SN/T 5214-2020Bole tree identification method (English PDF)

3.1 Terms and definitions

The following terms and definitions apply to this document.

3.1.1

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.

3.1.2

Axial placentation

The pistil is composed of multiple carpels. The carpels are connected to each other to form a central axis and a septum in the ovary. The number of ovary chambers is the same as the number of carpels.

The ovule is implanted on the central axis.

3.1.3

Raceme

A kind of infinite inflorescence. Its characteristic is that the flower axis is not branched, long, and there are many stalked florets from bottom to top, and the flower stalks of each floret, etc.

Long, flowering order from bottom to top.

3.1.4

Pinnate compound leaf pinnate A compound leaf with small leaves arranged in a feather shape on both sides of the leaf shaft is called a pinnate compound leaf.

3.1.5

Picolenticel

There are some crack-like protrusions visible to the naked eye on the surface of tree branches. It is a pore for the stem to exchange gas with the outside world.

3.2 Abbreviations

The following abbreviations apply to this document.

6.1 Equipment and appliances

Stereo microscope, electronic balance, magnifying glass, scalpel, dissecting needle, forceps, finger tube, petri dish, white porcelain plate, cotton, Sample bag, specimen holder, label, recording paper, specimen bottle, specimen box, conventional PCR instrument, micro spectrophotometer, electrophoresis instrument, gel Imaging system, high-speed refrigerated centrifuge, biological safety cabinet, oven, autoclave, 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, 1000 µL), centrifuge tube (2 mL), PCR reaction tube (0.2 mL, 0.5 mL).

6.2 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 triphosphate Acid (dNTP), Taq polymerase, 10×PCR buffer, insect repellent, camphor extract, desiccant, CTAB extraction reagent (see Appendix B).

8.1 Morphological identification method

According to the morphological characteristics of Bole tree, the identification key of Bole tree based on morphological characteristics of seeds and similar genera and species identification key (see Appendix C) and leaf Species identification of the suspected samples with the Bole tree and similar species retrieval table (see Appendix D and Appendix E) with morphological characteristics of the slice.

8.1.1 Morphological characteristics of Bole tree family

8.1.1.1 Leaf

Leaves alternate, odd-pinnate compound leaves; leaflets opposite or lower alternate, with small petioles, entire, pinnate veins; no stipules.

8.1.1.2 Flowers

The flowers are large, bisexual, symmetrical on both sides, forming a terminal, upright raceme; the calyx is broadly campanulate, 5-lobed; the petals are 5, separated, Arranged in a tile-like shape, unequal, the latter 2 are smaller, and are inserted on the upper part of the calyx; 8 stamens, connected at the base, are inserted on the lower part of the calyx, Slightly shorter than petals, filiform filaments, anthers backed; 1 pistil, ovary sessile, superior, 3~5 locules, axillary placenta, hanging in each ventricle There are 2 down-hanging ovules, the style is slightly longer than the stamens, and the stigma is capitate and small.

8.1.1.3 Fruits and seeds

The fruit is a capsule with 3-valved to 5-valved, thick, woody petals; large seeds.

8.1.2 Bole tree morphological characteristics (see Appendix F)

8.1.2.1 Whole plant and stem

Trees, 10 m-20 m high, gray-brown bark; branchlets have obvious lenticels.

8.1.2.2 Leaf

Odd-pinnate compound leaves are usually 25 cm~45 cm long, with sparse pubescent or glabrous axis; petiole length 10 cm~18 cm, lobules 7~15 Sheets, paper or leather, narrowly elliptical, rhomboid oblong, oblong-lanceolate or ovoid-lanceolate, somewhat oblique, 6 cm~26 cm long, 3 cm~9 cm wide, entire, apex acuminate or abruptly acuminate, base obtuse or short-pointed, wedge-shaped, leaf surface green, glabrous, leaf back powder green or off-white with pubescent hairs, often denser on both sides of the midrib and lateral veins; leaf veins are obvious on the back of the leaf, with 8 to 15 pairs of lateral veins; petiole long 2 mm~10 mm, without hair.

8.1.2.3 Inflorescence

The raceme is 20 cm~36 cm long; there are brown short hairs outside the total pedicel,, and calyx; the flower is light red, about 4 cm in diameter, the pedicel is 2 cm ~ 3 cm long; the calyx is about 2 cm in diameter, 1.2 cm ~ 1.7 cm long, with 5 short teeth on the top, sparsely pilose or glabrous on the inner surface, the petals are broad spatulate or obovate-wedge-shaped, rounded at the top, 1.8 cm~2 cm long, 1 cm~1.5 cm wide, hairless, with red longitudinal stripes on the inner surface; flowers the silk length is 2.5 cm~3 cm, with small pilose at the base; the ovary has bright, white pilose, and the style is pilose.

8.1.2.4 Fruit

The capsule is elliptic, subglobose or broad-ovate, 3 cm ~ 5.5 cm long, 2 cm ~ 3.5 cm in diameter, covered by very short brown hairs and often Mixed sparse white pilose, with or without obvious yellowish-brown nodules, fruit petal thickness 1.2 mm~5 mm; fruit stalk length 2.5 cm~3.5 cm, with or hairless.

8.1.2.5 Seeds

The seeds are oval, smooth, about 1.8 cm long and 1.3 cm in diameter when mature.

8.2 Molecular Biology Identification Method

8.2.1 Sample processing

Put the Bole tree plant material (100 mg of fresh plant tissue and 10 mg of dried silica gel leaves) from which DNA needs to be extracted into in a 2 mL EP tube, add 2 steel balls to each tube, add liquid nitrogen and grind for 3 min (30 times/s) with a refrigerated mixing mill.

8.2.2 Preparation of nucleic acid

Use a commercial plant genome extraction kit or use a modified CTAB method to extract total plant genome DNA. improved refer to Appendix B for the CTAB method.

The ratio of OD260/OD280 of PCR-grade DNA solution should be 1.7~1.9.

8.2.3 Amplification of DNA barcode fragments

Use universal primers for rbcL gene to amplify rbcL gene. For primer sequence, PCR reaction program and PCR reaction system, please refer to the attachment Record G.

8.2.4 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.

Amplification with rbcL primers can obtain an amplified fragment of about 991 bp.

8.2.5 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.

8.2.6 Standard DNA barcode

Bole tree rbcL gene sequence GenBank accession number. MG792130.

9.1 Determination of morphological results

Based on the morphological characteristics of fruits, seeds, and leaves, conform to the morphological identification characteristics described in Chapter 8 and conform to the characteristics based on the morphological characteristics of seeds.

The identification key of Bole tree and similar species (refer to Appendix C) and the key table of Bole tree and similar species based on leaf morphological characteristics (See Appendix D) The characteristics of the Zhongbo Le tree can be identified as the Bo Le tree.

9.2 Determination of molecular biology results

10.1 Storage method

10.1.1 Specimen preservation

The part of the Bole tree plant detected by the identification was pressed into a dry specimen, and the Bole tree seeds detected by the identification were put into the specimen bottle and marked Identification, indicate the serial number, Chinese name, scientific name, department, place of origin, name of goods, date of entry and exit, and keep it properly after the signature of the person who handles it.

10.1.2 Sample storage

Preserved samples are stored separately according to serial number, Chinese name, place of origin, entry and exit date, and are identified and confirmed by the handler and the sample manager After registration, keep it in a safe place.

10.2 Save time

The detected Bole Tree samples should be properly stored for at least 6 months.

Appendix A

(Informative appendix)