KR20100065413A - Microsatellite markers in chasmagnathus convexus and primers - Google Patents

Abstract

PURPOSE: A microsatellite marker of Chasmagnathus convexus and a primer for amplifying the same are provided to quantitatively and qualitatively evaluate condition of endangered species. CONSTITUTION: A microsatellite marker of Chasmagnathus convexus comprises a nucleotide selected from sequence numbers 1-9. The microsatellite marker of Chasmagnathus convexus comprises nine pairs. A primer for amplifying the marker comprises a nucleotide selected from sequence numbers 10-27.

Description

Microsatellite Markers in Chasmagnathus convexus and primers}

The present invention relates to a number of microsatellite markers and primers for amplifying the same, and in particular, to a pair of microsatellite markers and a primer for amplifying them.

Biodiversity on Earth is rapidly decreasing due to the direct or indirect effects of human activity. Recently, however, as biodiversity has emerged at home and abroad, efforts have been made to conserve in various ways.

In Korea, 229 species of endangered wild animals and plants (1st class 50 species and 2nd class 179 species) have been designated by the Ministry of Environment's "Wild East and Plant Protection Act", and habitats, in particular, microhabitants, are used to preserve these endangered species. Research is underway to protect and restore the loss of biodiversity by protecting it.

However, the decline in populations undergoing these endangered species causes a decrease in inbreeding and fitness, resulting in a loss of genetic diversity in the species. This reduces the species' ability to adapt to environmental changes and increases its chances of extinction.

In order to preserve these endangered species, not only the in-situ conservation, which preserves wild habitats, but also keeps the population through breeding and returns them back to the wild to maintain the decreasing size of the natural population. Ex-situ conservation is also very important.

Re-emission techniques through breeding and multiplying species that are currently suffering from severe population shortages are of great value in preserving endangered species. However, the re-release method through this multiplication should be largely two things.

Firstly, breeding for multiple generations for multiplication can lead to 'virtualization' where individuals adapt to the breeding environment, which reduces the genetic diversity of the breeding population and releases it into the wild. It is difficult to achieve the goal of restoring genetic diversity in wild populations.

Second, in terms of preserving the genetic diversity within the species during re-emission, the populations captured and bred must be released to the same area of origin. Many of the natural populations have had their own independent evolutionary history and are therefore also genetically and ecologically independent. Therefore, differentiation between populations is one of the important processes of speciation, so releasing individuals with different genetic composition will artificially interfere with this process.

For this reason, genetic information as well as ecological information about the target species are essential for the restoration of species through capture, breeding and introduction.

Recently, in developed countries, conservation genetic techniques have been actively used to assess the condition of species and populations. Conservation genetics is the theory and practice of genetics aimed at minimizing the threat of extinction by allowing species to be preserved as beings capable of adapting to changes in the environment.

There are three main activities of conservation genetics: first, to maintain genetic diversity in small populations and to avoid inbreeding, and second, to resolve conservation uncertainty and to define conservation units. Finally, molecular genetics is applied to understand species biology.

At present, conservation genetics are applied to the conservation of endangered and commercially available species, mainly in Europe and the United States. Development of appropriate molecular markers for the species must be preceded in order to apply conservation genetic techniques to establish population assessment and conservation strategies.

However, in Korea, attempts to protect wildlife by applying conservation genetic techniques have been made only for a few species of large mammals such as goats, and research and development of appropriate molecular markers are urgently needed in more diverse species. Do.

The most preferred molecular marker for recent population analysis is microsatellite. Microsatellite refers to the repetition of 1-5 short sequences, which is characterized by a very high polymorphism in the number of repeat sequences. By applying simple PCR techniques, the molecular marker can use a number of loci that follow Mendel's law of genetics, enabling a variety of reliable assays. Therefore, it has very high discrimination power and information power compared to isozyme, protein polymorphism, restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP). In particular, the process of semi-automating processes such as scoring and genotyping has the advantage of being able to process large amounts of data.

The present invention has been carried out to develop a new microsatellite marker suitable for population evaluation through a number of molecular genetic techniques as a result of the development of a new pair of microsatellite markers for the development of nine pairs of microsatellite markers and The purpose is to provide a primer for amplification.

The present invention provides a number of microsatellite markers comprising the nucleotide sequence of any one of SEQ ID NOs: 1-9.

In addition, the present invention provides a primer for amplifying a number of microsatellite markers, characterized in that the base sequence of any one of SEQ ID NO: 10 to 27.

The present invention can be usefully used in establishing an effective conservation strategy since it is possible to quantitatively and qualitatively diagnose and evaluate the situation of an endangered sea crab population using a microsatellite marker.

Microsatellite markers and primers for amplifying them that can be used for conservation genetic evaluation of the Chasmagnathus convexus population according to the present invention are made as follows.

Example 1: Sample Preparation and DNA Extraction

It is forbidden to catch sea crabs, an endangered species designated by the Ministry of Environment. Therefore, the collected individuals were cut and released only for the left fifth walking leg for the experiment and transported to the laboratory in 100% ethanol. DNA was carefully extracted from tissue in the shell and extracted using the Qiagen DNeasy Blood & Tissue Kit (USA).

Example 2: Screening and Amplification of Microsatellite-Containing DNA Fragments

The extracted DNA was digested with BfucI enzyme and separated only DNA fragments of about 200 bp to 1 kb using Chroma spin column 400-TE and then SAULA (5'-GCG-GTA-CCC- GGG-AAG-CTT-GG). -3 '), SAULB (5'GAT-CCC-AAG-CTT-CCC-GGG-TAC-CGC-3') linker. The ligated DNA was amplified by PCR reaction using SAULA as a primer, and then 5 kinds of 5'-biotinylated probes ((CA) ₁₁ , (GA) ₁₁ , (CAG) ₁₅ and (AAT) ₁₅ consisting of repeat sequences. , (CCT) ₁₅ ). Next, Strept-avidin-coated magnetic beads (Dynabead M-280) were washed sequentially with 1.4 × SSC, 2 × SPEP, 6 × SSC, mixed with the hybridized DNA, rotated for 15 minutes at room temperature, and then again with 2 × SSC. Wash once with 1 × SSC to wash out any residual DNA that is not bound to magnetic beads. Thereafter, the procedure of incubation at 90 ° C. for 5 minutes was repeated twice to separate DNA fragments having microsatellite bound to magnetic beads. PCR is performed on the DNA as a template to amplify the DNA with the micro satellite once again and then undergo a transformation experiment with Promega pGEM-T Easy Vector (USA).

Example 3: Colony Selection and Sequencing with Microsatellite

LB medium containing Ampicillin (50 mg / ml), IPTG (200 mg / ml) and X-gal (20 mg / ml) was inoculated with E. coli containing DNA obtained in Example 3 and incubated at 37 ° C. for about 16 hours. After that, only white colonies were carefully selected and extracted using the QIAGEN Miniprep Kit (USA). Thus obtained DNA was PCR with SP6 and T7 primers, and the product was purified again using QIAGEN PCR Purification Kit (USA), followed by sequencing reaction using BigDye. Thermal analysis was performed using Applied Biosystems' 3730 DNA Analyzer, and the results are shown in SEQ ID NOs: 1-9 below.

C250 (SEQ ID NO: 1)

G CTAGCCATCCCTTGGGAAAT CGGAAAAAGTACTACACACACACACACACACACACACACACACACACACACACACACACACACACTCACACACACACACACACACACACACACACGTACTGCAATGGTTAGCACGCTCGACTCCTGCA GTGGTTATCAGGCTTGGCTC ACAGTCGAGACGAGAGA

C1006 (SEQ ID NO: 2)

GAGCAATGGTTAATGTGAAACAG TTGTTGCTAAAGGGAAAAAAAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTTGGGCTCTCCCCTTTT CCACAATATTCCGTACATTACCA

C1007 (SEQ ID NO: 3)

GGAAGGAGACGAAAGGAGGA GAGAAAAGGCAACCAAGTGTAACATTTCTTCTCCCTCTTCCTCCTCCTCTTCTCGGACTTCGTTAA TGTCTGCCTTTTTACTCCGGAGAAGG

C1017 (SEQ ID NO: 4)

CTACCTCGCCTTCTCCTTCC TCTCCCTCTCTCTTGCCTCCTCTCCCTATCCCTCTCCCTCTTCTCCCTGCTCTTCCTCTCCCTCCTCCTCCTCCTCCTCCTCTCAGAGCCGTGACAGTGATCGGGG AAGTTAATTACGACTTCATAAATATGCAAATTGG

CC1028 (SEQ ID NO: 5)

TTGTGGGAGGAAGAGAGCTG CAGGCGAAGGAGAAGAGGAGGTACAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGTTGGAG CCAAGACGCAGGAGGCAAG

CC1029 (SEQ ID NO: 6)

G GCCGGACAAACAGACGAT CGGATCCCATTGTCGGTGATGGTGCGTAATCCCCTTATGGAGGTTCAAGTGGCCCCGGGGGAGAAGGAGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAAGA CAAGAGAACACAGGAAAATGAATTAA

CC1031 (SEQ ID NO: 7)

TTGCAGTGTCTGTGCCTTTG CGCTTAGTTTAGCTTACCCTCCCAAACGTTTTCTTCTTCTTCTTCTTCTTCTTCTCCTCCTCC TCCTGAGGGTCTACCTGTGTG

CC1034 (SEQ ID NO: 8)

CCCGATGGCAAGTATGTTTT TGGTGCCCAATGTTTATTCTAGAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAACTACCTCTATTCCTTCCACTTTTTGTCCAGGA CCACATCTACTCTCGCTCC

CC1042 (SEQ ID NO: 9)

CGTTTGTATTTATGTCTACGTCGTG GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTCTA TTTGTCCGGTTTATAAAATTAGGAA

Example 4: Primer Design and PCR Condition Optimization

Primers were designed using the Primer3 program (http://frodo.wi.mit.edu/) on the basis of flanking regions within the nucleotide sequence including the microsatellite. Next, repeated PCR experiments were performed to find the microsatellite primer specific PCR reaction composition and PCR temperature conditions. As a result, a total of 25 μl PCR reaction mixture [0.2 μl of Promega go Taq (5 unit / μl), 5 μl 5 × taq buffer, 1.0 μl 2.5 mM dNTP, 0.5 μl 10 μM forward primer, 0.5 μl 10 μM reverse primer, 16.8 μl DW] Microsatellite primer optimized PCR temperature conditions [after 5 minutes reaction at 95 ℃, 38 cycle reaction at 95 ℃ 30 seconds, 54 ℃ 50 seconds, 72 ℃ 50 seconds, and finally 72 ℃ 30 minutes reaction] After conducting the experiment, electrophoresis was performed on 1.5% agarose gel for a total of 9 pairs of markers CC250 (SEQ ID NO: 10, 11), CC1006 (SEQ ID NO: 12, 13), CC1007 (SEQ ID NO: 14, 15), and CC1017 (SEQ ID NO: 10). No. 16, 17), CC1028 (SEQ ID NOs: 18, 19), CC1029 (SEQ ID NOs: 20, 21), CC1031 (SEQ ID NOs: 22, 23), CC1034 (SEQ ID NOs: 24, 25), CC1042 (SEQ ID NOs: 26, 27) Confirmed that the response was successful. The sequence of these microsatellite primers, primer specific annealing temperature, and PCR product size are shown in Table 1.

Microsatellite Primer Sequence, Annealing Temperature and PCR Product Size Locus Primer sequence (5'-3 ') Tm ( ℃ ) Size (bp) CC250 F: CTAGCCATCCCTTGGGAAAT (SEQ ID NO: 10)
R: GAGCCAAGCCTGATAACCAC (SEQ ID NO: 11) 56 133-187 CC1006 F: GAGCAATGGTTAATGTGAAACAG (SEQ ID NO: 12)
R: TGGTAATGTACGGAATATTGTGG (SEQ ID NO: 13) 55 104-186 CC1007 F: GGAAGGAGACGAAAGGAGGA (SEQ ID NO: 14)
R: CCGGAGTAAAAAGGCAGACA (SEQ ID NO: 15) 56 94-106 CC1017 F: CTACCTCGCCTTCTCCTTCC (SEQ ID NO: 16)
R: CAATTTGCATATTTATGAAGTCGT (SEQ ID NO: 17) 56 177-189 CC1028 F: TTGTGGGAGGAAGAGAGCTG (SEQ ID NO: 18)
R: CTTGCCTCCTGCGTCTTG (SEQ ID NO: 19) 57
125-149 CC1029 F: GCCGGACAAACAGACGAT (SEQ ID NO: 20)
R: TTAATTCATTTTCCTGTGTTCTCTTG (SEQ ID NO: 21) 54 144-168 CC1031 F: TTGCAGTGTCTGTGCCTTTG (SEQ ID NO: 22)
R: CACACAGGTAGACCCTCAGGA (SEQ ID NO: 23) 58 127-151 CC1034 F: CCCGATGGCAAGTATGTTTT (SEQ ID NO: 24)
R: GGGAGCGAGAGTAGATGTGG (SEQ ID NO: 25) 56 140-204 CC1042 F: CGTTTGTATTTATGTCTACGTCGTG (SEQ ID NO: 26)
R: TTCCTAATTTTATAAACCGGACAAA (SEQ ID NO: 27) 55 83-121

Example 5: Genotyping of Microsatellites

Genotypes of 20 regions were analyzed with 9 pairs of microsatellite primers. Sample analysis was performed using a 3130xl Genetic analyzer (Applied Biosystems) and GeneMapper v4.0. Table 2 shows the number of repeat motifs and alleles for each locus of the microsatellite.

Repetition motifs and alleles per microsatellite locus Locus Repeating motifs Allele count CC250 (CA) 38 18 CC1006 (GT) 36 21 CC1007 (CCT) 7 3 CC1017 (CCT) 16 3 CC1028 (GGA) 11 10 CC1031 (CCT) 8 8 CC1034 (GA) 33 23 CC1042 (GT) 23 16

Claims (2)

Number of microsatellite markers, characterized in that consisting of the base sequence of any one of SEQ ID NO: 1 to 9. A primer for amplifying a number of microsatellite markers, characterized in that consisting of the nucleotide sequence of any one of SEQ ID NO: 10 to 27.