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-6 Desaturase Enzymes and Their Cosegregation with QTL Affecting Oleate Content in Soybean
a Dep. of Crop Science, North Carolina State Univ., Campus Box 7620, Raleigh, NC 27695-7620
b USDA-ARS, Soybean and Nitrogen Fixation Research Unit, 3127 Ligon St., Raleigh, NC 27607
* Corresponding author (ebachla{at}ncsu.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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-6 desaturase enzymes, which catalyze the desaturation of oleic acid to linoleic acid during fatty acid biosynthesis, are encoded by the FAD2-1 and FAD2-2 genes in soybean [Glycine max (L.) Merr.]. Breeders aim to incorporate the high-oleate trait into soybean germplasm in order to improve the nutritional value and oxidative stability of soybean oil. The objectives of this study were to map the isoforms of the FAD2-1 and FAD2-2 genes and investigate the association of these genetic loci with the oleate phenotype in three populations segregating for oleate content. The populations were grown in replicated multienvironment field trials. According to linkage analysis conducted for two of the populations, FAD2-1A and FAD2-1B mapped on Linkage Groups O and I, respectively, while the closely linked FAD2-2A and FAD2-2B isoforms mapped on Linkage Group L. Oleate quantitative trait loci with minor effects were detected in the proximity of FAD2-1B and possibly FAD2-2B on Linkage Groups I and L. Quantitative trait loci affecting maturity were also detected on chromosomal regions adjacent to the FAD2 genes. The genotyping assays developed for the FAD2-1A, FAD2-1B, and FAD2-2B isoforms, as well as their linked simple sequence repeat markers, can be used in soybean breeding programs for the elevation of oleic acid seed content through marker-assisted selection.
Abbreviations: ASPE, allele-specific primer extension • BLUP, best linear unbiased predictor • bp, base pair • CAPS, cleaved amplified polymorphic sequence • FAD, fatty acid desaturase • QTL, quantitative trait locus • SSR, simple sequence repeat • SBE, single-base extension • SNP, single nucleotide polymorphism
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Fatty acids are the precursors for the synthesis of triacylglycerols (TAGs), which constitute the storage lipid reserves of the developing oilseeds. Polyunsaturated fatty acids of the seed TAGs are synthesized primarily by -3 and -6 desaturase enzymes located in the endoplasmic reticulum; however, a small percentage is also derived from comparable enzymes localized in the plastids. The desaturase enzymes of the endoplasmic reticulum act on the fatty acids esterified to the first and second backbone position of phosphatidylcholine (PC), whereas the plastid-localized desaturases predominantly utilize galactolipids as substrates (Ohlrogge and Browse, 1995).
Plant fatty acid desaturase genes were initially elucidated in Arabidopsis. The desaturation of oleic to linoleic acid in the endoplasmic reticulum was shown to be controlled by the fad2 gene, encoding the oleoyl-PC -6 desaturase activity (Miquel and Browse, 1992; Okuley et al., 1994). Subsequently, genes designated FAD2-1 and FAD2-2 were reported to encode comparable activities in soybean (Heppard et al., 1996). The FAD2-1 gene was expressed only during seed development and FAD2-2 was constitutively expressed during seed development and vegetative growth. Therefore, it was suggested that the seed-specific FAD2-1 is primarily responsible for the desaturation of oleic to linoleic acid for fatty acids destined for seed storage lipids, while FAD2-2 is involved in the desaturation of membrane lipids in vegetative tissues, playing a lesser role in storage oil biosynthesis. Moreover, the existence of at least two copies of each of the FAD2-1 and FAD2-2 genes was proposed (Heppard et al., 1996).
Recently, the existence of two distinct isoforms of the soybean FAD2-1 gene (designated FAD2-1A and FAD2-1B), encoding microsomal -6 desaturase enzymes that differ in stability at high temperatures, was confirmed (Tang et al., 2005). The FAD2-1A and FAD2-1B genes map on Linkage Groups O and I, respectively, in the soybean genome. Both FAD2-1A and FAD2-1B produce alternatively spliced transcripts that do not change the amino acid residues of the encoded -6 desaturase enzymes (Schlueter et al., 2007). For FAD2-2 genes, which also encode microsomal -6 desaturase enzymes, three isoforms have been reported, designated FAD2-2A, FAD2-2B, and FAD2-2C. FAD2-2A and FAD2-2B are localized on Linkage Group L (Schlueter et al., 2007).
Environmental factors affect the unsaturated fatty acid content of soybean oilseeds. Temperature exerts an effect on the unsaturated fatty acid composition during the pod-filling stage that coincides with oil deposition. Planting and maturity dates lead to changes in the fatty acid profiles due to different temperatures during oil deposition (Burton et al., 1983; Wilcox and Cavins, 1992). For example, the elevated-oleic lines N98-4445A and N97-3363-3, which were used in this study, are unstable across environments and their oleate variation is largely attributed to growth temperature differences (Oliva et al., 2006; Wilson et al., 2002). Although temperature effects during oil deposition were implicated in the altered fatty acid composition of soybean oilseeds, the expression patterns of the isoforms encoding the -6 desaturase enzymes, with the exception of FAD2-2C, showed no change in the level of transcript accumulation at different temperatures during seed development that could explain the changes in the levels of unsaturated fatty acids in seed triacylglycerols (Heppard et al., 1996; Schlueter et al., 2007). A recent study of the -6 desaturase enzymes encoded by the FAD2-1A and FAD2-1B genes, however, provided evidence of differential enzymatic stability at high temperatures (Tang et al., 2005). Apart from temperature effects, coordination of metabolite flux through the reactions of glycerolipid biosynthesis in the endoplasmic reticulum coupled with the de novo fatty acid biosynthesis pathway in the plastids underlie the complexity of the factors (both genetic and environmental) that can ultimately determine oleate composition of the storage oil.
Although the location of the isoforms encoding the -6 desaturase enzymes was recently revealed, there is minimal information regarding whether the isoforms of FAD2-1 and FAD2-2 genes cause the observed genotypic variation for oleate content in populations in which this trait is segregating. As an initial step toward unraveling the complexity of oleate biosynthesis and accumulation in soybean, the effects of the FAD2-1 and FAD2-2 isoforms (and their alleles), as well as their epistatic and environmental interactions, need to be investigated (Cardinal, 2008). The objectives of this study were to: (i) identify single nucleotide polymorphisms (SNPs) in the coding or upstream regions of the isoforms of FAD2-1 and FAD2-2 genes for the soybean lines under study; (ii) develop isoform- and allele-specific markers; (iii) map the individual isoforms of the FAD2-1 and FAD2-2 genes in the soybean genome; and (iv) investigate whether segregation at FAD2-1 and FAD2-2 loci is associated with changes in oleic acid seed content in three soybean populations segregating for oleate content.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Experimental Design
The FAE and FAF populations were planted separately in sets -within-replications experimental designs with two replications in each location. Fifteen sets of seven rows by seven columns were randomly assigned in each replication, location, and year for the FAE population. Five sets of five rows by five columns were randomly assigned in each replication, location, and year for the FAF population. In each population, before lines were randomly assigned to each set, they were divided into five arbitrary groups that differed in maturity according to the lines' maturity phenotypes in 2004. Each maturity group was represented at least once in each set of the FAE and FAF populations, and the maturity groups that included the majority of the segregating lines were represented two to three times in each set. Once lines were randomly assigned to a set, they remained in that set in all replications, locations, and years. Within each set, the maturity groups were randomly assigned to a column to facilitate mechanical harvest. Then the lines within each maturity group were randomly assigned to plots within each column. The parental lines N97-3363-3 and PI423893 in the FAF population and the parental line N97-3525 in the FAE population were randomly assigned to one set, to a maturity group within that set depending on their maturity date, and, finally, to a plot within the set, as explained above. The N98-4445A parental line of the FAE population was assigned to a maturity group in all the sets and randomly assigned a plot within each set as explained above. The FAE population was planted in four-row plots at Clinton, Kinston, and Plymouth, NC, in 2005 and 2006. The FAF population was grown at Clinton and Kinston in 2005 and at Clinton, Kinston, Clayton, and Plymouth, NC, in 2006. The FAF population was planted in four-row plots, with the exception of Clayton in 2006, which was planted in one-row plots.
The FAS population was planted in five-seed hills at Clayton, NC, in a sets-within-replications design with three replications in 2006. Sets were randomly assigned to each replication, and lines within each set were randomly assigned to hills. The parental lines, N98-4445A and PI423893, were randomly assigned within each of the five sets in each replication.
Phenotypic Evaluation
Maturity date was recorded for the FAE, FAF, and FAS populations at the R8 reproductive stage as days after planting (Fehr and Caviness, 1977). For the FAE and FAF populations, mature soybean seed was harvested mechanically from the two middle rows of each experimental plot or each single-row plot for the FAF population at Clayton in 2006. Approximately 10 g of seed was subsampled from each plot for the evaluation of fatty acid composition. For the FAS population, 5 g of seed was subsampled from the seed harvested from each hill. Fatty acid composition was evaluated by gas chromatography, as described by Burkey et al. (2007).
Statistical Analysis
Statistical analysis of the sets-within-replications design was conducted using Proc MIXED in SAS 9.1 (SAS Institute, 2004). Environments, sets, replications, and lines, as well as their interactions, were considered as random effects. Best linear unbiased predictors (BLUPs) were obtained for all traits and lines of the FAE, FAF, and FAS populations. The degrees of freedom for FAF and FAS populations were calculated using the Satterthwaite approximation, while for the FAE population they were derived with the containment method to reduce the computing power required. Analysis of the FAE and FAF populations was conducted separately for each environment, as well as combined across all the environments for fatty acid traits and maturity date. The phenotypic data of the FAF population from Clinton in 2005 were discarded due to numerous missing data points and heterogeneity of variance in comparison with the other environments. The analysis of the FAS population for a single environment was performed as described above.
Simple sequence repeat (SSR) markers and allele-specific markers were tested for association with fatty acid phenotypes, derived as BLUPs, in FAE, FAF, and FAS populations using single-factor analysis in Proc GLM of SAS 9.1. The additive genetic effects were obtained by the CONTRAST statement in Proc GLM of SAS 9.1. Dominance genetic effects were also estimated for the FAS population. The means of the homozygous genotypic classes for each trait in the FAE and FAF populations or the homozygous and heterozygous genotypes in the FAS population, as well as their standard errors, were obtained with the LSMEANS statement in Proc GLM of SAS 9.1. The difference of the means of the homozygous genotypic classes, as well as its standard error and its significance, were determined with the ESTIMATE statement in Proc GLM of SAS 9.1. To investigate whether maturity date affected the associations between fatty acid traits and molecular markers, single-factor analysis was also conducted, with maturity date included as a covariate in the model.
Genotypic Evaluation and Linkage Mapping
Genomic DNA was extracted from leaf tissue of approximately 5 to 10 plants for each of the lines of the FAF and FAE populations collected from Kinston (2005) and Clayton (2004), respectively. The DNA isolation was conducted in a 96-well plate format with the Gentra PureGene DNA purification kit (Gentra Systems, Minneapolis, MN). Genomic DNA was isolated from leaf tissue (Keim et al., 1988) of each line of the FAS population from all the plants in each hill grown at Clayton (2006). One hundred forty-seven polymorphic SSR markers of the consensus linkage map (Cregan et al., 1999; Song et al., 2004) were genotyped across the 20 linkage groups of the soybean genome in the FAF population. Only SSR markers flanking the genomic regions of interest, as discussed below, were genotyped in the FAE and FAS populations (Table 1
). The reactions were performed in a 384-well PTC-200 Peltier Thermal Cycler (MJ Research, Waltham, MA). Thermocycling conditions were 95°C for 2 min and 38 cycles of 92°C for 1 min, 49°C for 1 min, and 68°C for 1 min and 30 s. The amplification products were resolved on 4% super-fine resolution agarose gels (Amresco, Solon, OH) with ethidium bromide staining in 1x Tris-borate-EDTA buffer, or 6.5% polyacrylamide gels in a LICOR 4300 DNA analysis system (LICOR Biosciences, Lincoln, NE) using M13-tailed unmodified primers and IRD-700 or IRD-800 labeled M13 oligonucleotides.
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Database Search and Polymerase Chain Reaction Primer Design
The coding regions of FAD2-1A and FAD2-1B were initially amplified with isoform-specific primers designed from the GenBank (Benson et al., 2002) accessions AB188250 and AB188251, respectively. For the isoforms of FAD2-2, in silico analysis was conducted on the soybean expressed sequence tags (Shoemaker et al., 2002) deposited in GenBank using BLASTN searches. Three distinct isoforms were predicted and isoform-specific primers were designed. The specific primers that amplify the coding region of each isoform are: FAD2-1A_d_F (5'-GTG TGG CCA AAG TGG AAG TT-3') and FAD2-1A_b_R (5'-CAA AGC TCC CTT CAG CCA GT-3'); FAD2-1B_a_F (5'-GTG TGG CCA AAG TTG AAA T-3') and FAD2-1B_b_R (5'-AAG CAC CCC TCA GCC AAT-3'); and FAD2-2B_c_F (5'-CCT CAG TTT AGT CTC AGC CAG ATT-3') and FAD2-2B_c_R (5'-TGG GCT TTA TTG CCT TTG TC-3'). The specific primers that amplify the upstream region of the FAD2-2B isoform are: FAD2-2B_UP_1_F (5'-ACG CAG TGA ATC AAA TGA CAA-3') and FAD2-2B_UP_2_R (5'-CTT CTT AAT CTG GCT GAG ACT AAA CT-3'). Isoform and primer designations for the isoforms of FAD2-1 genes follow the nomenclature of Tang et al. (2005), while the isoforms of FAD2-2 genes follow the nomenclature of Schlueter et al. (2007).
Sequencing of FAD2 Genes, Single Nucleotide Polymorphism Detection, and Allele-Specific Marker Development
Genomic DNA was isolated from bulked leaf tissue (Keim et al., 1988) from approximately 10 plants of the N98-4445A, N97-3363-3, and PI423893 lines and Brim. The amplification of the various FAD2-1 and FAD2-2 genes was performed in a MyCycler thermal cycler (Bio-Rad, Hercules, CA). The polymerase chain reactions (PCRs) were performed using the Expand High Fidelity PCR system (Roche Applied Science, Indianapolis, IN). Each 50-µL reaction contained 0.8 U polymerase, 20 pmol of each of the forward and reverse primers, 187 µmol L–1 deoxynucleotide triphosphates (dNTPs), and 100 ng of genomic template DNA. The cycling program included a 5-min step at 94°C, 30 amplification cycles of 30-s denaturation at 94°C, annealing at 55°C for 30 s, and extension at 72°C for 2 min, followed by an extension step for 7 min at 72°C. The amplification products were cloned into pCR 2.1 cloning vectors (Invitrogen, Carlsbad, CA) and sequenced (Iowa State DNA facility, Ames, IA). Pairwise alignments of the sequences obtained for the isoforms of FAD2-1 and FAD2-2 genes were performed for the detection of SNPs among the soybean lines N98-4445A, N97-3363-3, PI423893, and Brim. Single nucleotide polymorphisms leading to restriction site polymorphisms were identified, and cleaved amplified polymorphic sequence (CAPS) markers were developed for FAD2-1B and FAD2-2B using the HpyCH4III and AluI endonucleases, respectively.
The amplified fragments of FAD2-1B and FAD2-2B isoforms for CAPS analysis were obtained with the following primers: FAD2-1B_c_F (5'-ATC CAA AGT TGC ATG GTA CAC-3'), FAD2-1B_b_R (5'-AAG CAC CCC TCA GCC AAT-3'), FAD2-2B_UP_1_F (5'-ACG CAG TGA ATC AAA TGA CAA-3'), and FAD2-2B_UP_1_R (5'-TGC TAT CGT TAC CCA CAT ACG A-3'). Cycling conditions were the same as described above. Amplification of genomic DNA was conducted with a 12-µL PCR reaction containing 6 pmol of each of the forward and reverse primers, 0.5 U Taq polymerase (New England Biolabs, Ipswich, MA), 10x buffer (10 mmol L–1 Tris-HCl, 50 mmol L–1 KCl, 1.5 mmol L–1 MgCl2, pH 8.3), 234 µmol L–1 dNTPs, and 75 ng genomic DNA. Next, 10 µL of the amplified product was digested in a 22-µL reaction at 37°C for 2 h. For the digestion of FAD2-1B fragments, the reaction contained 2.5 U HpyCH4III, buffer (20 mmol L–1 Tris-acetate, 50 mmol L–1 potassium acetate, 10 mmol L–1 magnesium acetate, 1 mmol L–1 dithiothreitol, pH 7.9) and 5 µg bovine serum albumin (BSA). The FAD2-2B amplification products were digested in reactions containing of 5 U AluI, buffer (10 mmol L–1 Tris-HCl, 50 mmol L–1 NaCl, 10 mmol L–1 MgCl2, 1 mmol L–1 dithiothreitol, pH 7.9) and 5 µg BSA. Restriction fragments were resolved in 4% superfine-resolution agarose gels, as described above.
In the FAF population, the FAD2-1A isoform was genotyped with the allele-specific primer extension (ASPE) assay (University of California-Davis Genome Center, Davis, CA) in a Luminex platform (Luminex Corp., Austin, TX) using the primers FAD2-1A_ASPE1 (5'-TCA ATT ACT TCA CTT TAA TCC TTT gcc aaa gtg gaa gtt caa g-3') for N97-3363-3 and FAD2-1A_ASPE2 (5'-TCA TTC ATA TAC ATA CCA ATT CAT gcc aaa gtg gaa gtt caa c-3') for PI423893. The primers include the SNP-specific sequence (lowercase letters) coupled to the oligonucleotides associated with the fluorescent microspheres (uppercase letters). In the FAS population, FAD2-1A was genotyped with the single-base extension (SBE) assay in a Luminex platform (Luminex Corp., Austin, TX) according to Chen et al. (2000) (data not shown).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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-6 desaturase activity in our populations and was not studied further. Indeed, transcript analysis of FAD2-2A recently provided evidence that this isoform may not be expressed (Schlueter et al., 2007). Also, gene-specific primers designed for FAD2-2C amplified only part of the isoform's coding region, which did not reveal any SNPs (data not shown) among the genotypes used in this study. Consequently, further information for the FAD2-2C isoform could not be obtained.
Single Nucleotide Polymorphism Detection
The isoform-specific primers were used for the amplification of FAD2-1A, FAD2-1B, and FAD2-2B from genomic DNA of lines N98-4445A, N97-3363-3, and PI423893. Amplification products were cloned, sequenced, and aligned for the parental soybean lines of interest and the cultivar Brim, leading to the detection of SNPs in coding or upstream regions of each isoform. One SNP originating from PI423893 results in the change of the 22nd amino acid residue of the predicted protein of FAD2-1A from glycine to arginine due to a substitution of a guanine with a cytosine in the first position of the codon. This is one of the four amino acid residues that differ between the FAD2-1A and FAD2-1B enzymes in the N-terminal region. Interestingly, the location of the SNP in the coding region of FAD2-1A coincides with a region responsible for the instability of the enzyme at high temperatures (Tang et al., 2005).
For FAD2-1B, a total of five SNPs were detected in the coding region, two of which result in nonsynonymous amino acid changes. The substitution of a cytosine with a thymine in the second position of the 86th codon of FAD2-1B causes an amino acid change from serine to phenylalanine in PI423893. Also, the 126th amino acid residue of the FAD2-1B enzyme in N98-4445A and N97-3363-3 changes from valine to methionine due to the substitution of a guanine with an adenine at the first position of this codon. Three additional nucleotide polymorphisms that do not change the amino acid residues of the predicted FAD2-1B enzyme were also detected in PI423893 (Fig. 1 ). These are located in the third positions of codons 219, 223, and 228. All nonsynonymous SNPs in FAD2-1A and FAD2-1B were evaluated for potential implications in protein function using the SIFT program (Ng and Henikoff, 2003). None of the nonsynonymous amino acid changes, however, were predicted to be deleterious.
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Development of Allele-Specific Molecular Markers for FAD2-1B and FAD2-2B
The detected nucleotide substitutions were examined for possible restriction site polymorphisms coinciding with the location of the SNPs to develop molecular assays that could readily distinguish among the alleles found in N98-4445A, N97-3363-3, and PI423893. The substitution of a thymine with a cytosine in the 223rd codon of FAD2-1B generates an additional restriction site for the HpyCH4III endonuclease in PI423893 (Position 172, Fig. 1), and thus enabled the development of a CAPS marker. Digestion of a 395-bp FAD2-1B-specific fragment from PI423893 generates fragments of 172, 90, 70, and 63 bp, whereas the same product derived from N98-4445A or N97-3363-3 CAPS yields fragments of 235, 90, and 70 bp (data not shown). The additional HpyCH4III restriction sites that do not correspond to nucleotide substitutions within the amplified fragment serve as digestion controls.
The unique SNP in the upstream region of FAD2-2B results in the loss of an AluI restriction site in the fragment amplified from PI423893. The primers used for CAPS analysis amplify a 494-bp fragment in the upstream region of FAD2-2B. The AluI digestion of this amplification product from N98-4445A and N97-3363-3 generates fragments of size 173, 147, and 174 bp, in contrast to fragments of 320 and 174 bp when genomic DNA from PI423893 is used as a template (data not shown).
The SNP identified in the coding region of FAD2-1A did not alter restriction endonuclease cleavage sites. Therefore, an ASPE assay was designed with SNP- specific primers to distinguish between the N97-3363-3 and PI423893 FAD2-1A alleles in the FAF population. In the FAS population, an SBE assay was designed according to Chen et al. (2000) to distinguish between the N98-4445A and PI423893 FAD2-1A alleles using fluorescent microspheres in a Luminex platform. The segregation patterns of the CAPS markers for FAD2-1B and FAD2-2B, as well as of the ASPE and SBE assays for FAD2-1A, were utilized for linkage analysis and association tests with fatty acid content in the FAF and FAS populations.
Mapping the Isoforms of the FAD2-1 and FAD2-2 Genes
Linkage analysis was conducted for 147 polymorphic SSR markers genotyped in the FAF population, which cover the 20 linkage groups of the soybean genome, as well as the CAPS, ASPE, and SBE assays with both JoinMap 3.0 (Van Ooijen and Voorrips, 2001) and MAPMAKER/EXP 3.0 (Lander et al., 1987). Since, for the linkage analysis performed with MAPMAKER/EXP 3.0, the recombination frequency among markers and the map distances were slightly biased by analyzing the FAF F5-derived population as a recombinant inbred line population, only the linkage analysis performed with JoinMap 3.0 is presented here (Fig. 2
). It should be noted that linkage could not be declared among all markers on the same linkage group, probably due to the relatively small size of the FAF population. The FAS population was used to validate the localization of the FAD2-1A, FAD2-1B, and FAD2-2B isoforms.
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Associations between Molecular Markers and Fatty Acid Content or Maturity Date
Overview of Cosegregation Analysis
Segregation of the FAD2-1A, FAD2-1B, and FAD2-2B allele-specific markers, as well as SSR markers in the vicinity of the isoforms on Linkage Groups O, I, and L, was used to test for association with the content of unsaturated fatty acids in the oil and with maturity date (Table 1). Fatty acids and maturity date BLUPs were obtained for each line from the analysis of each environment separately and from the combined analysis across all environments using mixed models in SAS 9.1. Best linear unbiased predictors for each trait were used in single-factor analysis with FAD2-1A, FAD2-1B, and FAD2-2B allele-specific and SSR marker classes as the independent variables. Although maturity effects were significant for the majority of fatty acid traits and environments tested in the FAE, FAF, and FAS populations, they were not accounted in the computation of BLUPs. Instead, single-factor analysis was also performed with maturity date included as a covariate to determine whether the associations between fatty acid content and molecular markers were affected by maturity. All markers that were significantly associated (P < 0.05) with the unsaturated fatty acid content and with maturity date were included in Tables 2
, 3
, and 4
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Analysis of the FAE population indicated the existence of a minor oleate QTL in the proximity of the FAD2-2B isoform on Linkage Group L. The oleate QTL, which mapped on sat_340 (13.86 cM from FAD2-2B according to the linkage map of the FAS population) explained 2.2% of oleate genotypic variation and was detected in all six environments tested for the FAE population (data not shown). The high-oleate allele at this locus was inherited from the N98-4445A parental line and the means of the homozygous genotypic classes differed by 18.8 g kg–1 (Table 4). The minor oleate QTL on Linkage Group L was not detected in the FAF or FAS populations, possibly due to their smaller population size, which impacts the power of QTL detection and the magnitude of the QTL effect (Beavis, 1994). Quantitative trait loci with minor effects for oleic, linoleic, and linolenic acid traits have been reported on Linkage Group L at the genomic region where the FAD2-2A and FAD2-2B isoforms map (Hyten et al., 2004); however, the population used for that study did not segregate for major oleic acid genes, since it was derived from the cross of normal-oleate parental lines, and maturity effects were not taken into consideration.
On Linkage Group O, segregation of the FAD2-1A ASPE and SBE markers was not associated with changes in unsaturated fatty acid content in the FAF and FAS populations, which suggests the lack of oleate QTLs in the proximity of FAD2-1A.
Cosegregation Analysis for Other Genetic Loci Mapped on Linkage Groups O, I, and L
On Linkage Group O, analysis suggests a minor oleate QTL near the SSR markers sat_108 and satt153, which explained 2.4 and 0.6% of oleate genotypic variation in FAS and FAE populations, respectively (Tables 3 and 4). The satt153 locus coincided with a QTL for maturity explaining 8.9% of maturity genotypic variation in the FAE population. The late-maturity and low-oleate alleles were inherited from N97-3525, as expected by the negative genetic correlation between maturity and oleic acid seed content in the late-maturing FAE population (data not shown). After accounting for maturity effects in the model, however, it was determined that N97-3525 contributed the high-oleate allele (Table 4), which favors the hypothesis of two linked QTLs controlling maturity and oleate content near the SSR marker satt153.
Apart from the minor oleate QTL near FAD2-2B on Linkage Group L, minor QTLs for oleate content were also mapped on the SSR markers satt652 and satt462. The SSR marker satt652, which is located 65.47 cM away from FAD2-2B and 19.67 cM from satt462 according to the linkage map of the FAS population, explained 5.2% of the genotypic variation for the oleate trait in the FAF population (Table 2). The association between oleate and satt652 was significant at the P < 0.01 level in each of the five environments tested for the FAF population (data not shown), as well as in the combined analysis across all environments (Table 2). The oleate QTL near satt652 on Linkage Group L was further confirmed with the FAE population, where satt652 explained 0.5% of the genetic variation for the oleate trait (Table 4). The significant association between satt652 and oleate content was consistent across two of the six environments when maturity was used as a covariate (data not shown). The high-oleate allele of the detected QTL was inherited from the N97-3363-3 and N98-4445A alleles in the FAF and FAE populations, respectively. In addition, minor QTLs for linoleate and linolenate traits were detected near satt652 after accounting for maturity effects. These results confirm other oleate QTL mapping studies currently in progress (H.R. Boerma, personal communication, 2007). In the FAS population, satt652 was not associated with oleate content but explained 1.6% of linolenate genetic variation (Table 3).
Also, on Linkage Group L, the SSR marker satt462 explained 4.6% of the genotypic variation for the oleate trait in the FAF population (Table 2). The segregation of satt462 was significantly associated with oleate content at the P < 0.05 level in four of the five environments tested (data not shown), as well as in the overall environment analysis (Table 2). The FAE population provided additional evidence for an oleate QTL in the proximity of satt462. When maturity effects were taken into account, satt462 explained 1.2% of the genetic variation for oleic acid content in the seed oil (Table 4). The associations declared for the satt462 marker and oleate content in the FAE population were consistent across all six environments tested when maturity was used as covariate (data not shown). The high-oleate alleles of the QTL near satt462 on Linkage Group L were inherited from the N97-3363-3 and the N98-4445A parental lines in the FAF and FAE populations, respectively. The FAS population did not confirm the association of oleic acid content with the SSR markers sat_340 and satt462. In the FAF population, satt462 explained 7.9% of the genotypic variation for linolenate content (Table 2), but the QTL conditioning linolenate content could not be validated in the FAE or FAS populations.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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-6 desaturase enzymes. Therefore, the FAD2-1A, FAD2-1B, and FAD2-2B isoforms do not appear to encode for the major oleate genes that contribute to the elevated oleic acid content of the N98-4445A and N97-3363-3 lines. Further research on the FAD2-2C isoform as well as other candidate genes implicated in oleate biosynthesis, such as the plastidial desaturase enzymes, is critical to substantiate the results presented here and shed light on the genetic factors that control oleic acid content in soybean oilseeds. |
ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication October 14, 2007.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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 E. Bachlava, R. E. Dewey, J. W. Burton, and A. J. Cardinal Mapping and Comparison of Quantitative Trait Loci for Oleic Acid Seed Content in Two Segregating Soybean Populations Crop Sci., March 17, 2009; 49(2): 433 - 442. [Abstract] [Full Text] [PDF]
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M. J. Monteros, J. W. Burton, and H. R. Boerma Molecular Mapping and Confirmation of QTLs Associated with Oleic Acid Content in N00-3350 Soybean Crop Sci., November 24, 2008; 48(6): 2223 - 2234. [Abstract] [Full Text] [PDF]
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