Analysis of strawberry genetic collection ( Fragaria L . ) for Rca 2 and Rpf 1 genes with molecular markers

Strawberry (Fragaria × ananassa Duch.) varieties are susceptible to many fungal diseases. Identification of forms, carrying resistance genes, is an important stage in breeding programs leading to resistant varieties. The use of molecular markers allows to determine with high reliability the presence of the necessary genes in the genome and to identify promising forms. Some of the common strawberry’s diseases, causing significant damage to strawberry plantations, are anthracnose (Colletotrichum acutatum Simmonds) and red stele root rot (Phytophthora fragariae var. fragariae Hickman). Dominant Rca2 gene is involved in monogenic resis­ tance to C. acutatum pathogenicity group 2. Rpf1, Rpf2, Rpf3 genes are determined in monogenic resistance to red stele root rot. The purpose of this study was mole­ cular genetic testing genotypes of genus Fragaria L. to identify carriers of Rca2 allele anthracnose resistance and Rpf1 allele red stele root rot resistance. The objects of study were the wild species of the genus Fragaria L. and strawberry varieties (Fragaria × ananassa Duch.) of different ecological and geographic origin. To assess allelic state Rca2 anthracnose resistance gene the dominant SCAR marker STS­Rca2_240 was used, was linked to the resistance gene Rca2 with a genetic dis­ tance of 2.8 cM. Rpf1 gene red stele root rot resistance was identified with the dominant SCAR marker R1A, was linked to the resistance gene Rpf1 with a genetic distance of 3.0 cM. The resistant allele of the marker STS­Rca2_240 was identified in the Laetitia variety (Rca2Rca2 or Rca2rca2 genotype), which allows us to recommend it as a promising source in breeding for anthracnose resistance. The other studied forms have homozygous recessive state of the marker STS­ Rca2_240 (putative genotype rca2rca2). The resistant allele of the marker SCAR­R1A in the varieties and wild species of strawberry under study is absent, which presumably indicates their homozygous recessive genotype of Rpf1 gene (rpf1rpf1).

Plant gene pool and breeding Vavilov Journal of Genetics and Breeding • 2018 • 22 • 7 S trawberry (Fragaria × ananassa Duch.) is the most popular and economically important berry crop (Hummer, Hancock, 2009).The commercial plantations of strawberry are cultivated in 75 countries and it account for over 2/3 of the world berries production (FAOStat, 2018).At the same time, the strawberry is susceptible to many diseases and the mass development of them can lead to the 100 % death of the crop (Folta, Davis, 2006).
Anthracnose (Colletotrichum acutatum Simmonds) and red stele root rot (Phytophthora fragariae var.fragariae Hickman) are among the most harmful strawberry diseases causing significant damage to strawberry plantations in Europe and America (Smith, 2008;Newton et al., 2010).The most of commercial strawberry varieties of foreign breeding are susceptible to anthracnose and red stele root rot (Van de Weg, 1997a;DenoyesRothan et al., 2005).For the last years the pathogens of anthracnose and red stele root rot being tested in strawberry plantings of Russia and besides Phytophthora fragariae var.fragariae is included in the list of Quarantine pests, weeds and plant diseases in Russia (Aleksandrov et al., 2007;Golovin, 2014;Dudchenko et al., 2015).
Anthracnose affects the aboveground part of strawberry: fruits, flowers, petioles, leaves, stolons, causing a significant suppression of the plant, and sometimes there can be the death of plant.Pathogen can be located on a plant for a long time in latent state, this fact significantly complicates the reliable identification and protective measures (Leandro et al., 2001).The losses of marketable yield of strawberries because of anthracnose may reach 80 % (Dudchenko et al., 2015).
P. fragariae var.fragariae affect the root system, inhibiting the growth, causing withering and afterwards death of the plants.It is difficult to diagnose the pathogen visually because of similarity of symptoms of infection and stress factors (de los Santos et al., 2004;Aleksandrov et al., 2007).The occurrence of pathogen can be observed mainly after using of infected planting material, and the oospores can persist in soil in the absence of host plant according to the different data from 3 to 17 years (Duncan, Cowan, 1980ж Szkuta, 2006).
In connection with it, the development of new highly resistant to pathogens strawberry varieties is an important breeding task.The cultivation of varieties with genetic resistance to diseases will increase the profitability of plantations and as well as positively effect the ecological state of strawberry agrocenoses due to reduce to pesticide load (Korbin, 2011).
To improve the strawberry assortment it is very important: to deepen the genetic research, to reveal the patterns of loci inheriting economically important trait and to identify the donors.One of the promising trends to raise the efficiency of strawberry genetic studies became the use of contemporary techniques of molecular genetic analysis of genome applying DNA markers.At present time the molecular markers are wide applied in the analysis of genetic diversity, mapping, and strawberry variety genotype identification.But molecular markers are less involved in strawberry breeding (Whitaker, 2011).
The purpose of current study was to carry out molecular genetic testing of genus Fragaria L. genotypes.It was necessary to identify carrier of Rca2 anthracnose resistance gene and Rpf1 red stele root rot resistance gene.
To assess allelic state Rca2 anthracnose resistance gene was used the dominant SCAR marker STSRca2_240.The SCAR marker STSRca2_240 was multiplexed with the microsatellite marker EMFv020 used as the positive PCR control (LerceteauKohler et al., 2005).
The Rpf1 red stele root rot resistance gene was identified with the dominant marker SCARR1A (Haymes et al., 2000).
Primer sequences and product length are reported in Table 1.Used in this study primers were synthesized by Syntol (Russia).
Total genomic DNA was extracted from the fresh leaves using the Diversity Arrays Technology P/L (DArT) protocol.However, the use of the basic protocol did not allow obtaining DNA extract with the degree of purity necessary for PCR analysis because despite of the high concentration of DNA in solution, inhibition of test PCR was observed.To reduce the content of inhibitory impurities in the DNA solution, an additional double purification of DNA with 5M NaCl with reprecipitation with 70 % ethanol included the following steps was used.100 μl of 5M NaCl and 400 μl of 70 % ethanol were added to the DNA solution; mix centrifuged at 13,000 rpm for 5 min; the supernatant was removed; the precipitate was washed twice with 80 % ethanol, dried at room temperature and dissolved in deionized water.As a result of these steps, DNA extract with necessary of concentration and purity for PCR reaction was obtained.
The amplification was performed in T100 Thermal Cycler (BioRad).PCR conditions for the multiplex PCR reaction (STSRca2_240 and EMFv020) were as described by LerceteauKohler et al. (2005) as follow: 3 min denaturation at 95 °C, 35 cycles of 50 s at 95 °C, 50 s at 64 °C, and 1 min at 72 °C, and a final extension step of 5 min at 72 °C.
PCR conditions for the SCARR1A marker were as described by Haymes et al. (2000) as follow: 3 min denaturation at 94 °C, 25 cycles of 30 s at 94 °C, 45 s at 60 °C, and 1 min at 72 °C, and a final extension step of 7 min at 72 °C.
The amplification products were separated on a 2 % agarose gel and visualized by ethidium bromide staining.GeneRuler 100 bp DNA Ladder (Thermo Fisher Scientific) was used as a molecular weight marker.

results and discussion
Fragaria × ananassa Duch. is difficult object for molecular genetic analysis.The high ploidy level (8x), the combination of different basic genomes in the genotype, complex gene interactions, and polygenic control of traits make it difficult to study the genetics of strawberry.Disease resistance in strawberry in most cases is also controlled quantitatively.
Currently, however, monogenic resistance factors have been identified for some pathogens (C.acutatum, P. fragariae var.fragariae) (Van de Weg, 1997a, b;LerceteauKohler et al., 2002) and this allows effective screening of resistant genotypes using molecular markers.
The SCAR marker STSRca2_240 was linked to the resistance gene Rca2 with genetic distance of 2.8 cM.The PCR product associated with the resistance Rca2 allele has 240 bp.The linked inheritance of the marker alleles and the Rca2 gene allows, based on the presence or absence of the marker alleles, to identify the allelic state of the Rca2 gene (LerceteauKohler et al., 2005).
In studied collection of strawberry, the resistant allele of the marker STSRca2_240 was identified in the Laetitia variety (Rca2Rca2 or Rca2rca2 genotype).The other studied forms have homozygous recessive state of the marker STSRca2_240 linked to Rca2 resistance gene (putative genotype rca2rca2) (Fig. 1, Table 2).
The absence of a dominant allele of the STSRca2_240 marker in the anthracnose resistant variety Vicoda can be presumably due to two factors: the recombination that occurred in the variety (or its parental forms) between the Rca2 resistance gene and the marker STSRca2_240 (Lerceteau Kohler et al. (2005) noted similar recombination for the US292 genotype and its parent form Arking.) or the presence of other genetic resistance factors.
The recessive state SCAR marker STSRca2_240 in species F. virginiana ssp.platipetala and strawberry variety Elsanta confirmed in literature data (Njuguna, 2010;Sturzeanu et al., 2016).It should be noted that, according to W. Njuguna (2010), the dominant allele of the STSRca2_240 marker linked to the Rca2 gene is present in the Red Gauntlet variety, while, according to our research, in this variety the Rca2 gene is presumably represented by the recessive allele.Such a discrepancy in the results may be presumably due to an identification error and requires additional research.However, according to the results of artificial infection, the Red Gauntlet variety is susceptible to C. acutatum (Simpson et al., 1994), which indirectly confirms our data.
Monogenic resistance to red stele root rot of strawberry determined several genes.W.E. Van de Weg analyzing the interaction model of strawberry varieties and races of P. fragariae var.fragariae identified 5 resistance factors (R1-R5), corresponding to specific pathogen avirulence gene (Avr1-Avr5) (Van de Weg, 1997b).The main role in the formation of strawberry red stele root rot resistance belongs to three genes -Rpf1, Rpf2, Rpf3 (Whitaker, 2011).Rpf1 gene ensuring resistance of strawberry genotypes to 16 races of P. fragariae var.fragariae (Sasnauskas et al., 2007).The marker SCARR1A was linked to the resistance gene Rpf1 with genetic distance of 3.0 cM.The PCR product associated with the resistance Rpf1 allele has 285 bp.In genotypes with a homozygous recessive state of the gene Rpf1 (rpf1rpf1), this product is not amplified (Haymes et al., 2000).The molecular analyzes performed with SCARR1A marker showed the absent PCR product of 285 bp in studied strawberry varieties (Fig. 2), which presumably indicates their homozygous recessive genotype of Rpf1 gene -rpf1rpf1 (see Table 2).
The recessive state SCARR1A marker in the Elsanta and Kent varieties is confirmed in literature data (Haymes et al., 2000;Sturzeanu et al., 2016).According to the data of W. Njuguna (2010), the populations of F. virginiana ssp.platipetala of various growing sites also have not the dominant allele SCARR1 marker linked to the Rpf1 gene.

table 2 .
Presence (1)or absence (0) PCR products of the indicated markers, linked to с Rca2 anthracnose resistance gene and Rpf1 red stele root rot resistance gene in different strawberry varieties