KR101331740B1 - SSR primer derived from Paeonia lactiflora and use thereof - Google Patents

Abstract

The present invention relates to an SSR primer isolated from the peony and its use. More specifically, a kit for determining a breed of a peony comprising an SSR primer pair having two nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 28 and the SSR primer pair and the SSR primer pair It is about how to determine the breed.
The SSR primer pair provided in the present invention can effectively detect the DNA polymorphism of the peony, and can be usefully used to prepare the DNA profile of the peony. In addition, by efficiently evaluating the genetic resources of the peony through this, it is possible to effectively perform redundancy analysis and establishment of novelty with existing holding resources when introducing new genetic resources.

Description

SSR primer derived from Peony and its use {SSR primer derived from Paeonia lactiflora and use about}

The present invention relates to an SSR primer isolated from the peony and its use.

A large amount of genetic resources are being collected every year from most major crops and materials used for breeding. On the other hand, the evaluation of genetic resources has not been done sufficiently. Rapid identification of species and subspecies of collected genetic resources is very important for the management and protection of accurate genetic resources. In addition, identification of genotypes, identification and classification of genotypes, and identification of their relationship are crucial to the conservation of genetic resources, the creation of new varieties, and the improvement of crops.

Recently, the rapid development of molecular biology has led to the development of nucleic acid fingerprinting methods and various DNA markers that enable the study of bio-diversity at the level of DNA (DNA). Through this, it is possible to perform simple and rapid search of useful traits, identification of species of organisms, classification and identification of cultivars, and flexible relationship analysis of group populations with little effect on external environments. Fingerprinting using the polymerase chain reaction (PCR) developed so far includes randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphic DNA (AFLP), and simple sequence repeat (SSR) methods. Among these methods, the RAPD method has a disadvantage that the non-specific PCR products are amplified and thus the reproducibility is poor. The AFLP method is disadvantageous in that the appearance and analysis of bands having poor reproducibility are complicated because they are attracted by the detection of high DNA polymorphism. In contrast, the SSR method is a method of preparing a PCR primer based on the nucleotide sequence information of a microsatellite region, which is a DNA repeat sequence. The SSR method is easy for microsatellite analysis and has high reproducibility. It is often used for sympathy. Especially, the SSR method has an advantage that it is not affected by the external environment at all. Due to the superiority of the SSR method, studies are underway to develop SSR markers in several major crops and cultivated crops.

Accordingly, the present inventors have completed the present invention by developing SSR primer pairs that show a large number of polymorphic mutations in the peony, while continuing to develop SSR markers capable of efficiently evaluating the genetic resources of the peony.

Domestic Publication No. 10-2010-0112501

The present invention is to analyze the genetic diversity and flexibility of the peony using SSR primer pairs, to examine the possibility of breed discrimination by analyzing the genotype of the peony varieties, and to propose an efficient breed discrimination method.

The present invention provides an SSR primer pair having two nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 28.

The present invention also provides a kit for determining the variety of peony containing the SSR primer pair.

In addition, the present invention provides a method for determining the variety of peony using the SSR primer pair.

The SSR primer pair provided in the present invention can effectively detect the DNA polymorphism of the peony, and can be usefully used to prepare the DNA profile of the peony. In addition, by efficiently evaluating the genetic resources of the peony through this, it is possible to effectively perform redundancy analysis and establishment of novelty with existing holding resources when introducing new genetic resources.

Figure 1 shows the genetic relationship between the UPGMA phylogenetic tree collected 85 accessions from various regions of Korea.

The present invention provides an SSR primer pair having two nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 28.

The SSR primer pair is a primer pair represented by SEQ ID NO: 1 and 2; A pair of primers represented by SEQ ID NOS: 3 and 4; A pair of primers represented by SEQ ID NOS: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; A pair of primers represented by SEQ ID NOS: 9 and 10; A pair of primers represented by SEQ ID NOs: 11 and 12; A pair of primers represented by SEQ ID NOS: 13 and 14; A pair of primers represented by SEQ ID NOS: 15 and 16; A pair of primers represented by SEQ ID NOs: 17 and 18; A pair of primers represented by SEQ ID NOs: 19 and 20; A pair of primers represented by SEQ ID NOS: 21 and 22; Primer pairs represented by SEQ ID NOs: 23 and 24; Primer pairs represented by SEQ ID NOs: 25 and 26; It may be selected from the group consisting of primer pairs represented by SEQ ID NO: 27 and 28.

In addition, the SSR primer pair may be derived from Paeonia lactiflora. The peony is a perennial herb of the peony and is the origin of the horticultural species, called peony flower, in China . It is about 1m high and is divided into white peony and red peony according to the flower color. Because of its beautiful flowers, it has been widely cultivated for ornamental purposes since ancient times and grows well in half-shaded places. The root is treated as a valuable medicine in herbal medicine, and the root of Peony is used to treat anemia and painkillers, and the root of Peony is used as a blood pressure and antipyretic agent.

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

In addition, the present invention is the SSR primer pair; And a kit for determining a variety of peony, comprising a reagent for performing an amplification reaction.

In the kit of the present invention, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer and the like. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

In addition, the present invention comprises the steps of (a) extracting genomic DNA from the peony; (b) amplifying the target sequence by performing an amplification reaction using the SSR primer pair as a template of the extracted genomic DNA; And (c) provides a method for determining the variety of peony comprising the step of detecting the amplification product.

The method of the present invention comprises the step of separating genomic DNA from a peony sample. As a method for separating the genomic DNA from the sample, a method known in the art can be used. For example, the CTAB method may be used, or a wizard prep kit (Promega) may be used. Using the isolated genomic DNA as a template, the target sequence may be amplified by performing an amplification reaction using the SSR primer pair according to an embodiment of the present invention as a primer. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] 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 pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be, but is not limited to, a material that emits fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The primer pairs used to amplify the target sequence are as described above.

The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one method of detecting the amplification product, capillary electrophoresis can be performed. Capillary electrophoresis, for example, can use the ABi Sequencer. In addition, gel electrophoresis can be performed, and gel electrophoresis can utilize agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is performed, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement device such as a Geiger counter or liquid scintillation counter The radioactivity can be measured using a liquid scintillation counter.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example >

1. Materials and Methods

(1) plant material and DNA  extraction

A total of 85 accessions of Paeonia lactiflora were obtained from Milyang, Yeongcheon and Gimhae in Korea (Table 1). DNA was extracted from fresh leaves using Qiagen DNA extraction kit (Qiagen, Hilden, Germany). Purity and concentration of the extracted DNA was confirmed using NanoDrop ND-1000 (NanoDrop Technologies, Inc., Wilmington, DE, USA), the final DNA concentration was 20 ng / μL. Table 1 below shows 85 accessions of the peony used in this experiment.

NO ID Species Collection region NO ID Species Collection region One KUP001 YCH409 Gimhae 44 KUP044 YCH307 Yeongcheon 2 KUP002 YCH274 Miryang 45 KUP045 YCH320 Yeongcheon 3 KUP003 YCH277 Miryang 46 KUP046 YCH327 Yeongcheon 4 KUP004 YCH278 Miryang 47 KUP047 YCH328 Yeongcheon 5 KUP005 YH146-10 Miryang 48 KUP048 YCH329 Yeongcheon 6 KUP006 YH16-1 Miryang 49 KUP049 YCH331 Yeongcheon 7 KUP007 YH164-1 Miryang 50 KUP050 YCH333 Yeongcheon 8 KUP008 YH165-1 Miryang 51 KUP051 YCH337 Yeongcheon 9 KUP009 YH17-1 Miryang 52 KUP052 YCH338 Yeongcheon 10 KUP010 YH18-2 Miryang 53 KUP053 YCH340 Yeongcheon 11 KUP011 YH196-1 Miryang 54 KUP054 YCH341 Yeongcheon 12 KUP012 YH197-1 Miryang 55 KUP055 YCH345 Yeongcheon 13 KUP013 YH198-1 Miryang 56 KUP056 YCH351 Yeongcheon 14 KUP014 YH20-1 Miryang 57 KUP057 YCH353 Yeongcheon 15 KUP015 YH201-4 Miryang 58 KUP058 YCH354 Yeongcheon 16 KUP016 YH202-2 Miryang 59 KUP059 YCH355 Yeongcheon 17 KUP017 YH203-1 Miryang 60 KUP060 YCH356 Yeongcheon 18 KUP018 YH211-1 Miryang 61 KUP061 YCH357 Yeongcheon 19 KUP019 YH212-13 Miryang 62 KUP062 YCH361 Yeongcheon 20 KUP020 YH213-3 Miryang 63 KUP063 YCH362 Yeongcheon 21 KUP021 YH214-1 Miryang 64 KUP064 YCH363 Yeongcheon 22 KUP022 YH21-5 Miryang 65 KUP065 YCH364 Yeongcheon 23 KUP023 YH215-3 Miryang 66 KUP066 YCH365 Yeongcheon 24 KUP024 YH24-1 Miryang 67 KUP067 YCH366 Yeongcheon 25 KUP025 YH25-1 Miryang 68 KUP068 YCH369 Yeongcheon 26 KUP026 YH254-3 Miryang 69 KUP069 YCH370 Yeongcheon 27 KUP027 YH266-9 Miryang 70 KUP070 YCH372 Yeongcheon 28 KUP028 YH271-15 Miryang 71 KUP071 YCH373 Yeongcheon 29 KUP029 YH272-3 Miryang 72 KUP072 YCH376 Yeongcheon 30 KUP030 YH273-4 Miryang 73 KUP073 YCH377 Yeongcheon 31 KUP031 YH274-12 Miryang 74 KUP074 YCH378 Yeongcheon 32 KUP032 YH276-3 Miryang 75 KUP075 YCH392 Yeongcheon 33 KUP033 YH277-3 Miryang 76 KUP076 YCH393 Yeongcheon 34 KUP034 YH278-11 Miryang 77 KUP077 YCH399 Yeongcheon 35 KUP035 YH63-2 Miryang 78 KUP078 YCH403 Yeongcheon 36 KUP036 YH63-3 Miryang 79 KUP079 YCH405 Yeongcheon 37 KUP037 YH65-7 Miryang 80 KUP080 YCH407 Yeongcheon 38 KUP038 YH72-5 Miryang 81 KUP081 YH255-1 Yeongcheon 39 KUP039 YH275-15 Miryang 82 KUP082 YH256-11 Yeongcheon 40 KUP040 YCH261 Yeongcheon 83 KUP083 YH257-1 Yeongcheon 41 KUP041 YCH262 Yeongcheon 84 KUP084 YH258-1 Yeongcheon 42 KUP042 YCH281 Yeongcheon 85 KUP085 YH259-1 Yeongcheon 43 KUP043 YCH303 Yeongcheon

ID: identification number given by Kongju National University.

(2) SSR  motive enriched library Build

The SSR motif-enriched library from Peony's genomic DNA consisted of a modified biotin-streptavidin capture method. Total DNA was treated with seven restriction enzymes ( Eco RV, Dra I, Sma I, Pvu I, Alu I, Hae III, and Rsa I). After pooling, digested DNA was fractionated by size on 1.4% agarose gel. Fragments ranging from 300 to 1500 bp were dissolved from the gel, purified using a gel extraction kit (Qiagen), and adapters (AP11-5'-CTC TTG CTT AGA TCT GGA CTA-3 'and AP-12-5'-). TAG TCC AGA TCT AAG CAA GAG CAC A-3 '). The DNA ligated with the adapter was a long (40-45 nucleic acid) biotin-labeled repeat probe [(GA) ₂₀ , (CA) ₂₀ , (AGC) ₁₅ , (GGC) ₁₅ , (AAG) ₁₅ , (AAC) ₁₅ , (AGG) ₁₅ ]. Hybridized DNA fragments were obtained with magnetic beads coated with streptavidin. After thorough washing, the DNA fragments were separated from the beads with 50ul of distilled water, and PCR was amplified using AP11 primer. The amplified DNA product was inserted into pGEM-T Easy Vector (Promega) and E. coli Go to cells and replicate. Recombinant colonies were identified by blue / white colony selection.

(3) DNA sequencing  And SSR Primer  design

A total of 759 recombinant clones were randomly selected from primary transformation plates. DNA inserts in the plasmids were arranged using an ABI 3730xl DNA sequencer with the BigDye terminator kit (Applied Biosystems). SSR determination within replicated sequences and primer designs was performed using the SSR MANAGER program (Kim 2004). Primer pairs for polymorphism were evaluated in 8 bulk panels (5 accessions of peony per bulk) using the QIAxcel system (Qiagen). The size of the polymorphic PCR product was measured accurately according to Schuelke's (2000) M13 tail PCR method. PCR amplification was performed with 2 ul genomic DNA (10 ng / μl), 0.2 μl specific primer, 0.4 μl M13 universal primer (10 pmol / μl), 0.6 μl normal reverse primer, 2.0 μl 10 × PCR buffer, 1.6 μl DNTPs (2.5 mM), and 0.2 μl of taq DNA polymerase (5 unit / μl) in total volume of 20 ul. PCR amplification conditions are as follows: 3 minutes at 94 ° C., 1 step (30 seconds at 94 ° C., 45 seconds at 55 ° C., 1 minute at 72 ° C.), 30 times and 2 steps (at 94 ° C.) 30 seconds, 45 seconds at 53 ° C., 1 minute at 72) was repeated 10 times, and the extension reaction was carried out for 10 minutes at the final 72. SSR alleles were determined on an ABI PRISM 3500 Genetic Analyzer (Applied Biosystems) using GeneMapper 4.1 software and sized accurately using GeneScan 500 ROX (6-carbon-X-rhodamine) molecular size standards (50-500 bp). Measured.

(4) data analysis

The variability of each gene segment is measured using the genetic analysis package POPGENE version 1.31 to measure the number of alleles, observed heterozygosity, expected heterozygosity, and the genetic distance between accessions, respectively. It became. PowerMarker version 3.25 (Liu and Muse 2005) was used to measure the total number of alleles, major allele frequency, Gene Diversity, and Polymorphic Information Content. . The UPGMA algorithm was used to draw unrooted phylograms from the distance matrix using the MEGA4 software (Tamura et al. 2007).

2. Results

(One) SSR  Development

The enriched library for SSRs was run from Peony's genomic DNA. The development stages for constructing the enriched library and its structure and characteristics are summarized in Table 2. Table 2, below, relates to the screening process within the structure and properties of the microsatellite enriched library in the peony.

Screening steps Numbers (percentage) Sequenced clones 759 Redundant clones 35 (4.6%) Unique clones 724 (95.4%) SSR clones 321 (42.3%) Primer design 212 (27.9%) Checked markers 40 Polymorphic markers 14

A total of 759 recombinant clones were randomly selected and listed and found 35 (4.6%) extra clones. Of the remaining 724 (95.4%) unique clones, 321 (42.3%) had an SSR sequence.

Sequence analysis of all SSR-containing clones identified a total of 396 different repeat motifs. Dinucleotide SSRs (86.9%) were superior to trinucleotide SSRs (11.6%; Table 3). A small number (1.5%) of tetra- / penta- / gexanucleotide SSRs were also present in the library. Among the dinucleotide types, the AG / GA class of repeated motifs was most frequently identified (36.6%), GT / TG (24.1%) class, CT / TC (21.8%) class, and the rest (17.2%) classes. Followed. Of the trinucleotide SSRs, the GAA / AAG / AGA class of the repeated motifs was dominant (23.9%), GAG / AGG / GGA (17.4%) class, GTT / TTG / TGT (17.4%) class, and the remainder (41.3%). The class followed. Finally, 212 primer pairs were designed based on the flanking sequences of clones containing SSR. Of these, 40 primer pairs were used for screening for polymorphism. Only 14 primer pairs produced unique and reproducible polymorphic bands and were selected for later experiments. Table 3 below relates to the microsatellite sequences identified in the abundance of lively for the peony.

Repeat unit Repeat class Number (%) Di-nucleotide AG / GA 126 36.6 GT / TG 83 24.1 CT / TC 75 21.8 AC / CA 45 13.1 AT / TA 14 4.1 Total 344 86.9 Tri-nucleotide GAA / AAG / AGA 11 23.9 GAG / AGG / GGA 8 17.4 GTT / TTG / TGT 8 17.4 CAG / AGC / GCA 6 13.0 GCT / CTG / TGC 5 10.9 CAA / AAC / ACA 3 6.5 TGA / GAT / ATG 2 4.3 CCA / CAC / ACC 2 4.3 CTT / TTC / TCT One 2.2 Total 46 11.6 Other
(Tetra / Penta / Hexa) 6 1.5 Total repeat motifs 396

(2) SSR  Polymorphism

Variability in each gene segment is in terms of number of alleles, observed heterozygosity, major allele frequency, gene diversity, and polymorphic information content. Measured in The 14 SSR markers showed 103 alleles and 85 regions among 85 accessions (Table 1). Many degrees of alleles per gene portion varied between markers, ranging from four alleles (knu21, knu94) to 16 alleles (knu154) and an average of 7.4 alleles (Table 4). Of the 14 polymorphic gene moieties, 8 gene moieties consisted of complete repetition of dinucleotide or trinucleotide SSRs. The three gene segments (knu14, knu114, and knu124) consisted of complex repeats consisting of different repeat motifs (that is, composed of different repeat motifs), and the three gene parts (knu21, knu35, and knu115) are multiple repeats. (More than 1 SSR). The major allele frequency and the expected heterozygosity per gene segment have averages of 0.61 and 0.62, respectively, and 0.39 (knu14) to 0.88 (knu21) and 0.41 (knu21) to 0.78 (knu14), respectively. ) Varied. The values of genetic diversity had an average of 0.50, ranging from 0.21 (knu21) to 0.71 (knu14 and knu78), with PIC values of 0.43 on average and ranging from 0.19 (knu21) to 0.67 (knu78). Table 4 below relates to the general characteristics of the 14 new polymorphic markers developed for peony.

 (3) phylogenetic relationship

The proportion of covalent alleles was used to calculate the genetic distance between all pairwise combinations between the 85 accessions used in this experiment (Table 5). Genetic matrix distances measured by PowerMarker were used to draw the UPGMA tree using Mega4 software. The genetic distance between the 85 accessions of the Peony ranged from 0.10 to 0.95 with an average of 0.56. The diversity of genetic distances between the different accessions identified by SSRs reflected a high standard of polymorphism at the DNA level. All accessions could be grouped into 5 groups (groups I, II, III, IV, and V) within the UPGMA tree (FIG. 1). Table 5 shows Nei's genetic distances.

pop ID 1 2 3 One **** 2 0.6125 **** 3 0.9477 0.1074 ****

Attach an electronic file to a sequence list

Claims (7)

Primer pairs represented by SEQ ID NOs: 1 and 2; A pair of primers represented by SEQ ID NOS: 3 and 4; A pair of primers represented by SEQ ID NOS: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; A pair of primers represented by SEQ ID NOS: 9 and 10; A pair of primers represented by SEQ ID NOs: 11 and 12; A pair of primers represented by SEQ ID NOS: 13 and 14; A pair of primers represented by SEQ ID NOS: 15 and 16; A pair of primers represented by SEQ ID NOs: 17 and 18; A pair of primers represented by SEQ ID NOs: 19 and 20; A pair of primers represented by SEQ ID NOS: 21 and 22; Primer pairs represented by SEQ ID NOs: 23 and 24; Primer pairs represented by SEQ ID NOs: 25 and 26; And SSR primer pairs for discriminating Korean peony varieties consisting of 14 primer pairs of primer pairs represented by SEQ ID NOs: 27 and 28. The pair of SSR primers for discriminating Korean peony varieties according to claim 1, which are derived from peony. SSR primer pair for discriminating Korean peony varieties according to claim 1; And a reagent for carrying out an amplification reaction. The kit of claim 3, wherein the reagent for carrying out the amplification reaction comprises DNA polymerase, dNTPs, and a buffer. (a) extracting genomic DNA from the peony;
(b) amplifying the target sequence by using the extracted genomic DNA as a template and performing an amplification reaction using the SSR primer pair for discriminating Korean peony varieties according to claim 1; And
(c) a method of discriminating Korean peony varieties comprising detecting the amplification products. The method of claim 5, wherein the detection of the amplification product is carried out by capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.
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