A 5.2-kb insertion in the coding sequence of PavSCPL, a serine carboxypeptidase-like enhances fruit firmness in Prunus avium

Abstract

Fruit firmness is an important trait in sweet cherry breeding because it directly positively influences fruit transportability, storage and shelf life. However, the underlying genes responsible and the molecular mechanisms that control fruit firmness remain unknown. In this study, we identified a candidate gene, PavSCPL, encoding a serine carboxypeptidase-like protein with natural allelic variation, that controls fruit firmness in sweet cherry using map-based cloning and functionally characterized PavSCPL during sweet cherry fruit softening. Genetic analysis revealed that fruit firmness in the 'Rainier' × 'Summit' F₁ population was controlled by a single dominant gene. Bulked segregant analysis combined with fine mapping narrowed the candidate gene to a 473-kb region (7418778-7 891 914 bp) on chromosome 6 which included 72 genes. The candidate gene PavSCPL, and a null allele harbouring a 5244-bp insertion in the second exon that completely inactivated PavSCPL expression and resulted in the extra-hard-flesh phenotype, were identified by RNA-sequencing analysis and gene cloning. Quantitative RT-PCR analysis revealed that the PavSCPL expression level was increased with fruit softening. Virus-induced gene silencing of PavSCPL enhanced fruit firmness and suppressed the activities of certain pectin-degrading enzymes in the fruit. In addition, we developed functional molecular markers for PavSCPL and the Pavscpl^5.2-k allele that co-segregated with the fruit firmness trait. Overall, this research identified a crucial functional gene for fruit firmness. The results provide insights into the genetic control and molecular mechanism of the fruit firmness trait and present useful molecular markers for molecular-assisted breeding for fruit firmness in sweet cherry.

Keywords: BSA‐seq; PavSCPL; fruit firmness; molecular marker; sweet cherry.

Figure 1

Phenotypic characterization and statistical analysis of sweet cherry fruit firmness in the ‘Rainier’ (hard‐flesh fruit) × ‘Summit’ (hard‐flesh fruit) F₁ population. (a) Fruit phenotypes of the parents ‘Rainier’ × ‘Summit’ and F₁ individuals derived from the ‘Rainier’ × ‘Summit’ cross at dark red (DR) or dark colouring stages (double‐colour fruit). 1, 2 and 3 represent three different individuals of three fruit firmness types (extra‐hard‐flesh, hard‐flesh and soft‐flesh fruits, respectively) in the F₁ population. (b) Fruit firmness of two parents (‘Rainier’ and ‘Summit’) assessed at yellow‐white (YW), full red (FR) and dark red (DR) stages. (c) Fruit firmness of three extra‐hard‐flesh fruit individuals (EH 1, EH 2 and EH 3), three hard‐flesh fruit individuals (H 1, H 2 and H 3) and three soft‐flesh fruit individuals (S 1, S 2 and S 3) from F₁ population assessed at YW, FR and DR stages. Data are mean ± SD of three independent experiments. (d) Frequency distribution of fruit firmness in the F₁ population exhibiting a continuous and bimodal distribution with a distinct separation of the two groups of individuals in 2016–2019.

Figure 2

Identification of the candidate region for fruit firmness by BSA‐seq analysis and fine‐mapping in the ‘Rainier’ × ‘Summit’ F₁ population. (a) Initial mapping of the candidate region using BSA‐seq by calculation of the Δ(SNP index) value. (b) Fine‐mapping of the fruit firmness locus. The recombinant individuals were screened using the 15 SNP and InDel markers listed above the figure. The F₁ individuals are listed to the left of the figure, and the fruit firmness phenotype is specified to the right of the figure. Yellow and grey shading represent chromosome fragments corresponding to the extra‐hard‐flesh fruit and soft‐flesh fruit phenotypes, respectively. The candidate region was delimited to a 473‐kb region on chromosome 6 between the markers SNP‐7.418778 and SNP‐7.891914. Mb, million bases.

Figure 3

Identification and validation of the candidate gene controlling fruit firmness. (a) Expression analysis of 72 genes located in the 473‐kb genomic DNA region in the interval between SNP‐7.418778 and SNP‐7.891914 (upper element in b), based on the Prunus avium reference genome, in the Hard‐pool and Soft‐pool groups determined by RNA‐sequencing and quantitative RT‐PCR analyses, respectively. (b) Gene structure of PavSCPL and its allele. The exon–intron structure of PavSCPL and its allele in the extra‐hard‐flesh fruit and soft‐flesh fruit phenotypic groups is shown in the middle element. Green boxes represent exons and a black line between exons indicates an intron. ‘F1/R1’ and ‘F2/R2’ indicate the developed AFLP markers PavSCPL‐1‐F/R and PavSCPL‐2‐F/R based on the two PavSCPL alleles. The structure and sequence of the 5.2‐kb transposable element insertion are listed in Table S5.

Figure 4

Co‐segregation analysis for genotyping of the PavSCPL alleles with fruit firmness using the AFLP marker PavSCPL‐1‐F/R in the ‘Rainier’ × ‘Summit’ F₁ population and 118 sweet cherry accessions. (a) PCR analysis of three fruit firmness phenotypes (extra‐hard‐flesh, hard‐flesh and soft‐flesh fruits) in the F₁ population corresponding to three genotypes: Homozygous insertion genotype, AA (Pavscpl ^5.2‐kb /Pavscpl ^5.2‐kb , only a 5477‐bp long fragment); heterozygous insertion genotype, Aa (Pavscpl ^5.2‐kb /PavSCPL, two fragments, a 5477‐bp long fragment and a 233‐bp short fragment); and no‐insertion genotype, aa (PavSCPL/PavSCPL, only a 233‐bp short fragment) based on PavSCPL and Pavscpl ^5.2‐k alleles using the AFLP marker PavSCPL‐1‐F/R. (b) PavSCPL genotype analysis in sweet cherry 118 accessions by PCR using the AFLP marker PavSCPL‐1‐F/R. Number corresponding to each individual sweet cherry accession is listed in Table S7.

Figure 5

Characterization of the PavSCPL gene. (a) The fruit firmness of ‘Chunlu’, ‘Summit’ and ‘Ruby’ during different fruit developmental stages (initial degreening, yellow‐white, initial red, full red and dark red). Data are the mean ± SD from three biological replicates. (b) Expression patterns of PavSCPL in the leaf, flower and fruit at different developmental stages of three sweet cherry accessions differing in fruit firmness phenotype: ‘Chunlu’ (soft‐flesh fruit), ‘Summit’ (hard‐flesh fruit) and ‘Ruby’ (extra‐hard‐flesh fruit). Data are the mean ± SD from three independent replicates. (c) Quantitative real‐time PCR analysis of PavSCPL expression in F₁ individuals with soft‐flesh fruit or extra‐hard‐flesh fruit at the mature fruit stage. Pavactin (Pav_sc0002247.1_g030.1.mk) was used as the internal control. Data are the mean ± SD of three independent experiments.

Figure 6

Virus‐induced silencing of PavSCPL improved sweet cherry fruit firmness and suppressed pectin degradation. (a) PavSCPL transcript abundance relative to that of Pavactin in PavSCPL‐silenced fruit and control fruit at 15 days post‐infiltration (dpi), as detected by qRT‐PCR. (b) Phenotypes of PavSCPL‐silenced fruit and control fruit at 21 and 28 dpi. The red arrow indicates the location of vector injection. (c) Fruit firmness of PavSCPL‐silenced and control fruit at 21 and 28 dpi. (d) Change in activities of the cell wall modification enzymes pectin methyl esterase (PME), polygalacturonase (PG), β‐galactosidase (β‐GAL), pectate lyase (PL) and α‐L arabinofuranosidase (α‐AF) in PavSCPL‐silenced fruit at 21 and 28 dpi. (e) Expression analysis of the cell wall metabolism‐related genes PavPME (Pav_sc0000130.1_g870.1.mk), PavPG (Pav_sc0000254.1_g1300.1.mk), PavPGI (Pav_sc0005028.1_g040.1.mk), PavXTH14 (Pav_sc0000480.1_g990.1.mk), PavEXPA2 (Pav_co4016743.1_g010.1.br) and PavPL18 (Pav_sc0000229.1_g150.1.mk) in PavSCPL‐silenced fruit at 21 and 28 dpi. Data are the mean ± SD of six biological replicates. Statistical significance was determined using Student's t‐test. ** P‐value <0.01.