Primer composition and method for identifying shiitake Shenxiang 1845 variety by using polynucleotide polymorphism

Abstract

The invention discloses a primer composition and a method for identifying a shiitake Shenxiang 1845 variety by using polynucleotide polymorphism. The polynucleotide polymorphism primer combination disclosed by the invention has high specificity, is seven polynucleotide polymorphism sites screened based on a whole genome, and can effectively distinguish 'Shenxiang 1845' from other shiitake mushroom varieties (including all national affirmed varieties and plant new variety authorized varieties); according to the identification method, a high-throughput sequencing technology is adopted, the detection period is short, the detection accuracy is high, high throughput is achieved, and multi-target analysis and multi-sample analysis are completed through one-time detection; the identification method disclosed by the invention is wide in application and suitable for strain identification, infringement evidence collection and breeding material screening.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and specifically relates to a primer composition and method for identifying the Lentinula edodes variety Shenxiang 1845 by using polynucleotide polymorphism. Background Art

[0002] Lentinula edodes is one of the important edible mushroom cultivation varieties, and its variety is directly related to the quality of strains, yield and market rights. Traditional identification methods (such as morphological observation, antagonism test, etc.) have problems such as long cycle, easy to be interfered by the environment, and low accuracy. In recent years, molecular marker technologies such as SSR and SNP have been used for variety identification, but there are still limitations such as insufficient number of markers, low throughput, and cumbersome operation.

[0003] 'Shenxiang 1845' is a high-quality Lentinula edodes variety selected by hybridization, which has the characteristics of short cycle, easy to form flower mushrooms, high yield, strong stress resistance, and wide adaptability. However, due to the characteristics of asexual reproduction of edible mushrooms, problems such as abuse and misuse of varieties are likely to occur in actual production, and there is an urgent need to develop an efficient and accurate molecular identification method. Summary of the Invention

[0004] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments.

[0005] The preservation number of the Lentinula edodes variety Shenxiang 1845 of the present invention is GDMCC No: 65762, and it was preserved in the Guangdong Provincial Culture Collection of Microorganisms on January 13, 2025. The preservation address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences.

[0006] 1. Object of the Invention

[0007] The present invention provides a primer combination and a supporting identification method based on polynucleotide polymorphism, which can quickly and accurately identify the variety of 'Shenxiang 1845' and solve the problems of insufficient marker specificity and low detection efficiency in the prior art.

[0008] 2. Technical Solution

[0009] (1) Polynucleotide polymorphism primer combination:

[0010] The present invention conducts a whole-genome polymorphism analysis on the Lentinula edodes variety 'Shenxiang 1845' and other cultivated and wild varieties, and combines bioinformatics methods to screen out 7 polynucleotide polymorphism marker combination sites. The amplification products of the primers are DNA fragments with a length of 175 - 224 bp, and each fragment contains 3 - 16 tightly linked SNP sites, forming a unique polynucleotide polymorphism 'core molecular fingerprint'. Specific primer combinations are designed and synthesized, including 7 pairs of polynucleotide polymorphism marker primers, and the specific sequences are shown in Table 1.

[0011] The polynucleotide polymorphism marker combination sites have high polymorphism and variety specificity, and are stable under different generations and different cultivation conditions, and can be used as the core molecular markers for the identification of the 'Shenxiang 1845' variety.

[0012] Table 1 Sequence information of polynucleotide polymorphism primer combinations

[0013]

[0014] (2) Identification method

[0015] It includes the following steps:

[0016] ① Extract the genomic DNA of the Lentinula edodes sample to be tested;

[0017] ② Use the polynucleotide polymorphism site marker primer combination for multiplex PCR amplification;

[0018] ③ Purify the amplification product and add sequencing adapters;

[0019] ④ Perform high-throughput sequencing on the amplification product to obtain polynucleotide polymorphism site marker genotyping data;

[0020] ⑤ Compare the genotyping data with the'standard fingerprint map of Shenxiang 1845' (including the genotypes of 7 polynucleotide polymorphism sites), calculate the matching degree. If the matching degree is 100% (the genotypes of all 7 sites are exactly the same), it is determined to be the true variety. If there is 1 or more sites that do not match among the 7 sites, it is determined not to be the variety of the present invention.

[0021] The beneficial effects of the present invention: The polynucleotide polymorphism primer composition of the present invention has high specificity, and is based on 7 polynucleotide polymorphism sites screened from the whole genome, which can effectively distinguish 'Shenxiang 1845' from other existing Lentinula edodes varieties; the identification method of the present invention uses high-throughput sequencing technology, with a short detection period, high accuracy and high throughput, and can complete multi-target analysis and multi-sample analysis in one detection; the identification method of the present invention has a wide range of applications and is suitable for strain identification and breeding material screening. Description of the drawings

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for the description of the embodiments. Among them:

[0023] Figure 1 It is the MNP genotype amplified by 7 pairs of primer combinations of 'Shenxiang 1845';

[0024] Figure 2 It is the map of the polymorphic site marker allele genotypes of the primer MNP1 of the present invention in 277 Lentinula edodes strains;

[0025] Figure 3 It is the map of the polymorphic site marker allele genotypes of the primer MNP2 of the present invention in 277 Lentinula edodes strains;

[0026] Figure 4 It is the map of the polymorphic site marker allele genotypes of the primer MNP3 of the present invention in 277 Lentinula edodes strains;

[0027] Figure 5 It is the map of the polymorphic site marker allele genotypes of the primer MNP4 of the present invention in 277 Lentinula edodes strains;

[0028] Figure 6 It is the map of the polymorphic site marker allele genotypes of the primer MNP5 of the present invention in 277 Lentinula edodes strains;

[0029] Figure 7 It is the map of the polymorphic site marker allele genotypes of the primer MNP6 of the present invention in 277 Lentinula edodes strains;

[0030] Figure 8 It is the map of the polymorphic site marker allele genotypes of the primer MNP7 of the present invention in 277 Lentinula edodes strains. Specific Embodiments

[0031] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is made in conjunction with specific embodiments.

[0032] Example: Verification of the Specificity of the Primer Combinations for Polymorphic Site Markers

[0033] (1) Collection of strain population: Collect existing Lentinula edodes cultivated strains and wild strains, including a total of 277 strains including 'Shenxiang 1845'.

[0034] (2) Mycelium culture: Cultivate the mycelium of the collected strains on a PDA plate medium in a dark room at 25°C until the plate is covered with mycelium;

[0035] (3) DNA extraction: Batch extract DNA samples according to the operating steps of the DNA extraction kit, and dilute the DNA concentration to about 50 ng / μL;

[0036] (4) Perform DNA quality control, multiplex PCR amplification, library construction and purification according to the instructions of the multiplex PCR amplification and library construction kit. The specific operations are as follows:

[0037] Equally mix the 7 groups of polynucleotide polymorphism site identification primers in the present invention, and use multiplex PCR amplification to obtain amplification products of the target regions of a batch of strains.

[0038] The multiplex PCR amplification system is: the total volume is 30 μL, including: 10 μL GenoPlexs 3×T Master Mix, 4 μL primer combination, 1 μL DNA sample, 15 μL ddH2O.

[0039] The multiplex PCR amplification reaction procedure: 95°C for 4 min; (95°C for 20 s, 60°C for 4 min) × 17 cycles; 72°C for 5 min.

[0040] After magnetic bead purification of the multiplex PCR amplification products, construct a library. The PCR system is: 10 μL GenoPlexs 3×T Master Mix, 2 μL of each barcode primer, 16 μL ddH2O. The PCR amplification reaction procedure: 95°C for 4 min; (95°C for 15 s, 58°C for 15 s, 70°C for 30 s) × 8 cycles; 72°C for 5 min, and store at low temperature. When simultaneously identifying multiple strains to be tested, different adapters need to be ligated for easy sequencing identification; purify the products of different strains to obtain a sequencing library.

[0041] (5) High-throughput sequencing: Perform high-throughput sequencing according to the operation instructions of the high-throughput sequencing kit and high-throughput sequencer.

[0042] (6) Genotype identification: Compare the genotypes of the 7 target sequences contained in the sequencing results of all strains with the standard map of ′Shenxiang 1845′, calculate the number of markers that are consistent with the standard map among the 7 polynucleotide polymorphism marker sites, and calculate the genetic similarity between the strains;

[0043] (7) The identification results show that ′Shenxiang 1845′ (No. 37) is completely matched with the standard fingerprint map at 7 loci, while the matching degrees of the other varieties are all <50%, verifying the specificity of the primer combination. Table 27 shows the amplification products and allelic genotypes of the polynucleotide polymorphism marker primers (reference genome: GCA_001562095.1)

[0044]

[0045] Table 3 Comparison of Genotypes between Lentinula edodes Variety 'Shenxiang 1845' and Other Lentinula edodes Strains

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

Claims

1. A primer composition for identifying the Lentinula edodes variety Shenxiang 1845 using polynucleotide polymorphisms, characterized in that: The preservation number of the Lentinula edodes Shenxiang 1845 variety is GDMCC No: 65762, and it was preserved in the Guangdong Provincial Culture Collection of Microorganisms on January 13, 2025. The primer composition consists of 7 pairs of primers, and their sequences are shown in SEQ ID NO: 1-14.

2. A method for identifying the Lentinula edodes variety Shenxiang 1845 by using polynucleotide polymorphisms, characterized in that: The 7 pairs of primers are used for PCR amplification of the Lentinula edodes strain to be tested, and the PCR amplification products are sequenced.

3. The method for identifying the Lentinula edodes strain Shenxiang 1845 by using polynucleotide polymorphisms according to claim 2, wherein: The scaffold number corresponding to the PCR amplification product of the primers shown in SEQ ID NO: 1-2 is LSDU01000013.

1. In the PCR amplification product, the base at position 466261 is T, the base at position 466271 is C, and the base at position 466338 is G.

4. The method for identifying the strain Shenxiang 1845 of Lentinula edodes by using polynucleotide polymorphisms according to claim 2, characterized in that: The scaffold number corresponding to the PCR amplification product of the primers shown in SEQ ID NO: 3-4 is LSDU01000015.

1. In the PCR amplification product, the base at position 111427 is T / C, the base at position 111447 is G / A, the base at position 111453 is A, the base at position 111468 is C, and the base at position 111474 is C / A.

5. The method for identifying the Lentinula edodes strain Shenxiang 1845 by applying polynucleotide polymorphisms according to claim 2, wherein: The scaffold number corresponding to the PCR amplification product of the primers shown in SEQ ID NO: 5-6 is LSDU01000015.

1. In the PCR amplification product, the base at position 470509 is C, the base at position 470510 is A, the base at position 470517 is G, the base at position 470542 is T, and the base at position 470564 is A.

6. The method for identifying the strain Shenxiang 1845 of Lentinula edodes using polynucleotide polymorphisms according to claim 2, wherein: The scaffold number corresponding to the PCR amplification product of the primers shown in SEQ ID NO: 7-8 is LSDU01000022.

1. In the PCR amplification product, the base at position 1733425 is G, the base at position 1733429 is T, the base at position 1733445 is A, the base at position 1733455 is A, the base at position 1733461 is T, the base at position 1733467 is A, the base at position 1733477 is G, the base at position 1733486 is A, the base at position 1733497 is T, the base at position 1733502 is A, the base at position 1733511 is A, the base at position 1733514 is T, the base at position 1733515 is G, the base at position 1733530 is T, the base at position 1733548 is T, and the base at position 1733560 is G.

7. The method for identifying the Lentinula edodes strain Shenxiang 1845 by applying polynucleotide polymorphisms according to claim 2, wherein: The scaffold number corresponding to the PCR amplification product of the primers shown in SEQ ID NO: 9-10 is LSDU01000027.

1. In the PCR amplification product, the base at position 869187 is C, the base at position 869188 is A, the base at position 869191 is A, the base at position 869197 is A, the base at position 869209 is T, the base at position 869255 is G, the base at position 869263 is C, the base at position 869288 is G / A, and the base at position 869296 is C.

8. The method for identifying the strain Shenxiang 1845 of Lentinula edodes by using polynucleotide polymorphisms according to claim 2, wherein: The scaffold number corresponding to the PCR amplification product of the primers shown in SEQ ID NO: 11-12 is LSDU01000029.

1. In the PCR amplification product, the base at position 5155424 is C, the base at position 5155449 is G, the base at position 5155462 is G, the base at position 5155467 is A, the base at position 5155485 is T, and the base at position 5155488 is C.

9. The method for identifying the Lentinula edodes strain Shenxiang 1845 by applying polynucleotide polymorphisms according to claim 2, characterized in that: The scaffold number corresponding to the PCR amplification product of the primers shown in SEQ ID NO: 13-14 is LSDU01000030.

1. In the PCR amplification product, the base at position 3511199 is A, the base at position 3511210 is G, the base at position 3511219 is G, the base at position 3511229 is A, the base at position 3511230 is C, the base at position 3511251 is T, the base at position 3511283 is A, the base at position 3511286 is A, the base at position 3511307 is T, and the base at position 3511343 is A.

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