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United States Department of Agriculture

Agricultural Research Service

Title: Detecting the Presence of Downy Mildew and Genotyping Sunflower Host Plants in the Same Pcr Reaction

Authors
item Hu, Jinguo
item Chen, Junfang - NORTH DAKOTA STATE UNIV.
item Gulya, Thomas
item Miller, Jerry

Submitted to: Proceedings Sunflower Research Workshop
Publication Type: Proceedings
Publication Acceptance Date: March 5, 2004
Publication Date: March 15, 2004
Citation: Hu, J., Chen, J., Gulya Jr, T.J., Miller, J.F. 2004. Detecting the presence of downy mildew and genotyping sunflower host plants in the same PCR reaction. Proceedings Sunflower Research Workshop. Available: http://www.sunflowernsa.com/research/research-workshop/documents/148.pdf

Interpretive Summary: Downy mildew (DM) is a serious disease of sunflower worldwide. Various dominant genes conferring complete resistance to different races of DM were found in different Helianthus species and are being incorporated into cultivated sunflower breeding lines. Aiming at developing DNA-based markers associated with the resistance genes to accelerate the breeding progress, we are using the in-house developed TRAP (target region amplification polymorphism) technique to screen populations segregating for DM resistance. In this study, we used the F2 and BC1 populations derived from two breeding lines, HA434 (susceptible) and HA335 (resistant). The TRAP technique amplified 36 polymorphic fragments from the BC1 population. Among these, seven fragments segregated in the expected one to one ratio but independent from the resistant or susceptible phenotypes. The remaining 29 fragments were amplified exclusively from the seedlings scored as susceptible. The association between these 29 fragments and the susceptible phenotype also persisted in the F2 population. The hypothesis that these fragments were amplified from the DM fungus interpreted the data very well and was confirmed by a subsequent experiment. DNA samples were prepared from the DM fungus spores and subjected to PCR amplification with the TRAP protocol. Most of the fragments associated with susceptibility were amplified from the fungus DNA with the same primer combinations. Thus, we concluded that it is possible to detect the presence of downy mildew fungus and to genotype the host plants in the same PCR reaction. The immediate application of this technique is to help breeders to identify the susceptible seedlings by detecting the presence of the fungus in the cotyledon with a PCR reaction.

Technical Abstract: The in-house developed PCR-based TRAP (Target Region Amplification Polymorphism) protocol has been applied to develop markers associated with sunflower downy mildew resistance. The F2 and BC1 populations derived from the breeding lines HA434 and HA335 were used in the current study. For downy mildew resistance assay, we used the whole seedling immersion technique. TRAP amplifications were carried out with DNA samples extracted from the inoculated seedlings. The fixed primers were designed against the sunflower ESTs that are homologous to the conserved plant disease resistance gene components. Among the 36 polymorphic fragments amplified from the BC1 population, seven were segregating in the expected one to one ratio and independent from the DM resistant/susceptible phenotypes, but 29 fragments were amplified exclusively from the seedlings scored as susceptible. The association between these 29 fragments and the susceptible phenotype also persisted in the F2 population. Our hypothesis that these fragments were amplified from the DM fungus interpreted the data very well and was confirmed experimentally. Thus, we concluded that it is possible to detect the presence of downy mildew fungus and to genotype the host plants in the same PCR reaction. The immediate application of this technique is to help breeders to identify accurately susceptible seedlings by detecting the presence of the fungus in the cotyledon with a PCR reaction.

Last Modified: 10/20/2014
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