Abstract
One of the goals in oilseed rape programs is to develop genotypes producing oil with low linolenic acid content (C18:3, ≤3%). Low linolenic mutant lines of canola rapeseed were obtained via chemical mutagenesis at the Plant Breeding and Acclimatization Institute – NRI, in Poznan, Poland, and allele-specific SNP markers were designed for monitoring of two statistically important single nucleotide polymorphisms detected by SNaPshot analysis in two FAD3 desaturase genes, BnaA.FAD3 and BnaC.FAD3, respectively. Strong negative correlation between the presence of mutant alleles of the genes and linolenic acid content was revealed by analysis of variance. In this paper we present detailed characteristics of the markers by estimation of the additive and dominance effects of the FAD3 genes with respect to particular fatty acid content in seed oil, as well as by calculation of the phenotypic variation of seed oil fatty acid composition accounted by particular allele-specific marker. The obtained percentage of variation in fatty acid composition was considerable only for linolenic acid content and equaled 35.6% for BnaA.FAD3 and 39.3% for BnaC.FAD3, whereas the total percentage of variation in linolenic acid content was 53.2% when accounted for mutations in both genes simultaneously. Our results revealed high specificity of the markers for effective monitoring of the wild-type and mutated alleles of the Brassica napus FAD3 desaturase genes in the low linolenic mutant recombinants in breeding programs.
Keywords: Brassica napus L., FAD3 desaturase, Fatty acid composition, Genetic effects, Seed oil, SNaPshot analysis
Introduction
Oilseed rape (Brassica napus L.) is the second-most important oil crop of the moderate climate zone. Seeds of the double low varieties (canola, ‘00’, with very low glucosinolates content in seed meal and with no erucic acid in seed oil) produce oil containing, on average: 7% of saturated fatty acids – palmitic (C16:0) and stearic (C18:0), and polyunsaturated fatty acids – oleic (C18:1, 61%), linoleic (C18:2, 20%), linolenic (C18:3, 10%) and eicosenoic fatty acid (C20:1, 1%). Due to the food- and non-food use of the oil, rapeseed breeding forms characterized by diversified seed oil fatty acid content are of great value on the oil crop market, and one of the breeding goals is to develop genotypes producing oil with low linolenic acid content (C18:3, ≤3%). Such oil is desirable for deep frying, as low content of polyunsaturated linolenic acid prevents oxidation and rancidation of the oil, which is crucial for healty food production. In addition, oil with low linolenic acid content makes an important source of raw material for biofuel production, due to high stability of such oil. Genetic analyses revealed that in B. napus low linolenic acid content was controlled by the two fatty acid desaturase 3 genes (FAD3), named BnaA.FAD3 and BnaC.FAD3, according to the standardized gene nomenclature for the Brassica genus (Østergaard and King 2008). The genes are located in the A and C genomes of B. napus (AACC amphidiploid genome) (Barret et al. 1999; Jourdren et al. 1996), and were mapped by Hu et al. (2006) on the N4 (A4) and N14 (C4) linkage groups, respectively.
Low linolenic mutant lines of canola rapeseed were obtained via chemical mutagenesis at the Plant Breeding and Acclimatization Institute – NRI, in Poznan, Poland (Spasibionek 2006). Two statistically important single nucleotide polymorphisms were detected in two FAD3 desaturase genes: (1) a C to T substitution in the third position of the sixth codon of the seventh exon in the BnaA.FAD3 gene and (2) a G to A transition in the 5' splice donor site of the sixth intron in the BnaC.FAD3 gene (Mikolajczyk et al. 2010). Allele-specific SNP markers were designed involving a two-step detection of the wild-type and mutant alleles in both loci by SNaPshot analysis comprising an independent and specific PCR amplifications of B. napus BnaA.FAD3 and BnaC.FAD3 gene fragments comprising the mutation sites, and detection of the mutant alleles analysis using locus-specific primers (Mikolajczyk et al. 2010). Strong negative correlation between the presence of mutated alleles in the A and C genomes and linolenic acid content was revealed by analysis of variance (Mikolajczyk et al. 2010).
In this paper we report detailed characteristics of the developed allele-specific markers by estimation of the additive and dominance effects of the FAD3 genes with respect to particular fatty acid content in seed oil. In addition, percentage of phenotypic variation of seed oil fatty acid composition accounted by particular allele-specific marker was calculated for wild-type and mutated alleles of BnaA.FAD3 and BnaC.FAD3, respectively.
Materials and methods
Plant material consisted of 450 winter oilseed rape plants: 36 canola-type (00, 7-10% of linolenic acid in seed oil) cultivars and 26 doubled haploid (DH) mutant low linolenic (LL, ≤3% of linolenic acid) lines, as well as 26 DH lines developed from F1 hybrid obtained as a result of crosses between 00 and LL lines in addition to 362 plants of F2 and F3 segregating lines obtained from crosses of 00 and LL lines, from 2 to 10 plants of each line. Field experiments (2007/ 2008 and 2008/ 2009) were conducted in Karzniczka, Lagiewniki and Poznan, Poland (Mikolajczyk et al. 2010).
The fatty acid composition of mature seeds was determined by gas liquid chromatography of the methyl esters (Byczynska and Krzymanski 1969) using an Agillent Technologies 6890N gas chromatograph (DB 23 30 m, ID 025, 0.25 μm layer capillary column) equipped with a Chemstation integrator.
Allelic forms of the BnaA.FAD3 and BnaC.FAD3 genes were identified by independent and specific PCR amplifications of the gene fragments comprising mutation sites and detection of the mutant alleles by SNaPshot analysis using locus-specific primers (Mikolajczyk et al. 2010). Regression analysis was done for the mutations in the FAD3 genes on fatty acid composition in seed oil. The analysis was performed both – 1) individually, as well as 2) simultaneously for the two genes. The FAD3 genes observations were treated as independent variables and considered in individual models (the first case), as well as in multiple regression model (the second case). The additive and dominance effects of the presence or absence of mutations in the FAD3 genes for particular fatty acid content in seed oil were estimated. The additive effect of each allel was estimated as half of the difference between homozygotes and we tested null hypothesis that the difference between mean values for homozygotes is equal to zero by two-sample t-test. The dominance effect was estimated as deviation of mean value for heterozygotes from grand mean. We tested a null hypothesis that mean value for heterozygotes is equal to grand mean by two-sample t-test. The broad-sense heritability coefficients were calculated (Falconer and Mackay 1996). All calculations were carried out using GenStat v. 7.1 (Payne et al. 2003).
Results and conclusions
The presence of the mutated BnaA.FAD3 allele was associated with seed oil fatty acid composition with statistical significance, except for eicosenoic acid content (C20:1) (P = 0.129) – possibly due to little content of eicosenoic acid in oilseed rape oil. At the same time, the mutant form of the BnaC.FAD3 gene was associated with stearic (C18:0) (P < 0.001), linoleic (C18:2) (P < 0.001) and linolenic acid (C18:3) (P < 0.001) content (Table 1). The percentage of variation in fatty acid composition accounted for the presence of mutations in the BnaA.FAD3 and BnaC.FAD3 genes by the SNaPshot analysis was considerable only for the linolenic acid content and equaled 35.6% and 39.3%, respectively (Table 1), whereas the total percentage of variation in linolenic acid content was 53.2% when accounted for mutations in both genes simultaneously (Table 1). At the same time, the oleic acid content was markedly influenced by the presence of the mutated alleles of the FAD3 genes (Table 1) and it was not the case for the presence of the mutated BnaC.FAD3 alone (Table 1).
Table 1.
Regression analysis of the mutations in the FAD3 genes on particular fatty acid content in seed oil; C16:0, palmitic, C18:0, stearic, C18:1, oleic, C18:2, linoleic, C18:3, linolenic, C20:1, eicosenoic acid; SNP – genotyping of allelic forms of the FAD3 desaturase genes with the use of the SNaPshot assay, in the BnaA.FAD3 gene (FAD3A) and in the BnaC.FAD3 gene (FAD3C), respectively
| SNP | Trait | Estimates of regression coefficients | P-values | Percentage variation accounted | Standard error of observations | Heritability |
|---|---|---|---|---|---|---|
| FAD3A | C16:0 | 0.1355 | <0.001 | 3.0 | 0.550 | 0.50 |
| FAD3C | C16:0 | 0.065 | 0.051 | 0.6 | 0.557 | |
| FAD3A, FAD3C | C16:0 | 0.13, 0.02 | <0.001, 0.662 | 2.8 | 0.551 | |
| FAD3A | C18:0 | −0.1548 | <0.001 | 5.9 | 0.448 | 0.72 |
| FAD3C | C18:0 | −0.1213 | <0.001 | 4.1 | 0.452 | |
| FAD3A, FAD3C | C18:0 | −0.12, -0.07 | <0.001, 0.011 | 7.0 | 0.445 | |
| FAD3A | C18:1 | −0.587 | 0.027 | 0.9 | 4.140 | 0.56 |
| FAD3C | C18:1 | 0.402 | 0.106 | 0.4 | 4.150 | |
| FAD3A, FAD3C | C18:1 | −0.92, 0.75 | 0.002, 0.005 | 2.3 | 4.100 | |
| FAD3A | C18:2 | −0.98 | <0.001 | 3.4 | 3.730 | 0.79 |
| FAD3C | C18:2 | −1.91 | <0.001 | 15.5 | 3.490 | |
| FAD3A, FAD3C | C18:2 | −0.17, -1.85 | 0.490, <0.001 | 15.4 | 3.490 | |
| FAD3A | C18:3 | 1.606 | <0.001 | 35.6 | 1.580 | 0.88 |
| FAD3C | C18:3 | 1.5738 | <0.001 | 39.3 | 1.540 | |
| FAD3A, FAD3C | C18:3 | 1.10, 1.15 | <0.001, <0.001 | 53.2 | 1.350 | |
| FAD3A | C20:1 | −0.0186 | 0.129 | 0.3 | 0.191 | 0.44 |
| FAD3C | C20:1 | −0.0128 | 0.264 | 0.1 | 0.191 | |
| FAD3A, FAD3C | C20:1 | −0.02, -0.001 | 0.245, 0.588 | 0.1 | 0.191 |
Additive effects of the mutated alleles of the BnaA.FAD3 and BnaC.FAD3 genes were significant for stearic, linoleic and linolenic acid content and the signs of the effect values were the same for both genes (Table 2). Moreover, additive effect of the mutated allele of the BnaA.FAD3 gene was significant for the palmitic acid content (Table 2).
Table 2.
Additive (a) and dominance (d) effects of the BnaA.FAD3 (FAD3A) i BnaC.FAD3 (FAD3C) alleles on seed oil composition; particular fatty acid abbreviations (in the ‘Trait’ column) as in Table 1
| Trait | FAD3A | FAD3C | ||
|---|---|---|---|---|
| a | d | a | d | |
| C16:0 | 0.099a | −0.251b | 0.059 | −0.255b |
| C18:0 | −0.157b | −0.002 | −0.121b | 0.025 |
| C18:1 | −0.382 | 1.419b | 0.436 | 1.551b |
| C18:2 | −1.136b | −0.945a | −1.935b | −1.117b |
| C18:3 | 1.595b | −0.225 | 1.571b | −0.180 |
| C20:1 | −0.016 | 0.019 | −0.013 | 0.007 |
asignificant at 0.01 level; bsignificant at 0.001 level
Dominance effects assessed for the presence of the mutated alleles were significant for palmitic, oleic and linoleic acid content (Table 2). The absolute values of the dominance effects were larger than the absolute values of the additive effects for palmitic and oleic acids content. However, it was not the case for stearic, linoleic and linolenic acids content.
Additive effects are fixed in the population as the number of homozygotes increases in successive generations (Bocianowski and Krajewski 2009) and insignificant dominance effects at early generations indicate high selectivity of the markers, which proved very useful for monitoring wild-type and mutant alleles of FAD3 desaturase. The markers will be further applied in oilseed rape breeding programs concerning diversification of seed oil fatty acid composition.
Acknowledgments
Open Access
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