DNA-MARKERS OF SUNFLOWER RESISTANCE TO THE DOWNY MILDEW (PLASMOPARA HALSTEDII)

One of the areas of biotechnology sunflower is the development and testing of DNA markers of important agronomic traits and in particular markers of resis tance to downy mildew. Resistance of 16 Rf-lines of sunflower to the races 330 and 710 of Plasmopara halstedii has been studied. Genotyping of these lines was carried out using 9 STS-markers of three Pl-loci, Pl5, Pl6 and Pl8, associated with the resistance of sunflower to downy mildew. Only two out of nine STS-markers, НаР2 and НаР3 (locus Pl6), allowed us to identify the lines, which demonstrated resistance to the downy m ildew under the conditions of artificial infection.


INTRODUCTION
Downy mildew of sunflower is induced by the fungus Plasmopara halstedii (Farl.) Berl and de Toni. It is known to be one of the most harmful diseases of cultural sunflower. The level of crop lesion induced by this fungus may reach 70% (Sakr, 2014). Therefore, breeding of sunflower aimed at the resistance to downy mildew is considered to be one of the most priority tasks.
One of the main areas of modern biotechnology crops is to create disease resistant varieties and hybrids of a new generation using of post-genomic technologies or marker-assisted selection Kolosov et al., 2013;Rao et al., 2014). Breeding of crops resistance to diseases and pests is a laborious process, which deals with the creation of infectious backgrounds and vegetation studies under laboratory and field conditions (Karagodina et al., 2014;Klimenko et al., 2014;Stuthman et al., 2007). Use of specific molecular markers is considered to be promising for assessment of the selection material at early stages of plant development . This may decrease the amount of field works (Bouzidi et al., 2002;Behrouzi et al., 2012).
Seven races of the pathogen were identified in several region of Northern Caucasus using the standard set of sunflower line differentiators. It was found out that in majority of regions dominating position belonged to the race 330. In some fields the races 710 and 730 prevailed. Hence, in the South of Russia, including Rostov region, the selection of sunflower with respect to the resistance to downy mildew should be carried out mainly on the basis of races 330, 710 and 730.
The present study was aimed at the assessment of selected samples of sunflower to the downy mildew races 330 and 710 under laboratory conditions and genotyping of sunflower lines characterized by different level of resistance to downy mildew using STS-markers of three Pl-loci.

MATERIALS AND METHODS
The study was carried out on 16 lines of sunflower. These lines were developed as CMS PET1 pollen fertility recovery agents in the L.A. Zhdanov's Don

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Experimental Station of oil-Bearing Cultures ( Table 2). The criterion of sensitivity at fungus identification was the presence of conidial sporification on cotyledons and true leaves, necrosis and chlorosis; the criterion of resistance was absence of the disease symptoms. Resistance and sensitivity were assessed as the percentage of affected and healthy seedlings. All experiments were carried out in the climate controlling chamber (Binder, Germany) and repeated 3-5 times for 50-00 plants of each line.
To perform the molecular genetic analysis, genomic DNA was isolated from leaf tissue as described in (Boom et al., 1990). Primer sequences were taken from articles previously published by other authors in order to identify 9 STS-markers of three Pl-loci (Pl 5 , Pl 6 and Pl 8 ) associated with resistance to downy mildew (Bouzidi et al., 2002;Radwan et al., 2004) (Table 1). Polymerase chain reaction was carried out in 25 µL reaction mixture of the following composition: 67 mM Tris-HCl buffer, pH 8.8, 16 mM (NH 4 ) 2 SO 4 , 2.5 mM MgSO 4 , 0.1 mM mercaptoethanol, 0.25 mM of each dNTP (dATP, dCTP, dTTP and dGTP), 20 pM primers, 2.5 units of Taq-polymerase and 15 ng isolated DNA. Amplification was performed in the thermocycler PalmCycler (Corbett Research, Australia) (Markin et al., 2013).

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Thermal regime of the reaction was chosen individually for each pair of primers on the basis of their sequences. For majority of reactions the optimal thermal regime was as follows: (1) Denaturation at 95ºC for 3 min, (2) 35 cycles at the following thermal and time regime: Primer annealing at 60°C for 30 s, 2 min elongation at 72°C, denaturation at 95°C, 30 s, (3) 1 min final elongation. For other amplification reactions the regime was as follows: (1) Denaturation at 94°C, 3 min, (2) 33 Cycles at the following thermal and time regime: 94°C, 10 s, 60°C, 30 s and 72°C, 1 min 30 s, (3) final elongation at 72°C, 5 min. Amplification products were analyzed by electrophoresis in 1% agarose gel supplemented with ethidium bromide in Tris-Borate buffer (Kim et al., 2012). The obtained gels were photographed with the gel-documenting system (GelDoc 2000, BioRad, United States). Gene ruler 1 Kb DNA Ladder (Fermentas, Lithuania) was used as a molecular weight marker.
Resistance to a wide range of pathogens, including viruses, bacteria, fungi, insects and nematodes, is determined by the Resistance genes (R-genes). They contain conservative sequences that determine variety of functions. Presently, at least five classes of R-genes are known. These are intracellular protein kinases, receptorlike protein kinases, which contain Leucine-Rich Repeats (LRR), intracellular LRR-proteins, which contain Nucleotide Binding Site (NBS) and leucine-zipper motive, intracellular NBS-LRR-proteins, which carry Toll and Interleukin-1-Receptor (TIR) homologous domain and LRR-proteins, which provide binding of extracellular proteins to the plasma membrane (Bouzidi et al., 2002).
Study of genetic principles of sunflower resistance to the parasite Pl. halstedii allowed identifying of specific dominant genes called Pl-genes, which determine resistance to different races of Pl. halstedii. According to the previously published data, the world population of Pl. halstedii contains about 37 races, 6-7 of which may be considered as dominating ones (Liu et al., 2012). Genes Pl 1 and Pl 2 were identified in 1970 s and were found to be associated with resistance to races 100 and 300 of downy mildew (Zimmer, 1974). After successful introduction of these genes into the hybrid lines, in the following two decades, new races of this pathogen, which are tolerant to the previously found resistance genes, appeared. Later on, the gene Pl 6 , which determines resistance to 11 races of downy mildew causative agents, was identified in the wild form of H. annuus. It is presently shown that Pl 6 locus contains at least 11 tightly linked genes, each of which provides resistance to one of the races of downy mildew (Bouzidi et al., 2002). Gene Pl 8 is the analogue of the gene Pl 6 though identified in H. argophyllus rather than in H. annuus. Gene Pl 7 was found in H. praecox. Further analysis showed that genes Pl1/Pl2/Pl6 complex belong to the first linkage group and referred as the TIR-NBS-LRR resistance group and genes Pl5/Pl8 complex to the second linkage group and non-TIR-NBS-LRR group. The most recent publications report about the finding of new resistance genes, Pl 13 and Pl 16 , which were identified in the cultural sunflower line HA-R5 and determine resistance to the races 100, 300, 310, 330, 700, 710, 730, 731 and 770 (Liu et al., 2012;Mulpuri et al., 2009).
We analyzed known STS-markers of three Pl-loci (Pl 5 , Pl 6 and Pl 8 ) associated with the resistance of sunflower to downy mildew (Fig. 1), because this resistance is known to be controlled by dominant genes. The analysis was carried out on the sunflower lines, which demonstrated different level of resistance to the disease, in order to identify the lines most informative for the marker-assisted selection. These DNA-markers may be useful for fast identification of genotypes potentially resistant to this pathogen within vast samples. We believe that this would provide more effective solution for the problem of sunflower resistance to downy mildew.
Only two (НаР2 and НаР3) out of nine STS-markers that mark the Pl 6 locus were found to be informative. However, they allowed us to mark 4 out of 5 lines resistant to the downy mildew: J-6/1285, J-8/154, J-11/420 and J-7/465. In the genotypes of the resistant lines specific 1200 B.P. (НаР2) and 1800 B.P. (НаР3) PCR-fragments were found that is consistent with previously obtained data (Bouzidi et al., 2002). It is noteworthy that the third of the markers of the Pl 6 locus (HaP1), the primers of which flank a 2000 B.P. motive, which includes marker sequences of НаР2 and НаР3, did not provide identification of resistant genotypes. Tested STS-markers of the loci Pl 5 and Pl 8 did not allow us to identify genotypes resistant to the downy mildew, because they were equally present in all the lines studied.
It should be noted that in sunflower this disease may be not manifested at the early stages of plant growing (Sakr, 2014;Al-alawi and Obeidat, 2013;El-Shehawi et al., 2013). Moreover, it was shown that visually healthy plants, which were grown among the infected ones, produce seeds that are the carriers of downy mildew (Sakr, 2014;Pishdar et al., 2013). Therefore, plants of the line J-11/536, which demonstrated resistance to the downy mildew at early stages of growing, but were not marked with STS by the resistance loci, should be studied throughout the vegetation period and their seeds should be analyzed as well.

CONCLUSION
Resistance of Rf-lines of sunflower cultivated in the L.A. Zhdanov Don Experimental Station of Oil-Bearing Cultures to the most widely spread in Rostov region races of the downy mildew 330 and 710 was assessed. Lines contrastingly different by the resistance to these two races of the downy mildew were identified in laboratory conditions by the method of artificial infection. They were also genotyped with 9 STS-markers of three Pl-loci (Pl 5 , Pl 6 and Pl 8 ) associated with resistance of sunflower to the downy mildew. Only two out of nine STS markers (НаР2 and НаР3, locus Pl 6 ) provided successful identification of lines, which demonstrated resistance to the downy mildew in experiments with artificial infection. The studied DNA markers НаР2 and НаР3 may be especially promising for the marker-assisted selection of sunflower with respect to resistance to the downy mildew.

ACKNOWLEDGEMENT
This research was supported by the Russian Ministry of Education and Science, project no. 40.91.2014/K.