Background: Single nucleotide polymorphisms (SNPs) are widely used genetic markers with applications in human disease diagnostics, animal breeding, and evolutionary studies, but existing genotyping methods can be labor-intensive and costly. The aim of this study is to develop a simple and rapid method for identification of a single nucleotide change. Methods: A modified Polymerase Chain Reaction Amplification of Multiple Specific Alleles (PAMSA) and high resolution melt (HRM) analysis was performed to discriminate a bovine polymorphism in the NCAPG gene (rs109570900, 1326T > G). Results: The inclusion of tails in the primers enabled allele discrimination based on PCR product lengths, detected through agarose gel electrophoresis, successfully determining various genotypes, albeit with some time and labor intensity due to the use of relatively costly high-resolution agarose gels. Additionally, high-resolution melt (HRM) analysis with tailed primers effectively distinguished the GG genotype from the TT genotype in bovine muscle cell lines, offering a reliable way to distinguish SNP polymorphisms without the need for time-consuming AS-PCR. Conclusions: Our experiments demonstrated the importance of incorporating unique mismatched bases in the allele-specific primers to prevent cross-amplification by fragmented primers. This efficient and cost-effective method, as presented here, enables genotyping laboratories to analyze SNPs using standard real-time PCR.

Radish is an important root vegetable in the world, and many cultivars have been developed with various molecular marker systems to identify these cultivars. Recently developed markers for radish cultivar identification require only 11 primer pairs, but they still use conventional PCR with different annealing temperatures and time-consuming gel electrophoresis. To improve the genotyping method, we applied touchdown PCR with 11 primers with M13 tails among 105 radish cultivars. Touchdown PCR successfully generated amplicons in all 11 M13-tailed primers with a condition of annealing temperature starting from 55℃, decreased by 1°C and 33 cycles at 53°C. The 11 M13-tailed primers followed by fragment analysis produced 71 amplicons, which produced more amplicons than gel electrophoresis that produced 23 amplicons. Especially, simple sequence repeats produced more amplicons, 12 on average, than the other marker types. The present study requires less effort and provides more accurate results compared to genotyping using gel electrophoresis. Besides, a database can be established using digitized genotyping results among radish cultivars.

주변 국가인 태국, 캄보디아, 베트남, 라오스 등에서 벼멸구(Nilaparvata lugens)와 흰등멸구(Sogatella furcifera)를 채집하던 중, 벼멸구와 형태가 아주 유사한 이삭멸구(N. muiri)와 벼멸구붙이(N. bakeri), 그리고 흰등 멸구와 형태가 아주 유사한 흰등멸구붙이(S. kolophon), 피멸구(S. vibix) 그리고 애멸구(Laodelphax striatellus)가 동시에 채집이 되는 등 형태적 차이점이 거의 없어 전문가도 쉽게 구분하지 못하는 문제가 있음이 확인되었다. 따라서 형태가 유사한 상기 멸구류의 종 동정을 확실히 할 수 있는 PCR용 프라이머의 개발을 위해 벼멸구 및 흰등멸구의 미토콘드리아 내 COI 영역을 특이적으로 검출할 수 있는 프라이머 세트를 제작하고 종 동정 효과를 확인하였다.

메타바코딩을 이용한 환경 DNA 분석은 검출 감도가 높아 어류의 생물다양성 평가 및 멸종위기종의 검출에 유용 한 기술이다. 이번 연구는 메타바코딩을 이용해 우리나라 담수어류를 대상으로 높은 검출 효율을 보일 수 있는 적합 한 분석방법을 확인하기 위해 4가지 분석조건별, 즉 필터 (cellulose nitrate filter, glass fiber filter), 추출 키트 (DNeasy® Blood & Tissue Kit, DNeasy® PowerWater Kit), 프라이머 조합 (12S rDNA, 16S rDNA) 그리고 PCR 방법 (conventional PCR, touchdown PCR)로 나타나는 Operational Taxonomic Units (OTUs) 수와 종 조성을 비교하였다. Glass fiber filter와 DNeasy® Tissue & Blood Kit를 이용해 추출한 시료는 12S rDNA와 16S rDNA 프라이머 조합에서 담수어류 OTUs가 가장 많이 검출되었다. 모든 분석조건 중 프라이머 조합에서만 조기어강 (Class Actinopterygii) 평균 OTUs 수에서 통계적으로 유의한 차이를 보였고 (Non-parametric Wilcoxon Signed Ranks Test, p=0.005), 담수어류 평균 OTUs 수는 유의하지 않았다. 종 조성 비교 결과 역시 프라이머 조합에서 유의한 차이를 보였고 (PERMANOVA, Pseudo-F=6.9489, p=0.006), 나머지 분석조건에서는 유의한 차이를 보이지 않았다. NMDS 분석 결과 종 조성은 유사도 65% 기준에서 프라이머 조합에 따라 묶였고, 16S rDNA 프라이머 세트는 주로 멸종위기종인 모래주사 (Microphysogobio koreensis), 꼬치동자개 (Pseudogobio brevicorpus)가 기여하였고, 12S rDNA 프라이머 세트는 주로 일반종인 피라미 (Zacco platypus), 꺽지 (Coreoperca herzi) 등이 기여한 것으로 나타났다. 본 연구는 국내 하천에서 채취한 시료에 대한 메타바코딩을 이용한 종 다양성 분석의 기초정보를 제공한다.

Cold, salt and heat are most critical factors that restrict full genetic potential, growth and development of crops worldwide.. In this study, we applied an annealing control primer (ACP) based GeneFishing approach to identify differentially expressed genes (DEGs) in annual ryegrass (cv. Kowinearly) leaves under cold, salt and heat stresses. Two-week-old seedlings were exposed to cold (4°C), salt (NaCl 200 mM) and heat (42 °C) treatments for 6 h. A total 8 differentially expressed genes were isolated form ryegrass leaves. These genes were sequenced then identified and validated form National Center for Biotechnology Information (NCBI) database. We identified several promising genes encoding light harvesting chlorophyll a/b binding protein, alpha-glactosidase b, chromosome 3B, elongation factor 1-alpha, FLbaf106f03, complete genome, translation initiation factor SUI1, and glyceraldehyde-3-phosphate dehydrogenase. These genes were potentially involved in photosynthesis, plant development, protein synthesis and abiotic stress tolerance in plants. These genes might be useful for the enhancement of abiotic stress tolerance in fodder crops along with crop improvement under unfavorable environmental conditions.

유전분석을 위해서, CTAB buffer를 이용하여 가시나무류 4종의 genomic DNA를 분리하였다. CTAB buffer를 이용하여 분리한 genomic DNA의 순도는 Edward buffer를 이용했을 때보다 더 높게 나타났다. 가시나무(Q. myrsinaefolia)로부터적정 순도 gDNA는 2% CTAB에 0, 1 또는 2% PVP가 첨가된 buffer를 이용했을 때 얻어졌다. 종가시나무(Q. glauca)로부터 1% CTAB buffer와 1% PVP를 첨가한 buffer를 이 용하여 얻은 gDNA 순도는 1.84±0.02(A260/280)였다. 참가시나무(Q. salicina)의 적정순도 gDNA는 2% CTAB와 1 또는 5% PVP가 첨가된 buffer를 이용하여 분리할 수 있었다. 2% CTAB와 2% PVP가 첨가된 buffer를 이용했을 때, 졸가시나무(Q. phillyraeoides)의 gDNA의 순도는 1.85±0.01(A260/280)였다. 18개 의 RAPD primer를 사용하여 PCR을 수행하였을 때, 가시나무에서는 15개 primer에 의해 53개의 다형질 DNA가 발견되었다. 종가시나무에서는 11개의 primer에 의해 40개의 다형질 DNA가 나타났다. 참가시나무의 경우 16개의 primer에 의해 50개의 증폭된 DNA밴드를 확인 할 수 있었다. 졸가시나무에서 14의 primer가 증폭반응을 일으켰고, 53개의 다형질 DNA가 나타났다. 이 결과들은 가시나무류의 유전분석에 이용할 수 있다.

Non Governmental Organizations (NGOs), as major actors of the civil society, play a vital role in promoting conservation of natural resources, environmental protection, sustainable development, and environmental justice. While their location, size, organizational forms, scope, and impacts can vary widely, all of them operate towards the same mission of protecting the environment from degradation due to industrialization, uncontrolled development, depletion of bio-diversity, and over consumption of natural resources. Although environmental conservation has been a part of civil society involvement throughout history, environmental NGOs have emerged as a major sub-sector of the NGO sector during the past three decades. Their impacts are among the most visible contributions to humanity by the global NGO sector.

Salt stress is one of the most limiting factors that reduce plant growth, development and yield. However, identification of salt-inducible genes is an initial step for understanding the adaptive response of plants to salt stress. In this study, we used an annealing control primer (ACP) based GeneFishing technique to identify differentially expressed genes (DEGs) in Italian ryegrass seedlings under salt stress. Ten-day-old seedlings were exposed to 100 mM NaCl for 6 h. Using 60 ACPs, a total 8 up-regulated genes were identified and sequenced. We identified several promising genes encoding alpha-glactosidase b, light harvesting chlorophyll a/b binding protein, metallothionein-like protein 3B-like, translation factor SUI, translation initiation factor eIF1, glyceraldehyde-3-phosphate dehydrogenase 2 and elongation factor 1-alpha. These genes were mostly involved in plant development, signaling, ROS detoxification and salt acclimation. However, this study provides new molecular information of several genes to understand the salt stress response. These genes would be useful for the enhancement of salt stress tolerance in plants.

Copper (Cu) is a necessary microelement for plants. However, high concentrations of Cu are toxic to plants that change the regulation of several stress-induced proteins. In this study, an annealing control primer (ACP) based approach was used to identify differentially expressed Cu-induced genes in alfalfa leaves. Two-week-old alfalfa plants (Medicago sativa L.) were exposed to Cu for 6 h. Total RNAs were isolated from treated and control leaves followed by ACP-based PCR technique. Using GeneFishing ACPs, we obtained several genes those expression levels were induced by Cu. Finally, we identified several genes including UDP-glucuronic acid decarboxylase, transmembrane protein, small heat shock protein, C-type cytochrome biogenesis protein, mitochondrial 2-oxoglutarate, and trans-2,3-enoyl-CoA reductase in alfalfa leaves. These identified genes have putative functions in cellular processes such as cell wall structural rearrangements, transduction, stress tolerance, heme transport, carbon and nitrogen assimilation, and lipid biosynthesis. Response of Cu-induced genes and their identification in alfalfa would be useful for molecular breeding to improve alfalfa with tolerance to heavy metals.

The present research investigated copper and cadmium stress-induced differentially expressed genes (DEGs) using annealing control primers (ACP) with the differential display reverse transcription polymerase chain reaction technique in alfalfa (Medicago sativa L. cv. Vernal) leaves. Alfalfa leaves were subjected to 250 μM of copper and cadmium treatment for a period of 6 h. A total of 120 ACPs was used. During copper and cadmium treatment, 6 DEGs were found to be up or down regulated. During copper stress treatment, 1 DEG was up-regulated, and 3 novel genes were discovered. Similarly, during cadmium stress treatment, 1 DEG was up-regulated and 5 novel genes were identified. Among all 6 DEGs, DEG-4 was identified as the gene for trans-2,3-enoyl-CoA reductase, DEG-5 was identified as the gene for senescence-associated protein DIN1 and DEG-6 was identified for caffeic acid O-methyltransferase. All the up-regulated genes may play a role in copper and cadmium stress tolerance in alfalfa.

본 연구에서는 건강기능성 식품원료로서 이용 빈도가 증가하고 있는 하수오, 백수오 및 이엽우피소에 대한 종 특이 프라이머를 개발하였다. 개발된 종 특이 프라이머는 하수오, 백수오 및 이엽우피소에 대해 원물뿐만 아니라 육안 확인이 어려운 가공식품 등을 대상으로 사용원료의 진위여부를 빠르고 정확하게 확인할 수 있었다. 따라서 본연구에서 개발한 종 특이 PCR방법은 기존의 일반 프라이머를 사용하는 방법이 가지고 있는 식품 적용 한계를 극복할 수 있을 것이라고 판단하였다.