A New One-Tube Reaction Assay for the Universal Determination of Sweet Cherry (Prunus avium L.) Self-(In)Compatible MGST- and S-Alleles Using Capillary Fragment Analysis

Abstract

The sweet cherry plant (Prunus avium L.) is primarily self-incompatible, with so-called S-alleles responsible for the inability of flowers to be pollinated not only by their own pollen grains but also by pollen from other cherries having the same S-alleles. This characteristic has wide-ranging impacts on commercial growing, harvesting, and breeding. However, mutations in S-alleles as well as changes in the expression of M locus-encoded glutathione-S-transferase (MGST) can lead to complete or partial self-compatibility, simplifying orchard management and reducing possible crop losses. Knowledge of S-alleles is important for growers and breeders, but current determination methods are challenging, requiring several PCR runs. Here we present a system for the identification of multiple S-alleles and MGST promoter variants in one-tube PCR, with subsequent fragment analysis on a capillary genetic analyzer. The assay was shown to unequivocally determine three MGST alleles, 14 self-incompatible S-alleles, and all three known self-compatible S-alleles (S3', S4', S5') in 55 combinations tested, and thus it is especially suitable for routine S-allele diagnostics and molecular marker-assisted breeding for self-compatible sweet cherries. In addition, we identified a previously unknown S-allele in the 'Techlovicka´ genotype (S54) and a new variant of the MGST promoter with an 8-bp deletion in the ´Kronio´ cultivar.

Keywords: M locus-encoded glutathione-S-transferase; MAS; MGST; S-allele; fragment analysis; molecular marker assisted breeding; self-compatibility; self-incompatibility; sweet cherry.

Figure 1

The design of the assay for self-(in)compatibility determination in sweet cherries. The figure is divided into three parts showing: (1) overall S-allele detection, (2) discrimination of SC and SI S-alleles, and (3) MGST allele determination. Drawings based on the ‘Satonishiki’ cultivar reference sequence, BioProject No. PRJDB4877.

Figure 2

PaConsI-F-class primer binding site sequence analysis in detected S-alleles. The region of the primer is in the pink frame. Where used, the GenBank number is shown next to the genotype name; sequences without a reference were obtained in this study. (A) Upper—a consensus sequence of all S-RNases with known sequences, lower—a consensus sequence of all S-RNases with known sequences without the newly identified S54 allele (excluded due to the many mismatches present). The PaConsI-F primer sequence [19] is shown above the consensus. Nucleotides highlighted by yellow in the PaConsI-F primer are mutated in some S-alleles and were not compensated for by a previous degeneration of the primer. (B) S-alleles recognized by individual PaConsI-xxxx-F primers. Nucleotides highlighted in red indicate differences compared to the predominant nucleotide.

Figure 3

A representative output of fragment analysis in the ‘Halka’ cultivar (S1S4′; MGSTwt).

Figure 4

A representative output of fragment analysis for S3S3′ and S1S3 as a prototype of S3Sx genotypes.

Figure 5

A representative output of fragment analysis for S3′S3′ and S3′S4 as a prototype of S3′Sx genotypes.

Figure 6

A representative output of fragment analysis for S4′S4′ and S3S4′ as a prototype of S4′Sy genotypes.

Figure 7

Frequency of individual S-alleles found in the germplasm collection.