KR102290111B1 - Primer Sets for Detecting SSR markers for Grouping of Pyropia yezoensis Cultivas, and Method for Grouping of Pyropia yezoensis Cultivas Using the Composition - Google Patents

Abstract

The present invention relates to a primer set for detecting a simple sequence repeat (SSR) marker capable of stably and effectively discriminating cultivars using the genetic characteristics of Porphyra yezoensis Ueda, and a method for discriminating cultivars of Porphyra yezoensis Ueda using the same and, more specifically, to a primer set for detecting a SSR marker for group discrimination of Porphyra yezoensis Ueda cultivars (SEQ ID NO: 17 and SEQ ID NO: 18) and/or (SEQ ID NO: 19 and SEQ ID NO: 20) and a method for group discrimination of Porphyra yezoensis cultivars using the same.

Description

Primer Sets for Detecting SSR markers for Grouping of Pyropia yezoensis Cultivas, and Method for Grouping of Pyropia yezoensis Cultivas Using the Composition }

The present invention relates to a primer set for SSR marker amplification capable of grouping stably and effectively using the genetic characteristics of radiograss, and a method for classifying radiograss into several groups using the same.

Kim is the most important economic algae in the form of algae, which is grown in the country, the domestic radiation pattern Kim (P. yezoensis Ueda) has been the most cultivated, Seaweed Seaweed giant amphipods parent pattern (P. seriata Kjellman) and itbadi Seaweed ( P. dentata Kjellman) is also being farmed on a small scale. Until the introduction of artificial seedlings, most of the laver cultivated in Korea was black laver, but from 1968 to 1970, samples from each farm were collected and investigated. It has been confirmed that it is grown as the mainstream in the farms of This strong phenomenon of radioactive laver was accelerated with the spread of artificial seedlings, and radioactive laver is also the most cultivated laver in the three East Asian countries that produce laver.

Various seaweed varieties are being developed with the aim of adapting to the changed environment and increasing productivity and marketability due to seawater temperature rise due to global warming or other changes in the marine environment. In Korea, as a member of the International Federation for the Protection of New Plant Varieties (UPOV) in 2002, it is proposing various methods of developing laver varieties and developing new varieties through basic research to prepare for it. In Korea, a variety protection system has been implemented for seaweeds since 2012 (aquatic plant variety protection system http://www.nifs.go.kr/apvc/). As of April 2020, there are a total of 16 new types of laver registered in Korea, 2 cases of seed laver, 3 cases of black laver, and 11 cases of radish laver. There are a total of 11 laver varieties that have been applied for and are undergoing cultivation review, 10 cases of radish laver and 1 case of Itbodidol laver. Most of the laver varieties that have been registered or have been applied for and are undergoing cultivation review are radioactive laver, and 11 out of a total of 21 cases have been developed by the National Institute of Fisheries Science and distributed to fish households.

Until now, varieties have been identified and classified using phenotype-oriented morphological and physiological characteristics. However, in the case of seaweed including laver, it is not easy to distinguish morphologically unlike land plants. Therefore, the need to introduce and utilize more stable and effective techniques based on trait characteristics for genetic resource management, development of new varieties, and quality improvement and diversification of domestic laver plants in accordance with the Convention on Biological Diversity and the implementation of the variety protection system is emerging.

DNA markers for identification of laver varieties not only reflect the genetic characteristics of the variety intrinsically, but also can be applied to all varieties for characterization, and are very useful because they are not affected by the environment. Furthermore, the developed DNA marker can be used as basic data for the creation of a genetic map and molecular breeding. Registered Patent No. 10-1291609 and Registered Patent No. 10-1954583 disclose a primer set and a method for distinguishing seaweed species. However, this is for distinguishing the radish laver, cham laver, itbodidol laver, and mopatina laver from each other, and it is not enough to distinguish the varieties. Registered Patent No. 10-0769358, Registered Patent No. 10-1725316, and Registered Patent No. 10-1803819, etc., are for distinguishing between Japanese and native varieties of radish laver, and it was not possible to distinguish specific varieties of radioactive laver. .

So far, Korea, Japan, and China are the countries that mass-produce laver, and in Korea and China, in the case of radioactive laver, a considerable portion of Japanese varieties are distributed in the form of sasangs. In order to prevent the payment of royalties to Japanese varieties as much as possible in preparation for a new variety protection system, also called seed war, and to generate royalty income through normal variety management and quality improvement of laver, it is necessary to accurately identify the variety of radish laver. A possible method needs to be developed.

Registered Patent No. 10-1291609 Registered Patent No. 10-1954583 Registered Patent No. 10-0769358 Registered Patent No. 10-1725316 Registered Patent No. 10-1803819

An object of the present invention is to solve the problems of the prior art as described above, and an object of the present invention is to provide a primer set capable of grouping cultivars of radish laver more accurately and conveniently through nucleotide sequence analysis.

Another object of the present invention is to provide a kit for cultivar grouping of radish laver using the primer set and a cultivar discrimination method.

The present invention for achieving the above object
(SEQ ID NO: 17 and SEQ ID NO: 18) or (SEQ ID NO: 19 and SEQ ID NO: 20) relates to a primer set for detecting a simple sequence repeat (SSR) marker for group discrimination of cultivars.

Molecular markers such as the SSR marker of the present invention indicate polymorphism between individuals based on differences in DNA base sequence, and since they are not affected by the cultivation environment or growth period of crops, varieties can be more accurately distinguished.

The oligonucleotide used as the primer in the present invention may include a nucleotide analogue. Nucleotide analogues may include, for example, phosphorothioate, alkylphosphorothioate, or peptide nucleic acid (PNA), or may include an intercalating agent.

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The present invention also relates to a kit for distinguishing a variety of radioactive laver, characterized in that it includes a primer set for distinguishing the variety of radioactive laver. In addition to the primer set, the kit of the present invention may further include components necessary for the PCR reaction, for example, a buffer, a DNA polymerase, dNTPs, a DNA polymerase cofactor, and the like.

The present invention also comprises the steps of (A) isolating genomic DNA from a radiopattern sample; (B) amplifying a target sequence by performing an amplification reaction using the isolated genomic DNA as a template and the primer set of claim 1; and (C) detecting the amplification product. For the isolation of genomic DNA or PCR amplification reaction, a method known in the art may be used, and it will be easy for those skilled in the art to select an optimal method and conditions with reference to a known method, so a detailed description thereof will be omitted.

The detection of the amplification product may also be performed by a method known in the art, for example, capillary electrophoresis, gel electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement, but is not limited thereto. In the case of detecting an amplification product through fluorescence or phosphorescence measurement, it is natural to label the end of the primer with a fluorescent or phosphorescent material. For radioactivity measurement, an amplification product may be labeled by adding a radioactive isotope to the PCR reaction solution.

As described above, according to the present invention, varieties of radish laver can be divided into 3 or 4 groups, and maintenance and management of varieties with variety protection rights, selection of a control variety closest to the applied variety when testing new varieties, and infringement of variety protection rights and seed dispute resolution, etc., can be actively used to examine variety protection, protect intellectual property rights, and establish a laver seed distribution system.

1 is an electrophoresis photograph confirming the presence or absence of a primer for the SSR marker selected in an embodiment of the present invention.
2 is a view showing the size of the PCR amplification product for the SSR marker amplified using the primer set according to the present invention.
3 is a view showing the allelic profile and characteristics of each cultivar of Radistriae by the primer set of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, these embodiments are merely examples for easily explaining the content and scope of the technical idea of the present invention, and thereby the technical scope of the present invention is not limited or changed. It will be natural for those skilled in the art that various modifications and changes can be made within the scope of the technical spirit of the present invention based on these examples.

[Example]

1. Radiant pattern laver varieties and characteristics

Homozygote obtained from the Seaweed Bio Research Center was used for the radioactive laver samples. Table 1 summarizes the varieties of radioactive laver used in this example,

Using the Dellaporta method from the sporophyte of radish laver received for the following SSR analysis, Genomic DNA was extracted and used.

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2. Kim SSR Marker Selection

SSR is evenly distributed in the genomes of most advanced organisms, so there are many markers that can be analyzed, and it is highly reproducible and easy to analyze. Therefore, it was confirmed whether the SSR marker could be used to identify the laver variety. For the development of markers for SSR in the seaweed genome, the nuclear genome of Pyropia yezoensis , the nuclear genome, was analyzed and sequenced from the draft genome sequence.

For the preparation of SSR marker primers, about 500 bp sequences were obtained from each SSR up-stream and down-stream, and F and R-primers were prepared therefrom. The primer was designed so that the PCR product size was about 100-350 bp using primer3 (http://bioinfo.ut.ee/primer3-0.4.0/). The prepared primer was mapped to the entire seaweed draft-genome, the mapping site (5 bp agreement standard) was investigated, and specific primers were selected.

Based on the above criteria, 293 SSRs were selected, and the FAM, VIC or NED fluorescent dye sequence was designed to bind to the front part of the forward primer. The primers were again classified by the size of the PCR product and by fluorescence die to determine the reaction combination for PCR, and PCR was performed, and the operation of the primers was confirmed through agarose gel electrophoresis. PCR amplification was repeated 35 times by initial DNA denaturation at 95°C for 15 minutes, DNA denaturation at 95°C for 30 seconds, template DNA and primer conjugation at 60°C for 90 seconds, and DNA extension at 72°C for 60 seconds.

1 shows the PCR reaction results obtained in one PCR tube by simultaneously applying three SSR primers of different PCR produce sizes to the genomic DNA isolated from the filamentous body of one laver variety as a template.

The results of SSR markers, from which PCR products were successfully obtained, were mixed with formamide and DNA size standards, followed by fragment analysis, and SSR profiles were analyzed using ^{Applied biosystems' Peak Scanner TM Software.}

SSRs were analyzed to select 34 SSRs with two or more alleles. The obtained 34 SSRs were again analyzed on 13 cultivars, and finally, two SSRs with high genetic variation that could be applied to the grouping of rhizomes among SSRs were selected. Tables 2-3 show the characteristics of the two SSR markers, respectively, and the sequences of the primer sets used for PCR.

PCR was performed using, as a template, the primers of Tables 2 to 3, whose operation was confirmed by the above method, as a template, separated from the 13 varieties of radiographic filaments listed in Table 1. By measuring the size of the PCR product using a gene scan system, the SSR allele type for each radiograss cultivar was determined.

2 is a view showing the size of the PCR amplification product for the SSR marker amplified using each primer set, summarizing the allelic profile for the SSR, and FIG. The profile of the SSR allele is shown. 3, the number indicates the type of allele of each SSR. As can be seen in the figure, three types of alleles were found for SSR_10860 and SSR_7908.

Therefore, if the ⓐ marker SSR_10860 is used , the cultivar of Radial laver was classified into [Ebisu No. 6, Sugwawon 104, 105, 110, 112], [Jeonsu No. 2, Sugwawon 109, 111, 114, Hakgari, Haepung No. 1] and When the ⓑ marker SSR_7908 is applied to the three groups of [Orchard 106, 108], [Ebisu 6, Orchard 104, 105, 110, 112] and [Jeonsu No. 2, Orchard 109, 111, 114, Sea breeze 1] and [Orchard 106, 108, Hakgari] ⓒ When SSR_10860 and SSR_7908 are used at the same time , [Ebisu 6, Orchard 104, 105, 110, 112] and [Jeonsu No. 2, orchard 109, 111, 114, Haepung No. 1], [Orchard 106, 108], and [Haggari] can be accurately distinguished, and can be used as a stable tool in the process of identifying seaweed varieties.

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<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Primer Sets for Detecting SSR markers for Discrimination of Pyropia yezoensis Cultivas and Method for Discrimination of Pyropia Yezoensis Cultivas Using the Composition <130> P0520-201 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atcctatgag cgacctggtg 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cggcgaaaaa ttgaagtaat g 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggccatgatt aatgaacggt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atagtcatag ccgcgagcac 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tcagtctttg agagcacccc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gtttcgtgga ttcgcacac 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gggtcatggg atgtgtagga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggaaacgggt tgtttgatgt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gggtacctca aaaccgtgtg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctggaagaga ggagcgtcac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 agcattggtc agtcagaggg 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gttggtggtt gttccaacg 19 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 agaaacagca ctttccccct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tagctcttcc atttggtggg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 agtgaggttc gacagcgtct 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 catttttgct cccactaccg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gctgggagct aaggaggact 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ggcttagtcg aacgaccgta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 agcgacggta gcagttcagt 20 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cttcccctgg tggctctt 18 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cggagtttca tccacgattt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 cgagagacaa agtccccaag 20

Claims (10)

A primer set consisting of SEQ ID NOs: 17 and 18 for differentiating the radish variety into three groups having a PCR amplification product size of 340, 348 and 351.
delete delete delete delete delete delete delete A kit comprising the primer set of claim 1 for differentiating the radiograss variety into three groups having a PCR amplification product size of 340, 348 and 351.
(A) isolating genomic DNA from the radiopattern sample;
(B) amplifying a target sequence by performing a PCR amplification reaction using the primer set of claim 1 using the isolated genomic DNA as a template; and
(C) detecting the amplification product;
(D) determining the radiation pattern as belonging to any one of the three groups of PCR amplification product sizes 340, 348, and 351 according to the size of the PCR amplification product;
A method of distinguishing a group of radioactive laver varieties comprising a.

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