Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 201

acid), with traces of linolenic acid (Dorell and Vick 1997). The quality of
seed oil involves the modification of the fatty acid composition, which
determines the specific edible purpose (Pérez-Vich et al. 2000).
Stearic acid (C18:0), which is present in about 5% in sunflower oil, is
desaturated to oleic acid (C18:1) by the 9-stearoyl-acyl carrier protein

desaturase (SAD) (Ohlrogge and Browse 1995). A candidate gene approach
was used to isolate the corresponding gene(s) (Hongtrakul et al. 1998a). A
cDNA library was made from developing seeds and hybridized with a PCR
product obtained by using degenerate primers and genomic sunflower DNA.
The degenerate primers were designed from two near-consensus sequences
identified after aligning 15 plant SAD cDNA sequences from GenBank.
Nineteen SAD cDNA clones from sunflower were partially sequenced and
found to belong to two groups. Full-length cDNAs from each group (SAD6
and SAD17) were completely sequenced. SAD17 (U91340) was 1,426 bp
long with an ORF from bp 59 to 1,246, a 58 bp 5'-UTR and a 180 bp 3'-UTR
with an ATAAAA polyadenylation signal beginning at bp 1,380. SAD6
(U91339) was 1,335 bp long with an ORF from bp 84 to 1,271, an 83 bp 5'-
UTR and a 64 bp 3'UTR. The ORF of both cDNAs encoded 396 amino acid
proteins. Transit peptides of 32 bp were predicted from the transit peptide
cleavage site of a safflower cDNA (Thompson et al. 1991). Both genes are
strongly expressed in developing seeds, but only moderately expressed in
leaves and flowers, and weakly expressed in cotyledons, roots, and stem.
Oleoyl-phosphatidyl choline desaturase ( FAD2 ) is necessary for the
synthesis of linoleic (C18:2) from oleic acid (C18:1). Three FAD2 genes ( FAD2-
1 , FAD2-2 , and FAD2-3 ) have been isolated for the sunflower microsomal
oleate desaturase by a candidate gene approach from normal-type HA89
(Martinez-Rivas et al. 2001). OLD-7, a previously isolated FAD2 cDNA from
the sunflower cultivar Mammoth (Hongtrakul et al. 1998b), showed a 100%
identity to the FAD2-1 . In high oleic lines, Ol represents a chemically induced,
incomplete dominant mutation, which greatly increases oleic acid content
in developing seeds in sunflower. FAD2-1 cosegregates with Ol and has
been mapped to LG 14 (Lacombe and Bervillé 2001; Perez-Vich et al. 2002).
Hongtrakul et al. (1998b) developed dominant and codominant RFLP markers
and a codominant SSR marker for FAD2-1 . The codominant RFLP
distinguished between Ol locus genotypes and can be used to accelerate the
development of high oleic lines in sunflower.
The specificity of the acyl-acyl carrier protein (ACP) thioesterases plays
an important role in controlling the fatty acid composition of seed oil. These
enzymes are encoded by nuclear genes but are targeted to the plastid and
are usually categorized into two groups, FatA and FatB , according to their
sequence and acyl-ACP preference (Jones et al. 1995). The FatA genes encode
thioesterases with a preference for unsaturated acyl-ACPs and with
specificity for 18:1-ACP. On the other hand, FatB genes encode thioesterases