Supporting Information for Electrophoresis DOI /elps Sunil Archak, Vemireddy Lakshminarayanareddy and Javaregowda Nagaraju
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1 ELECTROPHORESIS Supporting Information for Electrophoresis DOI /elps Sunil Archak, Vemireddy Lakshminarayanareddy and Javaregowda Nagaraju High-throughput multiplex microsatellite marker assay for detection and quantification of adulteration in Basmati rice (Oryza sativa) ª 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2 Supplementary Table 1 Bulked samples of one traditional and one evolved Basmati cultivars from various places across growing area were collected and allele profiles at eight microsatellite loci were generated using multiplex assay. There was absolute homogeneity in the inter-population allele profiles. Populations are labeled after the place of sampling. Suffix B indicates that it is one of the multiple samples from a particular place. 1. Taraori Basmati Population RM72 RM202 RM348 RM44 RM55 RM171 RM241 RM1 Alewa B Ambala Amritsar Babain B Bahadurgarh B Bapauli B Barara B Bhikhiwindi Devigarh Gannoua B Gharunda B Gohana Guhla B Hansi Indri Israna B Jandialaguru Kaithal B Kalayat B Kharkhoda B Madlouda B Nariangarh B Narwana Nilokheri Panipat Patran Pundri B Radour B Rajaund Rajpura Ratia B Rohtak Safidon B Shahbad Sirsa Taran Taran Thanesar B Tohana B Uchanakalan
3 2. Pusa Basmati Population RM72 RM202 RM348 RM44 RM55 RM171 RM241 RM1 Ambala Assandh Babain Bapoli Barara Bhuna Bilaspur Chhachroli Ellnabad Fatiabad Gannour Gharonda Gulha Indri Israna Jagadhari Kaithal Kalayat Karnal Kathura Kharkhoda Ladwa Madlouda Mundlana Narayangarh Nissing Panipat Pehowa Pundri Rai Rajond Rania Ratia Sirsa Sonepat Thanesar
4 Supplementary Table 2. Inter-locus peak balance values for the multiplex loci. RM1 RM72 RM44 RM55 RM202 RM241 RM171 RM348 RM1 RM RM RM RM RM RM RM The peak balance values were derived from the peak height (rfu) ratios obtained by a factorial design experiment with a pair of loci amplified each time (duplex reaction). A ratio of 1 is considered the best combination, and ratios from 0.7 to 1.2 were considered practically adequate. Bold-faced values indicate compatible combinations. For PCR reactions and details of primers please refer to the main document.
5 Supplementary Figure 1. fgr sequence used for real-time PCR. Amplicon obtained from blue forward primer gives a 236 bp amplicon and red forward primer amplifies an 80 bp band. Reverse primer is common, and binds in the region deleted in the aromatic rices (GATTATGG) amplifying only in non-aromatic rices. >gi : Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 8, PAC clone:p0456b03 ATATATATATATATAATGATACACACCTCTCCTCCGTGTTAATGCAGCTCATTTGTTCTGTCCCGGTTCA AATATCTATTTTTCTCATATGTTGTCAGCATGATTCACTTAATTTAGTATATAGAAGATGCCATTATTTA TGTCTGGAATCTTACTGCAGAAGGGAAAACAATTGATAACGGAATTGATTGCATTCTAATTTGTTGTTTC TTTGTTATGTTCTTATCGACAATTACAAATTTGATTCTGAGAATCATGTTCGGGATGTGTATTTCTACTG CAGGAACTATCCTCTCCTGATGGCAACATGGAAGGTAGCTCCTGCCCTGGCTGCTGGCTGTACAGCTGTA CTAAAACCATCTGAATTGGCTTCCGTGTAAGTTTAACATGTTAACTTGTTAATGTCATACCCATGCTAGT TGCAATGACATTTGATTTTAAAATGTTGTGGCATGTCCATGCTGCAAGCAATGTAATTTGAAATCTCTCT CTATCATTAATTACCAGGACTTGTTTGGAGCTTGCTGATGTGTGTAAAGAGGTTGGTCTTCCTTCAGGTG TGCTAAACATAGTGACTGGATTAGGTTCTGAAGCCGGTGCTCCTTTGTCATCACACCCTGGTGTAGACAA GGTACAGCTATTCCTCCTGTAATCATGTATACCCCATCAATGGAAATGATATTCCTCTCAATACATGGTT TATGTTTTCTGTTAGGTTGCATTTACTGGGAGTTATGAAACTGGTAAAAAGATTATGGCTTCAGCTGCTC CTATGGTTAAGGTTTGTTTCCAAATTTCTGTGGATATTTTTTGTTCTCTTTCTACTAACTCTCTATTATC AATTCTCAATGTTGTCCTTTTCTTTTAACTCCTTTACTTTTTAGAATTGTGATCAAGACACTTTGAGCAT CATTCTAGTAGCCAGTTCTATCCTGTTTCTTACCTTTTTATGGTTCGTCTTTTCTTGACAGCCTGTTTCA CTGGAACTTGGTGGAAAAAGTCCTATAGTGGTGTTTGATGATGTTGATGTTGAAAAAGGTACATGCCACT TGCTATGATTAACTAATTCTGAAGTGCGGGACTTTGTAAAGCACTTAACTGAGCTGGATGCTAGACCCCC AAAAGCCCTTTTTGGTGTCTTGGGCTTGTTGCAGAAATACTGGTCCCAGACGAGCAGGATGCAAGAAAAT TAACTACTTTTGCCACTGATTAGTATTTCTTAGAAGTTACACCTCAAGGATTAGCAATACTTTCTTAAAA TGTGCTATTGATTAAAAAGATGTCCTGTATTATTTTGAGCAGATCTTGTACTGGTTGATCGGCTTGCATG AAAATATTGTTGAGGATTATAATGCCATGCCAACTGAGTAAAGAAAAGAGTTGTAAAATATGTTATGCAA CATGAATATATATGTGATTTCATTTTTCCTTTTTCTTTTCGTGGCAAGGAAGGCAGTTAGGAAGGACTGA TGTGAAAAGCACAAGTACTATTCTTAGTTCT M bp 100bp 50bp M: 50bp marker (MBI Fermentas), 1,6: Pusa Basmati, 2,7: Basmati370, 3,8: Sharbati, 4,9: IR64, 5,10: Jaya. Non-aromatic rices amplify a 236 bp band (lanes 3-5, blue primer) and an 80 bp band (lanes 8-10, red primer); Aromatic rices yield no amplicon.
6 Supplementary Figure. 2 Representative real-time PCR runs of RM348 and fgr loci Adulteration in the C T Value standard sample (%) (fgr locus) C T Value at RM ±0.25
7 Supplementary Figure 3. Aligned sequences of variety specific alleles of 8 microsatellite loci used in the multiplex assay
8 RM1 BASMATI370 : DEHRADUN : TARAORI : BASMATI386 : RANBIR : BASMATI217 : HARYANA : PUSA : SUPER : SHARBATI : JAYA : IR64 : * 20 * 40 * 60 -GAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG -GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG GAGAGAGAGAGAGAGAGAGAGAGag : 25 : 27 : 25 : 23 : 27 : 43 : 51 : 27 : 45 : 49 : 49 1
9 RM44 SUPER : BASMATI217 : HARYANA : SHARBATI : JAYA : IR64 : BASMATI370 : DEHRADUN : RANBIR : TARAORI : BASMATI386 : PUSA : * 20 * 40 * 60 ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG------CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG------CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG------CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG--CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG--CCGGA ATGGGATG-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG--CCGGA ATGGGATG GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG CCGGA : 50 : 50 : 50 : 50 : 50 : 50 : 56 : 56 : 56 : 60 : 60 : 60 2
10 RM55 BASMATI370 : DEHRADUN : RANBIR : JAYA : TARAORI : BASMATI386 : SHAARBATI : BASMATI217 : HARYANA : SUPER : IR64 : PUSA : * 20 * 40 * AAA-----GAGAGAGAGAGAGAGAGAGAGAGAGAGAGA----GGGGAGGGAGAGAGG AAA-----GAGAGAGAGAGAGAGAGAGAGAGAGAGAGA----GGGGAGGGAGAGAGG AAA-----GAGAGAGAGAGAGAGAGAGAGAGAGAGAGA----GGGGAGGGAGAGAGG AAA-----GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA--GGGGAGGGAGAGAGG AAA-----GAGAGAGAGAGAGAGAGAGA GGGAGAGAGG AAA-----GAGAGAGAGAGAGAGAGAGA GGGAGAGAGG AAAGAGT-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GG AAAGAGT-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GG AAAGAGT-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GG AAAGAGT-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GG AAAGAGT-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GG AAAGAGT-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GG AAA ************************************************* : 48 : 48 : 48 : 50 : 33 : 33 : 43 : 43 : 43 : 43 : 43 : 43 3
11 RM72 BASMATI370 : DEHRADUN : TARAORI : BASMATI386 : RANBIR : BASMATI217 : IR64 : HARYANA : PUSA : SUPER : SHARBATI : JAYA : * 20 * 40 * 60 * CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATT-AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATT-AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATT-AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATT-AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATT-AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATT----AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATTATTATT AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATT AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATT AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATT AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATT AT CG-TATTATTATTATTAT-C-ATTATTATTATTATTATTATTATTATTATTATTATTATT AT CG TATTATTATTATTAT C ATTATTATTATTATTATTATTATTATTATTATTATTATT AT
12 RM171 BASMATI370 : DEHRADUN : TARAORI : BASMATI386 : RANBIR : BASMATI217 : HARYANA : PUSA : SUPER : SHARBATI : JAYA : IR64 : * 20 * 40 AC-GATGGATGGATGGATGGATGGATG GGTT AC-GATGGATGGATGGATGGATGGATG GGTT AC-GATGGATGGATGGATGGATGGATG GGTT AC-GATGGATGGATGGATGGATGGATG GGTT AC-GATGGATGGATGGATGGATGGATG GGTT AC-GATGGATGGATGGATGGATGGATGGATGGATG-----GATT AC-GATGGATGGATGGATGGATGGATGGATGGATG-----GATT AC-GATGGATGGATGGATGGATGGATGGATGGATG-----GATT AC-GATGGATGGATGGATGGATGGATGGATGGATG-----GATT AC-GATGGATGGATGGATGGATG GATT AC-GATGGATGGATGGATGGATG GATT AC-GATGGATGGATGGATGGATGGATGGATGGATGGATG-GATT AC GATGGATGGATGGATGGATG**** G TT : 30 : 30 : 30 : 30 : 30 : 38 : 38 : 38 : 38 : 26 : 26 : 42 5
13 RM202 IR64 : BASMATI370 : DEHRADUN : TARAORI : BASMATI386 : RANBIR : BASMATI217 : PUSA : SHARBATI : SUPER : HARYANA : JAYA : * 20 * 40 * 60 TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA-GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA---GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA---GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA---GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA---GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA---GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA---GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA---GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA---GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GGA TT-AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GGA TT AAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAgaga GGA : 61 : 41 : 37 : 37 6
14 RM241 RANBIR : BASMATI370 : DEHRADUN : IR64 : TARAORI : BASMATI386 : BASMATI217 : HARYANA : PUSA : SUPER : JAYA : SHARBATI : * 20 * 40 * 60 * AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA-T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA-----T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA-----T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA T AA-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA T AA GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAga T : 75 : 71 : 71 : 63 : 57 7
15 RM348 BASMATI370 : DEHRADUN : TARAORI : BASMATI386 : RANBIR : BASMATI217 : HARYANA : PUSA : SUPER : SHARBATI : JAYA : IR64 : * 20 * 40 * AAGCCAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAGCAGCAGCAG-CGAA AAGCCAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAGCAGCAGCAG-CGAA AAGCCAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAGCAGCAGCAG-CGAA AAGCCAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAGCAGCAGCAG-CGAA AAGCCAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAGCAGCAGCAG-CGAA AAGCTAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAG CGAA AAGCTAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAG CGAA AAGCTAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAG CGAA AAGCTAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAG CGAA AAGCTAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAG CGAA AAGCTAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAG CGAA AAGCTAGAACAGCAAACACACACAGATTA-CAGCAGCAGCAG CGAA AAGC AGAACAGCAAACACACACAGATTA CAGCAGCAGCAG CGAA : 54 : 54 : 54 : 54 : 54 : 45 : 45 : 45 : 45 : 45 : 45 : 45 8
16 Supplementary Figure 4. Representative Multiplex allele profiles of traditional, evolved, and non-basmati rice varieties. Each peak of the multiplex profile represents the variety specific allele at that locus, with allele size annotated in the box. The loci (order: from left to right) are RM1, RM72 and RM171 (in blue); RM241 and RM202 (in green); RM44, RM348 and RM55 (in black). Please see Table 1 in the main document for details regarding the rice varieties and Table 2 for details regarding the loci.
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23 For Forensic and Research Use Only -1- Not for use in Diagnostic Procedures. Supplementary Figure 5. Quantification of Basmati370+IR64 standard samples at RM IR64 1% : Basmati370 99% Genotyper IR64 3% : Basmati370 97% IR64 5% : Basmati370 95% IR64 7% : Basmati370 93% IR64 10% : Basmati370 90% IR64 15% : Basmati370 85%
24 For Forensic and Research Use Only -2- Not for use in Diagnostic Procedures. Licensed to CDFD, CDFD Genotyper 3.7 IR64 30% : Basmati370 70% IR64 50% : Basmati370 50% IR64 60% : Basmati370 40% IR64 0% : Basmati % IR64 100% : Basmati370 0%
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