Analysis of strawberry promising varieties and selected forms by resistance to red stele root rot using molecular markers

The analysis of the allelic state of the Rpf1 red stele root rot gene in 14 promising foreign strawberry varieties and 6 selected forms of the I.V. Michurin FSC breeding was completed. The Rpf1 gene in a heterozygous state was identified in strawberry forms 61-15 (Bylinnaya × Olimpiyskaya nadezhda), 69-29 (Feyerverk × Bylinnaya), and 72-71 (Privlekatelnaya × Bylinnaya), which makes it possible to recommend them for involvement in breeding work to create resistant to P. fragariae var. fragariae strawberry varieties. Strawberry varieties Lebedushka, Elianny, Florence, Malwina, Monterey, Polka, Verona, Vima Tarda Asia, Chamora Turusi, Clery, Flamenco, Salsa and Symphony, and selected forms 56-5 (Gigantella Maxim × Privlekatelnaya), 69-42 (Feyerverk × Bylinnaya) and 35-16 (922-67 × Maryshka) have a recessive homozygous genotype.


Introduction
Resistance to fungal pathogens is one of the most important traits of any plant variety. Strawberry is susceptible to many diseases that affect all parts of plants: leaves, roots, flowers, fruits, etc. One of the most dangerous diseases of strawberry is diseases of the root system. Among them is red stele root rot, the causative agent of which is the quarantine phytopathogen Phytophthora fragariae var. fragariae Hickman [1][2][3]. In strawberry plants affected by red stele root rot, the death of lateral feeding and adventitious roots is observed with the formation of pathogen oospores in the affected tissues, inhibition of growth and wilting [4,5].
Strawberry resistance to red stele root rot is due to the presence in the genome of several race-specific genes, of which the Rpf1, Rpf2, and Rpf3 genes are the most important [6,7].
The purpose of this study was the molecular genetic analysis of the allelic state of the Rpf1 gene in promising strawberry varieties and selected forms for identification of resistant to P. fragariae var. fragariae genotypes.

Materials and methods
The studies were carried out in 2020-2021. Biological material was represented by widespread industrial strawberry varieties, introduced from various ecological and geographical regions of growth and promising strawberry hybrid seedlings obtained at the "I.V. Michurin Federal Scientific Center" (Table 1). The allelic state of the Rpf1 gene was identified by DNA analysis using diagnostic markers OPO-16C (RAPD) [8] and R1A (SCAR) [9].
The RAPD marker OPO-16C is represented by amplicons from 400 to 2000 bp. The 438 bp amplicon is linked to the Rpf1 gene (non-functional rpf1 allele). Other products of the OPO-16C marker are amplified regardless of the allelic state of the Rpf1 gene [8,10]. The SCAR marker R1A is represented by a 285 bp amplicon and linked to the functional allele Rpf1 [9]. The use of a combination of markers OPO-16C and R1A makes it possible to identify the allelic state of the Rpf1 gene: the presence of the R1A marker and the absence of the OPO-16C marker indicates a dominant homozygous genotype, the presence of both markers indicates a heterozygous genotype, the presence of the OPO-16C marker and the absence of the R1A marker, and the absence of both markers, indicates a recessive homozygous genotype [11].
Amplification products were separated by electrophoretic method in agarose gel (agarose concentration -2%, running buffer -1x TBE). Amplicon sizes estimated were performed using the Gene Ruler 100 bp DNA Ladder (Thermo Fisher Scientific).

Conclusion
Thus, as a result of the research, we identified the allelic state of the Rpf1 gene in 14 promising strawberry varieties of foreign breeding and 6 strawberry selected forms created in the I.V. Michurin FSC. The Rpf1 gene in a heterozygous state was identified in strawberry hybrid forms 61-15 (Bylinnaya × Olimpiyskaya nadezhda), 69-29 (Feyerverk × Bylinnaya) and 72-71 (Privlekatelnaya × Bylinnaya), which makes it possible to recommend them for involvement in breeding work to create resistant to P. fragariae var. fragariae strawberry varieties.