Marker controlled screening of resistant to red stele root rot ( Rpf1 gene) strawberry selected forms

. The results of the analysis of allelic polymorphism of the Rpf1 red stele root rot resistance gene in strawberry hybrid forms using diagnostic DNA markers were shown. The recessive homozygous genotype ( rpf1rpf1 ) was identified in strawberry seedlings 932-29 ( F. virginiana subsp. platypetala (Rydb.) Staudt × Feyerverk), 933-4 ( F. virginiana subsp. platypetala (Rydb.) Staudt × Rubinovyy kulon), 62- 7, 62-23 (Bylinnaya × Feyerverk), 65-2, 65-15 (Olimpiyskaya nadezhda × Bylinnaya) and 35-16 (922-67 × Maryshka). The heterozygous state of the Rpf1 gene was identified in strawberry forms 69-29 (Feyerverk × Bylinnaya), 62-41 (Bylinnaya × Feyerverk), 72-24, 72-71 (Privlekatelnaya × Bylinnaya), 65-17, 65-24 (Olimpiyskaya nadezhda × Bylinnaya), which allows them to be used in breeding for resistance to P. fragariae var. fragariae as valuable initial forms. The obtained data indicate the heterozygous state of the Rpf1 gene in strawberry forms 62-41 (Bylinnaya × Feyerverk), 72-24, 72-71 (Privlekatelnaya × Bylinnaya), 65-17, 65-24 (Olimpiyskaya nadezhda × Bylinnaya) and 69-29 (Feyerverk × Bylinnaya) (markers OPO-16C and SCAR-R1A are present in the genotype). Strawberry seedlings 932-29 ( F. virginiana subsp. platypetala (Rydb.) Staudt × Feyerverk), 933-4 ( F. virginiana subsp. platypetala (Rydb.) Staudt × Rubinovyy kulon) and 62-7 (Bylinnaya × Feyerverk) are characterized by the recessive homozygous state of the Rpf1 (only marker OPO-16C is present in the genotype). Strawberry seedlings 62-23 (Bylinnaya × Feyerverk), 65-2, 65-15 Olimpiyskaya nadezhda × Bylinnaya) and 35-16 (922-67 × Maryshka) also are characterized by the recessive homozygous state of the Rpf1 (both markers are absent in the genotype).


Introduction
Phytophthora fragariae var. fragariae Hickman is the causative agent of red stele root rot, one of the most dangerous strawberry fungal diseases [1,2]. According to the list of the European and Mediterranean Plant Protection Organization (EPPO), P. fragariae var. fragariae has been A2 quarantine pathogen status [3].
Symptoms of red stele root rot of strawberry plants include reddening of the axial cylinder and death of root tips, inhibition of growth, wilting and death of plants [4,5].
Resistance red stele root rot in strawberry varieties and forms is due to the presence of race-specific oligogens in the genome [6], of which the Rpf1, Rpf2, and Rpf3 genes make the greatest contribution to the formation of resistance [7].
Identification of genetic determinants of strawberry resistance to P. fragariae var. fragariae allows for targeted screening of resistant genotypes using diagnostic DNA markers [8,9].
The purpose of this study was the analyze allelic polymorphism of the Rpf1 red stele root rot resistance gene in strawberry hybrid seedlings for identify and involve valuable forms in the breeding programs to create resistant to fungal pathogens strawberry genotypes.

Materials and methods
The studies were carried out in 2020-2021. Biological material was represented by promising strawberry hybrid seedlings obtained at the "I.V. Michurin Federal Scientific Center".
Total genomic DNA of strawberry genotypes was extracted using the DArT method with modifications [10,11].
The SCAR-R1A marker (285 bp amplicon) corresponds to the Rpf1 resistance allele, and the target fragment is synthesized only if Rpf1 allele is present in the strawberry genome. The OPO-16C marker (438 bp amplicon) corresponds to the non-functional rpf1 allele. The combination of these markers makes it possible to identify the allelic state of the Rpf1 gene in the studied strawberry genotypes [13].
The amplification was performed in T100 Thermal Cycler (BioRad) according to the programs: -OPO-16C marker: 5 min denaturation at 94°C followed by 36 cycles of 30 s at 94°C, 45 s at 34°C and 60 s at 72°C, followed by a final extension step of 5 min at 72°C; -SCAR-R1A marker: 3 min denaturation at 94°C followed by 25 cycles of 30 s at 94°C, 45 s at 60°C and 60 s at 72°C, followed by a final extension step of 7 min at 72°C. 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).

Results and discussion
According to the research, the OPO-16C marker is identified in strawberry hybrid forms 932-29 (F.  Table).
In addition to the target fragment 285 bp, all studied strawberry genotypes also contain additional fragments of the OPO-16C marker. Amplification of additional fragments of the OPO-16C marker is also confirmed by other researchers. These fragments are synthesized in any strawberry forms and are not diagnostic signs of the allelic state of the Rpf1 gene [13,14].  Table.).   [8]. The identification of homozygous for the Rpf1 gene strawberry forms is of great practical importance, making it possible to obtain up to 100% resistant seedlings when used in hybridization. The use of homozygous forms in crossing will theoretically avoid the need for phenotypic or genotypic analysis of hybrid seedlings for a selective trait, which will reducing the analysis time, labor and financial costs. [15].
Analysis of the origin of the studied strawberry hybrid seedlings confirmed the data of molecular genetic testing. Strawberry seedlings 69-29 (Feyerverk × Bylinnaya), 62-41 (Bylinnaya × Feyerverk), 72-24, 72-71 (Privlekatelnaya × Bylinnaya), 65-17, 65-24 (Olimpiyskaya nadezhda × Bylinnaya), characterized by the presence of both markers in the genome and, therefore, the heterozygous state of the Rpf1 gene, were isolated in crossing combinations, where the donor of the functional Rpf1 allele is the Bylinnaya variety, and other parental genotypes (Olimpiyskaya nadezhda, Privlekatelnaya and Feyerverk) do not have the functional Rpf1 allele [16]. In this regard, strawberry hybrids with the identified Rpf1 gene can only have a heterozygous genotype (Rpf1rpf1). In the future, the selected hybrid seedlings will be crossed to obtain strawberry forms homozygous for the Rpf1 gene.