Transcriptome And NBS Gene Family Analysis To Identify Candidate Genes For Rust Resistance Of Orchardgrass(Dactylis Glomerata L.)

Orchardgrass?Dactylis glomerata L.?is an important grass forage with good regenerability and wide adaptability.It plays an important role in grassland husbandry and rocky desertification control in southwest China,but it is extremely susceptible to rust in spring and summer,which seriously affects its forage yield and quality,and seed production.The identification and analysis of NBS disease resistance genes and the analysis of the molecular mechanism of disease resistance by different resistant varieties can help the molecular breeding of the orchardgrass and reduce the harmful effects of rust on the orchardgrass.In this paper,based on the genome sequence of orchardgrass,we screened out the NBS disease resistance genes of orchardgrass by using the method of bioinformatics.Then,we inoculated the two materials of high resistance?PI251814?and high sensitivity?PI292589?to rust.We took RNA-seq samples from 7 and 14 days of inoculation and sRNA-seq from 7 days of inoculation to explore the disease resistance of orchardgrass from the transcription level,and finally combining with transcription level and NBS disease resistance genes,the rust resistance genes of orchardgrass were excavated to preliminarily analyze the molecular mechanism of rust resistance of orchardgrass.The main results are as follows: 1?Identification and analysis of 413 NBS disease resistance genesA total of 413 NBS disease resistance genes were identified through gene sequence comparison of orchardgrass,involving four major classes of N,NL,CN and CNL.Chromosome analysis showed that 413 NBS disease resistance genes were unevenly distributed on the 7 chromosomes,of which 99 genes were the most distributed on chromosome 5.Phylogenetic analysis showed that the number of NBS disease resistance genes in the two main branches differed greatly,revealing the complexity of the evolution of NBS disease resistance genes.Analysis of expression patterns of NBS disease resistance genes under abiotic stress showed that 11 NBS disease resistance genes were differentially expressed under waterlogging stress,and 5 NBS disease resistance genes were differentially expressed both under waterlogging and drought stress,with only 1 NBS disease resistance gene were differentially expressed under waterlogging and heat stress.2? Results of RNA-seq after being inoculated with rust fungusRNA-seq analysis showed that the number of differentially expressed transcripts in high resistance?HR?materials?3920?was significantly higher than that in high sensitive?HS?materials?1953?.KEGG analysis showed that "ath04626: plant-pathogen interaction" is significantly enriched in high resistance?HR?materials,but not in high sensitive?HS?materials.WGCNA analysis based on 7630 genes showed that 692 gene in yellow module was co-expressed.Heatmap analysis showed that most of yellow module gene expression patterns were significantly different in high resistance?HR?materials?over expressed on 7 and 14 days?and high sensitive?HS?materials?over expressed on 7 days and under expressed on 14 days?.KEGG analysis of 692 genes in yellow module showed that "ath04626: plant pathogen interaction" pathway was the most significant.3?Results of sRNA-seq after inoculation with rust fungusThrough sRNA-seq,348 conserved miRNAs were identified and 110 novel miRNAs were predicted,among which 123 miRNAs were expressed differently between the inoculated and non-inoculated materials.The analysis of differentially expressed miRNAs showed that 86 miRNAs belonged to 24 miRNAs families,and 29 miRNAs were specifically expressed in highly resistant materials?HR?.KEGG analysis of 29 miRNAs target genes with specific expression in high resistance?HR?materials showed that the target genes with specific expression in high resistance?HR?materials were significantly enriched in the "ath04626: plant-pathogen interaction" pathway.4 ? Combined analysis of RNA-seq,sRNA-seq and NBS disease resistance genesAmong the 7,630 differentially expressed genes found in RNA-seq,there were 65 NBS disease resistance genes,of which 38 NBS disease resistance genes were differentially expressed in high-resistance?HR?materials alone.There are two NBS disease resistance genes co-differentially expressed in abiotic stress and biotic stress.The target genes corresponding to the 123 miRNAs differentially expressed in sRNA-seq contained 207 NBS disease resistance genes,of which 43 were differentially expressed in high-resistance?HR?materials alone.The six genes differentially expressed in the RNA-seq analysis and the six target genes differentially expressed in the sRNA-seq analysis were co-differentially expressed.Four of these six genes?all four genes encode the RPM1 resistance protein expression?belong to the MIR399 family.The target genes corresponding to the miRNAs: zma-miR164h-5p,ath-miR399 b and novel₁48 are significantly down-regulated in high-resistance?HR?materials;the gma-miR5368,bdi-miR1432 and csi-miR399f-5p were significantly up-regulated in high-resistance?HR?materials,while these genes in high-sensitivity?HS?materials did not change at all.From the above results,it is speculated that genes that are significantly differentially expressed in high resistance?HR?materials are transduced by signals and activate related transcription factors?WRKY,bZIP,etc.?to regulate transcription and promote the expression of disease resistance genes;regulate the expression changes of disease resistance related pathways such as plant hormone signal transduction,plant pathogen interaction,etc.,so as to regulate the disease resistance and defense response of orchardgrass.