Identification And Expression Patterns Analysis Of RdDM Pathway Genes In Multiple Biological Processes In Grapevine

Grape(Vitis vinifera L.)is one of the oldest tree species in the world.It has a long history of cultivation and a wide cultivation area,with high nutritional and economic value.RNA-directed DNA methylation(RdDM)is an important epigenetic pathway in plants that mediates transcriptional silencing through siRNAs.It plays a crucial role in plant growth and development,including seed germination and dormancy,embryo and fruit development,flowering,morphogenesis,stress resistance,disease resistance,and hormone signal regulation.While some members of RdDM pathway genes have been deeply studied in plants such as Arabidopsis,tomato,rice,citrus,and apple,there has not been a comprehensive systematic study of the genes involved in the grape RdDM pathway.Therefore,this study carried out systematic identification of RdDM pathway genes,gene structure analysis,evolution analysis,and expression patterns analysis of VvRdDMs pathway genes in multiple biological processes in the whole genome of grapes,mining genetic resources related to biotic stress and abiotic stress,as well as growth and development regulation,and focused on the differential expression of VvRdDMs during seeded and seedless grape seed development.It lays the foundation for elucidating the gene function of grape RdDM pathway,and further provides new functional gene resources for grape molecular breeding.The main research results are as follows:1.By systematic bioinformatics research methods,a total of 60 VvRdDMs pathway genes in 14 different families(SHH,CLSY,NRPD,NRPE,DRM,RDM,RRP6L,DRD,DDM,IDN,HEN,DCL,RDR)were identified in the whole genome of grapevine,and the identified genes were unevenly distributed on 18 chromosomes,among which VvRdDM on chromosome 4 the highest number of genes(7).Gene structure analysis found that most genes had similar exon-intron distribution pattern in the same subfamily,indicating that VvRdDMs have a certain degree of conservation.Collinearity analysis found that among the 14 gene families,only 4 family members experienced gene duplication events during evolution,among them,50%of the members of the AGO family are the result of gene duplication events.All collinear gene pairs were purifying selection according to the Ka/Ks ratio of less than 1.Promoter element analysis found that VvRdDMs pathway gene contained 18 cis-acting elements,among which ABRE elements were the most abundant in hormoneresponsive cis-acting elements.Among stress response-related elements,except for VvNRPE3B,all VvRdDMs pathway genes contained at least one cis-acting element involved in abiotic stress.In terms of plant growth and development,CAT-box elements are the most abundant,while RY-element elements that play a role in seed-specific regulation are only found in VvDRMl and VvRDM3c genes.2.Using 8 sets of online transcriptome raw data,analyze the expression patterns of VvRdDMs pathway genes on abiotic stress(high temperature and low temperature),biotic stress(botrytis cinerea,downy mildew,and powdery mildew)and hormones(MeJA,SA,GA3,and ABA).It was found that the VvRdDMs pathway genes did not respond to powdery mildew infection,and five(VvRDR6,VvAG04a,VvDCL3,VvRDM3b,and VvNRPD1)were significantly down-regulated expression after botrytis cinerea infection,and VvDCL2,VvAGO3a,and VvAG03b significantly down-regulated expression after downy mildew infection.VvAGO3a showed significantly up-regulated expression to all temperature treatments(35,40,and 45℃).After low temperature stress,the expressions of VvIDN2c and VvAGO10b were significantly up-regulated,and the expression of VvDCL2 was significantly down-regulated.After GA3 treatment,the expression of VvDDMl and VvAGO6a was significantly up-regulated,and the expression of VvDCL2 was significantly down-regulated.After MeJA treatment,the expression of VvRDR3 and VvAGO7 was significantly up-regulated,and the expression of VvRDR1b was significantly down-regulated.The expressions of VvRDR1b,VvRDR1a,VvDCL2,and VvAGO5a were significantly downregulated after ABA treatment.Among the 14 families of RdDM pathway gene,the AGO family had the largest number of members that significantly responded to the above treatments,it is speculated that the VvAGO family genes plays an important role in grape stress resistance to botrytis cinerea,downy mildew and low temperature stress.3.Using 4 sets of online transcriptome raw data,the expression patterns of VvRdDMs pathway genes in different developmental stages of buds,inflorescences,seeds and fruits were analyzed,and some VvRdDMs pathway genes(VvRDM2b,VvAGO1,and VvAGO4a,etc.)had a broad spectrum of high expression in different organs at various stages,indicated that the VvRdDMs pathway genes may play biological functions in the formation of multiple tissues and organs in grapes;Most of the VvRdDMs pathway genes were significantly more expressed in seedless grape cultivars than in seeded grape cultivars during seed development.qRT-PCR analysis further indicated the potential role of VvRdDMs pathway genes in seed development(VvIDN2a and VvDRD1a)or ovule abortion(VvRDR1a and VvRDR6).In conclusion,a total of 60 VvRdDMs pathway genes belonging to 14 families were identified in the grapevine genome,and a comprehensive bioinformatics analysis and expression analysis were carried out.It was found that the VvRdDMs pathway genes respond to stresses such as downy mildew,botrytis cinerea,high temperature,gibberellin,abscisic acid,and various hormone treatments,indicating that the VvAGO family of the VvRdDM pathway plays an important role in responding to stress,and VvDCL2 has a potential function in adaptation to low temperature.And further proposed VvRDR1α and VvRDR6 genes as functional gene resources related to seed development.This study increases our understanding of VvRdDMs pathway genes,provides an important theoretical reference for grape epigenetic research,and provides new functional gene resources for grape seedless molecular breeding.