| DNA methylation is a critical and highly conserved epigenetic modification in plants and animals,which is essential for gene expression,transposon silencing,tissue development,environmental response and genome stability.As an important agricultural production area in China,the subtropical region provides an important guarantee for food security,fruit and vegetable supply for China.However,crop production in subtropical regions often suffers from multiple environmental and biotic stresses.In recent years,it was demonstrated that DNA methylation maintains genome stability and regulates gene expression in model plants,thereby enhancing crop resistance and improving traits.Compared with model plants,research on important crops in subtropics is relatively lagging behind,especially in genomics and epigenomics.Therefore,we hope to provide theoretical support and scientific basis for the breeding of new crop varieties with high resistance and high quality by conducting DNA methylation and multiple functional genomics studies on multiple crops in subtropical regions.In this study,three important crops in the subtropical region were selected:lemon(Citrus limon),honeysuckle(Lonicera japonica),and peanut(Arachis hypogaea).Through multiple sequencing technologies such as Pac Bio,Hi-C,Illumina and WGBS,and combined with molecular biology,we successfully assembled the genomes and epigenomes of lemon(Xiangshui variety),honeysuckle(Sijihua variety),and the methylome of tetraploid cultivated peanut and diploid wild peanuts.In ‘Xiangshui’ lemon,we investigated the epigenetic regulatory mechanism of citric acid synthesis during lemon development;in ‘Sijihua’ honeysuckle,we examined the epigenetic regulatory mechanism of flower color change;and in Peanut,we resolved the regulatory mechanism of DNA methylation on the differences in homologous gene expression and seed development process.The main findings are as follows:1)In this study,a high-quality reference genome of ‘Xiangshui’ lemon was constructed and anchored to 9 chromosomes,and a total of 380.14 Mb of reference genome was obtained.By annotating the genome,27,945protein-coding genes were obtained.Through comparative genomic and evolutionary analyses,a phylogenetic tree of the genus Citrus was constructed,and it was determined that lemons diverged in species from citron about 2.85 million years ago,and the amplification and contraction of lemon gene families were obtained.2)We constructed a single-base resolution DNA methylome during lemon fruit development.DNA methylation undergoes complex changes during lemon pulp development,in which the reduction of methylation in CG and CHG methylation environments is synergistically regulated by methyltransferases and DNA demethylases.The increase in CHH methylation levels is due to an increase in the activity of Rd DM pathway enzymes,which leads to an increase in the number of genomic methylcytosine,thus raising CHH and genome-wide methylation levels.The biosynthesis and accumulation of citric acid in lemon pulp is a complex process.By comparing the transcriptomic data of lemon with those of other citrus fruits(sweet orange and pomelo),this study identified key genes affecting the synthesis and accumulation of citric acid,such as MYB transcription factors(PH4/5)and ANTHOCYANIN 1(Cl AN1),and elucidated the roles of these genes in the regulation of DNA methylation.3)We constructed a high-quality reference genome of honeysuckle with a genome size of 886.04 Mb anchored to 9 pseudochromosomes.39,320protein-coding genes were obtained by genome annotation.We also performed the comparative genomic analysis with published honeysuckles to obtain important genomic structural variants.4)The constructed DNA methylome revealed that the DNA methylation level gradually decreased during the development of Honeysuckle and was regulated by the demethylase Lj DML3.The decrease of DNA methylation affected key genes in several important biological pathways of Honeysuckle,and we found that key genes in the carotenoid metabolism pathway controlling the flower color of honeysuckle were also regulated by DNA methylation.5)For tetraploid peanut and its ancestor diploid peanut,we constructed DNA methylome profiles.We found that during peanut evolution,the methylation levels of CG and CHG increased and CHH methylation decreased in tetraploids compared with their ancestral diploids.The biased and dominant expression of homoeologous genes occurring during polyploid peanut evolution is regulated by CHG methylation in gene regions that play important roles in peanut flowering,photosynthesis,synthesis of nitrogen and phosphorus compounds,and other important biological processes.6)In this study,we also analyzed DNA methylation during different developmental periods of tetraploid peanut seeds,and found that the methylation levels of CG and CHG remained essentially unchanged and CHH methylation was elevated during peanut seed development.We likewise found that subgenomic homoeologous gene expression bias was mainly regulated by CHG methylation.We also found that elevated CHH methylation affects the expression of key genes in many biological pathways during peanut seed development,such as WRI1,a key gene for peanut oil quantity,which is regulated by CHH methylation in the promoter region,thus changing its expression pattern during development.In summary,we reveal the regulatory mechanism of DNA methylation in three important crops in subtropical regions through genomics,comparative genomics,transcriptomics and epigenetics,and participate in the regulation of a variety of important biological processes,elucidating the key role of DNA methylation in the growth,development and genome evolution of crops,and providing theoretical basis and technical support for the future use of epigenetic perspective for variety improvement and crop breeding. |
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