SSR primers - Genetic variation and inheritance of phytosterol and oil content in winter oilsee

SSR primer sequences

SSR primer Forward primer (5’ - 3’) Reverse primer (5’→3’) BRAS014 CCCATTGACAACTCTTCTCTT CTGTGTTCGCCCATTATG CB10014 TGATACTAAAGGCAAGTCTA CTGACCATAACCCATCG CB10026 TCGTTCTGACCTGTCGTTAT GGAAATGGCTGCTCATGTT CB10028 CTGCACATTTGAAATTGGTC AAATCAACGCTTACCCACT CB10036 ATTCATCTCCTGCTCGCTTAG AAACCCAAACCAAAGTAAGAA CB10080 GCCCTCAACCTGTAAAGT TTGTTGGTGTGTGAATCATA CB10097 ACTTCGGTGGTTCTATTTCT CGACGGTTAATCAAGTTTCT CB10099 CTTCCCCTTTCATCGAACT TAGAAGCATTTGGAAACGCA CB10103 GACGGATGCCTAATAATGAT TCCTCAAAACTGCCTGTAAG CB10109 GTGTAGCCAGCTTGATCCT CTTCTTCTGATGCAGCAGTG CB10208 ACTACTGTTGCGGTTGGA GGCATTCATTACGTCTGC CB10415 GAACTCGTCGCGGTAGTA TCTCTTTCCTCGCAGATG CB10425 GGTGGCTTGTAGGGACTT GCTCCCGTAAACTCTTCC

CB10431 GGGTTTACTGGGTTCGTT GCAGAAGGGGAAACACTT

CB10493 TGACGTGTGAGCAACAGA CTGAGTCACAAGCCGAGT

CB10534 AGCTGCAACCACAACTCT GGAGCGCAAGAAAAG

CB10545 CTCGCAATAGTCGCAGAT TGCCCTACTGTCTCCTCA CB10572 AGTCACACAATGGCGTCT TTACGGTCTGAGCCTTGA MD38 AGCCTTACATATTCACCTACC TTAAGCTCCAACGTGTTC MR155 AGGCACTATATCAGACAACTG GACATATGCGATGACACTT

Markers associated with oil content in SGDH14×Express617 (Nina Behnke, personal communication).

No. Marker name

KASP Illumina Infinium Brassica 60K SNP 1 BNKS00092 Bn-A03-p14306440

Markers that were physically closely linked to candidate genes for phytosterol biosynthesis.

No. Marker name Candidate gene Gene ID

KASP Illumina Infinium Brassica 60K SNP

1 BNKS003008 Bn-A07-p21078249 HMG1 Bra015739

A.3 Candidate genes

List of candidate genes involved in phytosterol biosynthesis. BLAST search was performed withA. thaliana gene sequence againstB. rapav1.5 genome database (BRAD) andB. oleraceav1.0 genome database (Bolbase)

Gene annotation Species Gene ID Chr.

Sequence length E value

HMG1 A. thaliana AT1G76490 1 3034

3-hydroxy-3-methylglutaryl CoA reductase 1

B. rapa Bra008261 A02 2573 7e-22

Bra015739 A07 1945 2e-87

B. oleracea Bol039236 C02 998 e-129

Bol027626 C06 2730 6e-85

HMG2 A. thaliana AT2G17370 2 2427

3-hydroxy-3- methylglutaryl-CoA reductase 2

B. rapa Bra002053 A07 1910 2e-87

B. oleracea Bol017681 C07 1916 8e-93

HMGS A. thaliana AT4G11820 4 3566

3-hydroxy-3- methylglutaryl-CoA synthase

B. rapa Bra033126 A02 2835 1e-76

Bra014870 A07 2885 1e-70

B. oleracea Bol045227 C06 2849 2e-79

Bol008312 UNC^a 2820 9e-69

SMT1 A. thaliana AT5G13710 5 3364

Sterol

C24-methyltransferase 1

B. rapa Bra023430 A02 6937 e-156

Bra006211 A03 3102 e-136

B. oleracea Bol034248 C03 2746 e-135

Bol004256 UNC 2652 6e-48

SMT2 A. thaliana AT1G20330 1 1472

Sterol

C24-methyltransferase 2

B. rapa Bra205810 A06 1071 0

B. oleracea Bol026945 C05 1071 0

aUNC Unknown chromosome number

Alignment ofHMG1gene sequence fromA. thalianawith homologous copies fromB. rapaandB. oleracea.Yellow barindicates coding region whileline between bars indicates non-coding region.Red arrowindicates the locus-specific primer pair.Blue arrowindicates the allele-specific primer pair.

Additional 978 bp at the untranslated region was retrieved for locus Bol017626 fromB. oleraceagenome sequence.

Alignment ofHMG2gene sequence fromA. thalianawith homologous copies fromB. rapaandB. oleracea.Yellow barindicates coding region whileline between bars indicates non-coding region.Red arrowindicates the locus-specific primer pair.Blue arrowindicates the allele-specific primer pair.

interspecific SNP.

Alignment ofSMT1gene sequence fromA. thalianawith homologous copies fromB. rapaandB. oleracea.Yellow barindicates coding region whilelinebetween bars indicates non-coding region.Red arrowindicates the locus-specific primer pair.

region to search for more interspecific SNP.

A.5 Locus-specific markers

Locus-specific markers for phytosterol candidate genes

Chr. Gene ID Primer name Primer sequence (5’→3’)

A02 Bra008261 HMG1A02-2 Fwd CCTTCCTACTCCCTCGAGTCC 61 803 0

Rev TTCTCTAAACACGAAAACCCAAAAA

HMG1A02-4 Fwd CCTTTGTTGCTTGACGGGTAT 62 797 0

Rev CAGCCTCGCGAATCTACTTGA

A07 Bra015739 HMG1A07-4 Fwd GCATCGATTTCGTCCAGTCT 61 674 6

Rev GCCTCGCAAATCTACTCGAC

C06 Bol027626 HMG1C06-4 Fwd CTGTTGTTTTCAACAGGTCTGG 60 672 0

Rev TATGGATATCTTTCCCGTCGTT

C02 Bol039236 HMG1C02-1 Fwd GACTTCGTCCAATCGTTCATCTT 60 410 0

Rev AGCATTGCCCCAATATGGAT

HMG2

A07 Bra002053 HMG2A10-1 Fwd GACGCCTCTTCACGTCGTC 62 697 NA^a

Rev CAACGGGAGCTCTAGTCATGG

HMG2A10-2 Fwd TGGGACAGTGCTGCGAGA 63 799 NA^a

Rev CCACCAAGAGAACCAGCCATA

C07 Bol017681 HMG2C07-1 Fwd GACGCCTCTTCACGTCGTT 63 566 0

Rev GGAGCTCTAGTCATCGCATCC

HMG2C07-2 Fwd CAGAGGATGCAAGGCTATTCACTTA 64 557 0

Continued on next page

Chr. Gene ID Primer name Primer sequence (5’→3’)

HMG2C07-3 Fwd CAACGGACATGACAACTTGC 60 676 0

Rev CGAGGAGGAGCAGTGACAAA

HMGS

A07 Bra014870 HMGSA07-1 Fwd GCAGACTACTTTGTATTCCATTCTCC 64 544 0

Rev TGAGAACATAACCACCCTCTTCTCT

UNC Bol008312 HMGSUNC-3 Fwd AGTTCACTCCTTATTCGTCTTTGT 65 770 0

Rev CCTCATGTCTAGCCTTCAGTTTACCA

HMGSUNC-5 Fwd GCACAAATCAAATCGGTTCA 56 674 0

Rev CCAATATCCCGACGTTCTT

SMT1

A03 Bra006211 SMT1A03-2 Fwd AAGTCTGGAGCTATGGATCTCG 61 712 0

Rev AGCTCATTCAAAGGGTGAGAAG

UNC Bol004256 SMT1UNC-1 Fwd CTAAAGCCCGGACAATGTTTC 60 775 0

Rev CGGGCCAAGAAGAAATACAT

C03 Bol034248 SMT1C03-1 Fwd AATTAGCATCCCTGTTTTGGTC 61 752 0

Rev GAAGTCAGCCTACAAATTCATCAC

SMT2

A06 Bra205810 SMT2A06-1 Fwd TTCCACTTCTCTCCTTCCATCC 64 657 0

Rev CTTAGGCGCAACTCCAACG

SMT2A06-2 Fwd GACAGTGTCGCACTCTTCTTCACT 64 668 0

Rev GGTGACCCACTCGTACGATACT

C05 Bol026945 SMT2C05-1 Fwd CCAGATGCCCTTCGATGATAAT 64 448 0

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Appendix A.5:continued from previous page

Chr. Gene ID Primer name Primer sequence (5’→3’)

T_m (^◦C)

Sequenced fragment

size (bp) SNP

Rev ATCAGCAGTCTTAAACAACATCTCG

SMT2C05-2 Fwd GGCGTAGCATTCTTCTTCACT 63 650 0

Rev GATACGTACAAAGATCCGGGTTTC

SMT2C05-3 Fwd CATTGGAGGTCGTTGATCATTT 58 565 0

Rev AATGAGAAGAAGCCTGATTTGG

a No amplification was observed from Oase. Only fragment amplified from Sansibar was sequenced.

Allele-specific markers forHMG1andHMG2.

Chr. Gene ID Primer name Primer sequence (5’→3’)

(^◦C)

Fragment size (bp) A07 Bra015739 HMG1A07-O1 Fwd CAGAGGGTGCAAGGCTATGTA^a 62 618

Rev CAAAGAAGCCACGCTCGTC^b

A07 Bra002053 HMG2A10-2^c Fwd TGGGACAGTGCTGCGAGA 63 799 Rev CCACCAAGAGAACCAGCCATA

aDesigned based on intragenomic SNP at the 3’ end. See Appendix A.6

bDesigned based on intergenomic SNP at the 3’ end.

cHMG2A10-2 was designed based on intergenomic SNP but was shown to be also allele-specific

Allele-specific marker fordgat1.

Primer name Primer sequence (5’→3’)

(^◦C)

D120E-3 Fwd CTGCCTTTACCGTCGAGAAAC 60

Rev AGATCAAGCGGGCAAAAATGG

Dx-3 Fwd GTCTTCAGCTAATAGCATCAAACATTC 60

Rev GTGGAGGGTCAAAGCTAAATTTC

Alignment of the partial gene sequence ofhmg1fromB. rapa(Chiifu) with fragments amplified from Sansibar and Oase by locus-specific marker HMG1A07-4.Yellow barsindicate coding region.Brown barindicates the allele-specific primer designed based on intragenomic SNP at the 3’ end.Dotsindicates consensus nucleotides.

to phenylalanine (F) in Sansibar

A.7 Sequencing results

TTAGGGACTAAAGAGGTTATTTTGTGGTTTGCAGGTCGA

HMG2C07-1

AAAACATACAACTAATTATTAAATTATTTAGGAAAATAAAAAGAGAAAAATCCACCTAATATCTCTCCTGGATTTGTTATT

CATAAGACTTGCACCTAAAGGAAGCCAAAGGGTGTCAGATTTTCTGGAGAAGGCCGCAGAAGGATTAGTCGACGGTGGAAG

CAGTTGCTGCCACGAGCTTTCTTGTTGTTCCAATGTCGTACCTCATTTTATTTTGAGTACATTTCCATTTCCAAATATCAA ATGTTCTTTCAATGACACATCGTAGTGGAGAATGACAACGATTAAATTTTACAAATTACTCTGGATATAAATTTTTTGTTA AGTCTTGATCGCTTTAGATTTAAATTTAGATTTAGTGTTGTGAATATAAATGTTGTTGTGTAGAGTTCCCACAGCCGTTAC TAATCATTTTCATAGAGATTCTTTCGAAAATGCTCTGAACAGTACATATTAGAGGTTTGTTTCTTTAGATTAATTACTAAA CTGTAATTACACTGAATGATCTTTTGTTTCTTTAGATTAATTACTAGCAGTAAATTTTCATTTGACTAAGCGTTTTTTTTT TCTTCTTGGATATATACTTGTTCACTGAAAAAAAAAATCTTTTTAAATTGTTCAAGTCAAAGGCCAAATCAGGCTTCTT

A.8 Linkage map of SODH population

Linkage map of SODH population. The full map comprises of high fidelity markers (highlighted in grey), framework markers (indicated in bold and highlighted in grey), and placed markers (indicated in normal font). High fidelity markers are a subset of markers in which all marker orders are supported by a minimal log-likelihood difference of 3.0 with a maximal distance to the previous marker of 30 cM. Framework markers are selected from high-fidelity markers for QTL mapping. Placed markers are markers placed individually relative to the high-fidelity markers at their most likely position which are supported at a LOD score of less than three.

marker marker