Analysis Of RNA-Seq Data To Identify Functional Genes In Maize Kernel

The process of accumulation of kernel composition is one of the most important effects on maize yield and quality.With the development of sequencing technique and bioinformatics,genome-wide searching for functional genes related to accumulation of kernel composition based on high-throughput sequencing data might not only contribute to crop breeding and improvement,but also provide a new strategy for the study of function genomics in crop.In this study,we first build several platforms to analyze the RNA-Seq data of 368 maize diverse inbred lines,and then found that some genes related to accumulation of maize kernel composition were strongly selected during the maize development.Meanwhile,we also constructed the pan-transcriptome of maize whole kernel by De novo assembly,and explored the associations between transcriptomic variations and the process of accumulation of kernel composition.The main results are as follows:1.The analysis platforms of RNA-Seq data were developed with mainstream sof'twares in RNA-Seq studies and composed of a reference genome-based alignment analysis platform and a De novo assembly platform without the need of a reference genome.Among them,the reference genome-based platform contains three sub-platforms,such as alignment platform,SNP Calling platform and expression analysis platform,which can meet most requirements in RNA-Seq analysis for population.The De novo assembly platform makes a great contribution to find new genes or construct the maize pan-transcriptome.In addition,statistical and biological methods were performed to verify the results of the platform analysis,showing that these results were both accurate and reliable.2.368 maize inbred lines were divided into 190 temperate lines and 178 tropical lines according to their pedigree and adaptation.XP-EHH test was performed to detect the selection signals between the two populations with 558,579 high-quality SNPs obtained from RNA-Seq analysis.730 and 421 candidate selected regions that are specific to temperate maize and tropical maize were identified in this study,and the comparison of two selected regions suggested that temperate maize lines experienced more intensive selection than did the tropical lines.The selected genes were then annotated and found that regulatory genes with expression difference(P-value<0.05)were significantly enriched in selected regions.Furthermore,two genes,susl and incwl,which encodes two key enzymes in the sucrose degradation pathway were strongly selected in temperate maize(top 0.5%)and expressed more than that of tropical maize(P-value<0.01).Meanwhile,similar selected genes were also detected in maize oil synthesis pathway,which indicated that genes involved in accumulation of maize composition were obviously under strong artificial selection during the process of maize breeding.3.With an ePAV pattern transformation of gene expression and an "align-then-assembly" De novo assembly strategy,13,382 ePAV genes and 2,355 high-quality novel sequences were obtained from RNA-Seq data to construct the maize pan-transcriptome.Both the bioinformatics validation and resequencing experiments were performed and proved that the ePAV pattern could represent more expression differences and the "align-then-assembly" strategy was more suitable for De novo assembly of RNA-Seq data.Cooperating with Prof.Jianbin Yan and his group,genetic components in maize pan-transcriptome were then used for GWAS with 15 agronomic phenotypes and 526 metabolic traits to efficiently explore the association between tanscriptomic and phenomic variations.The ePAV pattern of related genes in flavonoid metabolic pathway was identified significantly associated(P-value<7.49E-5)with the content of many flavonoid metabolites,which suggested the ePAV genes and their ePAV patterns might be important to metabolic regulation in accumulation of maize composition.This study aims to use the RNA-Seq data of maize kernel to explore the functional genes that play important roles in accumulation of kernel composition,and try to explain their genetic mechanism and biological function to provide genetic resources for maize breeding.