SCREENING-UL UNOR SOIURI ROMÂNEȘTI DE MĂR PENTRU GENELE DE REZISTENȚĂ LA RAPĂN MOLECULAR SCREENING OF SOME ROMANIAN APPLE CULTIVARS FOR SCAB RESISTANCE GENES

Apple scab, incided by the fungus Venturia inaequalis (Cke.) Wint., is a devastating disease of apple reported from almost all apple producing Romanian areas, which causes up to 70% losses of production. Molecular markers were used for detection of scab resistance genes in 22 old and introduced apple cultivars ('Romus 3', 'Romus 5', 'Rebra', 'Rustic', 'Nicol', 'Colmar', 'Colonade', registered by Research Institute for Fruit Growing Pitesti; 'Generos', 'Iris', 'Irisem', 'Luca', 'Ciprian', 'Cezar', 'Remar', 'Valery', 'Real', registered by Research Station for Fruit Growing Voinesti, Dambovita; 'Aura', 'Starkprim', 'Ionaprim', 'Bistritean', registered by Research Station for Fruit Growing Bistrita and old cultivars: 'Domnesc', 'Cretesc'). The presence of scab resistance genes were detected using the molecular markers: AL-07 (SCAR), AM19 (SCAR), VfC for Rvi6 (Vf) gene, AD13 (SCAR) for Rvi4 (Vr1) gene, OPL19 (SCAR) for Rvi2 (Vh2) and Rvi8 (Vh8) genes and OPB12 (STS) for Rvi5 (Vm) gene. The Rvi6 gene was detected in 17 cultivars from different breeding center. The marker AD13 presents in genome of 8 cultivars, such as 'Romus 3', 'Romus 5', 'Generos', 'Iris', 'Irisem', 'Cezar', 'Remar', 'Aura'. The Rvi5 gene was revealed in 3 cultivars ('Nicol', 'Generos', 'Irisem'), only. Cuvinte cheie: Malus x domestica, marker, ameliorare.


Introduction
Apples (Malus x domestica Borkh.) are one of the most important horticultural crop grown in temperate areas and most commonly consumed fruits in the world (Ferree and Warrington, 2003; Muneer et al., 2017). Apple is host to a wide range of pests and diseases and most of the commercial cultivars are susceptible. Scab disease, caused by the ascomycete fungus Venturia inaequalis (Cke) Wint. (anamorph: Spilocaea pomi Fries), negatively affects fruit size and quality, due to blemishes and poor ripening. Managing apple scab requires numerous treatments during the growing season and, unfortunately, the pathogen has already developed resistance to many classes of fungicides.
Several resistance genes were identified from wild cultivars or other progenies. An important source of resistance to scab is Vf gene (renamed Rvi6), derived from Malus floribunda 821 (Bus et al., 2009). It has been used extensively as an important source of scab resistance in commercial cultivars (Crosby et al., 1992), but a new scab race, designated race 6, overcomes Vf resistance, although the resistance of M. floribunda 821 itself remains effective (Parisi et al., 1993). It is suggested that M. floribunda 821 contains an additional gene for hypersensitivity (Vfh), which was lost early in breeding efforts (Parisi and Lespinasse, 1996 (Dayton and Williams, 1970).
For durable resistance, new cultivars need to have several resistance genes incorporated. By traditional breeding, different sources of resistance can be introduced into one genotype.
The aim of this study was to screen 22 apple cultivars for the presence of five scab resistance genes employing a polymorphic sequence characterized amplified regions (SCAR) and sequence-tagged sites (STS) markers.

Material and methods
In our experiment, 22 apple cultivars were used for molecular screening. Two of all cultivars, 'Domnesc' and 'Cretesc', are known as old and scab susceptible cultivars (Table 1).
Young leaves from each cultivar were collected in plastic bags direct from an orchard at Genetic and Breeding Department of Research Institute for Fruit Growing Pitești, Romania. Total DNA was extracted from each cultivar using the kit extraction Isolate II Plant DNA (Bioline). Multi-locus genotyping of five apple scab resistance genes (Rvi2, Rvi4, Rvi5, Rvi6, Rvi8) was employed in all the cultivars using five SCAR markers and one STS marker (table 2).
The apple fresh grinded with liquid nitrogen was distributed into a tube of 2mL (100 mg) and kept at -80 0 C. The amplification reaction of DNA was carried out using the MyTaq TM Red Mix in 0.2-mL tubes with 25µL final reaction volume and 18-20 ng of genomic DNA. Amplifications were performed in a Termocycler PCR FastGene Ultra Cycler Gradient. The amplification was performed after the following programs: AL07 and AM19 markers (initial denaturation step at 95 0 C for 1 min., followed by 35 cycles of 1 min at 94 The amplified fragments were resolved in 2% agarose gel in a 10x TBE buffer, stained with midori green advance DNA Stain and visualization to combine Essential-V6WL26MX UV. The bands on the gels were transformed in binary data by being scored as "+" for the presence and "-" for absence of alleles for each cultivar per marker.

Results and discussions
Molecular markers linked to apple scab resistance would accelerate breeding by making it possible to select resistant individuals, regardless of the growth stage, interaction between resistance genes, source of inoculum or environmental conditions (Cheng et al., 1998).
To confirm the presence of Rvi6 (Vf) gene were used three markers: AL07, AM 19 and VfC. Marker AL07 produced two bands of 823 bp represents the recessive vf allele and of 570 bp represents the dominant allele Vf, so, it can be used for identification of homozygous and heterozygous genotypes. For all studied cultivars, primers for this marker were detected in 17 cultivars. It was absent in susceptible cultivars like: 'Nicol', 'Generos', 'Irisem', 'Domnesc' and 'Crețesc' (Figure 1, Table 3).
Primers for AM19 and VfC markers led to amplication of 526 bp ( Figure 2, Table 3

Conclusions
Romania has a large variety of apple cultivars released in different breeding programs for resistance to apple scab. The phenotypic evaluation of scab apple resistance is necessary to be complete with molecular screening using gene specific markers.
An examination of 22 Malus x domestica cultivars, including phenotypic susceptible and resistant cultivars, showed that they contained different PCR products. In our study, the frequencies of the detected resistance alleles were about 59% for Rvi2 and Rvi8, 36% for Rvi4, 13.5% for Rvi5 and 77% for Rvi6.