Development of molecular approaches in the study of lettuce downy mildew (Bremia lactucae) population biology
The project will investigate the use of molecular methods to study the population biology of B. lactucae infection on lettuce. Molecular microsatellite markers which can detect B. lactucae pathotype in lettuce are available. An important area of the project is to develop further molecular markers and use existing marker in epidemiological studies to aid our understanding of factors which contribute to pathogen population change within crop systems. Another important aspect would be factors affecting population change by B. lactucae in response to cultivar deployment. Lettuce is grown in glasshouses before being transplanted in field.
My research is based on Gene-for-Gene Theory which was raised by Harold H. Flor in 1940s. The symptom of B. lactucae is affected by the interaction between avirulence gene (Avr gene) and resistence gene (R gene). The expression of resistance or susceptibility of lettuce is conditional on the pathogen genotype .The degree of pathogen virulence is conditional on the host genotype (Crute & Pink, 1996). The population dynamics and equilibrium of the interacting species can be strongly influenced by the level of genetic variation in avirulence and virulence (Lebeda & Zinkernagel, 2003).
More information about my project can be found here:
The overall aim of the project is to develop and use molecular methods to study mechanisms which change population structure in B. lactucae on lettuce within agricultural ecosystems. An important area is to develop molecular markers for epidemiological studies and compare these with existing method for differentiating B. lactucae race. Population shifts by B. lactucae in response to cultivar deployment can be studied using neutral and driven molecular markers. The factors which cause changes in pathogen populations and the role of mutation also will be studied in this project.
- A. Phenotypic classification IBEB of isolates. 19 lettuces cultivars are used to identify the Spanish isolates.
- B. DNA extraction . Invitrogen ChargeSwitch gDNA plant kit is used to extracted better quality DNA.
- C. Cloning. Bremia DNA (CP24 and BL22) were cloned for sequencing (plasmid DNA sequencing and cloning PCR sequencing) by using AccepTor Vector Kit.
- D. Optimizing SSR primer. Three SSR primers (pv14, pv 16, pv39) trail on Bremia DNA (CP24 and BL22). But the banding patterns were not distinct. I run the samples on three different gel (Agrose (1.5% and 2.0%), EL800 and 6%POLY(NAT)), Agarose gel is used as a examination before run the sample on EL800 or Poly(nat). If the band is present I will run the sample on the other two. EL800 gives more clearly bands around 200-300bp. And CP24 were 1:10 diluted.
- E. SSR markers development.