Evaluation Of Buckwheat Germplasm Resources Resistant To Stem Canker And Molecular Mechanism Of Disease Resistance Based On Genome And Transcriptome

Stem Canker on Fagopyrum tataricum（Tartary buckwheat）has a great impact on the production of buckwheat,which affects the yield and quality of buckwheat directly.When the disease is serious,the yield and quality of buckwheat will be reduced greatly.The light occurrence of the disease would cause decreased yield of buckwheat,and the severe disease will cause buckwheat to wilt and die.At present,chemical control is the main way to control buckwheat stem canker.However,chemical pesticides have great limitations due to high residue,high toxicity and unfriendly to environment.Therefore,the breeding of disease-resistant germplasm resources is the most effective way to control disease.Breeding disease-resistant cultivars is an important and effective way to control buckwheat stem cancer.However,there are few reports on buckwheat germplasm resources and molecular mechanism of disease resistance.In this study,the pathogen identification of buckwheat stem cancer,the evaluation of buckwheat disease-resistant germplasm resources and the genome wide association study（GWAS）based on disease resistance were studied.Through transcriptome,plant genetic transformation,gene function research and other technical means,the resistance related genes were studied.The main results were as follows:1.The pathogen of buckwheat stem cancer was identified as R.solani AG-4 HGI according to four aspects:morphological characteristics,karyotype analysis,pathogenicity determination and molecular identification.The length of DNA fragment obtained by ITS sequence was 651 bp,which was uploaded to NCBI and obtained the accession number MT078642.2.Illumina Hiseq sequencing along with the second and third generation sequencing mode were used to analyze the Fungal genome sequencing.The number of Scaffolds was 405,the GC content was 48.16%,and the genome size was 65.36Mb by sequencing.A total of 18821 coding genes,1026 carbohydrate active enzymes（CAZymes）and 294 effectors were predicted.There were 35.844 kb repeats in genome,accounting for 0.05%of the genome,which were identified from 487elements of 23 families and 4 unknown repeat elements.3.A total of 500 buckwheat germplasm resources were evaluated for disease resistance.We obtained 11,136,177,138 and 38 immune,high resistance,disease resistance,medium resistance and susceptible varieties,accounting for 2.2%,27.2%,35.4%,27.6%and 7.6%of the total,respectively.Based on the resistance and susceptibility of germplasm resources,genome-wide association analysis was carried out.Forty nine significantly related genes,including FtPin G0302737900.01,FtPin G0808388800.01,FtPin G0302744900.01 were obtained under the condition of minus log₁₀p≥5.00,which may be involved in disease resistance in buckwheat.4.A total of 19140 genes were obtained by transcriptome sequencing of pathogen-host interaction.2102 genes were significantly up-regulated and 424CAZymes were up-regulated after 14 hours infection,indicating that 14 h of infection was the highest invasion period of pathogen.36613 genes were obtained by sequencing,of which 17745 genes were differentially expressed.The number of differentially expressed genes was more at 14h of infection.KEGG results showed that most of the differentially expressed genes were enriched in the process of hormone signal transduction,of which 62 differentially expressed genes belonged to jasmonic acid signaling pathway,indicating that jasmonic acid played an important role in the infection of buckwheat by R.solani AG-4 HGI.The content of hormones in buckwheat were determined after infection by R.solaniAG-4 HGI.The results showed that the content of jasmonic acid（JA）and JA Phe increased at 6 h,and increased significantly at 14 h.Gibberellins（GA）and SAG contents showed the same increasing trend at 14 h after infection.The main results of transcriptome analysis of buckwheat treated with salicylic acid （SA）,GA,ethylene（ET）and JA were as follows:there were 2783 up-regulated genes in SA transcriptome,943 up-regulated genes in GA transcriptome,1679 up-regulated genes in ET transcriptome,1515 up-regulated genes in ET&JA co-treatment transcriptome and 6790 up-regulated genes in JA transcriptome.JA transcriptome has the highest number of up-regulated genes compared to others,which were mainly enriched in flavonoid biosynthesis and hormone signal transduction processes.5.Based on genome sequencing of AG-4 HGI,GWAS analysis and multiple hormone transcriptomes,41 pathogenicity related genes of R.solani AG-4 HGI and 21resistance related genes of buckwheat were obtained.In this study,resistant related genes function of FtJAZ8 and FtCYP94 from buckwheat were verified from three aspects:disease resistance performance,physiological and biochemical index detection and molecular level of transgenic materials.The results showed that the transgenic materials of FtCYP94 and FtJAZ8 improved the disease resistance,with the disease index 23.49 and 19.07 respectively,and the control was 49.51.DAB（Diaminobenzidine）staining,MDA（Malonic dialdehyde）detection and PR1expression analysis showed that FtJAZ8 and FtCYP94 played positive regulatory role in the infection process of AG-4 HGI,and FtJAZ8 showed better disease resistance.6.The results of AtJAZ8 against plant pathogenic bacterium showed that AtJAZ8was involved in the process of Agrobacterium tumefaciens infecting Arabidopsis thaliana to produce root nodules.AtJAZ8 interacts with the virulence protein Vir E3 of Agrobacterium tumefaciens to regulate the formation of Arabidopsis nodules.The results revealed that AtJAZ8 could negatively regulate Agrobacterium infection.Therefore,JAZ8 negatively regulates the infection process of pathogen to host during the infection of fungal pathogen R.solani and bacterial pathogen Agrobacterium tumefaciens,so it is a broad-spectrum resistance factor.