Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 185

Another strategy for cloning resistance genes is the candidate gene
approach that has been described in detail in the Section 6.3.4. The number
of resistance gene analogs mapped in sunflower or used to develop PCR-
based markers had been very low at the beginning (Gentzbittel et al. 1998;
Gedil et al. 2001a; Plocik et al. 2004; Radwan et al. 2004). However, mining
of the sunflower EST database for NBS-LRR homologs proved to be very
efficient (Radwan et al. 2008).
The combination of disease resistance gene analogs (RGAs) and map-
based cloning strategies will hopefully allow resolving the structure and
the mechanisms behind the resistance to different Plasmopara halstedii races.
6.3 Candidate Gene Approach Based on Homology to Genes
of Other Species
Using candidate gene approach requires that the gene of interest has been
isolated in another species and sequences are available to be used as probes
or to design primers for PCR amplification in sunflower. The major problem
here is the degree of homology between genes in different species, which
either allows to start with specific primers from conserved regions or
degenerate primers if the sequence homology proves only moderate between
species. Primers are then used to amplify the candidate gene either from
genomic DNA or cDNA. The large amount of EST data (Chapter 3) now
available for sunflower will be very helpful for employing candidate gene
approach in sunflower. Candidate gene approach, although limited to genes
of known function, has been very successful in sunflower in the recent
years. Table 6-2 gives an overview of the genes that were isolated by a
candidate gene approach.
6.3.1 Herbicide Resistance in Sunflower
Wild biotypes of sunflower, native to North America (Rogers et al. 1982), are
weeds in corn, soybean and other crops in North America. These weeds are
commonly controlled by applying acetohydroxyacid synthase (AHAS)-
inhibiting herbicides as sulfonylurea (SU) and imidazoline (IMI). SU and
IMI are efficient inhibitors of acetohydroxyacid synthase (AHAS), an enzyme
that catalyzes the first step in the biosynthesis of the essential branched-
chain amino acids isoleucine, leucine and valine (Duggleby and Pang 2000).
To understand the mechanism behind the herbicide resistance, the AHAS
genes from sunflower were isolated by a candidate gene approach from the
two sources of resistance in wild sunflower populations ANN-PUR and
ANN-KAN (Kolkman et al. 2004). To design oligonucleotide primers for
amplifying AHAS gene fragments and isolating the AHAS genes from
sunflower several sources of DNA sequences were used. First, a cDNA probe