| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a Dep. of Crop and Soil Sciences, 3111 Miller Plant Sciences Building, Univ. of Georgia, Athens, GA 30602
b USDA-ARS and North Carolina State Univ., Raleigh, NC 27607
c USDA-ARS, National Center for Agricultural Utilization Research, Peoria, IL 61604
* Corresponding author (rboerma{at}arches.uga.edu)
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Abbreviations: GLM, general linear model • LG, Linkage group • QTL, quantitative trait loci • RFLPs, restriction fragment length polymorphisms • SSRs, simple sequence repeats.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
120 g kg-1 (Erickson et al., 1988a; Fehr et al., 1991; Burton et al., 1994). Reduction of palmitic acid content of soybean oil would lower the total saturated fatty acid content and improve the oil quality for human consumption. The manipulation of soybean oil quality by altering fatty acid composition is an important breeding objective in the USA (Wilson et al., 1981; Topfer et al., 1995). Soybean lines with reduced palmitic acid content have been developed through chemical mutagenesis, recurrent selection, and hybridization (Erickson et al., 1988a; Bubeck et al., 1989; Wilcox and Cavins, 1990; Burton et al., 1994). Previous studies have shown that reduced palmitic acid was conferred by at least two loci (Erickson et al., 1988a; Fehr et al., 1991; Wilcox et al., 1994), and no maternal effects were observed (Schnebly et al., 1994). The alleles conferring reduced palmitic acid from C1726(fap1) and A22(fap3) are at independent loci (Erickson et al., 1988b; Schnebly et al., 1994). Allelism studies for the lines C1726, N79-2077-12, and N90-2023 indicated that reduced palmitic acid alleles in N79-2077-12 and N90-2023 segregated independently of the fap1 allele in C1726 (Wilcox et al., 1994). However, the genes conditioning palmitic acid in N79-2077-12 and N90-2023 have not been assigned gene symbols. It was reported that the genes conditioning reduced palmitic acid in N87-2122-4 were inherited from N79-2077-12 (Burton et al., 1994; Wilcox et al., 1994), and the reduced palmitic acid content in N87-2122-4 was conditioned by a major gene and a genetic modifier (Rebetzke et al., 1998). N87-2122-4 is an important source of reduced palmitic acid genes being used by soybean breeders. Previous studies indicated that genes modifying the major palmitic acid loci could influence the genetic variation of palmitic acid content by increasing or reducing the palmitic acid content (Horejsi et al., 1994; Rebetzke et al., 1998). Modifier genes have been shown to influence palmitic acid by 2 to 23 g kg-1 (Horejsi et al., 1994). Understanding function and genomic location of genetic modifier genes would be useful to breeders in developing effective selection schemes to further reduce or stabilize the palmitic acid content in soybean.
Recent advances in molecular marker technology, especially the development of SSR markers in soybean and an integrated soybean genetic linkage map, have made possible the genetic mapping and dissection of qualitative and quantitative traits in soybean (Cregan et al., 1999). The SSR markers are highly amenable for automation and allele sizing which can provide for their use in high-throughput application and multiple trait selection (Diwan and Cregan, 1997; Mitchell et al., 1997). Using restriction fragment length polymorphism (RFLP) makers, Nickell et al. (1994) mapped fap2, an allele conferring elevated palmitic acid content from C1727 on LG-D of the public genetic linkage map (Cregan et al., 1999). Brummer et al. (1995) mapped the fan allele controlling reduced linolenic acid from C1640 on LG-B2. With a mapping population formed from Glycine max x Glycine soja Siebold & Zucc., Diers and Shoemaker (1992) mapped quantitative trait loci (QTL) conditioning five major fatty acids mainly on two linkage groups of the USDA/ISU map using RFLP markers. The objective of this study was to map the genes conferring reduced palmitic acid from N87-2122-4 with SSR markers.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
53 g kg-1), while Benning (123 g kg-1) and Cook (120 g kg-1) are cultivars with normal palmitic acid content (Boerma et al., 1992; Boerma et al., 1997; Burton et al., 1994). A F2 population consisting of 121 plants was derived from the cross of Cook x N87-2122-4. The F2 seeds were harvested from an F1 plant grown in the greenhouse. Each F2 seed was cut into a 1/4-seed fragment and 3/4-seed fragment with a razor blade. The 3/4-seed fragments, containing the embryonic axis, were used for planting, while the 1/4-seed fragments along with 10 seeds of each parent were used for fatty acid determination. The F2 seeds along with their parents, Cook and N87-2122-4, were planted in 0.95-L polystyrene cups (two seeds per cup) in the greenhouse. At maturity, each F2 plant was harvested individually. A bulk sample of 12 seeds from each F2 plant and their parents (10 12-seed bulk samples from each parent) were analyzed for fatty acid content in two independent laboratories. If fewer than 24 seeds were produced on an F2 plant, 12-half seed fragments were used for fatty acid analysis in each laboratory.
The seed fatty acid content was analyzed using gas-liquid chromatography of the methyl esters. The analyses were done in the USDA-ARS, Natl. Center for Agric. Utilization Res. at Peoria, IL (USDA-ARS/Peoria) for F2 seeds and in both the USDA-ARS/Peoria and Soybean Research Laboratory (USDA-ARS/North Carolina State Univ.) for F2:3 seeds.
DNA was extracted from leaf tissue of a single F2 plant by the modified CTAB procedure of Keim et al. (1988) and diluted to a concentration of 20 ng/µL for the PCR reaction. Leaves from each sample were ground in liquid nitrogen, and 700 µL of CTAB buffer [1.4 M NaCl; 100 mM Tris pH 8.0; 2% (w/v) CTAB; 20 mM EDTA; 0.5% (w/v) Na bisulfate; and 1% (v/v) 2-mercaptoethanol] were added to suspend the powdered materials. The samples were incubated in a water bath at 65°C for 1 h and then 500 µL chloroform/isoamyl alcohol (24:1, v/v) were added. After shaking for 30 min at room temperature, the samples were spun at 13 000 rpm (Beckman Microfuge E, Beckman Instruments, Carlsbad, CA) for 6 min. The supernatant was transferred to a new 1.5-mL tube. Eighty percent volume of isopropyl alcohol was added to precipitate DNA. The supernatant was decanted and the pellets were washed with 70% (v/v) ethanol. The DNA pellets were then dried and dissolved in 100 µL TE buffer
PCR reactions were prepared on the basis of the protocol by Diwan and Cregan (1997) with slight modifications. The 10-µL reaction mix contained 2 µL of 40 ng template DNA, 1.0x PCR buffer, 2.5 mM MgCl2, 100 µM of each dNTP, 0.2 µM each of forward and reverse primers, and 0.5 unit of Taq DNA polymerase. The reactions were performed in a dual 384-well or a 96-well GeneAmp PCR System 9700 Perkin Elmer Applied Biosystems (PE-ABI, Foster City, CA). Fluorescent dye-labeled primers were synthesized by PE-ABI (Foster City, CA). The primers were labeled with 6-FAM, NED, or HEX.
A loading sample for each lane was prepared with 2.5 µL of deionized formamide, 1.5 µL of loading buffer, 0.2 µL of Genescan Rox-500 (PE-ABI, Foster city, CA), and 1 to 3 µL of the pooled PCR products. Genescan ROX-500 is an internal size standard ranging in size from 35 to 500 basepairs. The mixture was denatured at 95°C for 2 min, and approximately 1.0-µL volume was loaded on each of 96 lanes on a 4.8% (w/v) acrylamide: bisacrylamide (19:1) gel with KLOEHN micro syringes (Kloehn Ltd., Las Vegas, NV). Electrophoresis was run with 120-mm well-to-read plate on ABI PRISM 377 DNA Sequencer at 750 V for approximately 1.5 h. Marker data were collected with PE ABI 377-96 DNA Sequencer Collection software. The marker fragments were analyzed with GeneScan and scored with Genotyper software (PE-ABI, Foster City, CA).
The fatty acid data were checked for the distribution and normality by means of SAS programs (SAS Institute, 1989). The broad-sense heritability was calculated on the basis of parent-offspring correlation (Fehr, 1987). The t test was used to test the difference in palmitic acid content between two parents and the 
2 procedure was used to evaluate segregation ratios of marker genotypes (SAS Institute, 1989).
Genetic linkage was estimated by the Kosambi mapping function of MAPMAKER/EXP (Lincoln et al., 1992a). The markers were assigned to linkage groups with the criteria of logarithm of odds (LOD) 
3.0 and maximum distance 
37.2 centimorgan (cM) between markers. The order of markers within the linkage group was determined by the ‘Compare’ command and confirmed by the ‘Ripple’ command.
The palmitic acid and marker data were analyzed for the presence of QTL. Interval mapping with MAPMAKER/QTL (Lincoln et al., 1992b) was used to estimate the positions of QTL. A minimum LOD score of 2.0 (default) was used for the determination of significance. Single factor analysis of variance (SF-ANOVA) was also used to determine the significance (P = 0.05) among SSR genotypic class means by means of General Linear Model (GLM) (SAS Institute, 1989). To detect the epistasis, two-factor ANOVA was performed on all pairs of significant markers. A multiple regression model with a FORWARD option was used for identifying the independent markers linked to the QTL among linkage groups at the 5% significance level.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
|
0.05) transgressive segregation of palmitic acid content in either the F2 or F2:3 was observed. The normality tests of F2 and F2:3 populations indicated that the palmitic acid was normally distributed. The broad-sense heritability was 0.94 based on the parent-offspring (F2 and F2:3) correlation method. The heritability estimates were consistent with previous reports from Fehr et al. (1991) and Wilcox et al. (1994). Six markers were selected from each linkage group on the basis of their approximate even distribution across the 20 linkage groups (Cregan et al., 1999). Of these markers, Satt276 on LG-A1 was found to have a common band in the two near-isogenic lines with reduced palmitic acid that differed from a common band in the two near-isogenic lines with normal palmitic acid content.
To search for additional genes conditioning reduced palmitic acid content, three to five SSR markers were initially selected from each linkage group to check the association between SSR markers and palmitic acid QTL in the F2 generation. The markers were chosen to cover the distance less than 40 cM on the public linkage map. On the basis of the SF-ANOVA, three markers, Satt276, Sat_368, and Satt684 on LG-A1 and one marker Satt175 on LG-M, were found to be associated with the variation of palmitic acid content in the F2 and F2:3 (Table 1). Linkage maps with additional SSR markers on LG-A1 and LG-M were constructed with MAPMAKER/EXP (Fig. 2 and 3)
. On the basis of 2 tests, all SSR markers segregated in the expected ratio (1:2:1). The order of the markers on both linkage groups is in close agreement with the integrated soybean genetic linkage map (Cregan et al., 1999).
|
|
|
|
0.05) from normality, visually it appeared discontinuous. The distribution for the F2:3 lines looked continuous and distinct phenotypes could not be clearly determined (Fig. 1). Therefore, the mapping was conducted by means of a quantitative trait approach. Results from MAPMAKER/QTL (Lincoln et al., 1992b) were similar to that found with SF-ANOVA. One major QTL was identified on the top of LG-A1 (LOD = 6.8 and R2 = 38% for F2; LOD = 4.8 and R2 = 31% for F2:3) (Fig. 2). Because no polymorphic DNA marker distal to Satt684 was available, it was not possible to determine the precise location or effect of the QTL for the reduced palmitic acid content. The gene action with this marker was additive, which agreed with the results of a classic genetic study (Wilcox et al., 1994). When marker Satt684 was homozygous for the N87-2122-4 allele, the predicted mean for palmitic acid content was 71.4 g kg-1. The LOD score of a minor QTL on LG-M, which explained 8% of the phenotypic variation, was very close to 2.0 based on the F2 phenotypes. It exceeded 2.0 and accounted for 12% of variation when based on F2:3 phenotypes (Fig. 3). The consistency of F2 and F2:3 data analysis supported the QTL locations on both linkage groups. Multiple regression models are useful in determining the relative importance of the markers associated with the phenotypic variation. The significant markers from LG-A1 and LG-M were included in a multiple regression model. Two markers, Satt684 from LG-A1 and Satt175 from LG-M, were retained in the model in both F2 and F2:3 (Table 1). The total variation accounted for by those two markers was 40% in the F2 and 34% in F2:3 generations based on the multiple regressions. Although the R2 for Satt175 was small, it significantly (P < 0.05) affected the palmitic acid content.
The interaction between markers represents an epistatic effect. To evaluate the importance of epistasis, the six pairs of the four markers detected by SF-ANOVA were tested for the interaction using a two-factor ANOVA (SAS Institute, 1989). The interaction between markers Satt684 and Satt175 was significant (P = 0.045) in the F2. The contrast test indicated that the interaction was from a quadratic term of two markers (Table 2). The allele from N87-2122-4 on LG-M only affected the variation in palmitic acid in the presence of the allele from N87-2122-4 on LG-A1. When both markers have homozygous alleles from N87-2122-4, palmitic acid was reduced to 60 g kg-1, which is near the palmitic acid content of N87-2122-4 (56 g kg-1) in the F2 experiment (Table 2). When the interaction term was included in a multiple regression model, two markers Satt684 and Satt175 accounted for 51% of total phenotypic variation in the F2 generation. In the F2:3 generation, the interaction between markers Satt684 and Satt175 approached significance (P = 0.1). Including the two markers in the regression model accounted for 43% of the variation in the F2:3 generation. Rebetzke et al. (1998) reported that one major gene and one modifier gene were responsible for conditioning of reduced palmitic acid content in N87-2122-4. The same phenomenon was also observed in palmitic acid content of soybean lines with fap1fap1fap3fap3 genotypes (Horejsi et al., 1994) and in the stearic content of soybean lines with major allele fas (Lundeen et al., 1987). The result from this study was consistent with the report from Rebetzke et al. (1998).
To simulate an approximate location of the major gene conditioning reduced palmitic acid from N87-2122-4 on LG-A1, three genotypic classes of palmitic acid (A1A1, A1A2, A2A2), were grouped on the basis of the average of both F2 and F2:3 data. The palmitic acid content of lines to include in each homozygous genotypic class was defined by the mean of palmitic acid content ±2SD of a parent grown in the same environments. The heterozygous class included lines with palmitic acid contents intermediate to the two homozygous classes. This approach was used by Stoltzfus et al. (2000) in the study of a fap5 allele. Specifically the families with palmitic acid less than 71 g kg-1 were classified as homozygous for the N87-2122-4 allele and greater than 109 g kg-1 as homozygous for the Cook allele. All other families (range of 72–108 g kg-1) were grouped as being heterozygous. Three genotypic classes of this simulated marker were scored as A (homozygous for Cook allele), B (homozygous for N87-2122-4 allele), and H (heterozygous for Cook and N87-2122-4 alleles) and mapped with the other SSR markers using MAPMAKER/EXP. The simulated marker mapped 14.5 cM distal to the Satt684 on LG-A1 and accounted for 87% of variation on the basis of the average of F2 and F2:3 data. Because of the effect of the minor gene on LG-M and the subjective determination of the phenotypic range to include in each genotypic class, the classification of three simulated marker classes might not be precise. However, this simulated marker could be used as a reference to indicate the approximate location of the major gene on the genomic map. This strategy was previously used by Tamulonis et al. (1997) to locate a soybean resistance gene to southern root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood], which has recently been verified with newly developed SSR markers (Li et al., 2001). These methods were also used to map a disease resistance gene in pea (Pisum sativum L.) and a K+ and Na+ discrimination QTL in wheat (Triticum aestivum L.) (Dirlewanger et al., 1994; Dubcovsky et al., 1996). Our results established the presence of a major gene conditioning palmitic acid content on LG-A1 and a minor gene on LG-M. Future efforts will be directed to identify other closely linked markers for the major gene on LG-A1 that will be useful for the marker-assisted selection in a breeding program.
|
ACKNOWLEDGMENTS |
|---|
Received for publication April 23, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
k. 1996. Mapping of the K+/Na+ discrimination locus Knal in wheat. Theor. Appl. Genet. 92:448–454.[Web of Science]This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
|
|
A. J. Cardinal, R. E. Dewey, and J. W. Burton Estimating the Individual Effects of the Reduced Palmitic Acid fapnc and fap1 Alleles on Agronomic Traits in Two Soybean Populations Crop Sci., March 19, 2008; 48(2): 633 - 639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Cardinal and J. W. Burton Correlations between Palmitate Content and Agronomic Traits in Soybean Populations Segregating for the fap1, fapnc, and fan Alleles Crop Sci., September 1, 2007; 47(5): 1804 - 1812. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Cardinal, J. W. Burton, A. M. Camacho-Roger, J. H. Yang, R. F. Wilson, and R. E. Dewey Molecular Analysis of Soybean Lines with Low Palmitic Acid Content in the Seed Oil Crop Sci., February 6, 2007; 47(1): 304 - 310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zhu, D. R. Walker, H. R. Boerma, J. N. All, and W. A. Parrott Fine Mapping of a Major Insect Resistance QTL in Soybean and its Interaction with Minor Resistance QTLs Crop Sci., March 27, 2006; 46(3): 1094 - 1099. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| The SCI Journals | Agronomy Journal | Vadose Zone Journal | |||
| Journal of Natural Resources and Life Sciences Education |
Soil Science Society of America Journal | ||||
| Journal of Plant Registrations | Journal of Environmental Quality |
The Plant Genome | |||
