Oil Composition Variations - Genetics, Genomics and Breeding of Crop Plants - page 270

olive, a partial cDNA for the stearate desaturase and complete cDNA for the
oleate desaturase were isolated in sunflower.
Further studies in sunflower revealed expression of these genes in the
developing embryos between 10 to 20 days after fertilization. Kabbaj et al.
(1996a, b) demonstrated with the stearate desaturase transcript as control
that the oleate desaturase transcript was lacking in HOAC sunflower,
whereas it was present in the control.
Hongtrakul et al. (1998a, b) showed by RT-PCR that the oleate desaturase
transcript was less expressed in HOAC sunflower than in traditional
sunflower and they also used the stearate desaturase transcript level in
both types as control. The absence of the transcript explains the absence of
the protein. However, this does not indicate whether the gene is present or
not. To look for the presence of the gene researchers have used RFLP, the
most available technique at this time.
8.2.3.2.4 DNA Changes Correlated with the Pervenets Mutation
Lacombe et al. (1998) and Hongtrakul et al. (1998b) reported an RFLP
between traditional and HOAC lines carrying the Pervenets mutation using
oleate desaturase cDNA as a probe. Signals have been interpreted as a
duplication of the oleate desaturase gene by Hongtrakul et al. (1998b),
whereas Lacombe et al. (2000) did not postulate a complete duplication of
the oleate gene.
Southern blots of genomic DNA from traditional sunflower restricted
separately with Eco RI, Hind III, Bam HI, or Sac I, and hybridized with the oleate
desaturase cDNA as a probe displayed RFLP profiles ( Fig. 8-1 ). Only one
fragment was clearly hybridized, suggesting that only one gene is present
in the sunflower genome. We cannot exclude that several copies could exist.
In contrast, all HOAC Pervenets lines displayed an RFLP profile that was
different from traditional sunflower ( Fig. 8-1b ). With Eco RI, an extra fragment
of 8 kb (Pervenets insertion) appeared in addition to the wild Eco RI 5.85 kb
fragment and was strongly hybridized with the probe. This means that it
should carry a sequence homologous to the oleate desaturase. With Hind III,
the wild 8.0 kb fragment disappeared and was replaced by a 16 kb fragment.
With Sac I and Bam HI, the wild fragments disappeared and were replaced by
another lengthened fragment. The double restriction Eco RI and Hind III
revealed a 2.1 kb fragment and the 8 kb Pervenets insertion. Taken all together,
these facts suggest that an insertion of 8 kb occurred on the 8.7 Hind III
fragment in the region of the Eco RI site.
Lacombe et al. (1999) attempted to clone genomic fragments from an
HOAC line that hybridized with the oleate desaturase probe corresponding
to the Pervenets RFLP. Unfortunately, they did not succeed in identifying
hybridizing clones that displayed the oleate desaturase rearrangement. They