Identification And Preliminary Gene Mapping Of Lesion Mimic Mutant Lm-NAU9918-1 In Wheat

Mutants are important materials for genetic research and functional genomic research,in which lesion mimic mutants are an ideal material for studying the mechanism of programmed cell death and disease resistance.Previously,a stable lesion mimic mutant obtained from EMS mutagenesis of wheat variety Nannong 9918 was selected and named as lm-NAU9918-1.In this study,the agronomic trait analysis of mutant,physiological analysis of mutant,preliminary genetic mapping of the mutant gene and analysis of the formation mechanism of the lesion mimic phenotype were carried out.The main results are as follows:1.Agronomic trait analysis of the lesion mimic mutant lm-NAU9918-1The mutant lm-NAU9918-1 showed lesion mimic phenotype on the leaves when the first leaf was not fully developed in the seedling stage,then the lesion mimic phenotype gradually spread to the leaf sheath in the later period,and the lesion mimic phenotype continued throughout the growth period.Light and low temperature promoted the emergence of lesion mimic phenotype.The results of agronomic trait analysis showed that the flag leaf length and grain length of the mutant increased significantly compared with the wild type,but the plant height,effective tiller number,spike length,spikelet number and thousand-grain weight decreased significantly compared with the wild type,while no significant difference was observed in second leaf and grain width.2.Physiological analysis of the lesion mimic mutant lm-NAU9918-1The methods of Typan blue and DAB staining were used to analyze the cell death and ROS accumulation in the mutant and wild-type.The results showed that cell death increased and ROS accumulated in the parts of lesion mimic;in addition,the CAT activity and MDA content in the mutant were significantly higher than those in the wild type.In the study,transmission electron microscopy was used to observe the ultrastructure of chloroplasts,and the relative content of chlorophyll was measured at the same time.The results showed that the chloroplast internal lamella structure in the lesion mimic parts of leaves from the mutant was disordered or even degraded,and the mitochondria appeared vacuolization,while the normal parts of the mutant and the wild type chloroplast were oval and arranged in order.SPAD content determination showed that the SPAD value of the mutant at the tillering stage and the mature stage was significantly lower than that of the wild type.The analysis of chlorophyll composition found that the chlorophyll a content of the mutant was significantly lower than that of the wild type at the tillering stage.In this study,the relationship between lesion mimic and resistance was analyzed.F_2:3population was constructed,using resistant mutant and varieties with highly susceptible powdery mildew as the parents,then the individuals showing lesion mimic phenotype but without Pm21 was identified by molecular marker detection and phenotypic identification,then these individuals were used for detecting the effects of lesion mimic on powdery mildew resistance.The results showed that the resistance of the mutant individuals has increased to varying degrees,indicating that this type of lesion mimic mutation may increase the resistance to the living parasite Bgt due to the increasing of cell death,but the specific mechanism still needs to be further analyzed.3.Preliminary genetic mapping of the mutant geneThe genetic population was generated by crossing the mutant with Nannong 0686,and the genetic analysis and preliminary mapping of the mutation site lm-NAU9918-1 were performed.The results showed that the phenotype of this type of lesion mimic was controlled by a single recessive gene;BSA combined with 55K chip and molecular markers analysis,the target gene is locked between the 6B chromosome SSR markers Xbarc198-6B and Xwmc398,and the genetic distances are 5.9c M and 8.4c M.4.Analysis of the formation mechanism of the lesion mimic mutantTranscriptome sequencing of mutant and wild-type plants at the seedling stage was performed with 3 biological repeats respectively.The average data size of each sample was6.70 G,and the mapping rate of clean reads of these samples on the reference genome reached 78.75%.So,the sequencing data met the requirements of follow-up analysis.The differential genes were screened and analyzed,and the results showed that there were 223differentially expressed genes between the mutant and the wild type;GO and KEGG analysis showed that the appearance of the lesion mimic phenotype may be due to the lack of Fe elements and abnormal chlorophyll synthesis,the accumulation of lipids,the abnormal signal transmission system and the activation of some resistance-related pathways in plants.