SN/T 5213-2020Agarwood identification method (English PDF)

3.1 Terms and definitions

The following terms and definitions apply to this document.

3.1.1

Inflorescence

The arrangement of flowers on the stalk. The simplest form of the inflorescence is a solitary flower. If there are multiple flowers arranged on the axis of the inflorescence, the inflorescence the type of inflorescence can be divided into infinite inflorescence and limited inflorescence.

3.1.2

Capsule

The fruit developed from the compound pistil of the connate carpel, the ovary has one or more compartments, and each compartment has multiple seeds.

3.2 Abbreviations

The following abbreviations apply to this document.

6.1 Instruments

Stereo microscope, electronic balance, autoclave, pH meter, constant temperature water bath, mortar, pure water meter, vortex oscillator, liquid nitrogen Tank, high-speed refrigerated centrifuge, nucleic acid protein analyzer, fluorescent quantitative PCR amplification instrument, adjustable pipette (1 000 µL,.200 µL, 20 µL, 10 µL, 2.5 µL) and so on.

6.2 Main reagents

CTAB buffer, phenol, chloroform, isoamyl alcohol, isopropanol, ethanol, RNase A, liquid nitrogen, real-time fluorescent PCR master mix, Primers and probes (see Table A.1 in Appendix A), unless otherwise specified, are of analytical or biochemical purity, and water is in accordance with GB/T 6682 Implementation.

7.1 Morphological methods

7.1.1 Morphological characteristics of the plant of Ruixiangceae

7.1.1.1 Deciduous or evergreen shrubs or small trees, rare herbs; stems usually have bast fibers.

7.1.1.2 Leaves. single leaves alternate or opposite, leathery or papery, sparsely herbaceous, margin entire, base jointed, pinnate veins, with short leaves Stem, without stipules.

7.1.1.3 Flower. The flowers are radiatively symmetrical, bisexual or unisexual, monoecious or different, head-shaped, spike-shaped, raceme, panic or umbel, Sometimes solitary or clustered, terminal or axillary; calyx is usually crowned, white, yellow or light green, sparse red or purple, often connected Bell-shaped, funnel-shaped, tube-shaped calyx tube, outer hairy or glabrous, lobes 4 to 5, arranged in a tile-like shape in the bud; petals missing, or Scale-like, the same number as the calyx lobes; stamens are usually twice or the same number as the calyx lobes, diminished to 2, mostly opposite to the lobes, or in another round with Lobes alternate, anthers ovate, oblong or linear, 2-chambered, split inward, sparsely laterally split; disks are ring-shaped, cup-shaped or scaly, rare;

Ovary upper, 2~5 carpels connate, sparse 1, 1 compartment, sparse 2 compartments, each room has 1 overhanging ovule, rare 2~3 ovules, close to the apex of the chamber is inverted, the style is long or short, terminal or near terminal, sometimes laterally, and the stigma is usually capitate.

7.1.1.4 Fruits. berries, stone fruits or nuts, dilute to 2-petal cracked capsules, with membranous, leathery, woody or fleshy skin.

7.1.1.5 Seed. The seed is drooping or anaphase; endosperm is abundant or has no endosperm, the embryo is erect, the cotyledon is thick and flat, and slightly raised.

7.1.2 Morphological characteristics of Agarwood plants

7.1.2.1 Trees or small trees.

7.1.2.2 Leaves. The leaves are alternate, with slender closed parallel veins.

7.1.2.3 Flowers. Flowers bisexual, axillary or terminal, usually composed of sessile or pedunculate umbels, without bracts; calyx tube campanulate, persistent, 5 lobes, extended, petals reduced to scales, 10, base united into a ring, inserted in the throat of the calyx, densely tomentose; stamens 2 Double the size of the sepal lobes, interspersed with scaly petals, very short filaments, anthers oblong, backed, and wide; the lower disk does not exist; the ovary is nearly absent Stem, coat, fully or incompletely 2-loculed, stigma capitate, style very short.

7.1.2.4 Fruit. Capsule stalked, flattened on both sides, obovate, and dorsal cracked; the fruit is leathery or woody, and the base is covered by the persistent calyx tube.

7.1.2.5 Seeds. Seeds are oval or elliptical, with tail-like appendages at the base, crisp seed coats, no endosperm, and embryos with thick, flat and concave cotyledons.

7.1.3 Morphological characteristics of Agarwood

7.1.3.1 Tree, dark gray bark, smooth, tough fiber; branchlets cylindrical, crepe, sparsely pilose when young, and then gradually shed Falling, glabrous or nearly glabrous (see Appendix B and Appendix C).

7.1.3.2 Leaf. The leaf is leathery, round, oval to oblong, sometimes nearly obovate, 5 cm~9 cm long, 2.8 cm~6 cm wide, first the tip is sharp or sharp and has a short pointed head, the base is wide and wedge-shaped, the top is dark green or purple-green, bright, the bottom is light green, both sides are hairless, side Veins 15-20 on each side, more obvious below, small veins are slender, nearly parallel, inconspicuous, and the edges are sometimes sparsely pilose; petioles are about 5 mm~7 mm, coat.

7.1.3.3 Flowers. fragrant flowers, yellow-green, multiple flowers, forming umbels; pedicels 5 mm-6 mm long, densely yellow-gray pubescent; calyx Tube shallow bell-shaped, 5 mm~6 mm long, densely pubescent on both sides, 5-lobed, lobes ovoid, 4 mm~5 mm long, apex rounded or obtuse Pointy, pubescent on both sides; petals 10, scaly, inserted in the throat of the calyx tube, densely hairy; stamens 10, arranged in 1 round, filaments about 1 mm, anthers oblong, about 4 mm long; ovary ovate, densely covered with off-white hairs, 2 locules, 1 ovule per locule, style very short or absent, column Head shape.

7.1.3.4 Fruit. Capsules have short stems, ovoid, green when young, 2 cm to 3 cm long, 2 cm in diameter, short pointed at the top, and gradually at the base.

Narrow, densely yellow pubescent, 2-valved, 2-chambered, with 1 seed per chamber.

7.1.3.5 Seeds. seeds are brown, ovoid, about 1 cm long and 5.5 mm wide, sparsely pilose, with appendages at the base, and appendages the length is about 1.5 cm, the upper end is wide and flat, the width is about 4 mm, and the lower end is shank-shaped.

7.2 Molecular biology methods

7.2.1 DNA extraction

Extract DNA according to the CTAB method, see Appendix A, or use a commercial DNA extraction kit.

7.2.2 Real-time fluorescent PCR identification method

See Appendix A for the real-time fluorescent PCR detection method of Agarwood, and the anti-pollution measures should comply with SN/T 1193.

7.2.3 Quality control

Positive control. Ct value is less than or equal to 35;

Negative control. Ct value is greater than or equal to 40;

Blank control. Ct value is greater than or equal to 40;

If one of the above indicators does not meet, the real-time fluorescent PCR amplification should be repeated.

9.1 Sample storage

See GB/T 14072 for the sample preservation method of species resources.

9.2 Results recording and data preservation

The complete experiment record includes. the name and number of the sample, source, type, time, time, location, method and result of the experiment Fruits, etc., and must have the signatures of the handlers and experimenters. Bioassays must have characteristic photos for classification and identification, and molecular biology tests must have the final experimental data. The original data should be archived and kept properly for re-inspection, negotiation and arbitration.

9.3 Review

The review work is in charge of the unit or personnel designated by the General Administration of Customs, which mainly inspects the completeness and truthfulness of experimental records, photos and other materials If necessary, experts will review the test results.

Appendix A

(Normative appendix) Real-time fluorescent PCR identification method A.1 DNA extraction A.1.1 Freeze the sample with a ball mill or mortar liquid nitrogen and grind it, weigh 50 mg-100 mg into a 1.5 mL sterile centrifuge tube, add Add 700 µL CTAB buffer, shake and mix, place in a 65 °C warm bath for 60 mg~90 min, keep mixing during the period; add after cooling 5 µL of RNase solution, put in a warm bath at 37 °C for 30 min.

A.1.2 Add an equal volume of phenol. chloroform. isoamyl alcohol (25.24.1), oscillate uniformly, 12000 r/min, and centrifuge for 15 min.

A.1.3 Take the supernatant and put it in another 1.5 mL new tube, add an equal volume of isopropanol, gently invert 3 to 5 times, and place in a refrigerator at -20 °C Leave it in the middle for 40 minutes.

A.1.4 Centrifuge at 12000 r/min for 10 min, and discard the supernatant.

A.1.5 Add 400 µL of 70% ethanol solution to wash, centrifuge at 12,000 r/min for 1 min, discard the supernatant, and dry the centrifuge tube at room temperature for 15 min.

A.1.6 Add 50 µL~100 µL TE or sterile deionized water, place in a 65 °C water bath for 40 minutes to fully dissolve the DNA, and measure the concentration of DNA.

After temperature and purity, store in the refrigerator at -20 °C for later use.

Note. The kit can also be used for DNA extraction, and the specific method is in accordance with the kit instructions.

A.2 Determination of DNA purity and concentration Measure the purity and concentration of DNA with a nucleic acid protein analyzer, obtain absorbance values at 260 nm and 280 nm, respectively, and calculate nucleic acid for purity and concentration, see formula (C.1) and formula (C.2).

DNA purity=OD260/OD280 (C.1) DNA concentration=50×OD260 µg/mL (C.2) the ratio of OD260/OD280 of PCR-grade DNA solution should be 1.7~2.0.

A.3 Real-time fluorescent PCR detection A.3.1 Primer probe sequence the sequence of primer probes identified by Agarwood real-time fluorescent PCR method is shown in Table A.1.

Note. The 5'end of the probe is labeled with FAM reporter fluorescent dye, and the 3'end is labeled with BHQ1 quenching fluorescent dye.

A.3.2 Testing system the detection system is shown in Table A.2.