• 원예과학기술지
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• 제32권4호
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• pp.535-543
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• 2014

Backcross breeding is the method most commonly used to introgress new traits into elite lines. Conventional backcross breeding requires at least 4-5 generations to recover the genomic background of the recurrent parent. Marker-assisted backcrossing (MABC) represents a new breeding approach that can substantially reduce breeding time and cost. For successful MABC, highly polymorphic markers with known positions in each chromosome are essential. Single nucleotide polymorphism (SNP) markers have many advantages over other marker systems for MABC due to their high abundance and amenability to genotyping automation. To facilitate MABC in hot pepper (Capsicum annuum), we utilized expressed sequence tags (ESTs) to develop SNP markers in this study. For SNP identification, we used Bukang $F_1$-hybrid pepper ESTs to prepare a reference sequence through de novo assembly. We performed large-scale transcriptome sequencing of eight accessions using the Illumina Genome Analyzer (IGA) IIx platform by Solexa, which generated small sequence fragments of about 90-100 bp. By aligning each contig to the reference sequence, 58,151 SNPs were identified. After filtering for polymorphism, segregation ratio, and lack of proximity to other SNPS or exon/intron boundaries, a total of 1,910 putative SNPs were chosen and positioned to a pepper linkage map. We further selected 412 SNPs evenly distributed on each chromosome and primers were designed for high throughput SNP assays and tested using a genetic diversity panel of 27 Capsicum accessions. The SNP markers clearly distinguished each accession. These results suggest that the SNP marker set developed in this study will be valuable for MABC, genetic mapping, and comparative genome analysis.

• 미생물학회지
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• 제45권2호
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• pp.170-176
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• 2009

제주도에 서식하는 2종의 해양 해면, Dictyonella sp.와 Spirastrella abata의 공생세균 군집구조를 16S rDNA-DGGE(denaturing gradient gel electrophoresis) 방법에 의해 분석하였다. 해면으로부터 total genomic DNA를 추출하여 GC clamp가 추가된 세균에 특이적인 341f primer와 518r primer를 이용하여 16S rRNA gene의 V3 부위를 증폭한 후 DGGE 전기 영동하고 재증폭하여 염기서열을 분석하였다. 그 결과 Dictyonella sp.에서 8개, Spirastrella abata에서 7개의 band를 확인할 수 있었다. 공통된 주요 band가 없는 패턴을 나타내었으며, DGGE band로부터 DNA를 추출하여 부분 염기서열을 분석한 결과, NCBI에 등록된 서열들과 93%~98%의 유사도를 나타내었다. Dictyonella sp.의 주요 해면 공생세균은 uncultured Gammaproteobacteria, Spirastrella abata의 경우 uncultured Alphaproteobacteria, Firmicutes에 각각 포함되어 해면 종에 따른 숙주 특이적 분포를 보이는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.462-469
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• 2012

A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from $50^{\circ}C$ to $55^{\circ}C$. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.

• 한국유기농업학회:학술대회논문집
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• 한국유기농학회 2009년도 하반기 학술대회
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• pp.302-302
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• 2009

유기농업에서 유기물은 양분의 공급, 토양의 이화학성 개선, 토양의 생물학적 건전성 유지 등 중요한 역할을 한다. 토양의 생물학적 건전성은 토양의 생태계적 기능을 지속적으로 유지시키는 토양미생물이 관여하고 있다. 따라서 본 연구는 유기물의 장기연용에 따른 밭토양 미생물의 다양성을 비교 분석하였다. 여러 가지 유기자원을 동일한 기준으로 매년 동일 장소에 처리하였다. 사용된 유기자원은 가축분퇴비, 채종유박인 유기질비료, 볏짚으로만 퇴비화한 볏짚퇴비와 겨울철 휴한기에 헤어리베치를 재배하여 이듬해 봄에 예취한 후 토양에 환원한 녹비처리구, NPK구, 가축분퇴비를 혼용처리한 NPK퇴비군, 양분을 전혀 시용하지 않은 무비구 등 총 7처리구였다. 각각의 처리구에서 토양(0-20 cm)을 채취하여 배양성 토양미생물은 희석평판법으로 해당 선백배지에 시료를 도말 하여 조사하였고 비배양성 미생물은 토양으로부터 genomic DNA를 추출하여 세균의 16S rDNA를 증폭시킨 후 denaturing gradient gel electrophoresis (DGGE)를 수행하여 분석하였다. 주요결과를 요약하면 밭토양에 서식하는 토양미생물의 균수는 처리별간의 차이를 보였으며 유기물처리구가 화학비료처리구보다 높았다. DGGE 분석을 통해 유기물 처리에 따른 군집의 다양성을 살펴본 결과 Fig. 1에서 보는바와 같이 Gel 상에서 다양한 위치의 밴드를 확인할 수 있었고 처리별로 특이 밴드가 있음을 확인할 수 있었다. Fig. 1에서 얻은 DGGE profile상의 밴드 강도와 수를 비교하여 Fig 2와 같은 dendrogram을 나타낼 수 있었다.

• 식물조직배양학회지
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• 제26권4호
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• pp.235-240
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• 1999

무릇 (S. scilloides Complex)에서 게놈 유형이 다른 AA, BB 및 AABB 게놈 식물의 조직배양을 통하여 유도된 재분화체를 대상으로 RAPD기법을 이용한 유전적 분석을 통해 게놈의 안정성과 게놈 유형에 따른 차이를 분석한 결과, 적용한 20가지의 primer중 Al, A2및 A3에서 게놈 유형에 따른 다형현상을 관찰할 수 있었다. RAPD분석 결과 AA게놈에서 39.2%로 다형성이 세 가지 유형 중 가장 낮게 나타났으며, BB 게놈에서는 72.3%로 가장 높은 다형성을 보였고, AABB 게놈에서는 45.7%의 다형성이 관찰되었다. AA유형에서 관찰된 총 110개의 밴드 중 특이 밴드가 A3에서 하나 나타나 0.9%의 변이율을 보였으며, BB 게놈의 경우 총 116개의 밴드 중 특이 밴드는 A3에서 5개 나타나 4.3%의 변이율을 보여 주었고, AABB유형에서는 총 103개의 밴드 중 특이 밴드가 Al에서 2개, A2에서 1개, A3에서 2개로 총 5개가 나타나 4.9%의 변이율을 나타내었다. RAPD 분석은 게놈 유형이 다른 무릇 체세포클론의 유전적인 안정성 분석에 유용하게 이용될 수 있다.

• Molecules and Cells
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• 제39권12호
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• pp.862-868
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• 2016

Goats (Capra hircus) are one of the oldest species of domesticated animals. Native Korean goats are a particularly interesting group, as they are indigenous to the area and were raised in the Korean peninsula almost 2,000 years ago. Although they have a small body size and produce low volumes of milk and meat, they are quite resistant to lumbar paralysis. Our study aimed to reveal the distinct genetic features and patterns of selection in native Korean goats by comparing the genomes of native Korean goat and crossbred goat populations. We sequenced the whole genome of 15 native Korean goats and 11 crossbred goats using next-generation sequencing (Illumina platform) to compare the genomes of the two populations. We found decreased nucleotide diversity in the native Korean goats compared to the crossbred goats. Genetic structural analysis demonstrated that the native Korean goat and cross-bred goat populations shared a common ancestry, but were clearly distinct. Finally, to reveal the native Korean goat's selective sweep region, selective sweep signals were identified in the native Korean goat genome using cross-population extended haplotype homozygosity (XP-EHH) and a cross-population composite likelihood ratio test (XP-CLR). As a result, we were able to identify candidate genes for recent selection, such as the CCR3 gene, which is related to lumbar paralysis resistance. Combined with future studies and recent goat genome information, this study will contribute to a thorough understanding of the native Korean goat genome.

• 한국발생생물학회지:발생과생식
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• 제18권4호
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• pp.275-286
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• 2014

To successful molecular breeding, identification and functional characterization of breeding related genes and development of molecular breeding techniques using DNA markers are essential. Although the development of a useful marker is difficult in the aspect of time, cost and effort, many markers are being developed to be used in molecular breeding and developed markers have been used in many fields. Single nucleotide polymorphisms (SNPs) markers were widely used for genomic research and breeding, but has hardly been validated for screening functional genes in olive flounder. We identified single nucleotide polymorphisms (SNPs) from expressed sequence tag (EST) database in olive flounder; out of a total 4,327 ESTs, 693 contigs and 514 SNPs were detected in total EST, and these substitutions include 297 transitions and 217 transversions. As a result, 144 SNP markers were developed on the basis of 514 SNP to selection of useful gene region, and then applied to each of eight wild and culture olive flounder (total 16 samples). In our experimental result, only 32 markers had detected polymorphism in sample, also identified 21 transitions and 11 transversions, whereas indel was not detected in polymorphic SNPs. Heterozygosity of wild and cultured olive flounder using the 32 SNP markers is 0.34 and 0.29, respectively. In conclusion, we identified SNP and polymorphism in olive flounder using newly designed marker, it supports that developed markers are suitable for SNP detection and diversity analysis in olive flounder. The outcome of this study can be basic data for researches for immunity gene and characteristic with SNP.

• Genomics & Informatics
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• 제10권3호
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• pp.194-199
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• 2012

In addition to single-nucleotide polymorphisms (SNP), copy number variation (CNV) is a major component of human genetic diversity. Among many whole-genome analysis platforms, SNP arrays have been commonly used for genomewide CNV discovery. Recently, a number of CNV defining algorithms from SNP genotyping data have been developed; however, due to the fundamental limitation of SNP genotyping data for the measurement of signal intensity, there are still concerns regarding the possibility of false discovery or low sensitivity for detecting CNVs. In this study, we aimed to verify the effect of combining multiple CNV calling algorithms and set up the most reliable pipeline for CNV calling with Affymetrix Genomewide SNP 5.0 data. For this purpose, we selected the 3 most commonly used algorithms for CNV segmentation from SNP genotyping data, PennCNV, QuantiSNP; and BirdSuite. After defining the CNV loci using the 3 different algorithms, we assessed how many of them overlapped with each other, and we also validated the CNVs by genomic quantitative PCR. Through this analysis, we proposed that for reliable CNV-based genomewide association study using SNP array data, CNV calls must be performed with at least 3 different algorithms and that the CNVs consistently called from more than 2 algorithms must be used for association analysis, because they are more reliable than the CNVs called from a single algorithm. Our result will be helpful to set up the CNV analysis protocols for Affymetrix Genomewide SNP 5.0 genotyping data.

• Animal cells and systems
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• 제16권1호
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• pp.50-56
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• 2012

Kelp grouper ($Epinephelus$ $bruneus$ Bloch 1793) is a commercially important fish in Korea. In recent years, the catch of kelp grouper in the coastal waters of Korea has significantly declined. Despite its importance, little is known about its genetic diversity and conservation efforts are hampered. In this study, we isolated and characterized 12 microsatellite loci using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. All loci were readily amplified and contained TG/CA denucleotide repeats. To characterize each locus, 30 individuals from a natural E. bruneus population in the coastal waters of Jeju Island, Korea, were genotyped. All loci except three, KEm118, KEm154, and KEm219, were polymorphic, with an average of 8.1 alleles per locus (range 2-18). The mean observed and expected heterozygosities were 0.47 (range 0.19-1.00) and 0.61 (range 0.29-0.92), respectively. A significant deviation from Hardy-Weinberg equilibrium was observed at three loci (KEm134, KEm184, and KEm283). These findings will be useful for effective monitoring and management of genetic variation of kelp grouper as well as for the implementation of a fisheries conservation program.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 추계학술대회 및 정기총회
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• pp.49-49
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• 2014

In order to find indigenous beneficial fungal species from crop field soils of Nigeria, 23 soil samples were collected from various places of Nigeria in June, 2013 and fungi were isolated through serial dilution technique. Isolated fungi were purified and differentiated according to their morphological and microscopic characteristics. In total, 38 different representative isolates were recovered and the genomic DNA of each isolates was extracted using QIAGEN$^{(R)}$ Plasmid Mini Kit (QIAGEN Sciences, USA) and the identification of fungi was carried out by sequence analysis of internal transcribed spacer (ITS) region of the 18S ribosomal DNA (18S rDNA). Recovered isolates belonged to 9 fungal genera comprising Fusarium, Aspergillus, Chaetomium, Coniothyrium, Dipodascaceae, Myrothecium, Neosartorya, Penicillium and Trichoderma. Aspergillus spp., Penicillium spp. and Trichoderma spp. were the most dominant taxa in this study. The antagonistic potentiality of species belonged to Trichoderma against 10 phytopathogenic fungi (F. oxysporum, C. gloesporoides, P. cytrophthora, A. alternata, A. solani, S. rolfsii, F. solani, R. solani, S. sclerotiorum and P. nicotiana) was assessed in vitro using dual culture assay. The dual culture assay results showed varied degree of antagonism against the tested phytopathogens. The potential Trichoderma spp. will be further evaluated for their antagonistic and plant growth promotion potentiality under in vivo conditions.