Flanking Sequence and Copy-Number Analysis of Transformation Events by Integrating Next-Generation Sequencing Technology with Southern Blot Hybridization

친양,우희종,신공식,임명호,조현석,이성곤 한국육종학회 2017 Plant Breeding and Biotechnology Vol.5 No.4

With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment.

형질전환 베타카로틴 강화 콩 계통 선발 및 도입유전자 특성 분석

친양(Yang Qin),권순종(Soon-Jong Kweon),정영수(Young-Soo Chung),하선화(Sun-Hwa Ha),신공식(Kong-Sik Shin),임명호(Myung-Ho Lim),권택(Taek-Ryoun Kwon),조현석(Hyun-Suk Cho),박순기(Soon Ki Park),우희종(Hee-Jong Woo) 한국육종학회 2015 한국육종학회지 Vol.47 No.2

The β-carotene biofortified transgenic soybean was developed recently through Agrobacterium-mediated transformation using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). GM crops prior to use as food or release into the environment required risk assessments to environment and human health in Korea. Generally, transgenic plants containing a copy of T-DNA were used for stable expression of desirable trait gene in risk assessments. Also, information about integration site of T-DNA can be used to test the hypothesis that the inserted DNA does not trigger production of unintended transgenic proteins, or disrupt plant genes, which may cause the transgenic crop to be harmful. As these reasons, we selected four transgenic soybean lines expressing carotenoid biosynthesis genes with a copy of T-DNA by using Southern blot analysis, and analyzed the integration sites of their T-DNA by using flanking sequence analysis. The results showed that, T-DNA of three transgenic soybean lines (7-1-1-1, 9-1-2, 10-10-1) was inserted within intergenic region of the soybean chromosome, while T-DNA of a transgenic soybean line (10-19-1) located exon region of chromosome 13. This data of integration site and flanking sequences is useful for the biosafety assessment and for the identification of the β-carotene biofortified transgenic soybean.

DH 집단을 이용한 벼멸구 저항선 연관 QTLs 분석

김석만,친양,손재근 한국육종학회 2009 한국육종학회지 Vol.41 No.3

벼멸구 저항성 유전자의 mapping, 벼멸구 저항성 관련 QTL 분석, 주요 농업형질과 벼멸구 저항성과의 관계 분석 등에 관한 연구를 수행 하여 얻은 결과는 다음과 같다. 벼멸구 저항성을 가진 통일형 벼인 '삼강'과 자포니카형 벼인 '낙동'을 교배하여 양성된 F_1의 약을 배양하여 육성된 120 DH 계통을 유전자 지도 작성을 위한 mapping 재료로 이용 하였다. Maker 검정에서 양친에 다양성을 나타낸 SSR 73, STS 89개 등 총 162 This study was conducted to develop a japonica-type rice cultivar with brown planthopper (BPH) resistance using DNA markers. A doubled haploid (DH) population consisting of 120 pure-lines was established by anther culture of F_1 hybrids between 'Samgang', a Tongil type BPH resistance cultivar, and 'Nagdong', a japonica cultivar. To determine the map position of genes responsible for BPH resistance in rice, a genetic map was constructed based on 120 DH lines. A total of 162 molecular markers were classified into 12 linkage groups, covering 1,884 Kosami centimorgan (cM) with an average of 11.6 cM. Five QTLs (qBPR3, qBPR6, qBPR7, qBPR8, and qBPR12) associated with BPH resistance were identified and mapped on chromosomes 3, 6, 7, 8, and 12, respectively, using the genetic map constructed in this study. To analyze the relationship between BPH resistance and agronomic traits, a total of eight QTLs related to the agronomic traits were detected on 12 rice chromosomes. In an analysis of relationships, three QTLs (qBPR3, qBPR7, and qBPR8) showed a linkage with tested agronomic traits. A QTL (qBPR3) located on chromosome 3 (RM282-3023) was closely linked to culm length (qCL3). The QTL (qBPR8) for BPH resistance on the short arm of chromosome 8 also overlapped the region detected in culm length (qCL8).

LM 유채의 현장 검출을 위한 RPA(recombinase polymerase amplification)기반 진단 기술 개발

박선하,친양,천민관,김양하,정아름,이범규 한국육종학회 2023 한국육종학회 공동학술발표집 Vol.2023 No.-

벼멸구 저항성과 도복관련 형질과의 관계분석

김석만,친양,손재근 한국작물학회 2010 Korean journal of crop science Vol.55 No.2

도복과 관련된 주요 형질인 초장, 3절간장, 좌절중 등에 대한 QTL을 분석을 수행하였으며 도복과 벼멸구 저항성과의 연관 관계에 대한 QTL 및 유의성을 분석하였다. 1. 초장, 3절간장, 모멘트, 도복지수, 좌절중과 같은 도복관련 형질에 대한 QTL을 분석하여 총 13개의 유의성 있는 QTL이 6개의 염색체상에서 확인되었다. 2. 벼멸구 저항성과 주요 농업형질관련 QTL과의 연관관계를 분석한 바, 7번과 8번 염색체 상의 qBPR7, qBPR8은 각각 모멘트(qMo7)와 도복(q3rdin8) 관련 QTL에 연관되어 있었다. 3. Mapping 집단을 저항성 계통군과 감수성 계통군으로 나누어 8개 형질에 대한 차이를 조사한 바, 3절간장과 도복 지수에서 두 집단간에 유의성이 인정되었다. 4. 저항성을 가지는 계통은 간장 및 3절간장 등이 ‘삼강’의 영향으로 ‘낙동’에 비하여 짧은 경향이었으며 이는 신장과 관련된 도복성향에 있어 본 집단에서는 벼멸구 저항성이 강하게 연관이 되었다는 분석결과를 찾을 수 없었다. This study was conducted to analyze the relationships between resistance of brown planthopper and traits related to the lodging in rice. For the linkage analysis of traits tested in this study, a genetic linkage map was created with 162 DNA markers spanning 12 rice chromosomes based on 120 doubled haploid (DH) lines, which were derived from a cross between Samgang', a Tongil type cultivar with BPH resistance, and ‘Nagdong’, a japonica cultivar. QTLs were identified to analyze the agronomic traits including lodging by composite interval mapping. Thirteen QTLs were detected for five traits comprised of plant length (PL), 3rd internode length (3rdIL), moments (Mo), lodging index (LI), and breaking weight (BW). The relationships between the BPH resistance and agronomic traits including lodging revealed that two QTLs (qBPR7, qBPR8) were linked to traits related to lodging. Two QTLs, qBPR7 and qBPR8 on chromosome 7 (RM531-7042) and 8 (RM1148- RM544) showed associations with moments and 3rd internode length, respectively.

식품안전관리를 위한 제초제 glufosinate 특이적 GM 작물 검출마커 개발

송민지,친양,조윤성,박태성,임명호 한국식품과학회 2020 한국식품과학회지 Vol.52 No.1

Over 500 genetically modified organisms (GMOs) have been developed since 1996, of which nearly 44% have glufosinate herbicide-tolerant traits. Identification of specific markers that can be used to identify herbicide-tolerant traits is challenging as the DNA sequences of the gene(s) of a trait are highly variable depending on the origin of the gene(s), plant species, and developers. To develop specific PCR marker(s) for the detection of the glufosinate-tolerance trait, DNA sequences of several pat or bar genes were compared and a diverse combination of PCR primer sets were examined using certified reference materials or transgenic plants. Based on both the qualitative and quantitative PCR tests, a primer set specific for pat and non-specific for bar was developed. Additionally, a set of markers that can detect both pat and bar was developed, and the quantitative PCR data indicated that the primer pairs were sensitive enough to detect 0.1% of the mixed seed content rate.

GM벼 OsCK1의 도입유전자 구조분석: Whole Genome Shotgun Sequencing을 이용한 다중 도입된 T-DNA 구조 분석

안홍일,이진형,친양,우희종,신공식,권순종,임명호 한국육종학회 2014 한국육종학회지 Vol.46 No.3

GM벼 OsCK는 벼 유래의 OsCK1 유전자를 벼에 형질전환 하여 벼흰잎마름병 및 벼도열병에 대한 저항성을 높게 한벼로 농촌진흥청에서 개발하였다. 형질전환 벡터의 구성은 양쪽 border (LB, RB) 상간에 2개의 MAR 염기서열이 서로 마주보는 형태로 위치하고 있으며, 제초제 저항성 유전자 PAT는 CaMV 35S promoter에 의하여 발현이 유도되고, 목표 유전자인 choline kinase (OsCK)는 actin promoter에 의하여 발현이 조절되며 left border 기준으로 역방향으로 배치되었다. 도입유전자 확인을 위하여 adaptor ligation PCR을 수행하였는데, MAR 영역에 위치하는 제한효소로 GM벼 genomic DNA를 절단한 후 adaptor를 붙였다. 염기서열 분석을 위하여 T-DNA의 양 말단에서 primer를 제작한 후 sequence 분석을 하였다. 분석한 결과, T-DNA의 right border 인근의 MAR sequence가 벼 genome의 10번 염색체 129971번 염기와 연결되어 있음을 확인하였다. Left 영역의 삽입위치는 이후 실시한 Illumina NGS 시스템을 이용하여 확인할 수 있었으며, GM 벼에는 2개의 T-DNA가 도입되었음을 알 수 있고, 첫 번째 T-DNA는 벼 10번 염색체 BAC클론 OSJNBa0014J14의 128947번째 염기와 129970째 염기에 위치하고 벼 genome 염기 1024 bp가 결실됨을 확인하였다. 이 과정에서 첫 번째 T-DNA left border와 첫 번째 MAR sequence 일부(370 bp)가 결실되었고 right border와 두 번째 MAR 영역 199 bp가 결실되었음도 확인하였다. 두 번째 T-DNA는 right border가 결실된 형태로 첫번째 T-DNA의 35S promoter 중간에 삽입되었음을 확인하였다. The purpose of this study was to characterize the T-DNAs introduced into the transgenic OsCK rice, as part of a biosafety evaluation. Choline Kinase (CK) gene is upregulated in the transgenic OsCK rice. We identified the insertion sites, flanking sequences, structures and sequences of the inserted T-DNAs. Based on the adaptor-ligation PCR, we found that the right border of the T-DNA was inserted at position no. 129971, on BAC clone OSJNBa0014J14 of chromosome 10. The flanking sequences of the left border region of the T-DNA (which was later identified as a region harboring a 1-kb long deleted sequence), could not be identified by various PCR-based trials. However, it was finally identified with whole-genome shotgun sequencing, using an Illumina sequencer. The result indicated that one of the T-DNAs was inserted into the CaMV 35S promoter region, whereas the other T-DNA was introduced at position 128947 on OSJNBa0014J14 clone, with an inverse orientation. During the insertion process, a 1024-bp-long chromosome sequences flanked by the right border of the T-DNA region was deleted. A 370-bp long left border region and 199-bp long right border region corresponding to the matrix attachment region (MAR) sequences of the T-DNA were also deleted. Collectively, these results indicate that whole-genome shotgun sequencing is a useful tool to reveal the detailed sequences and structures of the introduced T-DNAs, especially in the case of multiple T-DNA insertions. The expenses incurred on genome sequencing can be easily compensated by minimizing the time and efforts invested in conventional molecular analyses.

해충저항성 유전자변형 벼 Agb0101에 대한 PCR 검정

신공식,이진형,임명호,우희종,친양,서석철,권순종,조현석 한국식물생명공학회 2013 식물생명공학회지 Vol.40 No.1

Genetically modified (GM) rice Agb0101, which expresses the insecticidal toxin modified cry1Ac (mcry1Ac1)gene, was developed by the Rural Development Administration in Korea. To monitor the probable release of Agb0101 in the future, it is necessary to develop a reliable detection method. Here, we developed the PCR detection method for monitoring and tracing of GM rice. The primer pair (RBEgh-1/-2) from a starch branching enzyme (RBE4) gene was designed as an endogenous reference, giving rise to an expected PCR amplicon of 101 bp. For the qualitative PCR detection, construct- and event-specific primers were designed on the basis of integration sequence of T-DNA. Eventspecific PCRs amplified specifically 5’- or 3’-junction region spanning the native genome DNA and the integrated gene construct, while none of amplified product was shown on crops, rice varieties, and other insect-resistant transgenic rice lines. The event-specific real-time PCR method was performed using TaqMan probe and plasmid pRBECrR containing both rice endogenous gene RBE4 sequence and 5’-junction sequence as the reference molecule. The absolute limit of quantification (LOQ) of real-time PCR was established with around 10 copies for one plasmid molecule pRBECrR. Thereafter, the different amounts of transgenic rice (1, 3, 5, and 10%, respectively) were quantified by using the established real-time PCR method, with a range below 19.55% of the accuracy expressed as bias, 0.06-0.40 of standard deviation (SD) and 3.80-7.01% of relative standard deviations (RSD), respectively. These results indicate that the qualitative and quantitative PCR methods could be used effectively to detect the event Agb0101 in monitoring and traceability.

자포니카 벼멸구 저항성 벼 신품종 "드리미 2호" 육성

손재근,이현숙,박영희,김태헌,친양,조신화,박희연,김나혜 한국육종학회 2010 한국육종학회 학술발표회 발표요지 Vol.42 No.1

레스베라트롤 합성 GM 벼 검정을 위한 계통특이 마커 개발

우희종(Hee-Jong Woo),친양(Yang Qin),백소현(So-Hyeon Baek),여윤수(Yunsoo Yeo),신공식(Kong-Sik Shin),임명호(Myung-Ho Lim),조현석(Hyun-Suk Cho) 한국육종학회 2016 한국육종학회지 Vol.48 No.2

A variety of genetically modified (GM) crops have been developed in Korea. In these crops, the resveratrol-enriched transgenic rice plant (Agb0102) has moved ahead to generate the dossier for regulatory review process required for commercialization of GM crop. The resveratrol-enriched transgenic rice plant could be released to farmers for cultivation after national regulators have determined that it is safe for the environment and human health. Here, we developed a PCR-based DNA marker based on flanking sequences of transgene for the discrimination of resveratrol-enriched transgenic rice plant. This DNA markers will be useful for identifying of resveratrol-enriched transgenic rice plant, and can also be used to estimate transgene movement occurred by pollen transfer or seed distribution. Moreover, it is helpful for prompt screening of a homozygote-transgenic progeny in the breeding program.