Mining And Functional Study Of Chilling Requirement Major Genes In Peach

Peach(Prunus persica L.)is one of the most important economic tree species in China,its cultivation area and yield are among the top in the world.Bud dormancy is the result of plant adaptation to the environment conditions,which is crucial for survival in winter.During this period,bud meristem is insensitive to growth promotion signals;timely dormancy release is a necessary stage for normal growth and development of plants.Chilling requirement(CR)is the most important factor to break the bud dormancy of deciduous fruit trees.If the chilling requirement cannot be met,the flowering period will be prolonged,the flowers and fruits will be seriously lost,and even the fruit flavor and marketability will be affected.Under the background of global warming,the bud dormancy of perennial deciduous fruit trees was studied to further deepen the understanding of the regulation of chilling requirement on dormancy process.It is of great significance for peach cultivars with different chilling requirements and analyzing the dormancy regulation mechanism.However,the genes,functional characteristics and regulatory mechanisms controlling the chilling requirement of peach bud are still unclear.For the purpose of the above cognitive limitations,this paper carried out related research and obtained the following results:1.To understand the molecular response dynamics of low temperature accumulation controlling dormancy of peach flower buds,we took flower buds of two peach cultivars with different chilling requirement as the research objects and conducted chromatin immunoprecipitation sequencing(Ch IP-Seq)and RNA-seq analysis,the results revealing that H3K27me3 modification accumulated earlier in cultivars with low chilling requirements.Besides,the modification strength of H3K27me3 increased in both varieties during the process of cold exposure.Flower bud differentiation continued at the same time,which may be related to the transcriptional abundance of key genes for flower bud development;the distribution of H3K27me3 in the key genes was studied and related genes involving in dormancy regulatory pathways were proposed.2.During the process from dormancy induction to dormancy release,the content of plant hormone ABA decreased,while the content of bioactive GAs increased.This is related to the decreased expression level of ABA biosynthetic genes and the increased expression level of degradation genes,as well as the increased expression level of GA biosynthetic genes and the decreased expression level of degradation genes;the increased GA/ABA ratio,which is the key factor for the sustained differentiation of dormant buds.The key roles of DAM-like genes and stress response genes in the dormancy process of peach bud were also analyzed.3.In this study,345 peach natural populations with different chilling requirement were used for genome resequencing,structural variations(SVs)associated with chilling requirement were screened across the whole genome.Based on these variants,genomewide association studies(GWAS)were conducted,and a 15.8 kb block contained only one complete gene(Prupe.1G531700,PpDAM6)was found.We acted this gene as the major gene in the regulation of chilling requirement of peach bud.Transient silencing of this gene in peach flower bud and heterologous overexpression in apple confirmed its function in regulating bud dormancy transition.Additionally,combining with the transgenic results of Arabidopsis thaliana,tobacco(NC89)and apple,as well as the tissue-specific expression in Arabidopsis thaliana,PpDAM6 plays an important role in regulating bud dormancy cycle,vegetative and reproductive development.4.PpDAM6 gene was highly expressed in high chilling requirement accessions and low expressed in low chilling requirement accessions,the gene expression level was positively correlated with its protein expression level.Further analysis found that30-bp indel located in the PpDAM6 promoter region was associated with the expression level of PpDAM6,which further concluded that the deletion was closely related to the low-chilling requirement levels.Therefore,we developed this 30-bp indel as a molecular marker for distinguishing high-and low-chilling requirement peach accessions,and verified it in 136 peach natural populations with different chilling requirement.The development of this marker laid a genetic foundation for the identification and breeding of different flowering time varieties in different geographical areas.5.PpDAM6 has the characteristic of seasonal expression and is regulated by low temperature signal,which increases gradually during the dormancy induction period and decreases to the minimum when the dormancy is released along with the prolonged cold exposure.The expression level of PpDAM6 was not affected by H3K27me3 modification.PpDAM6 was positive regulation by cold response gene Pp CBF,this regulation mechanism provided protection for peach bud from low temperature damage.The interaction between PpDAM6 and Pp TIFY protein was verified through Y2 H screen library and bimolecular fluorescence complementation assays,which indirectly proved that jasmonic acid signaling pathway was involved in dormancy regulation.6.PpDAM6 involved in the signaling circuit of ABA-mediated dormancy regulation.The expression trend of PpDAM6 is positively correlated with ABA content and is induced by the ABA pathway gene Pp ABF.PpDAM6 induces the expression of downstream gene Pp NCED1,a key enzyme in downstream ABA biosynthesis,and CALS1/2,a callose synthase gene.It was revealed that PpDAM6 regulates the release of dormancy by mediating ABA signaling pathway and the permeability of intercellular plasmodesmata in dormant buds.This study provides a theoretical basis for the regulation of dormancy cycle in perennial plants.