BACKGROUND
the african oil palm  is the major oil crop in the world today  <cit> .
the wide range of applications  for mesocarp oil is due to its fac which is suitable for making common consumable products , pharmaceuticals and animal feedstocks.
in addition, palm oil has industrial applications, e.g.
making biodiesel, oleochemicals, cosmetics and textiles.
palm oil has roughly equal proportions of saturated and unsaturated fas.
the saturated fas are palmitic , stearic , myristic , arachidic  and lauric .
the unsaturated fas include  <dig>  % oleic ,  <dig>  % linoleic ,  <dig>  % linolenic  and  <dig>  % palmitoleic   <cit> .
in comparison, the mesocarp oil from the american oil palm, e. oleifera, is much more unsaturated with,  <dig> – <dig>  % c18: <dig>   <dig> – <dig>  % c18: <dig> and only  <dig> – <dig>  % c16: <dig> and  <dig> – <dig>  % c18: <dig>  as observed in the mpob colombian germplasm collection  <cit> .
as the current world demand is for less saturated edible oils, it would be advantageous if the e. guineensis oil can be selected to have a composition closer to e. oleifera oil  <cit> .
the desirable fac in colombian e. oleifera oil makes the palm an ideal material for introgression into elite e. guineensis such as the mpob nigerian germplasm , which is already known for its higher unsaturated oil content  <cit> .
the t <dig> germplasm has been distributed as a high iv material  and extensively used in various interspecific breeding programs by the oil palm industry  <cit> .
therefore, it is important to capture the favourable alleles linked to high iv in the successive hybrids and backcrosses.
the resulting e. oleifera × e. guineensis interspecific hybrid population was found to be segregating for iv and major fa traits which allowed for identification of qtls linked to these traits.
a number of qtls for iv and fac located on the t <dig> parental genetic map and mostly flanked by amplified fragment length polymorphism  and restricted fragment length polymorphism  markers were reported by singh et al.
<cit> .
the current study reports an extension of the work initiated by singh et al.
<cit>  by identifying qtls on a higher density simple sequence repeat  and single nucleotide polymorphism -based genetic map  <cit> .
in plants, the fa and triacylglycerol  biosynthesis pathways occur in separate compartments.
de novo fa synthesis occurs in the plastid and the growing fa chain is held by acyl carrier protein .
subsequently, acyl-acps are hydrolysed by acyl-acp thioesterases and the resulting non-esterified fas exported to the endoplasmic reticulum  for assembly into tags  <cit> .
recently, oil palm transcriptome data from developing fruits  were used to investigate the regulatory mechanisms of genes and transcription factors  governing the synthesis of fa and tag  <cit> .
the formation of fa destined for oil accumulation starts around 110 days after pollination  and reaches its peak at  <dig> dap.
it is during this period that tags begin to accumulate in the mesocarp and reach a peak at  <dig> dap  <cit> .
the transcriptome data have also opened up new avenues to develop candidate markers for fa biosynthesis genes with oil palm orthologues identified for β-ketoacyl-acp synthases , acyl-acp thioesterases  and stearoyl-acp desaturase .
the data was exploited by montoya et al.
<cit> , where snp markers were developed from these candidate genes and  <dig> of them were polymorphic and located on the e. oleifera × e. guineensis pseudo-backcross-one  genetic map.
among the  <dig> snp markers, four located within the confidence intervals of qtls linked to iv and fac  <cit> .
taking a slightly different approach, potential candidate genes and a tf associated with biosynthesis of fa and tag were identified in the major qtl regions revealed in this study.
this was done by comparing the qtl regions  to the oil palm genome assembly  <cit> .
markers based on these candidate genes were developed to saturate the qtl intervals.
the saturated qtl regions revealed closely linked markers and, if validated across different genetic backgrounds, these markers could have utility in a mas program.
a similar approach has been applied with great success in rice and even oil palm, in identifying candidate genes linked to mapped qtls  <cit> .
the second part of this study focused on validating the consistency of qtls linked to iv and fac in two independent pseudo-bc <dig> populations of e. guineensis × e. oleifera .
the validation families were derived from different genetic backgrounds compared to the populations described by montoya et al.
<cit> .
in addition to validating some of the previously reported qtl locations, this study revealed additional genomic regions influencing iv and fac.
compilation of different favourable alleles of qtls obtained from various genetic backgrounds will help to develop effective strategies for the application of mas in an interspecific hybrid breeding program.
in soybean, wang et al.
<cit>  described mas for c16: <dig> and c18: <dig> through pyramiding of two to three qtls, detected across various genetic backgrounds, exhibiting significant cumulative effects.
similarly, this study aimed to uncover a set of markers that can be used to select for favourable alleles linked to unsaturation, at least in the genetic backgrounds examined.
methods
mapping population
the mapping population ‘oxg’ is an interspecific cross between a colombian e. oleifera  and an nigerian e. guineensis tenera  as reported by singh et al.
<cit>  and ting et al.
<cit> .
the population consists of  <dig> hybrids and was created and is maintained by united plantations berhad , perak, malaysia.
of these, eight palms were excluded from the linkage analysis due to relatively high recombination frequencies  <cit> .
an additional two palms died and therefore, only  <dig> palms were used in this study.
validation crosses
two bc <dig> crosses from an independent breeding program at upb were used to attempt to validate the qtls detected in the oxg mapping population.
the breeding approach used in creating the bc <dig> crosses is illustrated in fig.
<dig>  the first cross was between a la mé e. guineensis  and a colombian e. oleifera  and is termed ‘gxo’.
the resulting gxo f <dig> hybrid  was then backcrossed with palm t <dig>  from the nigerian germplasm to produce the bc <dig>  pollen was then obtained from a selected bc <dig> palm  to cross-pollinate two e. guineensis palms.
the first e. guineensis  was derived from a cross between the palm t <dig> and a serdang pisifera and the second palm  was derived from a self-pollination of the original t <dig> palm.
the two bc <dig> families are named progenies ‘ <dig> –1’ and ‘ <dig> –5’, consisting of  <dig> and  <dig> palms, respectively.
they were field planted in the year  <dig>  the female parent of  <dig> – <dig>  died before any leaf and fruit could be sampled.
therefore, two siblings to 320/tt113/ <dig>  were genotyped to help with scoring and phase configuration of the markers used for the construction of genetic maps.fig.
<dig> the oxg mapping population and bc <dig> validation crosses used in this study.
the  <dig> oxg interpecific hybrids  were created by crossing a maternal colombian e. oleifera  with a paternal palm t <dig> , from the nigerian germplasm.
the same t <dig> palm was also used in creating the bc <dig> validation crosses .
the la mé e. guineensis  was crossed with a colombian e. oleifera  and the resulting gxo f <dig> hybrid  was then backcrossed with the t <dig> palm to produce the bc <dig>  pollen from a selected bc <dig> palm  was used to cross-pollinate two e. guineensis palms.
the first e. guineensis  was derived from a cross between the t <dig> and a serdang pisifera and the second palm  was derived from a self-pollination of the t <dig> palm.
the two bc <dig> families namely,  <dig> – <dig> and  <dig> – <dig> consisted of  <dig> and  <dig> palms, respectively
extraction and analysis of palm oil
the procedures for sampling ripe fruits and extracting mesocarp oil were as described by singh et al.
<cit> .
the porim test method  <cit>  was applied to measure the iv and fac in the palm oil.
candidate snp markers and genetic linkage map construction
candidate snp markers  flanking various genes associated with fa and oil biosynthesis were mined from the p <dig> genome build.
the oil palm snp assay design and genotyping were performed by a service provider, agena bioscience, inc.  using the iplex ® biochemistry on massarray® system  <cit> .
a custom two-multiplexed genotyping assay was designed and optimized for a panel of  <dig> snps using the assay design suite  <dig>  software .
primary pcr primers  were designed to contain a common 5’ 10-mer tag  and synthesized by idt .
the primary pcr reaction was carried out to amplify dna fragments of approximately 100 bp containing targeted snps from the good quality  oil palm genomic dna .
the pcr conditions included an initial denaturation at 95 °c for 2 min, 45 cycles of 95 °c denaturation , 56 °c annealing  and 72 °c extension , and a final extension at 72 °c for 5 min.
the remaining unincorporated dntps in the pcr product was inactivated using shrimp alkaline phosphatase  treatment .
this was followed by an allele-specific single base primer extension reaction performed using the iplex gold reaction kit .
reaction cocktail containing extend primer, buffer, enzyme, and mass-modified ddntps  was added to the primary pcr product and proceeded to extension reaction at 94 °c initial denaturation , 40 cycles of 95 °c denaturation , 52 °c annealing  and 80 °c extension , and a final extension at 72 °c for 3 min.
the extension reaction product was desalted using the spectroclean  resin treatment and transferred onto the spectrochip® array  for data analysis using matrix-assisted laser desorption/ionization time-of-flight  mass spectrometry.
the snp genotyping data and report were generated using the typer  <dig>  software .
the genotyping data were incorporated into the existing dataset  <cit>  for construction of the genetic map.
furthermore, the aflp and rflp markers reported by singh et al.
<cit>  to be flanking the qtl regions were also incorporated into the existing linkage map which was re-constructed using joinmap®  <dig>   <cit>  as described previously  <cit> .
the genetic maps for the two validation crosses,  <dig> – <dig> and  <dig> – <dig>  were similarly constructed using the same set of snp and ssr markers .
comparison of the genetic maps for oxg,  <dig> – <dig> and  <dig> – <dig> was carried out using mapchart  <dig>   <cit> .
quantitative trait loci  analyses
in the oxg population, the qtl analysis was performed using interval mapping , the multiple-qtl model  and the kruskal-wallis non-parametric ranking tests  with the default parameters in mapqtl  <dig>  <cit> .
the 95 % genome-wide lod significance threshold for each trait was determined by the permutation test option, with  <dig>  permutations.
each candidate qtl interval was further analyzed using g model   <cit> .
the gm analysis first estimated the genome-wide background effects of markers  from all the lgs  using a random effects model .
the background effects were used to correct and determine significant markers by backward elimination  on each lg.
finally, the effect of each significant marker was estimated using the multiple regression coefficients.
similar qtl analysis approaches were also applied in the  <dig> – <dig> and  <dig> – <dig> crosses to validate the detected qtls.
development of candidate ssr markers within qtl confidence regions
development of ssr markers  for candidate fa genes and tf was carried out by aligning contigs and clone sequences  containing the snps and ssrs mapped in the qtl confidence intervals, using blastn  <cit> .
markers from another independent genetic map   <cit>  that localize in the qtl-syntenic regions were also included in the similarity search.
scaffold regions linked to qtl confidence intervals were extracted from the p <dig> genome build and searched for sequence similarity  against the ncbi databases .
sequences with significant similarity  to genes and tfs associated with fa and tag syntheses were selected to attempt to mine ssrs using the microsatellite identification tool   <cit> .
four percent super fine resolution  agarose  gel electrophoresis was used to screen for potential polymorphism of the candidate ssrs against a panel of six randomly selected oxg hybrids and the two parental palms.
the informative ssrs were then pcr-amplified using the m13-tailed primer  approach as described by ting et al.
<cit> .
fragment analysis of the pcr products was performed by a service provider at the centre for marker discovery and validation , malaysia using an abi 3730xl dna analyzer  with fluorescent dye sets comprising fam, ned, pet, vic and genescan™- <dig> liz® size standard.
sizing of the ssr alleles was performed using the genemapper®  <dig>  software  and genotype scoring was as described previously  <cit> .
RESULTS
mapping candidate snps for fa genes onto the oxg genetic map
in the oxg interspecific population, the existing paternal t <dig>  and partially integrated  maps had  <dig>  markers/ <dig> lgs/ <dig>  cm and  <dig> markers/ <dig> lgs/ <dig>  cm, respectively  <cit> .
the two genetic maps were constructed mainly with snp and ssr markers replacing the traditional aflp and rflp markers used in the earlier version   <cit> .
the new marker systems are technically more user-friendly, cheaper and give better reproducibility than the aflp and rflp markers.
however, to anchor the previously  <cit>  detected qtls, the aflp and rflp markers flanking each qtl interval were also incorporated into this study.
an additional  <dig> snps  were identified flanking various fa genes distributed across  <dig> scaffolds of the p <dig> genome build.
of these, seven for palmitoyl-acp thioesterase , oleoyl-coa desaturase , linoleoyl-coa desaturase , enoyl-acp reductase  and stearoyl-acp desaturase  – were polymorphic in the oxg mapping population.
these candidate snp markers were mapped onto lgs ot <dig>  t <dig>  ot <dig>  ot <dig> and t <dig>  and used in the subsequent qtl analysis.
for the oxg mapping population, as only  <dig> integrated lgs  were available, six lgs  from the t <dig> paternal map were also included in the qtl analysis.
bc <dig> genetic linkage maps
in the bc <dig> validation crosses, two independent integrated maps with  <dig>  markers/ <dig> lgs/ <dig> .5 cm  and  <dig>  markers/ <dig> lgs/ <dig> .7 cm  <cit>  were constructed for the  <dig> – <dig> and  <dig> – <dig> populations, respectively.
resolution of the two genetic maps was good and the linear order of markers comparable to that in oxg with an average gap of  <dig>  cm  and  <dig>  cm .
both the high density genetic maps were used for qtl analysis.
iv and fac quantitative phenotypic data
in the oxg cross, palm oil was obtained from 85 f <dig> palms and measured for iv, c14: <dig>  c16: <dig>  c16: <dig>  c18: <dig>  c18: <dig>  c18: <dig> and c18: <dig>  the remaining palms could not be sampled due to sterility issues, abortive bunches and crown disease.
also  <dig> palms had died before phenotyping.
the present phenotypic data set includes an additional four palms to that reported by singh et al.
<cit>  and is summarised in table  <dig>  overall, the distribution of phenotypic data was similar to that reported previously.
the iv  and major fas, c16: <dig> , c18: <dig>  and c18: <dig>  showed a normal distribution .
however, the four minor components, c14: <dig> , c16: <dig> , c18: <dig>  and c18: <dig>  did not follow a normal distribution, also previously observed by montoya et al.
<cit> .
for c18: <dig>  the data was converted to a normal distribution  by log <dig> transformation, but the approach did not convert c14: <dig>  c16: <dig> and c18: <dig> to a normal distribution.
for these non-normally distributed traits, kw analysis was used.
correlation analysis gave similar results to those by singh et al.
<cit>  .table  <dig> summary of phenotypic data in the oxg mapping population and two bc <dig>  validation crosses
means , ranges and variances measured for iodine value and various fatty acid contents in palm oil
for the two bc <dig> crosses, data were collected for  <dig> and  <dig> palms of the  <dig> – <dig> and  <dig> – <dig> crosses, respectively.
the other palms did not bear any fruit or had died before sampling could be performed.
in  <dig> – <dig>  the widest data range observed  was for c18: <dig>  and c16: <dig> .
all the data showed normal distributions except for iv, c14: <dig> and c16: <dig>  however, log <dig> transformation  and the discarding of one or two outliers  returned the distributions to normality.
outliers were identified by using a boxplot and comparing the observed and expected mean  values.
significant positive correlations were observed between c14: <dig> and c16: <dig> , iv and c18: <dig>  and, iv and c18: <dig> .
strong negative correlations  were observed for c16: <dig> and c18: <dig>  iv and c16: <dig>  iv and c14: <dig> and, c16: <dig> and c18: <dig> while, c14: <dig> and c18: <dig>  c14: <dig> and c18: <dig> and, c18: <dig> and c18: <dig> showed moderate negative correlations of − <dig>  to – <dig>  .
in  <dig> – <dig>  c18: <dig> and c16: <dig> also showed the widest data distribution of  <dig> – <dig>  %  and  <dig> – <dig>  % , respectively.
the phenotypic data  demonstrated a normal distribution.
for c14: <dig> and c16: <dig>  normality was improved after removing one outlier.
the phenotypes iv and c18: <dig>  c14: <dig> and c16: <dig>  c14: <dig> and c18: <dig> and, c14: <dig> and c16: <dig>  showed moderate correlations  while, negative correlations were obtained for iv and c16: <dig>  c16: <dig> and c18: <dig>  c18: <dig> and c18: <dig>  c14: <dig> and c18: <dig> and, c16: <dig> and c18: <dig> .
oxg: qtls linked to iv and fac
this study aimed to identify the qtls linked to iv and fac in the improved snp and ssr-genetic map of the oxg cross  <cit> .
ten genome-wide significant qtls and two putative qtls  linked to iv, c14: <dig>  c16: <dig>  c16: <dig>  c18: <dig>  c18: <dig> and c18: <dig> were identified on lgs ot <dig>  t <dig>  t <dig>  ot <dig>  ot <dig> and t <dig> using a combined qtl detection method – im, mqm and kw.
only markers or regions consistently linked to the specific qtl using all three approaches were considered to be significant qtl.
subsequently, gm was used to estimate the effects of closely flanked markers using multiple regression coefficients after backward elimination and adjustment for the background marker effects  <cit> .
on the improved lgot <dig> , three major qtls  and two minor qtls  were revealed in the same map interval previously reported between markers pop-cb00075a and eagg/mcat- <dig> .
on the current lgot <dig>  the significance interval between the two markers  was filled by  <dig> snps and three ssrs.
of these, six snp markers, including a pate candidate snp , were within the qtl confidence interval .
this genomic region was of interest and further investigation was carried out with the additional results presented below in the ‘fine-mapping of the qtls on lgot1’ section.fig.
<dig> qtl profiles linked to iv and fac on the improved genetic linkage map of oxg.
the graphs  present qtls  significant at 95 % genome-wide  and chromosome-wide  thresholds in linkage groups  ot <dig>  t <dig>  ot <dig>  t <dig>  t <dig> and ot6
for group  <dig> , three genome-wide significant qtls for c14: <dig>  c16: <dig> and c18: <dig> were identified and the interval was flanked by markers eaag/mcat- <dig> and p4t8i, as reported previously.
the current map position for eaag/mcat- <dig> was determined using a less stringent mapping parameter .
the interval between eaag/mcat- <dig> and p4t8i contained three snp markers, which reduced the gap to  <dig>  cm/markers from the previous  <dig>  cm.
for c16: <dig> and c18: <dig>  the qtl peak co-localized with markers snpm <dig> and snpm <dig> at map position  <dig>  cm.
using gm, the marker effect was estimated to be  <dig>  %  and − <dig>  %  for c16: <dig> and c18: <dig>  respectively.
when the genotypes of the two markers were analysed, the ab and aa profiles showed significant differences  for c16: <dig> and c18: <dig> contents in the oxg hybrids .
for snpm <dig> , the homozygous aa  genotype showed an average  <dig>  %  for c16: <dig> and  <dig>  %  for c18: <dig> content which differed from the heterozygous ab  genotypes .
however, for c14: <dig>  the closest marker was snpm <dig>  with a minor effect of − <dig>  %  and which also showed a significant difference in c14: <dig> content between the aa  and ab  genotypes .fig.
<dig> distribution of phenotypes categorized based on the genotypes of the closest markers linked to the observed qtls in lgt <dig>  the upper panel  is of hybrids with genotype ab  while the lower panel  shows the phenotypes observed in genotype aa  in snpm <dig>  for c16: <dig>  and c18: <dig>  and, snpm <dig> for c14: <dig>
for lgot <dig>  qtl was detected for c18: <dig> in the  <dig> – <dig>  cm interval flanked by snpm <dig> and snpm <dig> .
the closest marker snpm <dig> at  <dig>  cm, had a positive effect of  <dig>  %  on the trait, as estimated by gm.
on lgt <dig> , three markers  showed significant association with c14: <dig> .
these markers, co-localized with the qtl peak at map position  <dig>  cm, were found at a distance  <dig>  cm from the previously detected qtl .
in addition, two putative qtls for c18: <dig> were detected on lgs t <dig>  and ot <dig>  .
the putative  qtls were considered in this case as the regions concerned also explained similar qtls in other studies .
a summary of the qtl results is presented in table  <dig> table  <dig> qtls linked to iodine value  and fatty acid composition  in the oxg interspecific mapping population.
qtls identified using interval mapping , the multiple-qtl model , the kruskal-wallis non-parametric tests  and g model
ot1a
pa5_ossra
pa3_ossra
spsc00328a
ot1a
pa5_ossra
pa3_ossra
spsc00328a
ot1a
pa5_ossra
pa3_ossra
spsc00328a
ot1a
ot1a
pa5_ossra
pa3_ossra
spsc00328a
aafter fine mapping
gw: 95 % genome-wide significant lod threshold
cw: 95 % chromosome-wide significant lod threshold
fine-mapping of the qtls on lgot1
the map interval  <dig> – <dig>  cm  on lgot <dig>  revealed multiple qtls  with a relatively large proportion of phenotypic variation explained  and was of particular interest.
admittedly, the phenotypic variation could have been overestimated due to the limited size of the mapping family, leading to a beavis effect  <cit> .
the markers in this region were mapped to the p <dig> genome build  <cit> .
to improve the efficiency of finding the qtl corresponding region in the genome build, additional markers that fit into the region were also obtained from an independent e. guineensis map   <cit> .
this resulted in  <dig> markers  mapping to scaffolds p5_sc <dig> and p5_sc <dig> with high identities  and e-values  <dig> – 1e- <dig>  the total physical coverage of the qtl interval on p5_sc <dig>  and p5_sc <dig>  were  <dig> ,438 bp  and  <dig> ,644 bp , respectively.
the blast results for the qtl corresponding region identified significant similarity to one tf and a number of genes associated with the fa and tag biosynthesis pathways  <cit> .
this included diacylglycerol acyltransferase  and long chain acyl-coa synthetase  in p5_sc <dig>  in p5_sc <dig>  sodium/metabolite cotransporter , palmitoyl-acp thioesterase , 3-hydroxyisobutyryl-coa hydrolase-like protein  <dig>  and ap2-like ethylene-responsive tf  were found.
for fine mapping the qtl confidence interval, ssrs  were mined and developed from the candidate gene introns  and regions  flanked at  <dig> – <dig>  bp before the 5’ start codon  and 351– <dig>  bp downstream  of the genes.
in addition, six ssrs were developed specifically to fill in the gaps in the identified interval and therefore were not located near to any candidate gene.
of the  <dig> developed candidate ssrs,  <dig> were polymorphic and mapped back to the qtl interval on lgot <dig>  unfortunately, five ssrs for dgat <dig> were not polymorphic and could not be mapped onto lgot <dig>  refined qtl intervals for iv, c14: <dig>  c16: <dig>  c18: <dig> and c18: <dig> as well as the map positions of the candidate genes and tf are illustrated in fig.
<dig> fig.
<dig> fine-mapping of qtl intervals with candidate markers and cross-validation of qtls in two independent bc <dig> crosses.
similar qtls between lgot <dig>  and lgs  <dig> and 1b of bc <dig> validation crosses  are indicated by common markers  and candidate ssr markers
after fine-mapping with candidate markers, slight increases in the lod scores were observed for markers in the qtl intervals.
for iv, significant qtl with lods  <dig> – <dig>  were revealed in the 113– <dig>  cm interval on lgot <dig>  two candidate genes, hibch and pate/fatb, together with three candidate ssrs  and six existing snps  co-localized directly with the qtl peak .
no recombination event was observed between the nine markers, possibly due to the limited size of the oxg population and the short distance  on the physical map.
using gm, the nine markers were detected to have a significant effect close to  <dig>   .
this result indicated that the maternal up <dig> genotype  had an increasing effect on iv compared to the t <dig> genotype  for snpm <dig>  snpm <dig>  spsc <dig>  snpm <dig> or snpe <dig>  in contrast, aa showed reduced levels of iv compared with ab when genotyped with snpm <dig>  snpm <dig>  pa5_ossr or pa3_ossr .
the difference in mean iv between the two genotypes improved to  <dig>   compared to  <dig>  estimated previously using the rflp marker, pop-cb00075a by singh et al.
<cit> .fig.
<dig> distribution of iv and fac phenotypes in oxg hybrids genotyped by the closest markers linked to the qtls on lgot <dig>  the upper panel  is of hybrids with genotype ab  while the lower panel  shows the phenotypic distribution of genotype aa  in snpm <dig>  snpm <dig>  spsc <dig>  snpm <dig> or snpe <dig>  for iv , c18: <dig> , c16: <dig>  and c14: <dig> .
for c18: <dig> , the ab and aa genotypes are observed in spsc00314
the same group of markers were also closely linked to the qtl for c18: <dig>  with similar allelic effects as observed on iv .
these markers detected a greater difference  in c18: <dig> content compared to the  <dig>  % explained by pop-cb00075a previously.
another qtl revealed by the same group of markers was for c16: <dig> content.
the estimated marker effect of  <dig>  %  had increasing alleles from the other parent, compared to that for iv and c18: <dig> which agrees with the strong negative correlation between the levels of saturated  and unsaturated fas .
the difference in the c16: <dig> mean values of the homozygous and heterozygous genotypes also increased to  <dig>  % from the  <dig>  % previously reported .
in addition to the three major qtls, these markers also had a minor effect of  <dig>  %  on c14: <dig> with the increasing alleles inherited in the same direction as c16: <dig>  the difference in c14: <dig> content between genotypes aa  and ab  was also relatively low at  <dig>  %.
with respect to c18: <dig>  the refined qtl interval  revealed a new candidate ssr marker spsc <dig> at the qtl peak with a negative effect of  <dig>  % .
a difference of  <dig>  % in c18: <dig> was observed between the aa and ab genotypes of spsc <dig> .
on the genome scaffold , spsc <dig> was located between pate/fatb and bass <dig> with an estimated distance of ~  <dig> kbp and  <dig> kbp, respectively.
therefore, it is still unclear at this point which of the genes, if any, is influencing the minor qtl for c18: <dig>
validation of qtls for iv and c16: <dig> in bc2
in this study, the qtls detected for iv and c16: <dig> in the oxg mapping population were tested for validation in the two bc <dig> crosses− <dig> – <dig> and  <dig> – <dig>  in  <dig> – <dig>  a major qtl was detected for iv with lod <dig>  on lg <dig>  the  <dig>  cm confidence interval  containing one ssr and  <dig> snps was then aligned to lgot <dig>  using nine transferable markers  which clearly showed that the same region was affecting iv in both crosses .
of these markers, snpm <dig> underlying the qtl peak revealed three genotypes – aa, ab and bb – in the  <dig> - <dig> palms.
the aa genotype, similar to that observed in oxg, differentiated the iv mean  from the bb genotype  with p =  <dig>  but, no significant difference observed in genotypes aa vs. ab  and bb vs. ab .
the same marker snpm <dig>  was also detected flanking a putative qtl peak for c16: <dig> with minor effect.
the qtls with overlapping confidence intervals for iv and c16: <dig> were also identified in the  <dig> – <dig> cross on a subgroup of lg <dig> .
when compared with lgot <dig> in oxg, the same qtl region encompassing the common marker spsc <dig> was also located at the end of the lg1b.
for iv, the qtl peak showed a high lod score  with the confidence interval  and contained two candidate ssr markers, spsc <dig> and spsc <dig>  with estimated effects of  <dig> – <dig>  iv units.
for the closest marker spsc <dig>  the maternal genotype  showed significantly  lower unsaturation  –  <dig>   compared to  <dig>   in the paternal genotype .
the spsc <dig> also showed a significant effect of  <dig>  %  and the aa and ab genotypes revealed a significant  mean difference  in c16: <dig> content.
additional qtls detected in the bc2
the  <dig> – <dig> and  <dig> – <dig> crosses revealed additional qtls for c18: <dig> on lg <dig> which were not detected in oxg.
for  <dig> – <dig>  the qtl in the confidence interval  <dig> – <dig>  cm enclosed seven markers .
of these, the closest flanking markers to the qtl peak  were snpm <dig> and snpm <dig> with estimated effects of  <dig>  %  and  <dig>  % , respectively.
in the  <dig> – <dig> cross, similar qtl  although significant at the chromosome-wide level rather than the genome-wide level were also detected on lg <dig> with lods ranging from  <dig> – <dig> .
the left-right flanking markers were snpm <dig>  snpm <dig>  snpm <dig>  snpm <dig> and snpm <dig> at  <dig>  cm and snpm <dig>  snpm <dig> and snpm <dig> at  <dig>  cm .
in comparison, more new qtls were detected in  <dig> – <dig> than in  <dig> – <dig>  these include i. c18: <dig> , ii.
c18: <dig> , iii.
iv , iv.
c16: <dig>  and, v. c18: <dig> .
all were minor qtls, significant at the 95 % chromosome-wide level.
discussion
this study extended the mapping of qtls linked to iv and fac in the oxg interspecific mapping population initiated by singh et al.
<cit> .
the preliminary map constructed with  <dig> markers localized  <dig> qtls mostly loosely flanked by aflp and rflp markers.
therefore, in this study, a  <dig> x more saturated genetic map was used to localize the qtls and improve coverage.
a total of  <dig> genome-wide and two putative qtls for iv and various fas were identified on six improved lgs , which included  <dig> of the qtls detected previously.
the results indicated that the current ssr and snp-based genetic map was effective in revealing qtls with the only exception being the minor qtl for c14: <dig> reported earlier on group  <dig> .
however, a new putative qtl was revealed for c14: <dig> at the nearby region of the same lg.
the shift in location could indicate a more precise estimation of the qtl with the improved map resolution on lgt <dig>  with a similar situation having been reported in eggplant  <cit> .
the detected qtls at this stage can only be considered to be specific to the oxg cross which would limit application of the markers for mas.
mapping families created via a sequential series of backcrosses and self-pollination would ideally help validate the qtls as reported in rapeseed  <cit> , maize  <cit>  and soybean  <cit> .
however such mapping families are not readily available for a perennial crop like oil palm and it would take about 10 years to create each generation, with initial phenotyping  <cit> , apart from the huge investment required in land, labour and management.
it is for this reason that, to date, qtls for iv and fac in oil palm have only been reported in single crosses involving a bc <dig>  tenera x dura f <dig>  <cit>  and an oxg hybrid  <cit> .
in this study, we used two independent bc <dig> pseudo-crosses  created by introgression of e. oleifera into t <dig> germplasm materials to try to validate the qtls detected in the oxg hybrid population.
to our knowledge, this is the first attempt to validate qtls for fac in oil palm of similar genetic backgrounds.
the two bc <dig> crosses are related by pedigree and also genetically linked with the t <dig> germplasm .
here we adopted a strategy to evaluate the qtls through an independent analysis with subsequent comparison of their locations.
however, our results showed that only the major qtls for iv and c16: <dig> were successfully cross-validated through a common location, and all similarly revealed a large proportion of phenotypic variance  in both  <dig> – <dig> and  <dig> – <dig>  the common qtls in bc <dig> revealed that the different genotypes of closely linked markers also showed distinct levels for iv and c16: <dig> content.
the finding is of interest as iv represents the cumulative unsaturation in palm oil and it has always been used as an indicator in selecting for higher unsaturation overall in breeding programs  <cit> , whereas the qtl for c16: <dig> can complement the prediction by indicating the palmitic  level.
it is however noted that the qtl for c18: <dig> could not be cross-validated in the two bc <dig> populations even though c18: <dig> is a major component contributing to iv and in this study was highly correlated with iv and negatively correlated with c16: <dig>  the small size of the mapping families could have limited the detection power for the qtl analysis, resulting in the lack of detection of the qtls in the bc <dig> crosses.
a similar observation was made in maize about the effect of population size when a sub sample  only revealed about 1/ <dig> of the qtls observed in larger samples  <cit> .
in addition to validating qtls in the interspecific bc <dig>  eight of the identified qtls were similar to those reported previously using either a bc <dig> or a tenera × dura cross  <cit> .
multiple qtls for c14: <dig>  c16: <dig>  c18: <dig> and c18: <dig> were reported previously in the respective intervals 0– <dig> , 0– <dig> , 0– <dig>  and  <dig> –  <dig>  cm on lg <dig> in the tenera x dura mapping family.
the intervals were aligned with the current qtls on lgt <dig> using three common markers, megcir <dig>  megcir <dig> and megcir <dig>  pointing to a similar map region.
in the previous studies by montoya et al.
<cit> , the intervals in lg <dig>  were reported to be linked to c18: <dig> and c18: <dig>  respectively.
similar regions affecting the qtls were also detected in the present oxg  and  <dig> – <dig>  crosses, determined using the common markers megcir <dig>  megcir <dig> and megcir <dig> mapped in both the studies.
another possible common qtl  was c14: <dig> on lgt <dig> which was also previously reported on the same lg in the bc <dig>  <cit> .
the putative qtl linked to c18: <dig> on lgt <dig> and lgot <dig> were similar to that reported previously.
the qtl on lgt <dig> matched the genomic region reported previously for a bc <dig> population  <cit> , as revealed by the common marker megcir <dig>  mapping in the same region.
with respect to lgot <dig>  the qtl region was very close to that reported previously for the same trait by singh et al.
<cit> .
the above comparison has provided useful information for comparing qtls linked to fac by different research groups.
we compiled and compared a total  <dig> qtls linked to iv and fac  across the different mapping families.
however, very low percentages of the qtls were common across two  to three  of the crosses and none at all in more than three crosses.
this highlights that in qtl analysis, even for a highly heritable trait such as fac, favourable alleles are likely to be population-specific requiring careful selection for implementation in mas.
nevertheless, this study revealed a core set of markers  that can be useful for mas, especially in genetic backgrounds involving the t <dig> and e. oleifera parental palms.
as the palm materials are commonly available and accessible to the oil palm industry, the markers will have practical application for selecting favourable alleles for unsaturation  in interspecific hybrids and their backcrosses.
detection of similar qtls for iv and c16: <dig> in both oxg and bc <dig> allowed the underlying qtl interval to be compared to the recently published oil palm genome build  <cit> .
this study is also the first to describe attempts at fine-mapping of targeted qtl regions for iv and various fac, by exploiting the whole genome sequence data of oil palm.
the genomic region of about  <dig> kbp showed the presence of an interesting gene with high similarity to pate/fatb in oil palm  and coconut .
comparison of genetic maps using the common markers megcir <dig>  megcir <dig> and megcir <dig> estimated that the current map position of pate/fatb in lgot <dig> is in accordance with montoya et al.
<cit> .
however, the authors did not associate the gene or region around the gene with any qtl for iv or fac.
in oil palm, pate/fatb encodes the palmitoyl-acp thioesterase that actively releases c16: <dig> from c16:0-acp and allows the resulting non-esterified fa to be incorporated into tag  <cit>  and is one of the main factors responsible for the relatively high saturation levels in palm oil  <cit> .
a similar role was reported for pate/fatb in oil biosynthesis in cotton seed  <cit> .
apart from a strong substrate preference for c16: <dig>  the substrate specificity of palmitoyl-acp thioesterase also extends to other saturated acyl-acps, including c14:0- and c18:0-acps  <cit> .
this was shown in other species by over-expression of pate/fatb in arabidopsis thaliana  <cit>  and canola seeds  <cit>  which also had an elevated concentration of c14: <dig>  this is in agreement with the strong positive correlation observed in this study between the c16: <dig> and c14: <dig> phenotypes.
in contrast, the phenotypic data for levels of c16: <dig> had a strong negative correlation with c18: <dig> levels which indicates that a reduction of c16: <dig> is associated with increased c18: <dig>  this was also observed in maize seeds where a mutated fatb allele caused a dramatic reduction of c16: <dig> in the oil and increased the level of unsaturated fas  <cit> .
the truncated fatb was reported responsible for the non-functioning of a critical catalytic domain and this could have resulted in the channelling of more c16:0-acp for elongation and subsequent desaturation into unsaturated acyl chains, such as c18:1-acp.
it was also suggested that at least one of the palmitoyl-acp thioesterase isoforms in oil palm can hydrolyze c18: <dig> efficiently  <cit> .
this could also explain the co-localization of qtls for iv, c14: <dig>  c16: <dig>  c18: <dig> and c18: <dig> on lgot <dig> found in this study.
therefore, the next step is to evaluate the identified pate/fatb gene in palms with varying contents of c16: <dig> and c18: <dig>
another interesting region was at ~  <dig> kbp  from pate/fatb .
this genomic region  contained a gene with significant similarity to hibch in both oil palm  and date palm .
in oil palm, information about the role of hibch  associated with fa and tag biosynthesis is very limited.
this enzyme is reportedly involved in the degradation of fas  in camelina sativa  <cit>  which takes place in mitochondrial and the peroxisomes.
in coconut, hibch was reported as being down-regulated during the production and accumulation of oil  <cit> .
several other interesting genes and tf encoding key enzymes involved in both the fa and tag pathways, including bass <dig>  lacs <dig>  dgat <dig> and wri <dig>  were also found in proximity to the qtl peak.
bass <dig>  is reported to play a major role in channelling pyruvate into plastids  <cit>  for conversion into acetyl-coa and malonyl-coa – the main substrates to initiate synthesis of fa.
at the final stage of fa biosynthesis, non-esterified fas are converted into acyl-coa by long chain acyl-coa synthetase and later transferred through the plastid envelope into the er for synthesis of tags  <cit> .
on the current lgot <dig>  the long chain acyl-coa synthetase family member  <dig>   was identified at  <dig>  cm close to the end of the qtl interval.
in a. thaliana, lacs <dig> showed greatest preference for the c16: <dig> substrate when expressed in e. coli.
substrate preference for various fas was reported in the following order: c16:0 > c16:1 > c18:2 > c18:1 > c20:1 > c18: <dig>  <cit> .
this may again explain the co-localization of qtls for c16: <dig> with other fas on lgot <dig> where genes, such as pate/fatb and lacs <dig>  show pleiotropic effects contributing to the complex interactions with the substrates in fa synthesis.
in this study, dgat is the only gene detected in the qtl region that is involved in formation of tags in the acyl-coa-dependent  pathway  <cit> .
the ~  <dig> kbp genomic region  has high sequence similarities to dgat <dig> in oil  and date palms .
in oil palm, dgat <dig> is known to interact with other acyltransferases, in particular, dgat <dig>  to synthesize tag  <cit> .
it is generally agreed that dgat <dig> plays an important role in one of the oil palm lipid production pathways.
however, information on the specificity and selectivity of each paralogue of dgat in oil palm is still lacking and requires further study to establish their function and effect on the composition of fa in palm oil.
in addition to the key enzymes mentioned above, we identified a wri <dig> tf co-localized with two markers  at  <dig>  cm in the qtl region on lgot <dig>  the oil palm wri <dig>  has high similarity to that in date palm , plum , euphrates poplar  and grape .
this tf has long been considered a master regulator directly influencing a number of enzymes in the fa and tag synthesis pathways in plants , including oil palm  <cit> .
CONCLUSIONS
this study for the first time extensively compared the qtls linked to iv and fac across various interspecific genetic backgrounds.
the detected qtls, to some extent, are population specific although the major qtls can be observed across related genetic backgrounds.
a core set of markers with practical application in selection for higher unsaturation in fa levels were described.
the candidate markers linked to the qtls revealed significant allelic and genotypic differences associated with iv and fac, suggesting that the markers are potentially useful in mas, at least in the genetic backgrounds described.
the fine-mapping approach employed in this study also proved effective in identifying candidate genes and a transcription factor affecting iv and fac in palm oil.
the identified genes and transcription factor, hibch, pate/fatb, bass <dig>  lacs <dig>  dgat <dig> and wri <dig> underlying the overlapping qtl confidence intervals for iv, c14: <dig>  c16: <dig>  c18: <dig> and c18: <dig>  have provided valuable information on several potential candidate genes and a transcription factor which are known in other species to strongly influencing the biosynthesis of fa and tag.
thus, to fully understand the interaction and effects of these genes, the listed candidate genes will be further studied to determine their expression levels in palms with different iv and fac.
availability of supporting data
the sequence information for the snp and ssr markers is available at http://genomsawit.mpob.gov.my
additional files
additional file 1: mapping candidate markers for various fatty acid related genes in the oxg linkage map.
candidate snp markers  for palmitoyl-acp thioesterase , oleoyl-coa desaturase , linoleoyl-coa desaturase , enoyl-acp reductase  and stearoyl-acp desaturase  mapped onto lgs ot <dig>  t <dig>  ot <dig>  ot <dig> and t <dig>  candidate ssr markers  also developed for ketoacyl-acp synthase i , acetoacetyl-coa thiolase , long chain acyl-coa synthetase , ap2-like ethylene-responsive tf , 3-hydroxyisobutyryl-coa hydrolase-like protein  <dig> , sodium/metabolite cotransporter , pate/fatb and sad.
additional file 2: pearson’s correlation coefficients for iodine value  and fatty acid composition  in palm oil of oxg mapping population.
additional file 3: pearson’s correlation coefficients for iodine value  and fatty acid composition  in palm oil of bc <dig>  validation cross.
additional file 4: pearson’s correlation coefficients for iodine value  and fatty acid composition  in palm oil of bc <dig>  validation cross.
additional file 5: co-linearity of markers between the oxg and bc <dig>  populations as observed in six linkage groups hosting qtls .
abbreviations
blastnsimilarity search of the ncbi nucleotide database using a nucleotide query
blastxsimilarity search of the ncbi protein database using a translated nucleotide query
cmcentimorgan map distance
lodlogarithm of odds
ntncbi nucleotide collection
nrncbi non-redundant protein sequences database
p5e.
guineensis  5th genome build
p5_scxxxxxe.
guineensis  5th genome scaffolds
pax_ossrcandidate ssr marker for palmitoyl-acp thioesterase
pcrpolymerase chain reaction
refseq_proteinncbi reference proteins database
sdstandard deviation
snpmxxxxxsingle nucleotide polymorphism marker
snpexxxxxcandidate single nucleotide polymorphism marker
spscxxxxxcandidate simple sequence repeat marker
competing interest
the authors declare that they have no competing interests.
authors’ contributions
rs, rs, sm, fm, n-ct, zy and kk conceived and designed the experiments.
n-ct, zy and kk carried out molecular experiments and data analyses.
llet, n-ct and rs participated in the snp experimental design and coordination.
jj assisted with the statistical analysis of the phenotypic and marker data.
llet, na, rr and k-lc provided bioinformatics support and involved in mapping the qtls to genome build.
jj, sm, fm, rs, rn, mda, mi, ak, xa, ks and lcc advised on the design of the study and contributed materials as well as reagents.
n-ct, zy and kk drafted the manuscript.
rs, sm, rs and fm assisted with interpretation of data and revised the manuscript critically.
all authors read and approved the final manuscript.

 the african oil palm is the major oil crop in the world today. the wide range of applications for mesocarp oil is due to its fac which is suitable for making common consumable products, pharmaceuticals and animal feedstocks. in addition, palm oil has industrial applications, e.g. making biodiesel, oleochemicals, cosmetics and textiles. palm oil has roughly equal proportions of saturated and unsaturated fas. the saturated fas are palmitic, stearic, myristic, arachidic and lauric. the unsaturated fas include  % oleic,  % linoleic,  % linolenic and  % palmitoleic. in comparison, the mesocarp oil from the american oil palm, e. oleifera, is much more unsaturated with, –  % c18: –  % c18: and only –  % c16: and –  % c18: as observed in the mpob colombian germplasm collection. as the current world demand is for less saturated edible oils, it would be advantageous if the e. guineensis oil can be selected to have a composition closer to e. oleifera oil. the desirable fac in colombian e. oleifera oil makes the palm an ideal material for introgression into elite e. guineensis such as the mpob nigerian germplasm, which is already known for its higher unsaturated oil content. the t germplasm has been distributed as a high iv material and extensively used in various interspecific breeding programs by the oil palm industry. therefore, it is important to capture the favourable alleles linked to high iv in the successive hybrids and backcrosses. the resulting e. oleifera × e. guineensis interspecific hybrid population was found to be segregating for iv and major fa traits which allowed for identification of qtls linked to these traits. a number of qtls for iv and fac located on the t parental genetic map and mostly flanked by amplified fragment length polymorphism and restricted fragment length polymorphism markers were reported by singh et al.. the current study reports an extension of the work initiated by singh et al. by identifying qtls on a higher density simple sequence repeat and single nucleotide polymorphism -based genetic map. in plants, the fa and triacylglycerol biosynthesis pathways occur in separate compartments. de novo fa synthesis occurs in the plastid and the growing fa chain is held by acyl carrier protein. subsequently, acyl-acps are hydrolysed by acyl-acp thioesterases and the resulting non-esterified fas exported to the endoplasmic reticulum for assembly into tags. recently, oil palm transcriptome data from developing fruits were used to investigate the regulatory mechanisms of genes and transcription factors governing the synthesis of fa and tag. the formation of fa destined for oil accumulation starts around 110 days after pollination and reaches its peak at dap. it is during this period that tags begin to accumulate in the mesocarp and reach a peak at dap. the transcriptome data have also opened up new avenues to develop candidate markers for fa biosynthesis genes with oil palm orthologues identified for β-ketoacyl-acp synthases, acyl-acp thioesterases and stearoyl-acp desaturase. the data was exploited by montoya et al., where snp markers were developed from these candidate genes and of them were polymorphic and located on the e. oleifera × e. guineensis pseudo-backcross-one genetic map. among the snp markers, four located within the confidence intervals of qtls linked to iv and fac. taking a slightly different approach, potential candidate genes and a tf associated with biosynthesis of fa and tag were identified in the major qtl regions revealed in this study. this was done by comparing the qtl regions to the oil palm genome assembly. markers based on these candidate genes were developed to saturate the qtl intervals. the saturated qtl regions revealed closely linked markers and, if validated across different genetic backgrounds, these markers could have utility in a mas program. a similar approach has been applied with great success in rice and even oil palm, in identifying candidate genes linked to mapped qtls. the second part of this study focused on validating the consistency of qtls linked to iv and fac in two independent pseudo-bc populations of e. guineensis × e. oleifera. the validation families were derived from different genetic backgrounds compared to the populations described by montoya et al.. in addition to validating some of the previously reported qtl locations, this study revealed additional genomic regions influencing iv and fac. compilation of different favourable alleles of qtls obtained from various genetic backgrounds will help to develop effective strategies for the application of mas in an interspecific hybrid breeding program. in soybean, wang et al. described mas for c16: and c18: through pyramiding of two to three qtls, detected across various genetic backgrounds, exhibiting significant cumulative effects. similarly, this study aimed to uncover a set of markers that can be used to select for favourable alleles linked to unsaturation, at least in the genetic backgrounds examined. methods mapping population the mapping population ‘oxg’ is an interspecific cross between a colombian e. oleifera and an nigerian e. guineensis tenera as reported by singh et al. and ting et al.. the population consists of hybrids and was created and is maintained by united plantations berhad, perak, malaysia. of these, eight palms were excluded from the linkage analysis due to relatively high recombination frequencies. an additional two palms died and therefore, only palms were used in this study. validation crosses two bc crosses from an independent breeding program at upb were used to attempt to validate the qtls detected in the oxg mapping population. the breeding approach used in creating the bc crosses is illustrated in fig.  the first cross was between a la mé e. guineensis and a colombian e. oleifera and is termed ‘gxo’. the resulting gxo f hybrid was then backcrossed with palm t from the nigerian germplasm to produce the bc pollen was then obtained from a selected bc palm to cross-pollinate two e. guineensis palms. the first e. guineensis was derived from a cross between the palm t and a serdang pisifera and the second palm was derived from a self-pollination of the original t palm. the two bc families are named progenies ‘ –1’ and ‘ –5’, consisting of and palms, respectively. they were field planted in the year the female parent of – died before any leaf and fruit could be sampled. therefore, two siblings to 320/tt113/ were genotyped to help with scoring and phase configuration of the markers used for the construction of genetic maps.fig. the oxg mapping population and bc validation crosses used in this study. the oxg interpecific hybrids were created by crossing a maternal colombian e. oleifera with a paternal palm t, from the nigerian germplasm. the same t palm was also used in creating the bc validation crosses. the la mé e. guineensis was crossed with a colombian e. oleifera and the resulting gxo f hybrid was then backcrossed with the t palm to produce the bc pollen from a selected bc palm was used to cross-pollinate two e. guineensis palms. the first e. guineensis was derived from a cross between the t and a serdang pisifera and the second palm was derived from a self-pollination of the t palm. the two bc families namely, – and – consisted of and palms, respectively extraction and analysis of palm oil the procedures for sampling ripe fruits and extracting mesocarp oil were as described by singh et al.. the porim test method was applied to measure the iv and fac in the palm oil. candidate snp markers and genetic linkage map construction candidate snp markers flanking various genes associated with fa and oil biosynthesis were mined from the p genome build. the oil palm snp assay design and genotyping were performed by a service provider, agena bioscience, inc. using the iplex ® biochemistry on massarray® system. a custom two-multiplexed genotyping assay was designed and optimized for a panel of snps using the assay design suite software. primary pcr primers were designed to contain a common 5’ 10-mer tag and synthesized by idt. the primary pcr reaction was carried out to amplify dna fragments of approximately 100 bp containing targeted snps from the good quality oil palm genomic dna. the pcr conditions included an initial denaturation at 95 °c for 2 min, 45 cycles of 95 °c denaturation, 56 °c annealing and 72 °c extension, and a final extension at 72 °c for 5 min. the remaining unincorporated dntps in the pcr product was inactivated using shrimp alkaline phosphatase treatment. this was followed by an allele-specific single base primer extension reaction performed using the iplex gold reaction kit. reaction cocktail containing extend primer, buffer, enzyme, and mass-modified ddntps was added to the primary pcr product and proceeded to extension reaction at 94 °c initial denaturation, 40 cycles of 95 °c denaturation, 52 °c annealing and 80 °c extension, and a final extension at 72 °c for 3 min. the extension reaction product was desalted using the spectroclean resin treatment and transferred onto the spectrochip® array for data analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. the snp genotyping data and report were generated using the typer software. the genotyping data were incorporated into the existing dataset for construction of the genetic map. furthermore, the aflp and rflp markers reported by singh et al. to be flanking the qtl regions were also incorporated into the existing linkage map which was re-constructed using joinmap® as described previously. the genetic maps for the two validation crosses, – and – were similarly constructed using the same set of snp and ssr markers. comparison of the genetic maps for oxg, – and – was carried out using mapchart. quantitative trait loci analyses in the oxg population, the qtl analysis was performed using interval mapping, the multiple-qtl model and the kruskal-wallis non-parametric ranking tests with the default parameters in mapqtl. the 95 % genome-wide lod significance threshold for each trait was determined by the permutation test option, with permutations. each candidate qtl interval was further analyzed using g model. the gm analysis first estimated the genome-wide background effects of markers from all the lgs using a random effects model. the background effects were used to correct and determine significant markers by backward elimination on each lg. finally, the effect of each significant marker was estimated using the multiple regression coefficients. similar qtl analysis approaches were also applied in the – and – crosses to validate the detected qtls. development of candidate ssr markers within qtl confidence regions development of ssr markers for candidate fa genes and tf was carried out by aligning contigs and clone sequences containing the snps and ssrs mapped in the qtl confidence intervals, using blastn. markers from another independent genetic map that localize in the qtl-syntenic regions were also included in the similarity search. scaffold regions linked to qtl confidence intervals were extracted from the p genome build and searched for sequence similarity against the ncbi databases. sequences with significant similarity to genes and tfs associated with fa and tag syntheses were selected to attempt to mine ssrs using the microsatellite identification tool. four percent super fine resolution agarose gel electrophoresis was used to screen for potential polymorphism of the candidate ssrs against a panel of six randomly selected oxg hybrids and the two parental palms. the informative ssrs were then pcr-amplified using the m13-tailed primer approach as described by ting et al.. fragment analysis of the pcr products was performed by a service provider at the centre for marker discovery and validation, malaysia using an abi 3730xl dna analyzer with fluorescent dye sets comprising fam, ned, pet, vic and genescan™- liz® size standard. sizing of the ssr alleles was performed using the genemapper® software and genotype scoring was as described previously.  mapping candidate snps for fa genes onto the oxg genetic map in the oxg interspecific population, the existing paternal t and partially integrated maps had markers/ lgs/  cm and markers/ lgs/  cm, respectively. the two genetic maps were constructed mainly with snp and ssr markers replacing the traditional aflp and rflp markers used in the earlier version. the new marker systems are technically more user-friendly, cheaper and give better reproducibility than the aflp and rflp markers. however, to anchor the previously detected qtls, the aflp and rflp markers flanking each qtl interval were also incorporated into this study. an additional snps were identified flanking various fa genes distributed across scaffolds of the p genome build. of these, seven for palmitoyl-acp thioesterase, oleoyl-coa desaturase, linoleoyl-coa desaturase, enoyl-acp reductase and stearoyl-acp desaturase – were polymorphic in the oxg mapping population. these candidate snp markers were mapped onto lgs ot t ot ot and t and used in the subsequent qtl analysis. for the oxg mapping population, as only integrated lgs were available, six lgs from the t paternal map were also included in the qtl analysis. bc genetic linkage maps in the bc validation crosses, two independent integrated maps with markers/ lgs/.5 cm and markers/ lgs/.7 cm were constructed for the – and – populations, respectively. resolution of the two genetic maps was good and the linear order of markers comparable to that in oxg with an average gap of  cm and  cm. both the high density genetic maps were used for qtl analysis. iv and fac quantitative phenotypic data in the oxg cross, palm oil was obtained from 85 f palms and measured for iv, c14: c16: c16: c18: c18: c18: and c18: the remaining palms could not be sampled due to sterility issues, abortive bunches and crown disease. also palms had died before phenotyping. the present phenotypic data set includes an additional four palms to that reported by singh et al. and is summarised in table  overall, the distribution of phenotypic data was similar to that reported previously. the iv and major fas, c16:, c18: and c18: showed a normal distribution. however, the four minor components, c14:, c16:, c18: and c18: did not follow a normal distribution, also previously observed by montoya et al.. for c18: the data was converted to a normal distribution by log transformation, but the approach did not convert c14: c16: and c18: to a normal distribution. for these non-normally distributed traits, kw analysis was used. correlation analysis gave similar results to those by singh et al..table summary of phenotypic data in the oxg mapping population and two bc validation crosses means, ranges and variances measured for iodine value and various fatty acid contents in palm oil for the two bc crosses, data were collected for and palms of the – and – crosses, respectively. the other palms did not bear any fruit or had died before sampling could be performed. in – the widest data range observed was for c18: and c16:. all the data showed normal distributions except for iv, c14: and c16: however, log transformation and the discarding of one or two outliers returned the distributions to normality. outliers were identified by using a boxplot and comparing the observed and expected mean values. significant positive correlations were observed between c14: and c16:, iv and c18: and, iv and c18:. strong negative correlations were observed for c16: and c18: iv and c16: iv and c14: and, c16: and c18: while, c14: and c18: c14: and c18: and, c18: and c18: showed moderate negative correlations of − to –. in – c18: and c16: also showed the widest data distribution of –  % and –  %, respectively. the phenotypic data demonstrated a normal distribution. for c14: and c16: normality was improved after removing one outlier. the phenotypes iv and c18: c14: and c16: c14: and c18: and, c14: and c16: showed moderate correlations while, negative correlations were obtained for iv and c16: c16: and c18: c18: and c18: c14: and c18: and, c16: and c18:. oxg: qtls linked to iv and fac this study aimed to identify the qtls linked to iv and fac in the improved snp and ssr-genetic map of the oxg cross. ten genome-wide significant qtls and two putative qtls linked to iv, c14: c16: c16: c18: c18: and c18: were identified on lgs ot t t ot ot and t using a combined qtl detection method – im, mqm and kw. only markers or regions consistently linked to the specific qtl using all three approaches were considered to be significant qtl. subsequently, gm was used to estimate the effects of closely flanked markers using multiple regression coefficients after backward elimination and adjustment for the background marker effects. on the improved lgot, three major qtls and two minor qtls were revealed in the same map interval previously reported between markers pop-cb00075a and eagg/mcat-. on the current lgot the significance interval between the two markers was filled by snps and three ssrs. of these, six snp markers, including a pate candidate snp, were within the qtl confidence interval. this genomic region was of interest and further investigation was carried out with the additional results presented below in the ‘fine-mapping of the qtls on lgot1’ section.fig. qtl profiles linked to iv and fac on the improved genetic linkage map of oxg. the graphs present qtls significant at 95 % genome-wide and chromosome-wide thresholds in linkage groups ot t ot t t and ot6 for group, three genome-wide significant qtls for c14: c16: and c18: were identified and the interval was flanked by markers eaag/mcat- and p4t8i, as reported previously. the current map position for eaag/mcat- was determined using a less stringent mapping parameter. the interval between eaag/mcat- and p4t8i contained three snp markers, which reduced the gap to  cm/markers from the previous  cm. for c16: and c18: the qtl peak co-localized with markers snpm and snpm at map position  cm. using gm, the marker effect was estimated to be  % and −  % for c16: and c18: respectively. when the genotypes of the two markers were analysed, the ab and aa profiles showed significant differences for c16: and c18: contents in the oxg hybrids. for snpm, the homozygous aa genotype showed an average  % for c16: and  % for c18: content which differed from the heterozygous ab genotypes. however, for c14: the closest marker was snpm with a minor effect of −  % and which also showed a significant difference in c14: content between the aa and ab genotypes.fig. distribution of phenotypes categorized based on the genotypes of the closest markers linked to the observed qtls in lgt the upper panel is of hybrids with genotype ab while the lower panel shows the phenotypes observed in genotype aa in snpm for c16: and c18: and, snpm for c14: for lgot qtl was detected for c18: in the –  cm interval flanked by snpm and snpm. the closest marker snpm at  cm, had a positive effect of  % on the trait, as estimated by gm. on lgt, three markers showed significant association with c14:. these markers, co-localized with the qtl peak at map position  cm, were found at a distance  cm from the previously detected qtl. in addition, two putative qtls for c18: were detected on lgs t and ot. the putative qtls were considered in this case as the regions concerned also explained similar qtls in other studies. a summary of the qtl results is presented in table  table qtls linked to iodine value and fatty acid composition in the oxg interspecific mapping population. qtls identified using interval mapping, the multiple-qtl model, the kruskal-wallis non-parametric tests and g model ot1a pa5_ossra pa3_ossra spsc00328a ot1a pa5_ossra pa3_ossra spsc00328a ot1a pa5_ossra pa3_ossra spsc00328a ot1a ot1a pa5_ossra pa3_ossra spsc00328a aafter fine mapping gw: 95 % genome-wide significant lod threshold cw: 95 % chromosome-wide significant lod threshold fine-mapping of the qtls on lgot1 the map interval –  cm on lgot revealed multiple qtls with a relatively large proportion of phenotypic variation explained and was of particular interest. admittedly, the phenotypic variation could have been overestimated due to the limited size of the mapping family, leading to a beavis effect. the markers in this region were mapped to the p genome build. to improve the efficiency of finding the qtl corresponding region in the genome build, additional markers that fit into the region were also obtained from an independent e. guineensis map. this resulted in markers mapping to scaffolds p5_sc and p5_sc with high identities and e-values – 1e- the total physical coverage of the qtl interval on p5_sc and p5_sc were,438 bp and,644 bp, respectively. the blast results for the qtl corresponding region identified significant similarity to one tf and a number of genes associated with the fa and tag biosynthesis pathways. this included diacylglycerol acyltransferase and long chain acyl-coa synthetase in p5_sc in p5_sc sodium/metabolite cotransporter, palmitoyl-acp thioesterase, 3-hydroxyisobutyryl-coa hydrolase-like protein and ap2-like ethylene-responsive tf were found. for fine mapping the qtl confidence interval, ssrs were mined and developed from the candidate gene introns and regions flanked at –  bp before the 5’ start codon and 351–  bp downstream of the genes. in addition, six ssrs were developed specifically to fill in the gaps in the identified interval and therefore were not located near to any candidate gene. of the developed candidate ssrs, were polymorphic and mapped back to the qtl interval on lgot unfortunately, five ssrs for dgat were not polymorphic and could not be mapped onto lgot refined qtl intervals for iv, c14: c16: c18: and c18: as well as the map positions of the candidate genes and tf are illustrated in fig.  fig. fine-mapping of qtl intervals with candidate markers and cross-validation of qtls in two independent bc crosses. similar qtls between lgot and lgs and 1b of bc validation crosses are indicated by common markers and candidate ssr markers after fine-mapping with candidate markers, slight increases in the lod scores were observed for markers in the qtl intervals. for iv, significant qtl with lods – were revealed in the 113–  cm interval on lgot two candidate genes, hibch and pate/fatb, together with three candidate ssrs and six existing snps co-localized directly with the qtl peak. no recombination event was observed between the nine markers, possibly due to the limited size of the oxg population and the short distance on the physical map. using gm, the nine markers were detected to have a significant effect close to. this result indicated that the maternal up genotype had an increasing effect on iv compared to the t genotype for snpm snpm spsc snpm or snpe in contrast, aa showed reduced levels of iv compared with ab when genotyped with snpm snpm pa5_ossr or pa3_ossr. the difference in mean iv between the two genotypes improved to compared to estimated previously using the rflp marker, pop-cb00075a by singh et al..fig. distribution of iv and fac phenotypes in oxg hybrids genotyped by the closest markers linked to the qtls on lgot the upper panel is of hybrids with genotype ab while the lower panel shows the phenotypic distribution of genotype aa in snpm snpm spsc snpm or snpe for iv, c18:, c16: and c14:. for c18:, the ab and aa genotypes are observed in spsc00314 the same group of markers were also closely linked to the qtl for c18: with similar allelic effects as observed on iv. these markers detected a greater difference in c18: content compared to the  % explained by pop-cb00075a previously. another qtl revealed by the same group of markers was for c16: content. the estimated marker effect of  % had increasing alleles from the other parent, compared to that for iv and c18: which agrees with the strong negative correlation between the levels of saturated and unsaturated fas. the difference in the c16: mean values of the homozygous and heterozygous genotypes also increased to  % from the  % previously reported. in addition to the three major qtls, these markers also had a minor effect of  % on c14: with the increasing alleles inherited in the same direction as c16: the difference in c14: content between genotypes aa and ab was also relatively low at  %. with respect to c18: the refined qtl interval revealed a new candidate ssr marker spsc at the qtl peak with a negative effect of  %. a difference of  % in c18: was observed between the aa and ab genotypes of spsc. on the genome scaffold, spsc was located between pate/fatb and bass with an estimated distance of ~  kbp and kbp, respectively. therefore, it is still unclear at this point which of the genes, if any, is influencing the minor qtl for c18: validation of qtls for iv and c16: in bc2 in this study, the qtls detected for iv and c16: in the oxg mapping population were tested for validation in the two bc crosses− – and – in – a major qtl was detected for iv with lod on lg the  cm confidence interval containing one ssr and snps was then aligned to lgot using nine transferable markers which clearly showed that the same region was affecting iv in both crosses. of these markers, snpm underlying the qtl peak revealed three genotypes – aa, ab and bb – in the - palms. the aa genotype, similar to that observed in oxg, differentiated the iv mean from the bb genotype with p =  but, no significant difference observed in genotypes aa vs. ab and bb vs. ab. the same marker snpm was also detected flanking a putative qtl peak for c16: with minor effect. the qtls with overlapping confidence intervals for iv and c16: were also identified in the – cross on a subgroup of lg. when compared with lgot in oxg, the same qtl region encompassing the common marker spsc was also located at the end of the lg1b. for iv, the qtl peak showed a high lod score with the confidence interval and contained two candidate ssr markers, spsc and spsc with estimated effects of – iv units. for the closest marker spsc the maternal genotype showed significantly lower unsaturation – compared to in the paternal genotype. the spsc also showed a significant effect of  % and the aa and ab genotypes revealed a significant mean difference in c16: content. additional qtls detected in the bc2 the – and – crosses revealed additional qtls for c18: on lg which were not detected in oxg. for – the qtl in the confidence interval –  cm enclosed seven markers. of these, the closest flanking markers to the qtl peak were snpm and snpm with estimated effects of  % and  %, respectively. in the – cross, similar qtl although significant at the chromosome-wide level rather than the genome-wide level were also detected on lg with lods ranging from –. the left-right flanking markers were snpm snpm snpm snpm and snpm at  cm and snpm snpm and snpm at  cm. in comparison, more new qtls were detected in – than in – these include i. c18:, ii. c18:, iii. iv, iv. c16: and, v. c18:. all were minor qtls, significant at the 95 % chromosome-wide level. discussion this study extended the mapping of qtls linked to iv and fac in the oxg interspecific mapping population initiated by singh et al.. the preliminary map constructed with markers localized qtls mostly loosely flanked by aflp and rflp markers. therefore, in this study, a x more saturated genetic map was used to localize the qtls and improve coverage. a total of genome-wide and two putative qtls for iv and various fas were identified on six improved lgs, which included of the qtls detected previously. the results indicated that the current ssr and snp-based genetic map was effective in revealing qtls with the only exception being the minor qtl for c14: reported earlier on group. however, a new putative qtl was revealed for c14: at the nearby region of the same lg. the shift in location could indicate a more precise estimation of the qtl with the improved map resolution on lgt with a similar situation having been reported in eggplant. the detected qtls at this stage can only be considered to be specific to the oxg cross which would limit application of the markers for mas. mapping families created via a sequential series of backcrosses and self-pollination would ideally help validate the qtls as reported in rapeseed, maize and soybean. however such mapping families are not readily available for a perennial crop like oil palm and it would take about 10 years to create each generation, with initial phenotyping, apart from the huge investment required in land, labour and management. it is for this reason that, to date, qtls for iv and fac in oil palm have only been reported in single crosses involving a bc tenera x dura f and an oxg hybrid. in this study, we used two independent bc pseudo-crosses created by introgression of e. oleifera into t germplasm materials to try to validate the qtls detected in the oxg hybrid population. to our knowledge, this is the first attempt to validate qtls for fac in oil palm of similar genetic backgrounds. the two bc crosses are related by pedigree and also genetically linked with the t germplasm. here we adopted a strategy to evaluate the qtls through an independent analysis with subsequent comparison of their locations. however, our results showed that only the major qtls for iv and c16: were successfully cross-validated through a common location, and all similarly revealed a large proportion of phenotypic variance in both – and – the common qtls in bc revealed that the different genotypes of closely linked markers also showed distinct levels for iv and c16: content. the finding is of interest as iv represents the cumulative unsaturation in palm oil and it has always been used as an indicator in selecting for higher unsaturation overall in breeding programs, whereas the qtl for c16: can complement the prediction by indicating the palmitic level. it is however noted that the qtl for c18: could not be cross-validated in the two bc populations even though c18: is a major component contributing to iv and in this study was highly correlated with iv and negatively correlated with c16: the small size of the mapping families could have limited the detection power for the qtl analysis, resulting in the lack of detection of the qtls in the bc crosses. a similar observation was made in maize about the effect of population size when a sub sample only revealed about 1/ of the qtls observed in larger samples. in addition to validating qtls in the interspecific bc eight of the identified qtls were similar to those reported previously using either a bc or a tenera × dura cross. multiple qtls for c14: c16: c18: and c18: were reported previously in the respective intervals 0–, 0–, 0– and –  cm on lg in the tenera x dura mapping family. the intervals were aligned with the current qtls on lgt using three common markers, megcir megcir and megcir pointing to a similar map region. in the previous studies by montoya et al., the intervals in lg were reported to be linked to c18: and c18: respectively. similar regions affecting the qtls were also detected in the present oxg and – crosses, determined using the common markers megcir megcir and megcir mapped in both the studies. another possible common qtl was c14: on lgt which was also previously reported on the same lg in the bc. the putative qtl linked to c18: on lgt and lgot were similar to that reported previously. the qtl on lgt matched the genomic region reported previously for a bc population, as revealed by the common marker megcir mapping in the same region. with respect to lgot the qtl region was very close to that reported previously for the same trait by singh et al.. the above comparison has provided useful information for comparing qtls linked to fac by different research groups. we compiled and compared a total qtls linked to iv and fac across the different mapping families. however, very low percentages of the qtls were common across two to three of the crosses and none at all in more than three crosses. this highlights that in qtl analysis, even for a highly heritable trait such as fac, favourable alleles are likely to be population-specific requiring careful selection for implementation in mas. nevertheless, this study revealed a core set of markers that can be useful for mas, especially in genetic backgrounds involving the t and e. oleifera parental palms. as the palm materials are commonly available and accessible to the oil palm industry, the markers will have practical application for selecting favourable alleles for unsaturation in interspecific hybrids and their backcrosses. detection of similar qtls for iv and c16: in both oxg and bc allowed the underlying qtl interval to be compared to the recently published oil palm genome build. this study is also the first to describe attempts at fine-mapping of targeted qtl regions for iv and various fac, by exploiting the whole genome sequence data of oil palm. the genomic region of about kbp showed the presence of an interesting gene with high similarity to pate/fatb in oil palm and coconut. comparison of genetic maps using the common markers megcir megcir and megcir estimated that the current map position of pate/fatb in lgot is in accordance with montoya et al.. however, the authors did not associate the gene or region around the gene with any qtl for iv or fac. in oil palm, pate/fatb encodes the palmitoyl-acp thioesterase that actively releases c16: from c16:0-acp and allows the resulting non-esterified fa to be incorporated into tag and is one of the main factors responsible for the relatively high saturation levels in palm oil. a similar role was reported for pate/fatb in oil biosynthesis in cotton seed. apart from a strong substrate preference for c16: the substrate specificity of palmitoyl-acp thioesterase also extends to other saturated acyl-acps, including c14:0- and c18:0-acps. this was shown in other species by over-expression of pate/fatb in arabidopsis thaliana and canola seeds which also had an elevated concentration of c14: this is in agreement with the strong positive correlation observed in this study between the c16: and c14: phenotypes. in contrast, the phenotypic data for levels of c16: had a strong negative correlation with c18: levels which indicates that a reduction of c16: is associated with increased c18: this was also observed in maize seeds where a mutated fatb allele caused a dramatic reduction of c16: in the oil and increased the level of unsaturated fas. the truncated fatb was reported responsible for the non-functioning of a critical catalytic domain and this could have resulted in the channelling of more c16:0-acp for elongation and subsequent desaturation into unsaturated acyl chains, such as c18:1-acp. it was also suggested that at least one of the palmitoyl-acp thioesterase isoforms in oil palm can hydrolyze c18: efficiently. this could also explain the co-localization of qtls for iv, c14: c16: c18: and c18: on lgot found in this study. therefore, the next step is to evaluate the identified pate/fatb gene in palms with varying contents of c16: and c18: another interesting region was at ~  kbp from pate/fatb. this genomic region contained a gene with significant similarity to hibch in both oil palm and date palm. in oil palm, information about the role of hibch associated with fa and tag biosynthesis is very limited. this enzyme is reportedly involved in the degradation of fas in camelina sativa which takes place in mitochondrial and the peroxisomes. in coconut, hibch was reported as being down-regulated during the production and accumulation of oil. several other interesting genes and tf encoding key enzymes involved in both the fa and tag pathways, including bass lacs dgat and wri were also found in proximity to the qtl peak. bass is reported to play a major role in channelling pyruvate into plastids for conversion into acetyl-coa and malonyl-coa – the main substrates to initiate synthesis of fa. at the final stage of fa biosynthesis, non-esterified fas are converted into acyl-coa by long chain acyl-coa synthetase and later transferred through the plastid envelope into the er for synthesis of tags. on the current lgot the long chain acyl-coa synthetase family member was identified at  cm close to the end of the qtl interval. in a. thaliana, lacs showed greatest preference for the c16: substrate when expressed in e. coli. substrate preference for various fas was reported in the following order: c16:0 > c16:1 > c18:2 > c18:1 > c20:1 > c18:. this may again explain the co-localization of qtls for c16: with other fas on lgot where genes, such as pate/fatb and lacs show pleiotropic effects contributing to the complex interactions with the substrates in fa synthesis. in this study, dgat is the only gene detected in the qtl region that is involved in formation of tags in the acyl-coa-dependent pathway. the ~  kbp genomic region has high sequence similarities to dgat in oil and date palms. in oil palm, dgat is known to interact with other acyltransferases, in particular, dgat to synthesize tag. it is generally agreed that dgat plays an important role in one of the oil palm lipid production pathways. however, information on the specificity and selectivity of each paralogue of dgat in oil palm is still lacking and requires further study to establish their function and effect on the composition of fa in palm oil. in addition to the key enzymes mentioned above, we identified a wri tf co-localized with two markers at  cm in the qtl region on lgot the oil palm wri has high similarity to that in date palm, plum, euphrates poplar and grape. this tf has long been considered a master regulator directly influencing a number of enzymes in the fa and tag synthesis pathways in plants, including oil palm.  this study for the first time extensively compared the qtls linked to iv and fac across various interspecific genetic backgrounds. the detected qtls, to some extent, are population specific although the major qtls can be observed across related genetic backgrounds. a core set of markers with practical application in selection for higher unsaturation in fa levels were described. the candidate markers linked to the qtls revealed significant allelic and genotypic differences associated with iv and fac, suggesting that the markers are potentially useful in mas, at least in the genetic backgrounds described. the fine-mapping approach employed in this study also proved effective in identifying candidate genes and a transcription factor affecting iv and fac in palm oil. the identified genes and transcription factor, hibch, pate/fatb, bass lacs dgat and wri underlying the overlapping qtl confidence intervals for iv, c14: c16: c18: and c18: have provided valuable information on several potential candidate genes and a transcription factor which are known in other species to strongly influencing the biosynthesis of fa and tag. thus, to fully understand the interaction and effects of these genes, the listed candidate genes will be further studied to determine their expression levels in palms with different iv and fac. availability of supporting data the sequence information for the snp and ssr markers is available at http://genomsawit.mpob.gov.my additional files additional file 1: mapping candidate markers for various fatty acid related genes in the oxg linkage map. candidate snp markers for palmitoyl-acp thioesterase, oleoyl-coa desaturase, linoleoyl-coa desaturase, enoyl-acp reductase and stearoyl-acp desaturase mapped onto lgs ot t ot ot and t candidate ssr markers also developed for ketoacyl-acp synthase i, acetoacetyl-coa thiolase, long chain acyl-coa synthetase, ap2-like ethylene-responsive tf, 3-hydroxyisobutyryl-coa hydrolase-like protein, sodium/metabolite cotransporter, pate/fatb and sad. additional file 2: pearson’s correlation coefficients for iodine value and fatty acid composition in palm oil of oxg mapping population. additional file 3: pearson’s correlation coefficients for iodine value and fatty acid composition in palm oil of bc validation cross. additional file 4: pearson’s correlation coefficients for iodine value and fatty acid composition in palm oil of bc validation cross. additional file 5: co-linearity of markers between the oxg and bc populations as observed in six linkage groups hosting qtls. abbreviations blastnsimilarity search of the ncbi nucleotide database using a nucleotide query blastxsimilarity search of the ncbi protein database using a translated nucleotide query cmcentimorgan map distance lodlogarithm of odds ntncbi nucleotide collection nrncbi non-redundant protein sequences database p5e. guineensis 5th genome build p5_scxxxxxe. guineensis 5th genome scaffolds pax_ossrcandidate ssr marker for palmitoyl-acp thioesterase pcrpolymerase chain reaction refseq_proteinncbi reference proteins database sdstandard deviation snpmxxxxxsingle nucleotide polymorphism marker snpexxxxxcandidate single nucleotide polymorphism marker spscxxxxxcandidate simple sequence repeat marker competing interest the authors declare that they have no competing interests. authors’ contributions rs, rs, sm, fm, n-ct, zy and kk conceived and designed the experiments. n-ct, zy and kk carried out molecular experiments and data analyses. llet, n-ct and rs participated in the snp experimental design and coordination. jj assisted with the statistical analysis of the phenotypic and marker data. llet, na, rr and k-lc provided bioinformatics support and involved in mapping the qtls to genome build. jj, sm, fm, rs, rn, mda, mi, ak, xa, ks and lcc advised on the design of the study and contributed materials as well as reagents. n-ct, zy and kk drafted the manuscript. rs, sm, rs and fm assisted with interpretation of data and revised the manuscript critically. all authors read and approved the final manuscript. 
the commercial oil palm produces a mesocarp oil with approximately equal proportions of saturated and unsaturated fatty acids. one way to manipulate the fatty acid composition in palm oil is through introgression of favourable alleles from the american oil palm, e. oleifera, which has a more unsaturated oil. in this study, a segregating e. oleifera x e. guineensis hybrid population for fac is used to identify quantitative trait loci linked to iv and various fas. clustering trait values according to the allelic forms of these candidate markers revealed significant differences in the iv and fas of the palms in the mapping and validation crosses. qtl analysis revealed major and two putative qtls for iv and six fas, c14: c16: c16: c18: c18: and c18: distributed across six linkage groups, ot t t ot ot and t the major qtls for iv and c16: on lgot explained –  % of the phenotypic trait variation and were validated in two independent bc populations. an increase in unsaturated fas content or iodine value as a measure of the degree of unsaturation would help to open up new markets for the oil. elaeis guineensiselaeis oleiferabackcross-two iodine value palmitic acid oleic acid malaysian palm oil boardissue-copyright-statement© the author 2016 using the whole genome sequence data, markers tightly linked to the candidate genes were also developed. the candidate gene approach described and exploited here is useful to identify the potential causal genes linked to fac and can be adopted for marker-assisted selection in oil palm.
 the commercial oil palm produces a mesocarp oil with approximately equal proportions of saturated and unsaturated fatty acids. an increase in unsaturated fas content or iodine value as a measure of the degree of unsaturation would help to open up new markets for the oil. one way to manipulate the fatty acid composition in palm oil is through introgression of favourable alleles from the american oil palm, e. oleifera, which has a more unsaturated oil.  in this study, a segregating e. oleifera x e. guineensis hybrid population for fac is used to identify quantitative trait loci linked to iv and various fas. qtl analysis revealed major and two putative qtls for iv and six fas, c14: c16: c16: c18: c18: and c18: distributed across six linkage groups, ot t t ot ot and t the major qtls for iv and c16: on lgot explained –  % of the phenotypic trait variation and were validated in two independent bc populations. the genomic interval contains several key structural genes in the fa and oil biosynthesis pathways such as pate/fatb, hibch, bass lacs and dgat and also a relevant transcription factor, wri the literature suggests that some of these genes can exhibit pleiotropic effects in the regulatory networks of these traits. using the whole genome sequence data, markers tightly linked to the candidate genes were also developed. clustering trait values according to the allelic forms of these candidate markers revealed significant differences in the iv and fas of the palms in the mapping and validation crosses.  the candidate gene approach described and exploited here is useful to identify the potential causal genes linked to fac and can be adopted for marker-assisted selection in oil palm. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords elaeis guineensiselaeis oleiferabackcross-two iodine value palmitic acid oleic acid malaysian palm oil boardissue-copyright-statement© the author 2016 