1、BSI Standards Publication Molecular biomarker analysis SSR analysis of maize PD ISO/TR 17623:2015National foreword This Published Document is the UK implementation of ISO/TR 17623:2015. The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal me
2、thods. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2015. Published by B
3、SI Standards Limited 2015 ISBN 978 0 580 77183 5 ICS 67.050 Compliance with a British Standard cannot confer immunity from legal obligations. This Published Document was published under the authority of the Standards Policy and Strategy Committee on 31 May 2015. Amendments/corrigenda issued since pu
4、blication Date Text affected PUBLISHED DOCUMENT PD ISO/TR 17623:2015 ISO 2015 Molecular biomarker analysis SSR analysis of maize Analyse molculaire de biomarqueurs Mthode danalyse SSR sur le mas TECHNICAL REPORT ISO/TR 17623 Reference number ISO/TR 17623:2015(E) First edition 2015-05-01 ISO/TR 17623
5、:2015(E)Foreword iv Introduction v 1 Scope . 1 2 Principle 1 3 Consumables and equipment 1 4 Procedure. 1 4.1 Sample preparation 1 4.2 DNA extraction and quantification 1 4.3 PCR amplification . 2 5 List of SSR-based maize markers validated through a GEVES intralaboratory study .2 5.1 Characteristic
6、s of the SSRs 2 5.2 SSR primer sequences 4 5.3 Observed SSR profiles of maize lines . 5 ISO 2015 All rights reserved iii Contents Page PD ISO/TR 17623:2015 ISO/TR 17623:2015(E) Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (I
7、SO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, gov
8、ernmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. The procedures used to develop this document and those intended for its further maint
9、enance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives). Atte
10、ntion is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introductio
11、n and/or on the ISO list of patent declarations received (see www.iso.org/patents). Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement. For an explanation on the meaning of ISO specific terms and expressions related to confor
12、mity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 16, Horizontal methods for
13、molecular biomarker analysis.iv ISO 2015 All rights reserved PD ISO/TR 17623:2015 ISO/TR 17623:2015(E) Introduction Varietal identification testing requires high-quality markers which are able to provide reproducible data using a variety of equipment, chemistries, and reagents. Accordingly, this Tec
14、hnical Report only addresses specific amplification methods for maize. The aims of this Technical Report are to provide a list of simple sequence repeat (SSR) markers and methods of analysis for maize. The SSR marker set has been validated through an intralaboratory study at GEVES (Laboratoire BioGE
15、VES, Domaine du Magneraud, BP.52, 17700 SURGERES). Properties and sequences of these SSR markers are publicly available on the website www.maizegdb.org. This Technical Report is linked to ISO 13495, which lists the different steps toward method validation and defined acceptance criteria. ISO 2015 Al
16、l rights reserved v PD ISO/TR 17623:2015 Molecular biomarker analysis SSR analysis of maize 1 Scope The methods and SSR markers included in this Technical Report are suitable for applications such as testing hybrid conformity, molecular fingerprinting of varieties, and checking variety identity. 2 P
17、rinciple Simple sequence repeat (SSR) analysis is based on the amplification and visualization of the polymorphism caused by variation in the number of repeats in a sequence motif that is two to five base pairs in length also known as a microsatellite. SSR analysis consists of the following steps: a
18、) sample preparation; b) DNA extraction; c) PCR amplification; d) separation; e) detection of the PCR products. 3 Consumables and equipment 96-well or 384-well microplate PCR reagents (DNA polymerase), buffer, MgCl 2 , dNTP, primers, etc. capillary electrophoresis reagents mixer/grinding mill microp
19、late centrifuge adjustable volume micropipettes micro-centrifuge for microtubes capillary electrophoresis system with fluorescence detection thermocycler 4 Procedure 4.1 Sample preparation For each sample, either individual seeds or seed mixes depending on the context are ground using a suitable mil
20、l (such as an IKA A10 or a Retsch MM301). 4.2 DN A e xtr action and quantification a) Obtain an aliquot of each homogenously ground sample. The amount required will depend upon the extraction protocol employed. TECHNICAL REPORT ISO/TR 17623:2015(E) ISO 2015 All rights reserved 1 PD ISO/TR 17623:2015
21、 ISO/TR 17623:2015(E) b) Extract DNA using in house protocol or equivalent. NOTE Collaborative study has been carried out with QIAGEN DNeasy 1)96 Plant Kit. c) The laboratory validates that the quantity of DNA extracted is appropriate to ensure a reliable result. 4.3 P CR amplification Conditions op
22、timised for ABI 9700 thermocycler. a) Mix preparation for simplex PCR (see Table 1). Table 1 Mix preparation for simplex PCR Concentration Volume for 1X H 2 O 3,125 L Buffer 10X 1 X 1 L dNTP (10 mM) 125 M 0,125 L MgCl 2(25 mM) 3 mM 1,2 L DNA polymerase (5 U/L) 0,25 U 0,05 L Forward primer (10 M) 0,2
23、5 M 0,25 L Reverse primer (10 M) 0,25 M 0,25 L Vol 1X mix 6 L DNA (2,5 ng/L) 4 L Final PCR vol 10 L b) Amplification conditions (see Table 2). A touchdown (TD) program is used. The hybridization temperature is lowered from 64 C to 55 C in decrements of 1 C per cycle. T a b l e 2 A m p l i f i c a t
24、i o n c o n d i t i o n s 10 cycles 30 cycles 94 C 94 C TD 94 C 10:00 0:30 72 C 0:30 72 C 72 C 64 C 0:30 55 C 0:30 10:00 10 C 0:30 0:30 NOTE Units for times in Table 2 are “minutes:seconds”. 5 List of SSR-based maize markers validated through a GEVES intralaboratory study 5.1 Characteristics of the
25、SSRs Data obtained with a 3130 Genetic Analyser (Applied Biosystems) (see Table 3). 1) QIAGEN DNeasy is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.2 ISO
26、 2015 All rights reserved PD ISO/TR 17623:2015 ISO/TR 17623:2015(E) Table 3 Characteristics of the SSRs No. SSR Bin/Chromosome Number of alleles recorded range of estimated allele sizes (bp) a Neis diversity index b 1 umc1147 1 4 6186 0,69 2 phi109275 1 6 121137 0,60 3 phi427913 1,01 5 119133 0,49 4
27、 umc1885 1,1 3 136142 0,63 5 phi064 1,11 8 75110 0,78 6 phi96100 2 4 275294 0,74 7 phi083 2,04 6 123136 0,76 8 umc1448 2,04 5 137161 0,77 9 phi102228 3,04 6 122133 0,54 10 umc1489 3,07 4 123135 0,51 11 umc1117 4,04 3 122135 0,67 12 umc1329 4,06 4 7492 0,63 13 phi093 4,08 7 281294 0,63 14 umc1180 4,1
28、 2 99102 0,47 15 nc130 5 5 139148 0,48 16 umc1478 5,01 4 134144 0,62 17 umc1792 5,08 5 115134 0,74 18 umc1153 5,09 5 101113 0,71 19 umc1143 6 5 7182 0,66 20 phi423796 6,01 5 125137 0,53 21 umc1133 6,01 3 91105 0,66 22 phi123 6,07 4 141147 0,66 23 phi089 6,08 4 8191 0,34 24 umc1545 7 6 7085 0,75 25 u
29、mc1134 7,03 4 7588 0,61 26 phi116 7,06 4 152173 0,70 27 umc1304 8,02 2 131136 0,50 28 phi233376 8,03 6 140159 0,68 29 bnlg1782 8,05 7 219236 0,73 30 phi015 8,08 7 82103 0,45 31 phi032 9,04 3 232239 0,53 32 bnlg1129 9,08 5 179202 0,72 33 umc1319 10,01 4 115124 0,65 34 phi050 10,03 3 8288 0,61 35 phi0
30、84 10,04 2 154157 0,50 36 umc1061 10,06 8 97107 0,46 aAllele sizes observed at the GEVES and illustrative data. bNeis diversity index was calculated based on several hundred maize lines already analysed at the GEVES. ISO 2015 All rights reserved 3 PD ISO/TR 17623:2015 ISO/TR 17623:2015(E) 5.2 SSR pr
31、imer sequences See Table 4. Table 4 SSR primer sequences No. SSR Forward primer sequence (5-3) Reverse primer sequence (5-3) 1 umc1147 GAGAAACCATCGACCCTTCCTAAC TTCCTATGGTACAGTTCTCCCTCG 2 phi109275 CGGTTCATGCTAGCTCTGC GTTGTGGCTGTGGTGGTG 3 phi427913 CAAAAGCTAGTCGGGGTCA ATTGTTCGATGACACACTACGC 4 umc1885
32、 TATACCAGCATCAGGTCTCGTCG GTAGAGTGACCGTGCTGTAGCAGA 5 phi064 CCGAATTGAAATAGCTGCGAGAACCT ACAATGAACGGTGGTTATCAACACGC 6 phi96100 AGGAGGACCCCAACTCCTG TTGCACGAGCCATCGTAT 7 phi083 CAAACATCAGCCAGAGACAAGGAC ATTCATCGACGCGTCACAGTCTACT 8 umc1448 ATCCTCTCATCTTTAGGTCCACCG CATATACAGTCTCTTCTGGCTGCTCA 9 phi102228 ATT
33、CCGACGCAATCAACA TTCATCTCCTCCAGGAGCCTT 10 umc1489 TTAATAGCTACCCGCAACCAAGAA CTGAGCCACAGTACCTTGCTGTT 11 umc1117 AATTCTAGTCCTGGGTCGGAACTC CGTGGCCGTGGAGTCTACTACT 12 umc1329 CCTCTCACATCTCCTCTCCCCT GTGTCGGTGTAGGTCTCCGTCTT 13 phi093 AGTGCGTCAGCTTCATCGCCTACAAG AGGCCATGCATGCTTGCAACAATGGATACA 14 umc1180 GAAGCC
34、CCTTGAAATGAATGAAC CGACGTACGTATAGACTCGCTCAG 15 nc130 GCACATGAAGATCCTGCTGA TGTGGATGACGGTGATGC 16 umc1478 GAAGCTTCTCCTCTCGCGTCTC CAGTCCCAGACCCTAGCTCAGTC 17 umc1792 CATGGGACAGCAAGAGACACAG ACCTTCATCACCTGCAACTACGAC 18 umc1153 CAGCATCTATAGCTTGCTTGCATT TGGGTTTTGTTTGTTTGTTTGTTG 19 umc1143 GACACTAGCAATGTTCAAA
35、ACCCC CGTGGTGGGATGCTATCCTTT 20 phi423796 CACTACTCGATCTGAACCACCA CGCTCTGTGAATTTGCTAGCTC 21 umc1133 ATTCGATCTAGGGTTTGGGTTCAG GATGCAGTAGCATGCTGGATGTAG 22 phi123 GGAGACGAGGTGCTACTTCTTCAA TGTGGCTGAGGCTAGGAATCTC 23 phi089 GAATTGGGAACCAGACCACCCAA ATTTCCATGGACCATGCCTCGTG 24 umc1545 GAAAACTGCATCAACAACAAGCTG
36、ATTGGTTGGTTCTTGCTTCCATTA 25 umc1134 AAAACTAACAGGCAGCAGACCAAC ATCAGCAAGTGACTGAATTCCTCC 26 phi116 GCATACGGCCATGGATGGGA TCCCTGCCGGGACTCCTG 27 umc1304 CATGCAGCTCTCCAAATTAAATCC GCCAACTAGAACTACTGCTGCTCC 28 phi233376 CCGGCAGTCGATTACTCC CGAGACCAAGAGAACCCTCA 29 bnlg1782 CGATGCTCCGCTAGGAATAG TGTGTTGGAAATTGACC
37、CAA 30 phi015 GCAACGTACCGTACCTTTCCGA ACGCTGCATTCAATTACCGGGAAG 31 phi032 CTCCAGCAAGTGATGCGTGAC GACACCCGGATCAATGATGGAAC 32 bnlg1129 GAGAGTATGCTACTCGCCGC GACGAGTTTGGAGTGCCATT 33 umc1319 TGAGAGCCACCTTCTTGAGCTACT TTCCTTGAAGGCGAAGGTAGGTAT 34 phi050 TAACATGCCAGACACATACGGACAG ATGGCTCTAGCGAAGCGTAGAG 35 phi08
38、4 AGAAGGAATCCGATCCATCCAAGC CACCCGTACTTGAGGAAAACCC 36 umc1061 AGCAGGAGTACCCATGAAAGTCC TATCACAGCACGAAGCGATAGATG4 ISO 2015 All rights reserved PD ISO/TR 17623:2015 ISO/TR 17623:2015(E) 5.3 Observ ed SSR pr ofiles of maize lines Molecular profiles of 10 maize lines are reported as estimated fragment siz
39、es in bp for each of 36 SSR markers. For a given SSR in a line, a single fragment size indicates that the marker is monomorphic within the line while two fragment sizes indicate that the marker is polymorphic within the line. T a b l e 5 S S R p r o f i l e s o b s e r v e d f o r 1 0 m a i z e l i
40、n e s d u r i n g i n t r a l a b o r a t o r y v a l i d a t i o n No. SSR Name of the lineA632 A641 CM7 EP1 F7 W64A B73 CO255 F2 MO17 1 umc1147 86 86 86 84 72 72 84 84 72 72 2 phi109275 137 126 126/137 121/126 126 137 137 126 121 121 3 phi427913 124 130 130 130/133 133 130 130 133 133 130 4 umc188
41、5 138 136 136 138 136 138 138 138 138 138 5 phi064 79 91 107 75 107/110 85 99 107 107 79 6 phi96100 278 278 278 294 294 288 278 288 294 287 7 phi083 123 123 123 125/131 129 129 125 123 132 125 8 umc1448 161 157 148 161 148 148 157 148 148 148 9 phi102228 130 126 122 122 130 126 122 122 122 130 10 um
42、c1489 123 123 123 123 131 123 123 123 123 131 11 umc1117 127 133 126 127 127 133 133 127 127 127 12 umc1329 74 83 83 7 7/8 3 83 83 83 83 77 83 13 phi093 281 281 286 286 286 282 288 281 286 281 14 umc1180 99/102 99 99 102 99 99 99 102 102 102 15 nc130 144 144 141/144 141 141 144 141 141 141 144 16 um
43、c1478 135 134 134 139 139 141 135 139 134 135 17 umc1792 125 119 123 116 115 116 115 115 115 116 18 umc1153 103/106 106 103 101/111 111 106 106 111 111 103 19 umc1143 80 80 74 71 71 80 80 71 71 76 20 phi423796 130 130 130 125/130 130 137 130 130 125 130 21 umc1133 98 98 101 91/105 91 98 98 105 101 9
44、1 22 phi123 147 141 147 141 141 145 141 141 141 141 23 phi089 82 81 90 90 90 90 81 90 90 82 24 umc1545 81 70 81/83 70/81 70 70 79 81 85 83 25 umc1134 84 84 81 81/88 81 84 81 81 81 88 26 phi116 173 173 152 162 162 169 169 152 162 173 27 umc1304 136 136 131 131 131 131 136 136 131 131 28 phi233376 144
45、 152 144 149/152 140 144 140 140 144 144 29 bnlg1782 230 219 219 226 221 219 219 226 219 219 30 phi015 97 97 97 84 97 82 103 97 97 97 31 phi032 232 232 238 232 232 232 232 232 232 238 32 bnlg1129 194/196 194 190 196/198 187 179 194 194 190 179 33 umc1319 115 124 115 121 121 121 115 121 115 115 NOTE
46、According to the repeated pattern of a SSR marker (di-, tri-, or tetranucleotide), allele sizes were mainly observed as expected. For few cases, discrepancies between expected and observed allele sizes were noticed and validated during the intralaboratory study. ISO 2015 All rights reserved 5 PD ISO
47、/TR 17623:2015 ISO/TR 17623:2015(E) No. SSR Name of the lineA632 A641 CM7 EP1 F7 W64A B73 CO255 F2 MO17 34 phi050 88 86 86 86 88 86 88 88 86 82 35 phi084 154 154 157 157 154 157 154 154 154 154 36 umc1061 104 104 104 101 101 104 104 101 101 101 NOTE According to the repeated pattern of a SSR marker
48、(di-, tri-, or tetranucleotide), allele sizes were mainly observed as expected. For few cases, discrepancies between expected and observed allele sizes were noticed and validated during the intralaboratory study.Table 5 (continued) 6 ISO 2015 All rights reserved PD ISO/TR 17623:2015This page deliber
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