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Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents

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Abstract

Modification of oleic acid (C18:1) and linolenic acid (C18:3) contents in seeds is one of the major goals for quality breeding after removal of erucic acid in oilseed rape (Brassica napus). The fatty acid desaturase genes FAD2 and FAD3 have been shown as the major genes for the control of C18:1 and C18:3 contents. However, the genome structure and locus distributions of the two gene families in amphidiploid B. napus are still not completely understood to date. In the present study, all copies of FAD2 and FAD3 genes in the A- and C-genome of B. napus and its two diploid progenitor species, Brassica rapa and Brassica oleracea, were identified through bioinformatic analysis and extensive molecular cloning. Two FAD2 genes exist in B. rapa and B. oleracea, and four copies of FAD2 genes exist in B. napus. Three and six copies of FAD3 genes were identified in diploid species and amphidiploid species, respectively. The genetic control of high C18:1 and low C18:3 contents in a double haploid population was investigated through mapping of the quantitative trait loci (QTL) for the traits and the molecular cloning of the underlying genes. One major QTL of BnaA.FAD2.a located on A5 chromosome was responsible for the high C18:1 content. A deleted mutation in the BnaA.FAD2.a locus was uncovered, which represented a previously unidentified allele for the high oleic variation in B. napus species. Two major QTLs on A4 and C4 chromosomes were found to be responsible for the low C18:3 content in the DH population as well as in SW Hickory. Furthermore, several single base pair changes in BnaA.FAD3.b and BnaC.FAD3.b were identified to cause the phenotype of low C18:3 content. Based on the results of genetic mapping and identified sequences, allele-specific markers were developed for FAD2 and FAD3 genes. Particularly, single-nucleotide amplified polymorphisms markers for FAD3 alleles were demonstrated to be a reliable type of SNP markers for unambiguous identification of genotypes with different content of C18:3 in amphidiploid B. napus.

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Acknowledgments

The authors thank Mr. Bo Gertsson, Lantmännen SW Seed, for providing the seeds of SW Hickory. We are grateful to Miss Christy Zhou at Duke University for critical reading of the manuscript. This research was financially supported by China National High-tech R&D Program (2006AA101A113) and the earmarked fund for Modern Agro-industry Technology Research System (nycytx-00503) to YZ.

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  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China

    Qingyong Yang, Chuchuan Fan, Zhenhua Guo, Jie Qin, Jianzhong Wu, Qingyuan Li, Tingdong Fu & Yongming Zhou

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Communicated by R. Visser.

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Supplementary material 1 (DOC 37 kb)

122_2012_1863_MOESM2_ESM.doc

Supplementary Fig. S1 Alignment of the genomic nucleotide sequences of FAD2 genes from Arabidopsis (AT3G12120.1), B. rapa, B. oleracea and B. napus. All gene nomenclature followed the rules proposed by Østergaard and King (2008), identical nucleotides among all the sequences are indicated with asterisks (DOC 44 kb)

122_2012_1863_MOESM3_ESM.doc

Supplementary Fig. S2 Alignment of putative amino acids sequences of FAD2 genes from Arabidopsis (AT3G12120.1), B. rapa, B. oleracea and B. napus as presented in Fig. S1 (DOC 173 kb)

122_2012_1863_MOESM4_ESM.doc

Supplementary Fig. S3 Alignment of the genomic nucleotide sequences of FAD3 genes from Arabidopsis (AT2G29980.1), B. rapa, B. oleracea and B. napus. All gene nomenclature followed the rules proposed by Østergaard and King (2008). The putative CDS or cDNA sequences are shown in capital letters; the introns are in lowercase letters, identical nucleotides among all the sequences are indicated with asterisks. ←: The copy-specific primers binding sites, ▲: The SNP sites between BnaA.FAD3.b and BnaC.FAD3.b, ▽: Additional mismatch base sites in the copy-specific primers (DOC 191 kb)

122_2012_1863_MOESM5_ESM.doc

Supplementary Fig. S4 Diagrammatic presentation of the gene structure of FAD3 identified in Brassica napus. Black boxes and solid lines represent exon and intron, respectively. Arrow heads mark the positions for designing primers used in cloning the FAD3 genomic DNA in B. napus. The identity for each primer is supplied above the arrowhead and the primer sequences are provided in Supplementary Table S1 (DOC 214 kb)

122_2012_1863_MOESM6_ESM.doc

Supplementary Fig. S5 Alignment of putative amino acids sequences of FAD3 genes from Arabidopsis (AT2G29980.1), B. rapa, B. oleracea and B. napus (DOC 98 kb)

122_2012_1863_MOESM7_ESM.doc

Supplementary Fig. S6 The genetic linkage map and QTLs for C18:1, C18:2 and C18:3 detected with SSR markers and allele-specific markers in the SJ DH population in three years. The bar to the left of the LG indicates the 1-LOD confidence interval for the QTL and the triangle indicates the QTL peak position. (DOC 545 kb)

Supplementary Table S1 (DOC 44 kb)

Supplementary Table S2 (DOC 60 kb)

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Yang, Q., Fan, C., Guo, Z. et al. Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents. Theor Appl Genet 125, 715–729 (2012). https://doi.org/10.1007/s00122-012-1863-1

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