Appendix A1 SNP genotyping using iPLEXTM Gold reaction

iPLEXTM Gold reaction based on MALDI-TOF MS developed by SEQUENOM was used as per the iPLEXTM Gold Application Guide for scoring polymorphisms. SEQUENOM’s Assay Design Software was used to design amplification primers for multiplex reactions. This software checks for possible dimer formation. A generic 10-mer tag (ACGTTGGATG) was added to the 5’ end of each amplification primer. Primers were designed to produce 80 to 120 bpamplicons (Table 1). Extension primers were designed with 3’ends immediately adjacent to the targeted polymorphic site.

Eleven to fourteen plex reactions were designed. Polymerase Chain Reaction (PCR) was performed in 5 μL reactions. Each reaction contained 1 μL of appropriate genomic DNA (5- 10 ng/μL), 1.25 x PCR buffer (15mM MgCl2), 1.625 mM MgCl2, 500 μM dNTP mix, 100 nM primer mix and 0.5 U Taq. Thermocycling conditions were: an initial denaturation step at 94°C for 15 minutes, 45 amplification cycles at 94° C for 20 seconds, 56° C for 30 seconds and 72°C for one minute and a final extension step at 72°C for 3 minutes.

Post PCR, reactions were treated with SAP (Shrimp Alkaline Phosphatase) to neutralise unincorporated dNTPs. Two microlitres of SAP mix containing 0.5 units of SAP and 1x SAP buffer was added to the PCR product. This mix was then incubated at 370 C for 40 minutes before denaturation of SAP via incubation at 850 C for five minutes.

To the SAP treated PCR product, 9 μL of iPLEX Gold reaction master mix was added. This mix contained 0.222x iPLEX buffer plus, 0.5x iPLEX terminator mix, 0.625 μM primer mix and 0.5x iPLEX enzyme. This mix was thermocycled as follows: an initial denaturation step at 94°C for 30 sec followed by an annealing step at 52º C for five seconds and extension step at 80º C for five seconds. The annealing and extension cycle was repeated four more times for a total of five cycles and then looped back to a 94º C denaturing step for five seconds before entering the five cycle annealing and extension loop again. The five annealing and extension steps with the single denaturing step were repeated an additional 39 times (a total of 40 times). A final extension followed at 72º C for three minutes.

iPLEX Gold reaction products were cleaned using resin. The cleaned iPLEX Gold reaction products were nanodispensed onto a spectroCHIP. Spectra were then acquired using the MassARRAYAnalyzer Compact mass spectrometer.

SNP / Forward primer / Reverse primer / Sequencing primer
4CL_1787 / ACGTTGGATGTCTGAAGATTGCTCGAGCAC / ACGTTGGATGACCCGATGCGGAAATTAGAG / GAAATTAGAGGATCGTCTTATTCA
4CL_1920 / ACGTTGGATGCAGTGTCAACCCATGAGTAG / ACGTTGGATGAGTTTTCAGCCGCTACTATG / tTTTTCAGCCGCTACTATGTGTCCT
4CL_1955 / ACGTTGGATGTGACAGGGGAAAACAAAAGG / ACGTTGGATGTAGTAGCGGCTGAAAACTCC / GGCTGAAAACTCCGGCTAC
4CL_2121 / ACGTTGGATGGCTTGACTTGTAACAAATGGG / ACGTTGGATGAAGGAAAAGGACTTTTCAGG / GACTTTTCAGGCCCCTC
4CL_2219 / ACGTTGGATGCCGGCAATTTCTAAATCCGC / ACGTTGGATGCTAGAAGCCAAGCCACAAAG / CAAAGAAAGATGGTCCAAG
4CL_222 / ACGTTGGATGCTCACGCGAAATTGAAGAAG / ACGTTGGATGTGGGTATGCGATTGAAATCC / ATTGAAATCCTTCGAGATCGC
4CL_2232 / ACGTTGGATGGCCACAAAGAAAGATGGTCC / ACGTTGGATGCCGGCAATTTCTAAATCCGC / aTGCGATAAACAAAACATTAAATAA
4CL_294 / ACGTTGGATGTCGCGTGAGGTAGATTTTTC / ACGTTGGATGAACACGGTCAAACACGCTTG / TCCCTCCTGTCACGACATTTCC
4CL_3817 / ACGTTGGATGCAACATCTAATGCACCCTCC / ACGTTGGATGTCTTGAATTATTGTGGACCG / gggTTGTGGACCGTCGAAAC
4CL_4464 / ACGTTGGATGGGGCAGGGTTGAAAAAGAAA / ACGTTGGATGTTGAGGAAGGACCTAAGAGC / GTTGGCCTCCGGTGTTT
4CL_485 / ACGTTGGATGTTCCTCCTTCGTTAGGTTGG / ACGTTGGATGAGATGGAAAAGAGGCATGCG / ATGCGGATCAAACTATTG
4CL_713 / ACGTTGGATGAAGGGACAGAAAAGGCCATC / ACGTTGGATGACCCGAGAGTGATATCAAGG / AGGAGGGGAAAGAAAATTAA
AQP1_2977 / ACGTTGGATGTAGAGAAGCTAACTGTACCG / ACGTTGGATGCATCCCTTTCATAAACCTGC / gACCTGCTATTTGTTTTACATTTAT
AQP1_3084 / ACGTTGGATGAGGGCGACAGAAAACAATAG / ACGTTGGATGAGATCCTCCTGTTCTAACCC / TAACCCAAATGTTAGCAGA
AQP1_3111 / ACGTTGGATGAGGGCGACAGAAAACAATAG / ACGTTGGATGAGATCCTCCTGTTCTAACCC / GCCTTCTCTTCCGTGTAATCT
AQP1_3146 / ACGTTGGATGCCCAAGTGTAATCTAACAGC / ACGTTGGATGGCCTTCTCTTCCGTGTAATC / TTGTTTTCTGTCGCCCTAA
CADp_183 / ACGTTGGATGAGGTATTGCTTGCTGAGGTG / ACGTTGGATGGAAACGAGACGGACCAATAG / gAGGAAGAATCCAAAAGAGAGAC
CADp_282 / ACGTTGGATGGGTTGCTGGTAGCGTTTTTG / ACGTTGGATGGTAGGAGCATAGATGAGGAG / AGGAGTGACGGGGATACATATGGA
CCoAOMT1_834 / ACGTTGGATGGACACAACACCTCAAGTTGG / ACGTTGGATGGCTTTTGTTGGAATCGATGG / GGAATCGATGGATGATTCT
CCoAOMT2_1669 / ACGTTGGATGTGTTGTCGTATCCGATCAGG / ACGTTGGATGCCGACAAGGACAACTACATC / GACAACTACATCAACTACCACA
CCoAOMT2_1722 / ACGTTGGATGATCGACCTGGTCAAGGTTGG / ACGTTGGATGGTAGTACCGGACGTACTTGC / tCCACGGAGCCGTTCCACA
CCoAOMT2_258 / ACGTTGGATGCTGTTATTTCCCTGAGCTCC / ACGTTGGATGCTTGGGATTTGCCTGTTTGC / aagGCGTCTACCCAAGAGAGCCAGAG
CCoAOMT2_920 / ACGTTGGATGTGGCCATGGACATCAATAGG / ACGTTGGATGAATCGATCTTGTGGGCAAGG / TCTTGTGGGCAAGGCCGGCCTTCT
COBL4_1391 / ACGTTGGATGTACTGCAGGTTGTAAGGGAC / ACGTTGGATGACTGCTCCAAGTTCAAAGCC / CAAAGCCGCTATACCTCAT
COBL4_1925 / ACGTTGGATGGTCACATTCCATGTCACTGC / ACGTTGGATGCCGAAGAACCATGGTGTAAC / GTGTAACACTGAAACATGTGGTTG
COBL4_1995 / ACGTTGGATGCACAGCAGCTGGGATTTTTC / ACGTTGGATGCAGTGACATGGAATGTGACC / ATGTGACCTGCACTTATTCCCA
COBL4_2214 / ACGTTGGATGACTCCTGCATCACCAATAGC / ACGTTGGATGGAGCGCTTATGTGCTCAATG / CACCCGAAGCCTGTTTAC
COBL4_2233 / ACGTTGGATGGATAAATCACCCGAAGCCTG / ACGTTGGATGTTTGGAGTCACTCCTGCATC / TGCATCACCAATAGCCG
COBL4_2655 / ACGTTGGATGTTCCATACGCAGATGACACG / ACGTTGGATGACTGAACATTCCCAAGTGGC / ATTCCCAAGTGGCCCCGCTTCCAT
COBL4_2725 / ACGTTGGATGTTGGGAATGTTCAGTCCGAG / ACGTTGGATGTTCCTTGGAAATGCCCAACC / ATGCCCAACCTTGCTTAA
COBL4_2828 / ACGTTGGATGATGAGAAGGACCCAAGAGAC / ACGTTGGATGGATTCATACCCTTTTCTGCC / CTGCCAAATTCAGCACGTC
COBL4_723 / ACGTTGGATGAATCAATCGCGTCTATATGG / ACGTTGGATGCACCATATATCTTGCGAGTTC / TGCGAGTTCGCTTCAGCAG
COBL4_781 / ACGTTGGATGATCTTGCGAGTTCGCTTCAG / ACGTTGGATGCGAGAAAGACAAATCAATCGC / AAATCAATCGCGTCTATATGG
CSA1_2516 / ACGTTGGATGCGACTTCTTCGTGAGTACAG / ACGTTGGATGAGCAAGGATGGAAAGCACTG / TGATCAAAGGTGGTTCTTTCA
CSA1_913 / ACGTTGGATGTGCCATTGTCGATCGACTTC / ACGTTGGATGGGTTTCCTTTTTATTCAGCG / cCCTTTTTATTCAGCGAACTCAATG
CSA3_1101 / ACGTTGGATGCGGGACAAAGTTTTGCTCAC / ACGTTGGATGTCCTGAGGATGACGATAACG / ctcACGCTCAATTTCCATCAGTTAT
CSA3_1767 / ACGTTGGATGGAATGCAGGATAGATGAGGC / ACGTTGGATGCCGGTATGGATTGATCTTGC / TTGCTCGATGCAATTGG
CSA3_1872 / ACGTTGGATGCGCTACAGGATATTGAACCC / ACGTTGGATGATCCAGGATCCAGGAGAAAG / AACCAAAGACCAAATGC
CSA3_2724 / ACGTTGGATGGAAAATGGGTACCCTTCTGC / ACGTTGGATGGGCTGGACTTTGTCTTTGAG / ggGAAGTACATTTCAGGGGCTCGGGG
CSA3_3734 / ACGTTGGATGTGATTGCTGCCCATGCTTTG / ACGTTGGATGGATCTGCAGCATCCCCATTG / GCACTATGCTTAGAATACTT
CSA3_4186 / ACGTTGGATGCTATAGAGGCACCCACTTAG / ACGTTGGATGGCAACAAGGTAGCAAAGCTC / TAGCAAAGCTCTGGGTATT
KOR_187 / ACGTTGGATGAACGGCTAGTGAAACGTTGC / ACGTTGGATGCCACACCGATTTACCAAGTC / TTACCAAGTCTCCCTCC
KOR_2328 / ACGTTGGATGATCTCGGCAGCAAGATCTGA / ACGTTGGATGGATCATTACTGCTGGATGCG / atACATTGATTATAAACGGCCTGTC
KOR_2493 / ACGTTGGATGCATCCCAATAACTAGTGGAG / ACGTTGGATGCATTTTTCGAGGCAACAGCG / GGAGATACAGTGCTGGT
KOR_2496 / ACGTTGGATGCATCCCAATAACTAGTGGAG / ACGTTGGATGCATTTTTCGAGGCAACAGCG / CGAGGGAGATACAGTGCTGGTTCA
KOR_2863 / ACGTTGGATGATTGAAAGTCTCAACGGCCA / ACGTTGGATGAGGTGTATAATTTGCCACTC / cccGCCACTCTTTCAGATCTAAAAGTA
KOR_4184 / ACGTTGGATGAAACATGGGAGGAACCGCAG / ACGTTGGATGAGGGAATGCAGGACTGGTTG / cccCCTTATCAGGCGACGGGA
KOR_742 / ACGTTGGATGTCTCTCTCACCCACTGTCG / ACGTTGGATGGCTTCAGCTCCCAGCAATAG / gATATCAAGAAAATCAAGAACAGG
KOR_801 / ACGTTGGATGGTACATGCTCATCCTCGCTC / ACGTTGGATGTTGCTGGGAGCTGAAGCAGA / CTGAAGCAGACCCAGCAGTT
KOR_813 / ACGTTGGATGCTGAAGCAGACCCAGCAGTT / ACGTTGGATGGTACATGCTCATCCTCGCTC / CCACAGTGGATGCAGAA
LIM_1106 / ACGTTGGATGTACACCAGGAAATCCTTACC / ACGTTGGATGACGCGAGACAAATGTGTAGG / ATGTGTAGGCTGTAAGAG
LIM_1157 / ACGTTGGATGCTCAATGAAGCAGCCTTTGG / ACGTTGGATGAAGGTAAGGATTTCCTGGTG / TTTCCTGGTGTATTTATAGAAC
LIM_1159 / ACGTTGGATGAAGGTAAGGATTTCCTGGTG / ACGTTGGATGCTCAATGAAGCAGCCTTTGG / GTCATGTCCTCGCATCA
LIM_1343 / ACGTTGGATGAGAGCTGCTTCAAATGCACC / ACGTTGGATGCAGTAGAGTTTCCCCTCGTG / ACGTAGTTGGATGGGCTGATCAC
LIM_444 / ACGTTGGATGGGGCTGATCTTAAGGTTCTC / ACGTTGGATGCTGAATGATATTCGCTTGGG / cTGTAGAAATGTGTGGGGGC
LIM_537 / ACGTTGGATGGCCTTTTGACTGATCATCCG / ACGTTGGATGTAAATCATGCGTGACCCCAG / tATCATGACAATTCATCATAACT
LIM_564 / ACGTTGGATGGCCTTTTGACTGATCATCCG / ACGTTGGATGTAAATCATGCGTGACCCCAG / CGTGACCCCAGCTTCTT
LIM_795 / ACGTTGGATGCAGCTCTTCAAGAGAACTGG / ACGTTGGATGAACAGAGTGCATGACTTGCG / TTTTACCTTCAAAGCTTTTTTC
LIM_901 / ACGTTGGATGTCTGGCTTTGCAATCTTGGG / ACGTTGGATGATGCACTCTGTTTCTGTCCC / TTCCTTCATACAAAATGTCCTCT
LIM_977 / ACGTTGGATGGACTGATTTAGCCTAACTGC / ACGTTGGATGTTGCAAAGCCAGAGAAACCC / cccCCGCGACTATGGTCTTGCCT
LIM_978 / ACGTTGGATGTTGCAAAGCCAGAGAAACCC / ACGTTGGATGGACTGATTTAGCCTAACTGC / cTTTAGCCTAACTGCAAAACTT
LTP_211 / ACGTTGGATGTGTAGATGACCAGGGAAACG / ACGTTGGATGACACTGGCTTATAGCTAGCG / ACTAAACTCATCTCGATCT
LTP_410 / ACGTTGGATGCAGAATCATTACTGCTGTGC / ACGTTGGATGTACAACCCCACATCAGCATC / CACATCAGCATCACCGAAAGCA
LTP_430 / ACGTTGGATGCAGAATCATTACTGCTGTGC / ACGTTGGATGCACATCAGCATCACCGAAAG / aAGTGTTGCGGAAAGTTG
LTP_507 / ACGTTGGATGCTGCACAGCAGTAATGATTC / ACGTTGGATGTGAGGATCAGCATGCTCAAG / ATCAGCATGCTCAAGATGCCCTTT
LTP_676 / ACGTTGGATGAGTGGCAGATCTCATTCCAG / ACGTTGGATGGGGTCAGCGTGATGCATTTG / tgaagTTATAGGAGCCAAATTGGTGA
LTP_730 / ACGTTGGATGAGGAGGATGTGAGGAGAAAG / ACGTTGGATGTGGCTCCTATAAATTCATGC / cACCAAAGCCAAATGCATCAC
LTP_754 / ACGTTGGATGTGGCTCCTATAAATTCATGC / ACGTTGGATGAGGAGGATGTGAGGAGAAAG / TGTGAGGAGAAAGAACTAAGAGTG
LTP_835 / ACGTTGGATGCAAAGCAGAAGCAATGAGGG / ACGTTGGATGATGTCACAGCGGCCTCAAC / GGAGCAGCACCGCCAGAAGA
LTP_925 / ACGTTGGATGTCTGCTCCTTGATCTTGCTG / ACGTTGGATGTGACATGCAGGCCGATAGAG / ATAGAGCTGAGCGCGTGCGTGA
MYB1_474 / ACGTTGGATGAGGTAATTGATCCACCGGAG / ACGTTGGATGTTGCCTACATAAGAGCGCAC / cCCGCGGGCCTCCTCCGCTG
MYB2_1380 / ACGTTGGATGGCACGTCTTCCTGGAAGAAC / ACGTTGGATGATCGCTCTCGTTTGACGAAG / gagCGTTTGACGAAGATGTAG
MYB2_1436 / ACGTTGGATGCCATGCATGTAGCTAAGACG / ACGTTGGATGTCCAATCCACAAGACATAGC / CAAGACATAGCTGCAGG
MYB2_1625 / ACGTTGGATGGTTTTTCACCTTCCCCTCAG / ACGTTGGATGAGTCCCACTTTTGCATGCTC / TAACTTGAATCATCTCCCATACT
NAP1_221 / ACGTTGGATGGAATGAAGACAGCCGTTCTC / ACGTTGGATGAGTTTTGGCCTTGAGCTCTG / aTTGAGCTCTGTGGTAATTGTT
NAP1_286 / ACGTTGGATGATGAACTTGGGGCAGTACAC / ACGTTGGATGAGAGCTCAAGGCCAAAACTC / TCATCACCGTAAGTGATTTTCTCT
NAP1_317 / ACGTTGGATGCAACTGTGTACTGCCCCAAG / ACGTTGGATGAACTGATTTGACCTGGGTCG / gCTTCTTAAGAATGAGAAGAAGATA
NAP1_346 / ACGTTGGATGAACTGATTTGACCTGGGTCG / ACGTTGGATGCTGTGTACTGCCCCAAGTTC / cccgTCTTCTCATTCTTAAGAAGTTAC
PCBR_1601 / ACGTTGGATGCAGATCACCCTGTGCCAATC / ACGTTGGATGATCATTCGGCCGGTAGAGAG / AGAGAGTCAAGAGGATTAACGTGT
PCBR_1651 / ACGTTGGATGTGATTGGCACAGGGTGATCT / ACGTTGGATGACTGTCGATATCACCACGTC / aATCGCCTTCACCAAGCTCTC
PCBR_1714 / ACGTTGGATGGTGGTGATATCGACAGTGGG / ACGTTGGATGACCTTAACGTTGCCAGCTTC / cccTCTTGGTCTGATCCGCCAT
CDPK_516 / ACGTTGGATGGAGATTCTCACAACTTCCCC / ACGTTGGATGTGGGATGCTGGGATTTAGAC / GCTGGGATTTAGACAGTGGGGCT
CDPK_589 / ACGTTGGATGCCCTTTGTAGTAGTAGTTTGG / ACGTTGGATGAGACAGGATAAGATAGTGGC / tGATAAGATAGTGGCCAGTCA
SAMS_744 / ACGTTGGATGCTTGTCAGGAATCTTTCCAG / ACGTTGGATGTCTCGTACGCCATTGGAGTG / GAGCCTCTTTCTGTTTTCGTTGA
SAMS_819 / ACGTTGGATGAAGGAGAGCTTTGACTTCAG / ACGTTGGATGTTCAAGAATCTGCCACTGCC / GATCCAGGTTGATGGAAATCAT
Susy1_1310 / ACGTTGGATGTGGAGGGAGAACACATTCTG / ACGTTGGATGGCGATCTGTTTTTGGCTTATG / CTGCAGAACATGATGGTGAAT
Susy1_1510 / ACGTTGGATGAGATGATCCAGCTCCTGTTG / ACGTTGGATGTGGGAACCCTATCCAAGAAC / CAAGAACTTCTCGAGAGTGCACGG
Susy1_1684 / ACGTTGGATGGCAAATGAACAAGTTGAATGG / ACGTTGGATGGGTGGCCAGGTACTTGTTTT / ggTGATTTCTTGATTAGGAGGATAT
Susy1_1733 / ACGTTGGATGCCATTCAACTTGTTCATTTGC / ACGTTGGATGATTTCTTCCTCTAGGGCACG / CAGGATGTAAACAACCTATAAA
Susy3_1008 / ACGTTGGATGTGAGAGGATTCAGACATGGC / ACGTTGGATGATTACGATGAGCCAAGAGGG / CAAGAGGGTCTCATCCAAACGTTC
Susy3_1641 / ACGTTGGATGGCAAGTGACCGAGTTTCTTC / ACGTTGGATGCGTCCATGTGGTGCTAAAAG / cctccAAAAGAAAGACTTACTTTCCAT
Susy3_4612 / ACGTTGGATGAGGGAAAAAGAGCACAGTTC / ACGTTGGATGATTTGCCATGTATACCTGCC / CACCACATTATACCCACT
Susy3_593 / ACGTTGGATGCCTTAACAGTCACGTTGCAC / ACGTTGGATGGTTTAATACCGTGAGCGAAC / GGTTGCCCTGTTCAGGTCGC
Susy3_64 / ACGTTGGATGCGAACTTTGACCTTGGAGT / ACGTTGGATGACAGTCAGATCAGCCAGAAG / ACGATTACTCCCACAGAGAT
Susy3_886 / ACGTTGGATGCGGTTTTGGTGGGATTTGAC / ACGTTGGATGCTGCAATACAGAAACGACAG / GGATCCAAATAACCCAACGCC
Susy3_909 / ACGTTGGATGCGGTTTTGGTGGGATTTGAC / ACGTTGGATGCTGCAATACAGAAACGACAG / AGAAACGACAGTAGTATAAGAAAC
TUB_1475 / ACGTTGGATGAAGTTCTGGGAGGTTGTGTG / ACGTTGGATGTAGTACACGTTCACTCGCTC / cGTCACCAGTATAGTTCCCCG
TUB_1543 / ACGTTGGATGTGCGCAGGCTGTCCATAGTC / ACGTTGGATGAGCGAGTGAACGTGTACTAC / gGTACTACAATGAGGCGAG