• 한국균학회지
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• 제49권4호
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• pp.455-467
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• 2021

Macrofungi are valuable resources as novel drug candidates, new biomaterials, and edible materials. Recently, genetic approaches pertaining to macrofungi have been continuously growing for their identification, molecular breeding, and genetic engineering. However, purification and amplification of fungal DNA is challenging because of the rigid cell wall and presence of PCR inhibitory metabolites. Here, we established a direct PCR method to provide a rapid and efficient method for PCR-grade macrofungal DNA preparation applicable to both conventional PCR and real-time PCR. We first optimized the procedure of lysis and PCR using the mycelia of Lentinula edodes, one of the most widely consumed macrofungal species. Lysates prepared by neutralizing with (NH₄)₂SO₄ after heating the mycelia in a mixture of TE buffer and KOH at 65℃ for 10 min showed successful amplification in both conventional and real-time PCR. Moreover, the addition of bovine serum albumin to the PCR mixture enhanced the amplification in conventional PCR. Using this method, we successfully amplified not only internal transcribed spacer fragments but also low-copy genes ranging in length from 500 to 3,000 bp. Next, we applied this method to 62 different species (54 genera) of macrofungi, including edible mushrooms, such as Pleurotus ostreatus, and medicinal mushrooms such as Cordyceps militaris. It was found that our method is widely applicable to both ascomycetes and basidiomycetes. We expect that our method will contribute to accelerating PCR-based approaches, such as molecular identification, DNA marker typing, gene cloning, and transformant screening, in macrofungal studies.

• International Journal of Industrial Entomology and Biomaterials
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• 제45권2호
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• pp.115-125
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• 2022

In this study, we investigated the developmental characteristics and genetic diversity of seven populations of two-spotted crickets (Gryllus bimaculatus De Geer, 1773 (Orthoptera: Gryllidae)) raised in South Korea. Regarding the developmental characteristics of the species, we observed no statistically significant difference in the weight of the nymphs in the six populations we tested. After molting, although weight differences were observed between the populations in each stage of the developmental period, the average weight for each developmental stage was constant. We also analyzed mitochondrial COI gene sequences (DNA barcoding region) of the reared crickets collected from five insect farms and two national insect rearing facilities and the resultant sequences were analyzed together with the 12 sequences from foreign countries specimens obtained from public data. We detected six haplotypes from 111 specimens, indicating a low intraspecific genetic distance (~1.8%). The most dominant haplotype was overwhelmingly haplotype 1, which was found in all South Korean specimens and four specimens from China, Indonesia, and Germany. These findings indicate that the low genetic diversity of South Korean specimens can be explained by the fact that the G. bimaculatus population imported for feed from Japan in the early 2000s became a maternal group that spread throughout cricket farms in South Korea. In order to breed healthy cricket strains, it is necessary to increase genetic diversity by importing them from other countries through appropriate quarantine procedures.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2022년도 추계학술대회
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• pp.210-210
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• 2022

Silybum marianum is an annual or biennial plant from the Asteraceae family. It can grow in low-nutrient soil and drought conditions, making it easy to cultivate. From the seed, a specialized plant metabolite called silymarin (flavonolignan complex) is produced and is known to alleviate the liver from hepatitis and toxins damages. To infer the phylogenetic placement of a Korean milk thistle, we conducted a chloroplast assembly and annotation following by a comparison with existing Chinese reference genome (NC_028027). The chloroplast genome structure was highly similar with an assembly size of 152,642 bp, an 153,202 bp for Korean and Chinese milk thistle respectively. Moreover, there were similarities at the gene level, coding sequence (n = 82), transfer RNA (n = 31) and ribosomal RNA (n = 4). From all coding sequences gene set, the phylogenetic tree inference placed the Korean cultivar into the milk thistle clade; corroborating the expected tree. Moreover, an investigation the tree based only on the ycf1 gene confirmed the same tree; suggesting that ycf1 gene is a potential marker for DNA barcoding and population diversity study in milk thistle genus. Overall, the provided data represents a valuable resource for population genomics and species-centered determination since several species have been reported in the Silybum genus.

• Journal of Ecology and Environment
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• 제47권4호
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• pp.168-176
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• 2023

Background: Many insectivorous bats have flexible diets, and the difference in prey item consumption among species is one of the key mechanisms that allows for the avoidance of interspecies competition and promotes coexistence within a microhabitat. In Korea, of the 24 bat species that are known to be distributed, eight insectivorous bats use forest areas as both roosting and foraging sites. Here, we aimed to understand the resource partitioning and coexistence strategies between two bat species, Myotis ikonnikovi and Plecotus ognevi, cohabiting the Mt. Jumbong forests, by comparing the differences in dietary consumption based on habitat utilization. Results: Upon examining their dietary composition using the DNA meta-barcoding approach, we identified 403 prey items (amplicon sequence variants). A greater prey diversity including Lepidoptera, Diptera, Coleoptera, and Ephemeroptera, was detected from M. ikonnikovi, whereas most prey items identified from P. ognevi belonged to Lepidoptera. The diversity index of prey items was higher for M. ikonnikovi (H': 5.67, D: 0.995) than that for P. ognevi (H': 4.31, D: 0.985). Pianka's index value was 0.207, indicating little overlap in the dietary composition of these bat species. Our results suggest that M. ikonnikovi has a wider diet composition than P. ognevi. Conclusions: Based on the dietary analysis results, our results suggests the possibility of differences in foraging site preferences or microhabitat utilization between two bat species cohabiting the Mt. Jumbong. In addition, these differences may represent one of the important mechanism in reducing interspecific competition and enabling coexistence between the two bat species. We expected that our results will be valuable for understanding resource partitioning and the coexistence of bats inhabiting the Korean forests.

• Fisheries and Aquatic Sciences
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• 제26권12호
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• pp.752-761
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• 2023

Genetic diversity serves as the basis for selecting and genetically enhancing any culturable species in aquaculture. Here, two different strains of wild (River Ravi and River Kabul) and six captive-bred strains of Labeo rohita from various provinces were se- lected, and genetic diversity among them was evaluated using three different microsatellite markers, i.e., Lr-28, Lr-29, and Lr-37, and one mitochondrial CO1 (Cytochrome c oxidase subunit 1) gene. Different strains of L. rohita were collected, and part of their caudal fin was cut and preserved in ethanol for DNA extraction and determination of genetic diversity among them. Results in- dicated that selected markers were polymorphic with polymorphic information content (PIC) content values above 0.5 with the highest in Lr-28 followed by Lr-29 and then Lr-37. The observed heterozygosity (Ho) of all strains was higher (Avg: 0.731) but less than the expected heterozygosity (He). Moreover, TMs and WRs showed the highest He, while TKs showed the lowest, He. Over- all, inbreeding coefficient (FIS) values observed for all strains with selected markers were positive. The DNA barcoding with the CO1 gene revealed genetic variation among various strains, as demonstrated by the clades in the phylogenetic tree separating the strains into two distinct clusters that then divided into sub-clusters. In conclusion, TMs showed the highest heterozygosity as compared to other strains. Overall results provide the baseline data for the initiation of the genetic improvement program.

• The Plant Pathology Journal
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• 제40권2호
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• pp.171-191
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• 2024

Identification of Helicotylenchus species is very challenging due to phenotypic plasticity and existence of cryptic species complexes. Recently, the use of rDNA barcodes has proven to be useful for identification of Helicotylenchus. Molecular markers are a quick diagnostic tool and are crucial for discriminating related species and resolving cryptic species complexes within this speciose genus. However, DNA barcoding is not an error-free approach. The public databases appear to be marred by incorrect sequences, arising from sequencing errors, mislabeling, and misidentifications. Herein, we provide a comprehensive analysis of the newly obtained, and published DNA sequences of Helicotylenchus, revealing the potential faults in the available DNA barcodes. A total of 97 sequences (25 nearly full-length 18S-rRNA, 12 partial 28S-rRNA, 16 partial internal transcribed spacer [ITS]-rRNA, and 44 partial cytochrome c oxidase subunit I [COI] gene sequences) were newly obtained in the present study. Phylogenetic relationships between species are given as inferred from the analyses of 103 sequences of 18S-rRNA, 469 sequences of 28S-rRNA, 183 sequences of ITS-rRNA, and 63 sequences of COI. Remarks on suggested corrections of published accessions in GenBank database are given. Additionally, COI gene sequences of H. dihystera, H. asiaticus and the contentious H. microlobus are provided herein for the first time. Similar to rDNA gene analyses, the COI sequences support the genetic distinctness and validity of H. microlobus. DNA barcodes from type material are needed for resolving the taxonomic status of the unresolved taxonomic groups within the genus.

• Journal of the Korean Wood Science and Technology
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• 제52권4호
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• pp.343-362
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• 2024

Eurycoma longifolia (pasak bumi) is a popular medicinal plant in Indonesia and is widely used in various products. Its high economic value has caused illegal harvesting and product falsification. Using molecular techniques, the authentication and traceability of E. longifolia derivatives can be controlled to ensure consumer safety. Therefore, this study aimed to authenticate the products and derivatives of E. longifolia (pasak bumi) produced, marketed, and consumed in Indonesia using molecular identification techniques. Genomic DNA from 37 leaf samples collected from the Sumatran mainland and the Riau Islands and six E. longifolia products were amplified and sequenced using trnL-trnF and internal transcribed spacer (ITS) regions. The results revealed that all leaf samples were indeed E. longifolia based on the markers used, with the six products, only the herbal tea product (sample code TCPB) was most likely derived from E. longifolia based on the two regions, suggesting that not all products labelled as E. longifolia in the market are authentic. The results also indicated that several other plants species are used as substitutes or adulterants, including Simaba spp., Simarouba spp., Homalolepis spp., Vernonia gigantea, Elephantopus scaber, Gymnanthemum amygdalinum, Cyanthillium spp., Potentilla lineata, Ailanthus altissima, Geijera paniculata, Hannoa chlorantha, and Dalbergia spp. Klebsiella pneumoniae bacteria were also identified in this study on the outer wooden cup of E. longifolia products. Therefore, this molecular approach is effective in identifying the authenticity of E. longifolia products, with trnL-trnF and ITS as the recommended DNA markers.

• 한국환경생태학회지
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• 제29권5호
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• pp.693-702
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• 2015

동절기에 제주도 지역에서 도래하는 떼까마귀의 유전적 특성과 집단 간 유연관계를 구명하기 위해, 미토콘드리아 COI 유전자 서열의 다형성에 기반한 모계 계통 구조와 계통 유연관계를 분석하였다. 떼까마귀 DNA는 우도와 제주도 내에서 발견된 깃털과 사체 시료에서 분리하였다. 결정된 COI 서열들(n=41)은 떼까마귀(Corvus frugilegus)에서 기존에 보고된 서열들과 97.0% 이상 일치하였다. 제주도 떼까마귀 COI 서열들은 3가지 haplotype(J01-J03)으로 구분되었으나 지역-특이적인 양상을 보이지 않아, 이들이 하나의 모계 기원에서 유래한 집단임을 알 수 있었다. 떼까마귀 전체 COI 서열에서 8개의 COI haplotype들이 발견되었다. 이 중 3가지 haplotype들은 러시아 동부, 몽골, 한국 등 동북아시아의 COI 서열들을 포함하였고, 나머지 5가지는 중앙아시아, 중동아시아, 러시아 서부, 유럽국가의 떼까마귀에서 발견되었다. 계통수 상에서 떼까마귀의 COI 서열들은 측소적 종분화 단계인 2아종, C. f. frugilegus와 C. f. pastinator인 2개의 모계 계통으로 뚜렷하게 구분되었다. DNA barcoding 분석을 통한 연구결과는 모계 계통의 구조, 계통 유연관계 및 분자생태를 이해하는 데 중요한 정보를 제공할 것이다.

• 환경생물
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• 제40권4호
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• pp.464-476
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• 2022

가속화되는 기후변화로 인해 전 세계 각지의 연안에서 해조류 대발생(macroalgal bloom)이 빈번하게 일어나고 있다. 특히, 녹조류의 대량 증식으로 인한 녹조(파래) 대발생(green tide) 현상은 지역 경제뿐만 아니라 연안 생태계 환경에도 막대한 피해를 주고 있다. 우리나라의 경우 2000년대부터 제주도 동북부 해안을 중심으로 파래대발생이 연중 지속적으로 관찰되며, 최근에는 남해와 동해 일대에서도 국지적으로 관찰되고 있다. 파래대발생의 원인 종은 갈파래속(Chlorophyta; genus Ulva)으로 알려져 있으며, 기후변화의 영향으로 해수 온도의 상승과 담지하수 및 인근지역 오염수 배출로 인한 질소와 인의 대량 유입으로 인한 영양염류 증가가 주요 원인으로 추정되고 있다. 갈파래속은 환경변화에 의해 형태적 발현의 가소성이 높은 표현형 적응성(phenotypic plasticity) 때문에 형태적 종 동정은 거의 불가능하다. 갈파래류 종 판별을 위해서는 분자유전학적 분석이 수행되어야 하나 현재 분자데이터를 이용한 갈파래 종 분포, 군집구조와 같은 생태조사연구는 매우 미흡한 실정이다. 파래대발생 피해 저감을 위해서는 파래대발생 주요 종들을 분자계통학적 분석을 통하여 정확하게 파악하는 것이 우선이다. 선행 연구에서는 2015년 파래대발생을 일으키는 주요 종 파악을 위해 핵 DNA ITS와 엽록체 DNA tufA (chloroplast elongation factor Tu) 유전자를 이용하여 분자계통학적 분석을 수행하였으며, 종 동정에는 tufa 유전자가 더 정확한 결과를 나타냈다. 따라서 본 연구에서는 tufA 유전자를 이용해 2015~2020년 제주도 연안에서 파래대발생을 일으키는 주요 구성 갈파래 종의 군집구조 및 종 다양성을 파악하고 종 구성의 시간적 변이를 확인하고자 하였다. 본 연구에서는 온대와 아열대 해역에서 주로 생장하는 것으로 알려진 큰갈파래와 구멍갈파래 종이 파래대발생의 주요 구성 종임을 확인하였다. 구멍갈파래는 2015년(35.75%)에서 2020년(36.18%) 기간 상대빈도의 변화가 거의 없고 안정적으로 유지되었으나, 큰갈파래의 경우 2015년(20.77%)에 비해 2020년(36.84%) 빈도가 대략 16% 증가하였다. 이러한 결과는 기후변화와 연관된 평균 해수면 온도의 상승에 큰갈파래의 높은 성장률 및 적응력과 관련이 있을 수 있으며, 제주도의 갈파래 군집을 구성하는 종 수는 2015년에는 9종이었으나 2020년에는 7종으로 감소하는 것으로 나타났다. 또한, 유럽 원산지 외래종인 긴통갈파래와 굽은갈파래가 2015년과 2020년 모두 제주 연안에서 관찰되어 이 두 종에 대한 향후 지속적인 모니터링이 필요하다. 본 연구의 결과는 우리나라 연안에서 발생하는 파래대발생의 저감을 위해 주요 종인 갈파래속(genus Ulva)에 대한 분자유전학적 데이터에 대한 정보를 제공하고자 한다.

• 환경영향평가
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• 제21권6호
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• pp.823-839
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• 2012

Environmental Specimen Banks (ESBs) are playing pivotal role in monitoring the effect of environmental pollution on the ecosystem based on the retrospective analysis of the representative samples collected regularly and stored in cryogenic condition. In Korea, National Environmental Specimen Bank (NESB) was established in 2009 and the standard operating procedures (SOPs) for sampling, and cryogenic milling and storage had been prepared during 2007-2010. Since then, the tentative SOPs for the seven kinds of specimens (shoots of Red Pine (Pinus densiflora) and Korean Pine (Pinus koraiensis), leaves of Mongolian Oak (Quercus mongolica) and Zelkova Tree (Zelkova serrata), eggs of Feral Pigeon (Columba livia var. domestica), muscles and organs of Common Carp (Cyprinus carpio), and Freshwater Bivalve (Unio (Nodularia) douglasiae)) have been put to test in the field and laboratory as well against the practicality and feasibility. The SOPs were improved by reflecting the findings from the research and the following discussion regarding the selection of specimen (Feral Pigeon suffering from a control management), sample size (a problem of decreasing number of sampling trees related to increasing sampling time) and period (a problem related to a bud growth), and sampling methods etc.. In addition, barcoding system for the management of the specimen information, and monitoring system of the cryogenic storage to regulate the optimum temperature and the liquid nitrogen level were also developed for the efficient and effective control of the samples. Lastly, the safety guide and emergency protocol were augmented to guarantee a safe work environment with the cryogenic facility. These improvements of the SOPs are expected to contribute to more stable operation of the NESB.