Paper Para Juan Carlos

7/22/2019 Paper Para Juan Carlos

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Plant Molecular Biology Reporter15 (1) 1997 pages8-15

Commen t a r y

M o d i f ic a t i o n o f a C T B D N E x tr ac tio nP r o t o c o l f o r P l a n ts C o n t a i n i n g H i g h

P o ly sa c ch a r id e a n d P o l y p h e n o l C o m p o n e n t sSue Por eb sk i L . G r a n t B a i l ey a n d Be r n a r d R . B a um

Agriculture Canada, Eastern Cereal and Oilseed Research Centre, Ottawa,Ontario, K1A 0C6, CanadaKe y Wo r d s Fragaria DNA extraction, RAPD, strawberryAbstract A relatively quick, inexpensive and consistent protocol for extractionof DNA from expanded leaf material containing large quantities of polyphenols,tannins and polysaccharides is described. Mature strawberry leaves, whichcontain high levels of these secondary components, were used as a study group.The method involves a modified CTAB extraction, employing high salt concen-trations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) toremove polyphenols, an extended RNase treatment and a phenol-chloroformextraction. Average yields range from 20 to 84 ~tg/g mature leaf tissue for bothwild and cultivated octoploid and diploidFragariaspecies. Results from 60 plantswere examined, and were consistently amplifiable in the RAPD reaction with aslittle as 0.5 ng DNA per 25-~tL reaction. Presently, this is the first procedure forthe isolation of DNA from mature strawberry leaf tissue that produces consistentresults for a variety of different species, both octoploid and diploid, and is bothstable and PCR amplifiable before and after extended storage. This proceduremay prove useful for other difficult species in the family Rosaceae.D NA extraction from berry plants and other plants containing)sticky and resinous materials has been difficult in the past(Webb Knapp, 1990; Varadarajan Prakash, 1991; Fang et al.,1992; Collins et al., 1992). In strawberries, like other berry plants, thepresence of secondary metabolites, such as polyphenols, tannins, andpolysaccharides, inhibit enzyme action. Polysaccharides are visuallyevident in DNA extracted by their viscous, glue-like texture and make

Abbreviat ions CTAB,hexadecyltrimethylammonium romide; EtOH, ethanol;PVP, polyvinylpyrrolidone.


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D N A E x t r a c t i o n i n P r esen ce o f P o l ysa cch a rid es a n d P o lyp h en o l st h e D N A u n m a n a g e a b l e i n p ip e t ti n g a n d u n a m p l i f ia b l e in t h e p o ly -m e r a s e c h a i n r e a c t i o n (P C R) b y i n h ib i ti n g T a q p o l y m e r a s e a c t iv i ty ( F a n ge t al., 19 92 ). W h e n y o u n g , e x p a n d i n g l e a f a n d s h o o t m a t e r i a l i s l i m i t e d o rt h e p l a n t i s n o t u n d e r g o i n g a c t iv e s h o o t e l o n g a t i o n a t t i m e o f c o l le c t io n ,D N A p u r i t y b e c o m e s i n c re a s i n g l y d if fi cu l t. D N A o b t a i n e d f r o m m a t e -r ia l p a s t t h e b u d d i n g s t ag e h a s b e e n s h o w n t o b e d i ff ic u l t t o e x tr a c t a n du n s t a b l e f o r l o n g - t e r m s t o r a g e ( L o d h i e t a l., 1 99 4). W i t h m a t u r i t y , l e a v e sc o n t a i n i n c r e a s e d q u a n t it ie s o f p o l y p h e n o l s , t a n n i n s , a n d p o l y s a c c h a -r id e s . D e a l i n g w i t h s u c h c o m p o n e n t s i n m a t u r e l e a v e s b e c o m e s n e c es -s a r y w h e n y o u n g e r e x p a n d i n g l e a ve s a n d s h o o ts a r e n o t a v a il ab le d u r i n gt he t i m e o f co ll ec ti on .W e w e r e u n i f o r m l y u n s u c c e ss f u l i n o u r a t t e m p t s t o a m p l i f y Fragar iaD N A b y P C R u s i n g o t h e r r e p o r t e d m e t h o d s , i n c l u d i n g th o s e o f D e l l a p o rt ae t a l . (1984), Sag ha i -M aro of e t al . (1984), Do yle & D oy le (1990) , La Ro ch e(1992) , Oard & Dronaval l i (1992) , Wang e t a l . (1993) , R ichards e t a l .(1994), and D av i s e t al. ( 1995 ). A s a r e s u l t , w e f o un d i t nece s s a r y to d ev i s ea p r o to c o l f o r D N A e x t r a c ti o n f r o m f u l l y d e v e l o p e d e x p a n d e d l e a f t is s u et h a t w o u l d y i e l d D N A s u it a b le f o r P C R , e s p ec i a ll y f o r R A P D s , a n d t h a tw o u l d b e c o n s i st e n t f o r m a n y d i f f e r e n t w i l d a n d c u l t iv a t e d s p e c ie s o fs t ra w b e r r i e s, b o t h d i p l o i d a n d o c t o p lo i d . T h e p r o t o c o l d e s c r i b e d h e r e isr e l a ti v e l y q u i c k a n d i n e x p e n s iv e , a n d p r o v i d e s c l e a n D N A , c o n s i s te n t lya m p l i f i a b l e i n p o l y m e r a s e c h a i n r e a c t i o n u s i n g t h e R A P D t e c h n i q u e(Wil l iam et a l . , 1990) for a l l Fragar ia s p p . a s s a y e d . Fragar ia t is s u e a n dD N A s t o r e d fo r 21 m o n t h s a t -2 0 ~ h a s a ls o b e e n s h o w n t o b e s ta b le a n dampl i f i ab l e .

M a t e r i a l a n d M e t h o d sS o l u t i o n s R e q u i r edc h l o r o fo r m : o c t a n o 1 2 4 : 1 ( v / v )5 M N aC 1TE bu f fer 10 m M tris -H C 1, i m M E D T A , p H 8 .4R N a se A : 10 m g / m Lp r o te i n a se K : 1 m g / m L 1c h l o r o f o r m - p h e n o l s a t u r a t e d i n TEpo l yv i ny l py r r o l i d on e ( PV P) S i gm a P - 9003-39-8 (40,000 )

extract ion buf fer 100 m M t r is , 1 .4 M NaC1, 20 m M ED TA , p H 8 .0 , 2%C T A B , 0 .3 % ~ - m e r c a n t o e t h a n o l2PCR react ion bu f fer 100 m M t ri s-HC1, 100 m M KC1, p H 8 .3p r i m e r : s et 8 U B C , U n i v e r s i t y o f B r i ti sh C o l u m b i aS to ff el f r a g m e n t : r e a g e n t s s u p p l i e d b y P e r k i n E l m e r


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10 P o r e b sk i B a i le y a n d B a u mNotes1. M ade fresh befo re use.2 . A dd ed jus t before use.

S i x ty s t r a w b e r r y p l a n t s o f b o t h w i l d a n d c u l t i v a te d C a n a d i a n o c t o p -l o i d s p e c i e s (2 n = 5 6 ) i n c l u d i n g F. ch iloensis (L.) Dc ne. , F . v i r g i n i ana D c n e ,F . v i r g i n i ana f o r m a m ul t i c i p i t a ( F e rn . ) C a t l i n g & C a y o u e t t e , F. X ananas s anm . c une i f o l i a ( N u t t . ) S t a u d t , a n d o n e d i p l o i d s p e c i e s (2 n = 14 ), F. vescaL ., w e r e u s e d , 5 -1 2 p l a n t s p e r s p e c i e s . T h e m o d i f i c a t i o n o f th e C T A Be x t r ac t io n p r o c e d u r e o f D o y l e & D o y l e (1 99 0) i n c l u d e s h i g h N a C 1 c o n -c e n t r a t i o n s i n t h e b u f f e r t o r e m o v e p o l y s a c c h a r i d e s ( L o d h i e t a l., 19 95 ),a n d p o l y v i n y l p y r r o l i d o n e ( PV P ) t o r e m o v e p o l y p h e n o l s ( M a l i y ak a l ,19 92 ). A n e x t e n d e d , o n e - h o u r R N a s e t re a t m e n t w a s r e q u i r e d f o r t h eD N A p r o d u c t t o b e R N A f re e a n d P C R a m p l if ia b le , w i t h a n a d d i t i o n a lp h e n o l - c h l o r o f o r m s t e p ( S a g h a i - M a r o o f e t a l . , 1 9 8 4 ) t o r e m o v e a n ye x c e s s p r o t e i n s . T h e s e s te p s p r o v i d e t h e p r i n c i p a l k e y t o s u c c e s s f o rc o l le c ti n g D N A f r o m m a t u r e s t r a w b e r r y l e a v e s.

E x a m i n a t io n o f p o l y m o r p h i s m t h r o u g h u s e o f p r i m e r s w i t h a r b it ra r ys e q u e n c e s ( R A P D ) w a s d o n e s u c c e s s f u l l y w i t h 16 d i f fe r e n t p r im e r s a f te ri n it ia l s c r e e n i n g o f a p p r o x i m a t e l y 5 0 p r i m e r s . T h e 2 5 ~tL v o l u m e P C Rr e a c t io n s c o n t a i n e d P C R r e a c t io n b u f f e r , 2 m M M gC 12, 0.1 m M d N T P s ,2 .4 m M p r i m e r , 2 u n i t s o f S t of fe l f r a g m e n t , I n g D N A , a n d w e r e p l a c e di n a M J R e s e a r c h I n c . P T C 1 0 0 T h e r m o c y c l e r . T h e t h e r m a l c y c l i n g i n -c l u d e d o n e c y cl e a t 5 m i n u t e s o f d e n a t u r a t i o n a t 9 4 ~ o n e m i n u t ea n n e a l i n g a t 4 7 ~ t w o m i n u t e s o f p r i m e r e x t e n s io n a t 7 2 ~ t h e n 3 5c y c l e s o f o n e m i n u t e e a c h a t 9 4, 4 7, a n d 7 2 ~ w i t h m i n i m u m r a m p t im e .P C R a m p l i c o n s w e r e t h e n r u n o n a 1.5 % 3 - t o 4 - ra m t h i n a g a r o s e g e l f o rb e s t r e s u l t s .

T h e P r o t o c o l

D N A E x tr a ct io n

9 C o l l e c t l e a v e s o f F ragaria f r e e z e in l i q u i d n i t r o g e n , a n d s t o r e a t -7 0~ u n t i l u se . G r i n d 0 .5 g o f l e a v e s u s i n g m o r t a r a n d p e s t l e i n t h ep r e s e n c e o f l i q u i d n i t r o g e n u n t i l fi n e l y g r o u n d . T r a n s f e r f r o z e ng r o u n d l e af t is s u e to 1 5 - m L p o l y p r o p y l e n e c e n t r i fu g e t u b e s .9 A d d 5 m L o f 6 0 ~ e x t r ac t i o n b u f f e r a n d 5 0 m g P V P / 0 . 5 g le a ft is s u e. M i x b y i n v e r s io n a n d i n c u b a t e in 60 ~ o v e n ( w i t h s h a k in g )f o r 2 5 to 6 0 m i n u t e s .


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D N Extrac t ion in Presence of Polysaccharides and Polyphenols 119 R e m o v e f r o m h e a t , a n d l et c o o l t o r o o m t e m p e r a t u r e f o r 4 to 6m i n u t e s .9 A d d 6 m L o f c h l o r o f o r m : o c t a n o l (2 4:1 ) a n d m i x b y i n v e r s i o n t of o r m a n e m u l s i o n .9 A f t e r m i x i n g t h o r o u g h l y , s p i n a t 3 0 00 r p m f o r 2 0 m i n u t e s i n at a b le t o p c e n t r i fu g e a t r o o m t e m p e r a t u r e .9 T r a n s f e r t o p a q u e o u s s o l u t io n t o n e w 1 5 -m L c e n t r i fu g e t u b e s u s i n gw i d e - b o r e p i p e t t e t ip . R e p e a t c h l o r o f o r m - o c t a n o l e x t r a c t i o n t or e m o v e c l o u d i n e s s ( P V P ) in a q u e o u s p h a s e . 19 A d d 1 / 2 v o l u m e o f 5 M N a C 1 to t h e f in a l a q u e o u s s o l u t io nr e c o v e r e d . 2 M i x w e l l. A d d t w o v o l u m e s o f c o l d ( -2 0 ~ 9 5 %e t h a n o l . M i x b y i n v e r s i o n . I f r e q u i r e d , p l a c e i n f r e e z e r ( -2 0 ~ f o r1 0 m i n u t e s t o a c c e n t u a t e p r e c i p i t a ti o n . T h e s o l u t i o n m a y b e l e f t a t4 t o 6 ~ t o p r e c i p i t a t e o v e r n i g h t .9 S p i n a t 3 0 00 r p m f o r 6 m i n u t e s . 39 P o u r o f f s u p e r n a t a n t , a n d w a s h p e l l e t w i t h c o l d (0 t o 4 ~ 7 0 % v /v e th a n o l . D r y p e l le t i n 3 7 ~ o v e n o r v a c u u m u n t i l d r y ( ~1 h o u r ) .9 D i s s o l v e i n 30 0 ~tL T E o v e r n i g h t a t (4 t o 6 ~ 4 T r a n s f e r t o 1 . 5 - m LE p p e n d o r f t u b es , s9 A d d 3 ~tL R N a s e A (1 0 m g / m L ) a n d i n c u b a t e in 3 7 ~ w a t e r b a t hf o r a p p r o x i m a t e l y I h o u r . A d d 3 ~tL p r o t e i n a s e K ( l m g / m L ) , a n di n c u b a t e a t 3 7 ~ f o r 1 5 t o 3 0 m i n u t e s .9 A d d 1 50 ~tL o f p h e n o l a n d 1 50 ~tL o f c h l o r o f o r m t o e a c h E p p e n d o r ft u b e .9 V o r t e x b r i e f ly , a n d s p i n ( in m i c r o f u g e ) a t 1 4 ,0 00 r p m f o r 1 0 t o 1 5m i n u t e s . C o l l e c t u p p e r l a y e r i n n e w 1 .5 - t ub e . A d d 5 0 ~ L T E top h e n o l p h a s e . V o r t ex , s p in , r e m o v e u p p e r l a ye r , a n d a d d t o sa m p l e .9 A d d 1 / 1 0 v o l . 2 M N a a c e t a te a n d 2 v o l . a b s o l u t e e t h a n o l a n d m i x .9 L e a v e o v e r n i g h t i n f r e e z e r ( -8 0 ~ S p i n a t 1 4 ,0 0 0 r p m f o r 10 t o 2 0

m i n u t e s . D r a i n a n d w a s h w i t h 7 0% v / v E t O H . R e m o v e E t O H .9 V a c u u m - d r y t u b e s. A d d 1 00 t o 2 00 ~tL T E . A l l o w t i m e f o r c o m p l e t er e s u s p e n s i o n . 69 D N A c o n c e n t ra t i o n s w e r e m e a s u r e d u s i n g a H o e f e r Q u a n t a 2 00F l u o r o m e t e r a n d 0 .5 n g D N A d i l u t i o n s w e r e p r e p a r e d f o r P C R u s e .o t e s1. B e c a r e fu l to r e m o v e o n l y t o p a q u e o u s s o l u t i o n a n d n o t w h i t e b a n d a t i n te r fa c e .( R e p e at th i s s te p u n t i l n o l o n g e r c l o u d y d u e t o t h e p r e s e n c e o f P V P b o u n dpo lysa c c ha r ide s . Twic e i s u sua l ly su f f i c ie n t .

2 . I n som e p la n t s a s e c o nd p r o c e d ur e o f s a lt p r e c ip i t a t ion m a y be u se f u l .3 . DN A pe l l e t s shou ld no w b e v i s ib le i n a ll tube s .4 . W h e n d i s so l v e d , s o m e D N A m a y s ti ll b e s l i g h tl y v i sc o u s b u t e a s il y p i p e tt e d .5 . P r od uc t s a t t h i s po in t i n t he p r o toc o l a re u sua l ly no t P C R a m pl i fi a b l e.6 . R e c o m m e n d e d o v e r n i g h t a t 4 ~


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12 P o r e b s ki B a i l e y a n d B a u mR e s u l t s a n d i s c u s s i o n

D N A c o n c e n t r a t io n s w e r e m e a s u r e d f o r t h e s ix t y s a m p l e s , a n d t h ea v e r a g e y i e l d s a n d r a n g e s i n ~ t g / g l e a f t i ss u e a r e :F. vesca 20 10-30F . m u l t i c i p i t a 30 15-45F . v i r g i n i ana Ga sp6 20 10-25P e t a w a w a 8 4 2 0 -1 2 0F. ch i loens i s V.I. 71 35 -90B.C. 52 10 -90F . a n a n a s s a w i l d 32 30 - 45cu l t i v a t ed 28 15 - 45PC R w as s ucce s s f u l u s i n g q uan t i t i e s a s s ma l l a s 0.5 ng i n a 25-~ tL

r ea c ti on . W h e n a m o u n t s w e r e i n c re a s e d b e y o n d 5 n g , D N A b a n d sb e c a m e le ss d is ti nc t . A l t h o u g h s e c o n d a r y m e t a b o l i te s m a y n o t b e c o m -p l e t e l y r e m o v e d , t h e D N A t e m p l a t e w a s p u r e e n o u g h t o a l l o w P C Ra m p l i fi c at io n . In a d d i t io n , s u c h D N A y i e l d s a r e l a r g e e n o u g h t o p e r m i ta s m a n y a s 1 .4 x 105 P C R r e a c t i o n s o r a n u m b e r o f R F L P a p p l i c a ti o n s .

T h i s D N A e x t r a c t i o n p r o t o c o l i s r e l a ti v e l y fa s t, s in c e a l e n g t h y u l t r a -c e n t r i f u g a t i o n is n o t r e q u i r e d , a s i n s o m e o t h e r P V P p r o t o c o l s M a l iy a k a l ,1 99 2). P V P , a s o li d p o l y m e r t h a t h a s h i g h m o l e c u l a r w e i g h t a n d i s w a t e r -s o lu b l e a n d c h e m i c a l ly i n e rt , w a s s h o w n t o r e m o v e t h e p o l y p h e n o l s ,w h i l e m a i n t a i n i n g a h i g h e r y i e ld t h a n p o l y v i n y l p o l y p y r r o l i d o n e P V P P),a n i n s o l u b le c r o s s- li n k in g p o l y m e r . P V P f o r m s a c o m p l e x w i t h p o l y p h e -n o l s t h r o u g h h y d r o g e n b o n d in g , a l l o w in g t h e m t o b e s e p a r a t e d f r o m t h eD N A , a n d r e d u c i n g le v e ls o f p o l y p h e n o l in t h e p r o d u c t M a l i ya k a l,1 992 ). A l t h o u g h P V P P is u s e f u l i n i m p r o v i n g s ta b i li ty o f e n z y m e s b yr e m o v i n g p h e n o l i c i m p u r i t i e s S i g m a C h e m i c a l C o ., 19 93 ), i t y i e l d ss ig n i fi c a n tl y l es s D N A t h a n P V P f r o m f u l ly e x p a n d e d l ea v e s.T h e t e c h n i q u e o f L o d h i e t a l. 1 99 4) u t i li z i n g P V P P g a v e v e r y l o w D N Ay i e l d s , 2 t o 3 ~ t g / g l e a f t i s s u e , w i t h f u l l y e x p a n d e d l e a v e s , a n d P C Ra m p l i f ic a t i o n w a s n o t c o n s i s t e n t l y s u c c e s s f u l fo r a ll s t r a w b e r r y s p e ci e s.E v e n w h e n y o u n g e x p a n d i n g l e av e s a n d s h o o t m a t e r ia l w e r e e x a m i n e du s i n g t h is p r o t o c o l , y i e l d s r e p o r t e d b y L o d h i e t a l. 1 99 4) fo r o t h e r b e r r yp l an t s 546 -1130 ~ t g / g ) w er e no t r eac hed .Th e m e th o d o f Jobes e t a l . 1995) u t i l i z in g PVP , NaC1, L iC1, an d SDS ist i m e c o n s u m i n g . W e w e r e a b l e t o o b t a i n e q u i v a l e n t r e s u l t s w i t h t h ee x c l u s i o n o f t h e S D S c le a n i n g a n d L iC 1, w h i c h i s u s e d f o r R N A r e m o v a l


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D N A Extrac t ion in Presence o f Polysaccharides and Polyphenols 13

Fig. 1. PCR amplimers on a 1,5 agarose gel with primer UBC8-751. left toright). K + O, ~ digested wit h Hind III+ ~ X 174 digested w ith Hae III; vesca F. vescaL.; virginiana-multicipita F. virginiana forma multicipita (Fern.) Cafling & Cay oue tte- from Gasp6; virginiana Gasp6, F. virginiana Dcne., from Gasp6; virginianaPetawawa, F. virginiana Dcne., from Petawawa; chiloensis sVI, F. chiloensis (L.)Dcne., fro m S. Vanc ouver Island; chiloensis mBC, F. chiloensis (L.) Dcne., from mi dBC coast; ananassa wild, X F. ananassa rim. cuneifolia (Nutt.) Staud t, f rom V.I., wild;ananassa cult, F. ananassa rim. cuneifolia (Nutt.) Staudt, f rom V.I., cultivated.


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14 Porebski Bai ley and Bauma n d t o p r e v e n t r e s i d u a l r ib o n u c l e o s i d e s fr o m a c t in g a s p r i m e r s i n t h et h e r m a l r e a c t i o n (S to rt s, 19 93 ). W e f o u n d a n e x t e n d e d R N a s e t r e a t m e n to f i h o u r w a s s u ff ic i e n t t o d e g r a d e t h e R N A i n to s m a l l r i b o n u c l e o s id e st h a t w e r e n o t d e t e c t a b le b y g e l e le c t ro p h o r e si s. R e m a i n i n g r i b o n u c l e o -s id e s w o u l d b e in v e r y sm a l l a m o u n t s , a n d w o u l d h a v e t o c o m p e t e w i t hl a r g e q u a n ti ti e s o f a d d e d p r i m e r s i n t h e R A P D r e a ct io n , m a k i n g i n t e r fe r -e n c e b y r i b o n u c l e o s id e p r i m e r h i g h l y u n l i k e ly .

O t h e r p r o t o c o l s p r o v e d u n s u c c e s s f u l i n r e m o v i n g s e c o n d a r y i n h ib i t-i n g c o m p o n e n t s i n all s p e c ie s te s te d , in c l u d i n g a N a O H t e c h n i q u e ( W a n ge t a l. , 1993), a p r o t oc o l d ev e l o pe d f o r ma i ze ( D e l l apo r t a e t a l. , 1984; O ar dDro nav al l i , 1992), a CsC1 pu r i f i c a t io n (R ichard s e t a l ., 1994), the E lu-Q u i k ( S c h e ic h e r S c h u e l l) m e t h o d ( L a R o c h e , 1 99 2), a n d v a r i o u s C T A Bex t r ac t i on s ( D oy l e D o y l e , 1990; Sa gha i - M ar oo f e t a l., 1984; D av i s e t a l. ,1995)).U n l i k e a ll o t h e r p r o t o c o l s m e n t i o n e d a b o v e , o u r r e l a t i v e l y f a st , c o n s is -t e n t p r o t o c o l p r o d u c e d v e r y a c c e p t a b l e D N A y i e l d s f r o m e x t r a c t e dm a t u r e l e a f t is s u e f o r a ll Fragaria s p e c i e s a s s a y e d . A l l D N A w a s s ta b l ea n d c o u l d b e a m p l i f i e d b y P C R , r e q u i r i n g o n l y 0 . 5 n g D N A i n 2 5 - ~ t Lr e a c t io n s , b o t h b e f o r e a n d a f t e r 21 m o n t h s o f st o r a g e . I n a d d i t i o n , R A P Dp r o fi le s w e r e s h o w n t o b e r e p r o d u c i b l e f o r a t l e as t fi v e p l a n t s e x a m i n e df r o m e a c h l o c a t i o n fo r e a c h s p ec i es . T h is t e c h n i q u e h a s p o t e n t i a l t o b e a ne f f e c ti v e p r o t o c o l f o r D N A e x t r a c t i o n u s i n g m a t u r e l e a f t i ss u e f o r s t r a w -b e r ri e s, a n d p e r h a p s f o r o t h e r s p e c i es in t h e f a m i l y R o s a c e a e w i t h h i g hp o l y s a c c h a r i d e s a n d p o l y p h e n o l s , w h e n s u f fi c ie n t y o u n g l e a f t is s u e isno t ava i l ab l e .Acknowledgments Special thanks to Paul Catl ing and Linda Bonen for theircom m ents an d to Brian Wollenschlager fo r his graphical suppo rt .

ReferencesCollins, G.G. and R.H. Symons. 1992. Extraction of nuclear DNA from grape vine leaves bya modified procedure. Plant Mol. Biol. Rept. 10:233-235.Davis, T.M., H. Yu and K.M. Haigis and P.J. McGowan. 1995. Template mixing: A methodof enhancingdetection and interpretationof codominantRAPD markers. Theor. Appl.Genet. 91:582-588.Dellaporta, S.L., J. Wood and J.B. Hicks. 1985. Maize DNA miniprep, p. 36-37, in MolecularBiology o f Plan ts . Malberg, J. Messing and I.Sussex, eds. Cold Spring Harbor Laboratory,

Cold Spring Harbor, N.Y.Doyle J.J. and J.L. Doyle. 1987. A rapid DNA isolation procedure for small quantities offresh leaf tissue. Phytochem. Bull 19:11-15.Fang, G., S. Hamrnar and R. Grumet. 1992. A quick and inexpensivemethod for removingpolysaccharides from plant genomic DNA. Biofeedback 13 1):52-54.


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D N A Ex t rac t ion in Pre se nc e o f Po ly sac c haride s and P o ly phe no l s 1 5L a Ro c h e , J . 1 99 2. A n e a s y a n d e f f i c ie n t p r o c e d u r e f o r is o l a t i n g p l a n t D N A u s in g t h e E lu -Q u ik D N A p u r i f i c a t i o n k i t. S e q u e n c e s 3 6:3- 4.L o d h i , M .A . , G . - N . Y e , N .F . W e e d e n , B .I . Re i s c h . 19 94 . A s im p le a n d e f f i c ie n t m e th o d f o rD N A e x t r a c t i o n s f r o m g r a p e v in e c u l t i v a r s a n d V i t i s s p e c i es . P l a n t M o l . B io l. Re p t . 1 2 :6 -13.M a l i y a k a l, E .J. 1 99 2 . A n e f fi c ie n t m e t h o d f o r i s o la t i o n o f R N A a n d D N A f r o m p l a n t sc o n t a in in g p o ly p h e n o l i c s . N u c l e i c A c id Re s e a r c h 2 0 :2 38 1.M auro , M .-C. , M. S t re fe le r , N.F . W eed en and B. I. Re isch . 1992 . G ene t ic ana lys is o f re s t r ic -t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m s i n Vitis J . He red i ty 83 :18-21 .O a r d , J .H . a n d S . D r o n a v a l l i . 1 9 92 . Ra p id i s o l a t i o n o f ri c e a n d m a iz e D N A f o r a n a ly s is b yran do m -pr im er PCR . P lan t Mol . Bio l. Rep t . 10:236-241.R ic h a r d s E . , M . Re i c h a r d t a n d S . Ro g e r s . 1 99 4. P r e p a r a t i o n o f g e n o m ic D N A f r o m p l a n tt issue. Curren t Protocols in Molecular Biology W ile y I n t e r s c i e n c e .S a g h a i - M a r o o f , M .A . , K .M . S o l im a n , R .A . J o r g e n s e n a n d R .W . A l l a r d . 1 9 8 4 . R ib o s o m a lD N A s p a c e r - le n g t h p o l y m o r p h i s m i n b a r le y : M e n d e l i a n i n h e r it a n c e , c h r o m o s o m a lloca t ion , and po pu la t i on dyn am ics . P roc . Na i l . Acad . Sc i. US A. 81:8014 - 8019 .S i g m a C h e m i c a l C o m p a n y . 1 99 3. BiochemicalOrganicCompounds or Research and D iagnosticReagents Sig m a Diagno s t ic s . S t. Lou is , USA . 2256 pp .V a r a d a r a j a n , G .S . a n d C .S . P r a k a s h . 1 99 1. A r a p id a n d e f f i c ie n t m e th o d f o r t h e e x t r a c t i o no f t o t a l D N A f r o m th e s w e e t p o t a to a n d i t s r e l a t e d s p e c i e s . P l a n t M o l . B io l. Re p t . 9 1 ): 6-12.W a n g , H . M . Q i a n d A . C u t le r . 1 99 3. A s i m p l e m e t h o d o f p r e p a r i n g p l a n t s a m p l e s f o r PC R .Nu cl . A cid R es. 21:4153-4154.W e b b , D .M . a n d S . J . K n a p p . 1 99 0. D N A e x t r a c t i o n f r o m a p r e v io u s ly r e c a l c it r a n t p l a n tgenus. Plant Mol. Biol . Rept . 8 :180-185.W il l iams , J.G.K. , A.R. Kube l ik , K.J . L ivak , J .A. Rafa lsk i a nd S .V. T ingey . 1990 . D N Ap o l y m o r p h i s m a m p l i f i e d b y a r b i tr a r y p r i m e r s a r e u s e f u l g e n e t ic m a r k e r s. N u c l . A c i d sRes. 18:6531-6535.

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