Published online 30 July 2007
Published in Crop Sci 47:1705-1710 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
The Low Linolenic Acid Soybean Line PI 361088B Contains a Novel GmFAD3A Mutation
Andrew S. Chappell and
Kristin D. Bilyeu*
USDA-ARS, Plant Genetics Research Unit, 110 Waters Hall, Univ. of Missouri, Columbia, MO 65211. Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable
* Corresponding author (bilyeuk{at}missouri.edu).
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ABSTRACT
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Characterization of genetic mutations at the molecular level allows for the development of perfect genetic markers and rapid assays to detect those markers, which enables breeders to directly select for desired alleles and accelerate the breeding process. The objectives of this study were to identify genetic lesions found in the GmFAD3A gene in the low linolenic acid soybean [Glycine max (L.) Merr] line PI 361088B, which contains a fan allele, and to develop rapid molecular marker assays that distinguish between the wild-type and PI 361088B alleles. The entire genomic sequence of the GmFAD3A gene was determined for PI 361088B. Two thymine nucleotides are inserted into a run of four thymines from nucleotide position 307 to 310 of the GmFAD3A coding sequence, resulting in a frameshift and the introduction of a premature stop codon at nucleotide 328. Two molecular marker assays were developed that rely on polymerase chain reaction (PCR) amplification of the region of interest followed by restriction endonuclease digestion and agarose gel electrophoresis or melting curve analysis. A third assay is an allele-specific PCR-based assay that does not require any endonuclease step and requires only melting curve analysis. In conclusion, the low linolenic acid soybean line PI 361088B contains a coding mutation in GmFAD3A; this allele can easily be distinguished from the corresponding wild-type allele using any of three rapid molecular marker assays that were developed.
Abbreviations: PCR, polymerase chain reaction • SNP, single nucleotide polymorphism
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INTRODUCTION
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LINOLENIC ACID is responsible for the poor flavor and odor stability of soybean [Glycine max (L.) Merr] oil (Dutton et al., 1951; Shen et al., 1997). To increase its stability, soybean oil is often partially hydrogenated, which leads to the formation of trans fatty acids. This is a concern from a human nutrition standpoint since trans fatty acids are associated with an increased risk of coronary disease (Hu et al., 1997). To circumvent the need for hydrogenation, soybean breeders have developed low linolenic acid soybeans through genetic alteration. The most effective alteration method has been the use of mutagenesis. Examples of such low linolenic acid mutants include A5 (Hammond and Fehr, 1983), J18 and M5 (Anai et al., 2005), CX1512-44 (Bilyeu et al., 2005), and C1640 (Wilcox and Cavins, 1985). Phenotypic screening of available soybean germplasm has also proven valuable in the hunt for low linolenic acid soybeans. An example of this is the low linolenic acid line PI 361088B (Rennie et al., 1988).
The first low linolenic acid soybean mutant reported was C1640, which has an approximately 50% reduction in seed linolenic acid levels (Wilcox and Cavins, 1985, 1987). Previous work demonstrated that the reduction in seed linolenic acid levels is controlled by a single locus, designated the fan locus (Wilcox and Cavins, 1987). The low linolenic acid mutant A5 is deleted for the omega-3 fatty acid desaturase gene GmFAD3A (Byrum et al., 1997; Bilyeu et al., 2003) and is allelic with C1640 (Rennie and Tanner, 1991), suggesting that C1640 similarly contains a GmFAD3A mutation. We recently confirmed this when a premature stop codon was identified in the GmFAD3A gene of C1640 (Chappell and Bilyeu, 2006).
The genotype that is responsible for the low linolenic acid phenotype of PI 361088B is allelic with C1640, suggesting it too contains a mutation in the GmFAD3A gene (Rennie et al., 1988). The PI 361088B line was originally identified in a phenotypic screen for low linolenic acid levels in the seed oil fraction and was found to contain a fan allele and 38.1 g kg–1 of seed linolenic acid (Rennie et al., 1988). Here we set out to sequence the GmFAD3A gene in PI 361088B to determine the exact genetic lesion within this gene. A novel two nucleotide insertion in the coding sequence of GmFAD3A was identified that results in a frameshift and the introduction of a premature stop codon. Three molecular marker assays were developed to rapidly distinguish between the wild-type and PI 361088B alleles. Any of these three assays can easily be used by breeders to incorporate the PI 361088B low linolenic acid allele into other lines.
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MATERIALS AND METHODS
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Sequencing of the PI 361088B GmFAD3A Gene
To sequence the GmFAD3A gene from PI 361088B, the full-length gene was amplified from PI 361088B genomic DNA using primers identified from our analyses of soybean sequences in GenBank: 387start (5'-GCAATGGTTAAAGACACAAAG-3') and 387rev (5'-TCAGTCTCGTTGCGAGTG-3'). Genomic DNA template consisted of 2-mm diameter washed FTA (Whatman, Clifton, NJ) card punches prepared from leaves according to the manufacturer's instructions. Polymerase chain reaction (PCR) conditions were essentially as described (Bilyeu et al., 2005) with conditions set at 94°C for 1 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 3 min. ExTaq (TaKaRa Mirus Bio, Madison, WI) polymerase (0.5 U per reaction) was used. Polymerase chain reaction product was cleaned up using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA) before sequencing. The primer used to sequence the region of the gene containing the PI 361088B allele (2-bp insertion) was 387start. To confirm the presence of the 2-bp insertion, the full-length GmFAD3A gene was amplified again, but this time from genomic DNA that was isolated from a single seedling using the DNeasy Plant Mini Kit (QIAGEN). The sequencing of this PCR product confirmed the presence of the 2-bp insertion. The entire PI 361088B and ‘Williams 82’ GmFAD3A genomic sequences have been deposited in GenBank under the accession numbers EF175462 and EF175461, respectively.
Molecular Marker Assays
To test for the presence of the 2-bp insertion, a 200-bp fragment containing the 2-bp insertion site was amplified from either Williams 82 or PI 361088B genomic DNA that was prepared using the DNeasy Plant Mini Kit (QIAGEN, Valencia, CA). The primers FAD3A-fwd3 (5'-TCTCACACACTGCTTTGTTATGCC-3') and FAD3A-rev3 (5'-ACAAGAATTGAGGAATGCAAGATG-3') were used for amplification using 0.2x Titanium Taq (BD Biosciences, Palo Alto, CA) with 0.25x SYBR Green I (Molecular Probes, Eugene, OR) with PCR conditions of 94°C for 1 min followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s. Approximately 35% of the PCR products (7 µL) were combined with 1 µL of 10x buffer 2 (New England Biolabs, Beverly, MA), 1 µL 10x bovine serum albumin (New England Biolabs), and 20 U XmnI (New England Biolabs) in a 10-µL reaction. Digests were conducted at 37°C for 3 h to overnight, and then reaction products were resolved on a 2% agarose gel. Melting curve analysis was conducted using a DNA Engine Opticon 2 (MJ Research/Bio-Rad, Hercules, CA) by increasing temperature from 70 to 90°C reading every 0.2°C and holding every second with excitation at 470 to 505 nm and detection at 523 to 543 nm.
To assay for the presence of the 2-bp insertion using allele specific primers, PCR was conducted on either Williams 82 or PI 361088B genomic DNA that was prepared using the DNeasy Plant Mini Kit using the primers FAD3A-fwd5a (5'-GCGGGCAGGGCGGCCAGTGGCCATGGAAGCTTTTC-3'), FAD3A-fwd5b (5'-GCGGGCCAGTGGCCATGGAAGCTTTTT-3'), and FAD3A-rev5b (5'-ATGTGTCCCACCAGGCTATTTAGA-3'). Titanium Taq with SYBR Green I was used with PCR conditions of 94°C for 1 min, followed by 35 cycles of 94°C for 30 sec, 65°C for 30 sec, and 72°C for 30 sec. Melting curve analysis was conducted as described above. This experiment was conducted a total of six times, and melting curve results were averaged and a standard deviation was calculated.
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RESULTS AND DISCUSSION
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Identification of a GmFAD3A Mutation in PI 361088B
To determine if the PI 361088B GmFAD3A gene contained any genetic lesions, the full-length GmFAD3A gene was amplified and sequenced from genomic DNA that was isolated from PI 361088B material. Sequence analysis revealed a two nucleotide insertion in the coding sequence of GmFAD3A (compared to Pana GmFAD3A coding sequence, GenBank accession AY204710). Two thymine nucleotides are inserted into a run of four thymines from nucleotide position 307 to 310 of the coding sequence (Fig. 1A
). This two nucleotide insertion results in a frameshift and the introduction of a premature stop codon at nucleotide 328 (codon 110). Reamplification of the PI 361088B GmFAD3A gene and resequencing of the gene confirmed the presence of the 2-bp insertion.
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Figure 1. Two rapid molecular marker assays to distinguish between PI 361088B and wild-type GmFAD3A alleles. (A) Sequence of nucleotides 301 to 330 of the GmFAD3A coding sequence from ‘Williams 82’ and PI 361088B DNA. Amino acid sequence from 101 to 110 is displayed below their corresponding codons. The XmnI recognition site is indicated by a black bar above the sequence. Note that the XmnI recognition site is disrupted by the two nucleotide insertion in the PI 361088B sequence. (B) A 200-bp sequence encompassing the region shown in Fig. 1A was amplified from Williams 82 or PI 361088B genomic DNA and treated with or without XmnI endonuclease. Products were then resolved on a 2% agarose gel (shown). When XmnI cleaves the Williams 82 polymerase chain reaction (PCR) product, bands of 140 and 60 bp are generated. (C) Melting curve analysis on Williams 82 and PI 361088B PCR products that have been digested with XmnI. The melting curve average was 77.4°C for the Williams 82 product and 79.4°C for the PI 361088B product.
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Interestingly, analysis by sequencing and molecular marker assays (see below) revealed that two of the 30 (6.6%) PI 1361088B seeds from the USDA soybean germplasm collection that were analyzed did not contain the 2-bp insertion in GmFAD3A. This finding suggests that the seed stock is either contaminated or less likely, that the allele is still segregating.
Molecular Markers for the Identification of the PI 361088B GmFAD3A Allele
To assist breeders who are trying to incorporate the PI 361088B low linolenic acid trait into other lines, several molecular marker assays were developed that rapidly distinguish between the wild-type (Williams 82) and PI 361088B alleles. The first assay that was developed is a molecular assay that utilizes PCR, restriction endonuclease digestion, and electrophoresis (CAPS assay; Konieczny and Ausubel, 1993). A 200-bp PCR fragment encompassing the GmFAD3A region of interest (nucleotides 307–312 of the GmFAD3A coding sequence) is subjected to restriction endonuclease digestion by the enzyme XmnI. XmnI recognizes and cleaves the wild-type sequence whereas introduction of the 2-bp insertion found in the PI 361088B allele destroys this site and prevents digestion (see Fig. 1A). Therefore, the 200-bp PCR fragment from wild-type DNA is cleaved into a 140-bp fragment and a 60-bp fragment. In contrast, the PCR product from PI 361088B is not digested and the 200-bp fragment is retained. This is easily visualized when the digested PCR products are resolved on an agarose gel (Fig. 1B).
A second molecular assay was developed that utilizes the same PCR fragments described above but instead of using electrophoresis, melting curve analysis is used to distinguish between wild-type and PI 361088B PCR products. Using a real-time PCR instrument, melting curve analysis was conducted on digested PCR products that were initially amplified in the presence of the double-stranded DNA binding dye SYBR Green I (Ye et al., 2002). The two melting curves were easily distinguishable as they differed by 2°C (Fig. 1C).
A third molecular assay was developed that does not rely on restriction endonuclease digestion. In this assay, allele-specific PCR is conducted followed by melting curve analysis. A forward primer was designed that anneals specifically to the wild-type allele sequence and contains a "long GC" 5' tail (Wang et al., 2005; Fig. 2A
). A second forward primer was designed that anneals specifically to the PI 361088B allele sequence and contains a "short GC" 5' tail. Including GC-rich tails in both primers reduces the possibility of primer bias. The difference in length of the GC tails is sufficient to discriminate the allele-specific products in a melting curve analysis (Wang et al., 2005). A reverse primer was designed that anneals 12 nucleotides 3' to both forward primers (not shown). All three primers were used in reactions that contained either wild-type or PI 361088B genomic DNA as PCR templates. Melting curve analysis was then conducted on PCR products. Two distinct peaks were detected when wild-type or PI 361088B DNA was used as template (Fig. 2B). In the presence of wild-type DNA, presumably the long GC forward primer is utilized in the amplification reaction since it was designed to specifically anneal to the wild-type sequence. This is in contrast to when PI 361088B DNA is used as template. In this case, the short GC forward primer is presumably used in the PCR instead of the long GC primer. Since these two products are of different lengths due to the GC tail length differences, they display different characteristic melting temperatures. Thus, the longer product, amplified from wild-type DNA (71 bp), exhibits a melting temperature of 81.9 ± 0.3°C whereas the shorter product (65 bp), amplified from PI 361088B DNA, exhibits a melting temperature of 79.8 ± 0.8°C.
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Figure 2. An allele-specific assay to distinguish between PI 361088B and wild-type GmFAD3A alleles. (A) Sequence of nucleotides 301 to 330 of the GmFAD3A coding sequence from ‘Williams 82’ and PI 361088B DNA. Amino acid sequence is displayed below their corresponding codons. The position of the forward primers used in the assay is indicated by a black bar above the corresponding sequence. A dashed line is used where sequence is not shown. The "long GC" and "short GC" 5' tails are also indicated. (B) Melting curve analysis on Williams 82 and PI 361088B PCR products from the allele-specific polymerase chain reaction. The melting curve shows a melting temperature of 79.8°C for PI 361088B and 82.2°C for Williams 82.
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Our analyses did not include molecular marker assays on heterozygous plants because none were available to us. However, we used all three molecular marker assays described here to evaluate samples that contained a mixture of Williams 82 and PI 361088B template DNA. In each case, the mixed samples were distinguishable from both the Williams 82 and the PI 361088B templates in the assays, which would be the expected result for heterozygous samples (data not shown).
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CONCLUSIONS
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The GmFAD3A gene was sequenced from PI 361088B and this gene contains an insertion that ultimately results in the introduction of a premature stop codon. In particular, two thymine nucleotides are inserted into a run of four thymines from nucleotide position 307 to 310 of the coding sequence. This two nucleotide insertion results in a frameshift and the introduction of a premature stop codon at nucleotide 328. This mutation presumably results in a truncated and nonfunctional enzyme.
All the low linolenic acid lines that contain the mutant fan locus have so far been shown to also contain a mutation in GmFAD3A, suggesting the fan locus is a mutant GmFAD3A gene. All of the fan lines that have been characterized at the molecular level are shown in Fig. 3
. Although derived separately, the A5 and J18 soybean lines both contain full or partial deletions of GmFAD3A (Anai et al., 2005; Bilyeu et al., 2003; Byrum et al., 1997; Fig. 3). M5 is similar to PI 361088B in that it too contains a coding mutation that results in a frameshift (Rahman et al., 1998; Rahman and Takagi, 1997). In this mutant, a 19-nucleotide deletion at the very 3' end of GmFAD3A results in a frameshift and an extension of the open reading frame, resulting in a C-terminal 25–amino acid extension of the protein. The CX1512-44 soybean line contains a noncoding single base pair change in GmFAD3A (Bilyeu et al., 2005). This single base pair change disrupts a splice site at the boundary of exon–intron 6, which results in missplicing of the gene. C1640 also contains a single base pair change, but this change is in the coding sequence (Chappell and Bilyeu, 2006). A TGG tryptophan codon in exon 6 is changed to a TGA stop codon. Similar to the PI 361088B mutation, the M5, CX1512-44, and C1640 alleles presumably render nonfunctional enzymes. In contrast, the A5 and J18 alleles presumably result in no transcript, and thus, no enzyme being produced.
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Figure 3. An overview of GmFAD3A alleles and mutations found in low linolenic acid soybean lines. Schematic diagram of GmFAD3A alleles found in the low linolenic acid soybean lines A5, J18, M5, CX1512-44, C1640, and PI 361088B. Exons are indicated by black boxes and introns by horizontal lines. To the right of the gene schematics is a list of the amount (g linolenic acid kg–1 of oil) of linolenic acid levels in seeds of the various lines. Also indicated is whether a rapid molecular assay has been published to test for the presence of that particular allele. Relevant references for each line or mutant are also listed.
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Characterization of genetic mutations or alleles at the molecular level allows for the development of perfect genetic markers and rapid assays to detect these markers. The choice of molecular marker assay depends on a number of factors, including investment in labor and instrumentation. The assays described here range from low instrumentation but slightly higher labor costs (CAPS assay) to low labor costs but higher instrumentation costs (GC tail assay). In addition, use of the CAPS assay involves post-PCR processing of samples that increases the risk of future PCR contamination. Although the instrumentation necessary for the GC tail assay is relatively high, the assay does not require any post-PCR processing of samples. The final choice of the most appropriate molecular marker assay would necessarily depend on throughput and available resources.
Of the six GmFAD3A alleles discussed here, four can already be detected using rapid molecular marker assays (Fig. 3). The continuing accumulation of GmFAD3A allele information at the molecular level will ultimately enable direct selection for mutant alleles in early generations of segregating populations by breeders trying to incorporate low linolenic acid traits into other soybean lines.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication December 11, 2006.
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ABSTRACT
INTRODUCTION
