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SN/T 4876.8-2020 English PDF

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SN/T 4876.8-2020: (DNA barcoding method - Part 8: Cuscuta)
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Basic data

Standard ID SN/T 4876.8-2020 (SN/T4876.8-2020)
Description (Translated English) (DNA barcoding method - Part 8: Cuscuta)
Sector / Industry Commodity Inspection Standard (Recommended)
Classification of Chinese Standard B16
Classification of International Standard 65.020.01
Word Count Estimation 13,132
Date of Issue 2020-12-30
Date of Implementation 2021-07-01
Regulation (derived from) General Administration of Customs Announcement No. 136 [2020]
Issuing agency(ies) General Administration of Customs

SN/T 4876.8-2020: (DNA barcoding method - Part 8: Cuscuta)

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Method for DNA barcoding - Part 8.Cuscuta spp The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards Replace SN/T 1162-2002 Issued by the General Administration of Customs of the People's Republic of China 2020-12-30 release 2021-07-01 implementation

Foreword

This document is drafted in accordance with GB/T 1.1-2020. SN/T 4876 "DNA Barcoding Method" is a series of standards and has been published The following parts. --Part 1.Quarantine Cactitermites --Part 2.Quarantine beetle --Part 3.Quarantine Moth --Part 4.Quarantine Sorghum --Part 5.Datura --Part 6.The genus Tubula --Part 7.Poinsettia subgenus --Part 8.Cuscuta This document is part 8 of SN/T 4876 This document was proposed and managed by the General Administration of Customs of the People's Republic of China. This document was drafted by. Fuzhou Customs of the People’s Republic of China, Shanghai Customs of the People’s Republic of China, Ningbo Sea of the People’s Republic of China Customs, Chinese Academy of Inspection and Quarantine Sciences, Qingdao Customs of the People’s Republic of China, Guangzhou Customs of the People’s Republic of China, People’s Republic of China Nanjing Customs. The main drafters of this document. Yu Wentao, Yin Liping, Wang Nianwu, Yu Yun, Fu Yining, Xu Ying, Fan Xiaohong, Shao Xiuling, Wu Hai Rong, Xue Huajie, Fu Jianguo. Cuscuta

1 Scope

This document specifies the DNA barcode detection and identification method of Cuscuta. This document is applicable to phytosanitary laboratories conducting molecular identification of Cuscuta at border ports.

2 Normative references

The contents of the following documents constitute the indispensable clauses of this document through normative references in the text. Among them, dated quotations Only the version corresponding to that date is applicable to this document; for undated references, the latest version (including all amendments) is applicable Used in this document. SN/T 1385 Quarantine and identification method of Cuscuta

3 Terms and definitions

The following terms and definitions apply to this document. 3.1 DNA barcode A short, standardized gene segment used to distinguish different species. This fragment has obvious genetic variation and differentiation among species It is easy to carry out PCR amplification. DNA barcode technology is mainly used for species identification and new species discovery. 3.2 Voucher specimen A specimen that provides physical evidence for identification or research and has sufficient archived information (collection and identification information, etc.) for long-term preservation, It can be a complete organism or a part of it, or it can be the genetic material of an organism. The archived information of the voucher specimen record should have It is retrospective. The preservation of voucher specimens is very important. It is a physical reference voucher for DNA barcodes. The voucher specimen information includes collection information, Collect information and appraisal information for review. 3.3 Nuclear ribosome DNA nuclear ribosome DNA; nrDNA All the genetic information contained in the nuclear chromosomes of eukaryotes, namely base pairs, is different from plastids such as chloroplasts and mitochondria DNA, as well as other forms of DNA such as cosmids and plasmids. 3.4 Chloroplast DNA chloroplast DNA; cpDNA DNA present in the chloroplast. The DNA of higher plant chloroplasts is a double-stranded covalently closed circular molecule, and its length depends on the biological species. However, the size is between 120 kb and 217 kb, which is equivalent to the size of the phage genome. 3.5 Internal transcribed spacer; ITS In eukaryotic ribosomal DNA, 18S rRNA and 28S rRNA gene spacer sequences are called internal transcribed spacers. Inward turn The ribosomal genome sequence where the recording spacer is located from 5'to 3'are. external transcribed spacer (external transcribed spacer, ETS), 18S gene, internal transcribed spacer 1 (ITS1), 5.8S gene, internal transcribed spacer 2 (ITS2), 28S gene and genes Intergenic spacer (IGS). That is, the internal transcribed spacer contains ITS1, 5.8S gene and ITS2.Ribosomal RNA The 18S, 5.8S and 28S genome sequences tend to be conserved in most organisms, with little variation among organisms, while the internal transcribed spacer ITS1 and ITS2, as non-coding regions, bear less selective pressure, relatively change greatly, and can provide detailed systematic analysis The required heritable traits. 3.6 1.5-Ribulose bisphosphate carboxylase large subunit gene RubisCo_large; rbcL 1.5-Ribulose diphosphate carboxylase (RubisCo) is an enzyme with the dual functions of carboxylation and oxygenation. It plays an important role in photosynthesis and photorespiration. Sucking plays an important role. RubisCo enzyme is composed of 16 subunits, including 8 large subunits and 8 small subunits. Of which large subunit The gene (rbcL) is encoded by the chloroplast genome.

4 Basic information of Cuscuta

Chinese name. Cuscuta Scientific name. Cuscuta spp. Taxonomic status. Angiosperm (Magnoliophyta), Magnolia (Magnoliatae), Solanaceae (Solanales), Convolvulaceae (Convolvulaceae) Cuscuta is a parasitic herb, has no roots, and has no hair on the whole. The stem is entwined, slender, linear, yellow or red, not green, Fix the host with the aid of a suction device. No leaves, or degenerated into small scales. The flowers are small, white or light red, sessile or short-stemmed, spike-like, total Shaped or clustered into head-like inflorescence; bracts are small or absent; flowers are 5 to 4 out of number; sepals are nearly equal in size, more or less connected at the base; corolla tubular, Pot-shaped, globose, or bell-shaped, with marginally split or fringe-shaped scales under the stamens of the inner surface of the corolla tube; the stamens are born in the throat of the corolla Or adjacent corolla lobes, usually slightly protruding, with short filaments and inward anthers; pollen grains are oval, without spines; ovary 2 compartments, each Chamber 2 Ovule, style 2, completely separated or somewhat commissural, stigma globose or elongated. Capsule spherical or ovoid, sometimes slightly fleshy, perilobate or Irregular rupture. Seeds 1 to 4, glabrous; embryo in the fleshy endosperm, linear, disc-shaped curved or spiral, without cotyledons or thin With tiny scaly scars. There are about 170 species of plants in this genus, which are widely distributed in the warm temperate zone of the world, mainly producing America. Cuscuta plants are easy to spread with the host and its products. They are malignant weeds that can cause serious economic losses and are difficult to control. Therefore, Many countries in the world classify it as a prohibited or restricted pest.

5 General principles

The method established in this standard should not completely replace the morphological identification method, but should complement each other with the morphological identification method SN/T 1385. Mutual corroboration.

6 Principles of the method

According to the ITS sequence of the genus Cuscuta and the characteristics of the rbcL gene, the DNA barcoding technology was used to screen and identify the genus Cuscuta.

7 Instruments and reagents

7.1 Apparatus and utensils Conventional PCR instrument, micro spectrophotometer, electrophoresis instrument, gel imaging system, sequencer, high-speed refrigerated centrifuge, biological safety Cabinet, oven, autoclave, microscope, dissecting mirror, balance (1/10 000 g), mortar, shaker, water bath, ice maker, pure Water meter, vortex shaker, refrigerator, refrigerated mixing mill, micropipette (2 μL, 10 μL, 20 μL, 100 μL,.200 μL, 1 000 μL), centrifuge tube (2 mL), PCR reaction tube (0.2 mL, 0.5 mL). 7.2 Main reagents Unless otherwise specified, all reagents are analytical reagents or biochemical reagents. Plant genome extraction kit, ultrapure water, DNA Marker, agarose, nucleic acid gel dye, liquid nitrogen, deoxyribonucleoside Triphosphates (dNTPs), Taq polymerase, 10×PCR buffer. CTAB extraction reagent (see Appendix A in A.1).

8 Screening and identification methods

8.1 Sample processing Suspected Cuscuta plants and plant materials of similar genera that cannot be completely determined using morphological methods will require DNA extraction Plant materials (1~2 seeds for seeds, 10 mg for dry silica gel stems), put them into a 2 mL EP tube, and add 2 steel balls to each tube. After adding liquid nitrogen, grind for 3 min (30 r/s) with a refrigerated mixing mill. 8.2 Preparation of nucleic acid Use a commercial plant genome extraction kit or use a modified CTAB method to extract total plant genome DNA. improved For the CTAB method, refer to Appendix A in A.2. The ratio of OD260/OD280 of PCR-grade DNA solution should be 1.7～1.9. 8.3 Amplification of DNA barcode fragments Use the universal primers of ITS sequence and rbcL gene to amplify ITS sequence and rbcL gene, primer sequence, reaction program and reverse The response system is shown in Appendix B. 8.4 Detection of PCR products Take 5 μL of PCR product, 1.5% agarose gel electrophoresis, stain with nucleic acid gel dye, observe and take pictures in the gel imaging system. ITS primer amplification can obtain an amplified fragment of about 600 bp, and rbcL primer amplification can obtain an amplified fragment of about 700 bp. 8.5 Sequencing and sequence processing Take 45 μL of the remaining PCR amplification products and separate them by 1.5% agarose gel electrophoresis. Use DNA product purification reagents for the target fragments The cassette was purified and sequenced. Sequencing primers are the same as PCR amplification primers. Sequencing results are spliced and edited using sequence splicing software, the peak maps are compared, the sequence direction is judged, and the sequencing primer sequence is removed. End sequencing quality Q value less than 20 sequences are removed.

9 Results judgment

9.1 Judgment of China's quarantine pest DNA barcode identification system Navigate to the species identification page. After inputting the FASTA format (or plain text format) sequence in the authentication box, click "Submit Authentication", After the system performs BLAST, the identification result is automatically generated. For example, the 10 sequences with the highest similarity are Cuscuta sequences, and the highest similarity Above 98%, it can be clearly identified as Cuscuta (see Appendix C). 9.2 Judgment of plant pest quarantine and identification system Analysis software analyzes and compares the query sequence. By comparing the identification characteristics in the database, it will meet the identification characteristics of the group and the similarity is 100% of the species are judged to be this species. See Appendix D for the operation method of "Plant Pest Quarantine and Identification System". 9.3 Judgment of international general database Log in to the GenBank database BLAST identification system (https.//blast.ncbi.nlm.nih.gov/Blast.cgi), in the BLAST results View the species with the highest sequence similarity (Max ident or similarity), generally the species closest to the query sequence. Cuscuta is common See Appendix E for information on the gene sequence list of similar species. 10 Storage of voucher specimens and original data 10.1 Storage of voucher specimens The collected specimens should be stored in dry silica gel. The source of the goods, the host of the dodder, the collection time, location, and who collected it, and the storage period It is one year for re-inspection. If a trade dispute is involved, it should be kept until the dispute is resolved. After the shelf life expires, it needs to be sterilized. 10.2 Save the original data After the sample is tested, its original record sheet (including DNA sequence and sequencing peak diagrams, etc.) and inspection report or certificate should be returned Files and keep them properly for re-inspection, negotiation and arbitration.

Appendix A

(Informative) DNA extraction method (modified CTAB method) A.1 Reagents A.1.1 CTAB extract. CTAB 20 g/L, sodium chloride 1.4 mol/L, Tris 0.1 mol/L (pH 8.0), EDTA 0.02 mol/L (pH 8.0), Autoclave for later use. A.1.2 Trichloromethane. isoamyl alcohol. volume fraction 24.1. A.1.3 50×TAE buffer. 242 g Tris, 57.1 mL glacial acetic acid, 100 mL 0.5 mol/L EDTA (pH 8.0), dissolve and mix thoroughly After that, add water to make the volume to 1 L and autoclave for later use. A.1.4 10× loading buffer. 0.25% bromophenol blue, 40% sucrose. A.2 Extraction steps Add 600 μL of CTAB extract that has been preheated to 65 ℃ into a 2 mL centrifuge tube containing plant powder, shake and mix thoroughly; 65 ℃ Water bath for 30 minutes, mix upside down from time to time; centrifuge at 10 000 r/min for 5 minutes, take the supernatant, and use an equal volume of chloroform-isoamyl alcohol (chloroform. Isoamyl alcohol=24.1) Extract once; take the supernatant, add an equal volume of isopropanol, precipitate at 4 ℃ for more than 10 min, then 120...

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