Physiological Characteristics And Genetic Analysis Of Leaf Roll Mutant RL1 And Yellow Leaf Mutant YL3 In Wheat

Leaves are the main organ of photosynthesis in wheat.Leaf shape,posture and chlorophyll content determine the accumulation of photosynthetic products.Therefore,it is of great significance to explore related genes and understand their regulatory mechanisms in wheat.In this paper,we studied the phenotypic characteristics,agronomic traits,physiological characteristics and genetic mechanism of the leaf roll mutant RL1 and yellow leaf mutant YL3.The results are as follows:1.Phenotypic identification and physiological characteristics analysis of RL1:This study reported a stably rolled leaf mutant（RL1）by the ethyl methyl sulphonate（EMS）mutagenesis cultivar Jinmai 47.During the whole growth period,the leaves of RL1 were curled,and the primary leaves were slightly curled along the axial vein to the paraxial plane.With the growth of the leaves,the curling was accelerated until the leaves became tubular.Compared with the wild type,the plant height,ear length,flag leaf width and1000 grain weight of RL1 decreased significantly.According to the germination percentage of seeds,the germination rate of mutant RL1 seeds was reduced by 22%.The chlorophyll content of RL1 was basically the same as that of the wild type,and there was no significant difference in net photosynthetic rate,transpiration rate,stomatal conductance and intercellular CO₂concentration,while the water utilization rate of the wild type was higher.The mutant RL1 showed fewer large and small leaf/lobular veins through paraffin section observation.The number of sclerenchyma and parenchyma cells was reduced in the midrib region of RL1.As well as compared with the wild type,the area and number of vesicular cells at the edge of leaves were significantly reduced in mutant RL1,resulting in the extreme rolling of the entire leaf to the adaxial surface.In addition,the results of different environmental treatments showed that low temperature,high temperature and drought promoted the leaf rolling in RL1.2.Genetic analysis and gene mapping of RL1:The leaf phenotype of RL1 was consistent with that of Jinmai 47 and Linfen 5064 for forward and backward crosses.Chi-square test showed that in F₂population,spreading leaves:slightly rolled leaves（intermediate type）:high rolled leaves=1:2:1.This indicated that the mutant RL1 was controlled by a pair of incomplete dominant nuclear genes.The mutant RL1 was located on the short arm of chromosome 1D using wheat 660K SNP chip by constructing spreading leaves and highly rolled leaves pools.According to the labels in the interval,the KASP primers were designed.The target interval was located between RLI and RLK,with a physical distance of 9.42 Mb.3.Phenotypic identification and physiological characteristics analysis of YL3:This study reported a stably mutant YL3 by the ethyl methyl sulphonate（EMS）mutagenesis cultivar Aikang 58.The mutant YL3 appeared slight spots from seedling stage.As the leaves grow,the spots spread to the entire leaf and became dry,which was accompanied by the whole growth period.Compared with the wild type,the mutant YL3 matured earlier,and the plant height,effective tiller number,grain number per panicle and 1000 grain weight were significantly decreased.The contents of chlorophyll a and chlorophyll b in mutant YL3 were significantly decreased,and the chlorophyll a/b ratio was significantly different,but there was no significant difference in carotenoids.The results of photosynthetic parameters showed that the net photosynthetic rate had significant difference.The chloroplasts of mutant YL3 were disordered by transmission electron microscopy.The thylakoids were loosely arranged,almost without starch granules,and more plastid pellets appeared,which inhibited the photosynthetic electron transport rate.DAB and NTB staining and malondialdehyde content determination showed that the reactive oxygen scavenging system of the mutant YL3 was damaged.The antioxidant enzyme content indicated that CAT activity decreased,while SOD and POD activities increased.In conclusion,the growth of YL3 is weak,chloroplast degrades,thylakoid structure is destroyed,and a large number of ROS is produced,which exceeds the scope of antioxidant enzyme clearance and leads to membrane system damage.4.Genetic analysis and gene mapping of YL3:The mutant YL3 was cross pollinated with Aikang 58,and the leaf phenotype was consistent.Chi-square test for F₂population,normal green leaf:spotted green leaf（intermediate type）:spotted yellow leaf=1:2:1.This indicated that the mutant YL3 was controlled by a pair of incomplete dominant nuclear genes.Transcriptome sequencing was performed by constructing normal green leaf pool and spotted yellow leaf mixed pool.GO and KEGG enrichment showed that differentially expressed genes were mainly concentrated in photosynthesis,chlorophyll biosynthesis and metabolism.By mining high-quality SNP mutation loci and calculating genotype frequency,the gene controlling this trait was initially located on chromosome 1A.High quality SNP mutation sites were selected and 9 pairs of high quality primers with obvious genotyping and polymorphism were screened.The mutant YL3 was located on the left side of KASP3 marker,and the target gene localization interval was 9.64 Mb,which contained21 differentially expressed genes,laying a foundation for mapping cloning in the next step.