Comparison of anthocyanin accumulation patterns in different peach skin color types
We compared anthocyanin accumulation in ‘SYBT’ with that in a red-skin cultivar ‘ZhongTaoJinMi’. The fruit skin of ‘SYBT’ is white from first to ripening stages. ‘ZTJM’ fruit skin is white in the early stages and nearly red in stages 4, 5 and 6 (Fig. 1A). Total anthocyanin content was measured in fruit skin. As expected, white-skinned ‘SYBT’ did not show anthocyanin in the skin throughout fruit development (Fig. 1B). Anthocyanins were not detected at the beginning of ‘ZTJM’ fruit development, and it appeared at stage 3 and increased to a great extent at stage 4, which was the final step at maturity, the anthocyanin content was about 10 times higher in ‘ZTJM’ skin than ‘SYBT’ skin. This is in accordance with the red coloration observed in stages 4 5 and 6 of ‘ZTJM’ skin.
Expression Analysis Of Genes Related To Peach Skin Blush
Expression profiles of structural genes involved in anthocyanin biosynthesis were examined using quantitative real-time PCR. We measured the expression of three PpMYB10 genes, PpMYB10.1, PpMYB10.2, PpMYB10.3, which are highly correlated with anthocyanin biosynthetic in peach. Expression levels of PpMYB10.1/2/3 were extremely low in the skin during the six developmental stages of ‘SYBT’ (Fig. 2A). In ‘ZTJM’ skin, expression levels of PpMYB10.1/2/3 were also low at the beginning of fruit developmental, and then the expression levels of PpMYB10.1 increased dramatically at stages 3 to 6, while expression levels of PpMYB10.2/3 remained low throughout fruit maturation. High transcription levels of PpMYB10.1 found in stages 4, 5 and 6 were tightly correlated with anthocyanin content, which were only observed in these three ripening stages of ‘ZTJM’ fruit. These results suggest that PpMYB10.1 alone is responsible for anthocyanin accumulation in the skin of ‘ZTJM’. We also measured expression of downstream structural genes in anthocyanin biosynthesis pathway, including PpCHS, PpF3H, PpDFR, PpANS and PpUFGT, which showed similar expression patterns in the skin of ‘ZTJM’ and ‘SYBT’ during fruit development. The expression levels of all genes were lower in the ‘SYBT’ cultivar than in the ZTJM’ cultivar throughout fruit development (Fig. 2B).
Variation characterization of PpMYB10.1 promoter region
The mutation in MYB10.1 promoter that causes anthocyanin not accumulation in SYBT fruit skin. We investigated the genomic structure of MYB10.1 promoter in SYBT. As shown in Fig. 3, we found many single nucleotide polymorphisms (SNPs) and short insertion/deletions, which were homozygous or heterozygous, in the sequences of the promoter compared with the ‘Lovell’ peach cultivar(Verde et al. 2013). In particular, the promoter of the SYBT MYB10.1 had two long fragment insertions. One is 5243 bp insertion in the 1173 bp upstream of ATG, which is considered to be allelic variation of MYB10.1(Tuan et al. 2015), and named Ins1 in this study, the other is the results of this study, we found that the size of the fragments amplified from the SYBT genome was approximately 3.5 kb longer than those amplified from the ‘Lovell’ peach genome, which is due to a 3500 bp insertion in the 2706 bp upstream of ATG and named Ins2 in this study. The other SNPs found in the promoter region were also detected in red-skin peaches, indicating that these SNPs do not control the non-reded skin of peaches cultivars.
Development And Validation Of Molecular Markers For Peach Skin Blush
Two allelic types of MYB10.1, MYB10.1-1/MYB10.1-2, were identified in Japanese cultivars, and those with two MYB10.1-2 alleles had white-skinned fruits. However, we investigated the transposon insertion of MYB10.1 promoter in 61 peach cultivars, we found that the genotype and phenotype were not consistent of some cultivars (Fig. 4A). Among 36 non-reded skin peach cultivars, 13 cultivars carried two MYB10.1-2 alleles, and 4 carried MYB10.1-1/MYB10.1-2, and the genotype of the 19 cultivars were two MYB10.1-1 alleles, and the other 25 red skin peach cultivars were two MYB10.1-1 alleles (Fig. 4A). Interestingly, 19 cultivars carried two MYB10.1-1 alleles, which do not accumulate anthocyanin at all, for example, the skin color phenotype (lanes 11, 12, 13, 14, 15, 16) was white, but Ins1 was absent in the PpMYB10.1 promoter. The transposon insertion of 5243 bp explained non-red phenotype of some cultivars, while other phenotypes could not correspond to them, suggesting that the non-red phenotype may be caused by deletion or other variations.
Although the transposon insertion was the key mutation leadings to the color mutation of skin, there was a deletion at 570 nt near ATG on the PpMYB10.1 promoter, which would affect the transcriptional activity of MYB10.1 transcription factor. In the study, we identified the deletion of 61 materials, and amplified bands of 1053 bp or 487 bp (Fig. 4B). The results showed that most of the 25 materials with red skin (lanes 37 to 61) had homozygous or heterozygous deletion mutations, accounting for 88.0%. Among the 36 non-red skin (lanes 1 to 36) materials, 24 had no deletion genotypes (1053 bp/1053 bp), accounting for 66.7%, which may explain the deletion of PpMYB10.1 promoter associated with the red color indexes in skin of these cultivars. Combining transposon insertion and deletion mutation to explain the skin coloring basically suggested that transposon insertion and non-deletion mutation were the key to cause skin non-red coloring.
After determining the relationship between Ins1 and deletion mutations and phenotype, there were some varieties with genotypes inconsistent with the phenotype. It may be caused by other mutations, so the upstream of the MYB10.1 promoter sequence was further amplified to find mutations. The promoter region was extended and amplified to 2700 bp upstream of ATG. Among the various varieties, we found a large fragment insertion of approximately 3500 bp (Ins2) upstream of the MYB10.1 promoter. Detection of 61 various peach cultivars, 14 non-red materials had homozygous insertion, which was consistent with Ins1 insertion, and only two heterozygous materials differed. All other red-colored cultivars were homozygous or heterozygous without insertion (Fig. 4C), which was 96.7% consistent with Ins1. The identification results were generally consistent with of Ins1, indicating Ins2 is a variation on the MYB10.1 promoter similar to Ins1, but may be related to the Ins1 transposon, and cannot explain the special type of inconsistent with Ins1.These results suggest that there are other reasons for the non-red skin trait except transposon Ins1 and Ins2 mutation.
Molecular identification the hybrid populations in the promoter of MYB10.1
The fruit red/non-red trait is an important trait for peach peel, and red skin is one of the most remarkable and distinguishable traits. Genetic populations and gene structures have been analyzed in previous studies, and variations in the MYB10.1 promoter were found to lead to anthocyanin accumulation, but these variants were not really applied in the breeding process(Bretó et al. 2016; Tuan et al. 2015; Beckman et al. 2005). Therefore, to confirm that the variation in this locus is associated with the peach skin red/non-red trait, 92 progenies from three populations were subjected to targeted PCR. The results showed that 22 non-red peach populations contained a homozygous insertion (426bp/426bp), 4 peaches had a small amount of red peel, while red peach populations were heterozygous (609/426bp) or had no insertion (609/426bp) (Fig. 5 and File S2).