| Japanese apricot(Prunus mume Sieb.et Zucc.)is native to China,a perennial woody plant of the Prunus genus in the Rosaceae family.Its fruits are mainly used for processing crispy mei,mei sauce,mei wine,other mei leisure foods and deep-processed products,which have high health care value and economic benefits.Green and red-skinned group are the main species for production and cultivation in Japanese apricot.Fruit color affects the processing quality,but the formation mechanism of the red color trait on fruit skin is still unclear in Japanese apricot.In this study,representative varieties ’Qingjia No.2’,’Shinano Koume’,and’Yanglao’ of green-skinned group,and representative varieties ’Ruantiao Hongmei’,’Xiaoye Zhugan’ and ’Zhonghong’ of red-skinned group were used as test materials.The red color components of fruit were clarified by phenotypic difference analysis and anthocyanin determination,and the key differential genes in the anthocyanin synthesis pathway were detected at the DNA level variation by whole-genome resequencing.We further cloned and characterized the function of PmUFGT3 gene by Agrobacterium-mediated transient expression in fruit skin of Japanese apricot and overexpression in the Atufgt Arabidopsis mutants,and finally site-directed mutagenesis of the amino acid variation in the coding region of PmUFGT3 of green-skinned group in Japanese apricot,then identified the key SNP variation site that determined the function of PmUFGT3,clarified the mechanism of red color trait formation in fruit skin of Japanese apricot,and developed SNP molecular markers to provide a basis for improving the efficiency of Japanese apricot breeding.The main findings are as follows:1.Analysis of fruit phenotype,physiology and biochemistry differences between green and red-skinned group in Japanese apricot.Fruits are divided into green and red-skinned group according to whether the fruit skin is red pigmentation or not in Japanese apricot.In this study,each three representative varieties between green-and red-skinned group in Japanese apricot were used as test materials,color difference analysis,pigment content determination and fine structure observation were used to study the fruit phenotypic traits between green and red-skinned group in Japanese apricot.The results showed that the fruits of red-skinned group in Japanese apricot had red pigmentation from the hardcore stage and reached the peak of pigment accumulation at the mature stage,showing dark red coloration,among which‘Xiaoye Zhugan’showed the most red coloration area and the darkest coloration,with the coloration area accounting for about 75%of the fruit surface.The fruits of green-skinned group in Japanese apricot had no red coloration from the hardcore stage to the ripening stage.A total of eight major anthocyanin compounds were detected,with cyanidin-3-O-glucoside being the most dominant anthocyanin.The test results clarified that the red color trait of fruit skin in Japanese apricot is mainly the result of anthocyanin accumulation.2.Identification of key differential genes in anthocyanin synthesis pathway at the DNA level in Japanese apricot by whole genome resequencing technology.Three varieties of green and red-skinned group were used as test materials,genomic resequencing was performed using second-generation sequencing to detect genomic DNA variations.Six DNA libraries were constructed,yielding a total of 92.2 Gb clean data and assembled into 32,004 unigenes,with an average coverage of 83.1%of the reference genome.A wide range of genetic variation was detected,including 7,387,057 single nucleotide polymorphisms(SNPs),456,222 insertions or deletions(InDels)and 129,061 structural variants(SVs).A total of 23,698 genes were annotated to six public databases(NR,NT,KEGG,GO,Swiss-prot and COG)using homology searches.Comparison of sequencing data and fluorescence quantitative PCR(qRT-PCR)showed,a total of 12 candidate genes were found potentially involved in the biosynthesis of anthocyanins,including three anthocyanin biosynthesis structural genes(4CL,F3H and UFGT3),five transcription factors(MYB29,MYB114,bHLH30,WD40 and NAC29),four regulatory genes(GST,UGT85A2,ABC and MATE).Differential gene expression analysis showed that PmUFGT3 gene may be the key differential gene for anthocyanin content differences in fruit skin between green and redskinned group in Japanese apricot.3.PmUFGT3 functional analysis and site-directed mutagenesis study of key SNP variation sites.The PmUFGT3 coding region and promotor were cloned by RT-PCR based on the sequence of Japanese apricot reference genome,and their bioinformatics and expression characteristics were analyzed.The results showed that the CDS of PmUFGT3 gene was 1509 bp,with only one exon,encoding 502 amino acids,and with a conserved structural domain of UDP-glycosyltransferase and similar protein(GT1＿Gtf-like).There are 10 non-synonymous SNPs in the coding region of Pm UFGT3 between green and red-skinned group in Japanese apricot,resulting in 8 amino acid variations,the secondary structure variation is close to the active site of the enzyme,which may lead to altered enzyme activity,and there is no difference in the cis-acting elements in the promoter region.It is hypothesized that the PmUFGT3 gene plays a key role in the formation of red fruit skin.In order to investigate whether the variation in the coding region of PmUFGT3 gene can cause functional changes,we used techniques such as transient expression in fruit skin of Japanese apricot,Arabidopsis overexpression,and site-directed mutagenesis to explore its function.Studies had shown that the PmUFGT3 protein was localized in the cytoplasm and vacuoles,by constructing the pCAMBIA1301-PmUFGT3 overexpression vector,and transiently infiltrated in fruits skin at immature stage of green-skinned group in Japanese apricot,it was found that transient expression of red-skinned group PmUFGT3 could produce a circle-like red coloration in the infected part of green-skinned group fruits skin and restore red coloration in the hypocotyl of Atufgt Arabidopsis mutant which could not synthesize anthocyanin,causing the synthesis and accumulation of anthocyanin;In contrast,the transient expression of green-skinned group PmUFGT3 didn’t have red coloration,meanwhile the complementary mutants didn’t have phenotypic changes.Then referring to the red-skinned group PmUFGT3 sequence in Japanese apricot,the seven amino acids variations of green-skinned group PmUFGT3 were changed by site-directed mutagenesis.Transient expression in fruit skin and Arabidopsis overexpression experiments found that when the common non-synonymous SNP variation on 1332 bp of green-skinned group was transposed from guanine to thymine,and the 444th amino acid was restored from glutamicacid to asparticacid,the coloring function of green-skinned group PmUFGT3 restored.The results confirmed that the PmUFGT3 gene is a key gene for anthocyanin synthesis in fruit skin and identified its key SNP variant loci in Japanese apricot.4.Development of SNP molecular markers related to red color traits in Japanese apricot fruits.Based on genome resequencing data from the Illumina platform,the average number of alleles per SNP marker locus was 1.9643,the heterozygosity was 0.02-0.75,and the average Shannon index was 0.517 and 0.4420 for red and green-skinned group varieties in Japanese apricot,respectively,with high dispersion and diversity in the red-skinned breed group in Japanese apricot.All germplasm was divided into two(K=2)clusters,including 10 and 34 germplasms,and the majority of red-skinned varieties were classified in cluster Ⅰ,showing a large genetic difference between the two cultivar groups(Fst=0.54).Developed 28 SNPs molecular markers for marker-assisted breeding,according to PIC values,a total of seven SNPs markers showed high polymorphism(PIC>0.5),including PmSNP＿1(4-CL),PmSNP＿4(WD40).PmSNP＿5(MYB29),PmSNP＿18(UFGT3)and PmSNP＿27(UFGT6)were highly correlated with red color trait in fruit skin of Japanese apricot.Among them,PmUFGT3 exhibited the highest polymorphism,it is of great significance for the development and utilization of germplasm resources related to red fruit skin in the future. |
