KR102563613B1 - Development of SNP markers and use thereof for discrimination of Pyogo mushroom cultivar Chamaram - Google Patents

Abstract

The present invention relates to a SNP marker composition for distinguishing Sanjo 701 or Chamaram among shiitake mushroom varieties and its use. Accordingly, it is possible to determine whether Sanjo 701 or Cham Aram among shiitake mushrooms is heterotype or homotype of the curve using a primer that specifically binds to the shiitake mushroom nucleotide sequence and a melt curve.

Description

[Development of SNP markers and use thereof for discrimination of Pyogo mushroom cultivar Chamaram}

The present invention relates to a SNP marker composition for discriminating chamaram among shiitake mushroom varieties and its use.

The present invention relates to a SNP marker for distinguishing Sanjo 701 or Chamaram among shiitake mushroom varieties developed by the Forest Mushroom Research Center of the National Forestry Cooperative Federation and cultivated in Korea, and HRM-PCR analysis using the same. Primer sequences capable of amplifying RL-LE-202 and RL-LE-203, which are marker sequences containing Aram-specific SNPs, by polymerase chain reaction (PCR), HRM-PCR analysis using the marker sequences and primer sequences It is about how to determine the above varieties through the method.

Shiitake ( Lentinula edodes ) is a white rotting fungus belonging to the order Agaricales , which grows while decomposing cellulose, hemicellulose, and lignin of trees. Over the past 20 years, shiitake cultivation has greatly increased, and now accounts for about 22% of world mushroom production, and is known as the most cultivated edible mushroom. It is mainly cultivated in Asian countries such as Korea, China, and Japan, and is also commercially grown in Western countries such as the United States, Canada, and the Netherlands. As of 2019, shiitake mushrooms accounted for about 98% of domestic forest mushroom production, recording a production value of about KRW 201.8 billion. Shiitake is rich in various nutrients and polysaccharides, and has a unique taste and aroma, so it is not only used as a cooking ingredient, but also has medical values such as anticancer, immune regulation, and antiviral effects.

Korea joined UPOV in 2002, and since 2008, shiitake has been designated as a crop subject to variety protection in accordance with the Seed Industry Act. And as the Nagoya Protocol on Access to Genetic Resources and Benefit Sharing was adopted in 2010 and entered into force in Korea in 2017, the importance of securing distinction for new and developed varieties is increasing. Up to now, about 57 varieties of shiitake have been developed, applied for and registered, and the classification of varieties is based on the external morphological characteristics of shiitake mushrooms produced by the Korea Forest Service, making accurate identification very difficult.

Molecular markers show polymorphisms among individuals based on differences in DNA nucleotide sequences, so they have the advantage of being able to quickly discriminate without being affected by the cultivation environment or developmental stage. In addition, it is possible to shorten the breeding period and reduce costs for breeding through early selection and systematic analysis. UPOV recommends the use of SSR (simple sequence repeat) or SNP (single nucleotide polymorphism) markers with high reproducibility and polymorphism for each breed. In general, mutation analysis using SSR is expensive, and the length of the amplification product is measured and compared. Therefore, it has the disadvantage that it is difficult to identify nucleotide sequence changes without length differences, such as SNPs. HRM (high-resolurion melting) is a method of analyzing differences in GC ratio, length, and base sequence by measuring the change in dissociation temperature of amplification products, and it is possible to analyze a large number of samples in a short time at low cost.

Sanjo 701 and Chamaram, developed at the Forestry Mushroom Research Center of the National Forestry Cooperative Federation, are the most preferred sawdust medium spawn in Korea, and the preference rates for the two spawn in 2020 were 45.2% and 25.2%, respectively. Sanjo 701 has the characteristics of bright mushroom color, very hard flesh, and heavy individual weight. It has the advantage of being possible.

The present inventors developed a SNP marker and analysis method capable of distinguishing Sanjo 701 or Chamaram, which are the most cultivated shiitake varieties in Korea, using the genome sequence of shiitake mushrooms. The present invention was completed by confirming that the cultivars can be easily distinguished by HRM analysis of the dissociation temperature of the marker sequence amplified using each primer pair.

Korean Registered Patent No. 10-2032502

The present invention relates to a SNP marker composition for distinguishing shiitake mushroom variety Sanjo 701 or Cham Aram and its use, and to provide a biomarker capable of quickly and accurately identifying Sanjo 701 or Cham Aram. The present invention is characterized in that the varieties can be simply distinguished by HRM analysis of the dissociation temperature of a marker sequence amplified using a pair of shiitake mushroom primers.

The present invention provides a biomarker composition for discrimination of shiitake cultivar Sanjo 701, including a primer set represented by each of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3.

In one embodiment of the present invention, the primer is characterized in that it specifically binds to the marker sequence RL-LE-202 of Sanjo 701 represented by SEQ ID NO: 1.

In addition, the present invention provides a biomarker composition for discriminating shiitake cultivar Chamaram, including a primer set represented by each of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6.

In one embodiment of the present invention, the primer is characterized in that it specifically binds to the marker sequence RL-LE-203 of Chamaram represented by SEQ ID NO: 4.

In addition, the present invention provides a kit for discrimination comprising the above composition and a reagent for carrying out an amplification reaction.

In one embodiment of the present invention, reagents for performing the amplification reaction may include DNA polymerase, dNTPs, and a buffer, but are not limited thereto.

In addition, the present invention Sanjo 701 identification method comprising the following steps:

(a) extracting DNA from shiitake mushrooms;

(b) preparing an amplification product by performing an amplification reaction of the DNA separated in step (a) using the composition containing the primer set according to claim 1;

(c) measuring a melt curve in the amplification step;

(d) analyzing the measurement result of step (c); and

(e) determining that the heterotype is Sanjo 701 for the Rl-LE-202 marker based on the measurement result, providing a method for determining Sanjo 701.

In addition, the present invention is a true alarm determination method comprising the following steps:

(a) extracting DNA from shiitake mushrooms;

(b) preparing an amplification product by performing an amplification reaction of the DNA separated in step (a) using the composition containing the primer set according to claim 2;

(c) measuring a melt curve in the amplification step;

(d) analyzing the measurement result of step (c); and

(e) providing a method for determining whether the heterotype is true for the Rl-LE-203 marker based on the measurement result;

In one embodiment of the present invention, after the step of preparing the amplification product, after heating at 95 ° C for 7 to 13 seconds, the melt curve is measured while slowly raising the temperature from 60 ° C to 95 ° C by 0.025 ° C per second. It may be, but is not limited thereto.

Since the SNP marker according to the present invention is analyzed through HRM based on real-time PCR, Sanjo 701 and Chamaram can be quickly and easily identified, and the procedure of confirming PCR products such as agarose gel confirmation can be omitted. . In identifying SNPs, since only primers are used without specific probes, the cost of identifying varieties can be reduced.

Since accurate identification is possible even at the mycelial growth stage, specific varieties or strains can be identified early without having to cultivate them, prevention of variety mixing due to illegally distributed seed fungi, and use as variety certification technology during distribution of the above shiitake strains is possible. do.

Figure 1 relates to the base sequence and primers of Sanjo 701 and Cham Aram among shiitake mushroom varieties.
Figure 2 shows mutations by performing PCR and sequencing using primer sequences.
Figure 3 shows the results of performing PCR and HRM-PCR analysis on Sanjo 701 and Chamaram among shiitake mushroom varieties.

The present invention provides a biomarker composition for discrimination of shiitake cultivar Sanjo 701, including a primer set represented by each of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3.

In addition, the present invention is to provide a biomarker composition for discriminating shiitake cultivar Chamaram, including a primer set represented by each of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6.

In addition, the present invention is to provide a kit for discrimination including the above composition and a reagent for carrying out an amplification reaction.

Hereinafter, the present invention will be described in detail.

In the present invention, 'nucleotide' is deoxyribonucleotide or ribonucleotide that exists in single-stranded or double-stranded form, and includes analogs of natural nucleotides unless otherwise specified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990))

In the present invention, a 'single nucleotide polymorphism (SNP)' is a DNA sequence that occurs when a single base (A, T, C or G) in the genome is different between members of a species or between paired chromosomes of an individual. means the diversity of Single bases can be changed (replaced), removed (deleted), or added (inserted) to a polynucleotide sequence. SNPs can cause changes in the translational frame. Single nucleotide polymorphisms may be involved in coding sequences of genes, non-coding regions of genes or intergenic regions between genes. SNPs in the coding sequence of a gene do not necessarily cause changes in the amino acid sequence of a target protein due to degeneracy of the genetic code. SNPs that form the same polypeptide sequence, also called synonymous and silent mutations, are said to be non-synonymous in the case of SNPs that form different polypeptide sequences. Non-synonymous SNPs can be missense or nonsense, with missense changes giving rise to other amino acids while nonsense changes form immature stop codons. SNPs present in non-protein-coding regions can lead to gene silencing, transcription factor binding or non-coding RNA sequences.

In the present invention, 'primer' means an oligonucleotide, and conditions in which synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of nucleotides and a polymerizer such as DNA polymerase, and suitable It can act as an initiation point for synthesis under conditions of temperature and pH. Specifically, the primers are deoxyribonucleotides and are single-stranded. Primers used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also contain ribonucleotides.

In the present invention, 'primer' refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow for the synthesis of extension products to begin. The specific length and sequence of the primer may depend on the complexity of the DNA or RNA target required, as well as the conditions of use of the primer, such as temperature and ionic strength. The oligonucleotide used as the primer may also contain a nucleotide analog, such as a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid, or an intercalating material agent) may be included.

As the 'primer' used in the present invention, a sequence that is perfectly complementary to the sequence containing the SNP may be used, but a sequence that is substantially complementary within a range that does not interfere with specific hybridization may be used. there is. Specifically, the probe used in the present invention includes a sequence capable of hybridizing to a sequence containing 10-30 consecutive nucleotide residues including the SNP of the present invention. More specifically, the 3'-end or 5'-end of the probe has a base complementary to the SNP base. In general, since the stability of duplexes formed by hybridization tends to be determined by the matching of sequences at the ends, in probes having bases complementary to SNP bases at the 3'-end or 5'-end, the terminal portion hybridizes. Otherwise, these duplexes can be broken under stringent conditions.

When the 'kit' of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally include reagents necessary for PCR amplification, such as buffer, DNA polymerase, DNA polymerase cofactor, and dNTPs. The kit of the present invention may be manufactured in a number of separate packaging or compartments containing the reagent components described above.

In addition, the present invention Sanjo 701 identification method comprising the following steps:

(a) extracting DNA from shiitake mushrooms;

(b) preparing an amplification product by performing an amplification reaction of the DNA separated in step (a) using the composition containing the primer set according to claim 1;

(c) measuring a melt curve in the amplification step;

(d) analyzing the measurement result of step (c); and

(e) To provide a method for determining Sanjo 701, including the step of determining that Sanjo 701 is the heterotype for the Rl-LE-202 marker based on the measurement result.

Finally, the present invention is a method for determining whether to be true, including the following steps:

(a) extracting DNA from shiitake mushrooms;

(b) preparing an amplification product by performing an amplification reaction of the DNA separated in step (a) using the composition containing the primer set according to claim 2;

(c) measuring a melt curve in the amplification step;

(d) analyzing the measurement result of step (c); and

(e) To provide a method for determining whether the heterotype is true for the Rl-LE-203 marker as a result of the measurement, including the step of determining true true.

In the present invention, 'HRM-PCR' is one of the PCR methods based on the real-time PCR technique, and detects the difference in melting temperature inherent in the PCR product in real time to obtain one sequence of the PCR product. It corresponds to the PCR method that distinguishes even the difference of

Specifically, when a PCR product is created, the PCR product has a unique Tm value according to the DNA sequence, and the intercalating dye intercalating into the double strand intercalates into the PCR product and emits a fluorescence wavelength. At this time, when the temperature is gradually raised from a low temperature to a high temperature, the PCR product undergoes denaturation at its own Tm value (specific temperature), that is, changes from double-strand to single-strand, and the fluorescence value drops rapidly. The temperature at this time is called melting temperature, and it indicates a technology that detects the difference in melting temperature and distinguishes even one difference in sequence from the PCR product.

The method of the present invention uses the same method as the kit, uses primers, and measures the melting temperature of the PCR product, so the common content between the two is to avoid excessive complexity in the present specification. omit

<Example 1> Shiitake mushroom sequences RL-LE-202 and RL-LE-203 and primers

Figure 1 shows the marker nucleotide sequence amplified in shiitake mushroom cultivars Sanjo 701 and Chamaram by PCR using primer sequences. The genomic DNA of the shiitake strains in Table 1 was resequenced and compared with the sequence of the standard genome B17 of shiitake mushroom. The DNA extraction method is as follows.

Shiitake mushroom mycelium cultured in PDB medium was filtered through Miraclos, washed with PBS buffer (NaCl 135mM, KCl 2.7mM, Na ₂ HPO ₄ 4.3mM, KH _{2PO 4} 1.4mM), and dried with a kitchen towel. , The dried mycelia were frozen in liquid nitrogen, ground finely in a mortar, and genomic DNA was extracted using the GeneAll® GenEx™ Plant kit. Add 500ul of PL buffer to the mycelia transferred to the tube, heat at 65 ° C for 10 minutes, mix well using a pipette, centrifuge at 13000 rpm for 1 minute, separate the supernatant, transfer to a new tube, and transfer the PP buffer to the upper layer. Add 1/3 of the liquid and mix well using a vortexer. After leaving on ice for 5 minutes, centrifuge at 13,000 rpm for 5 minutes, separate the supernatant, transfer to a new tube, add the same amount of PCI (phenol: chloroform: isoamylalcohol = 25: 24: 1), and mix by inversion. After centrifugation at 13000 rpm for 10 minutes, the supernatant was separated and transferred to a new tube, and the same amount of isopropanol was added and mixed by inversion. After leaving on ice for 10 minutes, centrifuged at 13000 rpm for 1 minute, removing the supernatant, adding 1 ml of 70% ethanol to slightly float the DNA pellet, and centrifuged at 13000 rpm for 1 minute. After removing the supernatant, centrifuge once more for 1 minute at 13000 rpm. After removing all remaining ethanol and drying the DNA pellet at room temperature for 5 minutes, 100ul of RE buffer was added to dissolve the DNA pellet.

Through sequence comparison, a base sequence with specific mutations in Sanjo 701 and Chamaram was found, and primers were prepared using this.

number cultivar name geographical origin One Forest No. 10 korea 2 autumn scent korea 3 Ceiling No. 2 korea 4 Sanmaru 1 korea 5 Sanmaru 2 korea 6 mountain baekhyang korea 7 Suhyanggo korea 8 KFRI 31 japan 9 Sanjo 101 korea 10 Sanjo 102 korea 11 Sanjo 103 korea 12 Sanjo 109 korea 13 Sanjo 111 korea 14 Sanjo 502 korea 15 Sanjo 701 korea 16 Sanjo 704 korea 17 Sanjo 705 korea 18 Sanjo 706 korea 19 Sanjo 707 korea 20 Sanjo 708 korea 21 Sanjo 709 korea 22 Sanjo 710 korea 23 be patient korea

<Example 2> SNP identification of shiitake mushrooms ‘Sanjo 701 and Chamaram’ through sequencing

In FIG. 2, PCR is performed using the primer sequence of FIG. 1 and sequencing is performed to show the result of confirming the mutation. PCR was performed using 20ng of genomic DNA extracted from shiitake mushroom standard genome strain B17, shiitake mushroom variety Sanjo 701, and chamaram. As PCR conditions, 35 cycles of amplification were performed at 95°C for 3 minutes, again at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 20 seconds, and then further reacted at 72°C for 5 minutes.

As a result of comparing and contrasting the sequencing results, it was confirmed that a mutation specific to Sanjo 701 or Chamaram existed. In the case of the RL-LE-202 marker sequence, B17 was a homotype of G and Sanjo 701 was a heterotype of GA. A heterotype of AC was identified.

<Example 3> Identification of ‘Sanjo 701 or Cham Aram’ variety among shiitake mushrooms using the kit

Figure 3 is the result of performing PCR using primers that bind Sanjo 701 and Chamaram to marker sequences RL-LE-202 and RL-LE-203, and measuring the melting curve. HRM analysis was performed using Applied Biosystems® MeltDoctor™ HRM Master Mix. Real-time PCR was performed using a mixture of 20 μl in total with a composition of 1 μl of 20ng genomic DNA, 10 μl of 2X HRM Master Mix, 1 μl each of primers F and R at 10 pmole/μl, and 7 μl of DW. As PCR conditions, 40 cycles were performed at 95 ° C for 10 minutes, again at 95 ° C for 15 seconds, at 54 ° C for 30 seconds, and at 72 ° C for 30 seconds, then heated at 95 ° C for 10 seconds and then heated at 60 ° C to 95 ° C per second. The melt curve was measured while gradually increasing the temperature by 0.025 ° C.

As a result of HRM-PCR analysis on 23 strains including Sanjo 701 and Chamaram, Sanjo 701 was heterotyped for the Rl-LE-202 marker, and the remaining strains were homotyped. Regarding the RL-LE-203 marker, it was shown as a heterotype in the case of Chamaram and a homotype in the case of the other strains.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Development of SNP markers and use thereof for discrimination of Pyogo mushroom cultivar Chamaram <130> CBNU2022P0448 <150> KR 10-2021-0047781 <151> 2021-04-13 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 104 <212> DNA <213> artificial sequence <220> <223> RL-LE-202 <220> <221> mutation <222> (36) <223> Y is G or A <400> 1 gcttgatcaa ttcgttggaa gacaaygtca tccaaaccat tattcttcca agtgacagat 60 aatctgaacg ttgtcctctt tcaagatgtc caactttcca ttgc 104 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> RL-LE-202F <400> 2 gcttgatcaa ttcgttggaa 20 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> RL-LE-202R <400> 3 gcaatggaaa gttggacatc 20 <210> 4 <211> 104 <212> DNA <213> artificial sequence <220> <223> RL-LE-203 <220> <221> mutation <222> (69) <223> Y is A or C <400> 4 gcttgatcaa ttcgttggaa gacaaygtca tccaaaccat tattcttcca agtgacagat 60 aatctgaacg ttgtcctctt tcaagatgtc caactttcca ttgc 104 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> RL-LE-203F <400> 5 actcgttcgt gaagccatag 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> RL-LE-203R <400> 6 tcgggaatga agctgactta 20

Claims (6)

A composition for discriminating shiitake varieties Chamaram, comprising an agent capable of detecting a single nucleotide polymorphism (SNP) located at the 26th nucleotide sequence of SEQ ID NO: 4.
According to claim 1,
The preparation is characterized in that the primer set represented by each of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6, a composition for discrimination of shiitake varieties Chamaram.
A kit for identifying the shiitake cultivar Brasil, comprising the composition for identifying the shiitake cultivar Chamaram of claim 1 or 2 and a reagent for performing an amplification reaction.
According to claim 3,
Reagents for performing the amplification reaction are characterized in that they include DNA polymerase, dNTPs and buffers, the kit.
A method for determining shiitake cultivars, including the following steps:
(a) extracting DNA from shiitake mushrooms;
(b) preparing an amplification product by performing an amplification reaction using the DNA isolated in step (a) as a template using a primer set represented by the nucleotide sequences of SEQ ID NOs: 5 and 6;
(c) measuring a melt curve in the amplification step;
(d) analyzing the measurement result of step (c); and
(e) determining a single nucleotide polymorphism (SNP) marker for shiitake cultivar Chamaram located at the 26th base sequence of SEQ ID NO: 4 as Chamaram if the measurement result of step (d) is a heterotype;
According to claim 5,
After the step of preparing the amplification product, it is heated at 95 ° C for 7 to 13 seconds, and then the melt curve is measured while slowly raising the temperature from 60 ° C to 95 ° C by 0.025 ° C per second.