NY/T 1433-2014 Technical Specification for Rice Variety Identification SSR Marker Method NY/T1433-2014 Standard compression package decompression password: www.bzxz.net
This standard specifies the operating procedures, data recording and statistics, and judgment methods for rice variety identification using simple sequence repeat markers. This standard is applicable to the SSR fingerprint data collection and variety identification of rice varieties.
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
Please note that some contents of this document may involve patents. The issuing organization of this document does not assume the responsibility for identifying these patents.
This standard is a revised version of NY/T 1433-2007 "DNA Fingerprint Method for Rice Variety Identification". This standard replaces NY/T 1433-2007 for reference in variety identification. Compared with NY/T 1433-2007, this standard has the following major technical changes, except for editorial changes:
—— The number of recommended primers has increased to 48 pairs. Among them, 19 original primers have been retained and 29 new primers have been added;
—— The data record and statistical items of rice variety SSR markers have been added;
—— The denaturing polyacrylamide gel electrophoresis silver staining detection and capillary electrophoresis fluorescence detection methods have been added.
This standard was proposed by the Seed Management Bureau of the Ministry of Agriculture. This
standard is under the jurisdiction of the National Technical Committee for Standardization of New Plant Varieties Testing (SAC/TC 277).
Drafting units of this standard: China National Rice Research Institute, Science and Technology Development Center of the Ministry of Agriculture.
The main drafters of this standard: Xu Qun, Wei Xinghua, Zhuang Jieyun, Lü Bo, Yuan Xiaoping, Liu Ping, Zhang Xinming, Yu Hanyong, Du Yuanyuan.
The previous versions of this standard are as follows:
——NY/T 1433-2007.
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) applies to this document.
GB 4404.1 Cereals and Grains Seeds
GB/T 3543.2 Sampling Procedure for Inspection of Crop Seeds
GB/T 6682 Specifications and Test Methods for Water Used in Analytical Laboratories
GB/T 19557.7 Guidelines for Testing Specificity, Consistency and Stability of New Plant Varieties Rice

ICS67.060
Agricultural Industry Standard of the People's Republic of China
NY/T 1433—2014
Replaces NY/T14332007
Technical Procedure for Identification of Rice Varieties
SSR Marker Method
Protocol for identification of rice varieties--ssR inarker method2014-03-24Release
2014-06-01Implementation
Ministry of Agriculture of the People's Republic of China
Normative reference documents
Terminology definition
Instruments and reagents
Solution preparation
Primer information
Operation procedures
Data recording and statistics
10 Determination method
Appendix A (Normative Appendix)
Appendix (Normative Appendix)
Appendix C (Normative Appendix)
Supplementary Notes (Informative Appendix)
Instruments and reagents
Solution preparation
Recommended primers
Reference list
NY/T 1433—2014
NY/T1433--2014
This standard was drafted according to the rules given in G31.1:2609. Please note that some 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 a revised version of NY/T1433—20147 1NA fingerprint method for the identification of water citrus varieties: this standard replaces NY/11153207 in the reference numbering of variety cases. Compared with NYT1133-2007, this standard has the following major technical changes except for editorial changes: the number of primers has been increased to 8 pairs. Specifically: the original 13 pairs of primers are retained, and 29 pairs of primers are added: the data recording and statistical items of water citrus varieties are increased: reverse addition denaturing acrylamide gel electrophoresis silver staining detection, paper tube fluorescence detection method. The standard was proposed by the Agricultural Bureau of the Ministry of Agriculture.|| tt||This standard was issued by the National Standardization Technical Committee for the Testing of New Varieties of Plants (SACC277) H11. The drafting units of the standard are: China Small Rice Research Institute, Science and Technology Development Center of the Ministry of Agriculture. The main drafters of this standard are: Xu Qun, Wei Xinghua, Sheng Jieyun, Kou Bo, Yuan Xiaoping, Liu Ping, Zhang Xinming, Yu Hanlu, Jun Yuan. The previous versions of this standard are as follows:
-NY/1433—2007
1 Scope
Technical regulations for rice variety identification SSK marking method NY/T1433-2014
This standard specifies the operating procedures, data recording and statistics, and judgment methods for rice variety identification using simple sequence (ssR) markers. This standard is applicable to the SSR fingerprint data collection and varietal identification of rice varieties. 2 Normative references
The following documents are indispensable for the verification of this document. For any dated referenced document, only the version of the date is applicable to this document. For any undated referenced document: the latest version (including all amendments) is the same as this document, G4404.1 Grain crop species Cereals
GB/T3513.2 Inspection procedures for crop species GB/T6682 Specifications and test methods for water for analytical laboratories GB/T19557.7 Guidelines for specificity, consistency and stability testing of new plant varieties Rice
3 Terms and definitions
The following technical definitions apply to this document. 3.1
recommended primer
Recommended primers
Preferably, a set of SSR primers with high polymorphism in the detection sites and good repeatability of the detection results should be selected in variety identification. 3.2
reference variety
reference variety
a variety with different allelic variations at the SSR loci used. Reference varieties are used to assist in determining the allelic variations of the samples submitted for inspection and to correct the systematic errors of instruments and equipment.
4 Principle
Because different varieties have different genetic compositions: the number of repetitions of simple repeat sequences in the genome is different, this difference can be detected through PCR amplification and electrophoresis, so that different varieties can be distinguished. 5 Instruments and reagents
See Appendix A.
6 Solution preparation
See Appendix. All reagents used are analytically pure. The water used for reagent preparation should meet the requirements of grade water specified in (GB/T6582, among which the preparation of silver staining solution only needs to meet the requirements of grade III. 7 Introduction See Appendix (tt
for information on the test material, which is divided into groups II, II, and III. Other non-linked primers not recommended in this document may also be used. 8 Operation procedures 8.1 Sample preparation NY/T1433-2014 When the test sample is rice seed, its quality shall meet the requirements for rice seed purity in (404.1). The separation and preservation of rice seed samples shall comply with the provisions of (B/T3543.2). Each sample shall be tested for 20 individuals (seed, slice or other equivalent) of the mixed sample. 8.2 DNA Extraction
Put the leaves in water for 2~3 minutes, add DNA extract solution 0: grind, add XA extract solution 1:1, and transfer 0u1 of the mixture to a 1m centrifuge tube. Or cut the leaves directly and put them in a 2.0ml centrifuge tube, add 590ml DNA extract solution to the tube, grind, add 550ml chloroform isocyanate (21:1) to the centrifuge tube, shake and mix: centrifuge at 10000g for 5 minutes. Transfer 100L of the supernatant to another 1.5ml centrifuge tube, add 800-20℃ precooled diol to precipitate DNA 1000g centrifuge for 10min, discard the supernatant, and then wash with 70% TE solution for 2 minutes: dry under natural conditions, add 100% TE (pH8.0) solution to dissolve DNA, detect concentration, and store at 2℃ for future use. Note: The above is a selected DNA extraction method: other DNA extraction methods that can achieve PCR amplification quality are applicable to this standard. 8.3P CR amplification
8.3.1 Primer selection
First select the primer group 1 in the appendix for amplification. When the number of differential sites detected between the samples is less than 2, then select the primer group Ⅱ in the appendix for detection. If the cumulative number of differential sites detected between the samples is less than 2, then select the primers Ⅲ and Ⅲ in turn. 8.3.2 Reaction system
1l. Reaction volume: including 1ut.70×PCR reaction buffer, C.6Lmmol, LMg(l., 0.8μl2.smmul/1.dNTF solution: 1ul.μmo/forward substance-nl, I.antibody, (.2.2UlTaDNA polymerase-35l.supernatant water, L sample DNA. If the buffer contains Mg1, then add MgC solution. Replace with an equal volume of sterile water
8.3.3 Reaction procedure
%. Pre-denaturation 1 rim; 91 (denaturation 15 5). 50℃--67℃ (set annealing temperature according to the primers in Appendix C) anneal for 15s-72 1min, first 30 cycles: 72.8m, 4 storage. 8.4 PCR product detection
8.4.1 Non-denaturing polyacrylamide gel electrophoresis silver staining detection 8. 4.1.1 Gel preparation
Insert a 1.5 mm wide spacer between two glass plates, sew the glass plates together, inject the lipid solution into the seal, let it gel for 1 min to 1 min to seal. Then add 5 m] 5×TBF buffer in the beaker: 3. 75 m acrylamide and bis(acrylamide) were mixed, stirred with double distilled water to 25 m: 2 uL ammonium persulfate and 12 L TMELD were added, mixed and poured between the gel plates. Then insert the sample well and allow it to polymerize within 0.1-6 minutes. The gel height should not be less than 100 μl. 8.4.1.2 Electrophoresis Remove the sealed agarose gel and place the glass plate in the electrophoresis tank. Add 1 μl of buffer in the electrophoresis tank and carefully dispense the sample well. Add 2 μl of 6× phenol blue-xylene cyanol electrophoresis indicator to 1 μl of PCR sample, mix and add the sample well. At 15:00, add a certain amount of ions to a sample well and start electrophoresis. The selected gradient is 5V/cm. Generally, the electrophoresis is performed from 30V to 60V. The duration is calculated based on the estimated length of the fragment to be tested and the migration time of xylene cyanol. 8.4.1.3 Silver staining
After electrophoresis, turn off the power supply and remove the glass plate: carefully separate the two glass plates, take the small polyacrylamide gel, rinse with double distilled water for 30s-60s, put it into the staining solution, and gently select 5li1-19rin for staining: then take it out of the staining solution, rinse it quickly with water, put it into the developer, and gently stain it until clear bands appear: rinse it with effective distilled water, drain it: pull it out or take a picture to image it, 8.4.2 Denaturing polyacrylamide gel electrophoresis silver staining detection 2
8.4.2.1 Cleaning the glass plate
NY/T 1433—2014
Clean the glass plate 7 times: rinse it with double hot water and let it air dry. Scrub it with anhydrous ethyl alcohol twice, wipe it with absorbent paper 1, and apply 0.5ml of alkane working solution on the long plate. Apply 0.5mL of silane working solution to the short plate with grooves. Prevent the two glass plates from contaminating each other during the operation.
8.4.2.2 Assemble the electrophoresis plate
After the glass plates are completely dried, assemble them into an electrophoresis plate and level them with a level. The thickness of the gasket is 0.1mm. 8.4.2.3 Make gel
Add 500L of freshly made 1% ammonium persulfate solution to 130μm.6. (10 μm polyacrylamide gel, 50 μm EMF) and mix quickly before pouring the gel: When the gel fills the glass interlayer, gently insert the C4μm thick honey fish nail into the gel for about 2.4 hours. Prevent bubbles from appearing during the gel process. Allow the gel to polymerize for at least 1 hour. After the gel is closed: clean the glue liquid that has overflowed from the gel plate, gently remove the old foam, wash it with water and set it aside. 8.4.2.4 Pre-electrophoresis
Install the gel plate on the electrophoresis tank, add about ml of 0.5>TEBE grade flushing solution preheated to 6% to the upper tank - make it about 3cm above the top of the gel. Add 800ml.1TBE buffer to the lower tank, and perform pre-electrophoresis for 10min-20min at a constant power of 60W. 8.4.2.5 Sample preparation
Add 2ml of sample loading buffer to the 1Cl.PCR sample, mix, denature at 95℃ for 5min on the PCR instrument, take out the sample, and heat overnight. Place on ice
8.4.2.6 Electrophoresis
Pipette with a pipette Add the sample slot, remove bubbles and impurities, insert the tooth end of a shark-tooth comb into the gelatin 1mm~-2mm, and add 3 samples to each sample hole. In addition to the sample to be tested, a reference variety should be added at the same time. Perform electrophoresis at 30V/cm--V/cm, and keep the gel temperature at about 50: electrophoresis for 1.1~2.5h (the electrophoresis time depends on the size range of the amplified fragment). 8.4.2.7 Silver staining
After electrophoresis, put the long glass plate attached to the gel into the fixative so that the fixative covers the gel, shake gently for 20min~3min, take out the gel plate, rinse it with double distilled water 1~2 times for 2min each: take it out and put it in the staining solution so that the staining solution covers the gel: shake gently for 20min~30 min: Take the gel plate from the staining solution and rinse it quickly with double distilled water: do not exceed 10s; put the gel plate into pre-cooled developer, shake it gently until clear stripes appear, take it out and quickly put the gel into fixative to fix it: rinse with double distilled water for 1min: take it out and drain it, scan or expand the camera,
8.4.3 Capillary electrophoresis and fluorescence detectionwwW.bzxz.Net
8.4.3.1 Preparation of PCR product samples
Take equal volumes of diluted solutions of different carbon-labeled amplification products and mix them, draw 1L from the mixture and add it to the wells of the 36-well plate of the DNA analyzer: add 0.1 μl of NaOH and 8.9 μl of deionized water to each well of the plate, denature the samples at 95℃ for 5min on the IPR instrument, put them out and quickly put them on ice and cool them to ℃ min. Centrifuge for 1 second and place on 1NA analyzer.
8.4.3.2 Electrophoresis detection
Follow the instrument operation instructions. Edit sample table: execute the run program and save the data. 9 Data recording and statistics
The allelic variation of each SSR site in the sample is expressed in the form of amplified fragment length. For ordinary polyacrylamide gel electrophoresis silver staining detection method: compare the allelic variation of each amplified site with the allelic fragment of the subtracted reference variety in reverse order to determine the allelic variation of the submitted sample at this site. For capillary electrophoresis fluorescence detection method, by using the same reference material and A analyzer, possible systematic errors between different batches of the same model or between different types can be eliminated: use general analysis software to read the allelic variation of the submitted sample at this site.
NY/T 1433—2014
The genotype data of homozygous sites are recorded as X/Y, where X is the size of the allele variation at the site, and the genotype data of heterozygous sites are recorded as X,Y: where X and Y are the two identical allele variations at the site, with the small fragment data in front and the missing data in the back. The allele variation data of the deletion site is recorded as 0/
Example 1:
The variety "Bamboo" did not amplify a 128bp fragment at the RM21 point: The genotype of this variety at this site: is 125/128. Example 2:
The variety "Dengyou 12" has two equal-risk mutations at the RM21 locus, 117p and 68hp respectively: the basic mutation of this variety at the locus is 117:168
10 Judgment method
When the number of difference loci between samples is 2, it is judged as "different varieties": When the number of difference loci between samples is 1: It is judged as "similar variety": The number of difference loci between samples is one. For samples that are determined to be "very similar varieties or identical varieties" and for which no two differential sites are detected by the 18 pairs of primers in the sample, interspecies identification can be carried out according to the regulations of R/19557.
A.1 Instruments and equipment
A1.1PR amplification instrument
Appendix A
(Normative Appendix)
Instruments and reagents
NY/T 1433-2014
A.1.2 High-voltage electrophoresis instrument: the maximum voltage is not less than 000)V, with only constant voltage, phase current and constant power functions. A.1.3 Electrophoresis tank and matching gel making accessories A.1.4 Universal electrophoresis instrument.
A.1.5JDNA analyzer: DNA-DNA tube electrophoresis, with fragment analysis function and data analysis software. High-speed refrigerated centrifuge: maximum centrifugal force not less than 20000: 4. 1.6
A.1.7 Horizontal shaker
A.1.8 Film observation lamp.
Town balance.
Volume pipette: specifications are 10l, 20l, 100u, 200)ul, 1000: Continuously adjustable force stirrer.
Nucleic acid concentration meter or ultraviolet spectrophotometer. Microwave oven,
High pressure sterilizer.
Acidity meter,
Ordinary refrigerator
Low temperature refrigerator.
Sanitary foam machine.
Tissue disruptor:
Gel imaging system or UV transilluminator or camera.A.2 Reagents
A.2.1 Sodium dodecylbenzenesulfonate
Polyvinylpyrrolidine
A.2.3 Chloroform.
A4.2.4 Isoamyl alcohol,
A2.5 Disodium ethylenediaminetetraethylamine.
A.2.6 Trihydroxymethylamine
A.2.7 Concentrated argon.
A.2.8 Sodium hydride.
10XPR reaction buffer
NY/T14332014
A.2.10 Magnesium chloride.
A.2.11 Sodium chloride.
A.2.124 Deoxyribonucleic acid.
A.2.13 [1NA polymerase,
A.2. 14 SSR primers.
A.2.15 Paraffin oil:
A.2.16 Agarose
A.2.17 DNA quality standard: 1NA fragment distribution range is at least 0bp~509h, and the size of DNA fragments within this range is generally small and the difference is no more than 100p.
A.2.18 Deionized 71amide.
Blue blue.
"Toluene cyanol.
Double diacrylamide.
Urea:
Affinity silica.
Stripping base.
Absolute ethanol
Amine.
Perfused ammonium acid
Glacial vinegar.
A.2.31Silver nitrate.
Indole.
A.2.33Acrylamide glue for DNA analyzer A.2.34DNA analyzer internal standard 1.1750C.5TDNA analyzer spectroscopic calibration mass: including FAM, NEI), VIC and PEI41Purpose INA fragments verified by carbon chromatographic identification. A.2.35
LDNA analyzer electrophoresis buffer,
B.1 Preparation of DNA extraction solution
(Normative Appendix]
Solution preparation
B.1.10.5mol/L ethylenediaminetetraacetic acid disodium salt (EDTA) (pH8.0) solution NY/T1433-2014
Weigh 186ml of diaminetetraacetic acid sodium salt (Na2E1) 1A·2H2O2, add 800ml of water, then add 20g of solid sodium hydride (VaOH), stir. Wait for Na:EIT After A.2H2O2 is completely dissolved, cool to room temperature: adjust the H2O2 to 8.0 accurately with VaOH and adjust the volume to 1000ml. At 103.4kPa (12℃), 20ml of B.1.20.5mol/L hydrochloric acid (HCl) solution
Take 25ml of concentrated hydrochloric acid (36~38%) and adjust the volume to 500ml with water. B.1.31mcl/L trishydroxymethylaminomethane hydrochloric acid (Tris-HCl) (pH8.0) solution Take 25ml of concentrated hydrochloric acid (36:38), add water and adjust the volume to 500ml. Weigh 3.3 trishydroxymethylaminomethane (Tris base): dissolve in 400ml of water, 0.5ml/1. acid solution (1.1.2) to pH 8. Adjust the volume to 1000ml, at 13.
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