• 한국균학회소식:학술대회논문집
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• 2015.05a
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• pp.53-53
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• 2015

Agrobacterium tumefaciens-mediated transformation(ATMT) of Flammulina velutipes was used to produce a diverse number of transformants to discover the functions of gene that is vital for its variation color, spore pattern and cellulolytic activity. Futhermore, the transformant pool will be used as a good genetic resource for studying gene functions. Agrobacterium-mediated transformation was conducted in order to generate intentional mutants of F. velutipes strain KACC42777. Then Agrobacterium tumefaciens AGL-1 harboring pBGgHg was transformed into F. velutipes. This method is use to determine the functional gene of F. velutipes. Inverse PCR was used to insert T-DNA into the tagged chromosomal DNA segments and conducting sequence analysis of the F. velutipes. But this experiment had trouble in diverse morphological mutants because of dikaryotic nature of mushroom. It needed to make monokaryotic fruiting varients which introduced genes of compatible mating types. In this study, next generation sequencing data was generated from 28 strains of Flammulina velutipes with different phenotypes using Illumina Hiseq platform. Filtered short reads were initially aligned to the reference genome (KACC42780) to construct a SNP matrix. And then we built a phylogenetic tree based on the validated SNPs. The inferred tree represented that white- and brown- fruitbody forming strains were generally separated although three brown strains, 4103, 4028, and 4195, were grouped with white ones. This topological relationship was consistently reappeared even when we used randomly selected SNPs. Group I containing 4062, 4148, and 4195 strains and group II containing 4188, 4190, and 4194 strains formed early-divergent lineages with robust nodal supports, suggesting that they are independent groups from the members in main clades. To elucidate the distinction between white-fruitbody forming strains isolated from Korea and Japan, phylogenetic analysis was performed using their SNP data with group I members as outgroup. However, no significant genetic variation was noticed in this study. A total of 28 strains of Flammulina velutipes were analyzed to identify the genomic regions responsible for producing white-fruiting body. NGS data was yielded by using Illumina Hiseq platform. Short reads were filtered by quality score and read length were mapped on the reference genome (KACC42780). Between the white- and brown fruitbody forming strains. There is a high possibility that SNPs can be detected among the white strains as homozygous because white phenotype is recessive in F. velutipes. Thus, we constructed SNP matrix within 8 white strains. SNPs discovered between mono3 and mono19, the parental monokaryotic strains of 4210 strain (white), were excluded from the candidate. If the genotypes of SNPs detected between white and brown strains were identical with those in mono3 and mono19 strains, they were included in candidate as a priority. As a result, if more than 5 candidates SNPs were localized in single gene, we regarded as they are possibly related to the white color. In F. velutipes genome, chr01, chr04, chr07,chr11 regions were identified to be associated with white fruitbody forming. White and Brown Fruitbody strains can be used as an identification marker for F. veluipes. We can develop some molecular markers to identify colored strains and discriminate national white varieties against Japanese ones.

• Microbiology and Biotechnology Letters
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• v.46 no.3
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• pp.253-260
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• 2018

Beneficial microorganisms are widely used in the forestry, livestock, and, in particular, agricultural sectors to control soilborne diseases and promote plant growth. However, the industrial utilization of these microorganisms is very limited, mainly due to uncertainty concerning their ability to colonize and persist in soil. In this study, the survival of beneficial microorganisms in field soil microcosms was investigated for 13 days using quantitative PCR with B. subtilis group-specific primers. Bacterial community dynamics of the treated soils were analyzed using 16S ribosomal RNA (rRNA) gene amplicon sequencing on the Illumina MiSeq platform. The average 16S rRNA gene copy number per g dry soil of Bacillus spp. was $4.37{\times}10^6$ after treatment, which was 1,000 times higher than that of the control. The gene copy number was generally maintained for a week and was reduced thereafter, but remained 100 times higher than that of the control. Bacterial community analysis indicated that Acidobacteria ($26.3{\pm}0.9%$), Proteobacteria ($24.2{\pm}0.5%$), Chloroflexi ($11.1{\pm}0.4%$), and Actinobacteria ($9.7{\pm}2.5%$) were abundant phyla in both treated and non-treated soils. In the treated soils, the relative abundance of Actinobacteria was lower, whereas those of Bacteroidetes and Firmicutes were higher compared to the control. Differences in total relative abundances of operational taxonomic units belonging to several genera were observed between the treated and non-treated soils, suggesting that inoculation of soil with the Bacillus strains influenced the relative abundances of certain groups of bacteria and, therefore, the dynamics of resident bacterial communities. These changes in resident soil bacterial communities in response to inoculation of soil with beneficial Bacillus spp. provide important information for the use of beneficial microorganisms in soil for sustainable agriculture.

• Korean Journal of Ichthyology
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• v.32 no.2
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• pp.39-48
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• 2020

The slender shinner (Pseudopungtungia tenuicorpa), a tiny freshwater fish of about 8 to 10 cm belonging to Cyprinidae, is an endangered species found only in the Han and Imjin Rivers on the Korean Peninsula. During the breeding season, this species spawns in nests of Coreoperca herzi, a predator of this species, or small crevices on rocks. This unique reproductive ecology can make this species more vulnerable to anthropogenic perturbance that can further limit the places to spawn. Here, mtDNA and microsatellite loci were analyzed to identify the genetic diversity and structure of slender shinners and further to provide the basic data necessary for the conservation planning of this species. A total of 28 polymorphic microsatellite markers were developed using Illumina paired-end sequencing, and 67 slender shinners collected from three localities in the Han River were genotyped using these loci. This species showed a remarkably high level of genetic diversity with mean expected heterozygosity of 0.914 and mean allele number per locus of 27.9, and no signature of drastic demographic decline was detected. As a result of our microsatellite analysis, the genetic structure between the two stems of the Han River, North Han and South Han, was prominent. Such a genetic structure was also evident in the sequence analysis of 14 haplotypes obtained from mtDNA control region. Although slender shinners are only found in very limited areas around the world, the genetic structure indicates that there is a block of gene flow among the populations, which should be reviewed in the future if management and restoration of this species is needed.

• Journal of Microbiology and Biotechnology
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• v.30 no.8
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• pp.1169-1179
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• 2020

In this study, two soybean genotypes, i.e., aluminum-tolerant Baxi 10 (BX10) and aluminumsensitive Bendi 2 (BD2), were used as plant materials and acidic red soil was used as growth medium. The soil layers from the inside to the outside of the root are: rhizospheric soil after washing (WRH), rhizospheric soil after brushing (BRH) and rhizospheric soil at two sides (SRH), respectively. The rhizosphere bacterial communities were analyzed by high-throughput sequencing of V4 hypervariable regions of 16S rRNA gene amplicons via Illumina MiSeq. The results of alpha diversity analysis showed that the BRH and SRH of BX10 were significantly lower in community richness than that of BD2, while the WRH exhibited no significant difference between BX10 and BD2. Among the three sampling compartments of the same soybean genotype, WRH had the lowest community richness and diversity while showing the highest coverage. Beta diversity analysis results displayed no significant difference for any compartment between the two genotypes, or among the three different sampling compartments for any same soybean genotype. However, the relative abundance of major bacterial taxa, specifically nitrogen-fixing and/or aluminum-tolerant bacteria, was significantly different in the compartments of the BRH and/or SRH at phylum and genus levels, indicating genotype-dependent variations in rhizosphere bacterial communities. Strikingly, as compared with BRH and SRH, the WRH within the same genotype (BX10 or BD2) always had an enrichment effect on rhizosphere bacteria associated with nitrogen fixation.

• Molecules and Cells
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• v.41 no.11
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• pp.979-992
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• 2018

Potato (Solanum tuberosum L.) is the third most important food crop, and breeding drought-tolerant varieties is vital research goal. However, detailed molecular mechanisms in response to drought stress in potatoes are not well known. In this study, we developed EMS-mutagenized potatoes that showed significant tolerance to drought stress compared to the wild-type (WT) 'Desiree' cultivar. In addition, changes to transcripts as a result of drought stress in WT and drought-tolerant (DR) plants were investigated by de novo assembly using the Illumina platform. One-week-old WT and DR plants were treated with -1.8 Mpa polyethylene glycol-8000, and total RNA was prepared from plants harvested at 0, 6, 12, 24, and 48 h for subsequent RNA sequencing. In total, 61,100 transcripts and 5,118 differentially expressed genes (DEGs) displaying up- or down-regulation were identified in pairwise comparisons of WT and DR plants following drought conditions. Transcriptome profiling showed the number of DEGs with up-regulation and down-regulation at 909, 977, 1181, 1225 and 826 between WT and DR plants at 0, 6, 12, 24, and 48 h, respectively. Results of KEGG enrichment showed that the drought tolerance mechanism of the DR plant can mainly be explained by two aspects, the 'photosynthetic-antenna protein' and 'protein processing of the endoplasmic reticulum'. We also divided eight expression patterns in four pairwise comparisons of DR plants (DR0 vs DR6, DR12, DR24, DR48) under PEG treatment. Our comprehensive transcriptome data will further enhance our understanding of the mechanisms regulating drought tolerance in tetraploid potato cultivars.

• Journal of Animal Science and Technology
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• v.52 no.5
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• pp.357-366
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• 2010

The emergence of next-generation sequencing technologies has lead to application of new computational and statistical methodologies that allow incorporating genetic information from entire genomes of many individuals composing the population. For example, using single-nucleotide polymorphisms (SNP) obtained from whole genome amplification platforms such as the Ilummina BovineSNP50 chip, many researchers are actively engaged in the genetic evaluation of cattle livestock using whole genome relationship analyses. In this study, we estimated the genomic relationship matrix (GRM) and compared it with one computed using a pedigree relationship matrix (PRM) using a population of Hanwoo. This project is a preliminary study that will eventually include future work on genomic selection and prediction. Data used in this study were obtained from 187 blood samples consisting of the progeny of 20 young bulls collected after parentage testing from the Hanwoo improvement center, National Agriculture Cooperative Federation as well as 103 blood samples from the progeny of 12 proven bulls collected from farms around the Kyong-buk area in South Korea. The data set was divided into two cases for analysis. In the first case missing genotypes were included. In the second case missing genotypes were excluded. The effect of missing genotypes on the accuracy of genomic relationship estimation was investigated. Estimation of relationships using genomic information was also carried out chromosome by chromosome for whole genomic SNP markers based on the regression method using allele frequencies across loci. The average correlation coefficient and standard deviation between relationships using pedigree information and chromosomal genomic information using data which was verified using a parentage test andeliminated missing genotypes was $0.81{\pm}0.04$ and their correlation coefficient when using whole genomic information was 0.98, which was higher. Variation in relationships between non-inbred half sibs was $0.22{\pm}0.17$ on chromosomal and $0.22{\pm}0.04$ on whole genomic SNP markers. The variations were larger and unusual values were observed when non-parentage test data were included. So, relationship matrix by genomic information can be useful for genetic evaluation of animal breeding.

• Molecules and Cells
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• v.38 no.5
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• pp.466-473
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• 2015

Over the last 30 years, Hanwoo has been selectively bred to improve economically important traits. Hanwoo is currently the representative Korean native beef cattle breed, and it is believed that it shared an ancestor with a Chinese breed, Yanbian cattle, until the last century. However, these two breeds have experienced different selection pressures during recent decades. Here, we whole-genome sequenced 10 animals each of Hanwoo and Yanbian cattle (20 total) using the Illumina HiSeq 2000 sequencer. A total of approximately 3.12 and 3.07 billion sequence reads were mapped to the bovine reference sequence assembly (UMD 3.1) at an average of approximately 10.71- and 10.53-fold coverage for Hanwoo and Yanbian cattle, respectively. A total of 17,936,399 single nucleotide polymorphisms (SNPs) were yielded, of which 22.3% were found to be novel. By annotating the SNPs, we further retrieved numerous nonsynonymous SNPs that may be associated with traits of interest in cattle. Furthermore, we performed whole-genome screening to detect signatures of selection throughout the genome. We located several promising selective sweeps that are potentially responsible for economically important traits in cattle; the PPP1R12A gene is an example of a gene that potentially affects intramuscular fat content. These discoveries provide valuable genomic information regarding potential genomic markers that could predict traits of interest for breeding programs of these cattle breeds.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.7
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• pp.920-929
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• 2017

Objective: The bursa of Fabricius (BF) is a central humoral immune organ belonging specifically to avians. Recent studies had suggested that miRNAs were active regulators involved in the immune processes. This study was to investigate the possible differences of the BF at miRNA level between two genetically disparate duck breeds. Methods: Using Illumina next-generation sequencing, the miRNAs libraries of ducks were established. Results: The results showed that there were 66 differentially expressed miRNAs and 28 novel miRNAs in bursa. A set of abundant miRNAs (i.e., let-7, miR-146a-5p, miR-21-5p, miR-17~92) which are involved in immunity and disease were detected and the predicted target genes of the novel miRNAs were associated with duck high anti-adversity ability. By gene ontology analysis and enriching KEGG pathway, the targets of differential expressed miRNAs were mainly involved in immunity and disease, supporting that there were differences in the BF immune functions between the two duck breeds. In addition, the metabolic pathway had the maximum enriched target genes and some enriched pathways that were related to cell cycle, protein synthesis, cell proliferation and apoptosis. It indicted that the difference of metabolism may be one of the reasons leading the immune difference between the BF of two duck breeds. Conclusion: This data lists the main differences in the BF at miRNAs level between two genetically disparate duck breeds and lays a foundation to carry out molecular assisted breeding of poultry in the future.

• Journal of Ginseng Research
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• v.38 no.4
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• pp.278-288
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• 2014

Background: Panax ginseng Meyer is a traditional medicinal plant famous for its strong therapeutic effects and serves as an important herbal medicine. To understand and manipulate genes involved in secondary metabolic pathways including ginsenosides, transcriptome profiling of P. ginseng is essential. Methods: RNA-seq analysis of adventitious roots of two P. ginseng cultivars, Chunpoong (CP) and Cheongsun (CS), was performed using the Illumina HiSeq platform. After transcripts were assembled, expression profiling was performed. Results: Assemblies were generated from ~85 million and ~77 million high-quality reads from CP and CS cultivars, respectively. A total of 35,527 and 27,716 transcripts were obtained from the CP and CS assemblies, respectively. Annotation of the transcriptomes showed that approximately 90% of the transcripts had significant matches in public databases.We identified several candidate genes involved in ginsenoside biosynthesis. In addition, a large number of transcripts (17%) with different gene ontology designations were uniquely detected in adventitious roots compared to normal ginseng roots. Conclusion: This study will provide a comprehensive insight into the transcriptome of ginseng adventitious roots, and a way for successful transcriptome analysis and profiling of resource plants with less genomic information. The transcriptome profiling data generated in this study are available in our newly created adventitious root transcriptome database (http://im-crop.snu.ac.kr/transdb/index.php) for public use.

• Journal of Ginseng Research
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• v.41 no.1
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• pp.60-68
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• 2017

Background: Various Panax ginseng cultivars exhibit a range of diversity for morphological and physiological traits. However, there are few studies on diversity of metabolic profiles and genetic background to understand the complex metabolic pathway in ginseng. Methods: To understand the complex metabolic pathway and related genes in ginseng, we tried to conduct integrated analysis of primary metabolite profiles and related gene expression using five ginseng cultivars showing different morphology. We investigated primary metabolite profiles via gas chromatography-mass spectrometry (GC-MS) and analyzed transcriptomes by Illumina sequencing using adventitious roots grown under the same conditions to elucidate the differences in metabolism underlying such genetic diversity. Results: GC-MS analysis revealed that primary metabolite profiling allowed us to classify the five cultivars into three independent groups and the grouping was also explained by eight major primary metabolites as biomarkers. We selected three cultivars (Chunpoong, Cheongsun, and Sunhyang) to represent each group and analyzed their transcriptomes. We inspected 100 unigenes involved in seven primary metabolite biosynthesis pathways and found that 21 unigenes encoding 15 enzymes were differentially expressed among the three cultivars. Integrated analysis of transcriptomes and metabolomes revealed that the ginseng cultivars differ in primary metabolites as well as in the putative genes involved in the complex process of primary metabolic pathways. Conclusion: Our data derived from this integrated analysis provide insights into the underlying complexity of genes and metabolites that co-regulate flux through these pathways in ginseng.