SN/T 5130-2019Identification of Oryza granulata Nees et Arn. Ex Hook.f (English PDF)

3.1 Terms and definitions

The following terms and definitions apply to this document.

3.1.1

Flaky, bristle-like, scaly. Spikelets are then arranged into spikes, fingers, racemes or panicles.

3.1.2

Also known as chaff, bran. Part of the spikelet is composed of neatly arranged epidermal cells with papillary protrusions, which are further divided into inner and outer ridges.

3.1.3

It is good for receiving sunlight, and because of its growing part, it can prevent insects, germs, rain, etc. from entering the leaf sheath tube. The leaf tongue is an extension of the leaf sheath, its size, shape, and presence or absence of furry can be used as one of the classification basis. Gramineous leaf tongue, common torn leaf tongue, two deep-lobed tongue, short Leaf tongue, eyelash tongue and long leaf tongue.

3.1.4

Its catalytic function is highly conserved, and its molecular structure has not changed much in the long-term evolution of organisms. It is a single copy and direct line in rice.

Homologous genes can be used as important indicators in biological genetic differentiation and molecular evolution research.

3.2 Abbreviations

The following abbreviations apply to this document.

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CTAB. cetyltriammonium bromide, cetyltrimethylammonium bromide.

PCR. polymerase chain reaction, polymerase chain reaction.

DNA. deoxyribonuleicid, DNA.

dNTP. deoxyribonucleidetriphosate, deoxynucleotide triphosphate.

Ta q enzyme. aquatic bacteria DNA polymerase.

6.1 Instruments and equipment

Liquid nitrogen tank, pure water machine, electronic balance, water bath, autoclave, high temperature drying box, centrifuge, electrophoresis apparatus, PCR instrument, electrophoresis tank, display Micromirror, scanning electron microscope, gel imaging system, high-throughput mill, vortex shaker, adjustable pipette 20µL, 10µL, 2.5µL), adjustable pipette tip, centrifuge tube.

6.2 Reagents

Reagents commonly used for PCR reactions, electrophoresis reagents, and nucleic acid molecular weight standards are all analytical or biochemically pure (unless otherwise specified).

Implementation according to GB/T 6682.

7.1 Morphological identification

7.1.1 Morphological characteristics of Oryza plants

Annual or perennial herbs. Culms erect, clustered.

7.1.1.1 Leaf

Leaf sheath glabrous; leaf tongue long membranous or auricular; leaf blade linear flat and broad.

7.1.1.2 Flowers

Terminal panicles develop loosely and often droop.

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7.1.1.3 Spikelets

Spikelets contain an amphoteric florets, with two degenerate lemmas attached below them, which are very flattened on both sides; glumes degenerate, showing only a bi-moon-shaped scar on the top of the spikelet Pregnancy outer scales of hard paper, with small wart spots or fine hairs, with 5 veins, with long awns or pointed tips; the inner and outer ridges are homogeneous, with 3 veins, and the side veins are close the margins are gripped by the 2 veins of the lemma; the scales are 2; the stamens are 6; the stigmas are 2, broombrush-shaped, protruding from both sides of the spikelet.

7.1.2 Morphological characteristics of Verrucous oryzae (see Appendix A and Appendix B)

Perennial herbs. Sometimes with short rhizomes. The culm is 30-70 cm high, flattened, with 5-9 knots.

7.1.2.1 Leaves

Leaf sheath glabrous, 5-8 cm long, shorter than internodes; leaf tongue 1--2 mm long, glabrous, with distinct leaf ears; leaves linear-lanceolate, long it is 5-20 cm wide and 6-20 mm wide, with jagged rough edges along the veins, smooth underside, curled inward when dry, pointed at the tip, and rounded at the base.

7.1.2.2 Flowers

The panicles are simple, erect, 3-12 cm long, with 2-5 branches, rising, sparsely spikelets, and rough edges.

7.1.2.3 Spikelets

Spikelets oblong, about 6 mm long, about 3 times wider, light green or gray; glazes degraded leaving only traces; hooked papillae on inner and outer condyles and there are warts around.

7.2 Molecular biology identification

7.2.1 DNA extraction

Extract DNA according to the CTAB method (see Appendix C for details).

7.2.2 PCR detection

PCR was performed on the DNA extracted from the sample (see Appendix D for details). Anti-pollution measures shall comply with the provisions of Sn/T 1193.

9.1 Sample preservation

For the sample preservation method of species resources, please refer to the provisions of GB/T 14072.

9.2 Results Recording and Data Storage

Complete experiment records include. sample name and number, source, type, time, time, place, method and result of the experiment.

Signed by the operator and the experimenter. Detection requires final experimental data and electrophoresis results. Raw data should be archived and properly protected Management for re-inspection, negotiation and arbitration.

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9.3 Review

The unit or person designated by the General Administration of Customs of the People's Republic of China is responsible. Mainly investigate the completeness of data such as experimental records and photos Authenticity, review experiments if necessary.

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Appendix A

(Informative appendix) Retrieval form for main species of Oryza 1b. Spikelets longer than 2 mm, stem nodes usually glabrous 2 2a. The lemma is awnless, with wart grains or hooked mastoids on the inner and outer lemma 3 2b. Outer palate with mantle or small pointed head, without wart grains or hooked mastoids 4 4a. Sterile lemma needles or setae 5 4b. Infertile epiphyseal linear or linear lanceolate 7 5b. Perennial, tall plants, spikelets wider than 1.8mm 6 7a. Spikelets with curved spikelet shafts. The junction of spikelets and branches is oblique.

7b. Spikelets have straight spikelet axis, the junction of spikelet and branch is flat, and there is no joint between lemma and awn 8a. Basal tongue usually shorter than 10 mm, apex rounded or truncate, inflorescence rarely having secondary branches 9 8b. Basal leaves usually tongue longer than 10 mm, apex pointed or lobed, inflorescence usually having secondary branches 10 10a. Annual or biennial, anthers usually shorter than 2.5 mm 11 10b. Perennial, anthers usually longer than 3 mm 13 11b. Wild species, spikelets shed at maturity, usually with awns 12 13b. Plants usually do not have rhizomes 14a. The stem at the base of the plant is brittle, and the spikelets are 6.6-11mm long. The anthers occupy 2/3-3/4 of the space in the floret 15b. Sometimes rhizomes, inflorescence axis smooth or ciliated 16 16a. Plants are tall and leaves are usually wider than 2cm 17 16b. Plants are relatively small, with leaves narrower than 2 cm 19 17b. The infertility is much shorter than the fertile flowers 18 19a. Base of inflorescence usually with 2 or more (3-5) similar branches 20 SN/T 51330-2019 ????? ????, ????,?

19b. Base of inflorescence usually without verticillate branches, usually 1–2 branches 22 20b. Plants cluster, awn usually longer than 2cm 21 22b. Plants without rhizomes, inflorescences partially spread, spikelets usually shorter than 6 mm 23b. Plants cluster, spikelets usually longer than 5mm 24 SN/T 51330-2019 ????? ????, ????,?

Appendix B

(Informative appendix) Characteristics of lemma papillae of main species in Oryza Note. The picture is from "Comparative Analysis of Micromorphological Characteristics of Oryza" Plants SN/T 51330-2019 ????? ????, ????,?

Appendix C

(Normative appendix) DNA extraction method C. 1 reagent C. 1.1 CTAB buffer 20 mmol/L EDTA, 100 mmol/L Tris-HC1 (pH8.0), 1.4 mmol/L NaCl, 0.1% beta-mercaptoethanol, 2% CTAB.

C. 1.2 TE buffer solution (PH8.0) Tris-HC1 at 10 mmol/L (pH 8.0) and EDTA at 1 mmol/L.

C. 1.3 Other reagents Chloroform. isoamyl alcohol (24. 1), isopropanol, 70% ethanol.

C. 2 Extraction steps C. 2.1 Take 0.1g powder sample, add 300µL CATAB buffer solution, grind it in a high-throughput mill to homogenate, and then add 300µL CATAB buffer solution, mix it upside down and transfer to a 1.5mL centrifuge tube; Hours, cool in a refrigerator at 4 ° C to below 15 ° C.

C. 2.2 Add 250 µL of chloroform. isoamyl alcohol (24. 1), mix up and down for 5 minutes, mix thoroughly, centrifuge at 2,000 rpm for 10 min at room temperature, transfer 300 µL of supernatant to another centrifuge tube pre-loaded with.200 µL of isopropyl alcohol , Gently mix upside down, place 30min at 4 ° C, 1200rpm, centrifuge at 4 ° C for 10min, discard the supernatant.

C. 2.3 The precipitate was washed with 70% ethanol, centrifuged again, the ethanol was decanted, the DNA was dried, and 100 µL TE buffer solution was added to dissolve the DNA. The water was incubated at 37 ° C for 30 min or at room temperature for 1 h.

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Appendix D

(Normative appendix) PCR detection method D. 1 Primer synthesis Primer1 5'-TCAGCCCCCCCTCTATTGTTGTC-3C Primer2 5'-TGTGCGAGCACACACATACCT-3 ' Primer3 5'-CATGGTGTAGAGCGCACGAAAT-3 ' Primer4 5'-GCTTAATAGAGGCGAGAGCG-3 ' the first round of amplification was performed using genomic DNA as a template and Primer1/Primer2 as primers. The amplified product was diluted 30-fold to make as a template, Primer3/Primer4 was used as a primer for the second round of amplification, and the PCR product size was 163bp, which is a specific expansion for verruca wild rice.

Primers.

D. 2 PCR reaction

In this method, the total volume of the PCR reaction is 25 µL, and the reaction system is shown in Table D. 1.

Table D. 1

The original component concentration 25µL reaction system loading volume (µL) Ultra-pure water 17.4 10 × PCrbufer 2.0 dNTPs 2.5mmmol/L 1.6 Forward primer 10µmol/L 0.8 Reverse primer 10µmol/L 0.8 Tazyme 2.5U/µL 0.4 DNA 25ng/µL 2.0 Primer1/Primer2 reaction conditions are. denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 30s, annealing at 55 ° C for 30s, and extension at 72 ° C.

1 min, 30 cycles, 72 min extension at 10 ° C, and stored at 4 ° C.

Primer3/Primer4 reaction conditions are. denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 30s, annealing at 60 ° C for 30s, and extension at 72 ° C.

30s, 24 cycles, 72min extension for 10min. Results of 2% agarose gel electrophoresis.

The DNA of verruca rice leaves was used as a positive control, the fake rice leaf DNA was used as a negative control, and the sterilized double-distilled water was used as a blank control.

D. 3 agarose electrophoresis Mix TAE and electrophoresis-grade agarose at 2%, add SBRGRENEN nucleic acid dye to a final concentration of 0.5 µg/mL, mix well, and make gel. Use an appropriate amount of 6 × loading buffer to mix with 10 µL of the sample, and then add it to the appropriate DNA molecular weight standards into the sample wells, and electrophoresis. At the end, place the entire gel on a gel imager and observe.

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D. 4 Results observation Through imaging observation, if a corresponding size product band appears at 163bp, it can be judged as positive (the test sample is verruca rice) no amplification band appeared, and it was judged as negative.

The test should be performed in parallel, and the results of two parallel test samples should be consistent. If the result of a test sample is positive when the other is negative, the test should be repeated. Can increase the amount of DNA template in PCR reaction to make two parallel test samples the results are consistent. The detected target fragment was amplified and the PCR product was confirmed. All the control results were normal and the result was determined to be positive;

The detected target fragments did not appear amplified, and all the control results were normal, and the results were determined to be negative.

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references

[1] Fan Shuguo, Zhang Zaijun, Liu Lin, et al. History and current status of taxonomic studies on Oryza. Wuhan Plant Research, 2000, 18 (18).

328-337.

[2] Lu Baorong, Ge Song, Sang Tao, et al. The current status and problems of the classification of Oryza. Journal of Plant Taxonomy, 2001, 39 (4). 373-388.

[3] Zhang Wenxu, Pei Xinde. Study on bimodal mastoids of rice noodles. Journal of Crop Science, 1998, 24 (6). 691-679.

[4] Zhang Wenxu, Tang Shengxiang. Scanning electron microscopy observation of exocondylar papilla of 20 species of Oryza. Chinese Journal of Crops, 1997, 3 (3). 280-289.

[5] Tang Shengxiang, Zhang Wenxu. Electron microscopic observation of the outer lamina structure of three wild rice grains in China. Journal of Plant Genetic Resources, 2003, 4 (2).

134-136.

[6] Song Yun, Chen Yan, Xu Yan, et al. Sequence analysis of single-copy nuclear gene TPI of Oryza and its application in identification of verruca wild rice. biological Technical Bulletin, 2012, 22 (12). 76-81.

[7] Xu Zhonggen. Comparative analysis of micromorphological characteristics of Oryza plants. Yangzhou University, 2010.

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