Transcriptome Sequencing Analysis Of Difference In Herbaceous Peony (Paeonia Lactiflora Pall.) Resistance To Gray Mold

Herbaceous peony (Paeonia lactiflora Pall.) is a well-known traditional flower in China and is widely used for landscaping and garden greening due to its high ornamental value. However, herbaceous peony is susceptible to gray mold in the production practice, which leads to wizened and necrotic leaves, sunken and broken stems, brown and rotten petals, and even death. These symptoms seriously affect the ornamental and commercial values of the plants. Thus, making clear the key genes and miRNAs of herbaceous peony and their molecular regulation mechansim resistance to gray mold has important theoretical values for breeding herbaceous peony cultivars of gray mold resistance. In this study, the pathogen of herbaceous peony gray mold in the Yangzhou region was identified based on morphological characteristics and molecular data. Then, the digital gene expression and small RNA sequencing analysis of the disease-resistant cultivar ’Zifengyu’and the disease-sensitive cultivar’Dafugui’were performed, the metabolic pathways related to disease resistance were identified, as well as the differentially expressed genes and miRNA related to disease resistance were excavated. The main results were as follows:(1) The infected leaves of herbaceous peony gray mold were collected to isolate the pathogen, and then the microscopic examination was conducted after sporulation. The preliminary identification was similar to Botrytis. The determination of pathogenic ity and host range revealed that the pathogen could not only infect herbaceous peony, but also gardenia {Gardenia jasminoides), tomato (Lycopersicon esculentum), Chinese rose (Rosa chinensis), camellia (Camellia japonica), green pepper (Capsicum annuum var. grossum), cucumber (Cucumis sativus) and Chinese cabbage (Brassica pekinensis). The rDNA-ITS sequence analysis indicated that the homology of this strain with Botrytis cinerea was 99%. The above results identified the pathogen of herbaceous peony gray mold in the Yangzhou region was B. cinerea Pers.(2) The leaves of disease-resistant cultivar’Zifengyu’and the disease-sensitive cultivar ’Dafugui’at four stages were gathered to construct eight cDNA libraries (’Zifengyu’-S1-’Zifengyu’-S4, "Dafugui’-S1-’Dafugui’-S4) for digital gene expression sequencing. After removing low quality reads, the clean reads all exceeded 11,680,333 each library. The gene expression leves were compared among different libraries, and the most differentially expressed genes (DEGs) screened were in’Zifengyu’-S1 vs.’Zifengyu’-S4 (6843, including 4916 up-regulated and 1927 down-regulated) and’Dafugui’-S1 vs.’Dafugui’-S4 (10355, including 5737 up-regulated and 4618 down-regulated). Then, the DEGs were annotated using the GO and KEGG database. The pathways plant-pathogen interaction, secondary metabolism synthesis and antioxidant system that related to disease resistance were screened, and 51,76, and 13 disease resistance-relevant candidate genes were identified, respectively. These candidates were almost up-regulated after the infection by gray mold, and their expression patterns differed between the two cultivars:their expression of the disease-resistant cultivar’Zifengyu’sharply increased during the early stages of infection, while it was relatively subdued in the disease-sensitive cultivar’Dafugui’. These results indicated that multiple disease resistance responses were inducted in herbaceous peony under B. cinerea stress, including activation of PTI and ETI, synthesis of secondary metabolites such as lignin, phytoalexins and benzoxazinoids, regulation of antioxidant enzymes expression level to scavenge ROS. Because the pathogen recognition, the consequent defense reaction and the up-regulation of disease-resistance genes all occurred rapidly in the disease-resistant cultivar’Zifengyu’, this cultivar was more resistant to the disease than the’Dafugui’cultivar.(3) The leaves collected of’Zifengyu’and’Dafugui’from four stages were equally mixed to construct two cDNA libraries for small RNA sequencing. After getting rid of low quality reads, 23,520,582 and 21,452,306 clean reads were obtained from’Zifengyu’and’Dafugui’, respectively, and then spliced to 4,592,881 and 5,809,796 small RNAs. Through screening against database and software predicting, a total of 271 and 298 conserved miRNAs,9 and 8 novel miRNAs were identified in’Zifengyu’and’Dafugui’, respectively. From the differential expression analysis of the two cultivars,237 conserved miRNAs (136 up-regulated,101 down-regulated) and 7 novel miRNAs (2 up-regulated,5 down-regulated) were screened, and the function annotations of target genes were also performed. In the KEGG annotations,9 disease resistant-relevant target genes corresponded to 7 candidate miRNAs were screened, which involved in disease resistant-relevant pathways including phenylpropanoid biosynthesis, diterpenoid biosynthesis, plant hormone signal transduction, and plant-pathogen interaction. Furthermore, the conjoint analysis of expression patterns of candidate miRNA and corresponding target genes were carried out, and we found that the expression levels of miR5254、miR165a-3p、 miR3897-3p and miR6450a were negatively correlated with their corresponding target genes, meaning that they might be involved in post-transcriptional regulation of herbaceous peony in response to B. cinerea stress.