Preliminary Study And Application Of 3D Genomics In Brassica Napus

Three-dimensional(3D) genomics is a research on three-dimensional chromatin structure based on the traditional one dimensional sequence. As the most quickly developing technology, chromatin conformation capture(3C) and its derivative techniques are used to measure high proximity and potential interactions between point to point in different chromatin regions. As rapid development of 3C and its derivative technologies, 3D genomics is not only applied to gene expression and transcription regulatory mechanism, but also to the genome assembly(traditional genome assembly, haploid type assembly, metagenome assembly, etc.). At present, the 3D genomics studies mainly focus on the human and model organisms such as mouse, Drosophila melanogaster and Saccharomyces cerevisiae, and have made a series of achievements. However, there were few reports about the 3D research on the plant, except a small number of researches on the model species Arabidopsis thaliana. Rapeseed is the largest oil crop in China and occupies an important position in cooking oil production. Recently, the draft genome of Brassica napus(B.napus) has been completed, which is the first complex genome of traditional polyploid crop. In order to study the 3D genomic application in the research of polyploid species, this study carried out the following work.In this study, we first selected the B.napus ZS11 the as the experimental object and adopt the high-throughput chromosome conformation capture(Hi-C) technology. Following the crosslinking DNA, cutting with restriction enzyme, filling ends and marking with biotin, ligateing, purifying and shearing DNA and pulling down biotin, we completed the library construction of the B.napus chromatin interaction. After high-thought sequenceing, we acquired 110 Gb paired-end sequences for chromatin interaction in B. napus. Through quality control, filtering noise and repeatability checking, the processing results showed that the data quality measures(unique mapping ratio, invalid ligation and repeatability, etc.) are in line with expectations, which shows feasibility of Hi-C technology in traditional polyploid plants. With the above processed chromatin interaction data, we constructed genome-wide interactive heatmap with high quality and high-resolution in B.napus, for subsequent study of genome assembly and transcription regulation research in 3D genomics.Then, to test feasibility of genome assembly based on chromatin interaction in polyploid plants, we mount the scaffolds that were not assigned and placed in B. napus with chromatin interaction for improving the integrity of reference genome sequence. After assembly, 91.05 Mb scaffolds can be assigned to the corresponding chromosomes, and 111.70 Mb scaffolds can be placed and oriented on the chromosomes successfully(In original sketch, there were 135.94 Mb unassigned scaffolds and 202.91 Mb unplaced scaffolds.). By predicting scaffolds that have been assembled in B. napus with the chromatin interaction, it is shown that the accuracy for the assgning, placeing and orientation predction were 98.85%, 90.15% and 90.39%, respectively. This research not only demonstrats feasibility and effectiveness of the approach that assisted assembly based on the 3D structure of chromatin in polyploid plants genome, but also opens the door for carrying out substantial assembly on polyploid plants.Finally, this research also explored the relationship between the 3D chromatin structure and the gene pairs involved protein-protein interactions(PPIs). Based on orthologous method, we predicted 163,475 PPIs in B. napus. Comparing with the random genes, it is shown that the interaction possibilities and interaction frequencies of genes involved in PPIs in B. napus were significantly greater than the randomly selected genes. The result shows that the genes involved in PPIs not only tend to be colocalization, but also share a closer spatial distance. The result suggests that the proteins encoded by the gene pairs of high DNA interaction in the 3D space have higher probability of forming PPI and it is also illustrated the connection between the 3D genome organization and PPIs formation, which provides a new opportunity for the study of gene transcriptional regulation in Brassica napus.