Identification of Downy Mildew Resistance Loci in Sunflower Germplasm

Downy mildew caused by Plasmopara halstedii is one of the most economically important fungal diseases on sunflower (Helianthus annuus). To date, several downy mildew resistance genes called Pl genes have been reported on sunflower genetic map. Previous findings have confirmed that Iranian sunflower germplasms are harbouring Pl resistance genes that may be used to control downy mildew. In the current study, there were investigated the Pl5 and Pl16 downy mildew resistance genes in 51 inbred lines of Iranian sunflower, using PCR-based method. Fifteen differential lines carrying Pl5 and Pl16 downy mildew resistance genes were used as positive control. DNAs from 51 sunflower inbred lines were used in PCR reactions using primer pair RS1008 and Hap3 previously reported to serve as tightly linked to Pl16 and P5 loci, respectively. The PCR results confirmed the presence of two Pl16 and Pl5 bands with the size of about 280 and 1,580 bp, respectively, in differential lines. The results indicated that 1 inbred line out of 51 was found to carry Pl5 gene and 10 lines were found to carry Pl16 gene across the studied Iranian sunflower genotypes. These findings may be used to assist breeders for conservation and selection of downy mildew resistant sunflower genotypes.


Introduction
Sunflower (Helianthus annuus L.), a diploid species (2n = 2x = 34), is the fourth most important oilseed crop in the world, with an annual production of 41.4 million tons of seed in 2014 (Faostat, 2014).Sunflower produces a healthy oil rich in unsaturated fatty acids as well as high vitamin E content (Gascuel et al., 2016).
Downy mildew is one of the major fungal diseases in most sunflower producing areas of the world (Gascuel et al., 2015).Plasmopara halstedii, a seed, air and soil-borne pathogen is the causal agent of the disease.It is assumed to be originated from central region of the North American continent (Leppik, 1966).The pathogen survives for up to 10 years in soil or on plants residues as sexual, thick-walled oospores.It can finally produce visible, characteristic downy hyphae on the leaves of host the plants (Agrios, 1988).Downy mildew damage may vary according to region, year, environmental conditions, cultivar and planting date.The systemic infection of downy mildew in sunflower may range from traces to 50% or even more up to 95% (Sackston, 1981) which cause up to 80% yield loss (Molinero-Ruiz et al., 2003).Control of downy mildew in sunflower production involves a combination of crop rotation, fungicide treatments and the use of downy mildew-resistant genotypes, which has been found through germplasms screening (Rahim et al., 2002;Gulya, 2005;Hulke et al., 2010).Breeding sunflower genotypes to resist downy mildew is a sustainable strategy to increase crop yield and reduce the use of fungicides.
To date, thirty six pathotypes of P. halstedii have been recognized (Gascuel et al., 2016).These pathotypes are often defined by an international nomenclature system, based on differential virulence profiles on a set of sunflower inbred lines containing different resistance gene called Pl (Tourvieille de Labrouhe et al., 2012;Gascuel et al., 2015).Modern sunflower genotypes carry one or more dominant Pl resistance genes.So far, more than 20 Pl gene (Pl1 to Pl 21, Pl Arg, Pl PMI3), conferring resistance to at least one pathotypes of P. halstedii, have been discovered in sunflower and wild species and 13 of them (Pl1, Pl 2, Pl 5-Pl 8, Pl 13-Pl 17, Pl 21, and Pl Arg) have been mapped in sunflower and assigned to six main clusters localized on five different linkage groups (LGs) (LG1, 2, 4, 8, and 13) (Mouzeyar et al., 1995;Roeckel-Drevet et al., 1996;Vear et al., 1997;Molinero-Ruiz et al., 2003;Yu et al., 2003;Mulpuri et al., 2009;de Romano et al., 2010;Bachlava et al., 2011;Liu et al., 2012;Qi et al., 2015), but none has been cloned so far (Gascuel et al., 2015;Qi et al., 2015Qi et al., , 2016)).Most of the Pl genes have originated from wild Helianthus annual species, for example, the Pl 1, Pl 2, and Pl 13 originated from a Canadian DNA extraction and PCR conditions Leaf samples were collected from the plants by detaching the leaves and freezing them in liquid nitrogen.Genomic DNA was isolated from the lyophilized tissues using the CTAB method (Li et al., 2007).The integrity of DNA samples were examined on 1% (w/v) agarose gel.
The PCR primer pairs of RS1008 and Hap3, previously reported to serve as tightly linked to Pl 16 and P 5 loci, respectively, were used in this study (Table 3).According to Liu et al.(2012), D7 differential line carries Pl 16 resistance gene, which is a ~280 bp band, so the Pl 16 was first amplified in D1, D7 and D15 differential lines using the primer pair ORS1008 and then, PCR amplifications were performed to detect Pl 16 in all inbred lines.In this study the D6 (803-1) differential line was used instead of YSQ differential line which carries Pl 5 resistance gene (a ~1580 bp band) (Radwan et al., 2004).The PCR program was set as follows: 4 min at 95 °C for pre-denaturation followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 60.1 °C for 45 s, extension at 72 °C for 70 min, and a final extension at 72 °C for 10 min.
Similarly, the Pl 5 resistance gene was first amplified in D1, D6 and D15 differential lines using the primer pair Hap3 and then PCR amplifications were performed to detect Pl 5 in all inbred lines.The PCR program was set as follows: 4 min at 95 °C for pre-denaturation followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55.3 °C for 45 s, extension at 72 °C for 70 min, and a final extension at 72 °C for 10 min.
The PCR products were separated on a 1% (w/v) agarose gel, at 100 W for 30 min (1× TAE).The gels were analysed after being stained with ethidium bromide and imaged with a Gel-Doc (Uvitec, UK).line 953-102-1-1, which is a selection involving wild H. annuus (Fick and Zimmer, 1974;Vear et al., 2008).The Pl 5 originated from H. tuberosus (Vranceanu et al., 1981), Pl 6 was derived from wild non-cultivated H. annuus and Pl 7 from H. praecox Englem and Gray (Miller and Gulya, 1991).Two Pl genes, Pl 8 and Pl Arg, were also derived from H. argophyllus Torrey and Gray (Seiler, 1991).The use of gene-for-gene hypothesis (Flor, 1955) is the most common breeding approach for resistance to downy mildew in sunflower.Thus, discovery of resistance loci and genes is one of the best strategies for increasing resistance against downy mildew in sunflower.Incorporating of the resistance genes into sunflower genotypes could help to mitigate the threat posed by downy mildew races.This can be obtained only once the identity and locus of a Pl gene is determined.
Therefore, molecular mapping of the resistance genes is necessary to conclude whether its resistance gene is different from known Pl genes.Thus, the objective of this study was to identify downy mildew resistance genes in 51 Iranian sunflower inbred lines.

Plant materials and growing conditions
Fifteen differential lines, carrying resistance genes against downy mildew, as positive control were kindly supplied by Dr Denis Tourvieille de Labrouhe, French National Institute for Agricultural Research, Department of Plant Health and Environment.The lines are listed in Table 1.In addition, 51 Iranian sunflower inbred lines (Table 2) were obtained from Department of Oilseeds, Seed and Plant Improvement Institute, Karaj, Iran.
Sunflower seeds were surface-sterilized by soaking in 10% sodium hypochlorite for 10 min and 70% ethanol for 4 min and then rinsed with sterile distilled water.A total of 66 seeds were sown in plastic pots and placed in controlled environmental room at 28/20 °C (day/night) with 70% relative humidity and a 12 h-photoperiod (light intensity of 350 μmol photons m -2 s -1 ) until four leaf stage.

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Table 1.Sunflower differential lines used as positive control to identify Pl5 and Pl16 resistance genes

Results
DNAs from sunflower differential lines were used as positive control in PCR reactions using RS1008 and Hap3 pair primers.The RS1008 and Hap3 pair primers have been previously reported to serve as tightly linked to locus Pl 16 and Pl 5, respectively.
According to the results, a PCR fragment ranging in size from 250 to 280 bp related to Pl 16 was amplified in D7 and D15 differential lines (Fig. 1).The DNA fragment was absent in D1 differential line (Fig. 1).Thus, this line could not dissect the resistant and susceptible lines.
Primer pairs Hap3 was used to amplify fragment linked to Pl 5.No DNA fragment with the expected sizes ranging from 1,500 to 1,580 bp was amplified in D1 differential line genomic DNA (Fig. 2), whereas PCR amplification using primer pair Hap3 used to amplify fragment linked to Pl 5, had resulted in a band from D6 and D15 differential lines genomic DNA (Fig. 2).These results may indicate that D1 differential line does not possess Pl 5 or Pl 16 genes.
PCR amplifications of Pl 16 gene using RS1008 pair primers showed bands ranging from 250 to 280 bp in 10 out of 51 inbred lines which means these lines carry Pl 16 gene (Fig. 3).The Pl 5 gene was present in only one of the 51 inbred lines i.e.A-19.According to data presented in Table 2, five inbred lines numbered as 2, 4, 12, 14 and 16, were found to be A-19 as the PCR results in terms of Pl 16 were quite similar amongst these inbred lines.However, Pl 5 PCR results indicated that the band was observed only in A-19 and not in other inbred lines (Fig. 4).In other words, Pl 5 band was not repetitive in all lines related to A-19.This finding might be due to distance between marker and gene as the further apart between two loci is the more likely crossing over will occur between them.It has been reported that there is 0.3 cM (centimorgan) distance between Pl 16 and RS1008 primer (Liu et al., 2012) whereas distance between Pl 5 gene and Hap3 primer has found to be 4.8 cM (Radwan et al., 2004).This may increase recombination between Pl 5 and Hap3 loci and reduce repetition.4,5,6,8,12,14,16 and 18 inbred lines (as indicated by asterisk) showed the expected band.M: 1Kb marker, PC: positive control, NC: negative control

Discussion
Using molecular markers efficiently accelerated crop breeding (Collard and Mackill, 2008).The use of molecular markers to investigate resistance genes has been reported in several studies (Gordon et al., 2007;Bipinraj et al., 2011;Kim et al., 2011).To date, several molecular markers associated with downy mildew resistance genes have been identified and mapped on the chromosomes (Mouzeyar et al., 1995).For instance, RFLP and RAPD markers have been used to map Pl 1 locus (Gascuel et al., 2015).Moreover, to identify Pl 13 resistance gene, SSR markers related to Pl 13 locus have been used (Mulpuri et al., 2009).STS marker was used to identify Pl 5 and Pl 8 (Radwan et al., 2004).In addition, SSR (ORS1008) marker was reported to detect Pl 16 locus (Liu et al., 2012).Genetic studies have confirmed that there are significant correlations between some resistance genes, for example, Pl 2 Pl 4 (Sackston, 1981), Pl 2 and Pl 1 (Mouzeyar et al., 1995) and Pl 1 and Pl 16 (Roeckel-Devert et al., 1996).The first report of Pl 2 was published by Zimmer and Kinman (1972) and Fick and Zimmer (1974), who showed that both HA61 and RHA274 harboured a single dominant gene Pl 2 giving resistance to race 300.Based on the gene for gene hypothesis, Gulya et al. (1991), suggested that RHA274 carried not only Pl 2 , giving resistance to race 300 but also Pl 9, giving resistance to race 310.However, they did not study genetic segregation patterns for the two genes.Molinero-Ruiz et al. (2003) observed segregations indicating a single gene for resistance to race 310 in RHA274 and RHA325, which they confirmed as Pl 9, but made no comparison with Pl 2. These authors also reported (Molinero-Ruiz et al., 2002) that both RHA274 and HA61 carried 2 complementary genes for resistance (Plw and Pl x) to race 330(SP) giving F2 segregations of 13R: 3S, meaning that the double recessive was resistant.Rahim et al., (2002) reported segregations indicating 2 independent genes in 518 RHA274 giving resistance to races 100 and 300 and since they considered that for each race, different genes are involved, they concluded that RHA274 carries Pl1 and Pl 11 giving resistance to race 100 and Pl 2 and Pl 12 giving resistance to race 300.

Conclusions
Overall, presence or absence of the band with size about 280 and 1,580 bp in sunflower lines might be used to differentiate the resistant and susceptible lines to downy mildew pathogen.The results obtained in the current study showed that molecular markers can be efficiently used in screening and breeding programs and the data suggest that application of molecular markers can facilitate breeding programs towards disease management.

Fig. 1 .
Fig. 1.PCR amplification of Pl16 with the expected size of 250 bp (as indicated by arrow) using ORS1008 primer pairs in D1, D7 and D15 differential lines.The D7 and D15 differential lines showed the expected band.M: 1Kb marker Fig. 2. PCR amplification of Pl5 with the expected size of 1,500 bp (as indicated by arrow) using Hap3 primer pairs in D1, D6 and D15 differential lines.The D6 and D15 differential lines showed the expected band.M: 1Kb marker

Table 2 .
Sunflower inbred lines used to identify Pl5 and Pl16 resistance genes M: Mutant line; A: Male sterile line; B: Maintenance line; R: Restorer line

Table 3 .
The primer sequences of ORS1008 and Hap3 primer pairs used in the study