Research On The Formation Of Heterokaryotic Hyphae And DNA Methylation During Hyphae Senescence In Morel

Morel(Morchella spp.)has high edible and medicinal value,and important progress has been made in artificial cultivation in recent years.However,the basic research on morel genetics is weak and the life history is not very clear,which has affected the innovation and development of morel breeding and cultivation technology.In addition,morel strains have the characteristics of rapid degradation.Strain degradation is jointly regulated by internal and external factors,and has a network of genetic and epigenetic co-regulation.This study used monospore homokaryotic strains M04M24(MAT1-1 mating type)and M04M26(MAT1-2 mating type)of Morchella importuna as materials,using Agrobacteriummediated genetic transformation(ATMT)to obtain transformants with fluorescent label of nucleus,and study the formation process of heterokaryotic hyphae.Whole genome bisulfite sequencing(WGBS)was used to carry out the whole genome methylation level and differential methylation region(DMRs)analysis of original strains and senile strains of M.importuna M04 and Morchella sextelata No.114.The main findings are as follows:(1)The histone H2 B gene of M.importuna was cloned to construct histone fluorescent protein fusion expression vector,and the homokaryotic hyphae genetic transformation of M.importuna was carried out using ATMT technology to realize the fluorescent labeling of nucleus.e GFP fluorescently labeled M04M24 positive transformants and m Cherry fluorescently labeled M04M26 positive transformants were obtained,and transformants of different mating types were hybridized on the culture medium.Fluorescence observation revealed that both e GFP and m Cherry fluorescence were expressed in nucleus of the hybrid hyphae.It is intuitively observed that the hyphae of different mating types of M.importuna can mate to complete the hybridization reaction and form heterokaryotic hyphae.(2)With the increase in the number of subcultures,the growth rate of M.importuna M04 and M.sextelata No.114 gradually slowed down.At the same time,obvious yellow pigment was produced and the strains were degraded.The overall methylation levels of the M04 original strain and the senescent strain were 5.95% and 6.80%,and the overall methylation levels of the No.114 original strain and the senescent strain were 7.97% and6.95%,respectively.In the four methylation libraries,the DNA methylation levels under different sequence backgrounds were all CG>CHH>CHG.Compared with other regulatory elements,the average methylation level of three sequences is the highest in repetitive sequences.Differential methylation analysis showed that 13781 and 16354 DMRs were detected in strain M04 and No.114,respectively,which involved 1043 and 1005 differentially methylated genes(DMGs),among which there were more DMGs in CHH sequence type.In strain M04,the proportion of hypomethylation in three sequences is higher.In strain No.114,the hypermethylation was more likely to occur in the CG and CHG sequences.Two fragments were randomly selected from strains M04 and No.114,and the methylation level of the fragments was verified by conventional bisulfite sequencing method,which was consistent with the WGBS results.This was the first study to observe the formation of heterokaryotic hyphae of M.importuna visually by using the nuclear fluorescent labeling method.It also carried out a preliminary study of the hyphae aging mechanism from the perspective of DNA methylation.On the one hand,it is helpful for in-depth understanding of the life history of morel.on the other hand,it lays a solid foundation for the development of morel genetics and the cultivation of high-quality strains.