KR101151109B1 - Identification method for cymbidium species of oriental orchid using by cross-species transferable microsatellite markers and primers for amplification thereof - Google Patents

Abstract

PURPOSE: A method for distinguishing Cymbidium species oriental orchids using a microsatellite marker is provided to enable phylogenetics research. CONSTITUTION: A method for distinguishing Cymbidium species comprises a step of amplifying a microsatellite marker using primer containing a pair of sequences among sequence numbers 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 19, and 19 and 10. The microsatellite marker comprises one sequence among sequence numbers 1 to 6. The Cymbidum species oriental orchids include Cymbidium goeringii, C RCHB, C, gykuchin, C, forrestii, C. kanran, C. sinense, and C. faberi.

Description

Identification method for Cymbidium species of oriental orchid using by cross-species transferable microsatellite markers and primers for amplification

The present invention relates to an egg identification method using a microsatellite marker, and in particular, Cymbidium goeringii , C. RCHB , C. gyokuchin , C. forrestii , Han ( A study on microsatellite markers commonly applied to symbiotic oriental eggs in C. kanran , C. sinense , C. faberi , etc. and methods of identifying oriental eggs in symbidium using the same will be.

)의 개화습성을 가지고 있으며 3-4월에 꽃을 피우고, 야산의 소나무 숲속에서 자라는 반음지성 식물로 잎이나 꽃이 작고 향기는 없으며 화려하진 않지만 변이에 따라 잎의 무늬가 다양하고 꽃의 색과 모양이 다양해서 애호가들에 의하여 재배되고 있다. 한국 춘란은 일반적으로 향이 진하지 않은 것으로 알려졌으나, 제주도나 전남 해안가에서 발견된 일부 춘란은 중국 춘란처럼 특이한 향이 있는 유향종이 동정되기도 하였다. Egg plants are the most advanced of the monocotyledonous plants, with 800 genera and 20,000-25,000 species worldwide. Among them, C. goeringii is an evergreen perennial herb that grows in Korea, China, Japan, and Taiwan. It grows mainly in the southern region and enjoys the condition of fresh wind in summer and relatively strong in winter cold. Plant (Lee et al., 2004). Chun-ran is a day off

It is a semi-negative plant that blooms in March-April and grows in the pine forests of Yasan. The leaf or flower is small, has no fragrance, and it is not gorgeous. Its variety is grown by lovers. In Korea, Chunlan is generally known to be not scented, but some Chunlan found on the coast of Jeju Island or Jeonnam have been identified with unique scents like Chinese Chunlan.

C. RCHB is widely distributed in China's Sichuan and Yunnan provinces. The leaves are 30 ~ 45 ㎝ long and 1 ~ 1.5 ㎝ wide. . Flowers bloom locally from the end of January to the middle of February, and there are more clusters than Chunlan, which blooms from 2 to 5 flowers per flowerbed.

C. gyokuchin is native to Taiwan, Fujian Province, China. The leaves are V-shaped, deep in bone, thick, 30 ~ 40 cm long, and 1 cm wide, which is larger than Chunlan.

Flowering season is autumn, with 5-9 flowers per flower, fragrance is very good, and petals are pale green. The petals are slightly pointed compared to the chunran, and the flowers are less than half the size of the chunran.

C. forrestii is native to the mountainous region of Zhejiang and Jiangsu provinces in China, and is limited to some areas compared to Chunlan. Leaves are 30 ㎝ long and 1 ㎝ wide, slightly larger than Chunlan. The flowering period is similar to Chunlan, and the scent of Chunran is very scented. Its appearance is similar to that of Chunlan, but the color of the portrait leaf is reddish purple. Petals and flowers are similar in size to Chunlan.

C. kanran is distributed in Korea, China, and Japan, and grows only in Seogwipo area, south of Halla Mountain. Leaves are 40 ~ 70 ㎝ long and 1 ~ 1.7 ㎝ wide. Flowering season is from November to January, with excellent fragrance, and 9 ~ 20 flowers bloom per season. The petals are sharper and wedge-shaped than the Chunlan, and the flowers are in full bloom.

C. sinense is native to southern China and Taiwan, and its leaves are 30 ~ 40 ㎝ long and 1.5 ~ 2.5 ㎝ wide and larger than Chunlan. The flowers of 10 ~ 15 flowers per circumference are in bloom, and the period of flowering is from late January to mid-February, with a dark and muddy scent, and the petals are purple. The petals are sharper than those of the Chunlan, but they are a little less sharp than the Clan, and the flowers are less than half the size of the Clan.

C. faberi is a kind of oriental eggs native to China's Jiangsu, Zhejiang, and Hubei provinces. The length of leaves is 30-50 cm and the width is 1-1.5 cm. Flowers bloom in April-May, a little later than Chunlan, and have very good scents. Flowers bloom 9-15 per flower. The petals are pointed wedge-shaped, slightly smaller than the Chunlan.

Orchids, including Chunlan, are high value-added industries that show a trend of continued market growth. The orchid industry has a much higher export performance than the major flowers such as roses, chrysanthemums, and orchids. It is the only major flower crop whose export performance is increasing. However, with increasing demand, distrust in the market has been spreading due to the distribution of fake varieties treated with pharmaceuticals, and cheap Chinese products are also traded as high-priced goods. In particular, the price of Chunlan, etc. varies greatly depending on the variability, so the temptation to produce and sell goods that make cheaper items expensive is high.

With the recent rapid development of biotechnology, research on the development of active genetic markers at the DNA level has led to various researches such as tracking the genetic relationship of important crops with important values and determining their position as a unique genetic resource. It's going on. In particular, the simple sequence repeat (SSR) locus, also called SSR (Simple Sequence Repeat), has been widely used for genetic breeding and breeding of crops. Genetic identification using microsatellite is also called DNA fingerprinting, a technique that analyzes hypervariable DNA that differs from one individual to another, like fingerprints. In other words, microsatellites are characterized by (1) very frequent and even distributions in the genome of organisms, (2) maintenance of many alleles and high discrimination, (3) high reproducibility and cross-applicability between species. Cross-species transferability makes it more discriminating than any genetic marker. Since research on microsatellite markers began with human genome research in the 1980s, microsatellite markers have now been developed for major horticultural and agricultural products.

However, most of the genetic markers developed for Chunran are based on random amplified polymorphic DNA (RAPD) or studies using ISSR (inter-SSR) markers. Recently, 14 types of SSR markers for chunran have been reported by Moe et al. (2010), and in Korean Patent Laid-Open No. 10-2011-0118199 as a prior application related to the inventor of the present invention, 10 kinds of microsatellites for chunlan And primers for amplifying them. However, Chunbi and Cymbidium Markers that can be applied simultaneously to the symbidium egg to distinguish different species of the genus have not been developed yet. Therefore, there is an urgent need to establish a microsatellite that can be applied to several species simultaneously in terms of recovering trust in the oriental egg market and protecting genetic resources of native eggs.

The present invention is Cymbidium ( Cymbidium ) including Chunlan The purpose of the present invention is to provide a marker and an identification method using the same, which can be applied simultaneously to the symbidium column to distinguish several species of the genus and to identify individuals. The present inventors conducted a study to develop microsatellite markers applied simultaneously to the symbidium eggs, and identified six new microsatellite markers with high polymorphism through the screening of SSR-enriched libraries in Chunlan. These markers confirmed polymorphisms in six other species in the symbidium, thus completing the present invention.

An object of the present invention is to provide a microsatellite marker that can be amplified by PCR at the same time to apply to the Symbidium ( Cymbidium ) eggs, especially chunran, chun gum, small heart eggs, Chinese chunran, Hanran, bonded eggs, symbidium such as sun-curing It is intended to provide microsatellite markers that can be applied simultaneously to the genus Oriental Egg to distinguish species from each other and to be used for individual identification and phylogenetic studies.

It is also an object of the present invention to provide a primer for amplifying the microsatellite marker of chunran.

It is also an object of the present invention to provide a method for identifying species and identifying individuals of genus Symbidium eggs using the microsatellite markers.

In the present invention, there is provided a microsatellite marker which is commonly applied to the oriental egg genus of Symbidium composed of the base sequence of any one of SEQ ID NOs: 1-6. The oriental eggs of the genus Cymbidium include, in particular, Chun-lan, Chun-gumran, Small-Shimran, Chinese Chunran, Hanran, Bonded egg, and Sun-curing flowers.

In addition, in the present invention, the base pair of any one of SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 19, and SEQ ID NO: 19 and 20 To provide a primer.

In addition, in the present invention, by using one or more microsatellite markers consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, a method for distinguishing species of oriental eggs of Cymbidium genus and identifying individuals This is provided.

In the present invention, for the first time, microsatellite markers simultaneously applied to oriental eggs in the genus Cymbidium were identified. The marker of the present invention has a comprehensive discrimination ability and an object identification accuracy of the spring eggs 3.97 X 10 ^-6 and 99.9996%, and a comprehensive discrimination power and the accuracy of the comprehensive object identification of the bonded eggs are 2.74 X 10 ^-5 and 99.9973%. appear. The identification method using the microsatellite marker of the present invention is used for species identification and individual identification of Symbidium species, and can be usefully used for phylogenetic studies on the regional distribution of Symbidium species, and furthermore, unique DNA ID for each individual species. The grant can also be useful for in-flight tissue culture and for the establishment of genetic validation systems for individuals of major spring eggs with high industrial or commodity value.

1A to 1C show genotyping results of six microsatellite markers of the present invention.
2 shows repeat units, repeat regions and nucleotide sequences of six microsatellite markers of the invention.

In the present invention, in order to develop a microsatellite marker that can be applied simultaneously to various species of symbidium, a microsatellite-concentrated library was produced and used. As a probe for separating microsatellites, a die-tri -And 12 kinds of tetranucleotide repeat sequences were used (see Table 1 below).

Screening was performed from the prepared Chunlan microsatellite-enriched libraries to isolate the positive clones, extract plasmids from them, analyze the direct sequencing of the inserted fragments, and analyze the location and characteristics of the microsatellite sequences. Of the analyzed microsatellite motifs, the base repeat unit was repeated five or more times and six loci with sufficient conserved sequences before and after the repeat region were selected and primers were prepared for them. Primer lengths ranged from 19-24mer and were designed to work for all seven symbidium species. The sequence of each primer is shown in Table 2 below. PCR amplification using these primers and genomic DNA of seven oriental eggs as a template, followed by genotyping of the PCR products and analysis of allelic distribution, showed diversity in most species and identified six microsatellite markers. Newly established.

The base sequences of the six microsatellite markers CG709, CG722, CG787, CG1023, CG1229 and CG1508 established in the present invention are the same as SEQ ID NOS: 1-6 in the sequence listing, respectively, and their repeat units and repeat regions As shown in FIG. In addition, primers for amplifying each of these microsatellite markers are shown in Table 2 and SEQ ID NOs: 9-20.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the examples are only for better understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Fabrication of Spring Satellite Microsatellite-Enriched Genome Library

In order to separate various microsatellite motifs from Chunlan, plasmid libraries were prepared in the following manner.

10 μg of the extracted genomic DNA was digested with Sau 3AI restriction enzyme. After electrophoresis of the cleaved genomic DNA, 0.2-1.0 kb fragments were selected from agarose gels, and the Sau 3AI-specific adapter consisting of Oligo-A and Oligo-B was attached to the DNA fragments. The sequence of the adapter is as follows.

Oligo-A 5'-GGCCAGAGACCCCAAGCTTCG (SEQ ID NO: 7)

Oligo-B 3'-CCGGTCTCTGGGGTTCGAAGC CTAG -PO4 (SEQ ID NO: 8) (underlined is Sau 3A1 cut)

Adapter-attached DNA was hybridized with biotin-oligonucleotides and selected with SA-PMP particles (Strepatavidin Paramagnetic Particle) (Promega, USA). The 5'-biotin-labeled oligomer used at this time is a total of 12 kinds composed of di-, tri-, and tetranucleotide repeat sequences as shown in Table 1 below. The isolated fragments were first amplified by PCR, inserted into a pGEM-T Easy vector, and transformed into E. coli DH10B strain.

[Table 1]

Example 2. Plasmid Isolation and Screening of Libraries

For library screening, the library was plated and incubated in agar medium containing X-GAL, and white colonies were randomly selected for liquid culture. Subsequently, the plasmid was purified using a Plasmid mini-purification kit (SolGent, Korea), and the insert size was determined by digestion with Eco RI restriction enzyme, followed by agarose electrophoresis. About 22% of the clones were determined to have SSR motifs, confirming that the fabricated library was suitably made.

In the entire SSR motif identified above, 2-bp repeat sequence was observed at the highest rate of 200 times, and 3-base repeat sequence was observed 86 times. In particular, two-base repeats of 'AG' repeats were observed 172 times, showing the highest ratio among all repeat units, four-base repeats seven times, and complex repeats were observed 53 times.

Example 3. Design of SSR Locus-Specific Primers

As the SSR marker for genotyping, 6 loci with base repeating units of 5 or more and sufficient conserved sequences before and after the repeat region were selected and primers were prepared using the Primer3 program to amplify them by PCR method ( http: //primer3.source forge.net). The length of the primer was about 19-24mer and the T _m was made to be 58 ℃ or more, and after PCR, the length of the fragment was determined to position the primer in the range of 100-400 bp. Primer sequences, annealing temperatures, and sizes of PCR products of the six markers selected are summarized in Table 2 below.

TABLE 2

Example 4. Amplification and Genotyping of SSR Markers

PCR for amplification of the SSR markers was carried out in 20 μl of the mixed solution. After the reaction, the size of the fragments was determined by electrophoresis on an agarose gel. In order to determine the exact allele of the amplified DNA, it was determined by confirming the nucleotide sequence of the amplified DNA fragment. PCR products amplified using FAM-, HEX-, NED- and VIC-fluorescent labeled primers were isolated by automated sequence analyzer (ABI 3130, Applied Biosystems, USA) and genotyped with GENEMAPPER program. . Genotyping results of the six microsatellite markers of the present invention are shown in FIGS. 1A-1C.

As a result of genotyping the specimens collected for each of the six markers identified in the present invention, the range of PCR size of each species is shown in Table 3, and the results of examining the allele frequency were shown in Table 4a. were: (observed heterozygosity Ho) -] [ Table 4f] kinds of alleles observed and the occurrence frequency as shown in FIG heterozygosity.

When the total discrimination ability and the object identification accuracy of the four or more specimens (n = 40) and the bonded eggs (n = 14) were calculated, the Chulens were 3.97 X 10 ^-6 and 99.9996% and the bonded eggs were 2.74 X 10 ^-5 and 99.9973%, respectively. The results show that the binding genotype obtained by simultaneously testing six markers is very accurate, with at least 99.99% accuracy.

[Table 3]

[Table 4a]

[Table 4b]

TABLE 4c

Table 4d

Table 4e

TABLE 4f

TABLE 5

Attach an electronic file to a sequence list

Claims (3)

A method for distinguishing and identifying individuals of species of oriental eggs of Cymbidium using at least one microsatellite marker consisting of the nucleotide sequence of any one of SEQ ID NOs: 1-6. The method of claim 1, wherein the Cymbidium genus Oriental Egg comprises chunran, chungeumran, smallsimran, Chinese chunran, hanran, bonded egg, sun-curing. The compound of claim 1 or 2, wherein any one of SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 19, and SEQ ID NOs: 19 and 20 Amplifying the marker using a primer consisting of a sequence pair.

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