This study aims to explore the molecular mechanisms underlying the formation of Non-red ground color in Xinjiang local peach (Prunus ferganensis and P. persica), providing a theoretical basis for the breeding of new peach varieties with Non-red skin color.

A phenotypic survey of the skin color of 85 progenies of Xinjiang peach and three cultivated varieties was conducted. The activity of various lengths of the PpMYB10.1 promoter was assessed using β-glucuronidase (GUS) staining. Predictive analysis of the cis-acting elements of the PpMYB10.1 promoter from different materials was performed using the PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Genotypes with a 5 243 base pairs (bp) transposon insertion and a 483 bp deletion in the PpMYB10.1 promoter of 88 materials were identified and cloned using polymerase chain reaction (PCR). The transcriptional activation of PpMYB10.1 by Pp.4G186800 was validated through dual-luciferase assays.

Based on skin color, 85 Xinjiang peach progenies were classified into Non-red and red types. The majority exhibited Non-red colors (green or light yellow) with minimal or no anthocyanin accumulation. Compared to the fully red variety Zhongtao Jinmi, these exhibited significantly lower levels of anthocyanins and reduced expression of PpMYB10.1. Using GUS staining to assess the effects of promoter mutations on activity, the results showed that a 5 243 bp insertion in the PpMYB10.1 promoter caused the fruit discs to display a lighter blue, indicating decreased activity. Conversely, a 483 bp deletion resulted in a darker blue, suggesting increased promoter activity. No such insertions or deletions were found in the PpMYB10.1 promoter of the Xinjiang peach progenies. The promoter sequences of PpMYB10.1 were cloned, and cis-acting elements were categorized into four groups: light-responsive, plant hormone-responsive, abiotic stress and growth development-responsive, and elements of unknown function. Due to the late maturation of the Xinjiang peach progenies, all were found to have a 0 bp/0 bp genotype for the maturity-regulating gene Pp.4G186800. Dual-luciferase assays showed that Pp.4G186800 could bind to the PpMYB10.1 promoter. Notably, when the promoter had a 483 bp deletion, Pp.4G186800 (+9 bp) formed a strong interaction, potentially enhancing early maturation and anthocyanin accumulation in the peach skin. However, in late-ripening peaches, Pp.4G186800 (-9 bp) had no enhancing effect on the promoter's activity; If the PpMYB10.1 promoter lacked the 483 bp deletion, its intrinsic activity was weak, even though Pp.4G186800 could still interact with it, the effect was minimal.

The presence of insertions and deletions in the PpMYB10.1 promoter is not directly related to the Non-red skin color of Xinjiang peach. In early and mid-ripening varieties, Pp.4G186800 can activate the transcription of PpMYB10.1, facilitating anthocyanin synthesis; however, its influence is reduced or absent in late-ripening peaches.

Fruit weight and soluble solid content (SSC) are two important quantitative traits in peach which are of importance to breeders. However, performing early prediction using a single marker is challenging as the traits are controlled by multiple minor genes. Genomic selection, a novel genome-wide tool, has been applied in fruit crops and can potentially enhance the breeding efficiency of these quantitative traits. However, its effects in peach and influencing factors require further investigation.

Establish a whole-genome selection technology system for peach single fruit weight and SSC, and laid a methodological foundation for the establishment of efficient molecular breeding technology system for peach.

The objectives of this study were to assess the accuracy of prediction of peach fruit weight and SSC in natural and hybrid populations using genomic selection. Here, a training population of 520 individuals was selected. Using genotypic data for 48 398 single nucleotide polymorphisms (SNPs) obtained from the resequencing results of the above training population, a total of 11 genome-wide prediction models were built to select the optimum model for fruit weight and SSC. Subsequently, the genomic breeding values of a small natural population of 56 individuals and 29 hybrid populations comprising a total of 1 145 seedlings were calculated.

The average sequencing data of each variety of the three groups was 1.95-3.52 Gb, and the sequencing depth was 5.29-10.79×. The sequencing data of the training natural population was aligned with the reference genome, and a total of 5 065 726 SNPs were obtained. After removing the SNPs with a high missing rate (>20%) and minor allele frequency of <0.05, a total of 48 398 SNPs on the genome were randomly selected for constructing whole-genome selection models for the training population. The models with the highest prediction accuracy for fruit weight and SSC were BayesA and randomforest, respectively. Using the above two models, it was found that the goodness of fit between the predicted breeding values and observed phenotype of fruit weight was 0.4767-0.6141, which was higher than that of SSC (0.3220-0.4329) in the natural populations. In hybrid populations, the prediction accuracy of fruit weight was 0.2319-0.4870, which was also higher than that of SSC (0.0200-0.2793). The results also showed that the prediction model constructed by training natural populations was more accurate in predicting natural populations than hybrid populations. Taking fruit weight as an example, it was also found that only 17.78% of the seedlings needed to be retained by genomic selection when targeting large fruit. Genomic selection was significantly more efficient than single and double marker selection. Furthermore, the effects of population dispersion, heritability and population structure on prediction accuracy are also discussed. The results indicated that prediction accuracy may vary and be affected by a combination of several factors.

In this study, a suitable genomic selection model for peach fruit weight and SSC was screened, and it was confirmed that the prediction efficiency of genomic selection was significantly higher than that of single marker selection. The results indicated the potential of genomic prediction in accelerating breeding progress of these two quantitative traits in peach.

The molecular markers and bioinformatics were used to identify the genotypes and combinations of the F-M locus, transposon insertion in YUC11 promoter region, and 9 bp insert in NAC72 coding region by 189 peach germplasms, in order to provide the theoretical foundation for the mechanism of peach flesh formation and the selection of breeding parents.

PCR amplification, KASP, and HRM were used to identify genotypes. Results of loci associated with the formation of flesh trait were further validated using bioinformatics by resequencing data in 189 peach germplasms.

Two genes encoding endopolygalacturonase in F-M locus, designated PGM and PGF, are associated with peach texture. Through primer amplification, 159 (84%) PGM and 99 (52%) PGF were detected in 189 peach germplasms. Four haplotypes (H0, H1, H2, and H3) were found in F-M locus, while H0 and H1 were the major genotypes. The haplotype combination of non-melting flesh, melting flesh, free-stone melting flesh were H3H3 and H2H3, H0H0 and H0H1, H1H1 and H0H1, respectively. In addition, it was found that 18 germplasms contain homozygous transposon insertion. HRM results demonstrated that 45 germplasms were homozygous insertion (early ripening), 71 germplasms were heterozygous insertion (middle ripening), and 73 germplasms were no insertion (late ripening). But six germplasm genotypes were inconsistent with the phenotypes. The most frequent genotypic combinations at the three loci of non-melting flesh, melting flesh, and free-stone melting flesh were mmffHdHdI, MMffHdHdI, MMFFHdHdL, respectively.

The existence of four haplotypes at the F-M locus further was confirmed, 18 germplasms containing stony hard genotype were identified, a molecular marker for the identification of maturity date was developed, and genotype combinations of different germplasms at flesh-related loci were identified.