DETECTING THE PRESENCE OF DOWNY MILDEW AND GENOTYPING SUNFLOWER HOST PLANTS IN THE SAME PCR REACTION JINGUO HU 1, JUNFANG CHEN 2, THOMAS J. GULYA 1 and JERRY F. MILLER 1 1 USDA-Agricultural Research Service, NCSL, Fargo, ND 58105 2 Department of Plant Sciences, North Dakota State University, Fargo, ND 58105 Introduction Downy mildew (DM) is one of the most important diseases of sunflower in North America (Gulya et al., 1997) and it has also been reported in all the sunflower growing regions except Australia (Roekel-Drevet et al., 2003). The causal agent is the obligate parasite Plasmopara halstedii (Farl). Berl. and de Toni. DM can affect sunflower plants at all growth stages. The affected young seedlings are stunted, leaves are chlorotic, and the capitulum of affected plants bears less seed. Substantial yield losses can result when the disease incidence is high and the climatic conditions favor disease development. Dominant genes conferring complete resistance to DM exist in both cultivated sunflower (Helianthus annuus L.) and related wild Helianthus species such as H. praecox Engleman and Gray and H. argophyllus Torrey and Gray. Sunflower breeders have successfully incorporated the resistance genes into modern sunflower varieties to combat DM. The dominant genes, Pl1 and Pl2, provide complete resistance to DM race 100 and race 300. New races of DM frequently overcome existing resistance genes necessitating the search for new resistance genes. So far, eleven Pl genes have been reported that confer resistance to one or all eight or nine identified DM races (Miller and Gulya 1991; Vear et al., 1997; Molinero-Ruiz et al., 2003). Genetic analysis revealed that some of the genes are tightly linked (Roeckel-Drevet et al.; 1996, Bert et al., 2001; Slabaugh et al., 2003). However, it is not clear whether these loci are single resistance genes conferring nonrace-specific resistance or clustered genes giving resistance to individual races following the gene-for-gene hypothesis. Our goal is to develop user-friendly DNA markers closely linked to the available Pl genes. These markers will be useful 1) in marker-assisted breeding to replace seedling
tests thereby accelerating breeding programs for the incorporation of new Pl genes, 2) for differentiating different Pl genes, and 3) for pyramiding different Pl genes into one breeding line. Using our in-house developed target region amplification polymorphism (TRAP) marker technique (Hu and Vick, 2003), we found that it is possible to detect the presence of the downy mildew fungus and genotype the host plants in the same PCR reaction. Materials and Methods The plant materials included the F 2 (HA434/HA 335) and the BC 1 (HA434//HA434/HA 335) populations. The donor parent carries the Helianthus annuus-originated Pl6 gene, which confers resistance to all known DM races, except race 304. The mixed DM race 770 was used for phenotypic resistance evaluation with the whole seedling immersion technique (Miller and Gulya, 1991). Germinated seeds with radicles of 1 to 2 cm in length were immersed in inoculum suspension containing 20,000 to 30,000 sporangia/ml for 3 hours, The inoculated seedlings were planted in flats containing 1 to 1 (v/v) mixture of perlite and sand and kept in the greenhouse for 12 to 14 days with temperature between 20 and 25 ºC and a 16-hour photoperiod. These flats were then transferred to a chamber with 100% relative humidity for 16 hours at 18 ºC. Susceptibility phenotyping was assessed by the presence of sporulation on the first true leaf and/or cotyledons. For DNA marker development, half of a cotyledon was taken from each seedling prior to high humidity incubation for DNA extraction. Total DNA samples from the sunflower tissues were isolated with the DNeasy 96 Plant Kit (QIAGEN), and DNA samples from the DM spores were isolated with the DNeasy miniprep kit (QIAGEN), following the manufacturer's instructions. The concentrations of DNA were determined with a DU7400 spectrophotometer (Beckman Coulter) and adjusted to 30 to 50 ng/µl for PCR reactions. For PCR amplification, the fixed primers were designed against the selected sunflower EST (expressed sequence tag) sequences in the Compositae Genomics database: http://compgenomics.ucdavis.edu. These selected ESTs have homology to the conserved
regions, such as NBS (nucleotide binding site) and LRR (leucine-rich repeat) domains, of most plant resistance genes. The arbitrary primers of 18 nucleotides long and labeled with IR-dyes were used in combination with the fixed primers in the PCR reactions following the TRAP protocol of Hu and Vick (2003). Results and Discussion Detecting the presence of downy mildew and genotyping the host plants in the same PCR reaction. The fixed primers designed against the sunflower ESTs homologous to the conserved R gene-components worked very well in this experiment. Each PCR reaction amplified about 50 scorable fragments with sizes ranging from 50 to 900 bases as reported earlier (Hu and Vick, 2003). The first three PCR reactions amplified seven markers that segregated in the expected one to one ratio in the BC 1 population, but none was associated with the resistance or susceptible phenotypes. These PCR reactions also amplified 29 fragments exclusively from the seedlings scored as susceptible. The association between these fragments and the susceptible phenotype also persisted in the F 2 population (Fig. 1). In the beginning, these data were very difficult to explain. Because the TRAP markers are dominant in nature, and if the fragments were amplified from the susceptible allele, r, of the resistance gene, they should be monomorphic in the BC 1 population which consists of two genotypes, Rr and rr, and should segregate in a three to one ratio in the F 2 population which contains RR, Rr, rr and rr genotypes. One possibility is that the fragments were amplified from the fungal DNA. The mechanisms of pathogenesis and resistance of DM have been well documented (Doken, 1989). Upon entering the host root, primarily through the zone of elongation, the fungus establishes a systemic infection by growing through the intercellular spaces between parenchyma cells of the root, hypocotyl, and stem (Mouzeyar et al., 1993). The resistance genes could prevent the pathogen from entering the root, or restrict the pathogen growth in the host tissue (Gulya et al., 1997). For our experiment, the
cotyledons were taken for DNA preparation just before the high humidity incubation Fig. 1. The amplification profile of inoculated seedlings and the DM fungus. Two fragments (sized 100 bp and 480 bp, indicated by arrows) from the 11 susceptible seedlings were from the DM fungus. Two segregating markers (sized 600 bp and 350 bp) are independent from the resistant phenotypes. for symptom development (sporulation). At this stage, the susceptible seedlings could contain a sufficient amount of DM mass such that the resulting DNA extract might contain DNA from both sunflower and DM fungus. The two fragments (100 bp and 480 bp) could have been amplified from the DM because the TRAP protocol uses a low annealing temperature of 35 ºC in the first five cycles of the PCR amplification. To test this hypothesis, we prepared DNA samples from DM race 770 spores and carried out PCR amplification. The result confirmed that those two fragments were amplified from the DM (Fig. 1). The differences between the amplified pattern of DNA from the
inoculated seedlings and the DNA from the DM fungus could be due to template DNA competition in the PCR reaction. From this electrophoretogram, two of the fragments in the susceptible seedlings can be traced to the fungus. Similar results were obtained with inoculated seedlings from six BC 1 and six F 2 populations segregating for DM resistance. Thus, it is possible to detect the presence of downy mildew and genotype the host plants in the same PCR reaction. Possible applications. The sunflower DM resistance to most races is controlled by single dominant genes (Vear et al., 1997 and Molinero-Ruiz et al., 2003). However, the accurate phenotype assessment can be very difficult because the incidence of the disease in susceptible control plants in an experiment does not usually reach 100%. Thus, a formula to calculate a correction factor was used to correct the observed susceptible seedling numbers in a DM resistance study (Molinero-Ruiz et al., 2003). In breeding practice, most breeders, including experienced breeders, sometimes make errors in selecting the healthy seedlings which do not carry the resistance genes from their segregating populations. Detecting the presence of DM pathogen in the seedling tissue with the TRAP technique will assist the breeders by eliminating the healthy, but susceptible seedlings, at an early stage. Acknowledgements The authors thank Angelia Hogness, Dale Rehder, and Scott Radi for their technical assistance. Mention of names of products and manufacturers does not imply endorsement by ARS-USDA or recommendation over other similar products. References Bert P. F., Tourvieille de Labrouhe D., Philippon J., Mouzeyar S., Jouan I., Nicolas P., Vear F. (2001) Identification of a second linkage group carrying genes controlling resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.). Theor. Appl. Genet. 103:992 997 Doken M. T., (1989). Plasmopara halstedii in sunflower seeds and their role of infected seeds in producing plants with systemic symptoms. J. Phytopathol. 124:23-26
Gulya T. J., Rashid K. Y., and Maservic S. M., (1997) Sunflower diseases. In: Sunflower Technology and Production, Ed. A.A. Schneiter, Soil Science Society of America, Madison, pp 263-379 Hu J., and Vick B. A. (2003) TRAP (target region amplification polymorphism), a novel marker technique for plant genotyping. Plant Mol. Biol. Reporter 21(3):289-294 Miller J. F. and Gulya T. J. (1991) Inheritance of resistance to race 4 of downy mildew derived from interspecific crosses in sunflower. Crop Sci. 31:40 43 Molinero-Ruiz M. L., Melero-Vara J. M., Dominguez J. (2003) Inheritance of resistance to two races of sunflower downy mildew (Plasmopara halstedii) in two Helianthus annuus L. lines. Euphytica 131:47-51 Mouzeyar S., Tourvieille de Labrouhe D., Vear F. (1993) Histopathological studies of resistance of sunflower (Helianthus annuus L.) to downy mildew (Plasmopara halstedii). J. Phytopathol. 139:289 297 Roeckel-Drevet P., Gagne G., Mouzeyar S., Gentzbittel L., Philippon J., Nicolas P., Tourvieille de Labrouhe D., Vear F. (1996) Colocation of downy mildew (Plasmopara halstedii) resistance genes in sunflower (Helianthus annuus L.). Euphytica 91:225 228 Roekel-Drevet P., Tourvieille J., Gulya T. J., Charmet G., Nicolas P., Tourvieille de Labrouhe D. (2003) Molecular variability of sunflower downy mildew, Plasmopara halstedii, from different continents. Can. J. Microbiol. 49: 492 502 Slabaugh M. B., Yu J. K., Tang S., Heesacker A., Hu X., Lu G., Bidney D., Han F. and Knapp S. J. (2003) Haplotyping and mapping a large cluster of downy mildew resistance gene candidates in sunflower using multilocus intron fragment length polymorphisms. Plant Biotech. J. 1:167-185 Vear F., Gentzbittel L., Philippon J., Mouzeyar S., Mestries E., Roeckel-Drevet P., Tourvieille de Labrouhe D., Nicolas P. (1997) The genetics of resistance to five races of downy mildew (Plasmopara halstedii). Theor. Appl. Genet. 95:584 589