• Journal of Microbiology and Biotechnology
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• 제29권5호
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• pp.785-793
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• 2019

Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is the most damaging disease in Brassica crops around the world. In this study, we developed a molecular marker specific to Xcc race 5. To do this, the available whole genome sequences of Xcc races/strains and Xc subspecies were aligned and identified a highly variable genomic region (XccR5-89.2). Subsequently, a primer set covering the 'XccR5-89.2' region was designed and tested against the genomic DNA of Xcc races/strains, Xc subspecies and other plant-infecting bacterial strains (Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora). The results showed that the 'XccR5-89.2' primer pair amplified a 2,172-bp fragment specific to Xcc race 5. Moreover, they also amplified a 1,515-bp fragment for Xcc race 1 and an over 3,000-bp fragment for Xcc race 3. However, they did not amplify any fragments from the remaining Xcc races/strains, subspecies or other bacterial strains. The 'XccR5-89.2' primer pair was further PCR amplified from race-unknown Xcc strains and ICMP8 was identified as race 5 among nine race-unknown Xcc strains. Further cloning and sequencing of the bands amplified from race 5 and ICMP8 with 'XccR5-89.2' primers revealed both carrying identical sequences. The results showed that the 'XccR5-89.2' marker can effectively and proficiently detect, and identify Xcc race 5 from Xcc races/strains, subspecies and other plant-infecting bacteria. To our knowledge, this is the first report for an Xcc race 5-specific molecular marker.

• Journal of Microbiology and Biotechnology
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• 제31권2호
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• pp.280-289
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• 2021

Genetic markers currently used for the discrimination of Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, our objective in this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. We evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genomes. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited at NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing the core and pangenomes. Genetic markers were confirmed to be specific for 59,196,562 genome sequences via in silico analysis. They were found in all strains of the same subspecies, but not in other subspecies or bacterial strains. These genetic markers also could be used to accurately identify genomes at the subspecies level for genomes known at the species level. A real-time PCR method for detecting three main subspecies (L. delbrueckii subsp. delbrueckii, lactis, and bulgaricus) was developed to cost-effectively identify them using genetic markers. Results showed 100% specificity for each subspecies. These genetic markers could differentiate each subspecies from 44 other lactic acid bacteria. This real-time PCR method was then applied to monitor 26 probiotics and dairy products. It was also used to identify 64 unknown strains isolated from raw milk samples and dairy products. Results confirmed that unknown isolates and subspecies contained in the product could be accurately identified using this real-time PCR method.

• Journal of Crop Science and Biotechnology
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• 제10권3호
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• pp.175-184
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• 2007

Two subspecies, japonica and indica, have been reported in rice, which differ in several ecotypic traits. However, reproductive barriers in hybrid progenies between subspecies have been major obstacles in breeding programs using inter-subspecific hybridization. As the first step to elucidate the reproductive barriers, we developed subspecies-specific(SS) STS markers in this study. A total of 765 STS primers were designed through comparing DNA sequences at every $2{\sim}3$cM interval between japonica and indica rices, which are available at Web DBs such as IRGSP, NCBI, TIGR, and GRAMENE, and tested for subspecies-specificity using 15 indica and 15 japonica varieties of diverse origin. Of them, 67 STS markers were identified as SS STS markers and their subspecies-specificity scores were estimated. The SS markers were dispersed throughout the genome along chromosomes. Of them, 64 SS markers were mapped on an RIL population derived from a Dasanbyeo(indica)/TR22183(japonica) cross. Genomic inclination of RILs was evaluated based on the genotyping with different types of markers. Association test between markers and segregation distortion revealed that segregation distortion might not be the cause of generating SS markers. The SS markers will be applicable to estimate the genomic inclination of varieties or lines and to study the differentiation of indica and japonica, and ultimately to breed true hybrid rice varieties in which desirable characters from both subspecies are recombined.

• 한국육종학회지
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• 제41권2호
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• pp.101-105
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• 2009

전 세계에서 광범위하게 수집한 벼 유전자원 320개를 63개의 아종특이적마커로 분석하여 유전자원의 다양성, 유연관계 및 유전집단의 구조분석을 하여 아종특이적마커의 아종판별 효율을 검정하고 아종의 게놈 구성을 검토하고자 본 시험을 수행하였다. 1. 본 연구에서 사용한 63개의 아종특이적 마커는 벼 품종을 인디카와 자포니카 두 아종으로 구분하는데 효과적으로 이용할 수 있었다. 2. 실험에 사용한 320개의 벼 유전자원들은 자포니카군(128개)과 인디카군(178개)으로 나눌 수 있었고 Basmati370, Shennong27 등 14개 품종은 중간형 admixture형태의 품종으로 구분할 수 있었다.

• 대한본초학회지
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• 제29권6호
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• pp.35-43
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• 2014

Objectives : Official Arisaematis Rhizoma is described only three species, Arisaema amurnse, Arisaema erubescens, and Arisaema heterophyllum, in national Pharmacopoeia. However, other Arisaema species, Arisaema ringens, Arisaema takesimense and Arisaema serratum, also have been distributed as an inauthentic Arisaematis Rhizoma in the herbal market. To develop a reliable molecular authentication method for Arisaematis Rhizoma in species level, we analyzed DNA barcode regions using six Arisaema species. Methods : Thirty-eight samples of six Arisaema plants species (A. amurense, A. amurense f. serratum, A. heterophyllum, A. takesimense, and A. serratum) were collected from different habitate and nucleotide sequences of DNA barcode regions (rDNA-ITS, matK, and rbcL gene) were analyzed after PCR amplification. The species-specific sequences and phylogenetic relations were estimated using entire sequences of three DNA barcodes based on the analysis of ClastalW and UPGMA, respectively. Results : The comparative analysis of DNA barcode sequences were revealed inter-species specific nucleotides to distinguish the medicinal plant of Arisaema Rhizoma in species levels excluding between A. amurense and its subspecies (A. amurense f. serratum) and A. takesimense and A. serratum, respectively. However, we obtained sequence differences enough to discriminate authentic and inauthentic Arisaematis Rhizoma. Therefore, we suggest that these SNP type molecular genetic markers were an reliable method avaliable to identify official herbal medicines. Conclusions : These marker nucleotides could be useful to identify the official herbal medicines by providing definitive information that can identify original medicinal plant and distinguish from inauthentic adulterants and substitutes.

• 생명과학회지
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• 제33권10호
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• pp.808-819
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• 2023

본 연구에서는 미생물균총에서 B. licheniformis K12 균주의 양상을 확인하기 위한 선발마커 개발을 시도하였다. Bacillus licheniformis는 바위게 내장에서 유산생산균주로써 분리되었다. 본 균주는 중온에서 성장이 양호할 뿐만 아니라 유전체 분석결과 protease, amylase, cellulase, lipase, protease, xylanase 등 다양한 고분자 물질들을 분해할 수 있는 효소류들을 보유하고 있는 것으로 나타났다. 선발마커 발굴을 위해 recombinase, integrase, transposase, phage-related genes, bacteriocin 등 유전자 변이 유발이 쉬운 게놈상의 영역에 대해 탐색을 실시하였다. 그 결과, 선발마커로써 가능성이 높은 5개 부위를 확보하였다. 후보마커는 recombinase 부위 3개(BLK1, 2, 3) integrase 부위 1개(BLK4), phase 관련 1개(BLK5)로 나타났다. 후보 선발마커로써 Bacillus species가 다른 B. licheniformis, B. velezensis, B. subtilis, B. cereus 등에 대해 PCR 분석을 실시하였다. 그 결과 BLK1 recombinase, BLK2 recombinase family protein, BLK4 site-specific integrase 등에서 B. licheniformis에서 특이적인 위치에 PCR 산물이 나타나는 것을 확인하였다. 또한, B. licheniformis 표준균주로써 subspecies에 대한 PCR을 수행한 결과 BLK1 및 3이 선발마커로써 양호한 것으로 나타났다. 따라서 BLK1이 미생물균총에서 종(species) 및 아종(subspecies) 선발마커로써 활용 가능할 것으로 판단된다.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2022년도 춘계학술대회
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• pp.110-110
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• 2022

Rice is the world's most important food crop and a major source of nutrition for about two thirds of populations. Northern Vietnam is one of the most important centers of genetic diversity for cultivated rice. In this study, we determined the genetic diversity and population structure of 79 rice landraces collected from northern Vietnam and 19 rice accessions collected from different countries. In total, 98 rice accessions could be differentiated into japonica and indica with moderate genetic diversity and a polymorphism information content of 0.382. We also detected subspecies-specific markers to classify rice (Oryza sativa L.) into indica and japonica. Additionally, we detected five marker-trait associations and rare alleles that can be applied in future breeding programs. Most interestingly, analysis of molecular variance (AMOVA) found genetic differentiation was related to geographical regions with an overall PhiPT (analog of fixation index FST) value of 0.130. More emphasis was given to provide signatures and infer explanations about the role of geographical isolation and environmental heterogeneity in genetic differentiation among regions in landraces from northern Vietnam. Our results suggest that rice landraces in northern Vietnam have a dynamic genetic system that can create different levels of genetic differentiation among regions, but also maintain a balanced genetic diversity between regions.

• Molecules and Cells
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• 제39권2호
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• pp.141-148
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• 2016

Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family.

• 생명과학회지
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• 제33권11호
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• pp.897-904
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• 2023

본 연구는 Bacillus velezensis K10의 유전체 분석결과에 따른 유전자의 고유한 특성을 이용하여 유전자마커를 탐색하고 확립하여 산업현장에서 활용하기 위해서 수행하였다. B. velezensis K10은 총 4,159,835 bps를 유지하였으며, 5,136개의 단백질을 발현하는 것으로 조사되었다. B. velezensis K10은 표준균주에 비교하여 전반적으로 외부요인에 기인되는 유전자의 이동이 훨씬 많은 것으로 나타났다. 이와 같은 양상에 기인하여 B. velezensis K10은 특유의 유전적 서열을 유지할 수 있는 것으로 추정되었다. 선발마커 발굴을 위해 recombinase, integrase, transposase, phage-related genes, 등 유전자 변이 유발이 쉬운 게놈상의 영역에 대해 탐색을 실시하였다. 그 결과, 선발마커로써 가능성이 높은 9개 부위를 확보하였다. 후보마커는 일부 다른 영역에서 특이성을 보이는 영역들이 존재했지만, phage 연관 유전자들에서 매우 특이성이 높았기 때문에, 모두 phage 관련 부위에서 모두 탐색되었다. 종간 후보 선발마커로써 B. licheniformis K12, B. velezensis K10, B. subtilis, B. cereus 등에 대해 PCR 분석을 실시하였다. 그 결과 BV3, BV5~9까지 총 6 프라이머 세트에서 B. velezensis K10 특이적인 PCR 산물이 형성되는 것을 확인하였다. 다른 한편으로, subspecies 수준에서 분석 결과, BV3, 5, 8, 9 등 4 프라이머 세트에서 B. velezensis K10 특이적인 PCR 산물이 형성되는 것을 관찰했다. 분석된 마커 중에서 BV5와 8가 가장 특이성이 높았기 때문에 종(species) 및 아종(sub-species) 수준에서 B. velezensis K10 유전자 선발마커로써 BV5와 8을 활용 가능할 것으로 제의된다.