Mapping and Tagging of Simply Inherited Traits - Genetics, Genomics and Breeding of Crop Plants - page 124

amplified polymorphic site (CAPS) markers were developed, which
completely co-segregated with the Pl gene conferring resistance to race 730.
Hewezi et al. (2006) aimed to clarify the function of the RGA PLFOR48
using a transgenic loss-of-function approach with expression of antisense
cDNA in RHA266 ( Pl 1 ) sunflower line. PLFOR48 is a full-length RGA obtained
with RACE-PCR from the clone HA-NBSR3 of Gentzbittel et al. (1998). The
transgenic sunflower lines showed developmental abnormalities and thus
could not be used for infection studies with P. halstedii . The sequence of
PLFOR48 showed homology to sequences in Nicotiana tabacum var. Samsun
NN, thus transgenic tobacco was developed with the same antisense cDNA
and the susceptibility to Phytophthora parasitica was investigated. A higher
susceptibility was observed in plants, which showed higher transgene
expression. The authors concluded that PLFOR48 seems to play a role in
both pathogen resistance and normal plant development. Genetic
transformation of susceptible sunflower lines with the PLFOR48 sense
construct is now being considered as an additional approach towards
demonstrating the functional role of this gene in downy mildew resistance.
Gedil et al. (2001) used degenerate oligonucleotide primers (Leister et
al. 1996; Yu et al. 1996) targeted to conserved amino acid sequences in
known NBS-LRR genes to amplify RGA fragments from sunflower genomic
DNA of HA89. PCR products were cloned, sequenced and analyzed by
single-strand conformation polymorphism (SSCP) gel electrophoresis to
identify redundant clones. Six RGA probes (RFLP) detected fragments that
were polymorphic between HA370 ( Pl 1 ) and HA372 ( pl 1 ) and mapped to
three linkage groups on the HA370 x HA372 map. Four RGAs belonging to
the non-TIR-NBS-LRR subclass were mapped on LG 13 (HR-1W23, HR-
1B39, HR-1W41, HR-1W53) and the two others belonging to the TIR-NBS-
LRR subclass were mapped on LG 8 (HR-4W2) and 15 (HR-1W22). Gedil et
al. (2001) developed a CAPS marker for Ha-4W2 on LG 8, which was linked
but did not completely co-segregate with Pl 1 . According to Ha-4W2, Slabaugh
et al. (2003) demonstrated a multigene family of HaRGC1 (= Ha-4W2; Gedil
et al. 2001) in the Pl 1 - Pl 2 - Pl 6 region, all belonging to the TIR-NBS-LRR
subclass and were assessed by multilocus intron fragment length
polymorphism (IFLP). Twenty-four HaRGC1s were mapped in three
populations spanning 2-4 cM on LG 8 and three SSRs (ORS166, ORS299,
ORS1043) were identified segregating within this cluster. This was further
evidence that the Pl 1 - Pl 2 - Pl 6 region is a large cluster of resistance genes. In
addition, the method of IFLP fingerprinting is suitable to search for new
downy mildew resistance genes from wild sunflowers. Altogether, 63
germplasm accessions (elite inbred lines, partially isogenic inbred lines,
open-pollinated populations, Native American land races, wild H. annuus
populations) were screened with the specific markers for HaRGC1 observing
48 unique markers. Only half of the total HaRGC1 copies were present in