Identification And Analysis Of Homologous Sequences Of Maize Pangenomic Disease Resistance Genes

Maize disease resistance genes play an important role in protecting maize from various pathogens in nature and in initiating its own defence system.Identification of maize disease resistance genes and their functional mechanisms is important for understanding the disease resistance mechanism of maize,obtaining key maize disease resistance genes and breeding superior maize disease resistance lines.The use of pan-genomes in plant research has now become a trend with the rapid development of sequencing technologies and the increasing availability of high-quality,population-scale genomes.Pan-genomes allow a more comprehensive discovery of genetic variation and domestication resources in plants.However,the identification and analysis of disease resistance gene systems in maize pan-genomes has rwerely been reported.Therefore,in this study,we propose to systematically identify the resistance gene analogues(RGAs)in the maize pangenome and conduct an in-depth analysis of the distribution,homology groups,phylogeny,evolutionary rates and expression patterns of maize RGA gene family members.The results of the study provide a wealth of data for the study of maize disease resistance genes and provide guidance for the selection and breeding of superior maize varieties.The specific findings are as follows:1.Pan-genomic screening of 31 maize inbred lines using RGAugury softwwere yielded 4,193 NBS-LRR genes,20,650 LRR-RLK genes,1,583 RLP genes,4,579 TM-CC genes and 114 RPW8 genes.The number of members of each RGA family was unevenly distributed among the 31 inbred lines,and the number of members of the LRR-RLK and TM-CC families varied widely among the inbred lines,with the number of genes of the LRR-RLK family ranging from 590-703 and the number of genes of the TM-CC family ranging from 133-182,whereas the number of genes of the NBS-LRR,RLP and RPW8 families varied less.The NBS-LRR,LRR-RLK and TM-CC families were more variable,with an average of 1.6 proteins per gene,whereas the RLP and RPW8 families could encode an average of 1.2 and 1.3 proteins per gene,respectively.The more disease-resistant inbred lines had fewer transcript isoforms and,correspondingly,the less disease-resistant inbred lines had more transcript isoforms.2.Chromosomal mapping of RGA genes revealed that the distribution of RGA genes on chromosomes with relatively distinct clustering is mainly on chromosomes 1 and 2,with a total of 7,874 RGA genes.Gene cluster analysis showed that RGA families can form a large number of gene clusters,with a total of 4,569 RGA gene clusters,but only about 23%of RGA genes were distributed in gene clusters.RGA genes and RGA gene clusters were similarly distributed in different inbred lines.3.Ortholog group analysis of RGA family genes using orthofinder softwwere identified 1196 ortholog groups,including 16,160 core genes,14,806 variable genes,44 specific genes,and 109 unassigned genes.The number of core genes in families NBS-LRR,LRR-RLK,TM-CC,and RPW8 was 2,180,11,403,2,049,and 61,respectively,and the number of non-core genes was 2,015,9,248,2,430,and 53,respectively,indicating that the copy number variation and gene presence/absence variants of these four family members were low.467 core and 1,116 non-core genes for the RLP family.The number of non-core genes was higher than the number of core genes,indicating the number of copy number variation and gene presence or absence variants was higher in the RLP family.4.The evolutionary rate of RGA family members was analyzed by ParaAT and KaKs_Calculator software.It was found that the Ka/Ks values of RPW8 family genes were all distributed between 0-0.2,which was under strong purification selection pressure.The LRR-RLK family is under strong purifying selection pressure,with Ka/Ks values distributed between 0-0.2,0.2-0.4,and 0.4-0.6 for 54%,25%,and 11%of LRR-RLK core genes,respectively,and 41%,28%,and 16%of LRR-RLK variable genes,respectively.The TM-CC family is also under strong purifying selection pressure,with Ka/Ks values distributed between 0-0.2,0.2-0.4,and 0.4-0.6 at 54%,31%,and 6%,for TM-CC core genes,respectively,and 49%,29%,and 10%,for TM-CC variable genes,respectively.NBS-LRR familiy were under less purifying selection pressure,with Ka/Ks values distributed between 0-0.2,0.2-0.4,0.4-0.6,and 0.6-0.8 in 22%,26%,23%,and 14%of NBS-LRR core genes,respectively,and 12%,22%,24%,and 19%of NBS-LRR variable genes,respectively.The RLP family was also under less pressure from purifying selection,with 43%,38%and 11%of RLP core genes and 21%,29%and 26%of RLP variable genes having Ka/Ks values between 0-0.2,0.2-0.4 and 0.4-0.6,respectively.Analysis of all RGA core genes and variable genes found that variable genes were subject to significantly weaker purifying selection pressure than core genes.5.Expression pattern analysis of RGA genes revealed that RGA genes were differentially expressed in response to infection by pathogenic fungi with different life histories and in resistant and susceptible inbred lines.Differential expression analysis revealed 28 candidate resistance genes and 9 candidate susceptibility genes in response to infection by the biotrophic fungus U.maydis;32 candidate resistance genes and 22 candidate susceptibility genes in response to infection by the hemibiotrophic fungus Setosphaeria turcica.qRT-PCR analysis showed that the candidate disease resistance genes Zm00001eb124900,Zm00001eb163440,Zm00001eb304670 and Zm00001eb334630 were up-regulated in the resistant inbred lines in at least one of the three periods after S.turcica infestation.The candidate susceptibility gene Zm00001eb275120 was up-regulated 24 h after infestation with S.turcica and down-regulated at all times after infestation with the resistant inbred lines.