Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 203

MT). BLAST searches of the Compositae Genome Program Database (CGPdb;
http://cgpdb.ucdavis.edu / ) and National Center for Biotechnology Information
(NCBI) GenBank Databases ( http://www . ncbi.nlm.niv . gov / ) were performed
using cDNA and amino acid sequences for Arabidopsis MPBQ/MSBQ-MT
(GenBank accession No. AB0542571 and At3g63410; Cheng et al. 2003;
Motohashi et al. 2003) as query templates to identify sunflower MPBQ/
MSBQ-MT homologs (Tang et al. 2006). Putative MPBQ/MSBQ- MT ESTs
identified in the initial search were subsequently used as templates in BLAST
searches and identified several additional sunflower ESTs homologous to
Arabidopsis MPBQ/MSBQ-MT. MT-1 and MT-2 cDNA ends were isolated
using 5'- and 3'-rapid amplification of cDNA ends (RACE) on developing
seed RNAs (Tang et al. 2006). Full-length MT-1 and MT-2 cDNA sequences
were isolated using RT-PCR. Full-length MT-1 and MT-2 genomic DNA
sequences were subsequently isolated by sequencing amplicons produced
by long-distance PCR (Tang et al. 2006). MT-1 and m cosegregated and
mapped to LG 1 (Tang et al. 2006). MT-1 represents a non-lethal knock-out
mutation caused by the insertion of a 5.2 kb Ty3/ gypsy -like retrotransposon
in exon 1 (Tang et al. 2006). MT-1 is not transcribed in the mutant
homozygotes ( mm ). MT-2 and the cryptic codominant mutation d
cosegregated with the m locus on LG 4 being epistatic to the d locus. The MT-
2 mutant allele carried a 30 bp insertion at the start of the 5'-UTR which
caused a reduced transcription in seeds and leaves in the homozygous
mutant ( dd ) compared to the wild type (Tang et al. 2006).
The g ( Tph2 ) mutation knocks out a -tocopherol methyltransferase ( -

TMT) activity and causes an accumulation of > 90% -T) in

sunflower seeds (Hass et al. 2006). Using a candidate gene approach as
described for the MT gene isolation, two

-tocopherol (

-TMT paralogs starting with

Arabidopsis -TMT (GeneBank accession nos. AF104220 and At1g64970;

Shintani and DellaPenna 1998; Bergmüller et al. 2003) could be identified
and sequenced in sunflower. The g mutation greatly decreased -TMT-1

transcription, caused alternative splicing of -TMT-2

transcription, and knocked out one of two transcription initiation sites
identified in the wild type. Both

-TMT-1 , disrupted

-TMT paralogs ( -TMT-1 and -TMT-2 )

mapped on LG 8, cosegregate with the g locus (Hass et al. 2006).
In addition, the tocopherolcyclase (TC) was cloned and isolated starting
from the GenBank accession nos. AL022537 and At4g32770 (Porfirova et al.
2002; Sattler et al. 2003). This gene also mapped to LG 8, but segregated
independently from the g locus (Hass et al. 2006).