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CROP BREEDING, GENETICS & CYTOLOGY

Phenotypic and Molecular Analysis of Oleate Content in the Mutant Soybean Line M23

Dep. of Agronomy, Iowa State Univ., Ames, IA 50011-1010

^* Corresponding author (wfehr{at}iastate.edu)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Soybean [Glycine max (L.) Merr.] oil with elevated oleate content would be useful for food and industrial applications that require increased oxidative stability. The first objective of this study was to determine if molecular selection for the Fad2-1 deletion associated with the ol allele in the mid-oleate mutant line M23 could be used to identify mid-oleate individuals in a breeding program. The second objective was to determine if modifying genes affect phenotypic expression of oleate content in individuals homozygous for the deletion from a cross between M23 and Archer, a cultivar with normal oleate content. The segregation among 88 F₂ plants from the cross satisfactorily fit a ratio of 1:2:1 homozygous normal (OlOl)/heterozygous (Olol)/homozygous (olol) for the Fad2-1 deletion on the basis of Southern analysis. A PCR-based marker Fad2-1-ol identified the same olol individuals as the Southern analysis. The PCR-based marker would be a more rapid and less labor intensive method for molecular selection of olol individuals than Southern analysis. The olol individuals had the highest mean oleate content, the Olol individuals were intermediate, and the OlOl individuals had the lowest mean oleate content. There was significant variation among the olol individuals and their distribution overlapped that of the OlOl and Olol individuals, which indicated that modifying genes had an important influence on the trait. It would be necessary to test the fatty acid profile of olol individuals to select those with the highest oleate content.

Abbreviations: bp, base pair • kbp, kilobase pair

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
VEGETABLE OILS with increased oleate have greater oxidative stability during high temperature heating and frying than normal oleate oils (Warner et al., 1994; Warner and Knowlton, 1997; Warner et al., 1997). Oleate has been demonstrated to lower total and low-density lipoprotein cholesterol, which is beneficial in reducing cardiovascular disease in humans (Kris-Etherton, 1999).

The mid-oleate mutant soybean line M23 was developed by X-ray irradiation of dry seeds of Bay, a cultivar with normal oleate content (Rahman et al., 1994). M23 had an oleate content of 461 g kg^–1 as an M₂ plant compared with 223 g kg^–1 oleate for Bay. Rahman et al. (1994) hypothesized that X-ray irradiation caused a mutation that partially blocked the desaturation pathway from oleate to linoleate. Takagi and Rahman (1996) proposed a single locus with two alleles, Ol controlling normal oleate and ol controlling mid-oleate.

Linoleate is formed from oleate by desaturation at the -6 position mediated by the enzyme -6 fatty acid desaturase. Okuley et al. (1994) isolated the cDNA encoding the microsomal -6 fatty acid desaturase from Arabidopsis thaliana (L.) Heynh. as the Fad2 gene. Heppard et al. (1996) found two clones encoding -6 fatty acid desaturase in soybean that they designated Fad2-1 and Fad2-2. Heppard et al. (1996) found Fad2-1 to be strongly expressed in developing seeds while Fad2-2 was expressed in both vegetative tissue and developing seeds.

Kinoshita et al. (1998) used the cDNA of Fad2-1 and Fad2-2 as probes to screen 40 F₂ plants from the cross of Bay x M23. The population was analyzed using restriction fragment length polymorphisms with the restriction enzyme EcoRI. The Fad2-1 probe detected a 4.6-kbp fragment in Bay, but not in M23. The population screened with Fad2-1 satisfactorily fit the 1:2:1 segregation for the ol locus as proposed by Takagi and Rahman (1996). Kinoshita et al. (1998) concluded that the deletion in the Fad2-1 gene gave rise to the ol allele in M23.

Genetic analyses of soybean mutants with major genes for altered fatty ester content have demonstrated that modifying genes can influence the segregation observed when a mutant line is crossed with conventional cultivars (Graef et al., 1988; Horejsi et al., 1994). The possible role of modifying genes in the oleate content of olol individuals has not been reported.

One objective of this study was to determine if molecular selection for the deletion in Fad2-1 could be used to identify lines with mid-oleate content in a breeding program. A second objective of this study was to determine the role of modifying genes in the segregation of progeny from the cross of M23 and a soybean cultivar with normal oleate content.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Population Development
The mutant line M23 developed by Rahman et al. (1994) was crossed to Archer, a cultivar of maturity group I with normal oleate content developed by Iowa State University and the Puerto Rico Agricultural Experiment Station (Cianzio et al., 1991). The cross was made during July 2002 at the Iowa State University Agricultural Engineering and Agronomy Research Center near Ames, IA. The F₁ seeds and seeds of each parent were planted during October 2002 at the Iowa State University–University of Puerto Rico soybean breeding nursery at Isabela, PR, on a Coto clay (very-fine, kaolinitic, isohyperthermic Typic Eutrustox). Hybrid F₁ plants were confirmed using the simple sequence repeat marker Satt173 developed by Cregan et al. (1999).

A random sample of 300 F₂ seeds and 24 seeds of each parent were planted during February 2003 at Isabela. A single trifoliolate leaf of each F₂ and parent plant was harvested for DNA extraction. Genomic DNA was isolated by the method described by Anderson et al. (1992).

The F₂ plants were harvested individually to obtain F_2:3 lines. There were 88 F₂ plants with sufficient DNA to perform Southern analysis. From 88 F₂ plants and 10 plants of each parent, 11 individual seeds were split with a razor blade and the identity of each seed was maintained for analysis and planting. The one-third of the seed without the embryonic axis was analyzed for fatty ester content. The remaining part of the seed was saved for planting. All of the fatty ester analyses for the study were conducted by the method described by Hammond (1991). The fatty ester content of each F₂ plant used for statistical analysis was the average fatty ester content of its 11 split F₃ seeds.

In May 2003, the 88 selected F_2:3 lines and 10 entries of each parent were planted in a randomized complete-block design with two replications at the Iowa State University Research Center near Ames, IA, on a Nicollet loam (fine-loamy, mixed, superactive, mesic Aquic Hapludoll). Two replications also were planted at the University of Missouri– Columbia Delta Research Center near Portageville, MO, on a Tiptonville silt loam (fine-silty, mixed, superactive, thermic Oxyaquic Argiudoll). The Portageville location was used because M23 is of maturity group V and was likely not to mature before frost at Ames. The split seeds were planted in one of the replications at the Iowa State University Research Center. The remaining plots were planted with random whole seeds. The plots at Ames were a single row 0.76 m long with 1.02 m between rows and 1.07 m between the ends of plots. For the hill plots at Portageville, there was 0.91 m between plots within a row and 0.76 m between rows. The seeding rate was 11 seeds in all plots. Maturity was taken when 95% of pods on the main stem were mature. After the plants matured naturally or were killed by frost, they were harvested and threshed individually. The identity of each plant originating from a split seed was maintained. A five-seed bulk from each F₃ plant was analyzed for fatty ester content.

Southern Analysis
The cDNA for Fad2-1 was obtained from Richard Dewey, USDA-ARS, Raleigh, NC. The probe was amplified to obtain sufficient quantity for Southern analysis using the forward primer 5'-ATG GGT CTA GCA AAG GAA AC-3' from Kinoshita et al. (1998) and the reverse primer 5'-GAG CAA CCA ATG GGC CAT AG-3'. The reverse primer was designed from the published cDNA sequence of Fad2-1. The final polymerase chain reaction (PCR) volume was 50 µL consisting of 50 ng Fad2-1 DNA, 2 mM MgCl₂, 200 µM dNTPs, 1.25 U of BIOLASE DNA polymerase, 1x BIOLASE 10x NH₄ buffer, and 0.25 µM of each primer (Bioline USA Inc., Boston, MA). PCR was conducted on programmable thermal controllers (MJ Research Inc., Waltham, MA) model PTC-100. The PCR procedure was 94°C for 2 min, 11 cycles starting at 60°C for 30 s (–1°C per cycle) and 72°C for 2 min, 35 cycles of 94°C for 30 s, 48°C for 30 s, and 72°C for 2 min followed by a final extension of 8 min at 72°C.

From the 88 F₂ plants and each parent, a 5-µg sample of genomic DNA was digested using the restriction enzyme EcoRI. Probe preparation and Southern hybridization were performed according to the method described by Sandhu et al. (2001). All of the F₂ plants and parents had a 2.1-kbp band. There was segregation for a 4.6-kbp band associated with Fad2-1. The F₂ plants were placed into three classes based on visual evaluation of the 4.6-kbp band intensities: similar to Archer (double intensity), heterozygous (single intensity), or similar to M23 (absent).

Analysis of the PCR-Based Marker Fad2-1-ol
Fad2-1-ol is a PCR-based marker developed by D. Sandhu (unpublished data, 2005). The 88 F₂ plants and parents were evaluated to determine if using a PCR-based marker would be a more efficient method of selecting for the Fad2-1 deletion in the ol allele of M23. The forward primer was 5'-GGG CCA TAG TGG GAG TTA TGG AAG-3' and the reverse primer was 5'-GCT ATA AGC AGA ACA CTT TCC ACA T-3'. The final PCR volume was 20 µL consisting of 25 ng DNA, 0.25 µM of each primer, 200 µM dNTPs, 2 mM MgCl₂, 0.5 U BIOLASE DNA polymerase, 1x BIOLASE 10x NH₄ Buffer (Bioline USA Inc., Boston, MA). The PCR procedure was 94°C for 2 min, 6 cycles starting at 55°C for 30 s (–1°C per cycle) and 72°C for 2 min, 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 2 min followed by a final extension of 8 min at 72°C. All plants had a 162-bp band. Segregation occurred for the 183-bp band. The plants were scored as the same as Archer (presence of 183-bp band) or M23 (absence of the 183-bp band).

Statistical Analysis
The 88 F₂ plants used for Southern analysis and their corresponding F_2:3 lines were used for statistical analysis of fatty ester content. The effect of environments, genotypes, and the genotype x environment interaction was determined for oleate using F_2:3 lines by the general linear model (PROC GLM) of SAS software (SAS Institute Inc., 1999). Environments, replications, and genotypes were considered random effects.

To determine significance among lines within the three genotypic classes, the data were analyzed using general linear model (PROC GLM) by SAS software (SAS Institute Inc., 1999). Environments and replications were considered random and genotypes were considered fixed. Contrasts were used to determine if there was a significant difference between the genotypic classes.

Phenotypic correlation coefficients were calculated for fatty esters between F_2:3 lines, between environments, and between F₂ plants and F_2:3 lines using the correlation procedure (PROC CORR) by SAS software (SAS Institute Inc., 1999).

Single-seed heritability estimates were obtained by regressing the fatty ester content of each F₃ plant from the single replication grown at Ames on the corresponding split F₃ seed from which it was derived using the regression procedure (PROC REG) by SAS software (SAS Institute Inc., 1999) and adjusting for inbreeding according to Nyquist (1991). Single-plant heritability estimates were obtained by regressing the oleate content of the F_2:3 lines grown at Ames and Portageville on the oleate content of the F₂ plants grown at Isabela using the regression procedure (PROC REG) by SAS software (SAS Institute Inc., 1999). Heritability estimates on a plot and entry-mean basis were calculated using variance components determined from the analysis of the F_2:3 lines at Ames and Portageville (Hallauer and Miranda, 1988).

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The ol allele conditioned by the Fad2-1 deletion was segregating in the Archer x M23 F₂ population on the basis of Southern analysis. The segregating EcoRI fragment was approximately 4.6-kbp, which was consistent with the results observed by Kinoshita et al. (1998). The F₂ plants segregated into three molecular classes on the basis of band intensity: 24 were OlOl (double intensity), 49 were Olol (single intensity), and 15 were olol (no band) (Table 1). The segregation satisfactorily fit a 1:2:1 ratio (P > 0.23), which was consistent with the single-gene model for inheritance of oleate in M23 proposed by Takagi and Rahman (1996).

Southern analysis and the PCR-based marker were used to evaluate two other soybean germplasm sources of mid-oleate. FA22 is a mutant line with mid-oleate content developed by Iowa State University. N98-4445A is a mid-oleate line developed by the United States Department of Agriculture and North Carolina State University (Wilson, 2004). Neither of the lines had the Fad2-1 deletion exhibited by M23 and could not be distinguished from Archer by Southern analysis or the PCR-based marker. The results indicated that mid-oleate content in FA22 and N98-4445A was not conditioned by the Fad2-1 deletion as in M23.

The olol F₂ plants and their F_2:3 lines had the greatest mean oleate content and the OlOl individuals had the least oleate (Table 2). The two genotypic classes were different (P < 0.01) on the basis of entry means across environments. The mean oleate of the Olol individuals was greater than that of the OlOl individuals (P < 0.1) and less (P < 0.01) than that of the olol individuals on the basis of entry means. The mean of the Olol individuals was less than the midpoint between the means of the OlOl and olol individuals, which indicated that the Ol allele exhibited partial dominance. Partial dominance for lower oleate in crosses with M23 also was reported by Takagi and Rahman (1996).

Narrow-sense heritability estimates for oleate based on the 88 F₂ individuals were 0.33 on an F₃ seed basis and 0.44 on an F₂ plant basis. Broad-sense heritability estimates were 0.46 on a plot basis at Ames, 0.37 on a plot basis at Portageville, and 0.82 on an entry-mean basis. The heritability estimates were lower than those reported by Hawkins et al. (1983) who analyzed 20 experimental lines with normal oleate at Ames and Isabela. They reported broad-sense heritability estimates for oleate of 0.50 on a seed basis, 0.52 on a plant basis, 0.58 on a plot basis, and 0.92 on an entry-mean basis.

The genotype x environment interaction for oleate between Ames and Portageville was significant (P < 0.01) for the 88 F_2:3 lines. The phenotypic correlation for oleate between the 88 F_2:3 lines at Ames and Portageville was 0.70 (P < 0.01) and the correlation for the 15 olol lines at Ames and Portageville was 0.60 (P < 0.05). The significant correlations occurred even though there were major differences in maturity between M23 and Archer, which resulted in a broad segregation for the trait among the olol lines. At Ames, M23 and 11 of the olol lines did not mature before frost; whereas M23 and all the olol lines matured before frost at Portageville. Failure of M23 and some of the F_2:3 lines to mature at Ames was considered responsible for the lower mean oleate at that environment (Table 2). Thomas et al. (2003) found that oleate concentration in soybean increased with higher temperatures during seed development. Nevertheless, four of the five olol lines with the greatest oleate at Ames were in the top five lines at Portageville. These results and the magnitude of the phenotypic correlation among the two environments suggested that initial selection for oleate in a limited number of environments may be possible. Additional research with lines adapted to appropriate production environments and from multiple crosses should be conducted to further evaluate alternative selection strategies for oleate content in olol lines.

Oleate content of the 88 F_2:3 lines was significantly negatively correlated (P < 0.01) with palmitate (–0.47), stearate (–0.38), linoleate (–0.97), and linolenate (–0.45) on the basis of entry means across Ames and Portageville. Hawkins et al. (1983) reported significant negative correlations (P < 0.01) of oleate with stearate (–0.57), linoleate (–0.95), and linolenate (–0.84), but no correlation with palmitate (0.00). As oleate increases among lines, all of the other fatty esters are likely to decrease, particularly linoleate. The importance of the reductions in the other fatty esters will depend on the oil that is desired for a particular end use.

The results of the study indicated that the deletion of Fad2-1 at the ol locus of M23 segregated as a single gene in the Archer x M23 F₂ population. Use of the PCR-based marker should facilitate molecular selection of segregates with the olol genotype in populations derived from M23. The range of oleate among olol genotypes indicated that modifying genes have an important influence on oleate content of olol individuals, and that analysis of fatty ester content of the seed will be necessary to identify olol individuals with the highest oleate content.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Silvia Cianzio for growing the F₁ and F₂ plants at Isabela, Dr. J. Grover Shannon for growing the F_2:3 lines at Portageville, Dr. Richard Dewey, USDA-ARS, Raleigh, NC, for providing cDNA of Fad2-1, and Dr. Kendall Lamkey for assistance with the statistical analyses.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
This journal paper of the Iowa Agric. and Home Econ. Exp. Stn., Ames, IA, Project 3732 was supported by the Hatch Act, State of Iowa, Iowa Soybean Promotion Board, Raymond F. Baker Center for Plant Breeding, and the United Soybean Board.

Received for publication November 17, 2004.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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