SN/T 5212-2020Water pine identification method (English PDF)

3.1 Terms and definitions

The following terms and definitions apply to this document.

3.1.1

Seminiferous scale

The gymnosperm megaspore leaf ball is composed of megaspore leaves, arranged in a spiral on the longitudinal axis, the upper part is larger and the top part is called Seed scales, also called pearl scales or fruit scales. It is generally believed that the pearl scales develop from megaspore leaves.

3.1.2

Bract scale

The gymnosperm megaspore leaf ball is composed of megaspore leaves, arranged spirally on the longitudinal axis, and the smaller thin slices below are bract scales, generally the bracts are considered to be megaspores that have lost their reproductive capacity.

3.1.3

Terminal bud

The general term for the buds formed at the top of the stem. When the immature embryo undergoes polar differentiation, the apical buds are initially formed, and the apical buds are the most active growth.

Longer one.

3.1.4

Axillary bud

A kind of lateral buds, especially the fixed buds that emerge from the leaf axils.

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 Triphosphates (dNTPs), Taq polymerase, 10×PCR buffer, insect repellent, camphor extract, desiccant. Extract with modified CTAB method Reagents (see Appendix B).

8.1 Morphological identification method

Based on the morphological characteristics of Chinese fir family and water pine, the key to the genus of water pine and its related genera (see Appendix C), based on the morphology of seeds and fruits the key to the characteristic water pine and its similar genera and species (see Appendix D) is used to identify the species of suspected samples.

8.1.1 Morphological characteristics of Chinese fir family

8.1.1.1 Stem

Evergreen or deciduous trees, with straight trunks, large branches or nearly whorls.

8.1.1.2 Leaf

The leaves are arranged spirally, scattered, rarely cross-opposed (Metasequoia), lanceolate, subulate, scaly or striped, the same leaves on the same tree Type or Type II.

8.1.1.3 Ball flower

The cones are unisexual, monoecious, and the stamens and scales of the cones are spirally arranged, rarely cross-opposed (Metasequoia); the male cones are small, Single or-clustered branch tops, or arranged in panicles, or leaf axils, stamens with (2-9) (usually 3-4) anthers, pollen without Air sacs; female cones are terminal or born on last year's branch near the top of branch, pearl scale and bract scale semi-connate (only the top is separated) or completely connate, or pearl scale Very small (Cunninghamia lanceolata), or degenerated bract scales (Taiwan fir genus), with 2 to 9 erect or anatrophic ovules at the base of the ventral surface of the pearl scale.

8.1.1.4 Cones

The cones are mature in the year and open when they are ripe. The seed scales (or bract scales) are flat or shield-shaped, woody or leathery, spiral-shaped or cross-opposed (Metasequoia spp.), which will gradually fall off after persisting or ripening. The ventral surface of the breeding scale (or bract scale) has 2 to 9 seeds.

8.1.1.5 Seed

The seeds are flat or triangular, with narrow wings around or on both sides, or long wings at the lower part; the embryo has 2-9 cotyledons.

8.1.2 Morphological characteristics of water pine (see Appendix E)

8.1.2.1 Whole plant and stem

The arbor is 8 m~10 m high and 25 m high. Those born in a wet environment, the base of the trunk swells into a columnar trough shape, and there is a protruding soil surface or absorption roots on the water surface, the column groove is as high as 70 cm, the diameter of the dry base is 60 cm~120 cm, and the trunk has twisted patterns; the bark is brown or off-white with bands Brown, longitudinally split into irregular long strips; branches are sparse, large branches are nearly flat, and upper branches are oblique; short branches are more or more from the top buds of biennial branches the axillary buds of the annual branches protrude, 8 cm~18 cm long, and fall off in winter; the main branches extend from the perennial and biennial apical buds and do not fall off in winter.

8.1.2.2 Leaf

Leaf multi-type. thick scaly leaves or raised dorsal and abdomen, spirally born on the main branch of perennial or current year, about 2 mm long, white Stomatal points, do not fall off in winter; strip-shaped leaves are flat on both sides, thin, often arranged in two rows, apex is pointed, base is gradually narrow, 1 cm~3 cm long and wide 1.5 mm~4 mm, light green, with stomatal bands on both sides of the dorsal midrib; strip-shaped diamond-shaped leaves are flat on both sides, dorsal-ventral bulge rises, apex acuminate or obtuse, Slightly curved outwards, 4 mm~11 mm long, radiating out or in three rows; strip-shaped leaves and strip-shaped subulate leaves together with lateral spur branches in winter Fall off.

8.1.2.3 Cones

Cones obovate, 2 cm~2.5 cm long, 1.3 cm~1.5 cm in diameter; seed scales are woody, flat, obovate in the middle, wedge-shaped at the base, the apex is round, and there are 6 to 10 slightly outwardly reversed triangular pointed teeth near the edge of the back of the scale; the bract and seed scales are all conjoined, and only the apex is separated.

Triangular, curved outwards, located in the middle or upper middle of the back of the seed scales.

8.1.2.4 Seed

The seeds are oval, slightly flat, brown, 5 mm to 7 mm long, 3 mm to 4 mm wide, with long wings at the bottom, 4 mm to 7 mm long.

8.1.2.5 Seedlings

Cotyledons 4 to 5, strip needle-shaped, 1.2 cm to 1.6 cm long, less than 1 mm wide, without stomatal lines; primary leaves are strip-shaped, about 2 cm long, 1.5 mm wide, whorled, opposite or alternate, with small white spots on the main stem.

8.2 Molecular Biology Identification Method

8.2.1 Sample processing

Put the water pine plant materials that need to extract DNA (1 to 2 seeds, 100 mg for fresh leaves, 10 mg for dried silica gel leaves), Put it into 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, see Appendix F.

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

GenBank accession numbers of the rbcL gene sequence of P. chinensis. MF893589 and MF893590.

9.1 Determination of morphological results

Based on the morphological characteristics of water pine cones, seeds and leaves, it meets the morphological identification characteristics described in Chapter 8 and conforms to the genus and the key to its related genera and genus (see Appendix C), the key to the genus and similar species based on seed and fruit morphological characteristics (see Appendix D) the characteristics of mid-water pine can be identified as water pine.

9.2 Determination of molecular biology results

Log in to the GenBank database BLAST identification system (http.//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. When sequence check when the species with the highest rate is Glyptostrobus pensilis, it can be used as the identification basis for water pine. Gene Sequence List Information of Common Approximate Species of Taxodium See Appendix G.

10.1 Storage method

10.1.1 Specimen preservation

Press the cork pine plants detected in the identification into dry specimens, and put the cork pine seeds detected in the identification into the specimen bottle, mark them, and indicate the serial number, Chinese name, scientific name, department, place of origin, name of the goods, and date of entry and exit shall be properly stored after being signed by the person in charge.

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 cork samples should be properly stored for at least 6 months.

Appendix A

(Informative appendix)