Appendix A1 SNP genotyping using iPLEXTM Gold reaction iPLEX
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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 bp amplicons (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 37 0 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 MassARRAY Analyzer Compact mass spectrometer. SNP 4CL_1787 4CL_1920 4CL_1955 4CL_2121 4CL_2219 4CL_222 4CL_2232 4CL_294 4CL_3817 4CL_4464 4CL_485 Forward primer ACGTTGGATGTCTGAAGATTGCTCGAG CAC ACGTTGGATGCAGTGTCAACCCATGAG TAG ACGTTGGATGTGACAGGGGAAAACAA AAGG ACGTTGGATGGCTTGACTTGTAACAAA TGGG ACGTTGGATGCCGGCAATTTCTAAATC CGC ACGTTGGATGCTCACGCGAAATTGAAG AAG ACGTTGGATGGCCACAAAGAAAGATGG TCC ACGTTGGATGTCGCGTGAGGTAGATTT TTC ACGTTGGATGCAACATCTAATGCACCC TCC ACGTTGGATGGGGCAGGGTTGAAAAAG AAA ACGTTGGATGTTCCTCCTTCGTTAGGTT Reverse primer ACGTTGGATGACCCGATGCGGAAATTA GAG ACGTTGGATGAGTTTTCAGCCGCTACTA TG ACGTTGGATGTAGTAGCGGCTGAAAAC TCC ACGTTGGATGAAGGAAAAGGACTTTTC AGG ACGTTGGATGCTAGAAGCCAAGCCACA AAG ACGTTGGATGTGGGTATGCGATTGAAA TCC ACGTTGGATGCCGGCAATTTCTAAATC CGC ACGTTGGATGAACACGGTCAAACACGC TTG ACGTTGGATGTCTTGAATTATTGTGGAC CG ACGTTGGATGTTGAGGAAGGACCTAAG AGC ACGTTGGATGAGATGGAAAAGAGGCAT Sequencing primer GAAATTAGAGGATCGTCTTATT CA tTTTTCAGCCGCTACTATGTGTC CT GGCTGAAAACTCCGGCTAC GACTTTTCAGGCCCCTC CAAAGAAAGATGGTCCAAG ATTGAAATCCTTCGAGATCGC aTGCGATAAACAAAACATTAAA TAA TCCCTCCTGTCACGACATTTCC gggTTGTGGACCGTCGAAAC GTTGGCCTCCGGTGTTT ATGCGGATCAAACTATTG 4CL_713 AQP1_2977 AQP1_3084 AQP1_3111 AQP1_3146 CADp_183 CADp_282 CCoAOMT1_83 4 CCoAOMT2_16 69 CCoAOMT2_17 22 CCoAOMT2_25 8 CCoAOMT2_92 0 COBL4_1391 COBL4_1925 COBL4_1995 COBL4_2214 COBL4_2233 GG ACGTTGGATGAAGGGACAGAAAAGGC CATC ACGTTGGATGTAGAGAAGCTAACTGTA CCG ACGTTGGATGAGGGCGACAGAAAACA ATAG ACGTTGGATGAGGGCGACAGAAAACA ATAG ACGTTGGATGCCCAAGTGTAATCTAAC AGC ACGTTGGATGAGGTATTGCTTGCTGAG GTG ACGTTGGATGGGTTGCTGGTAGCGTTTT TG ACGTTGGATGGACACAACACCTCAAGT TGG ACGTTGGATGTGTTGTCGTATCCGATCA GG ACGTTGGATGATCGACCTGGTCAAGGT TGG ACGTTGGATGCTGTTATTTCCCTGAGCT CC ACGTTGGATGTGGCCATGGACATCAAT AGG ACGTTGGATGTACTGCAGGTTGTAAGG GAC ACGTTGGATGGTCACATTCCATGTCACT GC ACGTTGGATGCACAGCAGCTGGGATTT TTC ACGTTGGATGACTCCTGCATCACCAAT AGC ACGTTGGATGGATAAATCACCCGAAGC CTG GCG ACGTTGGATGACCCGAGAGTGATATCA AGG ACGTTGGATGCATCCCTTTCATAAACCT GC ACGTTGGATGAGATCCTCCTGTTCTAAC CC ACGTTGGATGAGATCCTCCTGTTCTAAC CC ACGTTGGATGGCCTTCTCTTCCGTGTAA TC ACGTTGGATGGAAACGAGACGGACCAA TAG ACGTTGGATGGTAGGAGCATAGATGAG GAG ACGTTGGATGGCTTTTGTTGGAATCGAT GG ACGTTGGATGCCGACAAGGACAACTAC ATC ACGTTGGATGGTAGTACCGGACGTACT TGC ACGTTGGATGCTTGGGATTTGCCTGTTT GC ACGTTGGATGAATCGATCTTGTGGGCA AGG ACGTTGGATGACTGCTCCAAGTTCAAA GCC ACGTTGGATGCCGAAGAACCATGGTGT AAC ACGTTGGATGCAGTGACATGGAATGTG ACC ACGTTGGATGGAGCGCTTATGTGCTCA ATG ACGTTGGATGTTTGGAGTCACTCCTGCA TC AGGAGGGGAAAGAAAATTAA gACCTGCTATTTGTTTTACATTT AT TAACCCAAATGTTAGCAGA GCCTTCTCTTCCGTGTAATCT TTGTTTTCTGTCGCCCTAA gAGGAAGAATCCAAAAGAGAGA C AGGAGTGACGGGGATACATATG GA GGAATCGATGGATGATTCT GACAACTACATCAACTACCACA tCCACGGAGCCGTTCCACA aagGCGTCTACCCAAGAGAGCCA GAG TCTTGTGGGCAAGGCCGGCCTT CT CAAAGCCGCTATACCTCAT GTGTAACACTGAAACATGTGGT TG ATGTGACCTGCACTTATTCCCA CACCCGAAGCCTGTTTAC TGCATCACCAATAGCCG COBL4_2655 COBL4_2725 COBL4_2828 COBL4_723 COBL4_781 CSA1_2516 CSA1_913 CSA3_1101 CSA3_1767 CSA3_1872 CSA3_2724 CSA3_3734 CSA3_4186 KOR_187 KOR_2328 KOR_2493 KOR_2496 KOR_2863 ACGTTGGATGTTCCATACGCAGATGAC ACG ACGTTGGATGTTGGGAATGTTCAGTCC GAG ACGTTGGATGATGAGAAGGACCCAAGA GAC ACGTTGGATGAATCAATCGCGTCTATA TGG ACGTTGGATGATCTTGCGAGTTCGCTTC AG ACGTTGGATGCGACTTCTTCGTGAGTA CAG ACGTTGGATGTGCCATTGTCGATCGAC TTC ACGTTGGATGCGGGACAAAGTTTTGCT CAC ACGTTGGATGGAATGCAGGATAGATGA GGC ACGTTGGATGCGCTACAGGATATTGAA CCC ACGTTGGATGGAAAATGGGTACCCTTC TGC ACGTTGGATGTGATTGCTGCCCATGCTT TG ACGTTGGATGCTATAGAGGCACCCACT TAG ACGTTGGATGAACGGCTAGTGAAACGT TGC ACGTTGGATGATCTCGGCAGCAAGATC TGA ACGTTGGATGCATCCCAATAACTAGTG GAG ACGTTGGATGCATCCCAATAACTAGTG GAG ACGTTGGATGATTGAAAGTCTCAACGG ACGTTGGATGACTGAACATTCCCAAGT GGC ACGTTGGATGTTCCTTGGAAATGCCCA ACC ACGTTGGATGGATTCATACCCTTTTCTG CC ACGTTGGATGCACCATATATCTTGCGA GTTC ACGTTGGATGCGAGAAAGACAAATCAA TCGC ACGTTGGATGAGCAAGGATGGAAAGCA CTG ACGTTGGATGGGTTTCCTTTTTATTCAG CG ACGTTGGATGTCCTGAGGATGACGATA ACG ACGTTGGATGCCGGTATGGATTGATCTT GC ACGTTGGATGATCCAGGATCCAGGAGA AAG ACGTTGGATGGGCTGGACTTTGTCTTTG AG ACGTTGGATGGATCTGCAGCATCCCCA TTG ACGTTGGATGGCAACAAGGTAGCAAAG CTC ACGTTGGATGCCACACCGATTTACCAA GTC ACGTTGGATGGATCATTACTGCTGGAT GCG ACGTTGGATGCATTTTTCGAGGCAACA GCG ACGTTGGATGCATTTTTCGAGGCAACA GCG ACGTTGGATGAGGTGTATAATTTGCCA ATTCCCAAGTGGCCCCGCTTCC AT ATGCCCAACCTTGCTTAA CTGCCAAATTCAGCACGTC TGCGAGTTCGCTTCAGCAG AAATCAATCGCGTCTATATGG TGATCAAAGGTGGTTCTTTCA cCCTTTTTATTCAGCGAACTCAA TG ctcACGCTCAATTTCCATCAGTTA T TTGCTCGATGCAATTGG AACCAAAGACCAAATGC ggGAAGTACATTTCAGGGGCTCG GGG GCACTATGCTTAGAATACTT TAGCAAAGCTCTGGGTATT TTACCAAGTCTCCCTCC atACATTGATTATAAACGGCCTG TC GGAGATACAGTGCTGGT CGAGGGAGATACAGTGCTGGTT CA cccGCCACTCTTTCAGATCTAAA KOR_4184 KOR_742 KOR_801 KOR_813 LIM_1106 LIM_1157 LIM_1159 LIM_1343 LIM_444 LIM_537 LIM_564 LIM_795 LIM_901 LIM_977 LIM_978 LTP_211 LTP_410 CCA ACGTTGGATGAAACATGGGAGGAACCG CAG ACGTTGGATGTCTCTCTCACCCACTGTC G ACGTTGGATGGTACATGCTCATCCTCG CTC ACGTTGGATGCTGAAGCAGACCCAGCA GTT ACGTTGGATGTACACCAGGAAATCCTT ACC ACGTTGGATGCTCAATGAAGCAGCCTT TGG ACGTTGGATGAAGGTAAGGATTTCCTG GTG ACGTTGGATGAGAGCTGCTTCAAATGC ACC ACGTTGGATGGGGCTGATCTTAAGGTT CTC ACGTTGGATGGCCTTTTGACTGATCATC CG ACGTTGGATGGCCTTTTGACTGATCATC CG ACGTTGGATGCAGCTCTTCAAGAGAAC TGG ACGTTGGATGTCTGGCTTTGCAATCTTG GG ACGTTGGATGGACTGATTTAGCCTAAC TGC ACGTTGGATGTTGCAAAGCCAGAGAAA CCC ACGTTGGATGTGTAGATGACCAGGGAA ACG ACGTTGGATGCAGAATCATTACTGCTG TGC CTC ACGTTGGATGAGGGAATGCAGGACTGG TTG ACGTTGGATGGCTTCAGCTCCCAGCAA TAG ACGTTGGATGTTGCTGGGAGCTGAAGC AGA ACGTTGGATGGTACATGCTCATCCTCGC TC ACGTTGGATGACGCGAGACAAATGTGT AGG ACGTTGGATGAAGGTAAGGATTTCCTG GTG ACGTTGGATGCTCAATGAAGCAGCCTT TGG ACGTTGGATGCAGTAGAGTTTCCCCTC GTG ACGTTGGATGCTGAATGATATTCGCTTG GG ACGTTGGATGTAAATCATGCGTGACCC CAG ACGTTGGATGTAAATCATGCGTGACCC CAG ACGTTGGATGAACAGAGTGCATGACTT GCG ACGTTGGATGATGCACTCTGTTTCTGTC CC ACGTTGGATGTTGCAAAGCCAGAGAAA CCC ACGTTGGATGGACTGATTTAGCCTAAC TGC ACGTTGGATGACACTGGCTTATAGCTA GCG ACGTTGGATGTACAACCCCACATCAGC ATC AGTA cccCCTTATCAGGCGACGGGA gATATCAAGAAAATCAAGAACA GG CTGAAGCAGACCCAGCAGTT CCACAGTGGATGCAGAA ATGTGTAGGCTGTAAGAG TTTCCTGGTGTATTTATAGAAC GTCATGTCCTCGCATCA ACGTAGTTGGATGGGCTGATCA C cTGTAGAAATGTGTGGGGGC tATCATGACAATTCATCATAACT CGTGACCCCAGCTTCTT TTTTACCTTCAAAGCTTTTTTC TTCCTTCATACAAAATGTCCTCT cccCCGCGACTATGGTCTTGCCT cTTTAGCCTAACTGCAAAACTT ACTAAACTCATCTCGATCT CACATCAGCATCACCGAAAGCA LTP_430 LTP_507 LTP_676 LTP_730 LTP_754 LTP_835 LTP_925 MYB1_474 MYB2_1380 MYB2_1436 MYB2_1625 NAP1_221 NAP1_286 NAP1_317 NAP1_346 PCBR_1601 PCBR_1651 PCBR_1714 ACGTTGGATGCAGAATCATTACTGCTG TGC ACGTTGGATGCTGCACAGCAGTAATGA TTC ACGTTGGATGAGTGGCAGATCTCATTC CAG ACGTTGGATGAGGAGGATGTGAGGAG AAAG ACGTTGGATGTGGCTCCTATAAATTCAT GC ACGTTGGATGCAAAGCAGAAGCAATGA GGG ACGTTGGATGTCTGCTCCTTGATCTTGC TG ACGTTGGATGAGGTAATTGATCCACCG GAG ACGTTGGATGGCACGTCTTCCTGGAAG AAC ACGTTGGATGCCATGCATGTAGCTAAG ACG ACGTTGGATGGTTTTTCACCTTCCCCTC AG ACGTTGGATGGAATGAAGACAGCCGTT CTC ACGTTGGATGATGAACTTGGGGCAGTA CAC ACGTTGGATGCAACTGTGTACTGCCCC AAG ACGTTGGATGAACTGATTTGACCTGGG TCG ACGTTGGATGCAGATCACCCTGTGCCA ATC ACGTTGGATGTGATTGGCACAGGGTGA TCT ACGTTGGATGGTGGTGATATCGACAGT ACGTTGGATGCACATCAGCATCACCGA AAG ACGTTGGATGTGAGGATCAGCATGCTC AAG ACGTTGGATGGGGTCAGCGTGATGCAT TTG ACGTTGGATGTGGCTCCTATAAATTCAT GC ACGTTGGATGAGGAGGATGTGAGGAGA AAG ACGTTGGATGATGTCACAGCGGCCTCA AC ACGTTGGATGTGACATGCAGGCCGATA GAG ACGTTGGATGTTGCCTACATAAGAGCG CAC ACGTTGGATGATCGCTCTCGTTTGACGA AG ACGTTGGATGTCCAATCCACAAGACAT AGC ACGTTGGATGAGTCCCACTTTTGCATGC TC ACGTTGGATGAGTTTTGGCCTTGAGCTC TG ACGTTGGATGAGAGCTCAAGGCCAAAA CTC ACGTTGGATGAACTGATTTGACCTGGG TCG ACGTTGGATGCTGTGTACTGCCCCAAG TTC ACGTTGGATGATCATTCGGCCGGTAGA GAG ACGTTGGATGACTGTCGATATCACCAC GTC ACGTTGGATGACCTTAACGTTGCCAGC aAGTGTTGCGGAAAGTTG ATCAGCATGCTCAAGATGCCCT TT tgaagTTATAGGAGCCAAATTGGT GA cACCAAAGCCAAATGCATCAC TGTGAGGAGAAAGAACTAAGA GTG GGAGCAGCACCGCCAGAAGA ATAGAGCTGAGCGCGTGCGTGA cCCGCGGGCCTCCTCCGCTG gagCGTTTGACGAAGATGTAG CAAGACATAGCTGCAGG TAACTTGAATCATCTCCCATACT aTTGAGCTCTGTGGTAATTGTT TCATCACCGTAAGTGATTTTCTC T gCTTCTTAAGAATGAGAAGAAG ATA cccgTCTTCTCATTCTTAAGAAGT TAC AGAGAGTCAAGAGGATTAACGT GT aATCGCCTTCACCAAGCTCTC cccTCTTGGTCTGATCCGCCAT CDPK_516 CDPK_589 SAMS_744 SAMS_819 Susy1_1310 Susy1_1510 Susy1_1684 Susy1_1733 Susy3_1008 Susy3_1641 Susy3_4612 Susy3_593 Susy3_64 Susy3_886 Susy3_909 TUB_1475 TUB_1543 GGG ACGTTGGATGGAGATTCTCACAACTTC CCC ACGTTGGATGCCCTTTGTAGTAGTAGTT TGG ACGTTGGATGCTTGTCAGGAATCTTTCC AG ACGTTGGATGAAGGAGAGCTTTGACTT CAG ACGTTGGATGTGGAGGGAGAACACATT CTG ACGTTGGATGAGATGATCCAGCTCCTG TTG ACGTTGGATGGCAAATGAACAAGTTGA ATGG ACGTTGGATGCCATTCAACTTGTTCATT TGC ACGTTGGATGTGAGAGGATTCAGACAT GGC ACGTTGGATGGCAAGTGACCGAGTTTC TTC ACGTTGGATGAGGGAAAAAGAGCACA GTTC ACGTTGGATGCCTTAACAGTCACGTTG CAC ACGTTGGATGCGAACTTTGACCTTGGA GT ACGTTGGATGCGGTTTTGGTGGGATTT GAC ACGTTGGATGCGGTTTTGGTGGGATTT GAC ACGTTGGATGAAGTTCTGGGAGGTTGT GTG ACGTTGGATGTGCGCAGGCTGTCCATA GTC TTC ACGTTGGATGTGGGATGCTGGGATTTA GAC ACGTTGGATGAGACAGGATAAGATAGT GGC ACGTTGGATGTCTCGTACGCCATTGGA GTG ACGTTGGATGTTCAAGAATCTGCCACT GCC ACGTTGGATGGCGATCTGTTTTTGGCTT ATG ACGTTGGATGTGGGAACCCTATCCAAG AAC ACGTTGGATGGGTGGCCAGGTACTTGT TTT ACGTTGGATGATTTCTTCCTCTAGGGCA CG ACGTTGGATGATTACGATGAGCCAAGA GGG ACGTTGGATGCGTCCATGTGGTGCTAA AAG ACGTTGGATGATTTGCCATGTATACCTG CC ACGTTGGATGGTTTAATACCGTGAGCG AAC ACGTTGGATGACAGTCAGATCAGCCAG AAG ACGTTGGATGCTGCAATACAGAAACGA CAG ACGTTGGATGCTGCAATACAGAAACGA CAG ACGTTGGATGTAGTACACGTTCACTCG CTC ACGTTGGATGAGCGAGTGAACGTGTAC TAC GCTGGGATTTAGACAGTGGGGC T tGATAAGATAGTGGCCAGTCA GAGCCTCTTTCTGTTTTCGTTGA GATCCAGGTTGATGGAAATCAT CTGCAGAACATGATGGTGAAT CAAGAACTTCTCGAGAGTGCAC GG ggTGATTTCTTGATTAGGAGGAT AT CAGGATGTAAACAACCTATAAA CAAGAGGGTCTCATCCAAACGT TC cctccAAAAGAAAGACTTACTTTC CAT CACCACATTATACCCACT GGTTGCCCTGTTCAGGTCGC ACGATTACTCCCACAGAGAT GGATCCAAATAACCCAACGCC AGAAACGACAGTAGTATAAGAA AC cGTCACCAGTATAGTTCCCCG gGTACTACAATGAGGCGAG
