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Structural organization of fatty acid desaturase loci in linseed lines with contrasting linolenic acid contents

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Abstract

Flax (Linum usitatissimum L.), the richest crop source of omega-3 fatty acids (FAs), is a diploid plant with an estimated genome size of ~370 Mb and is well suited for studying genomic organization of agronomically important traits. In this study, 12 bacterial artificial chromosome clones harbouring the six FA desaturase loci sad1, sad2, fad2a, fad2b, fad3a and fad3b from the conventional variety CDC Bethune and the high linolenic acid line M5791 were sequenced, analysed and compared to determine the structural organization of these loci and to gain insights into the genetic mechanisms underlying FA composition in flax. With one gene every 3.2–4.6 kb, the desaturase loci have a higher gene density than the genome’s average of one gene per 7.8–8.2 kb. The gene order and orientation across the two genotypes were generally conserved with the exception of the sad1 locus that was predicted to have additional genes in CDC Bethune. High sequence conservation in both genic and intergenic regions of the sad and fad2b loci contrasted with the significant level of variation of the fad2a and fad3 loci, with SNPs being the most frequently observed mutation type. The fad2a locus had 297 SNPs and 36 indels over ~95 kb contrasting with the fad2b locus that had a mere seven SNPs and four indels in ~110 kb. Annotation of the gene-rich loci revealed other genes of known role in lipid or carbohydrate metabolic/catabolic pathways. The organization of the fad2b locus was particularly complex with seven copies of the fad2b gene in both genotypes. The presence of Gypsy, Copia, MITE, Mutator, hAT and other novel repeat elements at the desaturase loci was similar to that of the whole genome. This structural genomic analysis provided some insights into the genomic organization and composition of the main desaturase loci of linseed and of their complex evolution through both tandem and whole genome duplications.

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Acknowledgments

This work was conducted as part of the Total Utilization Flax GENomics (TUFGEN) project funded by Genome Canada and other stakeholders including the Flax Council of Canada, Manitoba Flax Growers Association and the Province of Manitoba. Project management and support by Genome Prairie are also gratefully acknowledged. We are thankful to Dr. Raju Datla of the National Research Council, Saskatoon, Saskatchewan, Canada, for generating the RNA-Seq dataset for flax and making it available through the TUFGEN project website (http://linum.ca/downloads/RNAseq). We wish to acknowledge the contribution of Dr. Frank M. You for generating the BAM files and co-developing LTRAnnotator, Travis Bank for the 454 assemblies of the BAC clones, Andrzej Walichnowski for manuscript editing and Manoj Sekhwal for initial preparation of GenBank submission files.

Authors’ contribution

Author Dinushika Thambugala conducted this work as part of her PhD thesis. Dinushika Thambugala carried out the analysis, interpreted the data and co-wrote the manuscript. Dr. Raja Ragupathy provided the analysis of the repeat elements. Dr. Sylvie Cloutier designed the experiments, generated the sequence data, participated in the interpretation of the data and co-wrote the manuscript. The authors read and approved the final manuscript.

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Authors and Affiliations

  1. Department of Plant Science, University of Manitoba, 66 Dafoe Rd, Winnipeg, MB, R3T 2N2, Canada

    Dinushika Thambugala, Raja Ragupathy & Sylvie Cloutier

  2. Ottawa Research and Development Centre, 960 Carling Ave, Ottawa, ON, K1A 0C6, Canada

    Sylvie Cloutier

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  1. Dinushika Thambugala
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  2. Raja Ragupathy
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  3. Sylvie Cloutier
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Correspondence to Sylvie Cloutier.

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The authors declared that the work in this manuscript was carried out in accordance with the current laws and regulations in Canada. The work is original except where indicated by special reference in the text and no part of the manuscript has been submitted for publication elsewhere. Any views expressed in the manuscript are those of the authors.

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ESM 1

Primers used to identify BAC clones harboring the desaturase genes and names of the sequenced clones (PDF 9 kb)

ESM 2

Summary of 454 sequencing statistics of the 12 BAC clones from CDC Bethune and M5791 fatty acid desaturase loci sad1, sad2, fad2a, fad2b, fad3a and fad3b (PDF 12 kb)

ESM 3

Annotation of genes located of the 12 BAC sequences harbouring the fatty acid desaturase loci sad1, sad2, fad2a, fad2b, fad3a and fad3b of CDC Bethune and M5791 (PDF 937 kb)

ESM 4

Transposable elements identified in the 12 BACs harbouring sad1, sad2, fad2a, fad2b, fad3a and fad3b loci of CDC Bethune and M5791 (PDF 23 kb)

ESM 5

List of expressed genes identified in the 12 BAC sequences and their level of expression in different tissues of linseed variety CDC Bethune based on normalized RNA-Seq data and expressed as fragments per kb of transcripts per million mapped reads (FPKM) as previously described (Kumar et al. 2013). The RNA-Seq data was provided by Dr. Raju Datla, National Research Council, Saskatoon, SK, Canada and was extracted from the publically available website (http://linum.ca/downloads/RNAseq) (PDF 162 kb)

ESM 6

Dot plots of desaturase loci BAC sequences of CDC Bethune (x-axis) and M5791 (y-axis) performed using JDOTTER (Brodie et al. 2004) with default parameters. a sad1, b sad2, c fad2a, d fad2b, e fad3a and f fad3b (PDF 395 kb)

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Thambugala, D., Ragupathy, R. & Cloutier, S. Structural organization of fatty acid desaturase loci in linseed lines with contrasting linolenic acid contents. Funct Integr Genomics 16, 429–439 (2016). https://doi.org/10.1007/s10142-016-0494-z

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