| Apples are one of the main fresh fruits in the world,and the color of the peel is one of the important quality indicators that determine the quality and commodity value of apples.Fruit tree bud change is an important source of fruit tree breeding.It has the characteristics of short selection cycle and fast breeding process.It is an important way to innovate germplasm resources.The ‘Red Delicious’ line is the most prone to bud change.Genome mutation detection of mutants will provide a theoretical basis for the study of apple bud change mechanism.This experiment uses ‘Red Delicious’(G0)and mutants ‘Starking Red’(G1),‘Starkrimson’(G2),‘Campbell Redchief’(G3)and ‘Vallee spur Delicious’(G4)as materials.Through whole-genome resequencing(WGS),the genome sequence of ‘Red Delicious’ and its mutants were analyzed,and mutation-related sites and genes were screened out.In addition,the genes related to anthocyanin synthesis were cloned from the peel of the ‘Vallee spur Delicious’,in order to explore the coloring mechanism of the ‘Red Delicious’ apple peel and provide a theoretical basis for the genetic improvement of apple peel color.The main results are as follows:1.The growth characteristics of G0 to G4 are that the internode length decreased from G0 to G4.Except G1,there were significant differences in internode length between G0 and G2,G3 and G4;the leaf area of G2 to G4 was significantly smaller than that of G1 and G0.The single fruit weight increased from G0 to G4.The single fruit weight from G1 to G4 was significantly higher than that from G0,and the peel color from G0 to G4 was also different.G0 and G1 were striped red,G2 to G4 were full red,among which G4 was the deepest color.2.The WGS of ‘Red Delicious’ and its fourth generation mutants obtained a total of189.38 Gb of valid data.The GC content of samples from G0 to G4 was 38.19% to 38.30%,and the percentages of bases greater than or equal to 20% and 30% were 96.77%～97.23% and92.28%～93.19%,respectively.The structural variant(SVs)identified from G0 to G4 ranged from 56,484 to 66,431,and Copy number variation(CNVs)ranged from 27,620 to 28,985.The CNVs and SVs from G0 to G4 were identified and evaluated.In the data set of SVs,the proportion of deletion(DEL)was the highest,followed by interchromosomal transfer(CTX);the SVs of G0,G1,G3 and G4 had the highest distribution of 200-300 bp,followed by 1200 bp.The SVs of G2 have the highest distribution between 300-400 bp.From G0 to G4,the distribution of SVs on chromosomes 2,3,9 and 15 was the most,and that on chromosome 16 was the least.There were more than 4300 SVs on chromosomes 2,3,9 and 15,and less than2100 on chromosome 16;From G0 to G4,the number of CNVs on chromosome 15 was the most,while that on chromosome 16 was the least.3.Single nucleotide polymorphism site(SNPs)and Insertion/deletion site(InDels)of DNA polymorphisms of ‘Red Delicious’ and its fourth generations were detected and commented.The SNPs identified from G0 to G4 were 1,973,621-2,058,335,and InDels were377,275-392,815;The SNPs and InDels mutation sites from G0 to G4 were analyzed.The results showed that SNPs and InDels were the most distributed in the intergenic region,followed by introns,and the least in upstream/downstream and exons;Among the SNPs variation from G0 to G4,nonsynonymous variation accounted for 54% of exons.From G0 to G4,C:G type changed into T:A type and T:A type changed into C:G type.In the InDels mutation from G0 to G4,frameshift mutations accounted for 65% of exons.The SNPs variation of G0,G1,G3 and G4 were most distributed on chromosome 11,followed by chromosomes 9,2 and 15,while G2 was the most distributed on chromosome 15,followed by2,11 and 9;The largest number of InDels mutations in G0 to G4 was found on chromosome15,while the SNPs and InDels mutations were the least detected on chromosomes 4 from G0 to G4.Compared with G0,the frameshift mutation InDels from G1 to G4 detected 42 short branch-related genes and 18 anthocyanin synthesis-related genes,of which 4 short branch-related genes were detected in G1 and 34 in G2,10 in G3,12 in G4,8 genes related to anthocyanin synthesis were detected in G1,14 in G2,4 in G3,and 6 in G4.4.Using the cDNA synthesized by reverse transcription of RNA extracted from the peel of ‘Vallee spur Delicious’ as a template,the genes MdWD40 and Md4CL were cloned by PCR,and pCAMBIA1300-super::Md WD40-GFP and pCAMBIA1300-super::Md4CL-GFP overexpression vector were constructed.MdWD40 and Md4CL encode 532 and 458 amino acids,respectively.Bioinformatics analysis showed that the molecular weights of MdWD40 and Md4CL were 31.76 and 51.44 4kDa,and the theoretical isoelectric points were 9.49 and5.58,respectively.Onion subcellular localization shows that MdWD40 and Md4CL are localized in the nucleus and belong to nuclear proteins,which is consistent with the predicted results of bioinformatics.The results of Agrobacterium-mediated transient expression in apple peels showed that compared with empty pCAMBIA1300-super::GFP(GFP),apple peels injected with overexpression vector turned red,anthocyanin content increased,and the expression levels of MdWD40 and Md4CL significantly up-regulated;the relative expression levels of F3’H,CHS,MYB1,and PAL related to the pericarp anthocyanin synthesis of the injection hole were significantly up-regulated in the MdWD40-GFP overexpression vector,while the relative expression levels of MYB1,UFGT and C4H were significantly up-regulated in the Md4CL-GFP overexpression vector. |
