Characterization And Fine Mapping Of A Yellow-green Leaf 14(ygl14) Mutant In Rice

Leaf is the main organ for photosynthesis in rice,and its color change is closely related to photosynthetic efficiency and photosynthate accumulation.Yellow-green leaf mutation is an important source of rice functional genomics research,which is of great significance to reveal the mechanism of chlorophyll biosynthesis in rice.Therefore,identification of more leaf color mutants and systematic phenotypic identification and gene mapping are conducive to the discovery of more leaf color control genes in rice.An indica maintainer line Xinong 1B mutated by EMS was used to obtain a yellow-green leaf mutant,temporarily named yellow green leaf 14?ygl14?.In order to reveal the phenotypic and genetic characteristics of ygl14 and lay a foundation for the isolation and functional study of YGL14,phenotypic and photosynthetic characterization,cytological observation of ygl14,fine mapping of candidate gene and relative expression analysis of related genes were systematically carried out in this study.The main results are as follows:1.Phenotypic characterization of ygl14The leaves of ygl14 mutant plants were yellow-green during the whole growth period.Compared with the wild type?Xinong 1B,WT?,the plant height,panicle length,flag and second leaf length and seed setting rate increased significantly?P<0.01?,while the number of primary branches and grains per panicle increased significantly?P<0.05?,while the number of effective panicles and 1000-grain weight did not change significantly.2.Photosynthetic characterization of ygl14Chlorophyll content in leaves of WT and ygl14 mutants was determined at seedling stage,tillering stage and heading stage.The results showed that the contents of chlorophyll a?chla?,chlorophyll b?chlb?,total chlorophyll?chlT?and carotenoid?car?in the leaves of ygl14 mutant were significantly lower than those of WT?P<0.01?,and decreased by 31.9%-40.0%,36.0%-52.2%,34.3%-42.2%,27.6%-38.7%respectively.Compared with WT,the net photosynthetic rate,stomatal conductance and transpiration rate of ygl14 mutant at grain filling stage decreased significantly?P<0.01?,while the intercellular carbon dioxide concentration increased significantly?P<0.01?.The results showed that the chlorophyll content of ygl14 mutant decreased and photosynthetic capacity was affected.3.Cytological observation of ygl14The results of frozen slices showed that the number of chloroplasts in the leaves of ygl14 mutant was less than that of WT,and some chloroplast structures were destroyed.Transmission electron microscopy?TEM?showed that the chloroplasts in WT mesophyll cells were abundant and bulky,with thick matrix,normal stacking of grana lamellae and normal lamellar structure.In ygl14 mutant,the number and volume of chloroplasts decreased significantly,some chloroplasts did not have normal stacking of grana,and all chloroplasts had excessive accumulation of formed starch grains.The results showed that YGL14 mutation resulted in abnormal chloroplast development in some of its mesophyll cells.4.Genetic analysisThe phenotypic normal indica restorer line,Jinhui 10,was hybridized with ygl14.The F₁ generation plants showed green color,and the plants in the F₂ generation group were isolated and showed parental traits.There were 3975 normal single plants in the F2 generation group and 1,350 single plants in the yellow leaf phenotype.The chi-square test showed that the normal strain and the mutant strain met the theoretical separation ratio of 3:1(?²=0.35<?²_0.05,1=3.84),indicating that the ygl14 mutation trait was controlled by a pair of recessive nuclear genes.5.Fine mapping of candidate gene400 SSRs covered the entire rice genome were used to screen the polymorphism between Jinhui10-like and ygl14-like gene pools,and to carry out the primary mapping for the candidate gene.Results showed that SSRs of ZTQ48 and RM3664 on chromosome 5 linked with YGL14,with the corresponding distances of 23.86 cM and21.59 cM.More polymorphic markers were developed between ZTQ48 and RM3664,and were used to fine map YGL14.And finally,the candidate gene was located between newly developed markers of T9 and T10,with a physical distance of 70.7 Kb.15 open reading frames?ORFs?were annotated within this 70.7 Kb physical interval,among which 7 encode hypothetical proteins,7 encode express proteins,and a cloned gene,OsCOLE1,encoding hormone transporter.6.Relative expression analysis of related genesReal-time fluorescence quantitative analysis was used to detect the expression of20 genes in WT and ygl14 of rice photosynthetic pathway.The results showed that CHLD,CHLI,CHLM,HEMA1,DVR and PORA genes related to chlorophyll biosynthesis pathway were all down-regulated in ygl14 mutant.The transcriptional levels of psaB,psbD,petA and ndhB were significantly up-regulated in chloroplast development and photosynthesis-related genes,while those of psaD,psbA,petB,petC and petD were down-regulated,while those of rbcL,RbcS genes encoding Rubisco size subunits were down-regulated.It is suggested that YGL14 gene mutation may affect chlorophyll metabolism and the expression of some photosynthesis related genes in rice.