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LY/T 3191-2020Technical regulations of DNA barcodes in woody species (English PDF)

林木DNA条形码构建技术规程

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Issued by

State Forestry and Grassland Administration

Level / Type

National · Recommended

Issue date

March 30, 2020

Implementation date

October 1, 2020

Scope

LY/T 3191-2020 is the English-translated version of 林木DNA条形码构建技术规程.

LY/T 3191-2020 — This standard specifies the sample collection strategy in the construction of forest tree DNA barcodes, the selection criteria for core DNA barcode fragments, and DNA extraction Technical requirements for technology and preservation, sequence determination and editing methods, and phylogenetic relationship construction. This standard applies to the construction of forest tree DNA barcodes.

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Document preview — LY/T 3191-2020

National Standard of the People's Republic of China

ICS: 65.020
Classification: B 60

Issued by: State Forestry and Grassland Administration

Foreword
1 Scope
2 Normative references
3 Terms and definitions
3.1 DNA barcodes
3.2 Primer
3.3 Polymerase Chain Reaction (PCR)
3.4 DNA sequence DNA sequence
3.5 DNA sequencing DNA sequencing technology
3.6 Phylogeny
3.7 Phylogenetic Analysis Using Parsimony (and Other Methods) (PAUP)
3.8 Cyberinfrastructure for Phylogenetic Research (CIPRES)
3.9 Molecular Evolutionary Genetics Analysis (MEGA)
3.10 Phylotools R package for DNA barcode phylogeny
4 Sample collection strategy
5 Selection criteria for core DNA barcode fragments
6 DNA extraction technology and preservation method
7 PCR amplification and sequencing
7.3 Sequence determination strategy
8 Sequence editing method
9 Method of phylogenetic relationship construction
9.1 Unweighted paired arithmetic average method
9.2 Least-squares (LS)
9.3 Neighbor-joining (NJ)
9.4 Maximum parsimony (MP)
9.5 Maximum likehood (ML)
9.6 Bayesian Inference

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009.

This standard was proposed by the State Forestry and Grassland Administration.

This standard is under the jurisdiction of the National Forest Seed Standardization Technical Committee (SAC/TC115).

Drafting organizations of this standard. Institute of Tropical Forestry, Chinese Academy of Forestry, Hunan Academy of Forestry, South China Plants, Chinese Academy of Sciences Garden, Institute of Botany, Chinese Academy of Sciences, College of Forestry, Jiangxi Agricultural University.

1 Scope

This standard specifies the sample collection strategy in the construction of forest tree DNA barcodes, the selection criteria for core DNA barcode fragments, and DNA extraction Technical requirements for technology and preservation, sequence determination and editing methods, and phylogenetic relationship construction.

This standard applies to the construction of forest tree DNA barcodes.

2 Normative references

The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this document.

For undated references, the latest version (including all amendments) applies to this document.

SN/T 4625-2016 DNA barcode screening and quality requirements SN/T 4626-2016 DNA barcode species identification operating procedures SN/T 4714-2016 DNA barcode database technical specifications

3 Terms and definitions

The following terms and definitions apply to this standard.

3.1 DNA barcodes

It is a short universal DNA sequence screened from mitochondria, chloroplasts, or nuclear genome regions, in order to compare existing organisms in species Fast and accurate identification and identification at the level.

3.2 Primer

It is a small piece of single-stranded DNA or RNA, which serves as the starting point for DNA replication. During the nucleic acid synthesis reaction, it enters as each polynucleotide chain.

A polynucleotide chain that functions as the starting point for line extension.

3.3 Polymerase Chain Reaction (PCR)

A method for enzymatically synthesizing specific DNA fragments in vitro, which consists of several steps of high-temperature denaturation, low-temperature annealing, and suitable temperature extension.

Cycles,, so that the target DNA can be rapidly amplified, with the characteristics of strong specificity, high sensitivity, simple operation and time-saving.

3.4 DNA sequence DNA sequence

The sequence of nucleotides in a polynucleotide chain. The only possible letters are A, C, G and T, which represent the four nucleosides that make up DNA.

Acids-adenine, cytosine, guanine, thymine; arranged together without gaps, such as the sequence AAAGTCTGAC.

3.5 DNA sequencing DNA sequencing technology

The determination of the sequence of nucleotides in a DNA molecule is to determine the sequence of A, T, G, and C that make up the DNA molecule.

3.6 Phylogeny

The origin, evolution history and genetic relationship of biological organisms over time.

3.7 Phylogenetic Analysis Using Parsimony (and Other Methods) (PAUP)

A software used to construct evolutionary trees (phylogenetic trees) and carry out related tests, including many molecular evolution models and methods, which can be beneficial Analyze molecular data (DNA, protein sequence), morphological data and other types of data using maximum likelihood method, parsimonious method, distance method, etc.

3.8 Cyberinfrastructure for Phylogenetic Research (CIPRES)

A remote supercomputer platform designed and opened for scientific research institutions and researchers, which can meet the operation requirements of huge amounts of data, and can greatly this saves time and cost and improves the reliability of the calculation results.

3.9 Molecular Evolutionary Genetics Analysis (MEGA)

A convenient and powerful free open source software for molecular evolution genetic analysis, which can complement other online public supercomputing platforms.

3.10 Phylotools R package for DNA barcode phylogeny

A software that can automatically arrange and compare large-scale DNA sequences, and can construct a super matrix based on multiple barcode fragments to obtain Take the phylogenetic relationship at the community level. The phylogenetic tree transformed by the non-parametric rate smoothing NPRS method (r8s) can be used for the corresponding community Phylogenetic analysis. In addition, according to the actual situation, it is possible to increase the comparison software such as MAFFT and other algorithms with faster running speed to obtain more accurate Accurate analysis results.

4 Sample collection strategy

4.1 Collection site. Clean mature leaves are best. Under special circumstances, materials such as branches, roots and bark phloem can be collected; avoid collecting Collect leaves that are bitten by insects and have pathogens.

4.2 Sample weight. 5-10 pieces of broad-leaved tree leaves, 5-10 grams of thin pinnate compound leaves or conifer leaves; enough for DNA extraction.

4.3 Number of samples. Common species are 3-5 individuals from different populations, and rare species should be collected as much as possible; to avoid false identification in samples Set species.

4.4 Sample number. Combine the species number and tree grade in the sample plot for numbering; when there are multiple individuals, they need to be distinguished; for example, camphor tree-1 (tree grade), Camphor tree-2 (tree grade). Record the collection time in detail.

4.5 Sample preservation. Put the material directly in the sealed bag and pour clean silica gel into the sealed bag; or put the material in an empty tea bag, no direct contact with silica gel to ensure that the material is not contaminated. If the material has high water content, pay attention to timely replacement of silica gel to ensure the quality of the material;

Under normal circumstances, the color of silica gel is dark blue. If it absorbs more water, the color of silica gel will change to light purple red. At this time, it needs to be replaced with a new one. When conditions permit, for some leaf materials that are not easy to extract DNA samples can be stored in cold storage.

4.6 Voucher specimens. Photos and information of the whole/part of the plant, macro-habitat/micro-habitat, and record the associated species and latitude and longitude. Voucher specimen Branches with flowers or fruits are required; when there are no flowers or fruits, branches with leaves are used. Record the signs of the trees and the samples on the ziplock pockets Numbering. Collect more than 3 voucher specimens for each sample point and each species; suppress them on the day after collection, and finally deliver them to the herbarium for storage.

5 Selection criteria for core DNA barcode fragments

5.1 Evolution rate. Fragments with a slower evolution rate can anchor the target species to a higher classification level of the family/genus, and fragments with a faster rate of evolution Then the closely related groups can be identified and distinguished.

5.2 Resolving power. There is obvious genetic variation between species, and the intra-species variation is small enough; if the fragment contains more variation sites, the resolution is high;

Fragments with a faster evolution rate have higher resolution than fragments with a slower evolution rate; combined fragments provide more mutation sites than individual fragments.

5.3 Fragment length. Short enough to reduce sequencing work, and facilitate DNA extraction and PCR amplification, especially for materials with DNA degradation Materials (such as long-preserved wax leaf specimens, processed folk medicinal materials); reduce sequencing costs; and minimize sequence length variation among different species.

5.4 Fragment versatility. Conserved regions exist to facilitate the design of universal primers. You can also screen from published genome and transcriptome data Choose appropriate DNA fragments as barcodes for specific species.

6 DNA extraction technology and preservation method

6.1 CTAB method. For a few species rich in secondary metabolites or oils (such as Lauraceae, Moraceae), increase before the CTAB procedure Ice bath step to eliminate the impact. See Appendix I for details.

6.2 Kit method. The spin column type is generally recommended, and the specific operation steps can be obtained on the instructions of the kit used.

6.3 DNA preservation method. After all the extracted DNA is tested by electrophoresis, part of it is stored in -80°C freezer for long-term storage.

Used, a part of it is placed in a refrigerator at -20°C for subsequent experiments.

7 PCR amplification and sequencing

7.1 PCR amplification reaction system (see the table below).

7.2 PCR amplification reaction strategy. According to the primer sequencing length, PCR amplification reaction strategies can be divided into two categories; the details are as follows.

7.3 Sequence determination strategy

ABI series automatic sequencer (Applied Biosystems, Foster City, California, USA) was used for sequencing. Sequencing primers using PCR Amplification primers. PCR products and corresponding primers must be provided when sequencing (primers must provide positive and negative directions). In order to verify the accuracy of sequencing or materials to the accuracy of pollution, each sequence is aligned in GenBank. See Appendix II for details.

7.4 Commonly used DNA barcode fragment sequencing primer pairs. For the research of community level forest tree DNA bar code, the main internationally used ones are rbcL, for primer pairs such as matK and ITS (or ITS2), trnH-psbA can be added as needed. See Appendix III for details.

8 Sequence editing method

9 Method of phylogenetic relationship construction

The use of plant DNA barcode data can be based on the amount of data, the genetic relationship of species, and the accuracy of the required phylogenetic tree.

Pieces, choose the appropriate paper mulberry method. Distance-based methods are generally faster in calculations than trait-based methods and are easy to apply to analysis Different types of data. See Appendix V for details.

9.1 Unweighted paired arithmetic average method

A more commonly used cluster analysis method was first used to solve classification problems. When used to rebuild a phylogenetic tree, its assumed front the condition is. in the process of evolution, each lineage has the same number of divergence, that is, the replacement rate of nucleotides or amino acids is equal and constant of. The phylogenetic tree produced by the UPGMA method is a simple embodiment of the species tree. This method is based on the assumption of a molecular clock and generates A rooted tree can be used for group data.

9.2 Least-squares (LS)

Take the pairwise distance matrix as the given data, and estimate the branch length on an evolutionary tree by matching those distances as close as possible, that is, pair the sum of squares of the difference between the predetermined and predicted distances is minimized.

9.3 Neighbor-joining (NJ)

A fast clustering method that does not require assumptions about molecular clocks and does not consider any optimization criteria. The basic idea is to merge classes at the time, not only the classes to be merged are required to be similar, but also the classes to be merged are required to be far away from other classes.

The star evolution tree is decomposed to continuously improve the star evolution tree.

9.4 Maximum parsimony (MP)

It is a method often used in phylogenetic analysis, according to discrete traits including morphological traits and molecular sequences (DNA, protein Quality, etc.), construct a phylogenetic tree of organisms, and analyze the evolutionary relationship between biological species. Under the concept of maximum parsimony, Biological evolution should follow the principle of parsimony. The evolution tree that requires the least number of mutations (the least number of evolutionary steps) may be the one that best fits the natural situation.

System tree. In the specific operation, it is divided into non-weighted maximum parsimony analysis (or called equal weighting) and weighted maximum parsimony analysis. The latter is based on According to the evolutionary law of the trait itself (for example, the evolution rate of different sites in DNA is different), it is weighted differently.

9.5 Maximum likehood (ML)

A relatively mature statistical method of parameter estimation, belongs to a class of phylogenetic tree reconstruction methods based entirely on statistical tree Representative, this method explicitly uses the probability model of nucleotide substitution, considers each probability in each set of sequence alignment, and looks for a higher Probability produces a phylogenetic tree of observed data. Currently, there are mainly some maximum likelihood tree construction programs and software such as PhyML and RA×ML.

9.6 Bayesian Inference

Known class conditional probability density parameter expression and prior probability, use Bayes formula to convert into posterior probability, according to the magnitude of posterior probability Make decision classification. Such as MrBayes software.

appendix

Appendix I (normative). The technical steps of CTAB method to extract plant DNA

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This preview omits tables, figures, formulas and parts of the technical clauses. The complete document — 8 pages — is available in the English PDF.

Referenced standards

Normative references

SN/T 4626-2016

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