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a Dep. of Crop Science, North Carolina State Univ., Raleigh, NC 27695
b USDA-ARS, Plant Sci. Res., 3127 Ligon St., Raleigh, NC 27607
* Corresponding author (andrea_cardinal{at}ncsu.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: BLUP, best linear unbiased predictor • EST, expressed sequence tags • FATA, 18:1-ACP thioesterase gene • FATB, 16:0-ACP thioesterase gene • KAS II, 3-ketoacyl-ACP synthetase II • PCR, polymerase chain reaction • TAGs, triacylglycerols • 16:0, palmitic acid • 18:0, stearic acid • 18:1, oleic acid • 18:2, linoleic acid • 18:3, linolenic acid
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Soybean oil quality can be improved through genetic alteration of the plant. Several lines with reduced palmitic acid concentration have been developed by mutagenesis. C1726 (85.4 g kg–1 16:0) and ELLP2 (71.4 g kg–1 16:0) were developed by ethyl methanesulfonate (EMS) mutagenesis of the cultivars Century and Elgin87, respectively. A22 (78.4 g kg–1 16:0) was developed by N-nitroso-N-methyl urea mutagenesis of A1937, and J3 (57.4 g kg–1 16:0) was developed from X-radiation of cultivar Bay (Fehr et al., 1991a; Stoj
in et al., 1998; Wilcox and Cavins, 1990; Rahman et al., 1996). In addition, one natural mutation was discovered in line N79-2077 (64 g kg–1 16:0) and its derived line N79-2077-12 from the fifth cycle of recurrent selection for high oleic concentration (Burton et al., 1983, 1994). The alleles conferring low palmitic acid in these lines were given different names: fap1 (in C1726), fap3 (in A22), fapnc (in N79-2077), fap* or fapx (in ELLP2), and sop1 (in J3) (Schnebly et al., 1994; Stojin et al., 1998; Wilcox and Cavins, 1990; Kinoshita et al., 1998; Primomo et al., 2002). Of those five alleles, three are at independent loci from each other (fap1, fap3, and fap*) (Schnebly et al., 1994; Stojin et al., 1998; Wilcox et al., 1994; Kinoshita et al., 1998; Primomo, 2000; Primomo et al., 2002). fapnc is allelic to fap3 (Primomo, 2000; Primomo et al., 2002). sop1 is independent of the fap1 locus, but its allelic relationship to the other loci is unknown (Kinoshita et al., 1998).
In addition to the major genes mentioned above, minor-modifier genes influence the low palmitic acid content in soybean oil (Stojin et al., 1998; Rebetzke et al., 1998b, 2001; Li et al., 2002; Horejsi et al., 1994). Rebetzke et al. (1998b) observed heritability estimates for 16:0 content on a plot basis between 0.37 and 0.73 and on an entry-mean basis between 0.83 and 0.96. They concluded that selection based on evaluations in a few environments for both major and minor genes would be successful.
Correlated changes in the other saturated and unsaturated fatty acids in the soybean oil have been observed in low palmitic acid lines that carry the major and minor genes described above. For example, when lines homozygous for the fap1 and fap3 alleles (<41.5 g kg–1 16:0) were compared with normal lines (>100 g kg–1 16:0) derived from the same population, the low palmitic acid lines tended to have reduced stearic acid (18:0) and increased oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3) contents (Ndzana et al., 1994). Similarly, progenies that inherited the major fapnc allele from the cross of N87-2122-4 to two high yielding normal palmitate lines had a 6 to 15% reduction in stearic acid, (Rebetzke et al., 1998b, 2001) a 4 to 10% increase in oleic acid content in both crosses (Rebetzke et al., 1998a, 2001) and an increase in linolenic acid content in one cross (Rebetzke et al., 1998a). No differences in linoleic acid were observed between progenies that inherited the major fapnc allele and those that inherited the normal allele (Rebetzke et al., 1998a). However, there was significant genetic variation within each major fapnc group because of the segregation of minor genes (Rebetzke et al., 1998a). Within these groups, there was a significant negative genetic correlation between palmitate and oleate and a significant positive correlation between palmitate and linolenate (Rebetzke et al., 1998a). Palmitate content in soybean oil was negatively correlated with linoleate content and positively correlated with oleate content in a population that was segregating for the fap1 and fan loci (Nickell et al., 1991).
Several steps in the TAG biosynthetic pathway could affect the palmitate concentration. In the case of low palmitic acid mutants, changes in activity or substrate specificity in 3-ketoacyl-ACP synthetase II (KAS II), 16:0-ACP thioesterase, lysophosphatidic acid acyltransferase, glycerol-3-phosphate acyltransferase, or diacylglycerol acyl transferase enzymes could be responsible for their phenotype. A biochemical study comparing C1726 (fap1fap1), N79-2077-12 (fapncfapnc) and ‘Dare’ found no evidence that glycerolipid acyltransferase activity or the biosynthetic steps after the formation of acyl-CoA were altered in these lines. The TAG composition of these lines was related to the amount of 16:0 produced in the plastids (Wilson et al., 2001b, 2001c). Northern analysis revealed that fapncfapnc genotypes have reduced accumulation of transcripts corresponding to a 16:0-ACP thioesterase (FATB) gene and no difference in transcript accumulation for genes encoding 18:1-ACP thioesterase (FATA) or 18:0-ACP desaturase activities (Wilson et al., 2001b). No differences in transcript accumulation for fap1fap1 genotypes were observed for any of the genes assayed (Wilson et al., 2001b). Southern analysis using the FATB cDNA as a probe revealed that multiple copies of the 16:0-ACP thioesterase gene are present in the soybean genome and that the fapncfapnc genotype has a deletion in one of those copies (Wilson et al., 2001a). It is not known which of the FATB gene copies is deleted in the fapncfapnc genotype, where in the soybean genome these FATB genes are located, and how much of the palmitate phenotypic variation each isoform accounts for.
The objectives of this study are to (i) identify the specific FATB gene that is deleted in low palmitic acid soybean lines homozygous for the fapnc mutation, (ii) develop allele specific PCR markers corresponding to this gene, and (iii) estimate the amount of phenotypic variation in the 16:0, 18:0, 18:1, 18:2, and 18:3 composition that is explained by this locus in two adapted populations grown in replicated trials.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Sequence information from soybean ESTs available in GenBank was used to synthesize PCR primers corresponding to the 3' flanking regions of the GmFATB1a, GmFATB1b, and GmFATB2a genes. To obtain sequence information from the respective 5' flanking regions, reverse primers directed against individual GmFATB genes were used together with a vector-specific primer and a soybean cDNA library that we had previously cloned into a yeast expression vector as a template (Dewey et al., 1994). The following primers were designed to amplify full length cDNAs using the above described first-strand cDNA from Century as the template: forward primer 5'-TCTACCGGAGAAGCGACCT-3' and reverse primer 5'-CCCCCTTACCCACCAAAGTA-3' (GmFATB1a); forward primer 5'-TACCCTACAAGCTGCCGTAG-3' and reverse primer 5'-CCCCCTTACCCACCAAAGTA-3' (GmFATB1b); and forward primer 5'-GTAAGGCGTAGGGGGTTGTT-3' and reverse primer 5'-ACACCAAGCAGCTGCATAAC-3' (GmFATB2a). Nucleic acid and predicted protein sequences were aligned and compared using the CLUSTALW (Thompson et al., 1994) and GAP (University of Wisconsin Genetics Computing Group) algorithms.
Amplification of Genomic DNAs
Genomic DNA was prepared from young soybean leaf tissue as previously described (Murray and Thompson, 1980). Primers 5'-GACATAGTTCAAGTGGACACT-3' (forward) and 5'-TTCACAACACCAAAGTTGTTCAC-3' (reverse) were used to amplify a 1178-bp fragment of GmFATB1a genomic DNA. The corresponding region of GmFATB1b genomic DNA was amplified using primers 5'-GACGTAGTTCAAGTAGACACC-3' (forward) and 5'-TTCACAACACCAATGTTGTTCAT-3' (reverse). Three introns were observed within this specific region of the GmFATB1 genes. The partial genomic sequence information generated by these primers is shown in supplemental Fig. S1
and have been deposited in GenBank as Accession no. DQ861997 (GmFATB1a) and no. DQ861998 (GmFATB1b).
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Development of Populations
Two F4-derived populations were developed by single seed descent (Brim, 1966). One population (FAHH00) was derived from the cross of a high yielding line, ‘Corsica’, and a low linolenic, low palmitic acid line, N97-3681-11. A second population (FADD00) was derived from the cross of high yielding line, ‘Brim’, and a low linolenic, low palmitic line, N97-3708-13. N97-3681-11, N97-3708-13 and the cultivar Satelite are sister lines that were derived from different F3 plant selections from the cross ‘Soyola’ x {‘Brim’(2) x [N88-431(2) x (N90-2013 x C1726)]}. Line N88-431 was selected from the cross N88-1299 x N82-2037. N84-1299 was a selection from the first cycle of a high protein recurrent selection population (Holbrook et al., 1989). N82-2037 had Gasoy17 as its maternal parent and N77-940 as the paternal parent. C1726 is a reduced palmitic acid selection from EMS treated Century and is homozygous for the fap1 mutation (Wilcox and Cavins, 1990). N90-2013 is a reduced palmitic acid selection from the cross PI123,440 x N79-2077-12 (Burton et al., 1994). Soyola is a low linolenic acid line (Burton et al., 2004). N97-3681-11 and N97-3708-13 are F4-derived lines and are assumed homozygous for the low palmitic fapnc mutant allele from N79-2077-12, the low palmitic fap1 mutant allele from C1726, and the low linolenic fan(PI123440) allele. In addition, N97-3681-11 and N97-3708-13 are highly related to Brim, and the coefficient of parentage between these lines and Brim is 0.777 (data not shown). Therefore, in the Brim x N97-3706-13 population only 22.3% of the soybean genome is expected to be segregating.
Phenotypic Evaluation of Populations
Each population was evaluated in separate field experiments. Ninety-eight F4-derived lines and the parents of the FADD00 population and 99 F4-derived lines and one parent (N97-3681-11) of the FAHH00 population were grown at Plymouth, NC, in 2002 and Caswell and Plymouth, NC, in 2003. Within each environment, the experimental design for each population was a 10 x 10 lattice with two replications. At harvest time, a 30 g subsample of seed from each plot was analyzed for fatty acid content, by gas-liquid chromatography of the methyl esters, at the USDA-ARS laboratory, Raleigh, NC. Fatty acid content is reported in grams per kilogram of total lipids.
Genotypic Evaluation of Populations
DNA was extracted by a CTAB (hexadecyltrimethylammonium bromide) protocol from a bulk of leaf tissue from several plants of each line (Li et al., 2002). FATB1a-312 and FATB1a-411 allele specific primer markers were used to perform PCR analysis with genomic DNA from each line in the two populations. The PCR reactions consisted of: 0.8 µL of 5 pmol/µL forward and reverse primers, 0.5 µL of 3.125 mM dNTPs, 3 µL of 15 mM MgCl, 0.05 µL of Taq polymerase, 3.35 µL of ddH20, and 5 µL of 10ng/µL DNA. The PCR protocol included a first step of denaturation at 95°C for 5 min, then a second step of denaturation at 92°C for 1 min, a third step of annealing at 59°C for 1 min, a fourth step of extension at 69°C for 2 min, then steps two to four were subsequently repeated for 36 cycles. The reactions were performed in a 96-well PTC-100 thermal cycler (MJ Research, Inc., Waltham, MA) or a 384-well PTC-200 thermal cycler (MJ Research, Inc.). The amplified fragments were separated in 4% (w/v) SFR agarose (AMRESCO, Inc., Solon, OH) gels stained with ethidium bromide and visualized under UV light. The genotype of the FATB1a-312 and FATB1a-411 markers were scored as a presence or absence of a 312- or 411-bp band.
Statistical Analysis of Populations
The fatty acid data from each population were analyzed as a lattice design across environments by PROC Mixed, SAS 8.2 (SAS Institute, Inc., 1999). Locations, replications, incomplete blocks, and lines were considered random effects. Since lines were considered a random effect and a mixed model approach was used, then a best linear unbiased predictor (BLUP) for each line was obtained by adding the overall mean effect to the random effect of each line (SAS Institute Inc., 1996). The BLUPs for each fatty acid for each line were then used to perform a t test to compare the effect of inheriting one or two copies of the FATB1a-312 or FATB1a-411 marker alleles (heterozygous or homozygous wild type) versus zero copies (homozygous mutant). The FATB1a-312 or FATB1a-411 marker class was the independent variable, and the BLUPs for each line for each fatty acid were the dependent variable in the analysis. The mean of lines homozygous or heterozygous for the FATB1a-312 or FATB1a-411 allele (presence of wild-type allele), the mean of lines homozygous for the absence of the FATB1a-312 or FATB1a-411 allele (mutant allele), the standard deviation of those means, the difference between means, and the R-square for the model were obtained using SAS Proc GLM, SAS 8.2 (SAS Institute, Inc., 1999).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Using a modified 5'-RACE strategy, full-length cDNAs of GmFATB1a, GmFATB1b, and GmFATB2a were amplified from the soybean cultivar Century and sequenced. The nucleotide and predicted protein products of GmFATB1a and GmFATB1b both share over 96% sequence identity. In contrast, approximately 73% nucleotide sequence identity (79% predicted protein identity) is shared between either GmFATB1a or GmFATB1b and the GmFATB2a sequence. Although full-length sequence information for GmFATB2b is lacking, alignments of the partial sequences derived from GenBank suggest that this sequence is approximately 95% identical to GmFATB2a (data not shown). These results, the existence of four isoforms that can be assigned into two pairs by sequence similarity, are very similar to those reported for the omega-3 desaturase (FAD3 gene) that converts linoleoyl-PC to produce linolenoyl-PC in soybean (Bilyeu et al., 2003; Anai et al., 2005). Four similar FAD3 sequences were observed that were grouped as two closely related pairs on the basis of sequence similarity. Both the FATB and FAD3 gene sequence observations are congruent with the hypothesis that the soybean genome had undergone both an ancient and a more recent duplication event (Shoemaker et al., 1996; Schlueter et al., 2004; Pfeil et al., 2005). According to this model, some soybean genes would be expected to be represented by four copies within the genome that could be separated in two pairs that would be more similar within each pair that originated from the more recent duplication event.
In silico analysis of the EST database indicated that the GmFATB1a gene isoform is the most prevalent. In addition, the fapnc allele had the strongest low palmitic acid phenotype of all mutations characterized to date. From these observations, we speculated that GmFATB1a would be the best candidate for being the specific FATB isoform deleted in fapnc lines. To test this possibility, we attempted to generate gene-specific PCR primers that would uniquely amplify the GmFATB1a isoform. However, because of the high sequence identity shared between GmFATB1a and GmFATB1b, we had difficulty finding sufficient polymorphisms for producing primers that would enable reliable, unambiguous amplification of these highly homologous genes. To overcome this problem we amplified a region of GmFATB1a and GmFATB1b genomic DNA (Fig. S1). By designing PCR primers on the basis of polymorphic regions found within both exon and intron regions, gene-specific markers capable of distinguishing GmFATB1a and GmFATB1b were created. As shown in supplemental Fig. S1, marker FATB1a-312 is defined by primers that specifically amplify a 312-bp fragment of the GmFATB1a gene when genomic DNA is used as a template; marker FATB1a-411 is generated using a primer pair that amplifies a 411-bp genomic fragment of GmFATB1a. Similarly, markers FATB1b-314 and FATB1b-417 are the products of GmFATB1b-specific primers that amplify 314- and 417-bp products, respectively.
Expected amplification products were observed for all four FATB1 markers when genomic DNA from normal soybean cultivar Dare was used as a template (Fig. 1A ). Soybean lines C1726 and C1727, which possess the fap1 and fap2 mutations, respectively, that alter the palmitic acid content of the soybean seed, yet are nonallelic to fapnc, also yielded the expected PCR products for all four markers. In contrast, when genomic DNAs from two lines that are homozygous for the fapnc locus (N79-2077 and N93-2008) were used as template, only markers corresponding to GmFATB1b amplified a DNA segment (Fig. 1A). These results indicate that GmFATB1a is the specific FATB isoform that is deleted in soybean lines possessing the fapnc locus. Furthermore, PCR reactions using genomic DNA from the fapnc containing low palmitate lines N97-3681-11 and N97-3708-13 and the GmFATB1a specific markers also failed to yield amplification products (data not shown). Cumulatively, our results strongly suggest that the molecular basis for the low palmitic acid fapnc mutation is a deletion of all or a portion of the GmFATB1a gene.
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The number of observed homozygous lines for the fapnc allele was significantly less than expected for F4-derived populations. A Chi-square test (not shown) demonstrated significant segregation distortion for this locus in both populations. No significant difference in maturity was observed between fapnc fapnc and Fapnc_ palmitate lines (data not shown); therefore, unintentional selection for later maturing lines during the inbreeding procedure cannot account for the segregation distortion. Only F4-derived lines that produced enough seed to be grown in replicated yield trials were used in this study. Therefore, if fapncfapnc lines tend to yield less than normal FapncFapnc palmitate lines, as was observed by Rebetzke et al. (1998b, 2001), selection against low yielding lines may have been responsible for recovery of fewer than expected fapncfapnc lines.
Phenotypic Evaluation of Populations
The mean palmitic acid content of seed oil from lines homozygous for the fapnc allele is 48.2 g kg–1 in the Corsica x N97-3681-11 population and 54.3 g kg–1 in the Brim x N97-3708-13 population (Table 1). These means are lower than the expected from lines homozygous only for the fapnc, such as N79-2077 (64 g kg–1 16:0) or its derived line N79-2077-12, because of the independent segregation of the additional low palmitate locus fap1 in these populations (Burton et al., 1983, 1994). The mean palmitic acid content of lines heterozygous or homozygous for the presence of the FATB1a-312 or FATB1a-411 markers was 89.2 and 89.5 g kg–1, in the two populations, respectively. These means are lower than the mean of Brim (110 g kg–1 of 16:0) because in addition to the segregating fap1 locus, they also represent both the heterozygous and homozygous classes for the wild-type Fapnc allele. The differences between the two groups were highly significant, and they explained 70.4 and 61.8% of the genetic variation in palmitic acid oil seed content in the two populations, respectively. The amount of genetic variation in palmitate content explained by this locus in these two populations is twice the amount explained by the major QTL detected in an F2 derived population segregating for the fapnc allele only (Li et al., 2002).
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The influence of the fapnc allele on oleic acid content was less clear. Lines homozygous for the fapnc allele had significantly higher oleic acid content in the Brim x N97-3708-13 population, but not in the Corsica x N97-3681-11 population. Increased oleic acid content has previously been described in populations segregating for the fapnc allele (Rebetzke et al., 1998a, 2001). The inheritance of the fapnc allele had no effect on the linolenic acid content in either population (Table 1). This result is contrary to that observed by Ndzana et al. (1994) and Rebetzke et al. (1998a), but the populations studied in this project differ from those previous reports in that our populations are also segregating for the low linolenic acid fan(PI123440) allele. Previous studies have shown that the major low palmitic and low linolenic acid loci are independent from each other (Cherrak et al., 2003). It is possible that the populations used in previous studies by Ndzana et al. (1994) and Rebetzke et al. (1998a) were segregating for "normal" alleles that affect linolenate content but that are not allelic to fan so the correlation between palmitate and linolenate would differ in those populations.
Although the fapnc allele affects the composition of several fatty acids in the seed oil, the amount of variation explained differed considerably between populations (Table 1). For example, the fapnc allele accounts for 50.4% of the genetic variation for stearic acid content in the Brim x N97-3708-13 population but only 22.7% in the Corsica x N97-3681-11 population. Similarly, the fapnc allele accounts for 7.6% of the linoleic acid genetic variation in the Brim x N97-3708-13 population and 24.6% in the Corsica x N97-3681-11 population.
In summary, we have determined that lines homozygous for the fapnc mutation have a deletion in the GmFATB1a gene that encodes a 16:0-ACP thioesterase. The GmFATB1a gene product also influences 18:0 production because lines that have a deletion in this gene have reduced palmitate and stearate in their oil. The most likely explanation of this observation is that the GmFATB1a encoded enzyme also has some activity on 18:0-ACP substrates. Segregation at the fapnc locus explained 62 to 70% of the genotypic variation in palmitate content in two F4-derived populations using specific markers we developed that uniquely amplify the GmFATB1a isoform. Future research goals will be to map the GmFATB1a gene and compare its genomic location with the major QTL for palmitate observed by Li et al. (2002).
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication May 2, 2006.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-linolenic acid mutants and elucidation of their contribution to fatty acid biosynthesis in soybean seed. Plant Sci. 168:1615–1623.[CrossRef][Web of Science]in, D., G.R. Ablett, B.M. Luzzi, and J.W. Tanner. 1998. Use of gene substitution values to quantify partial dominance in low palmitic acid soybean. Crop Sci. 38:1437–1441.This article has been cited by other articles:
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  E. Bachlava and A. J. Cardinal Correlation between Temperature and Oleic Acid Seed Content in Three Segregating Soybean Populations Crop Sci., June 26, 2009; 49(4): 1328 - 1335. [Abstract] [Full Text] [PDF]
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P. Zhang, J. W. Burton, R. G. Upchurch, E. Whittle, J. Shanklin, and R. E. Dewey Mutations in a {Delta}9-Stearoyl-ACP-Desaturase Gene Are Associated with Enhanced Stearic Acid Levels in Soybean Seeds Crop Sci., November 24, 2008; 48(6): 2305 - 2313. [Abstract] [Full Text] [PDF]
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E. Bachlava, J. W. Burton, C. Brownie, S. Wang, J. Auclair, and A. J. Cardinal Heritability of Oleic Acid Content in Soybean Seed Oil and Its Genetic Correlation with Fatty Acid and Agronomic Traits Crop Sci., September 23, 2008; 48(5): 1764 - 1772. [Abstract] [Full Text] [PDF]
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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]
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E. Bachlava, R. E. Dewey, J. Auclair, S. Wang, J. W. Burton, and A. J. Cardinal Mapping Genes Encoding Microsomal {omega}-6 Desaturase Enzymes and Their Cosegregation with QTL Affecting Oleate Content in Soybean Crop Sci., March 19, 2008; 48(2): 640 - 650. [Abstract] [Full Text] [PDF]
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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]
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