Monitoring Three Plasmopara halstedii Resistance Genes in Iranian Sunflower Inbred Lines

1. Background
Downy mildew of sunflower caused by an oomycete, Plasmopara halstedii (Farl.) Berl. etde Toni, is a widespread destructive plant disease worldwide (1). Yield losses under conductive environmental conditions can be high and reported to be up to 50% (2). The disease first was reported in USA in 1888 (3) and subsequently was observed in Europe in 1960 (4). The rapid spread of the disease during the last decades in Europe has led to a serious concern, threatening sunflower production (4). The causal agent of the disease was subjected to quarantine regulation in European Union since 1992 (4). Many approaches have been used to minimize the effects of the pathogen, including the use of fungicides and use of resistant genotypes. Under favorable conditions, application of systemic fungicides has been a common practice to control the disease. Nevertheless, extensive fungicide application impose high selection pressure on the pathogen populations leading to the emergence of fungicide resistance strains that eventually compromise effective disease control (5, 6). Therefore, employment of resistant genotypes is by far the most cost-effective and environmentally-safe strategy to control the disease. Rapid spread and high population dynamic of causal agent of the disease (4) highlights the need that sunflower producing countries equip their sunflower germplasm with different resistance genes.To this aim, screening of sunflower genotypes for resistance to the disease is required prior to their release in high risk growing regions.
Existence of physiologic races in P. halstedii is evident in many reports. Similar to many plant-pathogen interactions, sunflower-P. halstedii interaction follows the gene-for-gene model (7). Up to now, at least 35 races from different parts of the world have been reported (8). Nomenclature of the races is based on their reactions to a set of differential lines, which are lines possessing different resistance genes and show differential responses to specific races (9). To date, a number of dominant genes designated as Pl, have been proven to confer complete resistance to the pathogen in sunflower cultivars or their wild relatives (10). Pl1 was the first resistance gene identified in sunflower that provides resistancetorace100 (11). Several otherresistance genes have been identified and introduced to sunflower cultivars conferring resistance against different physiological races (12, 13, 14). Pl2confers resistance to race 300 (15). Pl3 and Pl4 were designated independently, but it was shown that they are equal to Pl1 and Pl2, respectively (11, 16).  Pl5 shows resistance to race 700 (17). Pl6, Pl7, and Pl8 confer resistanceagainst races 100, 300, 310, 330, and 700 (18, 19). Resistance to races 310, 330, and 300 is conferred by Pl9, Pl10, and Pl11, respectively (19). It has been found that Pl12 provides resistance against races100, 300, and 700 (12). Pl13was also identified as a locus conferring resistance to races300, 700, 730, and 770 (20). The recent identified resistance gene, Pl14, confers resistance to race 730 (14). Although, several lines of evidence shows that Pl genes might have the coiled coils-nucleotide binding site-leucine-rich repeat (CC-NBS-LRR) signature, so far none of the Pl genes are cloned (21, 13, 22). Nevertheless, tightly linked molecular markers to a few Pl loci have been previously reported (23). For instance, toll/interleukin1 receptors-NBS-LRR classes of plant resistance genes were used to develop markers that are linked to Pl6 locus. Furthermore, CAPS markers linked to Pl6 was also developed (24). Two full length sequences belonging to CC-NBS-LRRclasses of plant resistance genes were cloned and their subsequent primers were designed to tag Pl5/Pl8 region (13). CAPS molecular marker linked to Pl1 locus was identified through cloning of candidate resistance genes belonging to NBS superfamily of genes (25). These markers have been successfully used by many research groups as a fast and reliable technique to screen sunflower genotypes.
Despite the fact that downy mildew is the major sunflower disease in Iran, limited information is available on the presence of physiological races as well as the presence or absence of resistance genes within cultivated sunflower genotypes. Recently, P. halstedii race 100 was identified as the dominant race infecting sunflower in Iran (26). The responses of several sunflower inbred lines to this race were also evaluated and several resistant genotypes were identified (26). The race identification and germplasm screening to P. halstedii have been performed using whole seedling immersion in zoospore suspension. Although the method is sensitive and accurate, it is cost effective, time-consuming and requires high experience to perform the experiment. Moreover, screening of F2 segregation populations using inoculation assays would eliminate some susceptible individuals with other useful traits. These limitations encourage breeders to take the advantages of molecular markers linked to disease resistance genes. In this study, we used previously published molecular markers linked to Pl1, Pl6 and Pl13 to screen the available sunflower genotypes in Iran.

2. Materials and Methods

2.1. Assessment of Sunflowere Resistance to P. halstedii
      Sunflower inbred lines (26 lines), were provided by Oil Seed Department, Seed and Plant Improvement Institute (SPII; Table 1).They are highly inbred lines, which have been produced by at least 20 rounds of selfing. The genotypes were previously evaluated for resistance to P. halstedii race 100 using whole seedling immersion method (Personal communication with Rahmanpur (27). Germinated seeds (2-3 days seedlings) were immersed in pathogen zoospore suspension.The seedlings were cultivated in humid and dark green house and the plantlets were evaluatedfor the resistance after two weeks (Table 1). 
     
2.2. Molecular Marker Analyses
The oldest leaf at five-leaf stage was cut andsnap-frozen in liquid nitrogen. Genomic DNA was extracted according to previously described procedure (28). Integrity of the extracted DNA was checked using 1% (w/v) agarose gel electrophoresis. PCR reactions were performed using primer pairs for Pl6, Pl13 and Pl1 (Table 2). PCR programs and ingredients were according to the earlier reports (20, 23, 25). Briefly, PCR reactions were performed in 20 mL containing 30 ng sunflower genomic DNA, 2 mL 10×PCR buffer, 0.5 mM dNTPs, 0.5 mM of each primer, 3.75 mM MgCl2 and 1 U Taq DNA polymerase (CinnaGen, Iran). The PCR conditions were 95ºC for 4 min, followed by 35 cycles of 94ºC for 30s, 52ºC for 30s and 72ºC for 30 to 90s (according to the expected fragment size), plus a final extension at 72ºC for 3 min. Cleaved amplified polymorphic sequences (CAPS) analysis was performed according to previously described method (25) using restriction enzyme Tsp509I (Fermentas, London). DNA fragments were separated on 2% (w/v) agarose gel, stained with ethidium bromide and visualized using a UV trans-illuminator. All the above steps were repeated twice.
3. Results
DNAs from sunflower inbred lines with different responses to P. halstedii race 100 were used as template in PCR reactions using primer pair STS10D6 previously reported to serve as tightly linked to locus Pl13. A PCR fragment ranging in size from 250 to 500 bp was amplified in almost all lines (n=20; Figure 1). The DNA fragment was absent in one susceptible and two resistant lines. Thus, this marker could not dissect the resistant and susceptible lines as differentiated by race 100. Primer pairs Hap1, Hap2, and Hap3 were used to amplify markers linked to Pl6. No DNA fragments (data are not shown) with the expected sizes ranging from 500 to 3000 bp were amplified as reported (23). This mayindicate that none of the genotypes possessed Pl6 locus. PCR amplification using primer pair 4W2, used to amplify marker linked to Pl1, had resulted in a band of 370 bp in size (25). As expected, the band was monomorph among all tested lines (Figure 2). PCR reactions were treated with restriction enzyme Tsp509I and separated on a 1.5% (w/v) agarose gel (Figure 3). Digestion of PCR products resulted in two monomorphic bands below 250 bp for all sunflower lines regardless of their reactions to P. halstedii. In addition to these two bands, all six resistant lines contained a sharp band with the size of 276 bp as reported previously (25). Only two exceptions were observed among all lines, including the susceptible line #28 that had a banding pattern similar to resistant lines and line #32 that had an extra band with the approximate size of 370 bp.

4. Discussion
Employing molecular markers, either gene-based or map-based markers, efficiently accelerated breeding (29). Molecular markers have been widely used to monitor resistant genes in many species (30, 31, 32,  33, 34, 35) and most importantly in the early generation testing (36, 37). In this study, the presence of molecular markers linked to three Pl loci was investigated using PCR-based method. DNAs from sunflower inbred lines with different responses to P. halstedii race 100 were used as template in PCR reactions using primer pair STS10D6 previously reported to serve as tightly linked to Pl13 locus. Results showed that Iranian genotypes possess markers linked to Pl13 that can serve as a resistance gene against races 300, 700, 730, and 770 in the future (20). As expected there was no correlation between the phenotypic reactions of these genotypes to P. halstedii race 100 and the presence of this marker in the genotypes. However our results revealed that these genotypes still could be sources of resistance to other races other than race 100. 
Pl6 was another locus that was investigated using three different markers. No PCR amplification product was observed in any of the genotypes tested in this study. It is reported that all of these markers are STS-based markers and, therefore, lack of these markers in Iranian lines might be a real reflection of lacking Pl6 resistance gene. Thus, to avoid the possible outbreak of the disease under Iranian growing conditions, breeding efforts must be made to incorporate Pl6 resistance gene into Iranian sunflower lines.
The primer pair 4W2 was used to amplify the marker linked to Pl1 locus. The amplified band with the approximate size of 370 bp appeared to be monomorph among all the tested lines. Nevertheless, single-stranded conformational polymorphism (SSCP) analysis has been used to determine the possible complexity of the resulting band (25). The results showed that 4W2 primers bind to multiple sites in the NBS regions in susceptible and resistant lines leading to amplification of a single band with approximate size of 370 bp that consists of several different DNA amplicons (25). They showed that treating the PCR products with Tsp509I would lead to three bands (88, 93, and 188 bp) in susceptible lines while it generates four bands (88, 93, 188, and 276 bp) in resistant lines (25). In our experiment, we observed two monomorphic bands in all sunflower lines. The lowest band was consisted of two DNA fragments (88 and 93 bp) that were not separated due to approximate similar sizes and the second band with the size of 188 bp. In resistant lines, a 276bp band was observed that was indicator of resistant lines. Line #32 had an extra band similar to undigested band (370 bp), which may represent partial digestion, a phenomenon observed when the digestion inhibitors are presented in the reactions. Another exception was line #28 (susceptible line) that had a banding pattern similar to resistant lines. Because the same result was obtained when the  experiment repeated, we concluded  that maybe line, #28, incorrectly been scored during phenotypic evaluation and might have a resistance response to P. halstedii race 100.  Alternatively, it is possible that the marker used to differentiate the resistant and susceptible lines is not completely linked to Pl1 locus and thus some rare exceptions can be present in marker-assisted selection process. Overall, presence or absence of the band with size about 276 bp in sunflower lines might be used to differentiate the resistant and susceptiblelines to P. halstedii race 100.
In this study we used molecular markers to track loci conferring resistance to P. halstedii, and compared with the phenotypic reaction of sunflower lines to race 100. Our data showed that molecular markers can be efficiently used in screening and breeding programs. Good correlation between the presence of markers linked to resistance loci and their phenotypic reactions to pathogen justify implementation of these markers in marker-assisted selection programs. Our data suggest that application of molecular markers can facilitate breeding programs towards disease management.

Acknowledgments
 The work was supported by SPII under the grant number 2-03-03-89170.