Specific primer combinations and their applications

Abstract

The present invention relates to a specific primer combination, which is any one of primer combination 1, primer combination 2, and primer combination 3, or a combination thereof, wherein primer combination 1 includes an upstream primer F with a sequence as shown in SEQ ID NO.1 and a downstream primer R with a sequence as shown in SEQ ID NO.2, primer combination 2 includes an upstream primer F with a sequence as shown in SEQ ID NO.3 and a downstream primer R with a sequence as shown in SEQ ID NO.4, and primer combination 3 includes an upstream primer F with a sequence as shown in SEQ ID NO.5 and a downstream primer R with a sequence as shown in SEQ ID NO.6. The primer combination of the present invention diagnoses the color of a strain through conventional PCR, has high specificity, can effectively reduce the workload of breeding, can complete preliminary screening at the mycelial stage, reduces the number of hybrid offspring that need to be cultivated and screened, improves breeding efficiency, and also saves costs.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a specific primer combination and application thereof. Background Art

[0002] Flammulina filiformis, also known as the "Gou Mushroom," "Pu Mushroom," "Pu Mushroom," "Dong Mushroom," and "Hairy-stalked Money Mushroom," is an important edible and medicinal mushroom. Flammulina is delicious, smooth, and nutritious, rich in essential amino acids, vitamins, unsaturated fatty acids, and dietary fiber. Furthermore, it contains numerous bioactive substances beneficial to health, including fungal polysaccharides, fungal immunomodulatory proteins, flavonoids, terpenes, glycosides, and phenolic compounds. These substances possess specific anti-tumor, anti-blood pressure, anti-cholesterol, antioxidant, and anti-aging properties, and are also effective in treating liver and allergic respiratory diseases.

[0003] Cultivated varieties of Flammulina velutipes mainly fall into two categories: yellow and white. White Flammulina velutipes, characterized by their uniform fruiting, resistance to browning, and long shelf life, command over 90% of the market share. However, the white Flammulina velutipes used in industrial production are primarily imported. Yellow Flammulina velutipes, a variety of edible mushrooms with independent intellectual property rights in my country, boast superior flavor and taste to white Flammulina velutipes. However, their short storage life and prone to browning of the stems limit their cultivation and market reach. Currently, traditional hybridization is still the primary method of Flammulina velutipes breeding. A number of high-quality new yellow Flammulina velutipes varieties have been developed through methods such as single-spore hybridization, multi-spore hybridization, and double-spore hybridization. However, traditional breeding methods are long and labor-intensive, and the subsequent stages require significant time and effort for cultivation, trait analysis, and identification. Therefore, the development of specific primer combinations for Flammulina velutipes color identification is warranted. Summary of the Invention

[0004] The object of the present invention is to provide a primer combination 1, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.2.

[0005] The object of the present invention is to provide a primer combination 2, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.4.

[0006] The object of the present invention is to provide a primer combination 3, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.6.

[0007] Another object of the present invention is to provide a specific primer combination, wherein the primer combination is a combination of two or three of primer combination 1, primer combination 2, and primer combination 3;

[0008] Primer combination 1, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO. 2;

[0009] Primer combination 2, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO. 3 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO. 4;

[0010] Primer combination 3 includes an upstream primer F having a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.6.

[0011] In a preferred technical solution of the present invention, the specific primer combination is primer combination 1 and primer combination 2.

[0012] In a preferred technical solution of the present invention, the specific primer combination is primer combination 2 and primer combination 3.

[0013] In a preferred technical solution of the present invention, the specific primer combinations are primer combination 1, primer combination 2 and primer combination 3.

[0014] Another object of the present invention is to provide a method for color screening of Enoki mushrooms, which specifically comprises the following steps:

[0015] (1) Using the DNA of the to-be-tested Flammulina velutipes mycelium as a PCR template, a PCR reaction is performed using any one of primer combination 1, primer combination 2, and primer combination 3, or a combination thereof;

[0016] (2) Detect whether the amplified product has a band of a specific size and determine the color of the enoki mushroom based on the band type;

[0017] Primer combination 1, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO. 2;

[0018] Primer combination 2, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO. 3 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO. 4;

[0019] Primer combination 3 includes an upstream primer F having a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.6.

[0020] In the preferred technical solution of the present invention, in the PCR amplification in step (1), each 20.0 μL amplification reaction system includes: 10 μL of Taq DNA polymerase premix, 8 μL of sterile distilled water, 1 μL of total primer volume, and 1 μL of DNA template.

[0021] In the preferred technical solution of the present invention, the PCR amplification program in step (1) is: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 30 cycles; final extension at 72°C for 5 min.

[0022] In a preferred technical solution of the present invention, the method for detecting the amplified product in step (2) is agarose gel electrophoresis.

[0023] In the preferred technical solution of the present invention, when the PCR reaction is carried out with primer combination 1 and primer combination 2, the PCR product after electrophoresis is detected to see whether there are bands at 104bp, 317bp and 335bp. If there is a band at 317bp, it is a yellow enoki mushroom; if there are bands at 104bp and 335bp, it is a white enoki mushroom; if there are bands at 104bp, 317bp and 335bp, it is a light yellow enoki mushroom.

[0024] In the preferred technical solution of the present invention, when the PCR reaction is carried out with primer combination 2 and primer combination 3, the PCR product after electrophoresis is detected to see whether there are bands at 104 bp and 262 bp. If there is a band at 262 bp, it is a yellow enoki mushroom; if there is a band at 104 bp, it is a white enoki mushroom; if there are bands at both 104 bp and 262 bp, it is a light yellow enoki mushroom.

[0025] In the preferred technical solution of the present invention, when the PCR reaction is carried out with primer combination 1, primer combination 2 and primer combination 3, the PCR product after electrophoresis is detected to see whether there are bands at 104bp, 262bp, 317bp and 335bp. If there are bands at 262bp and 317bp, it is yellow enoki mushroom; if there are bands at 104bp and 335bp, it is white enoki mushroom; if there are bands at 104bp, 262bp, 317bp and 335bp, it is light yellow enoki mushroom.

[0026] Another object of the present invention is to provide an application of the specific primer combination of the present invention in the hybrid breeding of Flammulina velutipes, wherein the specific primer combination is any one of primer combination 1, primer combination 2, primer combination 3 or a combination thereof.

[0027] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentages are volume / volume percentages; when the present invention relates to the percentage between liquids and solids, the percentages are volume / weight percentages; when the present invention relates to the percentage between solids and liquids, the percentages are weight / volume percentages; and the rest are weight / weight percentages.

[0028] Compared with the prior art, the beneficial technical effects of the present invention include:

[0029] 1. The present invention designs and screens three sets of specific primer combinations for diagnosing strain color through conventional PCR with high specificity. The primer sequences involved in the present invention are located in the promoter region of the protein tyrosine phosphatase gene. Yellow and white Flammulina velutipes and hybrid F1 and F2 generation strains can be distinguished through simple PCR detection combined with agarose gel electrophoresis at the mycelial stage, and the color presented after fruiting can be determined with an accuracy rate of 80-100%. This can reduce the workload of screening in the later stages of hybrid breeding, improve breeding efficiency, and significantly save costs.

[0030] 2. The present invention is simple to operate, low in cost, and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The process of hybridization and self-pollination between yellow and white Enoki mushrooms;

[0032] Figure 2 Identification of color-associated loci by BSA sequencing of extreme segregating populations;

[0033] Figure 3 Color-linked InDel region primer design mode;

[0034] Figure 4 Application of the specific primer combination of the present invention in the F1 generation;

[0035] Figure 5 The effect of the specific primer combination of the present invention in Example 1 was tested on the Flammulina velutipes strain.

[0036] Figure 6 In Example 2, the effect of the specific primer combination of the present invention on the Flammulina velutipes strain was detected.

[0037] Figure 7 In Example 3, the effect of the specific primer combination of the present invention on the Flammulina velutipes strain was tested. DETAILED DESCRIPTION

[0038] The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples.

[0039] The wild Flammulina velutipes XD2205 in the examples was purchased from Yuechi Wanpeng Fungus Development Co., Ltd.), and the pure white Flammulina velutipes material AW27 was purchased from Meishan Changhong Agricultural Biotechnology Co., Ltd.

[0040] Example 1 Design and synthesis of specific primer combinations of the present invention

[0041] After the wild Flammulina velutipes XD2205 and pure white Flammulina velutipes material AW27 were cultivated and fruited, spore prints were collected, and single spore strains were isolated. The F2 generation trait segregation populations were obtained by pairing and hybridization and self-pollination. Figure 1 ).

[0042] The dark yellow and pure white F2 offspring strains were selected to form extreme mixed pools for BSA sequencing analysis, and color-associated loci of 1.94 Mb and 0.002 Mb were identified on chromosomes 4 and 9, respectively. Figure 2 ).

[0043] Analysis found that the genome of the white mixed pool had a fragment deletion within 1000bp upstream of the protein tyrosine phosphatase gene on Chr4 (i.e., the promoter region) compared to the yellow mixed pool: 11 bases were deleted at the 256676-256686 site, and 29 bases were inserted at the 256697-256726 site. Primers were designed by extracting fragments containing the above sites from the genomes of the yellow and white progeny mixed pools ( Figure 3 ), as a molecular marker to distinguish yellow and white strains.

[0044] The primers of the present invention were designed based on the aforementioned BSA results, which showed that there were site differences between the DNA of white and yellow Flammulina velutipes. The primers were synthesized by a biological company (Shanghai Sangon Biotech Co., Ltd.). The results are shown in Table 1.

[0045] Table 1 Sequences involved in primers

[0046]

[0047] Example 2: Specific primer combination of the present invention is used for color screening of Flammulina velutipes

[0048] After the wild Flammulina velutipes XD2205 and pure white Flammulina velutipes material AW27 were cultivated and fruited, spore prints were collected, and single spore strains were isolated. The F1 generation trait segregation population was obtained by pairing and hybridization and self-pollination. Figure 1 ).

[0049] DNA from wild Flammulina velutipes XD2205, pure white Flammulina velutipes AW27, and their F1 offspring was used as template for PCR reaction and 3% agarose gel electrophoresis. Primers InDelChr4.1F / InDelChr4.1R / InDelChr4.1midR (i.e., primer combination 1 and primer combination 2) were reacted in the same system, followed by electrophoresis.

[0050] In PCR amplification, each 20.0 μL amplification reaction system includes: 10 μL of Taq DNA polymerase premix, 8 μL of sterile distilled water, 1 μL of total primer volume, and 1 μL of DNA template.

[0051] PCR amplification program: pre-denaturation at 94°C for 4 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 5 min.

[0052] The results are shown in Table 2.

[0053] Table 2 Bands amplified by different primer combinations

[0054] yellow White light yellow Primer combination 1 317bp 335bp 317bp+335bp Primer combination 2 —— 104 bp 104bp

[0055] The results showed that the wild yellow strain had only one 317bp band, the white strain had two bands of 335bp and 104bp, and the light yellow F1 offspring had all three bands, one from each parent, proving that they were true hybrid offspring ( Figure 4 ).

[0056] Spore prints were collected from the F1 generation strains and then self-pollinated to obtain F2 offspring. Further identification using DNA templates from the F2 generation isolated population revealed that all light yellow offspring had three bands, one from the yellow parent and two from the white parent; while pure white offspring had only two bands, meaning they did not contain the same band as the yellow parent. This indicates that after primer combination 1 and primer combination 2 reacted in the same PCR system, electrophoresis could distinguish white, yellow, and light yellow Flammulina velutipes ( Figure 5 ), with an identification accuracy rate of more than 85%.

[0057] Example 2: Specific primer combination of the present invention is used for color screening of Flammulina velutipes

[0058] Eleven wild yellow Flammulina velutipes strains (H1-H11) and 11 pure white Flammulina velutipes strains (B1-B11) from different regions were selected to verify the reliability of the InDel molecular marker primers. DNA from the mycelia of these 22 Flammulina velutipes strains was used as a PCR template, and PCR reactions were performed using primer combinations 2 and 3.

[0059] In PCR amplification, each 20.0 μL amplification reaction system includes: 10 μL of Taq DNA polymerase premix, 8 μL of sterile distilled water, 1 μL of total primer volume, and 1 μL of DNA template.

[0060] PCR amplification program: pre-denaturation at 94°C for 4 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 5 min.

[0061] The method for detecting the amplified product is agarose gel electrophoresis, and the PCR product after electrophoresis is detected to see if there are bands at 104 bp and 262 bp. The results are shown in Table 3. Figure 6 .

[0062] Table 3 Bands amplified by different primer combinations

[0063] yellow White Primer combination 2 —— 104bp Primer combination 3 262bp ——

[0064] Primer combination 2 amplified bands in all white strains and some yellow strains; primer combination 3 amplified bands in most yellow strains but not in white strains. Therefore, using these two primer combinations together can distinguish between yellow and white strains of Flammulina velutipes with an accuracy rate of up to 90%. Primer combination 2 and primer combination 3 can also be used to identify light yellow strains. If both 104bp and 262bp bands are amplified, the strain is light yellow, with an accuracy rate exceeding 80%.

[0065] Example 3: Application of the specific primer combination of the present invention in color screening of Flammulina velutipes

[0066] Eleven wild yellow Flammulina velutipes strains (Y1-Y11) and 11 pure white Flammulina velutipes strains (W1-W11) from different regions were selected to verify the reliability of the InDel molecular marker primers. DNA from the mycelia of these 22 Flammulina velutipes strains was used as a PCR template, and PCR reactions were performed using primer combinations 1, 2, and 3.

[0067] In PCR amplification, each 20.0 μL amplification reaction system includes: 10 μL of Taq DNA polymerase premix, 8 μL of sterile distilled water, 1 μL of total primer volume, and 1 μL of DNA template.

[0068] PCR amplification program: pre-denaturation at 94°C for 4 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 5 min.

[0069] The amplified products were detected by agarose gel electrophoresis to detect whether the PCR products had bands at 104 bp, 262 bp, 317 bp, and 335 bp. The results are shown in Table 4.

[0070] Table 4 Bands amplified by different primer combinations

[0071] yellow White Primer combination 1 317bp 335bp Primer combination 2 —— 104bp Primer combination 3 262bp ——

[0072] Primer combination 1 can amplify bands in both wild yellow and white Flammulina velutipes, with the band in white Flammulina velutipes being slightly larger; Primer combination 2 can amplify bands in all white strains and some yellow Flammulina velutipes; Primer combination 3 amplifies bands in most yellow strains but not in white strains. Therefore, using the above three primer sets simultaneously can distinguish yellow and white Flammulina velutipes strains, with an accuracy rate of 100% in identifying white Flammulina velutipes and an accuracy rate of over 90% in identifying wild yellow Flammulina velutipes ( Figure 7 The three primer combinations can also be used to distinguish light yellow Flammulina velutipes. If bands of 104bp, 262bp, 317bp, and 335bp can be amplified simultaneously, it is confirmed to be light yellow Flammulina velutipes with an accuracy rate of 100%.

[0073] The present invention applies specific primer combination 1, primer combination 2, and primer combination 3 to the color identification of the fruiting bodies of the F1 and F2 offspring strains of the hybrid of yellow and white Flammulina velutipes. The PCR detection using mycelial DNA as a template shows that the color determination is consistent with the color presented after the mushrooms are fruited, demonstrating the effectiveness of the specific primer combination of the present invention in hybrid breeding.

[0074] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of protection of the claims of the present invention.

Claims

1. Primer combination 1, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.

2.

2. Primer combination 3, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.

6.

3. Specific primer combination, wherein the specific primer combination is any two or three combinations of primer combination 1, primer combination 2, and primer combination 3, Primer combination 1, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO. 2; Primer combination 2, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO. 3 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO. 4; Primer combination 3 includes an upstream primer F having a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.

6. 4 . The specific primer combination according to claim 3 , wherein the specific primer combination is primer combination 1 and primer combination 2.

5. The specific primer combination according to claim 3, wherein the specific primer combination is primer combination 2 and primer combination 3. The specific primer combination according to claim 3 , wherein the specific primer combination is primer combination 1, primer combination 2 and primer combination 3.

7. A method for color screening of Enoki mushrooms, comprising the following steps: (1) Using the DNA of the mycelium of Flammulina velutipes to be tested as a PCR template, PCR reaction was performed using any two or three combinations of primer combination 1, primer combination 2, and primer combination 3. (2) Detect whether the amplified product has a band of a specific size, and determine the color of the enoki mushroom based on the band type; Primer combination 1, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO. 2; Primer combination 2, comprising an upstream primer F having a nucleotide sequence as shown in SEQ ID NO. 3 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO. 4; Primer combination 3 includes an upstream primer F having a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.

6.

8. The method according to claim 7, wherein the method for detecting the amplification product is agarose gel electrophoresis.

9. The method according to claim 7, wherein in the PCR amplification in step (1), each 20.0 μL amplification reaction system comprises: 10 μL of Taq DNA polymerase premix, 8 μL of sterile distilled water, 1 μL of total primer volume, and 1 μL of DNA template.

10. The method according to claim 7, wherein the PCR amplification procedure in step (1) is as follows: preliminary denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 30 cycles; and final extension at 72°C for 5 min.

11. The method of claim 7, wherein when the PCR reaction is carried out using primer combination 1 and primer combination 2, the PCR product after electrophoresis is detected to determine whether there are bands at 104 bp, 317 bp, and 335 bp.

12. The method of claim 11, wherein when a PCR reaction is performed using primer combination 1 and primer combination 2, the PCR product after electrophoresis is detected to determine whether there are bands at 104 bp, 317 bp, and 335 bp. If there is a band only at 317 bp, it is a yellow enoki mushroom; if there are bands at 104 bp and 335 bp, it is a white enoki mushroom; if there are bands at all of 104 bp, 317 bp, and 335 bp, it is a light yellow enoki mushroom.

13. The method of claim 7, wherein when the PCR reaction is carried out using primer combination 2 and primer combination 3, the PCR product after electrophoresis is detected to determine whether there are bands at 104 bp and 262 bp.

14. The method of claim 13, wherein when a PCR reaction is performed using primer combination 2 and primer combination 3, the PCR product after electrophoresis is detected to determine whether there are bands at 104 bp and 262 bp. If there is a band at 262 bp, it is a yellow enoki mushroom; if there is a band at 104 bp, it is a white enoki mushroom; if there are bands at both 104 bp and 262 bp, it is a light yellow enoki mushroom.

15. The method of claim 7, wherein when a PCR reaction is performed using primer combination 1, primer combination 2, and primer combination 3, the PCR product after electrophoresis is detected to determine whether there are bands at 104 bp, 262 bp, 317 bp, and 335 bp.

16. The method of claim 15, wherein when a PCR reaction is performed using primer combination 1, primer combination 2, and primer combination 3, the PCR product after electrophoresis is detected to determine whether there are bands at 104 bp, 262 bp, 317 bp, and 335 bp. If bands are present at both 262 bp and 317 bp, the product is yellow enoki mushroom; if bands are present at both 104 bp and 335 bp, the product is white enoki mushroom; and if bands are present at both 104 bp, 262 bp, 317 bp, and 335 bp, the product is light yellow enoki mushroom.

17. Use of the primer combination 1 according to claim 1, the primer combination 3 according to claim 2, or the specific primer combination according to any one of claims 3 to 6 in breeding for color traits of Flammulina velutipes.

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