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.

Synergistic approach of PCR-based fragment length analysis and amplicon deep sequencing reveals rich diversity of S-alleles in sweet cherries from the Caucasian region of origin

“…The reference set is structured in such a way that each S-haplotype is included at least once (Table 2). Further genotypes can be continuously added to this reference set to fill in missing or newly identified S-alleles, such as the recently published S 54 allele from the variety 'Techlovicka' (Cmejlova et al, 2023).…”

“…Furthermore, the marker PaConsI-F/R2 is not suitable for the amplification from the S 13 allele as no PCR product or eventually a SSR like trace peak is produced, which has been already reported (Sonneveld et al, 2006;Marchese et al, 2010;Lisek et al, 2015) and confirmed in this work. Although the primer binding sites are present in S 13 , a short highly variable tandem repeat, presenting up to 25 (AT) repeats in P. avium and up to 38 in P. cerasus (Marchese et al, 2010) in the amplified region probably inhibits the PCR, as already discussed by Cmejlova et al (2023). For three genotypes of the Caucasian collection, the allele S 13 was assigned on the basis that in these only one PCR product was detected and a diploid, selfincompatible genotype was assumed.…”

“…The use of S-allele-specific primer pairs and other markers within the S-locus is an alternative to overcome the problems mentioned above. Recently, a new one-tube PCR assay with subsequent fragment analysis for S genotyping of P. avium has been developed (Cmejlova et al, 2023), based on multiplex PCR with 27 different primers, 8 of which are labeled with a fluorescent dye. This complex assay contains sequence-optimized primers for the universal amplification of the first intron of the S-RNase gene based on the PaConsI-F/R2 binding sites, a set of several S-allele specific primers, and a marker for the detection of MGST alleles.…”

“…This system has become well-established and has been used extensively for S-allele genotyping of sweet cherry (Vaughan et al, 2008;Ganopoulos et al, 2012;Sharma et al, 2016;Cachi et al, 2017;Marchese et al, 2017;Azizi-Gannouni et al, 2018) and has also be transferred to other Prunus species (Halaśz et al, 2021a, Halaśz et al, 2021bNicolaś-Almansa et al, 2023). It further served as a basis for the further development of the genotyping method, such as the one-tube reaction assay described by Cmejlova et al (2023). Different S-alleles of sweet cherry were numbered consecutively and to date, 23 S-alleles were described in commercial sweet cherry cultivars (Schuster, 2020;Cmejlova et al, 2023).…”

“…It further served as a basis for the further development of the genotyping method, such as the one-tube reaction assay described by Cmejlova et al (2023). Different S-alleles of sweet cherry were numbered consecutively and to date, 23 S-alleles were described in commercial sweet cherry cultivars (Schuster, 2020;Cmejlova et al, 2023). Further S-alleles have been identified in wild sweet cherries accessions (Cachi et al, 2017), which have not yet been found in cultivated cherries.…”