An SSR molecular marker and its application in detecting Napier grass

Abstract

This invention relates to the field of plant molecular biology, and more particularly to an SSR molecular marker and its application in the detection of *Phragmites australis*. The invention provides primer pairs for detecting the SSR molecular marker, comprising: *P. setaceum*-1F: 5'-TCTGCAGAATGAGACGGAGA 3'; *P. setaceum*-1R: 5'-ATGTGCCATGCCATAATCAA 3'. The SSR molecular marker provided by this invention can be used for the identification of *Phragmites australis* varieties. PCR amplification using the primer pairs provided by this invention allows for the determination of whether the sample is *Phragmites australis*, and the specific species, based on the amplification results. The SSR molecular marker and primer pairs provided by this invention are of significant importance in aiding in the identification of *Phragmites australis* varieties.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and in particular to an SSR molecular marker and its application in the detection of Napier grass. Background Technology

[0002] Pennisetum alopecuroides, a species of grass belonging to the genus Pennisetum in the Poaceae family, is widely distributed and is an excellent forage grass, ornamental grass, and raw material for papermaking. Pennisetum alopecuroides boasts a beautiful plant shape, attractive inflorescences, and colorful leaves. In landscaping, it can be planted individually, in clumps, in groups, or in rows to create various landscaping effects, resulting in excellent aesthetic appeal and high ornamental value. It also possesses advantages such as strong adaptability, drought tolerance, and low maintenance costs. However, because different varieties of Pennisetum alopecuroides have similar morphologies at certain growth stages, morphological identification is difficult, leading to confusion in the market regarding variety names, and the existence of synonyms and homonyms. Furthermore, the authenticity and purity of varieties in the market are inconsistent, resulting in varying landscape effects and disrupting the normal market order.

[0003] In recent years, SSR fingerprinting has been widely used for the identification and classification of cultivated plant varieties, offering advantages such as high efficiency and accuracy in variety identification at the DNA level. Molecular markers are genetic markers based on nucleic acid polymorphism, unaffected by tissue type, developmental stage, or environmental conditions. They possess advantages such as high quantity and high polymorphism, making them ideal genetic markers. Molecular markers have been widely applied in germplasm resource research, hybrid identification, genetic map construction, target gene localization, and genetic diversity research. Among them, microsatellites, or simple sequence repeats (SSRs), are simple repetitive sequences uniformly distributed throughout the genome, consisting of 2-6 tandem repeat segments of nucleotides. They possess advantages such as high polymorphism, good stability, multiple alleles, codominance, abundant quantity, good genome coverage, and ease of operation. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an SSR molecular marker and its application in the detection of Napier grass.

[0005] This invention develops SSR markers through transcriptome sequencing of 'purple-leaf' Pennisetum setaceum 'Rubrum', and screens out a pair of primers with high polymorphism, which can be used to identify multiple Pennisetum varieties with a discrimination rate of 100%.

[0006] Specifically, the SSR molecular marker is located on the ATM gene, and the repeating sequence is a GCT triple repeat, repeated 5 times.

[0007] In a first aspect, the present invention provides a primer pair, comprising:

[0008] P.setaceum-1F: 5'-TCTGCAGAATGAGACGGAGA 3'

[0009] P. setaceum-1R: 5'-ATGTGCCATGCCATAATCAA 3'.

[0010] The present invention further provides a kit comprising the primer pair.

[0011] Secondly, the present invention provides a method for detecting *Phragmites australis*, comprising:

[0012] DNA is extracted from the sample to be tested, and PCR detection is performed using the primer pair or the kit described herein.

[0013] Furthermore, the PCR detection system includes the following components:

[0014] The total volume is 10 μL, including 4–6 μL of 2×Taq Master Mix, 0.4–1 μL of forward primer, 0.4–1 μL of reverse primer, and 0.5–1.5 μL of DNA, with the remainder being ddH2O.

[0015] Furthermore, the PCR detection procedure includes the following:

[0016] Pre-denaturation at 94℃ for 2–4 minutes; denaturation at 94℃ for 25–45 seconds, annealing at 56℃ for 25–45 seconds, extension at 72℃ for 1–1.5 minutes, repeat 35 times; extension at 72℃ for 10 minutes.

[0017] Furthermore, the method also includes determining whether the sample to be tested is Napier grass, and the specific Napier grass variety, through PCR detection results.

[0018] Furthermore, the PCR detection results include: all bands obtained from PCR amplification and their location information.

[0019] For example, the primer P. setaceum-1 provides a unique amplification result for each species of Napier grass, and the amplification results are different between each species. By recording the band information of each Napier grass species, and then comparing the detection results of the samples to be tested in sequence, the Napier grass species can be identified.

[0020] Furthermore, the varieties of Pennisetum include Pennisetum, Purple Pennisetum, Beauty Pennisetum, White-tipped Pennisetum, Jingcai Pennisetum, Feathered Pennisetum, Purple-leaved Pennisetum, Purple Elephant Grass, Snowy Pennisetum, Fluffy Pennisetum, Long-spike Pennisetum, Dwarf Pennisetum, Little Rabbit Pennisetum, Hahailin Pennisetum, Red Elephant Grass, East African Pennisetum, Purple-spike Pennisetum, Jinghong Pennisetum, or Liqiu Pennisetum.

[0021] The present invention has the following beneficial effects:

[0022] This invention develops an SSR marker from the transcriptome of 'Purple Leaf' Napier grass and screens for a pair of primers with high polymorphism, which can be used for the identification of Napier grass varieties. The primer pair provided by this invention can be used for the identification of multiple Napier grass varieties with a discrimination rate of up to 100%.

[0023] The method for detecting Napier grass based on this primer pair is simple to operate, inexpensive, highly accurate, and versatile, and can significantly improve the efficiency of identifying Napier grass varieties. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is a non-denaturing polyacrylamide gel electrophoresis image of 19 species of Napier grass using primer P. setaceum-1 provided in Example 1 of this invention;

[0026] Where A is the electrophoresis diagram and B is the E-electrophoresis diagram; the numbers 1-19 on the horizontal axis represent the varieties of Napier grass (numbers 1-19), and the numbers 100, 200, 300, and 400bp on the vertical axis represent the length marked by Maker.

[0027] Figure 2is the identification fingerprint and molecular identity of 19 Pennisetum species obtained by using the primer pair P.setaceum-1 provided in Example 1 of the present invention; the numbers 1-19 on the ordinate represent Pennisetum samples (Nos. 1-19), and the numbers on the abscissa represent the lengths of alleles; white squares indicate the presence of amplified bands, black squares indicate the absence of amplified bands, which are represented as "1" and "0" respectively in the binary molecular identity; the decimal molecular identity is converted from the binary molecular identity; 1, Pennisetum alopecuroides, 2, Pennisetum alopecuroides 'Ziguang', 3, Pennisetum alopecuroides 'Liren', 4, Pennisetum alopecuroides 'Baijian', 5, Pennisetum alopecuroides 'Jingcai', 6, Pennisetum alopecuroides 'Yurong', 7, Pennisetum alopecuroides 'Ziye', 8, Pennisetum purpureum, 9, Pennisetum alopecuroides 'Xuerong', 10, Pennisetum alopecuroides 'Rongmao', 11, Pennisetum alopecuroides 'Changsui', 12, Pennisetum alopecuroides 'Aizhu', 13, Pennisetum alopecuroides 'Xiaotuzi', 14, Pennisetum alopecuroides 'Hahailin', 15, Pennisetum purpureum 'Red', 16, Pennisetum alopecuroides 'Dongfei', 17, Pennisetum alopecuroides 'Zisui', 18, Pennisetum alopecuroides 'Jinghong', 19, Pennisetum alopecuroides 'Liqiu'. Detailed Description of the Embodiments

[0028] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described clearly and completely below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1

[0030] 1. Materials and Methods

[0031] 1.1 Plant Materials

[0032] Nineteen Pennisetum species were sampled from the National Precision Agriculture Demonstration Base, Anhui and Yunnan, including Pennisetum alopecuroides, Pennisetum alopecuroides 'Ziguang', Pennisetum alopecuroides 'Liren', Pennisetum alopecuroides 'Baijian', Pennisetum alopecuroides 'Jingcai', Pennisetum alopecuroides 'Yurong', Pennisetum alopecuroides 'Ziye', Pennisetum purpureum, Pennisetum alopecuroides 'Xuerong',

[0033] Pennisetum alopecuroides 'Rongmao', Pennisetum alopecuroides 'Changsui', Pennisetum alopecuroides 'Aizhu', Pennisetum alopecuroides 'Xiaotuzi', Pennisetum alopecuroides 'Hahailin', Pennisetum purpureum 'Red', Pennisetum alopecuroides 'Dongfei', Pennisetum alopecuroides 'Zisui',

[0034] Pennisetum alopecuroides 'Jinghong', Pennisetum alopecuroides 'Liqiu'.

[0035] Table 1 Details of 19 Pennisetum materials

[0036]

[0037]

[0038] 1.2 CTAB extraction of genomic DNA

[0039] Genomic DNA was extracted from 19 plant samples using the CTAB method. Young leaves of the plant samples were ground with 700 μL of CTAB solution and vortexed; then incubated at 65°C for 30 minutes; 350 μL of chloroform / isoamyl alcohol (chloroform:isoamyl alcohol = 24:1) and 350 μL of saturated phenol were added, and the mixture was incubated on ice for 10 minutes; the mixture was centrifuged at 12000 rpm for 10 minutes at 4°C; the supernatant was discarded, and an equal volume of 70% ethanol was added, followed by centrifugation at 12000 rpm for 5 minutes at 4°C; this step was repeated, discarding the supernatant, adding an equal volume of 70% ethanol, and centrifuging at 12000 rpm for 5 minutes at 4°C; the mixture was then air-dried and 30 μL of water was added. The extracted DNA concentration was determined and uniformly diluted to 20 ng / μL, and stored at -20°C.

[0040] 1.3 SSR Analysis

[0041] This invention utilizes second-generation transcriptome sequencing of *P. setaceum 'Rubrum'* to identify SSR loci. Fifty SSR markers were randomly selected, and primers were synthesized. Materials with significant phenotypic differences were screened to identify eight pairs of SSR primers with clear bands, high stability, and good polymorphism, which were then used in subsequent experiments. These primer pairs can be used for variety identification or fingerprinting of *P. setaceum*. Further experiments revealed that among these eight polymorphic molecular marker pairs, one primer pair, *P. setaceum-1* (F / R), could directly identify 19 *P. setaceum* species, demonstrating high identification efficiency.

[0042] The specific sequence of primer P. setaceum-1 (F / R) is as follows:

[0043] P.setaceum-1(F):5'-TCTGCAGAATGAGACGGAGA-3'

[0044] P.setaceum-1(R):5'-ATGTGCCATGCCATAATCAA-3';

[0045] 1.3.1 PCR reaction

[0046] PCR amplification was performed on 19 samples of *P. setaceum* using primer P. setaceum-1, which was synthesized by Ribotech Biotechnology Co., Ltd. (Beijing, China).

[0047] The optimized 10 μL PCR reaction system consisted of 5 μL 2×Taq Master Mix (Tiangen, Beijing, China), 0.6 μL forward primer, 0.6 μL reverse primer, 1 μL DNA, and 2.8 μL ddH2O.

[0048] The PCR reaction program was as follows: 94℃ pre-denaturation for 3 minutes, 94℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 1 minute, repeated 35 times with the 94℃ denaturation for 30 seconds followed by a 72℃ extension for 1 minute, and a final extension at 72℃ for 10 minutes. After PCR amplification, the PCR products were electrophoresed on an 8.0% non-denaturing polyacrylamide gel at 170V for 80 minutes. A 100bp marker (TransGold, Beijing, China) was used for labeling.

[0049] 1.3.2 The preparation steps of polyacrylamide gel are as follows:

[0050] Lay the plate flat, align the glass plates and secure them with clips; measure 8 mL of 40% acrylamide gel stock solution, 4 mL of 10×TBE buffer, 28 mL of pure water, add 40 μL of TEMED and 200 μL of 20% APS solution, mix thoroughly and pour the gel, insert the toothed side of the comb, and let it solidify for 30-40 minutes; after the gel has solidified, remove the clips securing the glass plates and fix the glass plates to the vertical electrophoresis tank.

[0051] 1.3.4 Electrophoretic separation

[0052] Add 1×TBE buffer to each electrophoresis tank until it covers the concave plate, and clean the impurities on the gel surface; remove the comb, and use a pipette to sequentially spot the PCR amplification products into the sample wells; electrophoresis for 80 minutes at 170V.

[0053] 1.3.5 Rapid Silver Staining Detection Using the Weak Alkali Method

[0054] Add 0.6g of silver nitrate to 800mL of water and stain for 13min. Discard the silver stain. Rinse 2-3 times with 800mL of distilled water, averaging 30s each time. Shake in 8g NaOH, 800mL of distilled water and 8mL of formaldehyde until developed. Cover the film with a resealable bag and take a picture.

[0055] 2. Results and Analysis

[0056] 2.1 Statistical band type

[0057] The results are as follows Figure 1 As shown, the primer P. setaceum-1 was read in Figure 1The banding pattern at different lengths shown in this invention is illustrated, where "0" indicates no amplification site and "1" indicates the presence of an amplification site. This is related to the binary molecular identity cards of 19 species of *Phragmites australis* (e.g.,...). Figure 2 The variety was identified by comparing the results shown in the image.

[0058] 2.2 Identification of 19 species of Napier grass using a single primer pair

[0059] The results are consistent with traditional classification methods. Through DNA extraction, SSR analysis, and variety identification, 19 species of Napier grass can be quickly and accurately identified.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer pair, characterized in that, include: P.setaceum -1F:5'-TCTGCAGAATGAGACGGAGA 3' P.setaceum -1R:5’-ATGTGCCATGCCATAATCAA 3’。 2. A reagent kit, characterized in that, The kit includes the primer pair as described in claim 1.

3. A method for detecting Napier grass, characterized in that, include: DNA is extracted from the sample to be tested and PCR detection is performed using the primer pair described in claim 1 or the kit described in claim 2.

4. The method according to claim 3, characterized in that, The PCR detection system includes the following components: The total volume is 10 μL, including 4–6 μL of 2×Taq Master Mix, 0.4–1 μL of forward primer, 0.4–1 μL of reverse primer, and 0.5–1.5 μL of DNA, with the remainder being ddH2O.

5. The method according to claim 3 or 4, characterized in that, The PCR detection procedure includes the following: Pre-denaturation at 94℃ for 2-4 minutes; denaturation at 94℃ for 25-45 seconds, annealing at 56℃ for 25-45 seconds, extension at 72℃ for 1-1.5 minutes, repeat 35 times; extension at 72℃ for 10 minutes.

6. The method according to claim 3, characterized in that, Also includes: The PCR test results are used to determine whether the sample to be tested is Napier grass, and the specific variety of Napier grass.

7. The method according to claim 6, characterized in that, The PCR detection results include: all bands obtained from PCR amplification and their location information.

8. The method according to claim 6 or 7, characterized in that, The varieties of Pennisetum include Pennisetum, Purple Pennisetum, Beauty Pennisetum, White-tipped Pennisetum, Jingcai Pennisetum, Feathered Pennisetum, Purple-leaved Pennisetum, Purple Elephant Grass, Snowy Pennisetum, Fluffy Pennisetum, Long-spike Pennisetum, Dwarf Pennisetum, Little Rabbit Pennisetum, Hahailin Pennisetum, Red Elephant Grass, East African Pennisetum, Purple-spike Pennisetum, Jinghong Pennisetum, or Liqiu Pennisetum.

Images