Research On Point Mutations Arising Spontaneously And Induced In Rice OsMsh6 Mutants

Mismatch Repair(MMR)is one of the important pathways in DNA repair system.MMR ensures high fidelity in DNA replication and genomic stability and is highly conserved in organisms.It is responsible for the recognization and correction of single-base mismatches and unpaired nucleotides.Rice is one of the most important food crops in the world and a model species for monocots plant.Rice OsMsh6(LOC Os09g24220)is a mismatch repair gene homologous to AtMsh6 in Arabidopsis,but so far,its function and its response to mutagenesis have not been reported.Therefore,the frequency and characteristics of point mutations in OsMsh6 homozygous insertion mutants and in mutagenesis lines of OsMsh6 mutants treated by ion beam irradiation were analyzed by SLAF-seq,and a sequencing-free method was developed for quick and convenient detection of single-base mutations in this research.1.Spontaneous point mutations in the OsMsh6 mutant.The point mutations arising spontaneously in rice OsMsh6 mutants were investigated by specific-locus amplified fragment sequencing.Totally 994 single-nucleotide mutations were identified in three mutants and the mutation density is about 1/136.72Kb per mutant line on average.These mutations were relatively randomly distributed in chromosomes and might be accumulated in generation-dependent manner.All possible base transitions and base transversions could be seen and the ratio of transitions to transversions was about 3.12.The nearest-neighbor biases around the mutated base were also observed.The result suggests that OsMsh6 is important in ensuring genome stability by recognizing and repairing mismatches that arise spontaneously and provides useful information for investigating the function of the OsMsh6 gene in DNA repair and exploiting MMR mutants in rice induced mutation breeding.2.Induced point mutations in the OsMsh6 mutant.The insertion mutant NF9010 of rice OsMsh6 gene was used as the target of ion beam mutagenesis and its frequency and characteristics of point mutations were compared with the control(Msh6WT).The frequency of induced point mutations in the mutant NF9010 was about 3 times as the control,suggesting that point mutation frequency can be increased by using the MMR-deficiency as the starting material for radiation mutagenesis.All possible base transitions and base transversions could be detected in both the mutant NF9010 and the control Msh6WT,showing a wide mutation spectrum.However,the frequency of base transitions in the mutant was higher than that of the control,and more point mutations were concentrated on the 8th,11th and 12th chromosome.The nearest-neighbor biases around the mutated base induced by N+ ion beam treatment were also observed as the situation of mutations spontaneously in OsMsh6 mutants.The result is significant to construct an efficient system for induced mutation breeding by using MMR-deficiency mutants and also demonstrates the important role of Msh6 gene in mismatch repair from another aspect.3.A quick detection method for point mutations.Using the unique properties of CEL I enzyme,combined with ligation of artificial adapters and specific amplification,a sequencing-free method was established for quick determination of point mutation location and type.The method consists of 6 steps:a)designing primers and performing PCR to amplify the target sequence of the sample to be tested and the control respectively;b)heteroduplex formation between the two sets of amplification products and digestion by CEL I;c)identification of single base mutations based on pattern of digestion products separated by agarose gel electrophoresis;d)isolation of fragments cleaved by CEL I and connection to four adapters with a sticky end of A,C,G or T respectively;e)specific amplification of ligated fragments by using combination primers for target fragment and adapters;f)characterization of mutated base according to the adapters used for amplification of ligated fragments.The method is free of sequencing and can be used to determinate the location and type of known or unknown single-base mutations,providing a new way for quick and economical detection of point mutations.