobserved in the overexpression line OE780^ZS and the repression line R780^ZS at theseedling stage.Therefore,Os01g65780 may affect the chlorophyll content of leafby the transcription,not by the amino acid variation.5.Phylogenetic analysis of homologous proteins from rice and Arabidopsis revealsthat Os01g65780.1 was clustered in the PGSIPA clades of GT8 family type II,andwas high homology with Arabidopsis At3g18660(GUX1/At PGSIP1)andAt1g77130(GUX3/At PGSIP2),thus,Os01g65780 was designated as OsPGSIP1.OsPGSIP1 containing the conserved motif(Dx D,Hxx Gxx KPW)of GT8 domain.The OsPGSIP1 protein was localized in the Golgi apparatus,which was consistentwith subcellular localization results of the homologous protein GUX1-GUX5 inArabidopsis.6.Overexpressed OsPGSIP1 significantly increased cellulose,hemicellulose andstarch content of flag leaf,but decreased soluble sugar content.However,suppressed this gene significantly decreased the content of cellulose,hemicelluloseand starch in flag leaf,and increased the soluble sugar content.Therefore,OsPGSIP1 may have the same function affecting the composition of the cell wallas the homologous proteins GUX1 and GUX3 in Arabidopsis.7.NILAC had lower chlorophyll b,nitrogen content and net photosynthetic rate thanin NILZS.However,NILAC showed higher cellulose,hemicellulose and starchcontent of flag leaf,and lower soluble sugar content compared with NILZS.Although the 1000-grain weight in NILAC was significantly higher than that ofNILZS,there was no difference in yield per plant.8.The cytology observation of the flag leaf tissue showed that the diameter of thesmall vascular bundle in NILAC was significantly larger than that of NILZS,butthe spacing of adjacent small vascular bundles was significantly lower than that inNILZS,and there was no difference in the area of adjacent small vascular bundlesbetween the two NILs.Transmission electron microscopy observation showed thatthe cell wall thickness of mesophyll cells in NILAC was significantly higher thanthat in NILZS,and the capsular layer of chloroplasts in NILAC was looselydistributed,which might be a reason for thelower chlorophyll content in NILACthan that of NILZS.9.The expression of OsPGSIP1 was induced by nitrogen.The SPAD value increasedwith the increase of nitrogen concentration.However,under different nitrogenconcentrations,the expression level of OsPGSIP1 in NILAC was significantlyhigher than that in NILZS,and the SPAD was significantly lower in NILAC.Thestem mechanical strength in NILAC was also significantly higher than that inNILZS.The results establish a foundation for exploring that OsPGSIP1 functions in the secondary cell wall components and the genetic basis of the chlorophyll content of flag leaves.These results also provide a new strategy improving nitrogen use efficiency and improving stem mechanical strength in rice.