Method for identifying MNP marker of a flammulina velutipes variety and application thereof

Abstract

The application discloses a MNP marker identification method for a Flammulina velutipes variety and application thereof, and through data of 319 Flammulina velutipes strains, 350 marker sites are determined to be used for Flammulina velutipes variety identification. Sequencing of each marker site is more than 500 times, and is accurate to a single base, the result is highly accurate, the accuracy is more than 99.98%, accurate digitalization is realized, and a basic technical problem of modern seed industry management such as informatization, automation, intelligentization, networking and block chain is broken through.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a MNP marker identification method for Flammulina velutipes varieties and application thereof. BACKGROUND

[0002] Flammulina velutipes is the first edible mushroom variety to realize factory production. In recent years, the rapid development of factory cultivation has made its yield increase by leaps and bounds, ranking first among edible mushroom varieties in factory cultivation. Factory Flammulina velutipes has become one of the varieties with the highest industrialization level and the most intense market competition.

[0003] Tissue (substrate) separation is a widely used method for obtaining strains in the edible mushroom industry, which has caused a large number of "same name different things" (different names for the same strain) in production, leading to confusion of production seed and difficulty in protecting the rights and interests of breeders.

[0004] Substantive derivative varieties are varieties derived from the original variety or derived from the substantive derivative varieties of the original variety, which are obviously different from the original variety and have the same basic characteristics as the original variety except for the differences in traits caused by derivation. The identification of substantive derivative varieties is one of the key points for the healthy development of Flammulina velutipes seed industry. SUMMARY

[0005] This part aims to summarize some aspects of the embodiments of the application and briefly introduce some preferred embodiments.

[0006] As one aspect of the application, the application provides a MNP marker identification method for Flammulina velutipes varieties, wherein: 350 pairs of MNP marker primers shown in SEQ ID NO: 1-700 are used to perform PCR amplification and sequencing on each Flammulina velutipes variety to be identified; when the genetic similarity GS between two Flammulina velutipes varieties to be identified is greater than or equal to 92% but less than 96%, it is determined that the Flammulina velutipes varieties to be identified are suspected to be substantive derivative varieties; and when the genetic similarity GS between the Flammulina velutipes varieties to be identified is greater than or equal to 96%, it is determined that the Flammulina velutipes varieties to be identified are suspected to be the same variety.

[0007] As a preferred scheme of the MNP marker identification method for Flammulina velutipes varieties, the genetic similarity GS = n / N x 100%, wherein N is the number of MNP sites amplified by the MNP marker primers for two Flammulina velutipes varieties to be identified, and n is the number of MNP sites with the same genotype in the MNP sites amplified by the MNP marker primers for the two Flammulina velutipes varieties to be identified.

[0008] As a preferred scheme of the MNP marker identification method for Flammulina velutipes varieties, the identification method comprises the following steps,

[0009] (1) extracting DNA of the to-be-tested Flammulina velutipes variety;

[0010] (2) performing PCR amplification by using the 350 pairs of MNP marker primers;

[0011] (3) purifying and sequencing the PCR amplification products;

[0012] (4) performing genetic similarity analysis on the sequencing results.

[0013] As a preferred scheme of the MNP marker identification method of the Flammulina velutipes variety, when the proportion of the detected MNP marker sites in a to-be-identified Flammulina velutipes variety is greater than or equal to 95%, it is determined that the sequencing data is qualified.

[0014] As a preferred scheme of the MNP marker identification method of the Flammulina velutipes variety, when the proportion of the detected MNP marker sites in a to-be-identified Flammulina velutipes variety is less than 95%, if the reproducibility of the detected MNP marker sites in two repeated experiments is greater than or equal to 95%, it is determined that the sequencing data is qualified.

[0015] The application further provides application of the method in Flammulina velutipes variety identification.

[0016] The application further provides application of the method in identification of whether to-be-tested Flammulina velutipes varieties are the same variety.

[0017] The application further provides application of the method in identification of whether to-be-tested Flammulina velutipes varieties are the same substantial derivative variety.

[0018] The application has the following beneficial effects: the application selects 21 white strains and 9 yellow strains for resequencing, and develops 530 MNP markers according to the principles of high polymorphism, conservative primer region, single copy, consistent annealing temperature and uniform distribution. Through the data of 319 Flammulina velutipes strains, it is determined that 350 marker sites are used for Flammulina velutipes variety identification. The sequencing of each marker site is more than 500 times, accurate to a single base, the result is highly accurate, the accuracy is more than 99.98%, precise digitalization is realized, and the basic technical problems of modern seed industry management such as informatization, automation, intelligentization, networking and block chain are broken through. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiment description, wherein:

[0020] Figure 1The detection rate and distribution sequence number of MNP markers in *Flammulina velutipes* are plotted in 319 samples.

[0021] Figure 2 This is a clustering diagram of the MNP results.

[0022] Figure 350721 shows the differential distribution of MNP marker sites among samples of Enoki mushroom.

[0023] Figure 4 The images show DNA electrophoresis patterns of samples JZG01 and JZG02.

[0024] Figure 5 Electrophoresis images of high-throughput sequencing libraries for samples JZG01 and JZG02. Detailed Implementation

[0025] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0026] 1. Development and screening of marker sites in *Flammulina velutipes*

[0027] To design MNP marker primers, we selected 9 yellow varieties and 21 white strains from the authorized and wild-type *Flammulina velutipes* strains library. The genomes of each strain were re-sequencing at a depth of 50-fold. SNP sites were screened by comparing with the *Flammulina velutipes* reference genome. Based on the principles of high polymorphism in the amplified region, conserved primer regions, single-copy amplified regions, amplified length not exceeding 250 bp, amplified regions containing two or more discontinuous SNP markers, consistent primer annealing temperatures, uniform distribution, and no interference between marker and primer sequences, a total of 530 MNP marker sites were screened, and primers were designed for experimental testing. Using data from 319 *Flammulina velutipes* strains, 350 core marker sites were identified for the identification of *Flammulina velutipes* varieties and substantial derivatives. The primer sequences are shown in Table 1.

[0028] Table 1. MNP marker primer sequences for *Flammulina velutipes*

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[0033]

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[0036]

[0037]

[0038] 2. Verification of MNP markers of Flammulina velutipes

[0039] Using the synthesized 350 pairs of MNP marker primers of Flammulina velutipes, 319 strains of Flammulina velutipes collected were subjected to multiplex amplification, second-generation high-throughput sequencing and data analysis, and the average coverage of each sample was 4791 times. The MNP-Seq data of the sample were analyzed, and the 350 markers screened could be detected, among which 326 (93.27%) MNP markers could be detected in each sample on average, and the samples with a detection rate of more than 90% accounted for 84.3%. Figure 1 The detection rate and distribution sequence fragment number of MNP markers of Flammulina velutipes in 319 samples (horizontal coordinate: variety).

[0040] 3. Setting of quality control parameter detection rates R1 and R2

[0041] R1 in the present application refers to the proportion of detected marker sites in the sample, and it is specified that when R1 is greater than or equal to 95%, the sequencing data is determined to be qualified. If R1 is less than 95%, the reproducibility of the marker sites detected by two experiments of the sample is used, and when R2 is greater than or equal to 95%, the sequencing data is determined to be qualified.

[0042] R1 in the present application refers to the proportion of detected marker sites in the sample, and it is specified that when R1 is greater than or equal to 95%, the sequencing data is determined to be qualified. If R1 is less than 95%, the reproducibility of the marker sites detected by two experiments of the sample is used, and when R2 is greater than or equal to 95%, the sequencing data is determined to be qualified.

[0043] Table 2 Influence of site detection rate on stability of identification conclusion

[0044]

[0045]

[0046] Setting of quality control parameter R2: R2 in the present application refers to the reproducibility of the detection of marker loci in two reproducibility experiments of a sample. The present application provides that when R2 is greater than or equal to 95%, the sequencing data is determined to be qualified. The influence of the stability of locus detection (the value of R2) on the stability of identification conclusion is shown in Table 3. When the value of R1 is low, it indicates that a large number of loci are not detectable, and it is assumed that the number of detectable loci is 280; when the value of R2 is 95%, then the number of non-commonly detected loci is 14; according to the binomial distribution, the maximum number of differential loci in the non-commonly detected loci is 5 at a probability of 95%, and the maximum observed genetic similarity is 80.64%, and the genetic similarity (GS) deviation is 0.98%. It can be seen that the value of R2 ensures the stability of the genetic similarity, and ensures that the influence of the missing rate of the detected loci on the reproducibility of the identification conclusion is small.

[0047] Table 3 Influence of locus detection stability on stability of identification conclusion

[0048]

[0049] 4. MNP marker genotyping accuracy

[0050] In order to test the accuracy of the developed Flammulina velutipes MNP marker loci and detection method, reproducibility experiments were performed on 5 samples, and 2 libraries were constructed for each variety of DNA, numbered -11 and -12; 2 libraries were sequenced twice at different times. The number of different loci in the first and second sequencing of different batches of libraries is very small, and the same sample between different libraries detects the same MNP marker genotype, which is reproducible. According to the reproducibility calculation formula r = n / N, n: the logarithm of the repeatable genotype, N: the total number of genotypes compared. The accuracy is 99.94%. The results show that the developed MNP marker loci and detection method have high accuracy.

[0051] Table 4 MNP marker loci and detection method accuracy

[0052]

[0053] 5. Polymorphism of MNP markers

[0054] The core function of a set of DNA marker combination is variety identification, and the higher the polymorphism of DNA markers, the stronger the variety discrimination ability. We used 319 Flammulina velutipes strains to detect the number of allelic genotypes of 350 MNP markers, and the results showed that in the 319 Flammulina velutipes strains, the average number of allelic genotypes of MNP markers was 17.05.

[0055] 6. Variety discrimination ability of MNP marker method

[0056] 319 samples of Flammulina velutipes were compared with each other, and the differences in MNP marker sites between samples were analyzed. 50721 pairs of comparison results were obtained, the differences in genetic distance of 50721 pairs of samples were between 0% and 100%, and the genetic similarity coefficients of 45,245 pairs (accounting for 89.20%) of varieties were less than 96%, which were determined as different varieties; the genetic similarity coefficients of 5,476 pairs of varieties were higher than 96%, accounting for 10.79%, which indicated that the MNP marker method had strong variety distinguishing ability. Figure 2 Figure 3 is a clustering diagram of MNP results of site AMPL1690064. Figure 3 Figure 4 is a distribution of differences in MNP marker sites between 50721 pairs of Flammulina velutipes samples.

[0057] 7. Determination threshold of Flammulina velutipes varieties and substantial derived varieties

[0058] In the present application, the varieties with genetic similarity (GS) greater than or equal to 92% are determined as “the to-be-tested varieties suspected to be substantial derived varieties of the control varieties”.

[0059] In the present application, the varieties with genetic similarity (GS) greater than or equal to 96% are determined as “the to-be-tested varieties suspected to be the same varieties as the control varieties”.

[0060] Example analysis:

[0061] 1. Operation procedure

[0062] 1.1 Sample preparation

[0063] The samples to be submitted are preferably fresh mycelium or fruiting bodies, and the test strains and the control strains are cultured under the same conditions, and the culture amount can be 3 parallel tests.

[0064] The number of individuals extracted from the Flammulina velutipes variety population should meet the requirements of NY / T 2594.

[0065] The extracted samples can be mixed for detection.

[0066] 1.2 DNA extraction and quality inspection

[0067] The DNA of the to-be-tested sample is extracted by using a nucleic acid extraction reagent. The product is placed in a-20℃ refrigerator to prevent DNA degradation. The absorbance ratio of the DNA solution of samples JZG01 and JZG02 at 260 nm and 280 nm is measured and calculated by using a spectrophotometer, and the absorbance ratio is 1.96 and 1.97 respectively. The absorbance ratio at 260 nm and 230 nm is 1.84 and 1.76 respectively; 4 μL of DNA is electrophoresed on a 1% agarose gel to detect whether the DNA band is complete, and the results are as follows: Figure 4The DNA concentration of JZG01 and JZG02 was measured by Qubit Fluorometer, which was 27.6 ng / μL and 20.8 ng / μL, respectively.

[0068] Figure 4 The DNA electrophoretogram of sample JZG01 and sample JZG02.

[0069] 1.3 Multiplex PCR amplification and library construction

[0070] 1.3.1 Multiplex PCR amplification

[0071] Since the experimental procedures of multiplex PCR amplification and library construction of JZG01 and JZG02 are exactly the same, the following only describes the experimental procedures of JZG01, except for special instructions.

[0072] 1) Preparation of multiplex PCR amplification system. Add 4 μL of multiplex PCR primer mixture (0.2 μM concentration of each primer) in Table 1, 4 μL of genomic DNA of sample JZG01, 10 μL of GenoPlexs 3×T Master Mix and 12 μL of water in a PCR tube, and shake well.

[0073] 2) Multiplex PCR amplification reaction. Multiplex PCR amplification program: 95℃, 3min; (95℃, 20s, 60℃, 4min) × 15 cycles; 72℃, 4min. After the reaction is completed, the multiplex PCR amplification product is obtained.

[0074] 1.3.2 Purification of multiplex PCR amplification product

[0075] 1) Add 12 μL of GenoPrep DNA Clean Beads to the obtained multiplex PCR amplification product, shake well, and then stand at room temperature for 5 min.

[0076] 2) Place the PCR tube on the magnetic stand to adsorb the magnetic beads until the solution is clear.

[0077] 3) Use a pipette to aspirate the supernatant into a new 1.5 mL centrifuge tube, avoiding the absorption of magnetic beads.

[0078] 4) Add 18 μL of GenoPrep DNA Clean Beads to the supernatant, shake well, and then stand at room temperature for 5 min.

[0079] 5) Use the magnetic stand to adsorb the magnetic beads until the solution is clear. Carefully aspirate the supernatant with a pipette, discard the supernatant, and leave the magnetic beads.

[0080] 6) Add 40 μL GenoPlexs BW10 Buffer, suspend the magnetic beads, and stand at room temperature for 5 min. Absorb the magnetic beads with a magnetic stand until the solution is clear. Carefully aspirate the supernatant with a pipette, discard the supernatant, and leave the magnetic beads.

[0081] 7) Add 100 μL 80% ethanol, and carefully remove the supernatant with a pipette to avoid absorbing the magnetic beads.

[0082] 8) Stand at room temperature until the ethanol evaporates completely, avoid over-drying, and obtain the purified multiplex PCR amplification product.

[0083] 1.3.3 High-throughput sequencing library construction

[0084] To the obtained purified multiplex PCR amplification product, 10 μL GenoPlexs 3xT Master Mix, 2 μL P5 primer with a concentration of 5 μM, 2 μL P7 barcode primer with a concentration of 5 μM, and 16 μL water. Shake the prepared reaction system to mix and centrifuge briefly, and perform PCR reaction according to the following program: 95°C, 3 min; (95°C, 15 s; 58°C, 15 s; 70°C, 30 s) x 8 cycles; 72°C, 5 min. After the reaction is completed, the high-throughput sequencing library of 30 μL sample JZG01 is constructed.

[0085] 1.3.4 High-throughput sequencing library purification

[0086] Figure 5 The electrophoretogram of the high-throughput sequencing library of sample JZG01 and sample JZG02. In the figure, the leftmost band is a molecular weight standard, and the molecular weight from bottom to top is 100 bp, 250 bp, 500 bp, 750 bp, 1,000 bp, and 2,000 bp, respectively.

[0087] 1.4 High-throughput sequencing

[0088] The obtained high-throughput sequencing library is subjected to high-throughput sequencing. The average coverage multiple of high-throughput sequencing is set to be more than 700 times, and the sequencing length is not less than 300 bp.

[0089] 2 Data analysis

[0090] 2.1 Sequencing data splitting

[0091] The high-throughput sequencer splits the sequencing data to sample JZG01 and sample JZG02 (sample JZG01 and sample JZG02 are the same variety). Since the double-end sequencing mode is adopted, each sequencing fragment of each sample includes forward and reverse sequencing sequences. Among them, the names of the files storing the forward and reverse sequencing sequences of sample JZG01 are JZG01_1.fq.gz and JZG01_2.fq.gz respectively; the names of the files storing the forward and reverse sequencing sequences of sample JZG02 are JZG02_1.fq.gz and JZG02_2.fq.gz respectively.

[0092] 2.2 Sequencing data alignment

[0093] The reference genome of the golden needle mushroom is Genbank accession number JBDPIE000000000, and the file name is LedB17.fna.

[0094] The data alignment software is Bowtie2 (version number 2.1.0), which needs the index construction module bowtie2-build and the sequence alignment module bowtie2 of the software.

[0095] In the Linux window, input the following command line: bowtie2-build LedB17.fna LedB17.fna, to construct the index of the reference genome of the golden needle mushroom.

[0096] In the Linux window, input the following command line: bowtie2-q-p 2-×Led B17.fna-1JZG01_1.fq.gz-2JZG01_2.fq.gz-S JZG01.sam, to align the sequencing data of sample JZG01 to the reference genome of the golden needle mushroom. Among them, the parameter "-q" indicates that the input file is in fastq format; the parameter "-p 2" indicates that 2 threads are used for alignment; the parameter "-×LedB17.fna" specifies the reference genome sequence for alignment; the parameters "-1JZG01_1.fq.gz-2JZG01_2.fq.gz" specify the sequencing result file of sample JZG01; and the parameter "-S JZG01.sam" inputs the alignment result into the JZG01.sam file.

[0097] In a similar manner, the sequencing data of sample JZG02 is aligned to the reference genome of the golden needle mushroom.

[0098] In turn, determine the N ij of sample JZG01 and sample JZG02. Among the 344 common detected loci, whether the genotype of each common detected locus is different. Count the number of marker loci that are detected in both sample JZG01 and sample JZG02 but have no difference in genotype n ij= 338, and the number of different sites is 6.

[0099] The genetic similarity of sample JZG01 and sample JZG02 was calculated:

[0100]

[0101] The results are expressed as:

[0102]

[0103] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method for identifying MNP markers of a Flammulina velutipes variety, characterized by: The 350 pairs of MNP marker primers shown in SEQ ID NO: 1-700 are used to perform PCR amplification and sequencing on each to-be-identified Tricholoma species; when the genetic similarity GS between two to-be-identified Tricholoma species is greater than or equal to 92% but less than 96%, it is determined that the to-be-identified species are substantially derived species; when the genetic similarity GS between the to-be-identified Tricholoma species is greater than or equal to 96%, it is determined that the to-be-identified species are the same species.

2. The MNP marker identification method of the Flammulina velutipes variety according to claim 1, characterized by: The genetic similarity GS = n / N x 100%, wherein N is the number of MNP sites amplified by the MNP marker primers for two to-be-identified Tricholoma species, and n is the number of MNP sites with the same genotype among the MNP sites amplified by the MNP marker primers for two to-be-identified Tricholoma species.

3. The method for identifying MNP markers of Flammulina velutipes varieties according to claim 1 or 2, characterized in that: The identification method comprises the following steps, (1) extracting DNA of the to-be-tested Tricholoma species; (2) performing PCR amplification using the 350 pairs of MNP marker primers; (3) purifying and sequencing the PCR amplification products; (4) performing genetic similarity analysis on the sequencing results.

4. The MNP marker identification method of the Flammulina variety according to claim 3, characterized in that: When the proportion of MNP marker sites detected in a certain to-be-identified Tricholoma species is greater than or equal to 95%, it is determined that the sequencing data is qualified.

5. The MNP marker identification method of the Flammulina variety according to claim 4, characterized in that: When the proportion of MNP marker sites detected in a certain to-be-identified Tricholoma species is less than 95%, if the reproducibility of MNP marker sites detected in two repeated experiments is greater than or equal to 95%, it is determined that the sequencing data is qualified.

6. Application of the method of claim 1 in the identification of Tricholoma species.

7. Application of the method of claim 1 in identifying whether the to-be-tested Tricholoma species are the same species.

8. Application of the method of claim 1 in identifying whether the to-be-tested Tricholoma species are the same substantially derived species.

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