Oil Composition Variations - Genetics, Genomics and Breeding of Crop Plants - page 271

Figure 8-1 Organization of the oleate-desaturase region and details on the Pervenets
mutation.
A: Thick line: region sequenced (17,770 bp); E, H: restriction site for Eco RI and Hind III,
respectively. Thick bar marked by the thick and light arrows pinpoints the Oleate
desaturase repeated sequence. Dotted line marks the unknown sequence with deduced
restriction sites.
B: Scheme for the location of the OD gene. The line indicates the oleate-desaturase
repeat. Dotted line indicates unknown sequence.
C: Enlargement for the OD gene, the spacer region and the oleate-desaturase repeat
portion.
The promoter region, exon 1(e_1), intron 1 with the SSR-OD1, exon 2 and spacer are
represented by boxes with motives and the bar below indicate the regions with the direct
repeat.
also used several library constructions without success. However, they
cloned an oleate desaturase gene belonging to a microsomal oleate
desaturase (according to the targeted sequence) that displays all the features
of a functional gene (Lacombe et al. 2002). The microsomal oleate desaturase
(MOD) gene displayed a 1,683 bp intron carrying an SSR motif (ATT) repeated
17 times in RHA 345 (HA-INRA-OD1). The motif is repeated 16 times in
83HR4, allowing the origin of the oleate desaturase allele in each RIL to be
determined. Moreover, with the hypothesis that Pervenets insertion is very
close to the oleate desaturase sequence, recombination between the SSR
locus and Pervenets was not expected.
Lacombe et al. (2002) turned to a PCR approach hypothesizing that the
distance between the wild oleate desaturase sequence and the oleate
desaturase sequence on the Pervenets insertion could be determined by
PCR. Forward and reverse primers designed on the oleate desaturase cDNA
were used in pairwise combinations to anchor the wild gene (F) and the
Pervenets sequence R. The reverse primers were designed assuming the
insert was in the same direction or in the opposite direction of the wild
oleate desaturase gene. Long PCRs were run and the amplification products
were hybridized (Southern blots) with the full oleate desaturase cDNA. A
4.3 kb fragment was revealed and cloned into a plasmid. It was sequenced