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w ³wz The Korean Journal of Mycology The Korean Society of Mycology Kor. J. Mycol. 36(2) : 130-137 (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 441-707, Korea 1 Department of Crop Science, KonKuk University, Seoul 143-701, Korea (Received November 11, 2008. 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 <E-mail : wskong@rda.go.kr>» 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

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 01 2851 Nongmin59 P. ostreatus Korea 2006 02 2830 Baekdu1 P. ostreatus Korea 2005 03 2829 Jangpung P. ostreatus Korea (MKACC53972) 2005 04 2828 Samgu9 P. ostreatus Korea 2005 05 2827 Sinnong26 P. ostreatus Korea 2005 06 2826 Sinnong22 P. ostreatus Korea (MKACC53909) 2005 07 2825 Sinnong14 P. ostreatus Korea 2005 08 2029 2029 P. ostreatus Korea (MKACC51372) 1978 09 2001 Nonggi2-1 P. ostreatus Korea (MKACC52243) 1971 10 2018 Nonggi201 P. ostreatus Korea (MKACC51362) 1978 11 2072 Nonggi202 P. ostreatus Korea (MKACC51410) 1980 12 2180 Wonhyeong P. ostreatus Korea (MKACC51493) 1990 13 2194 Aeneutari1 P. ostreatus Japan (MKACC51506) 1979 14 2183 Wonhyeong2 P. ostreatus Korea (MKACC51496) 1990 15 2240 Wonhyeong3 P. ostreatus Korea (MKACC52342) 1994 16 2228 Chunchu1 P. ostreatus China (MKACC51529) 1994 17 2344 Chunchu2 P. ostreatus Netherland (MKACC51632) 1995 18 2706 Heukpyeong P. ostreatus Korea (MKACC52328) 2001 19 2535 Byeongneutari1 P. ostreatus Korea (MKACC51778) 2000 20 2506 Gyunhyeop1 P. ostreatus Korea (MKACC360) 2000 21 2505 Oknong1 P. ostreatus Korea (KACC359) 2000 22 2477 Heukjinju P. ostreatus Korea (MKACC51724) 1999 23 2487 Cheongpung P. ostreatus Korea 1999 24 2488 Myeongweol P. ostreatus Korea (MKACC51732) 1999 25 2504 Suhan P. ostreatus China (KACC358) 2000

132 Table 1. Continued Lane no. ASI* no. Commercial name Species Source Year 26 2549 Sinnong94 P. ostreatus Korea (MKACC52282) 2000 27 2595 Suhan2 P. ostreatus Korea (MKACC51818) 2001 28 2596 Suhan3 P. ostreatus Korea (MKACC52325) 2001 29 2598 Sinnong46 P. ostreatus Korea (MKACC51820) 2001 30 2594 Ilseong2 P. ostreatus Korea (MKACC51817) 2001 31 2597 Sinnong8 P. ostreatus Korea (MKACC51819) 2001 32 2593 JanganPK P. ostreatus Korea (MKACC52326) 2001 33 2707 Kimjae9 P. ostreatus Korea (MKACC52311) 2001 34 2708 Kimjae10 P. ostreatus Korea (MKACC52312) 2001 35 2709 Jangan2 P. ostreatus Korea (MKACC52313) 2001 36 2710 Heungrim1 P. ostreatus Korea (MKACC52314) 2001 37 2711 Jangan3 P. ostreatus Korea (MKACC52315) 2001 38 2717 Nongmin1 P. ostreatus Korea (MKACC52320) 2002 39 2718 Kimjae7 P. ostreatus Korea (MKACC52321) 2002 40 2719 Kimjae8 P. ostreatus Korea (MKACC52322) 2002 41 2721 Jangan5 P. ostreatus Korea 2002 42 2722 Jangan6 P. ostreatus Korea (MKACC52324) 2002 43 2724 Nongong99 P. ostreatus Korea 2003 44 2725 DH1012 P. ostreatus Korea 2003 45 2726 Sinnong11 P. ostreatus Korea 2003 46 2727 Sinnong12 P. ostreatus Korea 2003 47 2728 Sinnong13 P. ostreatus Korea 2003 48 2729 Cheongdo21 P. ostreatus Korea 2003 49 2730 Cheongdo22 P. ostreatus Korea 2003 50 2731 Wangheukpyeong P. ostreatus Korea 2003 51 2732 Nongong98 P. ostreatus Korea 2003 52 2733 Chiak3 P. ostreatus Korea 2003 53 2734 Chiak4 P. ostreatus Korea 2003 54 2735 Bupyeongkirin2 P. ostreatus Korea 2003 55 2736 Bupyeongheukdan4 P. ostreatus Korea 2003 56 2737 Sodam P. ostreatus Korea 2003 57 2738 Heukbaek P. ostreatus Korea 2003 58 2785 Bupyeongsoyeop1 P. ostreatus Korea 2004 59 2786 Bupyeongbokhoe P. ostreatus Korea 2004 60 2788 Jangan7 P. ostreatus Korea 2004 61 2789 Samguhwanghak P. ostreatus Korea 2004 62 2790 SamguPJ P. ostreatus Korea 2004 63 2791 Samgu01 P. ostreatus Korea 2004 64 2794 Chiak5 P. ostreatus Korea 2004 65 2795 Chiak7 P. ostreatus Korea 2004 66 2796 Samgu5 P. ostreatus Korea 2004 67 2797 Samgu8 P. ostreatus Korea 2004 68 2787 Yeongnong1 P. ostreatus Korea 2004 69 2792 Hanra1 P. ostreatus Korea 2004 70 2793 Hanra2 P. ostreatus Korea 2004 71 2016 Sacheol P. florida Germany (MKACC51361) 1976 72 2181 Sacheol2 P. florida Thailand (MKACC51494) 1990 73 2070 Yeoreum P. sajor-caju India (MKACC52247) 1982 74 2333 Yeoreum2 P. sajor-caju Korea (MKACC51621) 1995 75 2479 Sambok P. sajor-caju Korea (MKACC52343) 1999 76 2079 Jeonbok1 P. abalonus Thailand (MKACC50312) 1982 77 2302 Keunneutari1 P. eryngii Japan (MKACC51595) 1995 78 2394 Keunneutari3 P. eryngii Japan (MKACC52327) 1997 79 2720 Baeksongi P. nebrodensis Korea (MKACC52323) 2002 80 2859 Noeul P. salmoneostramineus Korea 2007 81 2858 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

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.

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 2070 2139 ³ 2333 2070 65%. URP-PCR ƒƒ k groupü

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. 3 12 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

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.

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