Genetic Analysis And Identification Of Candidate Genes For Two Spotted-leaf Mutants(spl101 And Spl102)in Rice

Spotted-leaf mutantrefers to a kind of mutants which can spontaneously formspots on leaves or leaf sheaths of plants in gross observation under normal growth conditions.Because the spots caused by this kind of mutationare similar to the lesions caused by pathogen infection,spotted-leaf mutant is also known as lesion-likemutant.Screening and studying the spotted-leaf mutant of rice are of great importance for revealing the reaction mechanism and molecular mechanismof disease resistance of rice,which at the same time can also enrichrice germplasm resources.In the study,the two rice spotted-leaf mutants obtained through mutagenesis of the rice variety KYJ by EMS were named as spl101 andsp1102.The F2 generationpopulation planted inFuyang,Hangzhouin 2015 was analyzed,and the main results were as follows:(1)Under the field conditions in Fuyang,Hangzhou,severe lesion-like phenotypes were found on the leaves ofspl10l and spl102 mutants obtained through mutagenesis of the ricevariety KYJ by EMS after the heading stage of rice,while no lesion-like phenotype was found on the leaves of wild type KYJ.(2)The F1generationpopulation was obtained through the backcross between spl101 and spl102mutants and wild type KYJ.No spotted-leaf phenotype was found on the leaf surface of the F1 generationpopulation,while spotted-leaf phenotypes were found on the plants of the F2 generationpopulationinbred from the F1 generationpopulation.According to statistics,it wasfound that the proportion of normal phenotypestospotted-leaf phenotypeswas close to 3:1.The Chi-square test showed that both spl101 and spl102 mutants were controlled by single recessive genes.(3)With the Mutmap method,it was speculated after sequencing that the candidate geneof both spl101 and spl102 was OsEDRl.In order to verify the hypothesis,the expression level of OsEDR1 was detected through the fluorescent quantitative PCR and the results showed that the expression levelsof OsEDRlof spl101 and spl102mutants decreased compared with that of the wild type KYJ.T-test showed that the P values were respectively 0.42 and 0.95,indicating that the expression level of OsEDR1 was not significantly changed by the mutationsof spl101 and sp1102.In addition,it was hypothesized that the mutation of spl101mutantoccurs at the junction of the sixth exonand the sixth intron.Therefore,the primer which could amplify the sixth intronand bilateral exons was designed for the RT-PCRdetection of the spl101 mutant and wild type KYJ.The RT-PCRresults showed that the spl101 band was not larger than that of KYJ.Then the RT-PCR productswere sequenced and the results showed that OsEDR1mRNA had additional 4bp in spl101.The further analysis of the sixth intron found that after the GT sequencemutated to AT sequence on the left boundary,another GT sequence was found at the locations of 3bp and 4bp in the downstream of the mutated AT sequence.And the downstream GT sequence could be identified as a new left boundary of the intron,whichled to a frameshift mutation in the sixthintron.(4)The experimentfound that the OsEDRlmutation in the spl101mutantoccurred at the junction of the sixth exonand the sixth intron,which affected the shearing of the sixth intron.However,in the spl102 mutant,the mutation caused the missense mutation of an amino acid and then affected the function of OsEDRl.Previous studies reported that the lesion-like phenotype could be found on the leaves of mutant plants with the inhibition or knockout of OsEDRl.Therefore,two new OsEDRl allele mutations were identified in our experiments,which has a positive significance for the further study on the reaction mechanism and molecular mechanism of disease resistance of plants and for enriching rice germplasm resources.Also the effectiveness of the Mutmap method in cloning spotted-leaf mutant genes in rice was verified.