Variety genuineness and purity testing of main crops with SSR markers—Maize
Some standard content:
ICS 65.020.20
National Standard of the People's Republic of China
GB/T39914—2021
Variety genuineness and purity testing of main crops with SsR markers--Maize2021-04-30Release
State Administration for Market Regulation
National Standardization Administration
2021-11-01Implementation
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2Normative references
3Technical evidence, definitions and abbreviations·
Terms and definitions
Abbreviations
Testing plan
Testing platform
Testing conditions
6Instruments, reagents and solution preparation·
Instruments
Solution preparation| |tt||Authenticity detection procedure
Primer synthesis
DNA extraction
PCR amplification
Amplification product separation
Data analysis
8 Variety purity detection procedure
LNA extraction
Primer screening and synthesis
8.3PCR amplification
8.4Amplification product separation
8.5Data analysis
9Result calculation and presentation
Authenticity identification
Purity determination
Result report
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GB/T 39914—2021
GB/T 39914—2021
Authenticity identification
Purity determination
Appendix A (Normative Appendix)
Solution preparation
Appendix B (Informative Appendix)
Allele amplification fragment information
Table 1 Authenticity identification primers
Table 2 PCR amplification reaction system
Table 3 Purity determination candidate primers
Information on the amplification fragments of the main alleles of known varieties B.1
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This standard was drafted in accordance with the rules given in GB/T1.12009. GB/T39914—2021
Please note that some of the contents of this document may involve patents. The issuing agency of this document does not assume the responsibility for identifying these patents. This standard is proposed by the Ministry of Agriculture and Rural Affairs of the People's Republic of China. This standard is under the jurisdiction of the National Crop Seed Standardization Technical Committee (SAC/TC37). The drafting units of this standard are: National Agricultural Technology Extension Service Center, Corn Research Center of Beijing Academy of Agricultural and Forestry Sciences, and Beijing Seed Management Station.
The main drafters of this standard are: Gan Fengge, Zhi Juzhen, Yi Hongmei, Zhao Jianzong, Zhang Like, Tian Hongli, and Lu Baochun. rrKaeerkAca-
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1Scope
Main crop variety authenticity and purity SSR molecular marker detection corn
GB/T 39914—2021
This standard specifies the detection principles, detection schemes, detection procedures and result reports of SSR molecular markers for authenticity and purity of Zeum G.VsL. varieties.
This standard applies to the authenticity verification and authenticity identification of Dinkami varieties, and does not apply to the identification of essentially derived varieties (ELV) and genetically modified varieties.
This standard applies to the purity determination of Dinkami single-cross varieties, and the purity determination of white cross lines and double-cross varieties is implemented as a reference. 2 Normative reference documents
The following documents are essential for the application of this document. For all referenced documents with a phase II, only the version with a phase II is applicable to this document. For all referenced documents without a phase II, the latest version (including all amendments) is applicable to this document. G3/T35413.1 General principles for inspection procedures for crop seeds G3/T3543.2 Sampling for inspection procedures for crop seeds G3/T6682 Specifications and test methods for water for analytical laboratories 3 Terms, definitions and abbreviations
3.1 Terms and definitions
The following terms and definitions apply to this document. 3.1.1
Variety verification
Variety verification
Compare with the standard sample of the corresponding variety name to verify whether the variety name of the sample for inspection is authentic. 3.1.2
Variety identification
Variety identification
The real variety name of the sample for inspection is determined through SSR molecular marker detection and screening and comparison through the SSR fingerprint data comparison platform for approved varieties (3.1.4).
Standard sample
standard sample
Standard sample
Standard sample
Standard sample
Standard sample is a physical seed sample that is recognized to represent the characteristics of the approved variety and is preserved by the designated institution of the state. 3.1.4
SSR fingerprint data comparison platform
SsRfingerprintprinihlasiplatform
Use the standardized method of SSR molecular markers to detect the alleles of the standard sample of the variety, and use the computer database technology and network information technology to construct the retrieval and comparison carrier of the approved variety data information. 3.1.5
reference sample
referenceconirol sample
sample with defined amplification product fragment size. 1
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GB/T 39914—2021
primer
a short single strand of five complementary DNA strands bound to the template DNA strand, which can provide a 3-OH end as the starting point for I)NA synthesis. Extend the five complementary strands of the synthetic template DNA.
combination primer
a set of fluorescently labeled primers with different colors or the same color that can be combined together for electrophoresis and have different amplification product fragment sizes.
allele
allele
a pair of genes at the same locus on a pair of homologous chromosomes, note 1: For SSR detection, alleles are expressed as the size of the amplified product fragment. Note 2: For primers or photolabeled primers, the size of the amplified product fragment refers to the range of a defined fragment size. This standard refers to 13n. 3.2 Abbreviations
The following abbreviations apply to this document:
HP: HACSPair, base pairs.
CTAB: Cetyltrimethylammoniumbromide, deoxyribonucleic acid. DNA: deoxyribonucleic acid. dNTPs: deoxyribonucleosidetriphosphales. PAGE: polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis. PCR: polymerase chain reaction. SDs: sodium doclecyl sulfamate SsR: simple sequence repeat Tuq enzyme: Tuq-DNA polymerase. Thermostable DNA polymerase 4 General Principles
Different varieties of rice have different numbers of simple sequence repeats (SSR) in their genomes that can be stably inherited from generation to generation: This difference can be detected by extracting DNA from representative test samples, amplifying and electrophoresing with SSR primers, and distinguishing varieties by the different sizes of their amplified product fragments. According to the SSR detection principle, fixed-term SSR primers are used to verify or identify the authenticity of the variety by comparing with standard samples or SSR fingerprint data comparison platforms. Authenticity verification is determined based on the difference in the number of SSR markers of the specified number of primers. Variety authenticity identification is determined based on the principle that there is no difference in the number of SSR markers. The appropriate primers that can accurately identify the fingerprint data or map of abnormal individuals of the variety are used to estimate the purity of the variety by the number of abnormal individuals or the self-identification rate of a certain number of test samples, so as to evaluate the overall typical consistency: 5 Detection Scheme
5.1 General Principles
For authenticity identification and purity determination, the accuracy and precision of the test results may vary depending on the primers, detection platform, and sample conditions. Based on the principle of "suitable for the test", the scale and capacity of the test should be comprehensively considered, and appropriate primers, detection platforms, and sample conditions should be selected to formulate corresponding test plans.
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GB/T 39914—2021
Under strictly controlled conditions, synthesize the selected primers, and test the test samples according to the procedures of LDNA extraction, PCR amplification, electrophoresis, and data analysis in accordance with the determined detection platform: fill in the test results in accordance with the specified requirements, and the inspection report should indicate the key information that affects the test results selected by the detection scheme. 5.2 Primers
5.2.1 Authenticity identification
5.2.1.1 The more primers are tested, the lower the probability of missed detection and misjudgment, and the workload also increases. This requires a balance between the test results and the acceptable accuracy rate. After Liu Zhongzhong determined that the Cami variety had passed the comprehensive test, based on high resolution and chromosome uniformity According to the screening principles of distribution, this standard selects 40 pairs of detection primers for variety authenticity identification, as shown in Table 1, and constructs a SSR fingerprint data comparison platform for known varieties based on this. The varieties in Table 1 can be divided into two groups, I and II. Numbers PM01PM20 are group I, and numbers PM21~PM40 are group I, each group contains 20 pairs. 5.2.1.2 Variety authenticity verification is a negation of the result, and relatively speaking, the requirement for the number of primers is not high, and the detection can be carried out in a sequential manner. First, use one set of primers to perform the detection. If no difference sites are detected or the number of difference sites that can determine the inconsistent results is detected, the detection is terminated. For the detection using a set of primers, if the number of difference sites detected but not reaching the number of difference sites that can determine the inconsistent results is reached, Then continue to complete the detection of group II primers
5.2.1.3 Variety authenticity identification emphasizes the affirmation of the results. Under the premise of having a known SSR fingerprint data comparison platform for the identified varieties, the known detection information can always be used to screen and determine the specific varieties. In order to minimize misjudgment, the detection will require a high number of primers. You can use a sequential method, or you can use the 40 SSR primers in Table 1 to perform detection until it can be determined after comparison with the SSR fingerprint data comparison platform: if there is still no difference in sites with the known varieties after comparison and no conclusion can be drawn, it is allowed to use other distinguishable SSR molecular markers for detection. 5.2.2 Purity determination
5.2.2. 1. Purity determination should clarify the type of abnormal individuals to be detected. Different types of abnormal individuals require different primers and numbers; sometimes the IVA fingerprint data of the corresponding female parent is also needed. The main types of abnormal individuals in hybrids are crosses and outcrosses, and the abnormal individuals in mixed white hybrids are outcrosses and mixed.
5.2.2.2 The selected species for variety purity determination should be screened out through pre-testing of test samples to accurately identify the abnormal individuals of the sample variety. The heterozygosity of primers, rapid DNA extraction and the potential of multiple combination electrophoresis should also be considered during screening. The screening results show that samples with genetic instability at individual sites can be eliminated, and the purity determination can be terminated if the test samples have serious genetic instability. 5.3 Detection platform
5.3.1 Electrophoresis is the key link of detection. For authenticity identification, denaturing PAGE electrophoresis and capillary electrophoresis can be selected. However, it should be noted that the denaturing PAGE electrophoresis detection data and SSR fingerprint data comparison platform are difficult to compare, making it difficult to conduct authenticity identification: For purity determination, PAGE electrophoresis and capillary electrophoresis can be used. Agar disc electrophoresis can also be used under the premise of selecting primers with different allele amplification fragments.
5.3.2 For large sample quantities, sample crushers, DNA automatic extraction and liquid transfer stations, and high-quality PCR amplification can be used. Instruments, multi-material capillary electrophoresis are combined to improve the overall efficiency of the test. 5.3.3 Technical conditions for DVA extraction, PCR amplification, and electrophoresis. Under the premise of being suitable for the test month and not affecting the test quality, appropriate local improvements to the provisions of this standard are allowed in accordance with the requirements of the test platform. 5.4 Samples
5.4.1 Authenticity identification
5.4.1.1 The sample sent for inspection is a seed, and the mass should not be less than 200g or not less than 500 grains. Sampling is taken at the production base of the seed, and the sample sent for inspection can be 3
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GB/T 39914—2021
Bunch of fruits, the number is not less than 5 (the total number of grains is not less than 500): Note: Sampling is carried out at the cattle production base. The samples for inspection can be seedlings, leaves, bracts and other tissues or organs. At this time, please note that the inspection and comparison objects should be the seedlings, leaves or bracts, and the number of seedlings, leaves or bracts should contain at least 20 individuals. For mixed sample testing, first extract INA separately, and mix with an equal amount of DVA. 5.4.1.2 Take samples with substitution properties from the samples for inspection, and use mixed samples or single individuals for testing. The source of the mixed sample should contain at least 20 individuals, and the source of the single individual sample should contain at least 5 individuals. 5.4.2 Purity determination
5.4.2.1 The samples for inspection are seeds. For hybrids, the quality should be not less than 200g or not less than 500 grains; for inbred lines, the quality should be not less than 100g or not less than [000 grains. Carried out simultaneously with authenticity identification The samples to be tested can be the same sample submitted for inspection. 5.4.2.2 Take a specified number of samples from the sample submitted for inspection, and the samples are tested independently using a single individual. The sampling and sub-sampling of the sample submitted for inspection should be representative and comply with the provisions of GB/T3543.2. The number of samples, for hybrids, should contain at least 96 (including controls when applicable) × 2 seeds; for white crosses, should contain at least 96 (including controls when applicable) × 1 seed. 5.5 Testing conditions
Straightness identification or purity determination should be carried out under control conditions that facilitate the correct implementation of the test, including but not limited to the following conditions: seed inspectors have the knowledge and skills to be familiar with the testing technology used; all instruments are compatible with the technology used and have been regularly maintained, verified and calibrated; use reagents of appropriate grades and sterilized consumables; use appropriate reference samples that affect the evaluation of the test results. 6 Instruments, reagents and solution preparation
Instruments
6.1.1 DNA extraction
High-speed refrigerated centrifuge, water bath or metal bath, UV spectrophotometer or nucleic acid concentration meter, tissue grinder6.1.2 PCR amplification
PCR amplification instrument.
6.1.3 Electrophoresis
6.1.3.1 Capillary electrophoresis
DVA analyzer.
PAGE electrophoresis
High-voltage electrophoresis apparatus, vertical electrophoresis tank and gel-making accessories, horizontal shaker, film observation lamp, gel imaging system or digital camera, 6.1.3.3 Agarose gel electrophoresis
Electrophoresis apparatus, horizontal electrophoresis tank and gel-making accessories, gel imaging system or UV transilluminator 6.1.4 Other apparatus
Micropipette, electric balance, autoclave steel, magnetic stirrer, microwave oven, refrigerator, staining box, -riKacerKAca-
6.2 Reagents
6.2.1 DNA extraction
GB/T 39914—2021
CTAB, chloroform, isopentanol, disodium ethylenediaminetetraacetate (EDTAVa: 2IIO), Trisbase, hydrochloric acid, sodium hydroxide, sodium chloride, 6.2.2PCR amplification
clVTPs, Ta enzyme, 10× buffer, mineral oil ddH,O. Primers and Mg+. 6.2.3 Electrophoresis
6.2.3.1 Capillary electrophoresis
DNA analyzer-specific endoprosthesis gel, molecular weight internal standard, deionized formamide, electrophoresis buffer. 6.2.3.2 PAGE electrophoresis
Deionized formamide (Formamidle), bromophenol blue (rphluc), dimethylbenzene cyanol FF, isacrylamide (isacrylamide), acrylatmide (Acrylatmide), boric acid (BoricAcicl), urea, affinity silane (BindingSilae), hydrophobic silane (RepelSilane), DVA molecular weight standard, anhydrous ethanol, tetramethylethylenediamine (TEMED), ammonium persulfate (APS), glacial acetic acid, ammonium acetate, silver nitrate, formaldehyde, sodium hydride,
6.2.3.3 Agarose gel electrophoresis
Agarose, bromophenol blue (BrphBluc), nucleic acid stain (Goldvicw or ethidium bromide). 6.3 Solution preparation
The solutions for DVA extraction, IR amplification, elution and silver staining shall be prepared according to the requirements specified in Appendix A. All reagents used are analytical grade reagents and the water used for preparation shall meet the requirements of Grade I water specified in GB/T6682. Water that meets the requirements of Grade III can be used for the preparation of silver staining solution.
7 Authenticity Detection Procedure
Primer Synthesis
According to the requirements of authenticity verification or identity authentication, select the primers in Table 1, use denaturing PA (E electrophoresis, only need to synthesize ordinary primers, use single primer electrophoresis: use fluorescent capillary electrophoresis, need to label the 5' end of the stop primer with fluorescent dye to synthesize primers, use single primer or combination primer electrophoresis, the fluorescent markers listed in Table 1 are only examples and are only applicable to a certain detection platform. Table 1 Authenticity Authentication Primers
Primer Name
PM01hnlg439wl
umel335y5
Chromosome
Primer Sequence (53)
E ; lg1940k?
umc2105k3
phio53k2
phi072k4
hnlg2291k4
umc 1705wl
lnlg2305k4
bnlg161ks
bnlg1702kl
ume1545y2
umel125y3
lhnlg240kl
phio80k15
phi065kg
umc1492y13
ume1432y6
ume1506kl2
umc1147y4
chromosome
Table 1 (continued)
Primer sequence (5°—3)
Forward: TTACACAACGCAACACGAGGC|| tt||Forward: GCTATAGGCCGTAGCTIGGTAGACACF Intermediate: CGTTTAAGAACGGTTGATTGCATTCC Reverse: GCCTTTATTTCTCCCTTGCTTGCC Forward: GAAGGGCAATGAATAGAGCCATGAG Reverse: ATGGACTCTGTGCGACTTGTACCG Forward CCCTGCCTCTCAGATTCAGAGATTG Reverse: TAGGCTGGCTGGAAGTTTGTTGCE: GCTCGTCTCCICCAGGTCAGG
Reverse: CGTTGCCCATACATCATGCCTC
Forward Forward:GCACACCCGTAGTAGCTGAGACTTG Reverse:CATAACCTTGCCTCCCAAACCC Forward:GGAGGTCGTCAGATGGAGTTCG
Reverse:CACGTACGGCAATGCAGACAAG
Forward:CCCCTCTTCCTCAGCACCTTG
Reverse:CGTCTTGTCTCCGTCCGTGTG
Forward:TCTCAGCTCCTGCTTATTGCTTTCG Reverse:GATGGATGGAGCATGAGCTTGC
Forward:GATCCGCATTGTCAAATGACCACZ AGGACACGCCAICGICATCA
EH:AATGCCGTTATCATGCGATGC
Reverse:GCTTGCTGCTTCTTGAATTGCGTForward:GGATGATGGCGAGGATGATGTC
R:CCACCAACCCATACCCATACCAGForward:GCAGGTGTCGGGGATTTTCTC
Z:GGAACTGAAGAACAGAAGGCATTGATACForward:TGAACCACCCGATGCAACTTG
Reverse:TTGATGGGCACGATCTCGTAGTCForward:CGCCTTCAAGAATATCCTTGTGCC R direction:GGACCCAGACCAGGTICCACC
F direction:GCGGAAGAGTAGTCGTAGGGCTAGTGTAG direction:GAGAACAAGTTCTTCAGACGCTTCAGG
Positive direction:GAGAAATCAAGAGGTGCGAGCATCReverse direction:GGCCATGATACAGCAAGAAATGATAAGCPositive directionGAGGAATGATGTCCGCGAAGAAG
Reverse direction:TICAGTCGAGCGCCCAACAC
Positive direction:AAGAACAGGACTACATGAGGTGCGATACReverse direction:GTTTCCTATGGTACAGTTCTCCCTCGCrrKaeerkAcaGCACACCCGTAGTAGCTGAGACTTG Reverse: CATAACCTTGCCTCCCAAACCC Forward: GGAGGTCGTCAGATGGAGTTCG
Reverse: CACGTACGGCAATGCAGACAAG
Forward: CCCCTCTTCCTCAGCACCTTG
Reverse: CGTCTTGTCTCCGTCCGTGTG
Forward: TCTCAGCTCCTGCTTATTGCTTTCG Reverse: GATGGATGGAGCATGAGCTTGC
Forward: GATCCGCATTGTCAAATGACCACZ AGGACACGCCAICGICATCA
EH:AATGCCGTTATCATGCGATGC
Reverse:GCTTGCTGCTTCTTGAATTGCGTForward:GGATGATGGCGAGGATGATGTC
R:CCACCAACCCATACCCATACCAGForward:GCAGGTGTCGGGGATTTTCTC
Z:GGAACTGAAGAACAGAAGGCATTGATACForward:TGAACCACCCGATGCAACTTG
Reverse:TTGATGGGCACGATCTCGTAGTCForward:CGCCTTCAAGAATATCCTTGTGCC R direction:GGACCCAGACCAGGTICCACC
F direction:GCGGAAGAGTAGTCGTAGGGCTAGTGTAG direction:GACGAAGTCTTCAGACGCTTCAGG
Positive direction:GAGAAATCAAGAGGTGCGAGCATCReverse direction:GGCCATGATACAGCAAGAAATGATAAGCPositive directionGAGGAATGATGTCCGCGAAGAAG
Reverse direction:TICAGTCGAGCGCCCAACAC
Positive direction:AAGAACAGGACTACATGAGGTGCGATACReverse direction:GTTTCCTATGGTACAGTTCTCCCTCGCrrKaeerkAcaGCACACCCGTAGTAGCTGAGACTTG Reverse: CATAACCTTGCCTCCCAAACCC Forward: GGAGGTCGTCAGATGGAGTTCG
Reverse: CACGTACGGCAATGCAGACAAG
Forward: CCCCTCTTCCTCAGCACCTTG
Reverse: CGTCTTGTCTCCGTCCGTGTG
Forward: TCTCAGCTCCTGCTTATTGCTTTCG Reverse: GATGGATGGAGCATGAGCTTGC
Forward: GATCCGCATTGTCAAATGACCACZ AGGACACGCCAICGICATCA
EH:AATGCCGTTATCATGCGATGC
Reverse:GCTTGCTGCTTCTTGAATTGCGTForward:GGATGATGGCGAGGATGATGTC
R:CCACCAACCCATACCCATACCAGForward:GCAGGTGTCGGGGATTTTCTC
Z:GGAACTGAAGAACAGAAGGCATTGATACForward:TGAACCACCCGATGCAACTTG
Reverse:TTGATGGGCACGATCTCGTAGTCForward:CGCCTTCAAGAATATCCTTGTGCC R direction:GGACCCAGACCAGGTICCACC
F direction:GCGGAAGAGTAGTCGTAGGGCTAGTGTAG direction:GACGAAGTCTTCAGACGCTTCAGG
Positive direction:GAGAAATCAAGAGGTGCGAGCATCReverse direction:GGCCATGATACAGCAAGAAATGATAAGCPositive directionGAGGAATGATGTCCGCGAAGAAG
Reverse direction:TICAGTCGAGCGCCCAACAC
Positive direction:AAGAACAGGACTACATGAGGTGCGATACReverse direction:GTTTCCTATGGTACAGTTCTCCCTCGCrrKaeerkAca
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