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1 w ³wz The Korean Journal of Mycology The Korean Society of Mycology Kor. J. Mycol. 36(2) : (2008) Universal Rice Primer(URP) w DNA w w k m t Á Á Á Á½Ÿy 1 Áœ p w, 1 w w Differentiation Among Commercial Strains of Pleurotus spp. Based on DNA Fingerprinting Using Universal Rice Primer (URP) Kyoung-In Seo, Kab-Yeul Jang, Young-Bok Yoo, Soon-Young Park, Kwang-Ho Kim 1 Mushroom Research Division, National Institute of Horticultural & Herbal Science, Rural Development Administration, Suwon , Korea 1 Department of Crop Science, KonKuk University, Seoul , Korea (Received November 11, Accepted December 24, 2008) and Won-Sik Kong ABSTRACT: To distinct the commercial strains in Pleurotus spp., 81 strains in eight Pleurotus species were used. DNA fingerprinting using URP-PCR was conducted to determine the phylogenetic relationships among Pleurotus strains. DNA profiles of Pleurotus species obtained by twelve URP primers were analyzed for genetic similarity by NTSYS program. We could divide strains into ten clusters, in which three of them belong to P. ostreatus and the others to the different species, respectively. At the 76% similarity level, 70 P. ostreatus strains were distinguished into three clusters. Cluster I contained 35 strains and some of them showed almost 100% similarity, one strain closely related to Weonhyeong and six strains closely related to Wangheukpyeong. In cluster II, twenty-one out of 23 strains showed 100% to Suhan. Cluster III contained twelve strains, including six strains closely related to Chunchu-2. The results suggested that there are many same strains with different names in mushroom spawn market. KEYWORDS : Commercial strains, DNA fingerprinting, Phylogenetic relationship, Pleurotus ostreatus k 30 š P. ostreatus, P. sajor-caju, P. eryngii. P. ostreatus y š ƒ š k m k x s, w x t š (Peberdy et al., 1993). ù 20 k» t w ü k (Pleurotus spp. P. eryngii) 2007» 92,324m, 49.5% wš (, 2008). k 2000 l t y ù 2008 x t y t 22 wš t q š m š. t w» t yw. x t y q š y *Corresponding author < t w m. w t w». DNA y ù, w»» ù w w t w. w w» w ü w w» z y w. ü m ƒ RAPD, 10 base oligonucleotide random primer w x. w RAPD x» w ¼ 20 base URP primer 12~16 base ISSR primerƒ t w š. URP w RAPD, t x q (Seo et al., 2001) (Kang et al., 2001), w³ (, 2002) š. 130

2 Universal Rice Primer(URP) w DNA w w k m t 131 x primer l repetitive sequence l URP(Universal Rice Primer) primer primer w w sww w PCR w ƒ w universal primer p ƒ š (Kang et al., 2002). p, 20 mer 55 o C annealing PCR ww x ùkü ü p w w š. w ½ (2007) š k 65 t w 8 URP primer w t p šw ù t ùš t ƒ w ƒ š š, w primer w v w. ü š m š k t URP-PCR DNA w ww k t x t w» w ww. œ ³ URP-PCR w ³ x ¾ q š m š P. ostreatus 70³ P. florida 2³, P. sajor-caju 3³, P. cystidiosus 1³, P. eryngii 2³, P. nebrodensis, P. cornucopiae var. citrinopileatus, P. salmoneostramineus ƒƒ 1³ 81³ w (Table 1). Genomic DNA Genomic DNA w ³ PDA plate ³ w 26 o C incubator 7 w. z MCM PDA plate ³ w š, 7 w ³ k z x w. Genomic DNA Plant DNA purification kit(toyobo) w w. w ³ w ³ 500 mg lysis buffer 600 µl š 65 o C 10 k z, phenol : chloroform : isoamylalcohol(25 : 24 : 1) 600 µl ƒwš vortexingw z 13,000rpm 15 w w.» 600 µl 40 µl z trapper p ö DNAƒ w. 70% k 2 washingw z 10 g 100 µl 3 ³. trapper p ö DNAƒ w z DNA p 4 o C, PCR w template DNA w. Table 1. Pleurotus strains used in this study Lane no. ASI* no. Commercial name Species Source Year Nongmin59 P. ostreatus Korea Baekdu1 P. ostreatus Korea Jangpung P. ostreatus Korea (MKACC53972) Samgu9 P. ostreatus Korea Sinnong26 P. ostreatus Korea Sinnong22 P. ostreatus Korea (MKACC53909) Sinnong14 P. ostreatus Korea P. ostreatus Korea (MKACC51372) Nonggi2-1 P. ostreatus Korea (MKACC52243) Nonggi201 P. ostreatus Korea (MKACC51362) Nonggi202 P. ostreatus Korea (MKACC51410) Wonhyeong P. ostreatus Korea (MKACC51493) Aeneutari1 P. ostreatus Japan (MKACC51506) Wonhyeong2 P. ostreatus Korea (MKACC51496) Wonhyeong3 P. ostreatus Korea (MKACC52342) Chunchu1 P. ostreatus China (MKACC51529) Chunchu2 P. ostreatus Netherland (MKACC51632) Heukpyeong P. ostreatus Korea (MKACC52328) Byeongneutari1 P. ostreatus Korea (MKACC51778) Gyunhyeop1 P. ostreatus Korea (MKACC360) Oknong1 P. ostreatus Korea (KACC359) Heukjinju P. ostreatus Korea (MKACC51724) Cheongpung P. ostreatus Korea Myeongweol P. ostreatus Korea (MKACC51732) Suhan P. ostreatus China (KACC358) 2000

3 132 Table 1. Continued Lane no. ASI* no. Commercial name Species Source Year Sinnong94 P. ostreatus Korea (MKACC52282) Suhan2 P. ostreatus Korea (MKACC51818) Suhan3 P. ostreatus Korea (MKACC52325) Sinnong46 P. ostreatus Korea (MKACC51820) Ilseong2 P. ostreatus Korea (MKACC51817) Sinnong8 P. ostreatus Korea (MKACC51819) JanganPK P. ostreatus Korea (MKACC52326) Kimjae9 P. ostreatus Korea (MKACC52311) Kimjae10 P. ostreatus Korea (MKACC52312) Jangan2 P. ostreatus Korea (MKACC52313) Heungrim1 P. ostreatus Korea (MKACC52314) Jangan3 P. ostreatus Korea (MKACC52315) Nongmin1 P. ostreatus Korea (MKACC52320) Kimjae7 P. ostreatus Korea (MKACC52321) Kimjae8 P. ostreatus Korea (MKACC52322) Jangan5 P. ostreatus Korea Jangan6 P. ostreatus Korea (MKACC52324) Nongong99 P. ostreatus Korea DH1012 P. ostreatus Korea Sinnong11 P. ostreatus Korea Sinnong12 P. ostreatus Korea Sinnong13 P. ostreatus Korea Cheongdo21 P. ostreatus Korea Cheongdo22 P. ostreatus Korea Wangheukpyeong P. ostreatus Korea Nongong98 P. ostreatus Korea Chiak3 P. ostreatus Korea Chiak4 P. ostreatus Korea Bupyeongkirin2 P. ostreatus Korea Bupyeongheukdan4 P. ostreatus Korea Sodam P. ostreatus Korea Heukbaek P. ostreatus Korea Bupyeongsoyeop1 P. ostreatus Korea Bupyeongbokhoe P. ostreatus Korea Jangan7 P. ostreatus Korea Samguhwanghak P. ostreatus Korea SamguPJ P. ostreatus Korea Samgu01 P. ostreatus Korea Chiak5 P. ostreatus Korea Chiak7 P. ostreatus Korea Samgu5 P. ostreatus Korea Samgu8 P. ostreatus Korea Yeongnong1 P. ostreatus Korea Hanra1 P. ostreatus Korea Hanra2 P. ostreatus Korea Sacheol P. florida Germany (MKACC51361) Sacheol2 P. florida Thailand (MKACC51494) Yeoreum P. sajor-caju India (MKACC52247) Yeoreum2 P. sajor-caju Korea (MKACC51621) Sambok P. sajor-caju Korea (MKACC52343) Jeonbok1 P. abalonus Thailand (MKACC50312) Keunneutari1 P. eryngii Japan (MKACC51595) Keunneutari3 P. eryngii Japan (MKACC52327) Baeksongi P. nebrodensis Korea (MKACC52323) Noeul P. salmoneostramineus Korea Geumbit P. cornucopiae var. citrinopileatus Korea 2007 *ASI, Agricultural Sciences Institute, Suwon, Korea; KACC, Korea Agricultural Culture Collection; MKACC, Mushroom Korea Agricultural Culture Collection; Year, Collected year

4 Universal Rice Primer(URP)에 의한 DNA 핵산지문법을 이용한 느타리의 유통 품종간 구분 URP-PCR 조건 URP-PCR은 Bioneer PCR Premix kit를 이용하였고, premix kit에 genomic DNA 50 ng, primer 100 ng을 첨가 하여 총 20 µl의 반응액을 조성 하였다. PCR 증폭반응은 ABI PCR SYSTEM 9700을 이용하여 처음 DNA의 열변 성을 위하여 94oC에서 5분간 1 cycle, 그리고 94oC에서 1 총 35 cycle 실시 분, 55oC에서 1분, 72oC에서 2분간으로 o 하였으며, 최종 DNA의 합성은 72 C에서 10분으로 하였 다. 증폭된 PCR 산물은 1 TAE(40 mm Tris; ph 8.0, 20 mm acetic acid, 1 mm EDTA) 완충용액에서 1.5%의 agarose gel로 전기영동한 후 1 µg/ml ethidium bromide 용액으로 염색하여 UV transilluminator상에서 나타나는 133 DNA band를 확인하였다. Primer는 12종류의 URP Primer 로 구성된 SRILS UniPrimer Kit(서린바이오사이언스)를 사용하였다. 유전적 유연관계 분석 12개의 URP primer를 이용한 PCR fingerprinting 결과 에서 다형성을 보이는 band에 대해 각 band를 하나의 형 질로 취급하여 band가 있으면 1, band가 없으면 0 으 로 코드화하였으며 전체에 대한 자료행렬을 작성하였다. 유연관계분석은 NTSYS(Numerica Taxonomy and Multivariate Analysis System; 2.02) computer program을 사용 하였으며, 계통 상호간의 유사도 계수(Similarity coefficient) Fig. 1. Representative DNA fingerprinting by four URP-PCR amplification of eighty-one commercial strains in Pleurotus spp. Lane number from left to right is same with Table 1. Solid line and arrows in URP9 indicate P. ostreatus strains and a common band for these strains, respectively.

5 134 Sokal and Sneath(1963) w š,» UPGMA(Unweighted Pair-Group Method with Arithmetic average) w. š URP-PCR w k t x 12 URP primer w ü t k 81t w. 12 primer PCR s x band 100 bp 6000 bp s. Fig. 1 URP5, URP9, URP11 URP12 primer w s 81 k t PCR x band š. URP9 primer 1,100 bp P. ostreatus œm ù kù bandƒ. k 8 81³ DNA x band pattern URP primer ƒ yw ùkûš, URP9 primer band pattern ƒ w. URP1~URP11 primer annealing 55 C o s ù, URP12 primer w s ùkü annealing 5 o C û 50 C o w. k (P. sajor-caju) 3t, k (P. cystidiosus) 1t, j k (P. eryngii) 2t, š(p. nebrodensis), y k (P. salmoneostramineus), k (P. citrinopileatus) ƒƒ 1t k (P. ostreatus) 70t pw PCR x band x w ù k (P. florida) 2t P. ostreatus w DNA band pattern. ½ (2007) š ew. Polymorphic band w 12 primer. k 81³ s PCR w 12 primer band ƒ ƒ primer URP7 19, ƒ primer URP11 8. PCR band 156 s³ 13. ½ (2007) 65 k t x 6~ 14 PCR x band š w, ƒ ù œ ³ ƒ š, w Taq polymeraseƒ» x ƒ ù ƒ. k t PCR 12 URP primer ùkù x» band data matrix w NTSYSpc UPGMA program w dendrogram w Fig. 2. t URP-PCR x type ù 10 group ù, P. ostreatus w t Ix l IIIx¾ sw. Ix 59y, 2029, x, w 2y 35t w, IIx 1y, t, w, 7y 23t, IIIx 1y, 2y,, 8y 12t w. IVx P. florida, Vx P. abalonus, VIx P. sajor-caju, VIIx P. eryngii, VIIIx P. nebrodensis, IXx P. salmoneostramineus, Xx P. citrinopileatus. IVx l Xx¾. t š p w ù kù ³ Ix group ax group x x k 2yƒ, s 100% t bx group œ98y, e 3y, 22y, 21yƒ. x t s t 76%. IIx group cx group t, 9y, 14y, w, 94y, 46y, ½ 9y, 2y, ½ 7y, 5y, 12y, e 4y, e 5y, e 7y, 5y, 8yƒ w, groupü t 76% ùkþ. t ½ (2007) URP-PCR w ü k t p e w. IIIx group dx group 2y, t,, 8y, ½ 10y, 3y, ½ 8yƒ 100% w p š groupü t 80% š. ½ (2007) ü k t t 100% w š w x. t ³ m x w t. w ASI 2736 t s 4yƒ 2y group š w j x ASI 2736 t Ix group w. w s group w š w 2717 t 2y group w ùkû. VIIx group j k t w group t VIIIx group 34%. y k t 10% û. p k t œ w k ³ ƒ. œ ³ x w x(asi 2180) x2y(asi 2183) t, ³ x». w ³ %. URP-PCR ƒƒ k groupü

6 Universal Rice Primer(URP) w DNA w w k m t 135 Fig. 2. Phylogenetic relationship among different strains of Pleurotus species based on DNA fingerprintings by URP-PCR analysis. I, II, III, P. ostreatus; IV, P. florida; V, P. abalonus; VI, P. sajor-caju; VII, P. eryngii; VIII, P. nebrodensis; IX, P. salmoneostramineus; X, P. citrinopileatus; a, Weonhyeong similar strains; b, Wangheukpyeong similar strains; c, Suhan similar strains; d, Chunchu-2 similar strains. w ù 100% p t ùkû, wù t w t ƒ. w, t j w x ùk ù, w m 2y, x, s m ƒ. URP-PCR x m ü k t t w p t group ½ (2007) ew. k 81t w URP-PCR x, s, w, 2y t ƒ 100% ƒà ùkù t 36 w URP band pattern w t p y wš w. Fig primer x t (Ix group) w 2180, 2183 band, URP3 primer 700 bp, URP4 primer 1100 bp, URP7 primer 1400 bp x t p band. s t (Ix group) w 2826, 2729, 2731, 2732, 2733 t URP3 primer 300 bp

7 136 서경인 등 Fig. 3. Comparison of URP-PCR fingerprinting patterns of P. ostreatus strains which showed very close relationship amplified by primers URP1 (A) to 12 (L). Molecular weight marker are 1 kb DNA ladder (Bioneer). Lanes 1~36, ASI 2794, 2795, 2826, 2729, 2731, 2732, 2344, 2708, 2487, 2711, 2719, 2180, 2183, 2595, 2732, 2733, 2828, 2504, 2829, 2825, 2721, 2724, 2734, 2796, 2797, 2549, 2795, 2598, 2709, 2707, 2788, 2789, 2794, 2737, 2727, 2718; 1, Suhan similar strains; 2, Wangheukpyeong similar strains; 3, Chunchu-2 similar strains; 4, Weonhyeong similar strains. Arrow head indicates specific bands to Suhan similar strains, arrow to Wangheukpyeong ones, circle to Chunchu-2 ones, and box to Weonhyeong ones. 한 단일 band가 나타나 이들 왕흑평 유사 품종들을 구분 하는 유용한 marker가 될 수 있을 것으로 사료되었다. 춘 추2호 품종군(III형 group)에 속하는 2344, 2487, 2708, 2711, 2719 품종은 URP1, URP2 primer에서 약 800 bp, URP5, URP10 primer에서 약 750 bp, URP12 primer에서 는 약 1100 bp 부분에서 특이 band를 찾을 수 있었다. 또 한 수한과 동일한 band pattern을 보인 품종은 2829, 2828, 2825, 2598, 2707, 2709, 2718, 2721, 2727, 2734, 2794, 2795, 2796, 2797로서 URP3 primer 약 400 bp 부 분에서 다른 품종들과 확실히 구분되는 뚜렷한 단일 band 를 볼 수 있었다. 본 연구결과 같은 band pattern을 보인 품종들은 DNA 수준에서 상당한 공통점을 갖고 있는 것 으로 볼 수 있으며, 이러한 품종들이 동일균주일 가능성 도 있다고 판단되었다. 또한 본 연구에서와 같이 URP primer를 사용한 DNA 핵산지문법을 이용함으로써 느타 리 유통품종을 구분할 수 있으며 이를 토대로 품종을 구 분하는 유용한 marker를 개발할 수 있을 것으로 생각되 었다.

8 Universal Rice Primer(URP) w DNA w w k m t 137 k Ÿ w s w w w ƒwš w ƒ ƒwš. ü š k t q š e ³ wš, t p yw ƒ y ƒ, t š. w ü mt wš j m ƒ y ƒ. DNA x w w y w š, t š w» š, t d txx p w y ƒ š w. k k 8 81 t xk ƒ ƒ y w t ƒ yw t š. w w wš ü Á m š k 81 t DNA w w w 10 ù, k w 70t 3 ù. w ü ü t x, w, 2y, s w ù w ùk ù t. I w 35 t xt w ù t 4 t š, s 6 t w ùk û. II 23 t 21 t w. w II 12 t 2y w t 6. š x ½, y,, x,,, URP-PCR x w ü k t p. Kor. J. Mycol. 35:61-67., ½,, ½, x k w ³ Hypocrea sp. xk URP-PCR. Kor. J. Mycol. 30:86-94., œ,,,, xy w. pp 488. In: 2008 y w z. w». Kang, H. W., Park, D. S., Go, S. J. and Eun, M. Y Fingerprinting of diverse genomes using PCR with Universal rice primers generated from repetitive sequence of Korea weedy rice. Korean Society for Molecular and Cellular Biology 13: Kang, H. W., Park, D. S., Park, Y. J., You, C. H., Lee, B. M., Eun, M. Y. and Go, S. J Genomic differentiation among oyster mushroom cultivars released in Korea by URP-PCR fingerprinting. Mycobiology 29: Peberdy, J. F., Hanifah, A. M. and Jia, J. H New perspectives on the genetics of Pleurotus. In: Mushroom biology and mushroom products. The Chinese University Press. Chang, S. T., Buswell, J. A. and Chiu, S. W. Eds. pp Seo, H. W., Yi, J. Y., Cho, H. M., Park, Y. E. and Oh, S. E Discrimination of potato varieties by random amplified polymorphic DNA analysis. Kor. J. Hort. Sci and Technol. 19: Sokal, R. R. and Sneath, P. H. A Principle of numerical taxonomy. Freeman, W. H. and Company, San Francisco (on disk). pp. 359.

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