BACKGROUND
tocopherols are the main antioxidants present in seed oils.
they form a family of four fat-soluble compounds with vitamin e activity named alpha-, beta-, gamma-, and delta tocopherol.
tocopherols contain a 6-chromanol ring structure methylated to varying degrees at positions  <dig>   <dig>  and  <dig>  and an isoprenoid-derived c <dig> saturated side chain at position  <dig>  the four tocopherols differ by the number and positions of the methyl groups on the 6-chromanol ring.
alpha-tocopherol is trimethylated, beta- and gamma-tocopherol are dimethylated, and delta-tocopherol is monomethylated  <cit> .
tocopherols are important antioxidants operating both in vivo, protecting cells from oxidative damage, as well as in vitro, protecting oils and oil-based products from oxidation  <cit> .
the relative in vivo and in vitro antioxidant properties of the specific tocopherols is determined by their chemical structure.
the relative biological activity of the tocopherols is estimated as 100% for alpha-tocopherol,  <dig> to 27% for beta-tocopherol,  <dig> to 20% for gamma-tocopherol, and  <dig>  to 2% for delta-tocopherol  <cit> .
however, there is a widespread confusion concerning their relative potency in vitro <cit> , though it is generally accepted that alpha-tocopherol shows better antioxidant activity in fats and oils at lower concentrations, but at higher concentrations gamma-tocopherol is a more active antioxidant  <cit> .
studies conducted in sunflower seed oil, in which alpha-tocopherol accounts for more than >90% of the total tocopherols, concluded that substitution of alpha-tocopherol by gamma-tocopherol has a positive impact on the stability of the oil  <cit> .
four sunflower germplasms named lg- <dig>  t <dig>  iast- <dig>  and iast- <dig> in which alpha-tocopherol in the seeds was almost completely replaced by gamma-tocopherol have been developed  <cit> .
gamma-tocopherol in the novel germplasm accounts for more than 85% of the total seed tocopherols, compared with more than 90% alpha-tocopherol in conventional sunflower seeds.
the increased gamma-tocopherol levels are produced by recessive alleles at the tph <dig> locus  <cit> , which encodes a gamma-tocopherol methyltransferase  enzyme  <cit> .
this enzyme catalyzes the methylation of delta- and gamma-tocopherol to yield beta- and alpha-tocopherol, respectively  <cit> .
gamma-tmt mutation in sunflower disrupts the synthesis of alpha-tocopherol and causes the accumulation of gamma-tocopherol  <cit> .
in a detailed sequence analysis of the gamma-tmt gene in sunflower, hass et al.
<cit>  identified two gamma-tmt paralogs  and five different haplotypes .
both gamma-tmt paralogs  <dig> and  <dig> cosegregated with tph <dig> and were mapped to linkage group   <dig> of the sunflower linkage map.
even though the tph <dig> mutation reduced or disrupted the expression of the two paralogs in developing sunflower seeds, none of the dna polymorphisms found within the gamma-tmt tph <dig> gene were associated with the high gamma-tocopherol phenotype  <cit> .
the authors suggested that the tph <dig> mutation must be very tightly linked to the gamma-tmt locus on lg  <dig> and may disrupt regulatory sequences.
phenotypic studies of demurin et al.
<cit>  and garcía-moreno et al.
<cit>  concluded that the four high gamma-tocopherol lines lg- <dig>  t <dig>  iast- <dig>  and iast- <dig> possess the same allele at tph <dig>  as no transgressive segregations were observed in crosses involving the four lines.
lines lg- <dig>  t <dig> and iast- <dig> were isolated from germplasm or m <dig> mutant plants that segregated for high alpha-tocopherol  and high gamma-tocopherol , with no intermediate levels of both tocopherol forms being observed  <cit> .
conversely, iast- <dig> was isolated from an m <dig> plant that exhibited large variation for alpha- and gamma-tocopherol levels, with gamma-tocopherol content in m <dig> seeds showing a continuous variation from zero to  <dig> % and m <dig> seeds from selected m <dig> plants showing a variation from  <dig>  to  <dig> % gamma-tocopherol, which was uniformly high  in m <dig> seeds from selected m <dig> plants  <cit> .
garcía-moreno et al.
<cit>  suggested that the intermediate gamma-tocopherol levels observed during the isolation of iast- <dig> might be produced by the presence of modifying genes that determined unstable expression of mutated alleles at tph <dig> locus.
modifying genes have been found to influence important traits in sunflower such as high oleic acid content  <cit>  or broomrape resistance  <cit> .
in this study, the genetic analysis of a population that showed segregation from low to high gamma-tocopherol values obtained from the cross between the two high gamma-tocopherol lines iast- <dig> and nmst <dig> has been carried out.
in the course of this analysis, we identified two different alleles at the gamma-tmt tph <dig> locus at lg  <dig>  the allele tph <dig> was present in line nmst <dig>  whereas the allele tph2a was identified in line iast- <dig>  additionally, we found four modifying genes at lgs  <dig>   <dig>   <dig> and  <dig> that in most cases corresponded to duplicated gamma-tmt loci.
modifying genes influenced the expression of tph2a alleles, but did not affect tph <dig> alleles.
RESULTS
phenotypic segregations
seeds of sunflower lines nmst <dig>  and iast- <dig> showed uniformly high gamma-tocopherol content, from  <dig>  to  <dig> % in nmst <dig> and from  <dig>  to  <dig> % in iast- <dig>  f <dig> seeds from the cross between nmst <dig> and iast- <dig> had also high gamma-tocopherol content, from  <dig>  to  <dig> %.
however, large segregation for gamma-tocopherol content was observed in f <dig> seeds from some f <dig> plants.
particularly, the analysis of 192 f <dig> seeds from an f <dig> plant derived from an f <dig> seed with  <dig> % gamma-tocopherol revealed a continuous large segregation for the trait, from  <dig>  to  <dig> %, with no discrete phenotypic classes being observed and a non-normal distribution  .
variation for gamma-tocopherol content was smaller at the f <dig> plant generation , which segregated from  <dig>  to  <dig> % .
however, examination of variation of individual f <dig> seeds within each f <dig> plant showed that minimum gamma-tocopherol content in individual f <dig> seeds from the different f <dig> families ranged from  <dig>  to  <dig> %, whereas maximum gamma-tocopherol content was in all cases above 94%.
no discrete classes could be distinguished for minimum gamma-tocopherol content in the f2: <dig> population .
map construction and candidate gene mapping
one hundred and sixty three out of  <dig>  simple sequence repeat  and insertion-deletion  markers were polymorphic in the screening of the high gamma-tocopherol lines nmst <dig> and iast- <dig> and four randomly selected f <dig> individuals from their cross.
a set of  <dig> high quality, evenly spaced, and preferably co-dominant markers were genotyped in the f <dig> population.
final linkage map for qtl analyses comprised  <dig> marker loci  grouped on  <dig> linkage groups .
the total genetic distance covered by these marker loci was  <dig>  cm, with an average marker interval of  <dig>  cm .
linkage groups contained between  <dig> and  <dig> marker loci, with  <dig> % of the mapped genome being within 20 cm to the nearest marker.
no marker loci had significantly distorted segregation ratios .
1lg = linkage group.
2ssr = number of ssr loci; indel = number of indel loci.
genome coverage offered by the marker set used for qtl analysis in the nmst <dig> x iast- <dig> population.
distances are expressed in haldane cm.
indel markers for the gamma-tmt gene described by hass et al.
<cit>  were not polymorphic between nmst <dig> and iast- <dig> or showed polymorphism in secondary loci.
therefore, new primer combinations based on the full-lenght sequence of the gamma-tmt gene in nmst <dig> and iast- <dig>  were designed and tested.
a primer combination  based on the forward primers f <dig> and f <dig> placed upstream and dowstream, respectively, of the t <dig> transcription initiation site  <cit> , and a reverse primer r <dig> complementary to the dna sequence in exon  <dig> amplified a primary gamma-tmt locus that was polymorphic between iast- <dig> and nmst <dig> .
the polymorphic band was about 1460 bp in iast- <dig> and 1420 bp in nmst <dig> .
when screened against the individuals from the f <dig> population, the gamma-tmt-f1/f2/r <dig> locus was co-dominantly mapped to lg  <dig> at the previously described position of the gamma-tmt tph <dig> gene  <cit> .
other indel markers for the gamma-tmt gene were also tested.
the f9/r <dig> gamma-tmt indel marker showed three loci in the nmst <dig> and iast- <dig> parental lines .
a primary locus  corresponding to a band of about 1200 bp was not polymorphic with this primer combination .
this locus, however, was demostrated to co-segregate with tph <dig> and was co-dominantly mapped to lg  <dig> in the population cas- <dig> x iast- <dig>  also segregating for gamma-tocopherol content  <cit> .
a second locus  that showed a dominant polymorphism, with a band of about 750 bp present in nmst <dig> and absent in iast- <dig>  was mapped to lg  <dig> .
the locus was also mapped to lg  <dig> in the cas- <dig> x iast- <dig> population.
finally, a third locus  that also showed a dominant polymorphism, with a band of 375 bp absent in nmst <dig> and present in iast- <dig>  was mapped to lg  <dig> .
in addition to the gamma-tmt-f9/r24a, b and c loci, a fourth gamma-tmt locus was identified in populations other than nmst <dig> x iast- <dig>  this locus was named gamma-tmt-f9/r24d and was mapped to lg  <dig> in populations cas- <dig> x iast- <dig>  <cit>  and iast- <dig> x ha- <dig>  .
finally, the locus mt- <dig> of the 2-methyl-6-phytyl- <dig> -benzoquinone/2-methyl-6-solanyl- <dig> -benzoquinone methyltransferase  gene was mapped to lg  <dig> using the f24/r <dig> indel marker  <cit> .
none of the other markers for tocopherol biosynthesis genes tested were polymorphic between nmst <dig> and iast- <dig>
qtl analyses
one-factor analysis of variance revealed that the gamma-tmt-f1/f2/r <dig> locus on lg  <dig> was underlying a major qtl affecting gamma-tocopherol content.
this locus had a significant effect on gamma-tocopherol content of the f <dig> and the f <dig> generations  and corresponded to the gamma-tmt tph <dig> gene also mapped to the same position on lg  <dig> by hass et al.
<cit> .
the mean and the standard deviation for gamma-tocopherol content in the f <dig> seed generation of plants homozygous for the iast- <dig> allele were  <dig>  ±  <dig> %, whereas those for plants homozygous for the nmst <dig> allele were  <dig>  ±  <dig> % .
these results suggested the existence to two different alleles at the gamma-tmt tph <dig> locus in lines nmst <dig> and iast- <dig>  the allele at iast- <dig> was unstable, producing in homozygous condition a broad distribution ranging from  <dig>  to  <dig> % in the f <dig> seed generation, whereas the allele at nmst <dig> was highly stable and resulted in gamma-tocopherol values above 97%.
similar results were observed in the f <dig> seed generation .
it is interesting to note that only three f <dig> plants homozygous for the nmst <dig> allele produced sufficient number of f <dig> seeds for tocopherol analyses, compared to 26 f <dig> plants homozygous for the iast- <dig> allele .
this was probably caused by a close linkage between tph <dig> and ms <dig> loci at lg  <dig>  the latter responsible for male sterility in the nuclear male sterile  line p <dig>  <cit>  from which ms <dig> was introgressed to nmst <dig>
mean values within rows followed by the same letter are not significantly different at  <dig>  level of probability .
association between the gamma-tmt-f1/f2/r <dig> locus on lg  <dig> and gamma-tocopherol content  determined by variance analysis in the nmst <dig> x iast- <dig> population.
mean gamma-tocopherol ± standard deviation  are presented in different genotypic classes: a = homozygous with respect to the allele derived from iast- <dig>  b = homozygous with respect to the allele derived from nmst <dig>  h = heterozygous.
composite interval mapping analyses confirmed the existence of a qtl with a main effect centered on the gamma-tmt-f1/f2/r <dig> locus on lg  <dig> .
this qtl was named tph2-gamma-tmt and explained  <dig> % and  <dig> % of the f <dig> and f <dig> gamma-tocopherol phenotypic variance, respectively .
a second qtl peak on lg  <dig> 16 cm apart from tph2-gamma-tmt was identified only in the qtl analysis of the f <dig> data .
this adjacent peak might be a ghost qtl resulting from the distorted f <dig> data in this region and was not taken into consideration to protect against type i errors in declaring qtls.
** = significant at the  <dig>  and * = significant at the  <dig>  probability level.
1lg = linkage group.
2absolute position from the top of the lg in centimorgans .
3one-lod support interval in centimorgans: refers to the region flanking each qtl peak in which lod scores decline by one.
4markers flanking the likelihood peak for a putative qtl.
<dig> a = additive effect.
a positive sign means an increase of the trait value due to nmst <dig> alleles.
d = dominant effect.
a and d estimates, as well as total r <dig> and lod score values were obtained from a simultaneous fit of all putative qtl using multiple regression.
6qtl detected below the lod threshold.
besides tph2-gamma-tmt, four other qtl with a moderate effect on gamma-tocopherol content were observed at the f <dig> level.
these qtl were located at lg  <dig> ,  <dig> ,  <dig> , and  <dig> .
they individually accounted for  <dig> - <dig> % of trait variation .
the qtl on lg  <dig>   <dig> and  <dig> were also detected in the f <dig> analyses at practically identical positions .
the model with all the qtl explained  <dig> % and  <dig> % of the phenotypic variance for gamma-tocopherol content in the f <dig> and the f <dig> generations, respectively.
the qtl peaks of gamt <dig>  and gamt <dig>  were centered on the gamma-tmt loci mapped at these linkage groups  .
additionally, the qtl peak of gamt <dig>  was likely to correspond to the gamma-tmt-f9/r24d locus mapped to lg  <dig> in populations cas- <dig> x iast- <dig>  <cit>  and iast- <dig> x ha- <dig> , located  <dig>  cm from the zvg <dig> marker locus at this lg.
analysis for epistasis in the f <dig> generation through two-way anova revealed significant interactions for gamma-tocopherol content involving the tph2-gamma-tmt marker locus on lg  <dig> and markers on lg  <dig>  lg  <dig> and lg  <dig>  the most significant interactions involved the ors <dig> marker locus on lg  <dig> , the gamma-tmt_f9r24b locus on lg  <dig> , and the gamma-tmt_f9r24c locus on lg  <dig> , which were associated to the gamt <dig> , gamt <dig> , and gamt <dig>  qtl, respectively.
significant interactions involving the tph2-gamma-tmt qtl on lg  <dig>  the gamt <dig>  on lg  <dig>  and the gamt <dig>  qtl on lg  <dig> were also detected in the composite interval mapping analyses, with the percentage of explained phenotypic variance increasing from  <dig> % to  <dig> % in the f <dig> and from  <dig> % to  <dig> % in the f <dig> when epistatic interactions were included in the multiple-locus model .
genotypic means for f <dig> gamma-tocopherol content in allelic combinations of the epistatically interacting marker loci were calculated to detail these interactions.
since ors <dig> and gamma-tmt_f9r24b marker loci were dominant, genotypic means were computed using close co-dominant markers, ors <dig> on lg  <dig> and ors <dig> on lg  <dig> .
the results showed that qtl regions on lg  <dig> and lg  <dig> only had a significant phenotypic effect when the iast- <dig> allele was present at the tph2-gamma-tmt locus on lg  <dig> .
thus, no significant differences were observed between different genotypes at ors <dig> on lg  <dig> or ors <dig> on lg  <dig> in presence of the nmst <dig> allele at tph <dig>  however, when the tph <dig> locus was homozygous for the iast- <dig> allele, the presence of nmst <dig> alleles at ors <dig> or ors <dig> resulted in a drastic reduction of gamma-tocopherol content as compared to the presence of iast- <dig> alleles .
values within columns followed by the same letter are not significantly different at  <dig>  level of probability .
1mean and standard deviation  for f <dig> gamma-t content and number of f <dig> individuals per genotypic class .
genotypic means for f <dig> gamma-tocopherol  content  in allelic combinations at epistatically interacting marker loci.
a = homozygous with respect to the allele derived from iast- <dig>  b = homozygous with respect to the allele derived from nmst <dig>  h = heterozygous.
sequence analysis of gamma-tmt loci
several loci amplified by the gamma-tmt f9/r <dig> indel marker in iast- <dig> and nmst <dig>  were cloned and sequenced.
a band of about 1200 bp from the non-polymorphic locus a and a band of about 375 bp from the dominant locus c that mapped to lg  <dig> were sequenced from iast- <dig>  a band of about 1200 bp from the non-polymorphic locus a and a band of about 750 bp from the dominant locus b that mapped to lg  <dig> were sequenced from nmst <dig>  the locus d that mapped to lg  <dig> was sequenced from lines iast- <dig> and ha- <dig>
the locus a fragment isolated from nmst <dig> harboured alleles from both gamma-tmt paralogs, which showed showed 100% sequence identity to gamma-tmt haplotype  <dig>  and  <dig>  from hass et al.
<cit> .
for iast- <dig>  the locus a sequence showed 100% sequence identity to gamma-tmt haplotype  <dig>  the locus b consensus sequence was 785 bp long and showed a significant homology to gamma-tmt haplotypes  <dig> and  <dig> , with 94% maximum sequence identity spanning 61% of the locus b fragment coverage.
the consensus sequence for locus c  showed a significant homology to gamma-tmt haplotypes  <dig> and  <dig> , with 86% maximum sequence identity spanning 19% of the locus c fragment coverage.
the sequences of locus d from iast- <dig>  and ha- <dig>  were very similar, with a 96% of sequence identity between them.
the consensus sequence obtained from this alignment showed significant homology to gamma-tmt haplotypes  <dig>   <dig>   <dig> and  <dig> , with 89% maximum sequence identity spanning 18% of the fragment coverage.
nucleotide sequences from iast- <dig> locus a, nmst <dig> locus a, nmst <dig> locus b, iast- <dig> locus c, ha- <dig> locus d, and iast- <dig> locus d were aligned.
the sequence alignment tree  revealed great similarity between loci a and b  and between loci c and d .
both groups were more distantly related, with  <dig> to 58% of sequence identity between locus a and the cluster of loci c and d.
full-length gamma-tmt genomic dna sequences
agarose gels from pcr products obtained with the f <dig> and r <dig> primers flanking the gamma-tmt gene mapped to lg  <dig>  <cit>  revealed two bands in nmst <dig> and iast- <dig> lines that were identified as the two gamma-tmt paralogs  <dig> and  <dig> described by hass et al.
<cit> .
consensus nucleotide sequences for iast- <dig> gamma-tmt paralog  <dig>  and paralog  <dig>  were identical to gamma-tmt haplotype  <dig>  and  <dig> , respectively, from hass et al.
<cit>  .
consensus nucleotide sequence for nmst <dig> gamma-tmt paralog  <dig>  was identical to gamma-tmt haplotype  <dig>  <cit>  .
consensus nucleotide sequence for nmst <dig> gamma-tmt paralog  <dig>  was almost identical to gamma-tmt haplotype  <dig>  <cit> , with the exception of five snps, one in the 5´utr , one in intron  <dig> , and three in intron  <dig>  .
discussion
the results of this research suggest that high gamma-tocopherol lines iast- <dig> and nmst <dig> possess different mutated alleles at the gamma-tmt tph <dig> locus on lg  <dig>  the gamma-tmt catalyses the methylation step from gamma-tocopherol to alpha-tocopherol in sunflower seeds  <cit> .
mutated tph <dig> alleles disrupt the activity of gamma-tmt, which results in accumulation of gamma-tocopherol  <cit> .
the allele at nmst <dig>  is stable and not affected by modifying genes, whereas the allele at iast- <dig>  is unstable and affected by modifying genes.
putative modifying genes located at lg  <dig>   <dig>   <dig> and  <dig> were identified in the present research, being the effect of those on lg  <dig>   <dig> and  <dig> highly significant in the epistatic interaction with the tph <dig> locus on lg  <dig>  studies on expression of tph <dig> mutations in plant tissues other than seeds also pointed to differences between t <dig> and iast- <dig> lines, as the latter showed lower gamma-tocopherol content in leaves, roots, and pollen  <cit> .
high gamma-tocopherol lines t <dig> and iast- <dig> were isolated following different strategies.
t <dig> derived from an open-pollinated cultivar that showed variation for high gamma-tocopherol content  at the single-seed level  <cit> .
genetic study of the trait in t <dig> seeds indicated that the trait was controlled by recessive alleles at a single locus tph <dig> in such a way that tph2tph <dig> and tph2tph <dig> genotypes produced low levels of gamma-tocopherol  and tph2tph <dig> genotypes showed high levels of gamma-tocopherol , with no intermediate levels being observed  <cit> .
on the contrary, iast- <dig> derived from a mutagenesis program in which an m <dig> seed with intermediate gamma-tocopherol content  was identified.
the trait showed continuous variation for gamma-tocopherol levels  in the m <dig> generation, which allowed selection of genotypes with stable high gamma-tocopherol content in the m <dig> generation  <cit> .
a comparative genetic study between both lines concluded that they shared the same allele at tph <dig>  as no transgressive segregation was observed in the f <dig> and f <dig> generations from crosses between them  <cit> .
however, the evaluation of several f <dig> populations from crosses of iast- <dig> with t <dig> and the conventional sunflower line ha <dig> showed that intermediate gamma-tocopherol values  occurred in some f <dig> populations, whereas other f <dig> populations showed uniformly high gamma-tocopherol content  or segregated into well-defined low and high gamma-tocopherol classes   <cit> .
the results of this research suggest that intermediate gamma-tocopherol levels are produced by the effect of modifying genes on tph2a alleles from iast- <dig>  we hypothesize that modifying genes were present in the mutagenized population from which iast- <dig> was developed, where selection against negative alleles at modifying genes led to the isolation of iast- <dig>  and also in other lines such as ha <dig> and t <dig>  modifying genes have no phenotypic effect in absence of alteration in the major gene  <cit> .
in this research, it was also found that modifying genes have no phenotypic effect on the mutated allele at t <dig> .
accordingly, they are expected to be segregating in ha <dig> and t <dig> lines, which produced different segregation patterns in crosses with iast- <dig>  <cit> .
the occurrence of different segregation patterns depending on allelic configuration of modifying genes has been previously reported in sunflower for high oleic acid content  <cit>  and broomrape resistance  <cit> .
full length genomic gamma-tmt sequences corresponding to the tph <dig> locus on lg  <dig> have been obtained in this study for both iast- <dig> and nmst <dig>  both lines have two gamma-tmt paralogs.
paralog  <dig> is identical between the two lines and also to the gamma-tmt haplotype  <dig> from hass et al.
<cit> , which included sequences from both high and low gamma-tocopherol lines.
paralog  <dig> from iast- <dig> is identical to gamma-tmt haplotype  <dig> from hass et al.
<cit> , which also included sequences from both high and low gamma-tocopherol lines, while paralog  <dig> from nmst <dig> carries slight snp differences.
consequently, we have not found any sequence difference in the gamma-tmt gene from iast- <dig> that would explain why the tph2a allele present in this line is unstable and affected by modifying genes.
previous research did not identify sequence differences in the gamma-tmt gene related to the high gamma-tocopherol phenotype  <cit> .
the authors found identical gamma-tmt paralog  <dig> and  <dig> sequences in both high and low gamma-tocopherol lines and reported that mutation leading to the high gamma-tocopherol trait in the sunflower material derived from the high gamma-tocopherol line lg- <dig>  <cit>  may disrupt regulatory sequences of the gamma-tmt gene.
relating the nmst <dig> line, the slight sequence differences in gamma-tmt paralog  <dig> were changes to those nucleotides also present in paralog  <dig> sequences from iast- <dig> and nmst <dig>  or found in a repetitive sequence within an intron  and were not likely to be involved in determining a more stable gamma-tmt allele.
modifying genes affecting important traits for sunflower breeding such as high oleic acid content  <cit>  and broomrape resistance  <cit>  have been reported.
the discovery of modifying genes affecting high gamma-tocopherol content confirms that the occurrence of modifying genes is not a rare phenomenon in sunflower genetics.
thus far there was no indication on the nature and mode of action of modifying genes.
the results of the present research suggest that tph <dig> modifying genes are in most cases additional gamma-tmt loci duplicated in the sunflower genome.
this was confirmed for modifying genes at lg  <dig> and  <dig>  whereas there is also indication that the modifying gene at lg  <dig> might be an additional copy of a gamma-tmt.
duplicated gamma-tmt loci have also been found in safflower  <cit>  and rapeseed  <cit> .
in fact, the two gamma-tmt loci from safflower  were identified by using the f9/r <dig> primer combination from the sunflower gamma-tmt  <cit> , the same one used in this study to map different gamma-tmt loci.
these results suggested that the f <dig> and r <dig> primer sequences are within a highly conserved region of the gamma-tmt gene.
in sunflower, hass et al.
<cit>  mapped, in addition to gamma-tmt-tph <dig> on lg  <dig>  another gamma-tmt locus on lg  <dig>  although the authors considered this locus a randomly amplified polymorphic dna .
in this study, the locus on lg  <dig>  showed significant homology to the sunflower gamma-tmt gene and underlay a gamma-tocopherol qtl.
our results indicated that interaction between duplicated gamma-tmt loci revert the high gamma-tocopherol phenotype to intermediate-low gamma-tocopherol values.
this effect has also been reported for modifying genes affecting the high oleic acid phenotype in sunflower, where modifying genes suppressed the effect of the ol-fad2- <dig> allele that is essential for accumulating high oleic acid content  <cit> .
also, epistatically interacting duplicated mpbq/msbq-mt genes that modify beta-tocopherol levels have been reported in sunflower  <cit> .
it is well known that duplicate gene pairs can form negative epistasis due to their overlapping functions  <cit>  and that suppression of a mutant phenotype can be altered by gene dosage  <cit> .
however, additional biochemical, functional, and sequence analyses are required to determine the mode of action of duplicated gamma-tmt loci in sunflower.
from a breeding perspective, the existence of several modifying genes influencing high gamma-tocopherol content should not be a constraint for the development of cultivars with seeds rich in gamma-tocopherol, as this research revealed that modifying genes do not alter the phenotypic expression of the mutated allele tph <dig> present at nmst <dig>  on the other hand, the influence of modifying genes on expressivity of mutated tph2a alleles opens up the possibility of selection for balanced levels of alpha- and gamma-tocopherol content, provided that stable combinations of modifying genes can be obtained.
the development of mid oleic acid hybrids of sunflower was based on genetic stocks carrying modifying genes that limit the expression of high oleic acid content produced by mutated alleles at ol locus  to the mid-range level   <cit> .
in this sense, determination of allelic diversity at gamma-tmt loci underlying gamma-tocopherol modifying genes present in hybrid parental lines might be a useful tool to predict the presence of adequate allelic combinations giving rise to the desired levels of gamma-tocopherol content.
further studies should elucidate whether the other two gamma-tocopherol sources developed thus far, iast- <dig> and lg- <dig>  possess stable or unstable alleles at tph <dig>  demurin et al.
<cit>  reported differences in the expressivity of the recessive homozygotes of the tph <dig> gene from lg- <dig> in different genetic backgrounds, which might be indicative of allelic instability.
CONCLUSIONS
the results of this research suggest that high gamma-tocopherol in sunflower lines iast- <dig> and nmst <dig> is determined by different mutated alleles at the gamma-tmt tph <dig> locus on lg  <dig>  the allele at nmst <dig> is not affected by modifying genes, whereas the allele at iast- <dig> is unstable and affected by modifying genes.
putative modifying genes have been located at lg  <dig>   <dig>   <dig> and  <dig>  being the effect of those on lg  <dig>   <dig> and  <dig> highly significant in their epistatic interaction with the tph <dig> locus on lg  <dig>  finally, our results suggest that modifying genes correspond to gamma-tmt loci duplicated in the sunflower genome.
phenotypic effects of modifying genes altering the expression of important genes in crop plants are known  <cit> , but in most cases the genetic basis for modification remains unclear.
in this study, we shed light into the mode of action and nature of modifier genes in sunflower, and suggest the relevance of duplicated loci affecting the expression of seed quality specific mutations.

 tocopherols are the main antioxidants present in seed oils. they form a family of four fat-soluble compounds with vitamin e activity named alpha-, beta-, gamma-, and delta tocopherol. tocopherols contain a 6-chromanol ring structure methylated to varying degrees at positions and and an isoprenoid-derived c saturated side chain at position the four tocopherols differ by the number and positions of the methyl groups on the 6-chromanol ring. alpha-tocopherol is trimethylated, beta- and gamma-tocopherol are dimethylated, and delta-tocopherol is monomethylated. tocopherols are important antioxidants operating both in vivo, protecting cells from oxidative damage, as well as in vitro, protecting oils and oil-based products from oxidation. the relative in vivo and in vitro antioxidant properties of the specific tocopherols is determined by their chemical structure. the relative biological activity of the tocopherols is estimated as 100% for alpha-tocopherol, to 27% for beta-tocopherol, to 20% for gamma-tocopherol, and to 2% for delta-tocopherol. however, there is a widespread confusion concerning their relative potency in vitro, though it is generally accepted that alpha-tocopherol shows better antioxidant activity in fats and oils at lower concentrations, but at higher concentrations gamma-tocopherol is a more active antioxidant. studies conducted in sunflower seed oil, in which alpha-tocopherol accounts for more than >90% of the total tocopherols, concluded that substitution of alpha-tocopherol by gamma-tocopherol has a positive impact on the stability of the oil. four sunflower germplasms named lg- t iast- and iast- in which alpha-tocopherol in the seeds was almost completely replaced by gamma-tocopherol have been developed. gamma-tocopherol in the novel germplasm accounts for more than 85% of the total seed tocopherols, compared with more than 90% alpha-tocopherol in conventional sunflower seeds. the increased gamma-tocopherol levels are produced by recessive alleles at the tph locus, which encodes a gamma-tocopherol methyltransferase enzyme. this enzyme catalyzes the methylation of delta- and gamma-tocopherol to yield beta- and alpha-tocopherol, respectively. gamma-tmt mutation in sunflower disrupts the synthesis of alpha-tocopherol and causes the accumulation of gamma-tocopherol. in a detailed sequence analysis of the gamma-tmt gene in sunflower, hass et al. identified two gamma-tmt paralogs and five different haplotypes. both gamma-tmt paralogs and cosegregated with tph and were mapped to linkage group of the sunflower linkage map. even though the tph mutation reduced or disrupted the expression of the two paralogs in developing sunflower seeds, none of the dna polymorphisms found within the gamma-tmt tph gene were associated with the high gamma-tocopherol phenotype. the authors suggested that the tph mutation must be very tightly linked to the gamma-tmt locus on lg and may disrupt regulatory sequences. phenotypic studies of demurin et al. and garcía-moreno et al. concluded that the four high gamma-tocopherol lines lg- t iast- and iast- possess the same allele at tph as no transgressive segregations were observed in crosses involving the four lines. lines lg- t and iast- were isolated from germplasm or m mutant plants that segregated for high alpha-tocopherol and high gamma-tocopherol, with no intermediate levels of both tocopherol forms being observed. conversely, iast- was isolated from an m plant that exhibited large variation for alpha- and gamma-tocopherol levels, with gamma-tocopherol content in m seeds showing a continuous variation from zero to % and m seeds from selected m plants showing a variation from to % gamma-tocopherol, which was uniformly high in m seeds from selected m plants. garcía-moreno et al. suggested that the intermediate gamma-tocopherol levels observed during the isolation of iast- might be produced by the presence of modifying genes that determined unstable expression of mutated alleles at tph locus. modifying genes have been found to influence important traits in sunflower such as high oleic acid content or broomrape resistance. in this study, the genetic analysis of a population that showed segregation from low to high gamma-tocopherol values obtained from the cross between the two high gamma-tocopherol lines iast- and nmst has been carried out. in the course of this analysis, we identified two different alleles at the gamma-tmt tph locus at lg the allele tph was present in line nmst whereas the allele tph2a was identified in line iast- additionally, we found four modifying genes at lgs and that in most cases corresponded to duplicated gamma-tmt loci. modifying genes influenced the expression of tph2a alleles, but did not affect tph alleles.  phenotypic segregations seeds of sunflower lines nmst and iast- showed uniformly high gamma-tocopherol content, from to % in nmst and from to % in iast- f seeds from the cross between nmst and iast- had also high gamma-tocopherol content, from to %. however, large segregation for gamma-tocopherol content was observed in f seeds from some f plants. particularly, the analysis of 192 f seeds from an f plant derived from an f seed with % gamma-tocopherol revealed a continuous large segregation for the trait, from to %, with no discrete phenotypic classes being observed and a non-normal distribution. variation for gamma-tocopherol content was smaller at the f plant generation, which segregated from to %. however, examination of variation of individual f seeds within each f plant showed that minimum gamma-tocopherol content in individual f seeds from the different f families ranged from to %, whereas maximum gamma-tocopherol content was in all cases above 94%. no discrete classes could be distinguished for minimum gamma-tocopherol content in the f2: population. map construction and candidate gene mapping one hundred and sixty three out of simple sequence repeat and insertion-deletion markers were polymorphic in the screening of the high gamma-tocopherol lines nmst and iast- and four randomly selected f individuals from their cross. a set of high quality, evenly spaced, and preferably co-dominant markers were genotyped in the f population. final linkage map for qtl analyses comprised marker loci grouped on linkage groups. the total genetic distance covered by these marker loci was  cm, with an average marker interval of  cm. linkage groups contained between and marker loci, with % of the mapped genome being within 20 cm to the nearest marker. no marker loci had significantly distorted segregation ratios. 1lg = linkage group. 2ssr = number of ssr loci; indel = number of indel loci. genome coverage offered by the marker set used for qtl analysis in the nmst x iast- population. distances are expressed in haldane cm. indel markers for the gamma-tmt gene described by hass et al. were not polymorphic between nmst and iast- or showed polymorphism in secondary loci. therefore, new primer combinations based on the full-lenght sequence of the gamma-tmt gene in nmst and iast- were designed and tested. a primer combination based on the forward primers f and f placed upstream and dowstream, respectively, of the t transcription initiation site, and a reverse primer r complementary to the dna sequence in exon amplified a primary gamma-tmt locus that was polymorphic between iast- and nmst. the polymorphic band was about 1460 bp in iast- and 1420 bp in nmst. when screened against the individuals from the f population, the gamma-tmt-f1/f2/r locus was co-dominantly mapped to lg at the previously described position of the gamma-tmt tph gene. other indel markers for the gamma-tmt gene were also tested. the f9/r gamma-tmt indel marker showed three loci in the nmst and iast- parental lines. a primary locus corresponding to a band of about 1200 bp was not polymorphic with this primer combination. this locus, however, was demostrated to co-segregate with tph and was co-dominantly mapped to lg in the population cas- x iast- also segregating for gamma-tocopherol content. a second locus that showed a dominant polymorphism, with a band of about 750 bp present in nmst and absent in iast- was mapped to lg. the locus was also mapped to lg in the cas- x iast- population. finally, a third locus that also showed a dominant polymorphism, with a band of 375 bp absent in nmst and present in iast- was mapped to lg. in addition to the gamma-tmt-f9/r24a, b and c loci, a fourth gamma-tmt locus was identified in populations other than nmst x iast- this locus was named gamma-tmt-f9/r24d and was mapped to lg in populations cas- x iast- and iast- x ha-. finally, the locus mt- of the 2-methyl-6-phytyl- -benzoquinone/2-methyl-6-solanyl- -benzoquinone methyltransferase gene was mapped to lg using the f24/r indel marker. none of the other markers for tocopherol biosynthesis genes tested were polymorphic between nmst and iast- qtl analyses one-factor analysis of variance revealed that the gamma-tmt-f1/f2/r locus on lg was underlying a major qtl affecting gamma-tocopherol content. this locus had a significant effect on gamma-tocopherol content of the f and the f generations and corresponded to the gamma-tmt tph gene also mapped to the same position on lg by hass et al.. the mean and the standard deviation for gamma-tocopherol content in the f seed generation of plants homozygous for the iast- allele were  ±  %, whereas those for plants homozygous for the nmst allele were  ±  %. these results suggested the existence to two different alleles at the gamma-tmt tph locus in lines nmst and iast- the allele at iast- was unstable, producing in homozygous condition a broad distribution ranging from to % in the f seed generation, whereas the allele at nmst was highly stable and resulted in gamma-tocopherol values above 97%. similar results were observed in the f seed generation. it is interesting to note that only three f plants homozygous for the nmst allele produced sufficient number of f seeds for tocopherol analyses, compared to 26 f plants homozygous for the iast- allele. this was probably caused by a close linkage between tph and ms loci at lg the latter responsible for male sterility in the nuclear male sterile line p from which ms was introgressed to nmst mean values within rows followed by the same letter are not significantly different at level of probability. association between the gamma-tmt-f1/f2/r locus on lg and gamma-tocopherol content determined by variance analysis in the nmst x iast- population. mean gamma-tocopherol ± standard deviation are presented in different genotypic classes: a = homozygous with respect to the allele derived from iast- b = homozygous with respect to the allele derived from nmst h = heterozygous. composite interval mapping analyses confirmed the existence of a qtl with a main effect centered on the gamma-tmt-f1/f2/r locus on lg. this qtl was named tph2-gamma-tmt and explained % and % of the f and f gamma-tocopherol phenotypic variance, respectively. a second qtl peak on lg 16 cm apart from tph2-gamma-tmt was identified only in the qtl analysis of the f data. this adjacent peak might be a ghost qtl resulting from the distorted f data in this region and was not taken into consideration to protect against type i errors in declaring qtls. ** = significant at the and * = significant at the probability level. 1lg = linkage group. 2absolute position from the top of the lg in centimorgans. 3one-lod support interval in centimorgans: refers to the region flanking each qtl peak in which lod scores decline by one. 4markers flanking the likelihood peak for a putative qtl. a = additive effect. a positive sign means an increase of the trait value due to nmst alleles. d = dominant effect. a and d estimates, as well as total r and lod score values were obtained from a simultaneous fit of all putative qtl using multiple regression. 6qtl detected below the lod threshold. besides tph2-gamma-tmt, four other qtl with a moderate effect on gamma-tocopherol content were observed at the f level. these qtl were located at lg,,, and. they individually accounted for - % of trait variation. the qtl on lg and were also detected in the f analyses at practically identical positions. the model with all the qtl explained % and % of the phenotypic variance for gamma-tocopherol content in the f and the f generations, respectively. the qtl peaks of gamt and gamt were centered on the gamma-tmt loci mapped at these linkage groups. additionally, the qtl peak of gamt was likely to correspond to the gamma-tmt-f9/r24d locus mapped to lg in populations cas- x iast- and iast- x ha-, located  cm from the zvg marker locus at this lg. analysis for epistasis in the f generation through two-way anova revealed significant interactions for gamma-tocopherol content involving the tph2-gamma-tmt marker locus on lg and markers on lg lg and lg the most significant interactions involved the ors marker locus on lg, the gamma-tmt_f9r24b locus on lg, and the gamma-tmt_f9r24c locus on lg, which were associated to the gamt, gamt, and gamt qtl, respectively. significant interactions involving the tph2-gamma-tmt qtl on lg the gamt on lg and the gamt qtl on lg were also detected in the composite interval mapping analyses, with the percentage of explained phenotypic variance increasing from % to % in the f and from % to % in the f when epistatic interactions were included in the multiple-locus model. genotypic means for f gamma-tocopherol content in allelic combinations of the epistatically interacting marker loci were calculated to detail these interactions. since ors and gamma-tmt_f9r24b marker loci were dominant, genotypic means were computed using close co-dominant markers, ors on lg and ors on lg. the results showed that qtl regions on lg and lg only had a significant phenotypic effect when the iast- allele was present at the tph2-gamma-tmt locus on lg. thus, no significant differences were observed between different genotypes at ors on lg or ors on lg in presence of the nmst allele at tph however, when the tph locus was homozygous for the iast- allele, the presence of nmst alleles at ors or ors resulted in a drastic reduction of gamma-tocopherol content as compared to the presence of iast- alleles. values within columns followed by the same letter are not significantly different at level of probability. 1mean and standard deviation for f gamma-t content and number of f individuals per genotypic class. genotypic means for f gamma-tocopherol content in allelic combinations at epistatically interacting marker loci. a = homozygous with respect to the allele derived from iast- b = homozygous with respect to the allele derived from nmst h = heterozygous. sequence analysis of gamma-tmt loci several loci amplified by the gamma-tmt f9/r indel marker in iast- and nmst were cloned and sequenced. a band of about 1200 bp from the non-polymorphic locus a and a band of about 375 bp from the dominant locus c that mapped to lg were sequenced from iast- a band of about 1200 bp from the non-polymorphic locus a and a band of about 750 bp from the dominant locus b that mapped to lg were sequenced from nmst the locus d that mapped to lg was sequenced from lines iast- and ha- the locus a fragment isolated from nmst harboured alleles from both gamma-tmt paralogs, which showed showed 100% sequence identity to gamma-tmt haplotype and from hass et al.. for iast- the locus a sequence showed 100% sequence identity to gamma-tmt haplotype the locus b consensus sequence was 785 bp long and showed a significant homology to gamma-tmt haplotypes and, with 94% maximum sequence identity spanning 61% of the locus b fragment coverage. the consensus sequence for locus c showed a significant homology to gamma-tmt haplotypes and, with 86% maximum sequence identity spanning 19% of the locus c fragment coverage. the sequences of locus d from iast- and ha- were very similar, with a 96% of sequence identity between them. the consensus sequence obtained from this alignment showed significant homology to gamma-tmt haplotypes and, with 89% maximum sequence identity spanning 18% of the fragment coverage. nucleotide sequences from iast- locus a, nmst locus a, nmst locus b, iast- locus c, ha- locus d, and iast- locus d were aligned. the sequence alignment tree revealed great similarity between loci a and b and between loci c and d. both groups were more distantly related, with to 58% of sequence identity between locus a and the cluster of loci c and d. full-length gamma-tmt genomic dna sequences agarose gels from pcr products obtained with the f and r primers flanking the gamma-tmt gene mapped to lg revealed two bands in nmst and iast- lines that were identified as the two gamma-tmt paralogs and described by hass et al.. consensus nucleotide sequences for iast- gamma-tmt paralog and paralog were identical to gamma-tmt haplotype and, respectively, from hass et al.. consensus nucleotide sequence for nmst gamma-tmt paralog was identical to gamma-tmt haplotype. consensus nucleotide sequence for nmst gamma-tmt paralog was almost identical to gamma-tmt haplotype, with the exception of five snps, one in the 5´utr, one in intron, and three in intron. discussion the results of this research suggest that high gamma-tocopherol lines iast- and nmst possess different mutated alleles at the gamma-tmt tph locus on lg the gamma-tmt catalyses the methylation step from gamma-tocopherol to alpha-tocopherol in sunflower seeds. mutated tph alleles disrupt the activity of gamma-tmt, which results in accumulation of gamma-tocopherol. the allele at nmst is stable and not affected by modifying genes, whereas the allele at iast- is unstable and affected by modifying genes. putative modifying genes located at lg and were identified in the present research, being the effect of those on lg and highly significant in the epistatic interaction with the tph locus on lg studies on expression of tph mutations in plant tissues other than seeds also pointed to differences between t and iast- lines, as the latter showed lower gamma-tocopherol content in leaves, roots, and pollen. high gamma-tocopherol lines t and iast- were isolated following different strategies. t derived from an open-pollinated cultivar that showed variation for high gamma-tocopherol content at the single-seed level. genetic study of the trait in t seeds indicated that the trait was controlled by recessive alleles at a single locus tph in such a way that tph2tph and tph2tph genotypes produced low levels of gamma-tocopherol and tph2tph genotypes showed high levels of gamma-tocopherol, with no intermediate levels being observed. on the contrary, iast- derived from a mutagenesis program in which an m seed with intermediate gamma-tocopherol content was identified. the trait showed continuous variation for gamma-tocopherol levels in the m generation, which allowed selection of genotypes with stable high gamma-tocopherol content in the m generation. a comparative genetic study between both lines concluded that they shared the same allele at tph as no transgressive segregation was observed in the f and f generations from crosses between them. however, the evaluation of several f populations from crosses of iast- with t and the conventional sunflower line ha showed that intermediate gamma-tocopherol values occurred in some f populations, whereas other f populations showed uniformly high gamma-tocopherol content or segregated into well-defined low and high gamma-tocopherol classes. the results of this research suggest that intermediate gamma-tocopherol levels are produced by the effect of modifying genes on tph2a alleles from iast- we hypothesize that modifying genes were present in the mutagenized population from which iast- was developed, where selection against negative alleles at modifying genes led to the isolation of iast- and also in other lines such as ha and t modifying genes have no phenotypic effect in absence of alteration in the major gene. in this research, it was also found that modifying genes have no phenotypic effect on the mutated allele at t. accordingly, they are expected to be segregating in ha and t lines, which produced different segregation patterns in crosses with iast-. the occurrence of different segregation patterns depending on allelic configuration of modifying genes has been previously reported in sunflower for high oleic acid content and broomrape resistance. full length genomic gamma-tmt sequences corresponding to the tph locus on lg have been obtained in this study for both iast- and nmst both lines have two gamma-tmt paralogs. paralog is identical between the two lines and also to the gamma-tmt haplotype from hass et al., which included sequences from both high and low gamma-tocopherol lines. paralog from iast- is identical to gamma-tmt haplotype from hass et al., which also included sequences from both high and low gamma-tocopherol lines, while paralog from nmst carries slight snp differences. consequently, we have not found any sequence difference in the gamma-tmt gene from iast- that would explain why the tph2a allele present in this line is unstable and affected by modifying genes. previous research did not identify sequence differences in the gamma-tmt gene related to the high gamma-tocopherol phenotype. the authors found identical gamma-tmt paralog and sequences in both high and low gamma-tocopherol lines and reported that mutation leading to the high gamma-tocopherol trait in the sunflower material derived from the high gamma-tocopherol line lg- may disrupt regulatory sequences of the gamma-tmt gene. relating the nmst line, the slight sequence differences in gamma-tmt paralog were changes to those nucleotides also present in paralog sequences from iast- and nmst or found in a repetitive sequence within an intron and were not likely to be involved in determining a more stable gamma-tmt allele. modifying genes affecting important traits for sunflower breeding such as high oleic acid content and broomrape resistance have been reported. the discovery of modifying genes affecting high gamma-tocopherol content confirms that the occurrence of modifying genes is not a rare phenomenon in sunflower genetics. thus far there was no indication on the nature and mode of action of modifying genes. the results of the present research suggest that tph modifying genes are in most cases additional gamma-tmt loci duplicated in the sunflower genome. this was confirmed for modifying genes at lg and whereas there is also indication that the modifying gene at lg might be an additional copy of a gamma-tmt. duplicated gamma-tmt loci have also been found in safflower and rapeseed. in fact, the two gamma-tmt loci from safflower were identified by using the f9/r primer combination from the sunflower gamma-tmt, the same one used in this study to map different gamma-tmt loci. these results suggested that the f and r primer sequences are within a highly conserved region of the gamma-tmt gene. in sunflower, hass et al. mapped, in addition to gamma-tmt-tph on lg another gamma-tmt locus on lg although the authors considered this locus a randomly amplified polymorphic dna. in this study, the locus on lg showed significant homology to the sunflower gamma-tmt gene and underlay a gamma-tocopherol qtl. our results indicated that interaction between duplicated gamma-tmt loci revert the high gamma-tocopherol phenotype to intermediate-low gamma-tocopherol values. this effect has also been reported for modifying genes affecting the high oleic acid phenotype in sunflower, where modifying genes suppressed the effect of the ol-fad2- allele that is essential for accumulating high oleic acid content. also, epistatically interacting duplicated mpbq/msbq-mt genes that modify beta-tocopherol levels have been reported in sunflower. it is well known that duplicate gene pairs can form negative epistasis due to their overlapping functions and that suppression of a mutant phenotype can be altered by gene dosage. however, additional biochemical, functional, and sequence analyses are required to determine the mode of action of duplicated gamma-tmt loci in sunflower. from a breeding perspective, the existence of several modifying genes influencing high gamma-tocopherol content should not be a constraint for the development of cultivars with seeds rich in gamma-tocopherol, as this research revealed that modifying genes do not alter the phenotypic expression of the mutated allele tph present at nmst on the other hand, the influence of modifying genes on expressivity of mutated tph2a alleles opens up the possibility of selection for balanced levels of alpha- and gamma-tocopherol content, provided that stable combinations of modifying genes can be obtained. the development of mid oleic acid hybrids of sunflower was based on genetic stocks carrying modifying genes that limit the expression of high oleic acid content produced by mutated alleles at ol locus to the mid-range level. in this sense, determination of allelic diversity at gamma-tmt loci underlying gamma-tocopherol modifying genes present in hybrid parental lines might be a useful tool to predict the presence of adequate allelic combinations giving rise to the desired levels of gamma-tocopherol content. further studies should elucidate whether the other two gamma-tocopherol sources developed thus far, iast- and lg- possess stable or unstable alleles at tph demurin et al. reported differences in the expressivity of the recessive homozygotes of the tph gene from lg- in different genetic backgrounds, which might be indicative of allelic instability.  the results of this research suggest that high gamma-tocopherol in sunflower lines iast- and nmst is determined by different mutated alleles at the gamma-tmt tph locus on lg the allele at nmst is not affected by modifying genes, whereas the allele at iast- is unstable and affected by modifying genes. putative modifying genes have been located at lg and being the effect of those on lg and highly significant in their epistatic interaction with the tph locus on lg finally, our results suggest that modifying genes correspond to gamma-tmt loci duplicated in the sunflower genome. phenotypic effects of modifying genes altering the expression of important genes in crop plants are known, but in most cases the genetic basis for modification remains unclear. in this study, we shed light into the mode of action and nature of modifier genes in sunflower, and suggest the relevance of duplicated loci affecting the expression of seed quality specific mutations. 
sunflower seeds contain predominantly alpha-tocopherol, with maximum vitamin e effect but lower in vitro antioxidant action than other tocopherol forms such as gamma-tocopherol. the objective of this research was to determine the genetic basis of unstable expression of high gamma-tocopherol content in sunflower seeds. no sequence differences at the gamma-tmt gene were found associated to allelic unstability. the trait is controlled by recessive alleles at a single locus tph underlying a gamma-tocopherol methyltransferase. male sterile plants of nuclear male sterile line nmst with stable high gamma-tocopherol content, were crossed with plants of line iast- with stable high gamma-tocopherol content but derived from a population that exhibited unstable expression of the trait. unstable expression of high gamma-tocopherol content is produced by the effect of modifying genes on tph2a allele at the gamma-tmt tph gene. a genetic linkage map comprising linkage groups was constructed from this population using ssr and indel marker loci, including indel markers for tocopherol biosynthesis genes. qtl analysis revealed a major qtl on lg that corresponded to the gamma-tmt tph locus, which suggested that high gamma-tocopherol lines nmst and iast- possess different alleles at this locus. our results suggested that modifying genes are mostly epistatically interacting gamma-tmt duplicated loci. additionally, unstable expression of increased gamma-tocopherol content in the range from to 85% has been reported. f seeds showed continuous segregation for gamma-tocopherol content from to %. gamma-tocopherol content in f plants segregated from to %.
 tocopherols are natural antioxidants with both in vivo and in vitro activity. sunflower seeds contain predominantly alpha-tocopherol, with maximum vitamin e effect but lower in vitro antioxidant action than other tocopherol forms such as gamma-tocopherol. sunflower germplasm with stable high levels of gamma-tocopherol has been developed. the trait is controlled by recessive alleles at a single locus tph underlying a gamma-tocopherol methyltransferase. additionally, unstable expression of increased gamma-tocopherol content in the range from to 85% has been reported. the objective of this research was to determine the genetic basis of unstable expression of high gamma-tocopherol content in sunflower seeds.  male sterile plants of nuclear male sterile line nmst with stable high gamma-tocopherol content, were crossed with plants of line iast- with stable high gamma-tocopherol content but derived from a population that exhibited unstable expression of the trait. f seeds showed continuous segregation for gamma-tocopherol content from to %. gamma-tocopherol content in f plants segregated from to %. a genetic linkage map comprising linkage groups was constructed from this population using ssr and indel marker loci, including indel markers for tocopherol biosynthesis genes. qtl analysis revealed a major qtl on lg that corresponded to the gamma-tmt tph locus, which suggested that high gamma-tocopherol lines nmst and iast- possess different alleles at this locus. modifying genes were identified at lgs and corresponding in most cases with gamma-tmt duplicated loci.  unstable expression of high gamma-tocopherol content is produced by the effect of modifying genes on tph2a allele at the gamma-tmt tph gene. this allele is present in line iast- and is different to allele tph present in line nmst which is not affected by modifying genes. no sequence differences at the gamma-tmt gene were found associated to allelic unstability. our results suggested that modifying genes are mostly epistatically interacting gamma-tmt duplicated loci. 