Molecular Mechanism Of Maize Inbreeding Depression Mediated By DNA Methylation

Maize（Zea Mays L.）originated in Mexico,is one of the widely grown cereal crops in the world and a typical cross-pollinated plant.It shows a significant decline in growth vigor during inbreeding and is an ideal plant for studying inbreeding depression.Most studies on the mechanism of inbreeding depression are based on classical genetics,but the genetic hypothesis proposed cannot fully explain inbreeding depression,especially for the population with limited genetic diversity.As the rapidly growing understanding of epigenetics,biologists realize that the epigenetic may play an important role in inbreeding depression,but have not made substantial progress.It is essential for crop breeding programs to breed elite inbred lines and maintain their growth vigor.Therefore,the reveal of epigenetic regulation mechanism of maize inbreeding depression will provide an important theoretical basis for understanding and overcoming of inbreeding depression.In this study,one single plant from the 6^th selfing generation（S6）was self-pollinated to produce the S7 population.Thirty plants in S7 were randomly selected and continuously self-pollinated using the single-seed descent without selection bias to generate a S11population with 28 lines,excluding two lost during propagation.In S10 generation,randomly mating between different lines were used to generate a mating-with-sibling population m S11.High-throughput sequencing technologies,including Methyl C-sequencing（Methyl-seq）,mRNA-sequencing（mRNA-seq）,small RNA-sequencing（s RNA-seq）,transposase-accessible chromatin sequencing（ATAC-seq）and chromatin immunoprecipitation sequencing（Ch IP-seq）,were used to investigate changes in DNA methylation,chromatin accessibility and gene expression between different generations of inbreeding and between inbreeding and sibling mating,and the connection of these changes with inbreeding depression.Electrophoretic mobility shift assay（EMSA）,DNA affinity purification（DAP）-q PCR assays and methylation-sensitive enzyme digestion followed by PCR（Chop-q PCR）were used to study the biological functions of DNA methylation.Finally,DNA methylation of endogenous gene promoter was changed via the CMV-VIGS vector to verify the role of DNA methylation and reveal the mechanism of epigenetic regulation of maize inbreeding depression.The main results of our study are as follows:1.Increased CHH methylation is close to genes during maize inbreeding depression.Phenotypic analysis of S7（n=60）,S9（n=60）,S11（n=60）and m S11（n=75）populations showed that inbreeding resulted in reduced leaf area,aerial dry weight and100-grain weight,whereas these phenotypes in m S11 population were higher than in S11population,ranging from L（Low-vigour）to H（High-vigour）variation.Meanwhile,CHH methylation levels were higher in S11 than in S7（similar to L）lines,but decreased in the H line,and these methylation changes mainly occurred around genes.2.H3K9me2-dependent pathway mediates CHH hypermethylation during inbreeding depression.We identified 8879 CHH hypermethylated differentially methylated regions（S11 hyper-DMRs）between S11 and S7 lines.Ch IP-seq data showed higher H3K9me2levels of S11 hyper-DMRs in the S11 and L lines than in the S7 and H lines,consistent with the changes in CHH methylation,and these S11 hyper-DMR methylation levels were reduced more than genomic regions in the CMT-defective mutants zmet2 and zmet5,indicating inbreeding-induced CHH hypermethylation may depend on the H3K9me2pathway.However,24-nt si RNA was not associated with the CHH methylation changes during inbreeding and sibling mating.3.Inbreeding induced hypermethylation occurrs around TCP binding sites（TBS）and may be associated with reduced chromatin accessibility.S11 hyper-DMRs were located adjacent to MNase HS and DNase HS regions,and possessed motifs that resemble TBS and cis-regulatory elements that mediate chromatin accessibility.ATAC-seq and Ch IP-seq data showed that the levels of nucleosome occupancy and H3K27me2 and H3K27me3modifications were higher in S11 than in S7,but chromatin accessibility was lower in S11than in S7.Moreover,genes with a greater increase in DNA methylation during inbreeding showed a greater decrease in chromatin accessibility,indicating that the reduced chromatin accessibility may be related to the increased CHH methylation.4.Hypermethylation of TBS inhibits the expression of genes involved in mitochondrion,chloroplast,and ribosome functions.763 down-regulated genes in S11relative to S7 lines were enriched in S11 hyper-DMR-associated genes.Expression levels of these genes（Down-regulated DMR-genes）were increased more in the methylation-defective mutants zmet2 and zmet5 than other genes.In addition,S11hyper-DMRs of down-regulated genes were adjacent to TBS motifs and mitochondrion,chloroplast and ribosome related genes were overrepresented in these genes,indicating hypermethylation of TBS during inbreeding inhibits expression of these organelle-related genes.5.CHH hypermethylation of TBS affects the binding affinity of Zm TCP proteins and mediates maize inbreeding depression.EMSA showed that 4 of 6 Zm TCP proteins tested preferred to binding unmethylated TBS probes.We further confirmed that hypermethylation of TBS in S11 reduced the binding affinity of Zm TCP proteins compared to S7 using DAP-q PCR assays.The inverted repeat IR was introduced into maize plants（VIGS-IR）to modify the DNA methylation of Zm00001d015449（Zm DIM1A）promoter using CMV-VIGS vector through viral infection.We found that DNA methylation levels of VIGS-IR plants were increased,expression levels of Zm DIM1A and growth vigor of VIGS-IR were reduced compared to the VIGS-GFP control plants.6.Mating with sibling can reverse inbreeding-induced hypermethylation to restore the growth vigour.Most hypermethylation induced by inbreeding was decreased in sibling mating lines.25%of hyper-DMRs were reversed only in the H but not in the L lines（reversed DMRs）.In these reversed DMR-associated genes,increased histone modifications（H3K27me2 and H3K27me3）and decreased chromatin accessibility and gene expression during inbreeding were also reversed in the H line.These genes may be related to the restoration of growth vigor by sibling mating,and were also overrepresented in biological processes of mitochondria,chloroplasts,and ribosome biosynthesis.In conclusion,during successive inbreeding among highly inbred lines,thousands of genomic regions across TCP-binding sites（TBS）are hypermethylated through H3K9me2-mediated pathway.These hypermethylated regions are accompanied by decreased chromatin accessibility,increased levels of the repressive histone marks H3K27me2 and H3K27me3,and reduced binding-affinity of maize TCP proteins to TBS.Consequently,hundreds of TCP-target genes involved in mitochondrion,chloroplast,and ribosome functions are downregulated,leading to reduced growth vigor.Conversely,mating with siblings can reverse corresponding hypermethylation sites and TCP-target gene expression,restoring growth vigor.These results support a unique role of reversible epigenetic modifications in inbreeding depression.