유전자 친자확인시험법은 동일 종 내에서도 개체를 식별 할 수 있는 강력한 분자진단체계이다. 반달가슴곰 우수리아 종(Ursus thibetanus ussuricus)은 한반도를 비롯한 중국, 러시아 등지에 분포하나, 우리나라에서는 지역적으로 절멸된 것으로 추정되고 있으며, 현재는 멸종위기야생생물Ⅰ급으 로 지정되어 있고, 북한, 러시아, 중국 등에서 재도입되어 종복원이 진행 중에 있다. 본 연구는 우리나라에 재도입된 반달가슴곰집단의 친자확인, 가계도 작성, 미확인개체 추적 등 방사개체의 관리에 요구되는 분자진단체계의 구축을 위하여 차세대염기서열분석법을 통해 결정한 반달가슴곰 전 장유전체 서열(whole genome sequence, WGS)에서 미세 부수체(microsatellite, MS) 마커 개발을 목적으로 하였다. 전장유전체 서열은 총 12개체의 혈액 DNA를 이용하여 결정하였고, 결정된 서열에서 총 1,730,507개의 단순반복서열(simple tandem repeat, STR)의 정보를 발굴하였다. 이 중 반복단위(repeat unit)의 길이가 2-6 nt로 구성된 STR은 총 5,954개였다. WGS 상에서 검출된 STR의 관찰이형접합율(observed heterozygosity, Ho)을 고려하여 총 97종을 1차 선정하였다. 부-모-자 유전자형의 멘델유전(mendelian inheritance)을 만족하는 48종의 마커를 반달가슴곰 전체 집단에 적용하여 시험하였고, 중합효소연쇄반응(polymerase chain reaction, PCR)을 통해 유전자형을 결정하였다. 유전 자형의 다양성지수와다형정보량(polymorphic information content, PIC), 동일개체출현율을 고려하여 최종 15종의 MS 마커 세트를 구성하였다. 분석결과, 전체 집단에서 출현한 평균 대립유전자의 수는 6.267개였으며, 기대이형접합율(expected heterozygosity)는 평균 0.7242을 나타내었다. 평균 PIC는 0.6718, 동일개체 출현율은 1.867×10-14 수준으로 높았으나, 부권부정율은 0.00337로 다소 낮은 수준을 보였다. 재도입된 1세대집단 전체에서는 평균 대립유전자 수 5.867개, 평균 기대이형접합율 0.7217, 평균 PIC 0.6573을 나타내었고, 후손 전체에서는 평균 대립유전자 수 5.933개, 평균 기대이형접합율 0.7140, 평균 PIC 0.6554를 나타내었다. 하지만, 후손집단을 2세대와 3세대로 구분했을 때, 2세대는 평균 대립유전자 수 5.867, 평균 기대이형접합율 0.722, 평균 PIC 0.657, 3세대는 평균 대립유전자 수 4.467, 평균 기대이형접합율 0.694, 평균 PIC 0.601을 나타내어 세대가 진행될수록 대립유전자의 수와 기대이형접합율, PIC 모두 감소하는 경향을 나타내었다. 최종 선정된 15개의 MS 마커체계를 이용하여 반달가슴곰 집단에 대한 미확인 개체 추적, 친자확인 등을 시험하였다. 최근 포획된 개체들 중에서 개체명이 확인되지 않았던 4개체는 자연출생 2세대, 3세대, 전파발신기가 탈락된 방사 1세대로 확인 되었다. 또한 동일성검사 결과 올해 5월 교통사고를 당한 반달가슴곰은 KM-53이며, 올해 출생한 새끼 반달가슴곰들에 대한 검사를 통해 2개체가 인공수정에 의해 출생한 것으로 확인되었다. 이상의 결과들은 본 연구에서 확립한 MS 마커의 조합이 자연방사와 자연출생 개체들로 구성된 반달 가슴곰 집단의 개체관리를 위한 분자마커체계로 유용하게 이용될 수 있음을 보여주고 있다. 또한 본 연구를 통해서 확립된 MS 마커들은 현재 지리산과 수도산에 서식하고 있는 반달가슴곰 집단의 개체관리, 미확인 개체 추적, 친자확인 등 유전자분석에 활용될 수 있을 것으로 기대되며, 아울러 향후 유전적 다양성 증진, 집단간 교류개체 선정 등에도 필요한 유전 정보를 제공할 수 있을 것이다. 결론적으로 반달가슴곰 전장유전체 서열을 토대로 마련된 분자진단체계는 향후 반달가슴곰의 보호와 생태복원을 위한 종복원에 기여할 것으로 기대된다.

Insect-killing fungi have high potential in pest management. A deeper insight into the fungal genes at the whole genome level is necessary to understand the inter-species or intra-species genetic diversity of fungal genes, and to select excellent isolates. In this work, we conducted a whole genome sequencing of Beauveria bassiana (Bb) JEF-007 and characterized pathogenesis-related features and compared with other isolates including Bb ARSEF2860. A large number of Bb JEF-007 genes showed high identity with Bb ARSEF2860, but some genes showed moderate or low identity. The two Bb isolates showed a significant difference in vegetative growth, antibiotic-susceptibility, and virulence against Tenebrio molitor larvae. When highly identical genes between the two Bb isolates were subjected to real-time PCR, their transcription levels were different, particularly in heat shock protein 30 (hsp30) gene which is related to conidial thermotolerance. In several B. bassiana isolates, chitinases and trypsin-like protease genes involved in pathogenesis were highly conserved, but other genes showed noticeable sequence variation within the same species. Given the transcriptional and genetic diversity in B. bassiana, a selection of virulent isolates with industrial advantages is a pre-requisite, and this genetic approach could support the development of excellent biopesticides with intellectual property protection.

The red imported fire ant (Solenopsis invicta) is a species of ant native to South America. The fire ant was inadvertently introduced into USA, Australia, New Zealand, and other Asian countries including China and Taiwan. Since the first report of the fire ant in port city of Busan, Korea in 2017, it was found in many other cities of Korea in following year. To obtain the molecular information of this invasive species, total RNA was extracted from the abdominal segment of the ants collected in Incheon, and subjected to transcriptome sequencing. By using Illumina sequencer platform, 101 base pared-end sequencing generated 2 × 50,064,081 of raw reads to obtain 2 × 45.95 Gbase of quality filtered nucleotide sequences. The in silico cDNA library was constructed by Trinity de novo assembler followed by TransDecoder ORF finder and CD-HIT clustering program to streamline the library. The final version of cDNA library contains 20,442 contigs with protein coding capability. To survey the virome of this ant, these contigs were searched against the viral reference sequences from NCBI RefSeq database with BLASTN program. As a result, contigs which showed high sequence identities with several RNA viruses including previously reported SINV-2 were found from the fire ant. This virome information might give an idea of a shift of virological environment of this newly found ant isolate or population in Korea.

저어새의 먹이생물을 파악하기 위해 2010년 6월부터 2014년 6월까지 인천 남동유수지에서 저어새의 토사물 시료를 채집하여 현미경 관찰 및 차세대염기서열 (NGS) 기법으로 분석 하였다. 저어새의 먹이생물은 어류, 갑각류, 다모류, 곤충류로 구성되어 있었으며, 주로 저어새는 어류와 갑각류를 섭이하는 것으로 나타났다. 최우점 먹이생물은 풀망둑 (Acanthogobius hasta)이었으며, 이 외에도 길게 (Macrophthalmus abbreviates), 징거미새우류 (Macrobrachium sp.), 칠게 (Macrophthalmus japonicus), 각시흰새우 (Exopalaemon modestus), 참 갯지렁이 (Neanthes japonica)가 우점 먹이생물로 출현하였다. 이들 먹이생물은 번식지 인근지역인 송도갯벌과 시화호에서 흔히 발견되며, 저어새는 채식지로써 이들 지역에 대한 의존도가 높을 것으로 판단된다. 현미경과 NGS로 분석한 일부 먹이생물에서 차이를 보였는데, 이는 토사물 내 먹이생물은 저어새의 위 내에서 분해되어 현미경 분석을 통한 형태학적 분류 특징을 찾기 어려웠던 반면, NGS 분석은 유전자를 통해 분류가 가능하기 때문에 형태학적 분석의 결과보다 높은 종 다양성을 보인 결과이다.

The varroa mite, Varroa destructor, is a small ectoparasitic mite which attacks honeybee, Apis mellifera, and also known to harbor small RNA viruses which infect honeybees. To survey the transcriptome of varroa mite, total RNA of female adult mites was subjected to RNA-seq to construct an in silico cDNA library. 2 × 8.3 Gbase of quality filtered paired-end nucleotide sequences were obtained to construct 28,302 of protein-coding contigs by de novo assembly, and subsequent BLAST search revealed the viruses infect honeybee or associated with varroa mites. Six of the contigs showed high sequence identity to Iflavirus, picorna-like virus, rhabdovirus, and macula-like virus were discovered. It implies that the viral flora in varroa mites and honeybees might be more complex than previously studied, and suggests the importance of further virome studies for better understanding of honeybee health.

As the sequencing batch reactor process is a time-oriented system, it has advantages of the flexibility in operation for the biological nutrient removal. Because the sequencing batch reactor is operated in a batch system, respiration rate is more sensitive and obvious than in a continuous system. The variation of respiration rate in the process well represented the characteristics of biological reactions, especially nitrification. The respiration rate dropped rapidly and greatly with the completion of nitrification, and the maximum respiration rate of nitrification showed the activity of nitrifiers. This study suggested a strategy to control the aeration of the sequencing batch reactor based on respirometry. Aeration time of the optimal aerobic period required for nitrification was daily adjusted according to the dynamics of respiration rate. The aeration time was mainly correlated with influent nitrogen loadings. The anoxic period was extended through aeration control facilitating a longer endogenous denitrification reaction time. By respirometric aeration control in the sequencing batch reactor, energy saving and process performance improvement could be achieved.

Genotyping-by-sequencing (GBS) is a cost-efficient method which can be useful for SNP marker discovery in a population of interest. GBS is genome reduction sequencing method using restriction enzyme. The quality of DNA is a key factor which could have an influence in downstream analysis. However, there have not been many studies which investigated the impact of DNA degradation and the quality of the data on marker discovery. In this study, GBS data of 6 Hanwoo samples (H1~6) showing differing level of DNA degradation were compared. Re-sequencing pipeline was followed to investigate the impact of DNA degradation on marker discovery. As a result, we found that the quantity and quality of SNPs were not affected in the sample H5 and H6 with moderately degraded DNA. On the other hand, marker discovery was greatly affected in samples with severe DNA degradation (H3 and H4). The findings in this study support that GBS is a robust genotyping method towards moderate DNA degradation.

Several studies on the correlation between temperament and genetic diversity are conducted in animals as well as human. Horse temperament is especially important because it is important factor for horse riding and racing. In this study, we performed targeted exome sequencing to find single nucleotide polymorphisms (SNPs) served as genetic markers that can evaluate the aggressive and docile levels of horses. We selected 71 candidate genes related to animal and human temperament through previous researches and verified it on the human reference genome (hg38) and horse reference genome (equCab2). We found that 16 orthologous genes were present in horse reference genome and 17 homologous genes found in horses based on the human reference genome. Finally, we designed probes to find the genetic variation in selected 33 genes. The sequencing libraries were constructed using the designed probe and DNA samples extracted from the blood of 8 aggressive and 8 docile horses. The constructed libraries were sequenced using the Illumina Hiseq2500 platform. SNPs data obtained from targeted exome sequencing will be used for genome wide association study (GWAS) and Sanger sequencing validation. This study will help to assess the horse temperament and to select superior horses for riding or racing.

Recently, we published a microinjection method for generating transgenic cattle using the DNA transposon system and their analysis by next-generation sequencing (Yum et al. Sci Rep. 2016 Jun 21;6:27185). In that study, we generated transgenic cattle using two different types of DNA transposon system, sleeping beauty (SB) and piggybac (PB), carrying Yellow fluorescent protein with SB (SB-YFP, female) and green fluorescent protein with PB (PB-GFP, male) under the control of the ubiquitous CAG promoter, respectively. The female and male founder cattle have been grown up to date (the female age: 40 months old, the male age: 33 months old) without any health issues. In genomic instability and blood analysis, there was no significant differences between wild type and founder cattle. In the present study, we confirmed germ-line transmission of the transposon-mediated transgene integrations and ubiquitous and persistent expression of transgene in second generation of offspring (F1). The F1 was born without any assistance and expressed GFP in the eyes without UV light. The ubiquitous expression of GFP was detected in skin fibroblast from the ear tissue and confirmed by genomic DNA PCR, which suggest that the transgene from the PB-GFP was successfully transmitted. Unfortunately, no transgene from SB-YFP were identified. To confirm the transgene integration site, the genomic DNA from blood was extracted and performed next-generation sequencing (NGS). The GFP gene was integrated in chromosome 4 (two copies), and 6. As results, a total of two copies of paternal transgene transmitted into the F1. All the integrated position was not related with coding region and there was no significant difference in genomic variants between transgenic and non-transgenic cattle. To our knowledge, this is the first report of germ-line transmission through non-viral transgenic founder cattle. Those transgenic cattle will be valuable resource to many fields of biomedical research and agricultural science.

The Automobile HVAC system is a habitat for odor-associated fungal communities. We investigated the odorassociated fungal community in an automobile HVAC system using a high-throughput DNA sequencing method. The fungal community structure was evaluated via metagenome analysis. At the phylum level, Ascomycota and Basidiomycota were detected, accounting for 43.41% and 56.49% of the fungal community in the HVAC system, respectively. Columnosphaeria (8.31%), Didymella (5.60%), Davidiella (5.50%), Microxyphium (4.24%), unclassified Pleosporales (2.90%), and Cladosporium (2.79%) were abundant at phylum of Ascomycota and Christiansenia (36.72%), Rhodotorula (10.48%), and Sporidiobolus (2.34%) were abundant at phylum of Basidiomycota. A total of 22 genera of fungi were isolated and identified from the evaporators of the HVAC systems which support fungal growth and biofilm formation. Among them, Cladosporium, Penicillium, Aspergillus and Alternaria are the most representative odor-associated fungi in HVAC systems. They were reported to form biofilm on the surface of HVAC systems with other bacteria by hypha. In addition, they produce various mVOCs such as 3-methyl-1-butanol, acetic acid, butanoic acid, and methyl isobutyl ketone. Our findings may be useful for extending the understanding of odor-associated fungal communities in automobile HVAC systems.

담체가 투여된 침지형 막결합 연속회분식 반응기(SMSBR)를 사용한 하수의 고도처리에서 담체가 여과성능과 제 거효율에 미치는 영향을 조사하였다. 담체는 반응기 부피 기준으로 10% 투여하였고, 담체와 분말활성탄을 첨가하지 않은 반 응기, 분말활성탄(10 g/L)만을 첨가한 반응기 및 담체와 분말활성탄을 모두 첨가한 반응기를 대조군으로 하였다. COD, T-N 및 T-P에 대한 제거효율은 담체 및 분말활성탄 첨가 유무에 따라 큰 차이가 없었다. 그러나 담체를 첨가하지 않은 경우 막간 차압(TMP)은 급격히 증가하였으나, 담체를 첨가한 경우에 막간차압은 매우 서서히 증가하였다. 담체를 투여한 SMSBR를 사 용하여 하수를 고도처리 할 때, 91일 이상의 운전기간 동안 막 세정 없이 운전이 가능하였다. 담체만을 투여한 경우, 운전 80 일 경과 이후의 COD, T-N 및 T-P 평균 제거율은 각각 95.0, 69.3% 및 51.4%이었다.

Lentinula edodes, the popular shiitake mushroom, is one of the most important cultivated edible mushrooms. It is used as a food and for medicinal purposes. Here, we present the 46.1 Mb draft genome of L. edodes, comprising 13,028 predicted gene models. The genome assembly consists of 31 scaffolds. Gene annotation provides key information about various signaling pathways and secondary metabolites. This genomic information should help establish the molecular genetic markers for MAS/MAB and increase our understanding of the genome structure and function.

국내 경주마의 승용마로 전환에 있어서 가장 큰 문제 중 하나인 침착하고 온순한 성격의 말 을 조기에 판정이 필요하고, 특히 crib biting같은 행동을 보이는 사나운 성격의 말을 조기에 제 거하는 선발 기술이 필요하다. 본 연구에서는 성격적으로 민감하고 난폭한 그룹과 온순한 성격 의 말을 구분 지을 수 있는 유전적 특성을 파악하고자 연구를 수행하였다. 말의 성격적 특성을 구분 지을 수 있는 유전적 변이를 찾기 위해서 기존에 포유동물의 성격적 특징과 관련이 있는 것으로 알려진 유전자 후보군 71개를 선발하였다. 71개 유전자 중, 말 참조 유전체에 이미 존재 하는 16개 유전자와 인간 참조 유전체를 기반으로 말에서 찾은 orthologous gene 17개를 합하여 총 33개 성격관련 유전자를 선정하였다. 선정된 33개 유전자의 exon 부분에 probe를 제작하여 목표로 하는 유전자만 추출한 후 Illumina Hiseq2500을 이용하여 whole exome sequencing을 진 행하였다. Whole exome sequencing 결과로 얻어진 데이터를 이용하여 sanger validation기법을 통해 SNPs분석을 실시하였다. 그 결과 33개의 유전자 중 androgen recptor gene (AR)과 dopamine receptor D4 gene (DRD4)유전자에서 유전적으로 성격에 차이가 크게 나타난 결과를 확인할 수 있었다. 이러한 유 전자를 활용한다면 말 사육 농가의 조기 말 성격 검사를 통하여 사육 방향을 선택할 수 있는 경 제적 이득을 취할 수 있을 것으로 예측되며, 향후 추가적인 유전자 분석이 완료 된다면 현장에서 신속하게 사용할 수 있는 키트 개발등이 가능할 것으로 기대된다.

Varroa destructor is a devastating ectoparasitic mite which attacks Honeybee, Apis mellifera. V. destructor feeds on honeybee hemolymph, and often harbors small RNA viruses such as the deformed wing virus to transmit these viruses in the infested bee hive. To survey the genes of V. destructor, total RNA was subjected to high-throughput transcriptome sequencing to construct in silico cDNA library by using the Illumina HiSeq 2000 platform. Total of 2×107,748,792 paired-end short reads were obtained and quality filtered reads were subjected to Trinity de novo assembler followed by TransDecoder, and CD-HIT program to make a V. destructor reference cDNA library containing 28,023 of clustered contigs with protein coding capacity. These cDNA sequences will help us to understand the molecular biology of V. destructor.