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Crop Science 41:1927-1930 (2001)
© 2001 Crop Science Society of America

CELL BIOLOGY & MOLECULAR GENETICS

Genetic Mapping of a Macromutation and Quantitative Trait Loci underlying Fatty Acid Composition Differences in Meadowfoam Oil

Sureeporn Katengam, Jimmie M. Crane, Mary B. Slabaugh and Steven J. Knapp*

Dep. of Crop and Soil Science, Oregon State Univ., Corvallis, OR 97331

* Corresponding author (steven.j.knapp{at}orst.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The seed oil of meadowfoam (Limnanthes alba Hartw. ex Benth.) is a rich source of very long-chain fatty acids (VLCs), primarily 20:1{Delta}5, 22:1{Delta}5, 22:1{Delta}13 (erucic acid), and 22:2{Delta}5{Delta}13 (dienoic acid). Wild-type L. alba ssp. versicolor populations produce more erucic and less dienoic acid than wild-type L. alba ssp. alba populations, phenotypic differences that are caused by the macromutation E and possibly by quantitative trait loci (QTL). The aim of this study was to map the E locus and QTL affecting fatty acid concentrations among intersubspecific backcross progeny. The segregation ratio for the E locus (94 Ee to 86 ee progeny) was not significantly different from one to one (P = 0.84). The E locus was associated with 94 and 77% of the phenotypic variance for erucic and dienoic acid concentration, respectively. Erucic acid varied from 42 to 151 g kg-1 among Ee and 185 to 269 g kg-1 among ee progeny, while dienoic acid varied from 151 to 318 g kg-1 among Ee and 66 to 209 g kg-1 among ee progeny. The E locus mapped to Linkage Group 4 and pleiotropically affected every VLC. Composite interval mapping, performed with the E locus and 18 background markers as cofactors, was used to search the genome for QTL. Two significant QTL peaks were found for erucic and dienoic acid on Linkage Group 4. One QTL was centered on the E locus and produced a massive peak. The other QTL produced a marginally significant peak 30.4 cM downstream of the E locus and was associated with less than 1% of the phenotypic variance. The segregation of additional QTL in this population affecting dienoic acid cannot be ruled out.

Abbreviations: QTL, quantitative trait loci • AFLP, amplified fragment length polymorphism • VLC, very long-chain fatty acid


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
MEADOWFOAM SEED OIL is primarily comprised of unsaturated, very long-chain (C20 and C22) VLCs (Earle et al., 1959; Bagby et al., 1961; Smith et al., 1960; Miller et al., 1964; Higgins et al., 1971). Although rapeseed (Brassica napus L.), crambe (Crambe abyssinica Hochst. ex R.E. Fr.), and several other Cruciferae produce VLCs, primarily erucic acid (22:1{Delta}13) (Downey and Craig, 1964; Harvey and Downey, 1964), meadowfoam is one of the purest sources of VLCs known in the plant kingdom. The VLC concentrations of meadowfoam oil typically range from 940 to 960 g kg-1. Less than 20 g kg-1 of the total fatty acids are saturated (Knapp and Crane, 1995), which is significantly less than soybean (Glycine max L.) and other widely consumed seed oils (Weiss, 1983). The saturated fatty acid concentration of rapeseed oil, one of the richest sources of unsaturated fatty acids, is {approx}60 g kg-1.

The principal fatty acids of meadowfoam oil are erucic acid, cis-5-eicosenoic acid (20:1{Delta}5), cis-5-docosenoic acid (22:1{Delta}5), and cis-5, cis-13-eicosenoic acid (22:2{Delta}5{Delta}13) or dienoic acid (Earle et al., 1959; Smith et al., 1960; Bagby et al., 1961). The unique functionality of meadowfoam oil can be partly attributed to the presence and high concentration of {Delta}5 double bonds (Erhan et al., 1993; Isbell, 1997; Isbell et al., 1999; Isbell, 1999; Mund and Isbell, 1999). The mean concentration of {Delta}5 unsaturated fatty acids among L. alba germplasm accessions is 830 g kg-1 (Knapp and Crane, 1995). Although {Delta}5 unsaturated fatty acids are found in low concentrations in some Chenopodiaceae (Kleiman et al., 1972), meadowfoam is the richest known source of {Delta}5 unsaturated fatty acids, and is presently the sole commercial source of {Delta}5 unsaturated VLCs (Knapp and Crane, 1999). The position of the double bond and high concentration of {Delta}5 fatty acids has led to the development of novel chemical derivatives, often with high yields or processing efficiencies (Isbell, 1997, 1999). The oil has been proposed as a source of fatty acids and triglycerides for producing cosmetics, waxes, lubricants, surfactants, detergents, plastics, and other industrial products (Burg and Kleiman, 1991; Higgins et al., 1971; Isbell, 1997, 1999).

The synthesis of VLCs in plants is fairly well understood (Cassagne et al., 1994; Bao et al., 1998). The synthesis of fatty acids is carried out in the chloroplast and other plastids, where fatty acid synthase sequentially condenses two-carbon units into C16 and C18 fatty acyl chains. The fatty acids produced through this process are palmitic (16:0), stearic (18:0), and oleic (18:1{Delta}9) acids, which are released from acyl carrier protein by the activities of thioesterases. The acyl residues are exported to the cytoplasm and converted to acyl-CoA esters by acyl-CoA synthetase, thereby producing substrates that are further elongated in species that produce VLCs and further desaturated in species that produce polyunsaturated fatty acids. These processes and the assembly of triacylglycerols (seed storage lipids) are carried out in the endoplasmic reticulum (Bao et al., 1998).

Whereas the end products of de novo fatty acid synthesis are 16- or 18-carbon fatty acids in most seed oils, Arabidopsis thaliana (L.) Heynh., rapeseed, and several other species produce longer chains through the activities of elongases that elongate 18:1{Delta}9 to 20:1{Delta}11, and 20:1{Delta}11 to erucic acid (Bao et al., 1998; Harwood, 1996; Kunst et al., 1992; Casagne et al., 1994). The concentration of erucic acid in rapeseed (an allotetraploid) oil is primarily affected by two loci with multiple alleles (Downey and Craig, 1964; Harvey and Downey, 1964; Ecke et al., 1995; Thormann et al., 1996). Most of the alleles identified thus far additively affect erucic acid concentrations and are simply inherited (Kondra and Stefansson, 1965; Johnson, 1977; Ecke et al., 1995).

Meadowfoam has evolved a variant of the pathway found in A. thaliana and rapeseed. Pollard and Stumpf (1980) proposed two branches in the pathway underlying the synthesis of VLCs in meadowfoam. One branch produces 20:0 and 22:0 by elongating 16:0. The elongated fatty acids are desaturated by {Delta}5 desaturase, thereby yielding 20:1{Delta}5 and 22:1{Delta}5. The other branch produces 20:1{Delta}11, 22:1{Delta}13, and 22:2{Delta}5{Delta}13 by elongating oleic acid (18:1{Delta}9). The diene (22:2{Delta}5{Delta}13) is produced from 22:1{Delta}13 substrate by a {Delta}5 desaturase.

Two wild-type fatty acid profiles have been described in L. alba (Knapp and Crane, 1995). Wild L. alba ssp. versicolor populations typically produce more erucic and less dienoic acid than wild L. alba ssp. alba populations. The fatty acid profile difference between the subspecies is primarily caused by the E locus, a macromutation with a profound effect on erucic and dienoic acid concentrations (Knapp and Crane, 1998). L. alba ssp. alba germplasm seems to be homozygous for the dominant allele, whereas L. alba ssp. versicolor germplasm seems to be homozygous for the recessive allele. Nevertheless, differences in erucic and dienoic acid have been reported within the subspecies (Knapp and Crane, 1995), and erucic and dienoic acid concentrations vary continuously among E_ and ee progeny in segregating populations (Knapp and Crane, 1998). This fatty acid diversity could be caused by allelic variants of E, QTL, and nongenetic factors. Our aims were to study the effect and segregation of the E locus in a genetic background (intersubspecific cross) different from that reported by Knapp and Crane (1998), and to map the E locus and QTL underlying the fatty acid composition differences between the L. alba subspecies.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
This study was performed using 180 progeny from the backcross population [(OMF40-11 x OMF64) x OMF64] described by Katengam et al. (2001). The donor parent (OMF40-11) is an inbred line developed from the L. alba ssp. alba cultivar Mermaid. The recurrent parent (OMF64) is an inbred line developed from the L. alba ssp. versicolor accession PI 374801 (Knapp and Crane, 1997). Katengam et al. (2001) used 100 of the backcross progeny to construct a genetic map comprised of 103 AFLP markers. The distal AFLP markers from the arms of each linkage group (10 total) and 56 well-spaced AFLP markers dispersed across the original map were used to construct a framework map for QTL analyses.

The fatty acid concentrations (phenotypes) of the mapping population progeny and 80 additional backcross progeny were measured using gas chromatography, as described by Knapp and Crane (1995). These complete data (180 observations total) were used analyses of the segregation of the E locus, whereas only the phenotypes collected on the mapping population progeny (100 observations total) were used for the QTL analyses. Fatty acid methyl esters were extracted from the half-seed samples (Knapp and Crane, 1998). Standards with known 18:1{Delta}5, 18:1{Delta}9, 18:3 (linolenic acid), 20:0, 20:1{Delta}11, 20:1{Delta}5, 22:1{Delta}5, erucic acid, and dienoic acid concentrations (supplied by Dr. Thomas Abbott, USDA-ARS, NCAUR, Peoria, IL) were used to identify peaks and verify measurements.

Histograms (phenotypic distributions) were produced for the four principal fatty acids found in meadowfoam oil (20:1{Delta}5, 22:1{Delta}5, erucic acid, and dienoic acid), and a scatter plot was produced for displaying the erucic by dienoic acid distribution. Backcross progeny were assigned to Ee (low erucic and high dienoic acid concentration) and ee (high erucic and low dienoic acid) classes using the erucic acid distribution. The fit of the observed to the expected distribution for the segregation of a single gene in a backcross population (1:1) was tested using a {chi}2–test with one degree of freedom. The E locus was added to the AFLP map (Katengam et al., 2001) using the genotypes assigned on the basis of the observed phenotypic classes (Ee and ee).

Simplified composite interval mapping was performed using MQTL (Tinker and Mather, 1995) with the E locus and 18 additional background markers as cofactors. The background markers were spaced at {approx}30 cM intervals throughout the genome. The null hypothesis of no QTL was tested for positions throughout the genome by comparing test statistics to an empirical genome-wise significance threshold calculated from 1000 permutations for P = 0.05 (Dirge and Churchill, 1996).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Two distinct phenotypic classes were observed among the backcross progeny, one with low erucic and high dienoic acid, and another with high erucic and low dienoic acid (Fig. 1) . There were 96 progeny in the low erucic, high dienoic (Ee) class and 84 progeny in the high erucic, low dienoic (ee) class. The observed distribution fit the expected distribution for a single dominant macromutation segregating in a backcross population (P = 0.84). The erucic by dienoic acid distribution for the backcross population (Fig. 1) was nearly identical to that reported for another intersubspecific cross, Mermaid (L. alba ssp. alba) x UC-309 (L. alba ssp. versicolor) (Knapp and Crane, 1998). The phenotypic distributions for fatty acids other than erucic acid were continuous. The macromutation (E) produced two nonoverlapping erucic acid distributions (Fig. 1); thus, progeny were unambiguously assigned to genotypic classes (Ee and ee). The resultant E locus genotypes were used to map the E locus to Linkage Group 4 (near the AFLP Locus ACG_CAA_79) on the genetic map described by Katengam et al. (2001) (Fig. 2) .



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  		Fig. 1. Erucic and dienoic acid concentrations for 180 (OMF40-11 x OMF64) x OMF64 BC1 progeny.

 


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  		Fig. 2. The E locus and a subset of AFLP loci on Linkage Group 4 of the genetic map of meadowfoam.

 
OMF40-11, the EE genotype, produced 95 g kg-1 erucic and 204 g kg-1 dienoic acid, whereas OMF64, the ee genotype, produced 203 g kg-1 erucic and 92 g kg-1 dienoic acid (Table 1). Ee backcross progeny produced 90 g kg-1 erucic and 227 g kg-1 dienoic acid, whereas ee backcross progeny produced 227 g kg-1 erucic and 106 g kg-1 dienoic acid; thus, the phenotypes of the parents were reproduced by the phenotypes of the progeny. The erucic and dienoic acid concentrations of the progeny phenotypically transgressed the means of the parents (Table 1). The effects of the E locus on the various VLCs were estimated using linear contrasts between Ee and ee progeny means, and found to significantly affect every fatty acid (Table 2).


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  		Table 1. Mean seed oil fatty acid concentrations for OMF40-11 and OMF64 and minimum and maximum seed oil fatty acid concentrations for 180 (OMF40-11 x OMF64) x OMF64 BC1 progeny.

 

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  		Table 2. Seed oil fatty acid concentrations, probabilities of tests of significance of mean differences between Ee and ee progeny, and coefficients of determination (r2) for the effect of the E locus among 180 (OMF40-11 x OMF64) x OMF64 BC1 progeny.

 
We speculate that the E locus encodes a {Delta}5 desaturase. The proposed pathway for VLC synthesis (Pollard and Stumpf, 1980) in meadowfoam seeds predicts that erucic and dienoic acid are sequentially produced in one branch of the pathway from elongated 18:1 substrate, while 20:1{Delta}5 and 22:1{Delta}5 are sequentially produced in another branch of the pathway from elongated 20:0 substrate. Reduced activity of {Delta}5 desaturase on erucic acid might lead to increased erucic concentration. Such a phenotypic change is produced by the recessive genotype (ee) in L. alba ssp. versicolor. Erucic and dienoic acid concentrations were negatively correlated (-0.90) among the backcross progeny (Fig. 1). Whether or not a single {Delta}5 desaturase operates on both branches of the pathway and all substrates is still uncertain. The recent discovery and cloning of a {Delta}5 desaturase from L. douglasii (Cahoon et al., 2000) opens the way to testing this.

Although the E locus had a significant pleiotropic effect on the concentrations of 20:1{Delta}5 and 22:1{Delta}5, this locus was only associated with 14% of the phenotypic variance for 20:1{Delta}5 and 20% of the phenotypic variance for 22:1{Delta}5 (Table 2). By contrast, the E locus was associated with 94% of the phenotypic variance for erucic acid concentration and 77% of the phenotypic variance for dienoic acid concentration. There was greater dispersion in the dienoic than the erucic acid distribution (Fig. 1). Dienoic acid concentrations ranged from 151 to 318 g kg-1 among Ee and 66 to 209 g kg-1 among ee progeny, and the dienoic acid distributions of the two classes overlapped (Fig. 1).

Our hypothesis was that QTL were associated with the dienoic acid differences within the Ee and ee classes; however, composite interval mapping, performed using the E locus as a cofactor, only uncovered one QTL for 20:1{Delta}5, 22:1{Delta}5, erucic, and dienoic acid concentration, coincident with the E locus on Linkage Group 4, and a second putative QTL for erucic and dienoic acid concentration on Linkage Group 4 (Fig. 3) . The latter peaks were significant and identically positioned for both traits, but accounted for <1% of the phenotypic variance. The primary QTL peaks for 22:1{Delta}5, erucic, and dienoic acid concentration on Linkage Group 4 were centered on the E locus, while the QTL peak for 20:1{Delta}5 concentration was close to but not perfectly centered on the E locus, and was not statistically significant (when tested using composite interval mapping). Conversely, the 20:1{Delta}5 concentrations of Ee and ee progeny were significantly different when tested using a linear contrast between genotype means (Table 2).



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  		Fig. 3. Composite interval mapping test statistics for 20:1{Delta}5 (A), 22:1{Delta}5 (B), erucic acid (C), and dienoic acid (D) concentrations on Linkage Group 4 of the genetic map of meadowfoam. The significance thresholds (indicated by dashed horizontal lines) are based on an empirical genome-wise Type I error rate of 5%. The position of the E locus (72.3 cM) is indicated by the dashed vertical line in each plot.

 
The QTL analyses were primarily undertaken to search for genes affecting dienoic acid concentration. We speculated that QTL affecting dienoic acid (other than the E locus) might be segregating because 23% of the phenotypic variance was not explained by the E locus, and the phenotypes of the progeny transgressed the phenotypes of the parents (Table 1). Additional analyses and selection to fix transgressive phenotypes are needed to assess whether the unexplained phenotypic variance is partially or completely nongenetic. Although seed to seed differences in dienoic acid concentration were pronounced (Fig. 1), erucic and dienoic acid concentrations from bulk seed samples harvested from field-grown germplasm accessions usually differ by less than {approx}2% year over year (Crane and Knapp, 1995–2001, unpublished data).


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
This research was funded by grants from the USDA (#58-5114-8-1021 and #58-3620-8-107). Oregon Agric. Exp. Stn. Tech. Paper no. 11796.

Received for publication November 13, 2000.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





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