An est-sst molecular marker for identifying zingiber officinale var. officinale germplasm, a primer pair and an identification method

Abstract

The present application belongs to the field of biotechnology, and relates to an EST-SST molecular marker, in particular to an EST-SST molecular marker, a primer pair and an identification method for identifying rattan and shrub Zanthoxylum bungeanum germplasm. The present application first discovers an EST-SST molecular marker site for identifying rattan and shrub Zanthoxylum bungeanum germplasm, and designs a specific primer group, obtains a genotyping condition through fluorescence PCR and capillary electrophoresis detection of a peak diagram, the rattan Zanthoxylum bungeanum germplasm shows negative at 127bp, and shows positive at 133bp, 134bp and 136bp with 1 or 2 alleles. The shrub Zanthoxylum bungeanum germplasm shows positive at 127bp, and shows negative at 133bp, 134bp and 136bp. According to the characteristics, the germplasm of a sample to be tested can be rapidly and accurately determined.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to EST-SST molecular markers, specifically to an EST-SST molecular marker, primer pair, and identification method for identifying vine-like and shrub-like Zanthoxylum nitidum germplasm. Background Technology

[0002] Zanthoxylum nitidum is a well-known traditional Chinese medicine with a long history of medicinal use. It was first recorded as "Manjiao" in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) during the Qin and Han dynasties. It originates from the root of *Zanthoxylum nitidum* (Roxb.) DC., a plant belonging to the Rutaceae family and the *Zanthoxylum* genus, and is mainly produced in Guangxi and Guangdong provinces of China. This species includes one original variety, *Zanthoxylum nitidum* var. *nitidum* (Roxb.) DC., and one hairy-leaved variety, *Zanthoxylum nitidum* var. *tomentosum* Huang. The original variety is further divided into three types based on whether the plant parts are hairy, the amount of thorns, and the size and shape of the leaflets. All three types exhibit shrub-like characteristics in their seedling stage. In mature plants, type 1 becomes more woody and vine-like, while types 2 and 3 remain more shrub-like. In recent years, while significant success has been achieved in the artificial domestication and introduction of Zanthoxylum nitidum, problems have arisen during the domestication process, such as similar phenotypic characteristics, indistinct typological features, and unclear genetic backgrounds. These issues hinder the identification, evaluation, research, and utilization of germplasm resources. Shrub characteristics are one of the important traits of Zanthoxylum nitidum. Shrub-type germplasm has a higher degree of lignification and a more compact plant shape than woody vine germplasm, eliminating the need for scaffolding during artificial planting. Therefore, selecting shrub-type germplasm can increase planting density and save planting costs. However, existing literature lacks methods for quickly and accurately screening Zanthoxylum nitidum with shrub-type characteristics. Patent CN102191318A discloses a method for identifying medicinal plants based on the nucleotide sequence of the rDNA ITS-D3 region, providing an approach to identifying the phylogenetic relationships of different medicinal plants using the rDNA ITS-D3 region nucleotide sequence. This method requires extensive bioinformatics analysis in the later stages; however, this approach is not applicable to identifying vine-like and shrub-like Zanthoxylum nitidum germplasm.

[0003] SSR molecular markers have advantages such as high sensitivity, simple operation, wide distribution and rich polymorphism, which provide new ideas for the identification of functional genes, variety identification and molecular marker-assisted breeding of medicinal plants. However, there are no reports on SSR molecular markers for shrub and vine traits of Zanthoxylum nitidum. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes an EST-SST molecular marker, primer pair, and identification method for identifying vine-like and shrub-like Zanthoxylum nitidum germplasm.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides an EST-SST molecular marker for identifying the shrub and vine traits of Zanthoxylum nitidum. The SSR site targeted by the molecular marker includes ZYX31, and the repeating units contained in the nucleotide sequences at each site are (ATA)6.

[0007] The upstream and downstream primer sequences of the molecular marker are shown in SEQ ID No. 11 and SEQ ID No. 12, respectively.

[0008] This invention also provides a method for identifying vine-like and shrub-like Zanthoxylum nitidum germplasm, comprising the following steps: providing the variety to be identified and extracting genomic DNA from the sample; using the upstream and downstream primers of the above-mentioned molecular markers, performing fluorescence PCR amplification to obtain the amplification product; and performing electrophoresis detection on the amplification product to detect the fluorescence PCR amplification product.

[0009] The amplification product was then diluted, and deionized formamide containing dye fragment standards was added for capillary electrophoresis detection. The capillary electrophoresis results were analyzed to obtain an allele typing table and typing peak diagram. Based on the fingerprint pattern formed by the coding, the germplasm of the sample to be tested was determined.

[0010] Specifically, the reaction system for the fluorescent PCR is as follows: 1 μL of 10 ng / μL template, 0.5 μL of 10 pM upstream primer, 0.5 μL of 10 pM downstream primer, 5 μL of 2×Taq PCR Master Mix and 3 μL of ultrapure water.

[0011] Specifically, the amplification program for the fluorescent PCR is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, gradient annealing at 62-52℃ for 30 s, extension at 72℃ for 30 s, for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, extension at 72℃ for 30 s, for 25 cycles; 72℃ extension for 20 min.

[0012] Furthermore, the fluorescence capillary electrophoresis detection system comprises: 1 μL of 10 ng / μL diluted fluorescence PCR product, and 0.5 μL of dye fragment standard (GeneScan). TM 500 LIZ), 8.5 μL of highly deionized formamide (Hi-Di) TM Formamide).

[0013] When the sample to be tested was a vine-like Zanthoxylum nitidum germplasm, all alleles at 127bp were negative, while one or two alleles at 133bp, 134bp, and 136bp were positive.

[0014] When the sample to be tested was shrub-like Zanthoxylum nitidum germplasm, the allele at 127bp was positive, while the alleles at 133bp, 134bp, and 136bp were all negative.

[0015] The present invention has the following beneficial effects:

[0016] Compared with existing morphological identification techniques, this invention is the first to discover EST-SST molecular marker sites for identifying shrub and vine traits in *Zanthoxylum nitidum*, and designs a specific primer pair. Genotyping information is obtained by detecting peak patterns using fluorescent PCR and capillary electrophoresis. In vine-like *Zanthoxylum nitidum* germplasm, all alleles at 127 bp are negative, while one or two alleles at 133 bp, 134 bp, and 136 bp are positive. In shrub-like *Zanthoxylum nitidum* germplasm, alleles at 127 bp are positive, while alleles at 133 bp, 134 bp, and 136 bp are negative. Based on these characteristics, rapid and accurate identification of the germplasm of the sample can be achieved. The identification method of this application has better interspecific applicability and advantages such as speed, accuracy, high specificity, and reliability. It can also provide important tools and valuable information for genetic diversity analysis of *Zanthoxylum nitidum*, identification of phylogenetic relationships with closely related genera and species, and phylogenetic studies. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present 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 only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is an example of electrophoresis images of some of the samples tested in Example 2.

[0019] Figure 2 This is a gel electrophoresis image of PCR products from some samples in Example 2.

[0020] Figure 3 This is an example of XYZ31 detection data obtained from some vine germplasm in Example 2.

[0021] Figure 4 This is an example of XYZ31 detection data obtained from some shrub germplasm in Example 2. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1

[0024] Based on the simplified genome data of *Zanthoxylum nitidum*, the inventors designed and synthesized 192 pairs of SSR primers. Preliminary screening of the identification effects on *Zanthoxylum nitidum* germplasm from 10 different sources was conducted. The 192 primer pairs and identification results are summarized in the table below. The results showed that 107 pairs of SSR primers failed to amplify, 43 pairs of SSR primers amplified successfully with obvious single heterogeneous peaks, and only 39 pairs of SSR primers showed good polymorphism. Further screening from the polymorphic SSR primers yielded 15 pairs of primers, as shown in Table 1, which were used for amplification on 142 samples from 51 populations.

[0025] Table 1 Information on 15 pairs of SSR primers

[0026]

[0027]

[0028] The amplification results were analyzed using GenAIex software to calculate the genetic diversity of the 15 primer pairs, and the results are shown in Table 2:

[0029] Table 2. Indicators of SSR primer amplification products from 15 pairs of primers

[0030]

[0031] A total of 245 alleles (Na) were detected, with a minimum of 9 alleles, a maximum of 37 alleles, and an average of 16.333 alleles per locus. The total number of effective alleles (Ne) was 85.828, ranging from 2.271 (ZYX092) to 13.464 (ZYX137), with an average of 5.722 effective alleles per locus, representing 35.03% of the total. The Shannon index (I) ranged from 1.313 (ZYX092) to 2.994 (ZYX137), with an average of 1.984. The observed heterozygosity (Ho) ranged from 0.114 (ZYX092) to 0.681 (ZYX006), with an average of 0.481. The expected heterozygosity (He) ranged from 0.565 (ZYX092) to 0.926 (ZYX137), with an average of 0.777. The polymorphism information content (PIC) ranged from 0.531 (ZYX092) to 0.922 (ZYX137), with an average of 0.756. Literature indicates that indicators such as the polymorphism information index (PIC) are important indicators for measuring the polymorphism of SSR molecular markers. When PIC > 0.5, it indicates high polymorphism. In this study, the PIC ranged from 0.531 to 0.922, with an average of 0.756, indicating that the 15 primer pairs screened had high polymorphism and could effectively reveal the genetic diversity of Zanthoxylum nitidum.

[0032] Further screening of 15 pairs of SSR primers that can distinguish germplasm of Zanthoxylum nitidum revealed one pair of SSR primers capable of identifying both shrub and vine traits of Zanthoxylum nitidum. The specific operation method includes the following steps:

[0033] S1, Provide the sample to be tested:

[0034] Samples of Zanthoxylum nitidum, known to be vine-like and woody, were randomly selected from different regions (the germplasm numbers were used for easy recording during sampling). Forty-four individual samples from 16 woody vine Zanthoxylum nitidum populations were collected. The germplasm numbers and collection locations of the samples are shown in Table 3 below. Seventy-eight individual samples from 28 shrub-like Zanthoxylum nitidum populations were collected. The sample numbers and collection locations of the samples are shown in Table 4 below.

[0035] Table 3. Germplasm number and collection information of woody vine-like Zanthoxylum nitidum samples

[0036]

[0037] Table 4 Germplasm number and collection information of *Zanthoxylum nitidum* samples

[0038]

[0039]

[0040] S2, Extract genomic DNA:

[0041] After stacking each sample neatly, cut an appropriate amount of leaf material into a 2mL centrifuge tube. Freeze the collected samples in the centrifuge tubes at low temperature, then grind the leaves into a powder using a tissue homogenizer. Extract genomic DNA from the samples using the Tiangen Plant Genomic DNA Extraction Kit. Detect the DNA concentration using Nanodrop One, and dilute the DNA to 10 ng / μL with ultrapure water for later use. Electrophoresis images of some extracted genomic DNA samples are shown below. Figure 1 As shown.

[0042] S3, Fluorescent PCR amplification:

[0043] The PCR reaction system includes: 1 μL genomic DNA, 0.3 μL upstream and downstream primers (10 pM), 5 μL 2×Taq PCRMaster Mix, and ultrapure water to a final volume of 10 μL.

[0044] The reaction program is as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 62-52℃ annealing for 30 s, 72℃ extension for 30 s, run for 10 cycles, 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, run for 25 cycles, and finally 72℃ extension for 20 min.

[0045] S4, capillary electrophoresis:

[0046] Take 2 μL of the fluorescent PCR amplification product and use 1% agarose gel electrophoresis to check whether the fluorescent PCR amplification bands are normal (results of some sample sampling are shown below). Figure 2 (As shown), and referring to the standard DNA Marker, the fluorescent PCR products were diluted to the same concentration. 1 μL of each diluted fluorescent PCR product was added to 0.5 μL of Liz 500 internal standard (GeneScan). TM 500 LIZ), deionized formamide 8.5 μL (Hi-Di) TM Formamide was detected by capillary electrophoresis using an ABI 3730xl sequencer, and the results are shown in Table 5.

[0047] S5, Data Processing:

[0048] The fluorescence capillary electrophoresis results were analyzed using GeneMarker 2.0 peak plot analysis software to read the size of the fluorescence PCR amplified fragments, and allele typing tables and typing peaks were obtained. The peak plots of the ZYX31 locus in the above 44 vine-like germplasm samples and 78 shrub-like germplasm samples were analyzed one by one. It can be seen that there are 10 alleles of SSR primer ZYX31, which are ordered from smallest to largest as 126, 127, 128, 129, 130, 131, 132, 133, 134 and 136.

[0049] Table 5. Capillary electrophoresis results of PCR products amplified using ZYX31 primers.

[0050]

[0051] The vine-like germplasm of *Zanthoxylum nitidum* showed alleles negative at 127bp, but 1-2 alleles positive at 133bp, 134bp, and 136bp. Figure 3 Capillary electrophoresis peaks are visible in some vine-like germplasm. The shrub-like *Zanthoxylum nitidum* germplasm showed positive alleles at 127 bp, and positive alleles at 133 bp, 134 bp, and 136 bp. Figure 4 (See capillary electrophoresis peak diagram of some shrub-like germplasm).

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer pair for identifying vine-like and shrub-like Zanthoxylum nitidum germplasm, characterized in that: The primer pairs are shown as nucleotide sequences in SEQ ID No. 11 and SEQ ID No.

12.

2. A method for identifying vine-like and shrub-like Zanthoxylum nitidum germplasm using the primer pair described in claim 1, characterized in that, The steps are as follows: (1) Using the genomic DNA of the sample to be tested as a template, and the primer pair described in claim 1 as the upstream and downstream primers, fluorescent PCR amplification is performed; (2) The fluorescent PCR amplification products from step (1) are detected by electrophoresis to identify the target product band, and the target product is recovered; (3) After the target product of step (2) is detected by capillary electrophoresis, peak diagram analysis is performed to obtain the allele typing table and typing peak diagram. Based on the fingerprint pattern formed by the coding, the germplasm of the sample to be tested is determined. When the sample to be tested is a vine-like seed of Zanthoxylum nitidum, all samples showed negative results at 127bp, while one or two samples showed positive results at 133bp, 134bp, and 136bp. When the sample to be tested was shrub-like Zanthoxylum nitidum germplasm, it showed a positive result at 127bp, and a negative result at 133bp, 134bp, and 136bp.

3. The method for identifying vine-like and shrub-like Zanthoxylum nitidum germplasm according to claim 2, characterized in that: The fluorescent PCR system used for fluorescent PCR amplification in step (1) also includes 2×Taq PCR Master Mix and ultrapure water.

4. The method for identifying vine-like and shrub-like Zanthoxylum nitidum germplasm according to claim 3, characterized in that, The fluorescent PCR system specifically consists of: 1 μL of 10 ng / µL template, 0.5 μL of 10 pM upstream primer, 0.5 μL of 10 pM downstream primer, 5 μL of 2×Taq PCR Master Mix, and 3 μL of ultrapure water.

5. The method for identifying vine-like and shrub-like Zanthoxylum nitidum germplasm according to claim 3 or 4, characterized in that: The amplification program for the fluorescent PCR was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, gradient annealing at 62~52℃ for 30 s, extension at 72℃ for 30 s, for 10 cycles; denaturation at 95℃ for 30 s, annealing at 52℃ for 30 s, extension at 72℃ for 30 s, for 25 cycles; extension at 72℃ for 20 min.

6. The method for identifying vine-like and shrub-like Zanthoxylum nitidum germplasm according to claim 5, characterized in that: The capillary electrophoresis detection system in step (3) consists of: 1 μL of the target product diluted to 10 ng / µL, 0.5 μL of dye fragment standard, and 8.5 μL of highly deionized formamide.

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