KR102261896B1 - Primer set for discriminating genetic background of Perilla sp. and uses thereof - Google Patents

Abstract

The present invention relates to a primer set for distinguishing the genetic background of plants of the genus Perilla comprising five primer sets, a kit for distinguishing the genetic background of plants of the genus Perilla including the SSR primer set, and a method of distinguishing the genetic background of the genus Perilla using the SSR primer set. The SSR primer set of the present invention can efficiently evaluate the genetic resources of plants of the genus Perilla to provide useful information for molecular breeding studies of plants of the genus Perilla.

Description

A primer set for discriminating the genetic background of plants of the genus Perilla and its use {Primer set for discriminating genetic background of Perilla sp. and uses them}

The present invention relates to a set of primers and their use for differentiating the genetic background of plants of the genus Perilla.

Perilla crop ( Perilla frutescens Britt. ) is an annual self-fertilizing plant. According to morphological characteristics and uses, two varieties, perilla ( P. frutescens var. frutescens , 2n=40) and perilla ( P. frutescens var. crispa , 2n=40), and perilla oil crops and perilla perilla have been widely cultivated and used as medicinal crops since ancient times, mainly in East Asia.

Perilla seeds contain various useful ingredients and are used as medicinal crops, oil crops, and leafy vegetables. The seeds of perilla contain a large amount of linolenic acid, an omega-3 fatty acid, which inhibits the synthesis of eicosanoids that cause adult diseases such as high blood pressure and allergic diseases, and improves learning ability and extends life. It is known to have bioregulatory functions of The seeds of perilla seeds themselves are used as sesame for perilla tea or confectionery, and the oil extracted from the seeds is used for food, pharmaceutical additives, and industrial purposes. Perilla leaves contain perilla aldehyde, limonene, and perilla ketone, which have a unique scent as a vegetable essential oil. Its unique scent can stimulate the taste buds, so it is used as a fresh leafy vegetable, and perilla leaf extract is used as a cosmetic color or flavoring.

In addition, perilla leaves are similar in shape to sesame leaves, and one or both sides of the leaves are purple, so it is also called perilla leaf. It is already used as a herbal medicine for asthma, cough, phlegm, sore throat, indigestion, back pain, insomnia, and diabetes. The purple component of perilla leaves is anthocyanin, and it is rich in vitamin B1, vitamin B2, vitamin B6, niacin, vitamin C, vitamin E, and iron, calcium, magnesium, zinc, etc., so it is a vitamin and mineral supply food. In addition to antibacterial and fungal actions, a number of effects such as alleviating allergy symptoms, anti-aging and anticancer effects have been reported.

Perilla and perilla crops are mainly distributed around the Himalayas, Southeast Asia, and East Asia. There are Indian theories, Southeast Asian theories, and southern Chinese theories as to the origin of these crops, but since ancient times, they have been most cultivated and used in East Asia. It is presumed to be the origin of perilla and perilla crops. Wild species of perilla and perilla crops have not yet been reported in East Asia, but recently there have been reports of weedy types of these crops. It was reported that weed types of these crops occupy a very important position in phylogenetic in understanding the origin and differentiation process of cultivated perilla and cultivated perilla. In Korea, it has been reported that weed types of these crops mainly grow wild around farmhouses, fields, roads, streams, or fields. In general, perilla and perilla are distinguished by morphological characteristics such as plant height, plant flavor, and seed size, but these two crops have the same number of chromosomes and can hybridize with each other by artificial crossbreeding. And in the taxonomic study of perilla and perilla crops, it was reported that there are intermediate types that are difficult to identify morphologically between these two crops. In order to successfully carry out the conservation of genetic resources of a crop and the development of varieties of that crop, it is first necessary to collect and search for the genetic resources for the crop, and to measure, evaluate, and understand the diversity of genetic variation in the collected genetic resources. Above all, it is most important.

In particular, the simple sequence repeat (SSR) analysis method used in the present invention is an analysis technique using polymerase chain reaction (PCR) and has excellent reproducibility, and has many alleles at each gene locus in plant populations. Since the characteristics can be clearly indicated, the genetic diversity and phylogenetic relationship of perilla and perilla can be analyzed and used for fingerprinting and molecular mapping.

On the other hand, Korea Patent No. 1993289 discloses 'molecular marker for purple discrimination of perilla leaves and their use', but the primer set for distinguishing the genetic background of plants of the genus perilla of the present invention and its use are described. none.

The present invention was derived from the above needs, and in the present invention, 15,991 SSR loci were identified by performing RNA sequencing of Perilla frutescens var. frutescens culturing strain PF98095 and searching for SSR (simple sequence repeat). Then, PCR was performed on various perilla and perilla ( Perilla frutescens var. crispa ) lines using the primer set prepared based on the SSR loci, showing polymorphism between the perilla and perilla lineages and showing a clear amplification pattern. The SSR primer set was selected, and the present invention was completed by confirming that the selected SSR primer set could effectively discriminate the genetic backgrounds of 34 perilla and perilla lineages.

In order to solve the above problems, the present invention provides an oligonucleotide primer set of SEQ ID NOs: 1 and 2; oligonucleotide primer sets of SEQ ID NOs: 3 and 4; oligonucleotide primer sets of SEQ ID NOs: 5 and 6; oligonucleotide primer sets of SEQ ID NOs: 7 and 8; and oligonucleotide primer sets of SEQ ID NOs: 9 and 10; provides an SSR primer set for distinguishing genetic backgrounds of plants of the genus Perilla sp.

In addition, the present invention is the SSR primer set; And it provides a kit for distinguishing the genetic background of plants of the genus Perilla, comprising a reagent for performing an amplification reaction.

In addition, the present invention comprises the steps of isolating genomic DNA from a plant sample of the genus Perilla; amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the SSR primer set; and detecting the product of the amplification step; it provides a method for distinguishing the genetic background of plants in the genus Perilla.

The SSR primer set of the present invention can effectively detect the DNA polymorphism of perilla and perilla plants, which are varieties of perilla, efficiently evaluate the genetic resources of plants of the genus perilla to provide useful information, such as molecular breeding studies of plants of the genus perilla. It is expected that there will be

1 is a perilla cultivated type (cultivated type of var. frutescens ; lanes 1-24) using 5 SSR primer sets of the present invention (KNUPF78, KNUPF79 KNUPF80, KNUPF81 and KNUPF82) 24 strains, perilla weedy type (weedy type) of var. frutescens ; lanes 25-29) and 5 strains of the cultivated type of var. crispa (lanes 30-34) were subjected to PCR.
2 is a phylogenetic tree showing the relationship between perilla cultivation type, wild dog weed type and perilla cultivation type (see Table 1) system resources.

In order to achieve the object of the present invention, the present invention provides an oligonucleotide primer set of SEQ ID NOs: 1 and 2; oligonucleotide primer sets of SEQ ID NOs: 3 and 4; oligonucleotide primer sets of SEQ ID NOs: 5 and 6; oligonucleotide primer sets of SEQ ID NOs: 7 and 8; and oligonucleotide primer sets of SEQ ID NOs: 9 and 10; provides an SSR primer set for distinguishing genetic backgrounds of plants of the genus Perilla sp.

In the SSR primer set of the present invention, the plant of the genus perilla may be perilla ( Perilla frutescens var. frutescens ) and a perilla variety ( P. frutescens var. crispa ). It is not limited thereto. The perilla and perilla perilla are different varieties within the same species.

The primer may include an oligonucleotide consisting of a fragment of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more consecutive nucleotides in the sequence of SEQ ID NOs: 1 to 10, depending on the sequence length of each primer. . For example, the primer (22 oligonucleotides) of SEQ ID NO: 1 may include an oligonucleotide consisting of a fragment of 19 or more, 20 or more, 21 or more consecutive nucleotides in the sequence of SEQ ID NO: 1. In addition, the primer may also include an addition, deletion or substitution of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 10. The odd-numbered oligonucleotide primers in the SEQ ID NO: are forward primers, and the even-numbered oligonucleotide primers are reverse primers.

The SSR primer set of the present invention is based on a simple sequence repeat (SSR) loci in the genome sequence of the cultivated type of var. frutescens lineage PF98095. Among the primer sets, various perilla and perilla (P. var. crispa) ) was finally selected as the SSR primer set that showed polymorphism between lines and showed a clear amplification pattern.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and can serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the DNA or RNA target required.

As used herein, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included.

The present invention also provides the SSR primer set; And it provides a kit for distinguishing the genetic background of plants of the genus Perilla, comprising a reagent for performing an amplification reaction.

In the kit of the present invention, the SSR primer set is the same as described above, and the distinguishable plants of the genus perilla may be perilla and perilla, but is not limited thereto.

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

In one embodiment of the present invention, the kit may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout explaining how to use the kit, eg, how to prepare reverse transcription buffer and PCR buffer, and suggested reaction conditions. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information published or provided through electronic media such as the Internet.

The present invention also

isolating genomic DNA from a plant sample of the genus perilla;

amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the SSR primer set; and

It provides a method for distinguishing the genetic background of plants of the genus Perilla, including; detecting the product of the amplification step.

In the method according to one embodiment of the present invention, the plants of the genus perilla may be perilla and perilla perilla, but is not limited thereto, and the SSR primer set is the same as described above.

The method of the present invention comprises the step of isolating genomic DNA from a plant sample of the genus Perilla. A method for isolating genomic DNA from the sample may use a method known in the art, for example, a CTAB method may be used, or a Wizard prep kit (Promega) may be used. The target sequence may be amplified by performing an amplification reaction using the isolated genomic DNA as a template and using the oligonucleotide primer set according to an embodiment of the present invention as a primer. Methods for amplifying a target nucleic acid include a polymerase chain reaction (PCR), a ligase chain reaction, a nucleic acid sequence-based amplification, and a transcription-based amplification system. amplification system), strand displacement amplification or amplification via Qβ replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In one embodiment of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. The labeling material may be a material emitting fluorescence, phosphorescence, or radioactivity, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer during amplification of the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material. ^{In addition, when a radioactive isotope such as 32} P or ³⁵ S is added to a PCR reaction solution for labeling using a radioactive material, the amplification product is synthesized and radioactivity is incorporated into the amplification product, so that the amplification product can be radioactively labeled. The oligonucleotide primer sets used to amplify the target sequence are as described above.

In one embodiment of the present invention, the method for distinguishing the genetic background of the plant of the genus Perilla comprises the step of detecting a product of the amplification step, and the detection of the product of the amplification step is a DNA chip, gel electrophoresis, Capillary electrophoresis, radiometric measurement, fluorescence measurement or phosphorescence measurement may be performed, but is not limited thereto. As one of the methods for detecting the amplification product, capillary electrophoresis may be performed. Capillary electrophoresis may use, for example, an ABi Sequencer. In addition, gel electrophoresis can be performed, and agarose gel electrophoresis or acrylamide gel electrophoresis can be used for gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, when PCR is performed by labeling the 5'-end of the primer with Cy-5 or Cy-3, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorometer. can do. In addition, the radioactive measurement method is a ^{radioactive isotope such as 32} P or ³⁵ S is added to the PCR reaction solution during PCR to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter (Geiger counter) or liquid scintillation The radioactivity can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

1. Plant material and DNA extraction

Crabs of 24 strains of Cultivated type of car. frutescens , 5 strains of Weedy type of car. frutescens and 5 strains of Cultivated type of car. crispa (Table 1). The nomic DNA was extracted from fresh young leaves using the Plant DNAzol Reagent protocol (GibcoBRL Inc., USA). The 34 plant resources of the genus perilla are resources collected from domestic and foreign regions during the period from 2006 to 2015. In the Accession number in Table 1 below, PF is the abbreviation of the scientific name of perilla and perilla, and the next two digits are the year of collection. , and the next three digits indicate the collection number that was collected that year (Kangwon National University, Department of Biological Resources Science).

2. SSR primer set development and PCR amplification

To develop the SSR primer, RNA-seq data of perilla cultivated PF98095 was de novo assembly using Trinity software (http://TrinityRNA Seq.sourceforge.net), and Perl script MISA tool (http://pgrc.ipk) -gater slebe n.de/misa) was used to search for the SSR portion in each RNA sequence of PF98095. As a result, a total of 15,991 SSR loci having di-, tri-, and tetra-nucleotide repeat units were identified, and the SSR primer set was designed through the PRIMER 3 program. Among the 15,991 SSR loci selected in this way, an SSR primer set having a relatively large number of repeats of each repeat motif (unit) was first selected to confirm the amplification pattern and polymorphism.

PCR amplification was performed to confirm amplification patterns and polymorphisms in the SSR loci, and the composition of the solution was mixed with 20 ng of genomic DNA, 1x PCR buffer, 0.3 M of primer, 0.2 mM of dNTP and 1 unit of Taq Polymerase (Takara). to make a total of 30 μl. The PCR process was pre-denatured at 94°C for 5 minutes; A total of 30 cycles of denaturation at 94° C. for 1 minute, annealing at 65° C. for 1 minute, and extension at 72° C. for 2 minutes; Final elongation at 72° C. for 10 minutes; After PCR was performed, five SSR primer sets with a clear amplification pattern and polymorphism between perilla and perilla strains were finally selected (Table 2).

3. Electrophoresis

5 μl of PCR product was mixed with 10 μl of loading buffer (98% formamide, 0.02% BPH, 0.02% Xylene C and 5 mM NaOH), and then 0.5x on a 6% acrylamide-bisacrylamide gel (19:1). After filling the TBE buffer, 2 μl of the sample was loaded, and electrophoresis was performed at 250 V for 30 minutes. After the electrophoresis was completed, the DNA band was confirmed using EtBr (Ethidium bromide).

Example 1. Genetic background analysis of perilla and perilla plants using SSR primer set

Perilla and perilla strains using 5 SSR primer sets (KNUPF78, KNUPF79 KNUPF80, KNUPF81 and KNUPF82) targeting 24 perilla-cultivated lines, 5 perilla weed-type lines, and 5 perilla-cultivated lines (Table 1) Hepatic genetic polymorphisms were analyzed.

As a result, the KNUPF78 primer set amplifies a DNA fragment with a size of about 100-150 bp in 34 perilla and perilla lineages and the number of alleles was 4, and the KNUPF79 primer set amplifies a DNA fragment with a size of about 100-150 bp and alleles. The number of genes was 8, the KNUPF80 primer set amplifies a DNA fragment with a size of about 195-205 bp and the number of alleles was 7, and the KNUPF81 primer set amplifies a DNA fragment with a size of about 190-205 bp, and the number of alleles was 4, and finally, the KNUPF82 primer set amplifies a DNA fragment with a size of about 120 to 140 bp and the number of alleles was 4.

In addition, the major allele frequency measured in the five SSR primer sets ranged from 0.235 to 0.794 with an average value of 0.435, and the genetic diversity value was from 0.351 to 0.830 with an average value of 0.674. , polymorphic information content (PIC) values ranged from 0.328 to 0.808, with an average value of 0.643 (Table 3).

Example 2. Analysis of the genetic relationship between perilla and perilla resources

In order to analyze the relationship for 24 perilla cultivation-type lines, 5 perilla weed-type 5 lines and perilla perilla cultivation-type 5 lines (Table 1) using 5 SSR primer sets of the present invention, phylogenetic tree using the STRUCTURE program (phylogenetic tree) was prepared (Fig. 2).

Through this, it was considered that the SSR primer set of the present invention could be usefully utilized for the cultivation of plants of the genus Perilla by distinguishing the genetic background of various plants of the genus Perilla.

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Claims (7)

oligonucleotide primer sets of SEQ ID NOs: 1 and 2; oligonucleotide primer sets of SEQ ID NOs: 3 and 4; oligonucleotide primer sets of SEQ ID NOs: 5 and 6; oligonucleotide primer sets of SEQ ID NOs: 7 and 8; And oligonucleotide primer sets of SEQ ID NOs: 9 and 10; Perilla ( Perilla frutescens var. frutescens ) and Perilla ( Perilla frutescens var. crispa ) SSR primer set composition for distinguishing the genetic background of plants. delete The SSR primer set composition of claim 1; And a kit for distinguishing the genetic background of perilla ( Perilla frutescens var. frutescens ) and perilla ( Perilla frutescens var. crispa ) plants, comprising a reagent for performing an amplification reaction. The kit according to claim 3, wherein the reagents for performing the amplification reaction include DNA polymerase, dNTPs and a buffer. isolating genomic DNA from a plant sample of Perilla frutescens var. frutescens or Perilla frutescens var. crispa;
amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the SSR primer set composition of claim 1; and
A method of distinguishing the genetic background of perilla and perilla plants, including; detecting the product of the amplification step. delete The method according to claim 5, wherein the detection of the product of the amplification step is performed through a DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement or phosphorescence measurement.

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