The incidence of freshwater algal bloom has been increasing globally in recent years and poses a major threat to environmental health. Cyanobacteria are the major component of the bloom forming community that must be monitored frequently. Their morphological identities, however, have remained elusive, due to their small size in cells and morphological resemblances among species. We have analyzed molecular diversity and seasonal changes of cyanobacteria in Paldang Reservoir, Korea, using morphological and 16S rRNA pyrosequencing methods. Samples were collected at monthly intervals from the reservoir March-December 2012. In total, 40 phylotypes of cyanobacteria were identified after comparing 49,131 pyrosequence reads. Cyanobacterial genera such as Anabaena, Aphanizomenon, Microcystis and Synechocystis were predominantly present in samples. However, the majority of cyanobacterial sequences (65.9%) identified in this study were of uncultured origins, not detected morphologically. Relative abundance of cyanobacterial sequences was observed as high in August, with no occurrence in March and December. These results suggested that pyrosequencing approach may reveal cyanobacterial diversity undetected morphologically, and may be used as reference for studying and monitoring cyanobacterial communities in aquatic environments.

Radiotherapy (RT) is a mainstay in the treatment of head and neck squamous cell carcinoma (HNSCC). For locally advanced HCSCC, concurrent chemoradiotherapy (CCRT) benefits HCSCC patients in terms of better survival and loco-regional control. In this study, we evaluated changes in oral microbiota in patients, who received CCRT for head and neck cancer. Oral rinsed samples were weekly collected before and during CCRT and at 4 weeks following treatment from HNSCC patients, who had received 70 Gy of radiation delivered to the primary sites for over 7 weeks and concurrent chemotherapy. Oral microbiota changes in three patients were analyzed by next-generation sequencing using 16S rRNA 454 pyrosequencing. On an average, 15,000 partial 16S rRNA gene sequences were obtained from each sample. All sequences fell into 11 different bacterial phyla. During early CCRT, the microbial diversity gradually decreased. In a patient, who did not receive any antibiotics during the CCRT, Firmicutes and Proteobacteria were the most abundant phylum. During the early CCRT, proteobacteria gradually decreased while Firmicutes increased. During the late CCRT, firmicutes gradually decreased while Bacteroides and Fusobacteria increased. In all the patients, yellow complex showed a gradual decrease, while orange and red complex showed a gradual increase during the CCRT. At 4 weeks after CCRT, the recovery of oral microbiota diversity was limited. During CCRT, there was a gradual increase in major periodontopathogens in association with the deterioration of the oral hygiene. Henceforth, it is proposed that understanding oral microbiota shift should provide better information for the development of effective oral care programs for patients receiving CCRT for HNSCC.

This research is on the halophilic bacteria inhibiting in solar salterns. It was conducted by measuring the composition of cultivating ground in each step of salt production and targeting solar salterns during 2012 and 2013 in Jeungdo, Shinangun, JeollaNam-do, Korea. In each step of salt production, the succession of the group has been studied using pyrosequencing method and the new halophile variety has been separately identified in solar salterns. The results are as follows; The decomposition ability of protein, lipid, cellulose, and starch, has been experimented with separately identifying 33 bacteria that appeared as dominants in solar salterns. Overall, the bacteria with decomposition capability of all 4 components were Micrococcus luteus, Nocardioides daejeonensis in Actinobacteria group, Halobacillus trueperi whb45 in Firmicutes group, and Aurantimonas coralicida, Lutibacterium anuloederans, Nesiotobacter exalbescens in Alphaproteobacteria group. Analysis in the diversity of bacteria community of diluted solution with concentration of 3%, 6%, 11%, 20%, and 30% has been made using pyrosequencing method. In the Order level, SAR 11 dominated in 3% salt solution, Rhodobacterales group (18.9%) in 6% solution, Micrococcales group showed the highest ratio in 11% and 20% solutions, and Rhodobacterales group was dominant in the solution of 30%. In Phylum level, Proteobacteria group showed dominancy in 3% and 6% salt solutions, Actinobacteria group was dominant in 11% and 20% solutions, and Proteobacteria group was dominant in 30% solution. Among the isolated strains, seven novel strains (Kmb21, Kmb26, Kmb27, Kmb32, Kmb42, Kmb64, and Kmb80) were donate registered to NCBI and KCTC, JCM, each as new variety candidate.

Wolbachia is one of the most common endosymbionts best known to induce several reproductive alterations in its insect hosts. In some cases, the insect hosts harbor more than two strains of the bacterium. The Vollenhovia emeryi ant lives in dead trees and is morphologically subdivided into the long-winged and the short-winged. Interestingly the short-winged morph is free of Wolbachia, but only the long-winged morph is multiple- infected with the Wolbachia bacterium. We sampled four populations of the long-winged morph in Korea and performed pyrosequencing in Multilocus Sequence Typing (MLST), to determine the bacterial strain diversity. Six different gene regions (coxA, fbpA, ftsZ, gatB, hcpA and wsp gene) were targeted and amplified. However, the result shows that diversity of haplotypes is very high. The pyrosequencing approach in MLST, a new method of discriminating Wolbachia strains, is promising to effectively detect multiple infections and rare haplotypes.

Among hemipteran insects which is the most important insect vector of plant viruses, small brown planthopper, Laodelphax striatellus, transmits the rice stripe virus (RSV) causing rice stripe disease. For effective control of RSV, it is important to understand interaction between RSV and L. striatellus. Therefore, in this study, expressed sequence tag (EST) databases were generated based on 454 GS-FLX pyrosequencing for comparative transcriptome analysis between nonviruliferous and RSV-viruliferous L. striatellus. By comparing the two EST libraries, we showed that 108 host genes were significantly up-regulated and 28 host genes were significantly down-regulated in viruliferous insects. Interestingly, genes encoding ribosomal proteins were mainly up-regulated in viruliferous L. striatellus, whereas genes related to translation were concentrated in the downregulated cohort. These RSV-dependently regulated genes may have important function in the behavior of planthopper or the transmission of RSV.

국화과(Compositae)는 현화식물 중 세계에서 가장 넓게 분포하고, 쌍자엽식물 중 가장 진화된 식물분류군이며, 우리나라에는 약 300여종이 존재하는 것으로 알려져 있다. 구절초, 감국, 쑥, 쑥갓, 개미취, 참취, 곰취 등 국화과 식물들은 예로부터 민간에서 약용 및 식용 소재로써 다양하게 사용되어왔다. 본 연구는 국화 및 국화근연종 유용유전자원 선발을 통하여 육종 소재를 확대하고, 중간모본 및 신품종 육성기반을 구축하고자 DNA 마커시스템의 개발을 위해 수행되었다. 1. 화단국인 Smileball(Dendranthema grandiflorum) 품종을 사용하여 SSR-enriched library를 작성하였고, GS FLX 분석을 통해 18.83Mbp의 염기서열 결과를 얻었으며, read의 평균 길이는 280.06bp로 나타났다. 2. 단순반복염기서열(SSR) 부위를 포함하는 26,780개 clones 중 di-nucleotide motifs가 16,375개(61.5%)로 우세하였고, trinucleotide motifs(6,616개, 24.8%), tetra-nucleotide motifs(1,674개, 6.3%), penta-nucleotide motifs(1,283개, 4.8%), hexa-nucleotide motifs(693개, 2.6%) 순으로 나타났다. 3. 얻어진 di-nucleotide motifs들 중에서는, AC/CA class가 93.5%로 대부분이었고, tri-nucleotide motifs에서는 AAC class가 50.5%, tetra-nucleotide motifs는 ACGT class가 43.6%이고, pentanucleotide motif에서는 AACGT class 27.2%이며, hexa-nucleotide motif에서는 ACGATG class 21.8%였다. 4. 얻어진 염기서열 결과를 토대로 다양한 motif를 갖는 100개의 SSR 마커를 제작하였고, 차후 이를 활용하여 국화 유전자원의 다형성 및 유전자형 분석을 통해 분자유전학적 다양성 및 집단의 구조분석이 가능하고, 국화의 분자육종기반 구축을 위한 유용한 도구가 될 것 이다.

After pea aphid Acyrthosiphon pisum genome project, hologenome concept was applied to pea aphid and symbioant such as Buchnera aphidicola. Here we screened symbiotic microorganism in four lab strains (two genetically different insecticide susceptible strains, host plant: cucumber and two different host adopted imiacloprid resistance strains, host plants: cucumber and potato) and four field populations (Jeju, Goryeong, Gimjae and Muju, host plant: potato) of cotton aphid based on GS-FLX pyrosequencing which were conducted with universal primer amplified partial fragments of 16S rRNA from total DNA which was extracted from each strain and population. B. aphidicola occupied over 90% of all identified prokaryotic microorganisms which all tested samples. It’s interesting that the ratio of B. aphidicola occupied over 99% in all of the tested lab strains. However, specific enterobacteriaceae occupied six to seven percents of all field populations which closely related endosymbiont of Glycaspis brimblecombei. That means B. aphidicola occupied only 91~92% of all identified prokaryotic microorganisms. Futhermore, other actinobacteridae and bacillaceae also were detected in field populations. The results obtained for these ratios suggested that there has some interaction between symbioant and environment NOT in imidacloprid resistance.

본 연구에서는 퇴비화실험 재료인 복합유용미생물 슬러지와 일반슬러지의 미생물 군집 비교를 통해 복합유용미생물 슬러지에 대한 미생물학적 특성을 밝히고 일반슬러지 퇴비와 비교하여 복합유용미생물 우점 슬러지를 퇴비화 하였을 때 발현되는 미생물의 성상을 비교하여 복합유용미생물 우점 슬러지 퇴비가 토양에 시용 되었을 때 토양에 미치는 영향을 간접적으로 확인하고자 한다. 또한 현재 시판되고 있는 광합성균을 이용한 돈분퇴비와 같은 공정으로 본 실험에서 사용한 복합유용미생물을 우점 시킨 돈분퇴비를 제조하여 시판퇴비와의 비교는 물론 퇴비 제조 방법에 따른 복합유용미생물 군집의 차이점을 알아보고자 하였다. Pyrosequencing을 통해 연구에서 사용된 복합유용미생물 우점 슬러지의 bacteria 군집 문(Phylum)의 수준에서 분석한 결과 Proteobacteria(41.30%), Gemmatimonadetes(14.12%)로 우점 되어 있었다. Proteobacteria는 하폐수 처리공정에서 유기물 농도가 높은 환경에서 주로 발견되며, 광합성세균은 이산화탄소 및 황화수소를 흡수 이용하며, 오염 및 악취의 방지 효과를 나타낸다고 알려져 있다. Gemmatimonadetes는 활성화된 우수처리장에서 발견되는 호기성균으로 일반슬러지에서는 0.3%정도 발견되었으나 복합유용미생물 우점 슬러지에서는 14.12%로 복합유용미생물 우점 활성슬러지 공정이 활성도가 높다고 판단할 수 있다. 복합유용미생물 우점 슬러지 퇴비와 일반 슬러지 퇴비의 다양성분석을 한 결과 퇴비화 숙성이 진행되면서 미생물이 단순화 되고 퇴비화에 주로 발현되는 미생물인 Bacteroidetes, Proteobacteria, Firmicutes등의 우점도가 높아지는 타 연구결과와 일치하였다. 특히 복합유용미생물에서 4.17% 우점 되어 있는 Actinobacteria는 고정된 질소를 공급하는 역할을 하고 퇴비가 숙성이 진행되면서 온도가 뜨거워질 때 활성도가 높아지는데 복합유용미생물 슬러지 퇴비가 일반슬러지 퇴비보다 부숙 온도가 높고, 높은 온도 유지기간도 길었기 때문으로 보인다.본 연구에서 개발한 BCL의 bacteria 군집을 종(species) 수준 분석하기 위해 시판퇴비인 PMP와 BMP와 비교한 결과 PMP와 BMP에서는 기타 (<1%)가 각각 49%, 48% 였지만 BCL퇴비는 기타 (<1%) 15%로 BCL퇴비가 다른 시판 퇴비보다 종 다양성은 떨어졌지만 시판퇴비보다 퇴비 내 미생물이 우점 되어 공생역할을 하는 것으로 나타났다.

Methanogenic community shift and comparison were determined by 454 pryosequencing for two different full-scale anaerobic digesters treating municipal sludge. For monitoring long-term of microbial communities, samples were collected for two year at three-monthly basis. The two mesophilic AD bioreactor were operated at similar operating conditions, but different substrate streams. Methanospirillum were identified as the key drivers of methanogenesis in full-scale anaerobic digester treating municipal sludge. In Joongrang (JR) digester, Methanospirillum was dominant (48%±10.3) over almost all period, but the dominant genus move to Methanosaeta and Methanoculleus due to low acetate concentration (0.02 g/L), total ammonia nitrogen concentration, respectively. In Asan digester (AS), Methanospirillum also was dominant (41%±12.6) like JR digester, but methanogenic community shift was examined twice. One of those was from Methanospirillum to Methanophaerula due to pH sharply decrease (<5.5) and second shift was Methanosaeta increase due to low VFAs concentration (0.25 g/L).

For the development of SSR marker system in Vicia villosa Roth, an enriched library was constructed by using a modified biotin-streptavidin capture method and the selected clones were sequenced with GS-FLX(454). Of 37,794 sequenced reads, we found that 8,474 reads (22.4%) were redundant, leaving 29,320 unique ones (77.6%). Among the unique clones, 17,174 reads (58.6%) were having microsatellite repeating motifs. Sequence analysis of all SSR-containing reads revealed a predominance of the di-nucleotide SSRs (62.5%). The tri-nucleotide and the tetra-nucleotide SSRs were 5.7% and 22.5%, respectively. As the di-nucleotide type, the AG/GA class of repeat motif was most frequently identified (55.0% of the total di-nucleotide SSRs), followed by the CT/TC class (19.5%), and the TA/AT class (12.1%). Among the tri-nucleotide SSRs, the AGT/GTA/TAG class of repeat motifs was predominant (22.2%), followed by the ACT/CTA/TAC class (17.8%). Among the tetra-nucleotide SSRs, the CTTT/TTTC/TTCT/TCTT class of repeat motifs was predominant (31.2%), followed by the AAAG/AAGA/AGAA/ GAAA class (19.9%). Finally, we designed 779 primer pairs from the flanking sequences of SSR containing reads. We are undertaking the analysis of polymorphisms using the diverse collected accessions of Vicia villosa Roth now. This newly developed SSR marker set shall provide a very useful tool for implementing molecular diversity assessment and population structure studies of Vicia villosa Roth onward.