Metagenome Assembly In Rice Phyllosphere And Its GWAS Analysis Under Rice Blast Stress

The phyllosphere microbiota plays crucial roles in sustaining plant health and productivity.It has been shown that host genetics regulate the composition of plant microbiome.As yet,there is little information on shotgun metagenomic dataset and the interaction between rice and its phyllosphere microorganisms under rice blast stress.Based on this,in this study,some varieties of Rice Diversity Panel Ⅱ core collection(CRDP-Ⅱ)population infected by rice blast fungus were used.The metagenomic data was assembled by next-generation sequencing technology.GWAS analysis was carried out on the relative abundance data and rice genome.Candidate genes regulating phyllosphere microorganisms were screened.And a strain of Methylobacterium widely existing in phyllosphere was selected for sequencing analysis.The results are as follows:(1)The implemented 110 rice varieties were selected from C-RDP-Ⅱ and used to phenotype them in terms of their resistance to rice blast.For 110 varieties,the percentage of susceptible accession was 86.36%.For three main sub-population,the percentage of susceptible accession was 95.45%(Aus),84.78%(IND)and 73.33%(TRJ),respectively,which meet the condition of stress.(2)The phyllosphere microbial communities of different rice varieties were collected and their metagenomes were sequenced to construct a complete phyllosphere microbial library.A total of 1.34 terabases(Tb)of reads were generated and 569 medium-quality draft MAGs was recovered.The MAGs were primarily assigned to the following three classes:153 MAGs was assigned to Alphaproteobacteria,226 MAGs to Gammaproteobacteria,and 106 MAGs to Bacteroidia.In the diversity analysis,the relative abundance of Acinetobacter is greater than 50 in nearly half of the samples.And the relative abundance of Pantoea and Pseudomonas were also the highest.(3)According to the phenotypic results obtained in(1)and the metagenomic data and library obtained in result(2),combined with the rice genomic resources that have been sequenced,GWAS analysis was carried out.487 strains were screened,and the associated SNP covered about 235 loci on all chromosomes.Among them,Pseudomonas and Xanthomonas have most species associated with rice loci,while locus145,locus228,locus 143 and other loci are associated with a variety of bacteria and are in an important position in the association network.(4)The genome of Methylobacterium currus TP-3 which was isolated from tomato leaves before,was analyzed by genome sequencing.TP-3 has 2 chromosomes and 4 plasmids with a total 70.38%GC in 7 740 048 bp.Through the genome analysis of five basic databases,a total of 7588 genes were predicted.Through some specific functional databases or software,655 genes related to type Ⅲ secretory system effectors and 2196 genes involved in pathogen-host interaction were predicted.