| Peach(Prunus persica(L.)Batsch),belongs to Rosaceae plant,is one of the important fruit trees originating in our country.Peach has high budding rate,strong shoot sprouting and vigorous vegetative growth.Controlling tree vigor requires a lot of manpower and material resources.It is controversial about using plant growth regulators.So,adjusting tree architecture has always been one of the important research directions of peach.‘Zhongyoutao 14’(CN14)is a temperature-sensitive semi-dwarf(TSSD)cultivar of peach,whose shoots growth depends on surrounding temperature changing,and the tree architecture is semi-dwarf.In this research,The genome of CN14 was sequenced and de novo assembled using single-molecule real-time sequencing and chromosome conformation capture assembly.Based on the high quality genome of CN14.Eighty-six resequenced F1 individuals were used to identify candidate genes controlling TSSD by genome-wide association analysis(GWAS).Based on the reference of CN14 genome,334 peach resequencing data published were used to map 10 agronomic traits gene locus in peach by GWAS,such as the fruit hairiness/hairless,red colour around stone and so forth.Transcriptome analysis was employed to screen transcriptional regulators associated with the peach carotenoid metabolic pathway during the developmental stages of fruits with different flesh colours(yellow/white).The main findings are as follows:1,Two sequencing strategies and Hi-C-assisted assembly to obtain a high-quality peach genome of CN14.In this study,we combined the Pac Bio sequencing platform and the next generation sequencing platform to perform whole-genome sequencing of CN14,a temperature-sensitive cultivar of peach,with Pac Bio sequencing depth of 586×,161.7 Gb of sequencing data,and a reads N50 of approximately 21.675 Kb.After whole-genome assembly using Hi-C data,the CN14 genome was approximately 228.8 Mb of sequence anchored on eight The contig N50 was 6.86 Mb and the Scaffold N50 was 27.86 Mb.A total of 30,181 genes were annotated,with a BUSCO value of 98% and LAI value of 24.51,resulting in a high quality CN14 genome.2,Aquaporin protein gene PpTIP2 is a key gene for temperature-sensitive semi-dwarf traits in peach,and its promoter activity is regulated by temperature.Using the CN14 genome as the reference genome,GWAS analysis was performed using the resequencing data and phenotypic data of the F1 generation population of the CN14×HSM cross,and the SNP significantly associated with the temperature-sensitive semi-dwarf trait were located at Chr3:4,376,6037.The PpTIP2 gene promoter sequences differed between the different types,and SNP variation of the promoter showed co-segregation with the temperaturesensitive semi-dwarf trait,with five cis-acting elements differing between the promoter types.The subcellular localization results showed that PpTIP2 was co-expressed in the nuclear and membrane.3.PpCCD4 plays a key role in the formation of different flesh colours(yellow/white),PpWRKY33 can interact with PpCCD4 and participates in peach fruit colour formation.GWAS indicated that the locus associated with peach flesh color(yellow/white)trait was located on chromosome 1,near the PpCCD4 gene.Analysis of carotenoid content and expression of genes related to carotenoid metabolic pathway in CN14(white flesh)and HSM(yellow flesh)fruits at different developmental stages revealed that carotenoid content gradually increased in HSM fruits of yellow flesh peache,while gradually decreased in CN14 fruits of white flesh peach.In ripe fruit of HSM,purple xanthin,zeaxanthin and β-carotene were the main contents.Among the genes related to carotenoid synthesis,PSY,PDS,ZDS and LCYB were expressed in the same pattern in both fruit types,but among the genes related to the carotenoid degradation pathway,the carotenoid cleavage dioxygenases 4 gene(CCD4)was differentially expressed in the two different flesh colour peach,indicating that it was the main factor responsible for the difference in flesh colour,which was consistent with the results of GWAS-based gene mapping.Transcriptome analysis of CN14(white flesh)and HSM(yellow flesh)fruits at different developmental stages revealed a significant correlation between PpWRKY33 and PpCCD4 expression;Yeast-one hybrid and Dual luciferase complementation(LUC)experiments showed that PpWRKY33 suppressed the expression of PpCCD4.This indicates that PpWRKY33 regulates peach fruit colour formation by inhibiting the expression of PpCCD4.4.Based on the CN14 high quality genome,334 peach resequencing data published were used to map fruit hairiness/hairless,red colour around stone,fruit maturity date,double flowers,flesh colour(While/yellow),Pollen fertility,kernel taste,flower opening time(onset,bloom and end).The most relevant SNP locus for red colour around stone trait was located on Chr4,and three linked loci were also found on Chr3,6 and 8;the significantly relevant locus for fruit maturity date trait,Chr4:11550220,was near the Pp04G017800(Chr4:11558954,NAC transcription factor Prupe.4 G186800)gene;The loci significantly associated with fruit hairiness/hairless trait are in the CN14 genome Chr5:16,757,982 at exon 3 of the PpMYB25 gene;the SNP loci significantly associated with the double flowers trait are in Chr2: 24,849,526,near the AP2 gene,and Chr6: 19,298,780,near TOE transcription factor.The SNP loci for kernel taste(sweet/bitter)traits were located within Chr2 and Chr8,with three phenylpropanoid biosynthesis genes(Pp02G031770,Pp02G031580 and Pp02G032190)potentially associated with bitter taste production in peach kernels;The mapping interval of pollen fertility,traits of peach were located in the Chr6:1,708,346-2,288,022,which included ATP synthesis genes involved in chorionic cell and pollen development as well as TPR-like genes;The mapping intervals for the three traits of flower bloom date,full bloom date and bloom ending date were all located in the Chr1: 26,970,777-27,The SNPs associated with the flower opening time(onset,bloom and end)were located in the promoter region of the 9-cis-epoxycarotene dioxygenase 4(NCED4)gene,which encodes a pathway related to abscisic acid(ABA)synthesis,and in the coding region,and NCED4 may be a candidate gene for controlling flowering time in peaches.Pp01G032100 and Pp01G033110)and a WD40 transcription factor(Pp01G032170)may be associated with peach flowering traits at this locus. |
