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1 식물병연구 Res. Plant Dis. 19(3) : (2013) Note Open Access Research in Plant Disease The Korean Society of Plant Pathology RT-PCR 과 nested PCR 을이용한 Nepovirus 속식물검역바이러스 4 종의정밀진단 이시원 1,2 강은하 1,3 신용길 1 * 이수헌 4,5 ** 1 농림축산검역본부식물검역기술개발센터, 2 단국대학교미생물학과, 3 성균관대학교유전공학과, 4 경북대학교응용생명과학부, 5 경북대학교식물의학연구소 Development of RT-PCR and Nested PCR for Detecting Four Quarantine Plant Viruses Belonging to Nepovirus Siwon Lee 1,2, Eun-Ha Kang 1,3, Yong-Gil Shin 1 * and Su-Heon Lee 4,5 ** 1 Plant Quarantine Technology Center, Animal and Plant Qurantine Agency, Suwon , Korea 2 Department of Microbiology, Dankook University, Cheonan , Korea 3 Department of Genetic Engineering, Sungkyunkwan University, Suwon , Korea 4 School of Applied Biosciences, Kyungpook National University, Daegu , Korea 5 Institute of Plant Medicine, Kyungpook National University, Daegu , Korea (Received on March 25, 2013; Revised on September 13, 2013; Accepted on September 17, 2013) For quarantine purpose, we developed the RT- and nested PCR module of Tomato black ring virus (TBRV), Arabis mosaic virus (ArMV), Cherry leafroll virus (CLRV) and Grapevine fanleaf virus (GFLV). The PCR modules, developed in this study make diagnosis more convenient and speedy because of same PCR condition. And also, the methods are more accurate because it can check whether the result is contamination or not using the mutation-positive control. We discard or return the 27 cases of Nepovirus infection seed by employing the module past 3 years. This study provides a rapid and useful method for detection of four quarantine plant viruses. Keywords : Detection, Nepovirus, Nested PCR, Quarantine, RT-PCR 최근세계의기후변화는농업과생태계를포함하는우리의환경체계에영향을미치고있으며, 평균기온의지속적증가에따라바이러스를매개하는곤충이늘어나고, 식물바이러스병이확산되고있는추세이다 (Garrett 등, 2006). 또한자유무역의영향으로다양한종류의식물들이국내로수입되고있으며, 그에따라잠재적또는동정되지않은병원성식물바이러스들이농작물, 사료, 견목, 종자, 야채, 과일및한약재를통해서함께수입될가능성이높아지고있다. 검역병원체의지정은식물을자연 *Corresponding author Phone) , Fax) ) syg1286@korea.kr **Corresponding author Phone) , Fax) ) suheon@knu.ac.kr 기주로하는병원체로, 국내에분포하지않는병원체중검역적위험도가높은병원체를매년위험도평가를거쳐정도에따라 금지급, 관리급 및 규제비검역 으로구분하여총 100종으로관리하고있다 (Animal, Plant and Fisheries Quarantine and Inspection Agency, 2013). 우리는이중 Nepovirus속 4종의식물검역바이러스, Tomato black ring virus (TBRV), Arabis mosaic virus (ArMV), Cherry leafroll virus (CLRV) 및 Grapevine fanleaf virus (GFLV) 를선택하였다. TBRV 는약 28 nm의지름을가지는구형의바이러스로 (Murant, 1970), 선충을벡터로산딸기, 딸기및복숭아일부품종에감염된다. ArMV는약 30 nm의지름을가지고있고 (Francki 등, 1985), 자연상태에서기주범위가매우넓어채소및과수등다양한작물에발생하며, 국내에서는딸기바이러스병진단을위한프라이머가개발되어있다 (Choi 등, 2009). CLRV는

2 RT-PCR 과 nested PCR 을이용한 Nepovirus 속식물검역바이러스 4 종의정밀진단 221 약 28 nm의지름을가지고있고, 주로즙액, 접목, 종자및선충에의해전염된다. 자연상태에서대부분의기주는체리등의목본식물과완두등의콩과작물과관련이있으며, 실험적기주범위는 36개과 (family) 이상의넓은기주를가지고있다. GFLV 는약 30 nm의지름을가지는구형바이러스로자연상태에서주요기주는포도속식물과관련이있으나, 콩과를비롯한등다양한기주를가지 는것으로알려져있다. Enzyme-linked immunosorbent assay(elisa) 는항원또는항체를색의변화로측정, 진단하는방법으로 1977년식물바이러스검정에처음사용되었으며 (Clark과 Adams, 1977), 검사의정확성과간편성으로식물검역바이러스검사법으로오랫동안사용되었다. 그러나검사과정에소요되는시간, 낮은민감도및거짓양성반응등의문제점 Table 1. List of plant viruses used in this study Virus Genus Family Type Tomato black ring virus (TBRV) Nepovirus Secoviridae Tissue and RNA Arabis mosaic virus (ArMV) Nepovirus Secoviridae Tissue and RNA Cherry leaf roll virus (CLRV) Nepovirus Secoviridae Tissue and RNA Grapevine fanleaf virus (GFLV) Nepovirus Secoviridae Tissue and RNA Alfalfa mosaic virus (AMV) Alfamovirus Bromoviridae cdna sample Bean common mosaic virus (BCMV) Potyvirus Potyviridae cdna sample Bean yellow mosaic virus (BYMV) Potyvirus Potyviridae cdna sample Broad bean wilt virus 2 (BBWV 2) Fabavirus Secoviridae cdna sample Broad bean wilt virus (BBWV) Fabavirus Secoviridae cdna sample Carnation mottle virus (CarMV) Carmovirus Tombusviridae cdna sample Cowpea chlorotic mottle virus (CCMV) Bromovirus Bromoviridae cdna sample Cowpea mild mottle virus (CPMMV) Carmovirus Tombusviridae cdna sample Cowpea mosaic virus (CPMV) Comovirus Secoviridae cdna sample Cowpea severe mosaic virus (CPSMV) Comovirus Secoviridae cdna sample Cucumber green mottle mosaic virus (CGMMV) Tobamovirus Virgaviridae cdna sample Cucumber mosaic virus (CMV) Cucumovirus Bromoviridae cdna sample Cucumber necrosis virus (CNV) Tobamovirus Virgaviridae cdna sample Cymbidium ringspot virus (CymRSV) Tobamovirus Virgaviridae cdna sample Kyuri green mottle mosaic virus (KGMMV) Tobamovirus Virgaviridae cdna sample Lettuce mosaic virus (LMV) Potyvirus Potyviridae cdna sample Prune dwarf virus (PDV) Ilarvirus Bromoviridae cdna sample Pepper mottle virus (PepMoV) Potyvirus Potyviridae cdna sample Pepper veinal mottle virus (PYMV) Potyvirus Potyviridae cdna sample Raspberry ringspot virus (RpRSV) Nepovirus Secoviridae cdna sample Ribgrass mosaic virus (RMV) Tobamovirus Virgaviridae cdna sample Soybean mosaic virus (SMV) Potyvirus Potyviridae cdna sample Squash mosaic virus (SqMV) Comovirus Secoviridae cdna sample Tomato bushy stunt virus (TBSV) Tobamovirus Virgaviridae cdna sample Tobacco mild green mosaic virus (TMGMV) Tobamovirus Virgaviridae cdna sample Tobacco mosaic virus (TMV) Tobamovirus Virgaviridae cdna sample Tobacco necrosis virus (TNV) Necrovirus Tombusviridae cdna sample Tomato mosaic virus (ToMV) Tobamovirus Virgaviridae cdna sample Tomato ringspot virus (ToRSV) Nepovirus Secoviridae cdna sample Tobacco ringspot virus (TRSV) Nepovirus Secoviridae cdna sample Tobacco rattle virus (TRV) Tobamovirus Virgaviridae cdna sample Tobacco streak virus (TSV) Ilarvirus Bromoviridae cdna sample Tomato spotted wilt virus (TSWV) Tospovirus Bunyaviridae cdna sample Turnip mosaic virus (TuMV) Potyvirus Potyviridae cdna sample

3 222 이시원 강은하 신용길 이수헌 이발견되었으며 (Caruso 등, 2003; Priou 등, 2006) 최근에는항혈청검사법이핵산검사법으로변화되고있는추세이다 (Kim 등, 2000; Kim 등, 2005; Lee 등, 2011a, 2011b; Park 등, 2004). 따라서본연구에서는식물검역 Nepovirus속 4종의바이러스들을 RT-PCR과 nested PCR 을사용하여, 신속하고정확하게진단할수있는방법을개발하였다. 검사법개발대상바이러스 (TBRV, ArMV, CLRV 및 GFLV) 4종이감염되어있는식물의조직을국립식물검역원 ( 현농림축산검역본부 ) 의금지품수입허가를통해수 입하였다 (ADGEN, England). 실험에사용된 Alfalfa mosaic virus(amv) 외 34종의 cdna는국내기업 (PLUTOS, Korea) 에서구매하여실험에활용하였으며 (Table 1), 모든시료들은 80 o C 초저온냉동고에보관하였다. 식물검역바이러스 4종의진단용프라이머를설계하기위하여, 미국국립생물정보센터 (National Center for Biotechnology Information) 로부터검사법개발대상바이러스인 TBRV(AY157994), ArMV(NC_006056), CLRV (S84125) 및 GFLV(NC_003623) 와각각의바이러스와분류학적으로유사한바이러스들의염기서열을다운로드하 Table 2. Sequence information of final selection and nested primer sets used in this study Primer Sequence Length (bp) Expected size (bp) TBRV #1 TBRV #2 ArMV #1 ArMV #2 CLRV #1 CLRV #2 GFLV #1 GFLV #2 TBR-C30 5'-TGC CAG CCT CAA CGA AAT C-3' 19 TBR-N40 5'-ACT GTG TTA AAT CGC CCG CA-3' TBR-C70 5'-AGA TGC ACG GGG AGA TAA CT-3' 20 TBR-N20 5'-GCA GTC TGT TCA CCT GCG-3' TBRN-C30 5'-CAA CRG CAG TGA TTC CAA ACA T-3' 22 TBRN-N40 5'-TGG AAR GAG GCA AAG TCT GT-3' TBRN-C70 5'-TTC CAT CCC GCT TCA GCA AGT-3' 21 TBRN-N20 5'-AGT TTG TCG AGG GTT CTG A-3' ArM-C32 5'-ATT GGG GAA GGA CGG AAC AGA AAG A-3' 25 ArM-N24 5'-CCC TTT CGG TCC CTC ATT GGC T-3' ArM-C29 5'-GAC CCC GTT CCA TTC ACT A-3' 19 ArM-N30 5'-AAC GCG GGG TCT TGC TGG TA-3' ArM-C29 5'-GAC CCC GTT CCA TTC ACT A-3' 19 ArM-N25 5'-TGC CTC ACT GGA AAT AAC A-3' ArM-C28 5'-ACT CTT AGG ACT ATG CCT AAG T-3' 22 ArM-N24 5'-CCC TTT CGG TCC CTC ATT GGC T-3' CLR-C10 5'-GGA AAG ATT ACG TAA AAG GAA AAC-3' 24 CLR-N60 5'-GGT TTT CAG CCC AGG KYA GTC TT-3' CLR-C25 5'-CTT GCT AAC GCT ATC TAC CCA CAT-3' 24 CLR-N50 5'-TCT GGC GGG AAT AGT GRA GGT C-3' CLR-C20 5'-AAA MMC GAT CGG GGC AAC AA-3' 20 CLR-N80 5'-CAA TTC TGG CGA CCG TGT AAC-3' CLR-C30 5'-TRR RAT TCA AAG TCA CAG GTR TG-3' 23 CLR-N62 5'-TCA GCC CAG GKY AGT CTT ATT TYA-3' GFL-C30 5'-CTG CAA AAT TCC CAA CCA ACA ACT-3' 24 GFL-N120 5'-GTT GTG TTT TGG GTA TGG GAG GTA-3' GFL-C50 5'-TCA CCC GAA GGA GCG ATA GG-3' 20 GFL-N100 5'-RTC TCC AAG GTT GCA TTT CAC R-3' GFL-C40 5'-ATT CGG TGT TCA GAC TCC AYT-3' 21 GFL-N130 5'-CCC GGG GTG TAT GTG GAA GAG GAY-3' GFL-C60 5'-ACT TCC GTC CTC TTC CAC ATA CAC-3' 24 GFL-N110 5'-TAC TTG CCC TCC CAT ATT CTT TGA-3'

4 RT-PCR 과 nested PCR 을이용한 Nepovirus 속식물검역바이러스 4 종의정밀진단 223 였다. 다운로드한염기서열들은 DNAMAN DNA analysis software package(dnaman version 6.0; Lynnon Biosoft, Canada) 를사용하여 sequence alignment 후 annealing temperature o C를조건으로종특이적인염기서열을탐색하였다 (Pan 등, 2000) (Table 2). 감염시료에서 total RNA의추출과기주의 genomic DNA 추출은 SolGent RNA mini prep kit(solgent, Korea), RNA-spin TM IIp RNA extraction kit(intron, Korea) 및 DNeasy Plant mini kit(qiagen, Netherlands) 를사용하였다 (Lee 등, In press). RT-PCR은추출한 total RNA 1 μl를주형으로, 설계된정방향과역방향프라이머 (25 pmol) 를각각 1μl, DW(Sigma, USA) 7 μl 및 RT-PCR premixture (Plutos, Korea) 10 μl로전체 20 μl로수행하였다. PCR은위에서추출한기주의 genomic DNA 1 μl를주형으로정방향과역방향프라이머 (25 pmol) 를각각 1μl, DW 7 μl 및 nested-pcr kit(plutos, Korea) 10 μl로전체 20 μl로실시하였다. RT-PCR 조건은 42 o C, 60분의역전사반응과 95 o C, 10분의 denaturation 후 95 o C에서 45초, 55 o C에서 1분, 72 o C에서 1분과정을 35회반복후, 마지막으로 72 o C 에서 5분간반응시켰다. RNA를주형으로 cdna 합성은 42 o C에서 60분후 60 o C에서 10분반응하였으며, PCR 조건은 RT-PCR 의역전사반응을제외한나머지반응과같게하였다. PCR 산물은 1X TAE buffer를이용하여 1.2% agarose gel에서 80분전기영동후 ethidium bromide로염 Table 3. Final selection and nested primer sets used in this study to detect 4 Nepovirus species Virus 1 Final selection primer sets Nested primer sets Positive control (bp) Upstream Downstream Length (bp) Upstream Downstream Length (bp) TBRV ArMV CLRV GFLV TBR-C30 TBR-N TBRN-C30 TBRN-N TBR-N20/C10 TBR-C70 TBR-N TBRN-C70 TBRN-N (2,999) ArM-C32 ArM-N ArM-C29 ArM-N ArM-N17/C32 ArM-C29 ArM-N ArM-C28 ArM-N (1,443) CLR-C10 CLR-N CLR-C20 CLR-N CLR-N10/C10 CLR-C25 CLR-N CLR-C30 CLR-N (1,497) GFL-C30 GFL-N GFL-C40 GFL-N GFL-N60/C10 GFL-C50 GFL-N GFL-C60 GFL-N (1,656) 1 TBRV: Tomato black ring virus, ArMV: Arabis mosaic virus, CLRV: Cherry leafroll virus, and GFLV: Grapevine fanleaf virus. Fig. 1. Results of RT-PCR products and nested-pcr products. Circle, final selected nested primer pairs. M, 100 bp step DNA Ladder maker(genepia, Korea); lane 1, TBRV final selected primer set #5 (510 bp); lane 2, TBRV nested-pcr product from primer #5 (TBR- C30/N40, 313 bp); lane 3, TBRV final selected primer #18 (662 bp); lane 4, TBRV nested-pcr product from primer #18 (TBR-C70/ N20, 317bp); lane 5, ArMV final selected primer #8 (773 bp); lane 6, ArMV nested-pcr product from primer #8 (ArM-C29/N25, 385 bp); lane 7, ArMV final selected primer #9 (780 bp); lane 8, ArMV nested-pcr product from primer #9 (ArM-C28/N24, 487 bp); lane 9, CLRV final selected primer #8 (673 bp); lane 10, CLRV nested-pcr product from primer #8 (CLR-C20/N80, 313 bp); lane 11, CLRV nested-pcr product from primer #8 (CLR-C24/N80, 280 bp); lane 12, CLRV final selected primer #44 (633 bp); lane 13, CLRV nested- PCR product from primer #44 (CLR-C30/N62, 479 bp); lane 14, CLRV nested-pcr product from primer #44 (CLR-C30/N80, 230 bp); lane 15, GFLV final selected primer #25 (699 bp); lane 16, GFLV nested-pcr product from primer #25 (GFLV-C40/N130, 430 bp); lane 17, GFLV nested-pcr product from primer #25 (GFLV-C40/N140, 280 bp); lane 18, GFLV final selected primer #42 (582 bp); lane 19, GFLV nested-pcr product from primer #42 (GFLV-C60/N110, 289 bp); lane 20, GFLV nested-pcr product from primer #42 (GFLV- C60/N120, 164 bp).

RT-PCR과 nested PCR 결과, TBRV(set 1, 510 313; set 2, 662 307 bp), ArMV(set 1, 773 385; set 2, 780 487 bp), CLRV(set 1, 673 313; set 2, 633 479 bp) 및 GFLV(set 1, 699 430; set 2, 582 289 bp)

5 224 이시원 강은하 신용길 이수헌 색하여 UV 하에서확인하였다. PCR 증폭산물에대한 nested 프라이머조합을설계하였으며, 1차 PCR 산물을 1/100 희석하여 1 μl를주형으로사용하였고나머지조건은위의 PCR 조건과동일하게반응하였다. 또한최종합격한프라이머조합의민감도를확인하기위하여, total RNA를원액 10 7 까지희석하여 1μl를주형으로 one-step RT-PCR 로반응하였다 (Lee 등, 2013). 바이러스별로 2 sets 씩의 RT-PCR 을선발하였으며, 이에대한 nested 조합을선발하였다. RT-PCR과 nested PCR 결과, TBRV(set 1, ; set 2, bp), ArMV(set 1, ; set 2, bp), CLRV(set 1, ; set 2, bp) 및 GFLV(set 1, ; set 2, bp) 에각각특이적밴드를형성하였으며 (Table 3, Fig. 1), 염기서열유사바이러스, 공통기주에감염될수있는바이러스및기주의 genomic DNA 에서비특이적인반응이나타나지않았다 (data not shown). 또한 RT-PCR 조합들은 TBRV(set 1, 10 3 ; set 2, 10 1 ), ArMV(set 1, 10 3 ; set 2, 10 4 ), CLRV(set 1, 10 5 ; set 2, 10 4 ) 및 GFLV(set 1, 10 2 ; set 2, 10 3 ) 의민감도를보였다 (Fig. 2). 각각의바이러스에대하여두세트씩선발된프라이머조합들을모두포함하는 PCR 산물을증폭하였으며, 증폭산물은 TBRV(2,999 bp), ArMV(1,443 bp), CLRV(1,497 bp) 및 GFLV(1,656 bp) 였다. 증폭산물을 insert DNA로 pgem -T Easy Vector를사용하여클로닝하였다 (Promega, USA). 양성대조구로선발된클론을대상으로, nested 프라이머증폭부위안쪽으로제한효소인 Xho I이반응할수있는염기서열 (CTCGAG, 6 bp) 을 Site-Directed Mutagenesis Kit를사용하여삽입하였다 (Nelson과 McClelland, 1992). Fig. 3. Xho I restriction enzyme to cut mutation-positive plasmid. The digestion products were analyzed on a 1.2% agarose gel prestained with ethidium bromide. (A) Lane M, Maker; lane 1, TBRV nested PCR product for mutation-positive plasmid; Lane 2, Result of restriction enzyme Xho I treatment. (B) Lane M, Maker; lane 1, ArMV nested PCR product for mutation-positive plasmid; Lane 2, Result of restriction enzyme Xho I treatment. (C) Lane M, Maker; lane 1, CLRV nested PCR product for mutation-positive plasmid; Lane 2, Result of restriction enzyme Xho I treatment. (D) Lane M, Maker; lane 1, GFLV nested PCR product for mutation-positive plasmid; Lane 2, Result of restriction enzyme I treatment. Fig. 2. PCR sensitivity test for the detection of four quarantine viruses. 제작된플라스미드는염기서열분석결과프라이머부착부위가확인되었으며 (data not shown), 양성대조구로사용할돌연변이-양성대조구는, 제한효소 Xho I을처리하여 6개의염기서열에대한삽입여부를확인하였다 (Fig. 3). 본연구에서검사한방법으로, 최근 3년 ( 년), 화물, 우편및휴대로국내유입된식물에대하여검사를진행한결과, TBRV(7건 ), ArMV(19건 ) 및 CLRV(1건 ) 검출되어폐기또는반송되었으며, 최근 GFLV는검출되지않았으나 2007년수입콩종자에서 2건이검역된사례가있었다. TBRV 는양상추종자에서 3건검출되었으며, 토마토종자, 백일초종자, 콩종자및고추종자에서각각 1건씩검출하였다. ArMV는수선구근 8건, 종구용쪽파 3건, 무수카리구근, 백합구근, 벨레발리아구근, 양상추종자, 카마시아구근, 튤립구근, 하늘나리구근및히야신스

6 RT-PCR 과 nested PCR 을이용한 Nepovirus 속식물검역바이러스 4 종의정밀진단 225 구근에서 1건씩검출하였다. CLRV 는복숭아종자에서 1건검출되어검역처분하였다. 요약 본연구에서는식물검역바이러스 4종 (TBRV, ArMV, CLRV 및 GFLV) 을 RT-PCR 과 nested PCR 방법으로진단할수있는방법을개발하였다. 본연구에서개발한방법은모두같은 PCR 조건으로검사자에게편리성과신속성을높여줄뿐아니라, 돌연변이- 양성대조구의사용으로실험오염여부를확인할수있어더욱정확하다. 개발한방법으로최근 3년 Nepovirus속 4종의바이러스를검사한결과, 27건을검출하여검역처분하였다. 본연구결과들은앞으로도수출입식물에서해당바이러스들을신속, 정밀하게진단할수있는방법으로활용할수있을것으로기대된다. Acknowledgement This study was supported by a grant (Project No. Z ) from Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. References Animal, Plant and Fisheries Quarantine and Inspection Agency List of plant quarantine viruses in Korea in newly revised in Res. Plant Dis. 19: (In Korean) Caruso, P., Bertolini, E., Cambra, M. and López, M. M A new and sensitive co-operational polymerase chain reaction for rapid detection of Ralstonia solanacearum in water. J. Microbiol. Meth. 55: Choi, G. S., Lee, J. A., Cho, J. D., Chung, B. N., Cho, I. S. and Kim, J. S Strawberry virus diseases occurring in Korea, Res. Plant Dis. 15: (In Korean) Clark, M. F. and Adams, A. N Characteristics of the microplate method of enzyme linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34: Francki, R. I. B., Milne R. G. and Hatta, T Atlas of plant viruses. Vol. II, pp CRC Press, Boca Raton, Florida, USA. Garrett, K. A., Dendy, S. P., Frank, E. E., Rouse, M. N. and Travers, S. E Climate change effects on plant disease: Genomes to ecosystems. Annu. Rev. Phytopathol. 44: Kim, D., Hyun, J., Hwang, H. and Lee, S RT-PCR Detection of Citrus tristeza virus from early Satsuma mandarin and Cheju island. Plant Pathology J. 16: Kim, Y. J., Park, S., Yie, S. W. and Kim, K. H RT-PCR Detection of dsrna Mycoviruses Infecting Pleurotus ostreatus and Agaricus blazei Murrill. Plant Pathology J. 21: Lee, J. S., Cho, W. K., Lee, S. H., Choi, H. S. and Kim, K. H. 2011a. Development of RT-PCR based method for detecting five non-reported quarantine plant viruses infecting the family Cucurbitaceae or Solanaceae. Plant Pathology J. 27: Lee, J. S., Cho, W. K., Choi, H. S. and Kim, K. H. 2011b. RT-PCR Detection of Five Quarantine Plant RNA Viruses belonging to Poty and Tospoviruses. Plant Pathology J. 27: Lee, S., Kang, E. H., Kim, Y. J., Kim, S. M. and Shin, Y. G Detection of Carnation necrotic fleck virus and Carnation ringspot virus using RT-PCR. Res. Plant Dis. 19: (In Korean) Murant, A. F Tomato black ring virus. CMI/AAB Descriptions of Plant Viruses No. 38. Association of Applied Biologists, Wellesbourne, UK. Nelson, M. and McClelland, M Use of DNA methyltransferase/endonuclease enzyme combinations for megabase mapping of chromosomes. Methods Enzymol. 216: Nelson, M. and McClelland, M Use of DNA methyltransferase/endonuclease enzyme combinations for megabase mapping of chromosomes. Methods Enzymol. 216: Pan, Y. B., Burner, D. M. and Legendre, B. L An assessment of the phylogenetic relationship among sugarcane and related taxa based on the nucleotide sequence of 5S rrna intergenic spacers. Genetica 108: Park, M. R. and Kim, K. H RT-PCR Detection of three non-reported fruit tree viruses useful for quarantine purpose in Korea. Plant Pathology J. 20: Priou, S., Gutarra, L. and Aley, P An improved enrichment broth for the sensitive detection of Ralstonia solanacearum (biovars 1 and 2A) in soil using DAS-ELISA. Plant Patholgy J. 55:

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