Characterization And Fine Mapping Of Two Yellow-Green Leaf Mutants Ygl16 And Ygl17 In Rice(Oryza Sativa L.)

Rice?Oryza sativa L.?is one of the most important food crops in the world.The leaf is an important photosynthetic organ.Choloplast in rice is an active metabolic center,which converts solar energy to carbohydrates and store them into rice seeds through photosynthesis.Leaf-color mutations widely exist in plant kingdom.A large number of studies have shown that leaf-color mutations are ideal materials for studying leaf development.Two yellow-green leaf mutants,i.e.ygl16?yellow-greenleaf16?and ygl17?yellow-greenleaf17?,screened from the chemically induced mutant library of restorer line Xinong 1B via EMS?Ethyl Methane Sulfonate?in rice?Oryza sativa L.?were used to identify potential novel functional genes controlling rice leaf color.In the present study,we carried out series of experiments to dissect both ygl16 and ygl17,including phenotypic and photosynthetic characterization,subcellular structure discovering,genetic analysis and gene mapping via segregating population derived fron crosses of Jinhui 10/ygl16 and Jin10/ygl17,and gene expression analysis by qRT-PCR.The main results are as follows:?1?Phenotypic characteristics of ygl16 and ygl17The leaves of both mutants initiated to exhibit phenotypic performance of yellow-green since the 4th-developed leaf stage,and all the mutant plants shortened since tillering stage.For ygl16,the yellow-green phenotype began from leaf tip,and extended to the middle and base part.Compared to the wild type?WT?,the phenotypic performances of plant height,secedondary panicle branches,filled grains per panicle,and setting rate of both ygl16 and ygl17 statistically decreased?P<0.01?,and their1000-grain weight were significantly lower than that of WT?P<0.05?.Besides,for ygl16,significant differences were observed among the traits of effective panicles per plant,panicle length,and grain no.per panicle,while for ygl17,the same trends were observed among traits of length of the 1^stt nd 2ⁿdd internodes from the top,and total grain no.per panicle.?2?Synthetic characteristics of ygl16 and ygl17Results of chlorophyll contents of leaves at heading stage indicated that the content of carotenoid?Car?,chlorophyll a?Chla?,Chlb,total chlorophyll content?ChlT?of both ygl16 and ygl17 were statistically lower than those of WT?P<0.01?.In addition,the photosynthetic rate(P_n,?mol CO₂ m^-22 s^-1)of ygl17 was significantly lower than that of WT?P<0.01?,while the P_n,stomatal conductance(G_s,mmol H₂O m^-22 s^-1),and transpiration rate(T_r,mmol H₂O m^-22 s^-1)of ygl16 were significantly lower than those of WT?P<0.01?.On the other hand,the significant increasing trends were observed for the intercellular CO₂ concentration(C_i,?mol CO₂ m^-22 s^-1)for ygl16?P<0.01?.For ygl16,the decreasing of Gs might result in the low capacity of chlorophyll cells in capturing CO₂ for phytosynthesis,and as a result,leading to the accumulation of CO₂ between cells,and increased the performance of C_i.?3?Ultrastructural characteristics of ygl16 and ygl17Results from the cytological investigation showed that abundant basal granules were observed among the chloroplasts of WT,and the stromal lamellae exhibited closely arrangmant.Wehile for both mutants of ygl16 and ygl17,increased holophilic corpuscles were observed,the stromal lamellae presented loosly,and the grana presented low packaging density.These observeations indicated that both ygl16 and ygl17 possessed abnormally developed the chloroplasts among the leave cells.?4?Genetic analysis of yellow-green leaf performance of ygl16 and ygl17Leaves of both F1s of ygl16 and restorer line,Jinhui 10?J10?and ygl17 and J10presented normal green,similar to WT,while segregation were observed among the individuals of both F₂s.For F₂ derived from cross of ygl16 and J10,the corresponding observations of WT-like and mutant-like were 2713 and 866.The ratio of WT-and mutant-like individuals was 3.13?2713/866?,while the ratio for F2 derived from the cross of ygl17 and J10 was 3.12?4168/1335?,both matched well with the theoretical ratio of 3:1(?²=1.232<?²_0.05,1=3.84).The results of genetic analysises suggested that the target mutant performanece of both ygl16 and ygl17 were seperatedly controlled by recessive nucleic gene.?5?Molecular mapping and candidate gene identification of GYL16 and YGL17Molecular mapping results revealed that the target genes for both ygl16 and ygl17,i.e.YGL16,and YGL17 were located to the long arm of chromosome 2?Chr2?.For YGL16,the flankering markers,ZW2-30 and F90-52,formed a?48kb physical interval,including 5 open reading frames?ORFs?.Sequence alignment of these 5ORFs between WT and ygl16 detected a base changing of G?WT?to T?ygl16?at position 470 of LOC?Os02g51080 at the 1^stt exon,resulting in the changing of coding amino acid glycine?Gly,WT?to valcine?Val,ygl16?.So the gene of LOC?Os02g51080,initially identified as the geranyl reductase?CHL P?,among 5ORFs of was determined as the target gene of YGL16.And 62 ORFs were annotated within the 467 kb physical region formed by flankering markers,X8-SSR7 and RM1342,of YGL17.Alignment results detected no sequence differences of some annotated ORFs between WT and ygl17,and no related genes have been reported in this region.?6?Expression characterists of candidate genes and chlorophyll moletabolic pathway related genesResults of qRT-PCR showed that the expression level of YGL16 exhibited higher in leaf materials,whild lower in the organs of root,stem,leaf sheath,and young panicles.Additionally,compared to WT,significantly down-regulated expression trannds?P<0.01?were observed among PS?-encoding genes?PsaA,PsaB,PsaC?,PS?-encoding genes?PsbA,PsbB,PsbC?,and cytochrome b6f complex-encoding genes?PetA,PetB?for both ygl16 and ygl17.Similar trends were also observed between carotenoid metabolic pathway-related enzyme genes,i.e.SY2,in ygl16 and ygl17.Besides,the expression levels of 6 out of 7?6/7?chloryphyll metabolic pathway related genes,i.e.CHLD/I/M,DVR,CAO1,and PORA,presented significant lower level in ygl16,while HEMA exhibited contrary trend.In ygl17,only 3/7 genes,i.e.CAO1,HEMA,and PORA,presented statistical lower expression level,while the expression levels of the rest 4 exhibited significantly higher than those of WT.Summarily,both YGL16 and YGL17 were speculated to affect the metabolism of photosynthetic pigments and expression of photosynthesis related genes.