Genome Wide Spectrum Of Somatic Mutation And RIP In Neurospora Crassa

Mutation provides the raw material for organic evolution.Research of mutation is one of the key aspects of evolutionary biology.Spontaneous mutations naturally occur without a mutagenic agent.Exploring the characteristics of spontaneous mutations can reveal essential information for understanding the evolution of genes and species.Repeat-induced point mutation(RIP)is a specific mutation phenomenon during the sexual cycle in filamentous fungi.RIP usually occurs before meiosis and can mutate G:C into A:T when it detects duplications in the genome by some means.Current research on RIP are limited to artificial insertions of exogenous and endogenous duplication sequences;not enough valid evaluations and discussions were made about distributions of RIP in natural circumstances and whole-genome regions.These gave rise to the limited comprehension of the basic pattern of RIP mutations.In our study,Neurospora crassa,a model filamentous fungus with special genetic traits such as RIP,was used as experimental material.Our experiments systematically evaluated the frequency and distribution of mutations both in the asexual and sexual stages of N.crassa.To evaluate mutation rate in the asexual stage,8 parallel MA lines in which 1d to 36 d growth single-cell individuals were picked as samples.A total of295 individual samples were cultured and whole-genome high-coverage resequenced separately.To estimate mutation rate in the sexual stage,67 asci with 5 pairs of parents,which have different diversities between each pair,were picked under microscope.These asci were then isolated as 286 single ascospores for separate culturing and whole-genome resequencing.We found that the mitotic mutation rate of N.crassa is 1.92 × 10-6 per base per generations,which is significantly higher than evaluations in prior.The number of mitotic mutations accumulated during the asexual cycle was about 100 times lower than that in the sexual cycle.Moreover,mutations in both asexual and sexual cycles tend to be located in non-coding regions.Different from the spectrum and distribution of asexual mutations,mitotic mutations in the sexual cycle have strong spectrum G:C->A:T and most of them are located on duplication sequences by clustered in a “strand-biased” fashion.Characteristics of these mutations are highly consistent with RIP mutation,which suggests that these mutations are generated as a result of the RIP process.After dividing the whole genome into duplication regions and non-duplication regions,we found that:(1)The mutation rate of duplication regions(8.30 × 10-6 per base per generation)is much higher than that of non-duplication regions(2.10 × 10-7 per base per generation).(2)Even in duplication regions,the distribution of mutations has a significant difference between diverse duplication regions.(3)The mutation rate in non-duplication regions of N.crassa is still higher than other fungi such as yeast,indicating a high level of mutation rate in N.crassa overall.Further research showed that,under the condition of the basic premise for inducing RIP,the GC-content,identities,and copy number of duplications had no significant effects on RIP.Even duplications with near-same nucleotide backgrounds may have large differences in RIP frequencies between them.The length of duplications significantly affected RIP: longer the length of duplications,more RIP mutations can be detected in these sequences.Also,analysis of Hi-C data demonstrated that RIP has a high correlation with the pairing of duplicated sequences,indicating that the pairing of sequences may be one of the influences on initiating RIP.We also found that duplication regions in N.crassa have a higher methylation level than non-repeat sequence regions.Our study is the first quantitative measure of genome-wide spontaneous mutations of N.crassa during sexual and asexual cycles.We also explored the characteristics and influence factors of RIP.To observe a more natural distribution of mutations,the methods of our research do not involve transgene.The results of our study provide a new direction and references for future research and lay the foundation for further analysis of extreme high-frequency mutations.