Physical Mapping Of Barley Yd2, A Resistance Gene To Barley Yellow Dwarf Virus

Barley Yd2 gene is the most effective source of resistance to Barley Yellow Dwarf Virus(BYDV) in all kinds of resistance sources of cereal crops and their relatives, which has been used in the disease-resistant breeding programs worldwide for more than 40 years. The Yd2-mediated resistance shows no hypersensitive response, providing durable resistance against BYDV in host plants. Unlike many cloned resistance genes from plant genomes, Yd2 does not prevent the virus from spreading systematically from the point of the viral infection, rather it appears to act by reducing the rate of replication of the virus in the host plants. The previous reports indicated that it is not possible to isolate Yd2 by PCR-based and transfer tagging techniques used commonly in gene cloning. Instead, map-based cloning might be a better way for Yd2 isolation.Based on the high saturation genetic map of Yd2 region with molecular marker, this study was conducted for physical mapping of Yd2 area using the most closely linked markers as probes. The physical mapping generated the data for establishing the relationship between genetic and physical distance around the region of Yd2, which is critical to the success of map-based cloning of this target gene because the relationship between genetic and physical distance can vary over 100-fold in different regions of a genome.In the present study, four different methods for the preparation of genomic HMW DNA were trialed to analyze the quantity and quality of HMW DNA prepared so as to determine which method would be most suitable for barley physical mapping. Twenty two restriction enzymes such as EcoRI, NcoI , XbaI and HindIII were test for their restricted DNA fragments qualified for this study. Eleven probes (AWPH7D, BCD134, YLp etc.) for Southern hybridization were selected from the DNA markers which mapped within 1 cM of Yd2. PFGE electrophoresis was chosen to separate the restricted DNA fragments. The results showed that nuclei embedded in agarose plugs gave higher quantities and better quality of HMW DNA in comparison to the other three methods including leaf material and protoplasts embedded in ararose plugs and nuclei embedded in agarose microbeads. The HMW DNA prepared by nucleic embedded in agarose plugs was selected for subsequent physical mapping in the Yd2 region. Of 22 restriction enzymes tested, 10 enzymes (BssHII,Cla I ,Not I ,Nru I ,Pru I, Pru II ,Sal I , Sma I, Sac II ,Snab I) cut DNA into fragment greater than 100kb, which were able to produce good hybridization signal with 6 probes (BCD134, MWG802, MWG952, YLM, YLP, WG889). Three DNA probes YLM, YLP and BCD 134 hybridized to the same 180kb Pvul restriction fragments. Further restriction enzyme mapping of this 180 Kb fragment provides strong proof that this single 180kb Pvul restriction fragment encompasses both the YLP and BCD134 loci. The two loci YLP and BCD134 were separated on the genetic map by 0.5cM and shown to be a maximum distance of 180kb apart. Therefore, a ratio of physical to genetic distance around the Yd2 region can be calculated as 1cM being equivalent to 360kb (1cM=360kb). This information suggests that there is a high possibility to isolate Yd2 gene by map-based cloning approach. The data described in this paper shows a great potential in the comparative genomics of cereal crops and plant function genomics.