Supplementary information

Molecular Breeding

Isolation and characterization of the omega-6 fatty acid desaturase (FAD2) gene family in the allohexaploid oil seed crop CrambeabyssinicaHochst

Jihua Cheng1,2, E.M.J. Salentijn1, Bangquan Huang2, F.A. Krens1, A.C. Dechesne1, R.G.F. Visser1 and E.N. van Loo1*

1 Wageningen UR Plant Breeding, P.O. Box 16, 6700 AA Wageningen, The Netherlands

2 College of Life Science, Hubei University, P. R. China

* To whom correspondence should be addressed. E-mail: Robert.vanloo@wur.nl

 

Table S1. Primers list in this study

Category Primers combination Primers sequences (5’-3’) Size (bp) RemarkFAD2 degenerated primers FAD2-Fw1; FAD2-Rv1 AA(A/C)GC(A/T)(G/C)AATCCCT(A/C)GCTCTTT; C(A/G)TACCACGGTGTTCCATCFAD2 probe FAD2-probe-Fw; FAD2-probe-Rv CGCCACCATTCCAACACTG; CGAAGCAGCGTAACGGTAGA 310Genome walking Adaptor ACTCGATTCTCAACCCGAAAGTATAGATCCCA

Adpr1; Rv-GSP1 GTTTACTCGATTCTCAACCCGAAAG; AGGAGAAGTAAGGGACGAGGAGGAA Enzyme:EcoR V; Adpr2; Rv-GSP2 CAACCCGAAAGTATAGATCCCA; AGTAAGAGAGAGGGTGAGGGAGGAG Enzyme: EcoR V; Adpr1; Rv2-GSP1 GTTTACTCGATTCTCAACCCGAAAG; GACCATAACAAACGGCGAGGATACCAG Enzyme: Dra I; EcoR VAdpr2; Rv2-GSP2 CAACCCGAAAGTATAGATCCCA; GTAGATGGGAGCGTTAGGGTGGAAATG Enzyme: EcoR VAdpr2; FAD2A-Rv-GSP2 CAACCCGAAAGTATAGATCCCA; CGTGGTGGCCACATTCATGCAGAGG Enzyme: Dra IAdpr1; Fw-GSP1 GTTTACTCGATTCTCAACCCGAAAG; CTACGATTCATCTGAGTGGGATTGGTT Enzyme: Dra I; EcoR V; Ssp IAdpr2; Fw-GSP2 CAACCCGAAAGTATAGATCCCA; TGGCTACTGTTGACAGAGACTATGG Enzyme: Dra I; EcoR V; Ssp I

3’-RACE 3’Primer; Fw2-GSP1 GCTGTCAACGATACGCTACGTAACG; TTCCGCTTCTGATAGTCAACGGGTT3’Nested Primer; Fw-GSP2_C1-2 CGCTACGTAACGGCATGACAGTG; CACGCATCCTTCATTGCCTCACTAC 7003’Nested Primer; Fw2-GSP2_C3 CGCTACGTAACGGCATGACAGTG; TTCATCTGAGTGGGATTGGTTAACG 700

Flanking primers upFAD2-A; dnFAD2-A GTCTCCACCTTTTGACTCTT; ATAACTCTGTTCTTTCACCATC 1254upFAD2-B; dnFAD2-B TCATATCCACATGCCCCTAT; ACCAGTGGCTTTGACGA 1572upFAD2-C1; dnFAD2-C1 GCTATCGTTTATTTATTTTTCTTTC; AGCGATGAGAAGAACAATACAGAGA 1572upFAD2-C2; dnFAD2-C2 CTCAACGCTATCGTTTATTTCTTTC; AGATAAAAGTAGCTTCACAGG 1660upFAD2-C3; dnFAD2-C3 CCACGTACTATCCATTTTTGAAAGT; GGCACAACACAATGGATACTT 1393

RT-PCR G1F3; G1R4 TCCAACACTGGCTCCCTCGAG; AAGTAAGTGATCAAAACGGGA 383 CaFAD2-A specificG2F3; G2R5 TCCTGCTTCAACTACATCT; TGCGTTATAATGCGGCATT 800 CaFAD2-B specificC1F3; C1R3 CAAGGCTGTGTCCTAACA; AACACTTCGTCTCTTTCAACC 200 CaFAD2-C1 specificC2F3; C2R4 TGCCAAGGCTGTGTCCTAGGT; GCTAAGTACAAAGGCCAGCAA 310 CaFAD2-C2 specificC3F2; C3R2 CAACACTGGATCATTGGAA; GTAATGAGGCAATGAAGGATGA 411 CaFAD2-C3 specific18S F2; 18S R2 CGTGCCCTTTACGCCCAGTCATT; AGGTAGCTTGTCTCGCCCAGGAG 360

Real time PCR qRT-C1F5; qRT-C1R5 CAAGGCTGTGTCCTAAGA; CGTCGTCTCTTTCAAGCGGA 200 CaFAD2-C1 specific, Efficiency: 1.003qRT-C2F2; qRT-CF2 CGCCATTCCAACACTGGCTCC; GCGAAGCCGTCGTAAGGTCT 191 CaFAD2-C2 specific, Efficiency: 1.001qRT-C3F7; qRT-C3F7 CAAGGCTGTGTCCTAAGC; AACACTTCATCTCTTTCCAAT 200 CaFAD2-C3 specific,Efficiency: 0.923Actin2-F1; Actin2-R1 ATTCAGATGCCCAGAAGT; CTCATACGGTCAGCGATA 180 Efficiency: 0.823

454 amplicon 454-AM1-Fw; 454-AM1-Rv CTCCTCCCTCACCCTCTCTC; ACGTGCGTGTCCGTGATGT 724 Amplicon1454-AM2-Fw; 454-AM2-Rv TTCTACTACATCGCCACCAAC; ATTGGTAGTCGCTGAAAGCAT 153 Amplicon2454-AM3-Fw; 454-AM3-Rv TCACCCTCTCTCTTACTTAGCTT; AGACGTTCACGGTCGTTG 437 Amplicon3454-AM4-Fw; 454-AM4-Rv AGAAGAAATCAGACATCAAGTGGTAC; GAAGACCTTGTTCAACATCCCATA 448 Amplicon4454-AM5-Fw; 454-AM5-Rv GCTCAATTCCTCGCTCTTTC; GTCCAAGAGGGTTGTTGAGG 392 Amplicon5

Table S2. Deep 454-amplicon sequencing of the FAD2 gene family from C. abyssinica cv. ‘Galactica’. Five different amplicons were analysed for genetic variants of the FAD2 gene.

Amplicon1 Amplicon2 Amplicon3 Amplicon4 Amplicon5

Gene % % % % %

CaFAD2-A 20% / 15% 1% 15%

CaFAD2-B 25% 1% 16% 0% 1%

CaFAD2-C1 20% 30% 22% 57% 0%

CaFAD2-C2 22% 32% 23% 40% 40%

CaFAD2-C3 13% 27% 19% 1% 33%

CaFAD2-D / 11% / / /

CaFAD2-E/F / / 5% / 11%

Nseq total 43267 12883 15395 13562 5577 90684

Genotypes > 50 lines 5 lines 5 lines 5 lines 5 lines

Per line 865 2577 3079 2712 1115

Nseq total= total number of the sequence. %= frequency of a FAD2 variant in FAD2-gene family among the sequences of an amplicon. The symbol “/”=no sequences matched to certain FAD2 variant.

Figure S1.The nucleotide alignment of full length CrambeFAD2 (CaFAD2-A, -B, -C1, -C2 and -C3). The dots indicate identical nucleotides, and the deletions are highlightedinred. The sizes of CaFAD2-A and -Bare seven basepairs and one basepair shorter than the others (1152 bp) respectively. CaFAD2-C1,-C2 and -C3 share 95%-97% identity to each other.CaFAD2-A and -B share 90% identity to each other and 88%-90% to the others.

Figure S2.Deduced amino acids alignment of the FAD2 genes in C. abyssinica cv. ‘Galactica’.CaFAD2-A and -B encode for truncated FAD2 proteins because of harbouring internal stop codons (indicated by *). CaFAD2-C1, -C2 and -C3 encode for functional FAD2 proteins which share 98% to 99% identity. CaFAD2-C1 differsfromCaFAD2-C2 in four residues (highlighted inblack colour), and five residues (highlighted inred colour) are specific to CaFAD2-C3. The three conserved histidine-rich domains are highlighted in blue colour.

Figure S3.The nucleotide alignments of partial intron sequences (A) and 3’-UTR sequences (B) of crambeFAD2. The red arrow indicates an intron splice site (AG)

A

B

Figure S4. Schematic diagram on the locations of five amplicons in crambeFAD2 gene. Amplicon1 (226-950) = amplicon-1 starts from the position 226 to position 950.

Figure S5.The nucleotide alignment of seven FAD2 genes in C. abyssinica.CaFAD2-E and CaFAD2-F are considered to be one gene CaFAD2-E/F (see results). The symbols of “-” indicate unavailable sequences.

FigureS6.The amino acids alignment of FAD2 family in C. abyssinica. The amino acid sequences were deduced from DNA sequences of eight FAD2 variants. CaFAD2-A, -B, -D, -E and -F harbour internal stop codons (indicated by *) and encode for truncated FAD2 proteins. The amino acids shown in grey blocks are polymorphic residues among CaFAD2-C1, -C2 and -C3.

Figure S7.Genetic variation of the FAD2 family in C. abyssinica. An amplicon (142 bp) covering all CaFAD2 variants was used to test bootstrap of phylogeny with Neighbour-Joining method (MEGA4, USA).

FigureS8.Phylogenetic analysis of deduced amino acids of FAD2 in plants. A phylogenetic tree was constructed with Neighbour-Joining method (MEGA4). Accession numbers of different FAD2 in the analysis: Arabidopsis thaliana (AtFAD2, L26296.1); Arachishypogaea (AhFAD2, FJ768732.1); Brassica carinata (BcFAD2, AF124360.2); Brassica juncea (BjFAD2, gi|1212780); Brassica napus (BnFAD2, AF243045.1); Brassica rapa (BrFAD2, gi|20520623); Camelinasativa (CsFAD2-1, HQ008320.1; CsFAD2-2, HQ008321.1; CsFAD2-3, HQ008322.1); Crambeabyssinica (CaFAD2-C1, CaFAD2-C2 and CaFAD2-C3in this study); Gossypiumhirsutum (GhFAD2-1, gi|2578032; GhFAD2-2, gi|4490390; GhFAD2-3, AF331163.1); Glycine max (GmFAD2-1A, L43920.1; GmFAD2-1B, gi|59956943; GmFAD2-2A, gi|59956945; GmFAD2-4, gi|351725574); Sesamumindicum (SiFAD2, AF192486.1); Zea mays (ZmFAD2, EU965315.1)

FigureS9.Expression profile of CaFAD2 genes in vegetative and reproductive tissues of C. abyssinica cv. ‘Galactica’.The expression of CaFAD2-A, -B, -C1 to -C3 was measured using RT-PCR in different tissues.SL=seedling, LF=leaf, BD=flower bud, FL=flower, RT=root, S1=7 DAP seeds, S2=14 DAP seed, S3=21 DAP seed, S4= 28 DAP seed. DAP=Days after pollination. Lane A to C3 are positive controls with plasmid clones of the different variants; G=Genomic DNA from C. abyssinica used as a template for PCR with the set of 5 primer combinations for the detection of five FAD2 genes. RT-PCR-product of 18S ribosomal RNA (18S rRNA) was used as a reference for the amountof cDNA (reference).