Mining candidate genes of single fruit mass in peach by BSA-seq and RNA-seq analysis

【Objective】Single fruit mass is a critical target in crop production and the domestication of horticultural species. This study aimed to identify candidate genes influencing peach (Prunus persica L.) single fruit mass, providing valuable insights for breeding peach varieties with diverse single fruit mass characteristics.【Methods】Initially, a total of, 31 small fruit-type peach plants and 26 large fruittype peach plants from the F1 hybrid population were utilized to establish separate DNA libraries for small and large fruit types respectively. The BSA- seq (Bulk Segregant Analysis sequencing) involved two parental pools (Zhong You Tao 13 and Zheng 2007-4-28) and two extreme trait mixed pools representing small and large fruit types. Fresh leaves from each sample were collected, and genomic DNA was extracted using the CTAB method. The quality of the DNA was assessed by Shenzhen Huada Gene, followed by sequencing. SNP association analysis was conducted using the Delta SNP-index, the Euclidean Distance (ED) method, and G' values, which facilitated the identification of candidate genomic segments associated with the target traits through the integration of various analytical results. Subse-quently, mesocarp tissues from single plants of both large and small fruit types during the peach fruit development period were selected for transcriptome sequencing (RNA- seq) to identify candidate genes. To validate the expression profile of the Prupe.6G029300 gene, quantitative real-time PCR (qRT-PCR) experiments were performed using four different peach varieties. Finally, an expression vector for the Prupe.6G029300 gene was constructed, and its function was observed by heterologous transformation in tomato.【Results】The phenotypic data of 95 individual plants within the F1 population were subjected to analysis, revealing an average single fruit mass of 118.27 g, with observed maximum and minimum values of 180 g and 60 g, respectively. To identify genes associated with peach single fruit mass, DNA samples from the parental lines and two of their progeny were sequenced. The sequencing yielded a total of 44.38 Gb of raw data, which was subsequently filtered to 37.56 Gb, achieving quality scores of Q20 ≥ 97.45% and Q30 ≥ 93.02%, indicative of high sequencing quality. The GC content ranged from 39.95% to 44%. Employing the delta SNP-index, Euclidean distance algorithm (ED), and G' value for bulked segregant analysis sequencing (BSA- seq), the locus associated with single fruit mass was mapped to chromosome 6, spanning positions 2 191 165 to 3 794 466, encompassing a total of 1.6 Mb, and 261 genes. Additionally, 36 samples were collected from the mesocarp of both large and small fruit types at 30, 45, 60, 75, 90 and 105 days post- anthesis, with three replicates for each of the six time points. The transcriptomic sequencing of these samples resulted in 373.71 Gb of raw sequencing data, from which 364.06 Gb of clean data was obtained post- processing. Each sample yielded 9.41 Gb of clean data, with a GC content exceeding 44.99%. The range of clean reads aligned to the peach reference genome (LoveII) was between 93.96% and 98.16%. An analysis was conducted on 261 genes identified in this region, excluding those with no or low expression levels (FPKM＜2 across all stages in both large and small fruit types). This filtering resulted in 189 genes being retained for further analysis. The focus was primarily on genes exhibiting high expression during the early stages of fruit development and low expression in later stages. By comparing gene expression patterns between large and small fruit types, 38 genes were selected for subsequent analysis. The positions and functions of these 38 candidate genes were summarized, revealing that 7 lacked functional annotations. Among the remaining 31 genes, seven were potentially implicated in the development of single fruit mass traits in peaches. Notably, the gene Prupe.6G029300 exhibited four significant mutations (P = 5.60E-05) within its coding sequence (CDS) region. Using the large fruit type as a reference, the small fruit type displayed a T-to-C substitution at Chr.6: 2 273 054 bp, a C-to-T substitution at Chr.6: 2 273 696 bp, a G-to-T substitution at Chr.6 2 276 865 bp, and a C- to- T substitution at Chr.6 2 278 498 bp. The gene Prupe.6G029300 was validated in various peach varieties exhibiting distinct maturity and single fruit mass phenotypes. These varieties consistently demonstrated high expression levels of the gene during the early stages of fruit development, followed by reduced expression in the later stages. Stable transformation experiments revealed that overexpression of Prupe.6G029300 in tomato plants resulted in smaller fruit size compared with the wild type.【Conclusion】The locus associated with peach single fruit mass was located on chromosome 6 spanning 191 165-3 794 466 bp. The Prupe.6G029300 gene exhibited a pattern of elevated expression in the early stages and diminished expression in the later stages of peach fruit development. Overexpression of Prupe.6G029300 in tomato led to a reduction in single fruit mass, suggesting its involvement in the regulation of peach fruit development.