SSR molecular marker primer pair capable of accurately distinguishing wild jujubes from different producing areas and application of SSR molecular marker primer pair

Abstract

The invention provides an SSR molecular marker primer pair capable of accurately distinguishing wild jujubes from different producing areas and application of the SSR molecular marker primer pair, and belongs to the technical field of plant biology. The primer pair provided by the invention is C1, C2, C3 or C4; the nucleotide sequence of the C1 is as shown in SEQ ID NO.1-2; the nucleotide sequence of the C2 is as shown in SEQ ID NO. 3-4; the nucleotide sequence of the C3 is as shown in SEQ ID NO.5-6; and the nucleotide sequence of the C4 is as shown in SEQ ID NO. 7-8. The wild jujube specific gSSR marker developed by the invention can comprehensively reflect genetic information of detected species, and can be used for accurate identification and genetic diversity analysis of wild jujubes from different producing areas.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, and in particular to an SSR molecular marker primer pair that can accurately distinguish wild jujubes from different origins and their applications. Background Technology

[0002] Sour jujube seed is the fruit of the jujube plant (Ziziphus jujuba) in the Rhamnaceae family. Ziziphus jujuba The dried, mature seeds of *Mill. var. spinosa* (Bunge) Hu exH. F. Chou have the effects of nourishing the heart and liver, calming the mind and soothing the nerves, astringing sweat, and promoting the production of body fluids. Modern pharmacological studies have shown that jujube seed has a regulatory effect on the reduced immune function of insomnia patients and has the effect of protecting myocardial cells.

[0003] The distribution of jujube (Ziziphus jujuba) dates back to the Tang Dynasty, as recorded in the "Newly Revised Materia Medica," which states that "jujubes grow in the rivers and marshes of Hedong," referring to southwestern Shanxi Province. Currently, it is mainly distributed in Shanxi, Hebei, and Shandong provinces. Among the many producing areas, the jujube kernels from Shanxi and Hebei have the highest effective content. Shanxi Province is rich in wild jujube resources, exhibiting a certain degree of clustering, with the main producing areas concentrated in the central and southern parts of Shanxi. In 2020, Shanxi Province identified the "Top Ten Shanxi Medicinal Herbs," and jujube kernels were recognized as one of them, further promoting the development of the jujube industry in Shanxi Province.

[0004] Although jujube is increasingly used in the pharmaceutical industry, there are still significant differences in medicinal components and efficacy between different producing areas. Furthermore, due to the mixed germplasm resources of wild jujube and the prevalence of premature ripening, the quality of jujubes from different producing areas varies, and the content of medicinal components in the jujube kernels differs. Therefore, establishing accurate and effective identification methods for jujubes from different producing areas is crucial to ensuring the clinical use of jujube kernels as a medicinal material.

[0005] With the development of sequencing technology, more and more plant whole genomes have been sequenced and published. This has facilitated the development of gSSR markers; gSSR primers can be designed simply by downloading the whole genome sequence from a public database. SSR molecular markers, also known as microsatellite molecular markers, are molecular markers developed by utilizing the characteristics of highly conserved flanking sequences of simple sequence repeats and the rich differences between different alleles. SSR markers possess characteristics such as codominance, good reproducibility, rich polymorphism, and widespread distribution throughout the genome. Previous studies have proven that SSR markers are effective and reliable, and their operation and application are simple and easy to understand. They have shown outstanding performance in the construction of genetic maps, gene mapping analysis, and molecular-assisted breeding in most eukaryotes.

[0006] gSSR is a kind of DNA tandem repeat sequence in genome, which is composed of several nucleotides (usually 1-6 nucleotides) as repeat unit (motif) repeated multiple times (usually 10-50), these repeat sequences are throughout various segments of chromosomes, due to different genotypes in different chromosomal segment repeat sequence length, base composition and adjacent DNA base sequence at both ends of repeat sequence are also different, so that gSSR becomes a kind of molecular marker with good polymorphism.

[0007] The patent CN 116790790 B Zizyphus jujuba Mill. SSR molecular marker development method and application in Zizyphus jujuba Mill. germplasm resource identification provides a scheme for developing Zizyphus jujuba Mill. SSR molecular marker for germplasm resource identification, but the SSR molecular marker primer given must be in the form of combined primer to realize the identification of Zizyphus jujuba Mill. in multiple producing areas, and part of the producing area cannot be distinguished by one pair, for example, Zizyphus jujuba Mill. in Shaanxi and Hebei producing area can only be classified into one category, and cannot be accurately distinguished. Therefore, it is necessary to provide a kind of SSR molecular marker and identification method capable of accurately distinguishing wild Zizyphus jujuba Mill. in different producing areas.

[0008] Based on this, the present application is proposed. SUMMARY

[0009] In order to achieve the above-mentioned application purpose, the present application provides the following technical scheme: The present application provides a kind of SSR molecular marker primer pair capable of accurately distinguishing wild Zizyphus jujuba Mill. in different producing areas, the primer pair is C1, C2, C3 or C4; The nucleotide sequence of C1 is shown as SEQ ID NO.1~2; The nucleotide sequence of C2 is shown as SEQ ID NO.3~4; The nucleotide sequence of C3 is shown as SEQ ID NO.5~6; The nucleotide sequence of C4 is shown as SEQ ID NO.7~8.

[0010] The present application also provides the application of the SSR molecular marker primer pair in identifying wild Zizyphus jujuba Mill. in different producing areas.

[0011] Preferably, the producing area of the wild Zizyphus jujuba Mill. is Jizhou District of Tianjin City, Pinglu County of Shanxi Province and Quyang County of Hebei Province.

[0012] The present application also provides a kind of method for identifying wild Zizyphus jujuba Mill. in different producing areas by using the SSR molecular marker primer pair, comprising the following steps: (1) extracting the genomic DNA of the wild Zizyphus jujuba Mill. to be tested; (2) Taking the genomic DNA as a template, performing gSSR-PCR reaction; (3) performing gel electrophoresis on the de-amplification products, and different wild jujube from different producing areas can be effectively distinguished according to specific bands displayed in the electrophoresis results.

[0013] Preferably, the wild jujube from different producing areas is from Jizhou District of Tianjin, Pinglu County of Shanxi Province and Quyang County of Hebei Province.

[0014] Compared with the prior art, the present application has the following beneficial effects: The present application fills the blank of using jujube markers to identify the producing areas at present, and enriches the types of jujube molecular markers. The jujube specific gSSR markers developed in the present application can comprehensively reflect the genetic information of the detected species, and can be used for accurate identification and genetic diversity analysis of jujube from different producing areas. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The amplification results of primers C1, C2, C3 and C4 in jujube materials from three producing areas are shown in Table 1, M: Marker; 1: Jizhou District of Tianjin; 2: Pinglu County of Shanxi Province; 3: Quyang County of Hebei Province. DETAILED DESCRIPTION

[0016] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0017] Example 1

[0018] Jujube gSSR site analysis

[0019] MISA software was used to search for SSR sites in the jujube genome, and the software setting standards were as follows: base pair, bp 1, 2, 3, 4, 5, 6 bp, and the number of repetitions was not less than 10, 6, 5, 5, 5, 5, 5 times in turn, and the compound SSR was less than 100 bp. The results showed that there were 266713 gSSR sites in the jujube genome, of which 164207 were single nucleotide repeat motif sites, accounting for 61.56% of the total number of sites; the number of gSSR sites of di-nucleotide repeat motif and tri-nucleotide repeat motif was 74821 and 22376 respectively, accounting for 28.05% and 8.38% of the total number of gSSR sites respectively (Table 1).

[0020] Table 1 Different SSR repeat motifs in jujube genome

[0021] Jujube gSSR primer design and screening

[0022] The primer 5.0 was used to design gSSR markers, and the primer was adjusted manually. The design standard of the primer was: the GC content was 35%-65%, the product size was 100-300 bp, the Tm value was 55-65℃, the Tm value difference between the upstream and downstream primers was not more than 5℃, the primer length was 18-27 bp, and the primer GC content was 35%-65%. A total of 64 pairs of cpSSR primers were designed, and finally 4 pairs of primers were screened (Table 2).

[0023] Table 2 Primer sequences of gSSR markers of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu

[0024] Verification of gSSR markers of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu

[0025] The materials used in the experiment were collected from three wild Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu materials in Jizhou District of Tianjin, Pinglu County of Shanxi Province and Quyang County of Hebei Province, the genomic DNA was extracted by improved SDS method, and the concentration of DNA was detected by 1% agarose gel electrophoresis.

[0026] The total system of gSSR-PCR reaction was 15 μL, including 2 × Taq 10 μL of PCR Master Mix, 2 μL of 0.2 μM of gSSR marker upstream and downstream primers, and 1 μL of 40 ng of template DNA.

[0027] The gSSR-PCR reaction program was 5 min of pre-denaturation at 94℃, 1 min of denaturation at 94℃, 45 s of annealing at 50-60℃, 1.5 min of extension at 72℃, 34 cycles, and finally 5 min of extension at 72℃.

[0028] 2.5 μL of amplified product was mixed with an equal amount of 6×loading liquid, non-denaturing polyacrylamide gel electrophoresis was carried out at 180 V for 1 h, and silver staining was used for color development.

[0029] It was found that 4 pairs of gSSR-PCR could produce differences among the materials Figure 1 ).

[0030] C1: The wild Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu material from Jizhou District of Tianjin produced a specific band at 400 bp, the wild Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu material from Quyang County of Hebei Province produced a specific band at 750 bp, and the wild Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu material from Pinglu County of Shanxi Province did not produce a specific band.

[0031] C2: The wild Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu material from Jizhou District of Tianjin produced a specific band at 500 bp, the wild Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu material from Quyang County of Hebei Province produced a specific band at 350 bp, and the wild Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu material from Pinglu County of Shanxi Province did not produce a specific band.

[0032] C3: the wild jujube material from Jizhou District of Tianjin produces a specific band at 360bp, the wild jujube material from Quyang County of Hebei produces a specific band at 250bp, and the wild jujube material from Pinglu County of Shanxi does not produce a specific band.

[0033] C4: the wild jujube material from Jizhou District of Tianjin produces a specific band at 350bp, the wild jujube material from Pinglu County of Shanxi produces a specific band at 750bp, and the wild jujube material from Quyang County of Hebei does not produce a specific band.

[0034] It can be seen that the gSSR-PCR developed by the application can accurately identify and analyze the wild jujube materials from Jizhou District of Tianjin, Pinglu County of Shanxi and Quyang County of Hebei.

[0035] The above only describes the preferred embodiments of the present application, and it should be noted that the ordinary skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A SSR molecular marker primer pair capable of accurately distinguishing different provenances of wild Ziziphus jujuba, characterized in that, The primer pair is C1, C2, C3 or C4; The nucleotide sequence of C1 is shown as SEQ ID NO. 1~2; The nucleotide sequence of C2 is shown as SEQ ID NO. 3~4; The nucleotide sequence of C3 is shown as SEQ ID NO. 5~6; The nucleotide sequence of C4 is shown as SEQ ID NO. 7~8.

2. The application of the primer pair of the SSR molecular marker in identifying wild Ziziphus ju Jub from different producing areas.

3. Use according to claim 2, wherein the compound is ###0002### The wild Ziziphus ju Jub is from Jizhou District of Tianjin, Pinglu County of Shanxi Province and Quyang County of Hebei Province.

4. A method for identifying wild Ziziphus jujuba Mill. from different habitats using the primer pair of the SSR molecular marker of claim 1, characterized in that, The method comprises the following steps: (1) extracting the genomic DNA of the wild Ziziphus ju Jub to be tested; (2) taking the genomic DNA as a template to perform gSSR-PCR reaction; (3) performing gel electrophoresis on the amplified product, and effectively distinguishing the wild Ziziphus ju Jub from different producing areas according to the specific bands shown in the electrophoresis result.

5. The method of claim 4, wherein, The wild Ziziphus ju Jub is from Jizhou District of Tianjin, Pinglu County of Shanxi Province and Quyang County of Hebei Province.

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