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Biochemical Genetics, Vo l. 34, Nos. 7/8, 199 6

E x t e n s iv e C h r o m o s o m e A b e r r a t i o n s i n Spiroplasmacitri S t r a in B R 3F e n g c h u n Y e , 1 U l r i c h M e l c h e r , 2 J o h n E . R a s c o e , 1a n d J a c q u e l i n e F l e t c h e r 1,3Received 9Jan . 1996 - -Fina l lO May 1996

Genetic variations in the pl an t pathogen, Spi ropla sma c i t r i strain BR3, werecharacterized through physical genom e ma pping o f the original isolate , BR3 -3X,an d two d erivatives, BR 3- T a nd B R 3- G, obtained after several years o f differentma intena nce conditions. B R 3 -T w as transmitted fro m pla nt to pla nt via itsnatural insect vector, the leafhopper Circu l i f e r t ene l lus , whi le BR3-G wasm ain tain ed only in pla nts by periodic grafiing an d has lost its ability to be insecttransmitted. By pu lsed f ie ld gel electrophoresis (PF G E) analysis a nd D N Ahybridization, extensive changes in chr om oso m al D N A restriction patternsrelative to the parent, BR 3-3X , were observed in both B R3 - T and B R3 -G, each o fwhich also ha d a larger gen om e size than the paren t l ine. Genetic organizationwas relatively conserved between BR3-T and BR3-3X. In contrast , a largechro m oso ma l inversion a nd deletions o f approximately 10 kb near each o f theinversion borders we re observed in BR 3-G . O ne o f the deletions, w hich includedseveral possibly fun ction al g enes, was closely l inked to a SpV l-related trans-posa se gene . The locations o f the deletion borders we re also determined. Theresults o f this stu dy de m on stra ted remarkable genom e instability o f spiroplasmas.KEY WORDS: Spiroplasma citri; chromosome aberrations; SpVl-like sequences; transposaseand insect transmission.

I N T R O D U C T I O NSpi ropla smas a re wa l l - l e s s p roka ryote s (Mol l i cu te s ) hav ing mot i l i ty and ahe l ic a l mor pho l ogy . L i ke o t h e r m e m be r s o f t he c la ss M ol l i cu t e s, t he y a r e1 Department of Plant Pathology, Oklahoma State University, Stillwater, Oklahoma 74078.2 Departmen t of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater,Oklahoma 74078.3 To whom correspondence should be addressed.

2690006-2928/96/0800-0269509.50/0 1996 PlenumPublishingCorporation

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am o n g t h e sm a l l e s t an d s i m p l e s t o r g an i sm s . N ev e r t h e l e s s , t h ey a r e n o tp r i m i t i v e p r o k a r y o t e s b u t , ap p a r en t l y , w e r e d e r i v ed f r o m G r am - p o s i t i v eb ac t e r i a t h r o u g h su cces s iv e g en o m e r ed u c t i o n ( R az i n , 1 98 9) an d o t h e rg en e t i c ch an g e s su ch a s a r ed u ce d (2 5 t o o l % ) G + C co n t en t . T h e g en o m er ed u c t i o n i n t h e se o r g an i sm s m a y h av e l ed t o t h e e l i m i n a t io n o f g en esi nv o lv e d i n D N A r e p a i r s y s te m s th a t a r e c o m m o n l y f o u n d i n o t h e r b a c t e r i aan d a co n seq u en t h ig h f r eq u en cy o f g en e t i c m u t a t i o n i n m o l l i cu t e s (R az i n ,1989). Re m ark ab le ge ne t i c var i a t ion , pa r t i cu la r ly var i ab le ge no m e sizes , hasb e e n f o u n d i n m y c o p l a sm a s ( C h r i s ti a n s e n e t a l . , 1987), and a r ec en t su rveyi n d i ca t ed t h a t t h e g en o m e s izes o f sp i ro p l a sm a sp ec ie s a l so v a r y f r o m 9 90 t o2200 k i lobase pa i r s (kb) (Car le e t a l . , 1995). H ow ever , s ign if ican t gen e t i cv a r i a t io n w i t h i n a s in g l e iso l a te o f sp i ro p l a sm a o r g en e t i c ch an g es l i n k ed t o ap a r t i cu l a r p h en o t y p i c sw i tch h av e n o t y e t b e en r ep o r t ed .I n o u r l ab o r a t o r y , S . c i t r i s t r a i n B R 3 w as i so l a t ed f r o m h o r se r ad i shp l an t s w i t h b r i t t l e r o o t d i s ea se ( F l e t ch e r e t a l . , 1981). This isolate, t r iplyc l o n ed an d d e s i g n a t ed B R 3 - 3 X , w as m a i n t a i n ed i n s ev e ra l d i f f e r en t w ay sover a 10-year per iod . A BR3-3X der iva t ive ce l l l i ne , des igna ted BR3-G,o b t a i n ed t h r o u g h e x t en d ed p l an t - t o -p l an t t r an sm i s s i o n o f t h e o r ig i n a l iso-l a t e by g ra f ting , und erw en t a ph eno typ ic swi tch , los ing it s insec t t r ansmiss i -b i l i t y ( Way ad an d e e t a l . , 1 99 3). D u r i n g t h i s s am e t i m e p e r i o d , t h e B R 3 - 3 Xi so la t e w as a lso m a i n t a i n ed b y r ep e a t ed t r an sm i s s io n f r o m t u r n i p t o t u r n i pv i a it s n a t u r a l i n sec t v ec t o r , t h e l e a f h o p p e r C i r c u I i f e r t e n e l l u s . T h e r e i so l a t edS . c i t r i , des ig na ted B R3-T , w as still i nsec t t ransm iss ib le and l ac ked obv iousp h en o t y p i c ch an g es . I n t h i s s t u d y , w e r ep o r t t h e p h y s i ca l m ap p i n g o f t h eg e n o m e s o f S . c i t r i s t ra i n B R 3 an d i t s d e ri v a ti v e s an d d o c u m en t f o r t h e f i rs tt im e s ign i fi can t gen e t i c var i a t ions wi th in the sam e i so la te o f sp i rop lasmasf o ll o w in g m a i n t en an c e u n d e r d i f f e r en t co n d i ti o n s .

M A T E R I A L S A N D M E T H O D SPu lse d F ie ld Ge l E le c tr op h or e s i s (PFG E) An alys is an d Ph ys i c a l M ap p in g

o f t h e G e n o m e o f S . c i t r i BR3 L in e sS . c i t r i B R 3 l in e s B R 3 -3 X , B R 3 - T , an d B R 3 - G w e r e g ro w n t o lo g p h ase i nL D 8 b r o t h m ed i u m ( D av is , 1 9 79 ) a t 3 2 C . C e l ls w e r e h a r v es t ed b y cen t r i fu -g a t i o n ( 12 ,0 00 g, 3 0 m i n ) . I n t ac t ch r o m o so m a l D N A o f S . c i t r i w a s p r e p a r e di n l o w - m e l t in g p o i n t ag a r o se a s d e sc r i b ed p r ev io u s l y (Y e e t a l . , 1 9 9 2 ) an dd i g es t ed w i t h r e s t r i c t i o n en zy m es B s s H I I ( S t r a t ag en e , L a Jo l l a , C A ) an dS a l I ( P r o m e g a , M a d is o n , W I ) . T h e r e s u lt in g f ra g m e n t s w e r e s e p a r a t e d b yp u l sed f ie l d g e l e l ec t r o p h o r e s i s ( P F G E ) i n 1 % ag a r o se u s i n g a T A F E sy s t em( G en e l i n e T M I I, B ec k m an I n s t r u m en t s , I n c ., P o r te r v il le , C A ) , a s p r ev i o u s lyr e p o r t e d ( Y e e t a l . , 1992 , 1994a). In o rd er to over lap B s s H I I a n d S a l I

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C h r o m o s o m e A b e rr a ti o n s i n S p i r o p l a s m a c i t ri

T a b l e I . P r o b e s U s e d f o r G e n o m e M a p p i n g o f S . c i r r i B R 3 L i n e s

P r o b e D e s c r i p t i o n R e f e r e n c e a

271

X 2X 3X 5X 1 4X 1 8X 3 3A 1A 4A 5A 8A 9A l lE 4 6G 2 5B s HS I Io r /Cs p ip y r G - p u r A -

p u r B

1.8-kb E c o R I - S a l I f r a g m e n t3.1-kb E c o R I - S a l I f r a g m e n t2.5-kb E c o R I - S a l I f r a g m e n t2.8-kb E c o R I - S a l I f r a g m e n t2.0-kb E c o R I f r a g m e n t f r o m5.0-kb E c o R I f r a g m e n t f r om0.5-kb E c o R I f r a g m e n t f r o m1.5-kb E c o R I f r a g m e n t f r o m0.9-kb E c o R I f r a g m e n t f r o m2.0-kb E c o R I f r a g m e n t f r o m1.2-kb E c o R I f r a g m e n t f r o m0.5-kb E c o R I f r a g m e n t f ro m4.0-kb E c o R I f r a g m e n t f r o m

f r o m S . c i t r i B R 3 - 3 Xf r o m S . c i r r i B R 3 - 3 Xf r o m S . c i t r i B R 3 - 3 Xf r o m S . c i t r i B R 3 - 3 XS . c i r r i B R 3 - 3 XS . c i r r i B R 3 - 3 XS . c i t r i B R 3 - 3 XS . c i t r i B R 3 - 3 XS . c i t r i B R 3 - 3 XS . c i t r i B R 3 - TS . c i t r i B R 3 - TS . c i t r i B R 3 - 3 XS . c i t r i B R 3 - T0.8-kb E c o R I f r a g m e n t f r o m S . c i t r i B R 3 - G75-kb B s s H I I f r a g m e n t f r o m S . c i t r i B R 3 - 3 X83-kb S a l I f r a g m e n t f r o m S . c i t r i B R 3 - 3 XC h r o m o s o m a l r e p l ic a t io n or ig in o f S . c i t r i R 8 A 2Sp i ra l i n p r o t e i n g e n e o f S . c i t r i R 8 A 2r R N A ( 16 S ) g e n e o f S . c i t r i R 8 A 2C T P s y n t h e t a s e , a d e n y l o s u c c i n a t e s y n t h e t a s e , a n dlyase o f S . c i t r i R 8 A 2

Y e e t a L (1994 )C h e v a l i e r e t a l. (1990 )L aigr e t e t a l . (1990 )C i n i ( 1 9 92 )

a M a r k e r s w i t h o u t r e f e r e n c e a r e f r o m t h i s w o r k . A l l S . c i t r i B R 3 f r a g m e n t s w e r e c l o n e d i n B l u e -sc r ip t I1 SK + v e c to r a n d m a i n t a i n e d in E . c o l i DH5c~.

f r a g m e n t s o f e a c h l i n e t o a s s e m b l e t h e g e n o m e m a p , w e u s e d r a n d o m l ys e le c te d p l a sm i d s c o n ta in i n g c lo n e d f ra g m e n ts o f g e n o m i c D N A o f B R 3 - 3 X ,B R 3 - T , o r B R 3 - G in Escher ich ia co l i s t r a i n D H 5 a a n d s o m e k n o w n S. citrig e n e s ( T a b l e I) a s p r o b e s t o h y b r id i ze th e P F G E - s e p a r a t e d D N A . S o m es m a l l P F G E - s e p a r a t e d f r a g m e n t s , s u c h a s B s s H I I H ( 7 5 k b ) a n d S a l I I (83k b ) , w e r e a l s o p u r i f i e d f r o m a g a r o s e g e l s a n d u s e d a s p r o b e s . T h e r o d -s h a p e d s p i r o p la s m a v ir u s S p V 1 - R 8 A 2 w a s i s o la t e d f r o m S. citri s tr a in R 8 A 2b y p r e c i p i t a ti o n w i t h 3 .5 % P E G ( 6 0 0 0 ) c o n t a i n i n g 0 . 4 M N a C 1 . T h e 7 - k bc ir c ul ar s i n g le - st r a n d e d v ir a l D N A ( R e n a u d i n et aL, 1 9 9 0 ) w a s t h e n p u r i fi e db y p h e n o l e x t r a c ti o n a n d u s e d a s a p r o b e t o h y b r id i z e th e r e s t r ic t e d g e n o m i cD N A o f S. citri B R 3 . A l l th e p r o b e D N A s w e r e u s e d a s t e m p l a te s f orl a b e l in g w i t h [o L -3 2p ]d A T P , u s i n g a r a n d o m p r i m i n g l a b e l in g k it ( G I B C O -B R L , G a i th e r sb u r g , M D ) . F o r m a p a s s em b l y , B s s H I I a n d SaII f r a g m e n t sr e a c t i n g w i t h t h e s a m e p r o b e w e r e a s s u m e d t o o v e r l a p e a c h o t h e r . B s s H I If r a g m e n t s o v e r l a p p i n g t h e s a m e S a l I f r a g m e n t , o r t w o S a l I f r a g m e n t so v e r la p p i n g t h e s a m e B s s H I I f r ag m e n t , w e r e a s s u m e d t o b e l in k e d . D o u b l ed i g es ti o n s o f th e c h r o m o s o m a l D N A w i th B s s H I I a n d S a l I w e r e a l s oc o n d u c t e d u s i n g t h e m e t h o d o f P y le ( P y l e a n d F in c h , 1 9 8 8) . D a t a f r o m b o t h

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272 Ye, Melcher, Rascoe, and FletcherS o u t h e r n b l o t h y b r id i za ti o n s an d d o u b l e d i g e s t io n s w e r e u sed t o e s t ab li shp h y s ica l g en o m e m a p s f o r B R 3 -3 X , B R 3 - T , an d B R 3 - G .

C l o n i n g D N A s M a p p e d t o t h e D e l et i o n R e g i o n s o f t h e B R 3 - G G e n o m eD i f f e re n t ia l g e n o m i c D N A s c re e n in g s w e r e c a r r i e d o u t t o c l o n e f r a g m e n t sd e l e t ed o r r ea r r an g ed i n B R 3 - G . B r ie fl y, t o t a l D N A s o f B R 3 - 3 X an d B R 3 - Gw er e d i g e s t ed t o co m p l e t i o n w i t h E c o R I ( G I B C O - B R L ) a n d s e p a r at e d in0 .7% agarose . D N A s in d i f f e re n t s ize r anges (0 .5 -2 .0 , 2 .0 -4 .4 , 4 .4 -6 .6, an d6 . 6 - 9 . 3 k b p ) w e r e p u r i f i ed f r o m t h e g e l f o r b o t h B R 3 - 3 X an d B R 3 - G .P u r i f i ed B R 3 - 3 X D N A s o f d i f f e r en t s i z e s w e r e s ep a r a t e l y l i g a t ed w i t hE c o R I - d i g e s t e d an d ca l f i n t e st i n a l p h o s p h a t a se ( P r o m eg a ) - t r ea t ed p l a sm i dB l u e sc r ip t S K ( S t ra t a g e n e ) u s i n g T 4 D N A l ig a se ( G I B C O - B R L ) . A f t e rt r a n s f o r m a t i o n o f E. col i D H 5 oL , m o r e t h an 3 00 co l o n i e s f r o m each s izer a n g e D N A b a n k w e r e s c r e e n e d u s in g p ur if ie d B R 3 - G D N A o f t h e i d en ti ca ls ize r an g e a s p r o b e . N eg a t i v e c l o n es, w h i ch sh o u l d co n t a i n p l a sm i d s h av in gf r ag m en t s u n i q u e t o B R 3 - 3 X i n a sp ec if ic s iz e r an g e , w e r e s e l ec t ed . T h esep l a sm i d s w e r e t h en u sed a s p r o b es t o h y b r i d i ze E c o R I - a n d H i n d I I I( G I B C O - B R L ) - d i g e s te d g e n o m i c D N A o f t h e d i f fe r e n t c e ll li n es t o c o n fi rmt h e i r p r e s e n c e in B R 3 - 3 X a n d B R 3 - T a n d t h e i r a b s e n c e in B R 3 - G .

M a p p i n g a n d N u c l e o ti d e S e q u e n c i n g o f a D N A S e g m e n tC o r r e s p o n d i n g to O n e o f t h e B R 3 - G D e l e t io n R e g i o n s

" C h r o m o s o m a l w a l k " p r o c e d u r e s w e r e a d a p t e d t o e x t e n d t h e c l o ni n g o f o n eo f t h e r e g i o n s ( D e l e t i o n A r e a ] ) d e l e t e d i n B R 3 - G . T h e p l a s m i d p E 4 6 ,r e su l ti n g f ro m t h e ab o v e d i f fe r en t ia l s c r een i n g an d m ap p e d t o t h e d e l e t i o nr eg i o n , w as u sed a s t h e i n it ia l p r o b e . T h e c l o n ed f r ag m en t s m ap p e d t o t h ed e l e t io n w e r e s u b s e q u e n t l y " s h o t - g u n " s u b c l o n e d in t o M 1 3 m p 1 8 a n d / o rM 1 3 m p 1 9 v e c to r s fo r D N A s e q u e n c in g b y th e m e t h o d o f S a n g e r ( S a n g e r e tal., 1 97 7), u si n g t h e P R I S M R e a d y R e a c t i o n D y e D e o x y T e r m i n a t o r C y c leS eq u en c i n g k it ( P e r k i n E l m er A p p l i ed B i o sy s tem s D i v is io n , N o r w a l k , C T )a n d a n a u t o m a t i c s e q u e n c e r (3 73 D N A S e q u e n c e r S tr e tc h , P e r k in E l m e r ) .D N A s e q u e n c e s w e r e a s s e m b l e d u s i n g A s s e m b l y L i g n s o f t w a r e p r o g r a m s( IB M - K o d a k , R o c h e s t e r , N Y ) , a n d t h e c o n s e n s u s s e q u e n c e w a s a n a l y z edu s i n g D N A S t r i d e r ( M a r c k , 1 9 8 8 ) . T h e n u c l e o t i d e s e q u e n c e h a s b e e nd ep o s i t ed i n G en B an k a s acce s s io n U 4 44 05 . P o t en t i a l t r an sc r i p t io n t e r m i n a -t o rs w e r e i d en t if i ed u s i n g t h e T e r m i n a t o r ap p l ica t io n i n t h e G C G seq u en cean a ly s is p ack ag e ( M ad i so n , WI ) . P r o t e i n s eq u en ce d a t ab an k s w e r e s ea r ch edf o r s eq u en ce s s im i l a r t o t h o se co n cep t u a l l y t r an s l a t ed u s i n g B L A S T P( A l t sch u l et al., 1990) and B L IT Z (S tu r roc k and Col lins, 1993). Th e approx i -m a t e p o s i ti o n s o f t h e t w o S . citri B R 3 - G d e l e ti o n b o rd e r s w e r e d e t e r m i n e d

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C h r o m o s o m e A b e r r a ti o n s i n Spiroplasma citri 2 7 3

b y S o u t h e r n b l o t h y b r id i z at io n u s in g D N A p r o b e s o f t h e c o r r e s p o n d i n gr e g i o n s . U p o n h y b r i d i z a t i o n , p r o b e s l o c a t e d w i t h i n t h e d e l e t i o n s h o u l dr e v e al b a n d s p r e s e n t i n B R 3 - 3 X a n d B R 3 - T b u t a b s e n t i n B R 3 - G , w h e r e a sp r o b e s s p a n n i n g t h e d e l e t i o n b o r d e r s s h o u l d r e v e a l b a n d s n o t m i s s i n g b u th a v in g d i f f e r e n t s iz e s i n B R 3 - G . T h e B R 3 - G f r a g m e n t s w h i c h w e r e r e v e a l e db y t h e p r o b e s s p a n n i n g t h e d e l e t io n b o r d e r s w e r e t h e n c l o n e d a n d s e-q u e n c e d f o r d e t e r m i n a t i o n o f t h e e x a c t p o si t io n s o f t h e t w o d e l e ti o nb o r d e r s .

R E S U L T SAnalys i s by Pu l se d F ie ld Ge l E ie c tr ophor e s i s

o f G e no m i c D N A o f S . c i t r i B R 3 L i n e sAs sh ow n in F ig . 1A, B s s H I I an d S a l I r e st ri c ti o n p a t t e rn s o f b o t h B R 3 - T a n dB R 3 - G c h r o m o s o m a l D N A w e r e q u i t e d i f f e r e n t f r o m e a c h o t h e r a n d f r o mt h o s e o f t h e p a r e n t c e ll l in e , B R 3 - 3 X . T h e r e s t r i c ti o n p r o d u c t s r e s u l ti n gf r o m B s s H I I a n d S a l I d i g e s t i o n o f e a c h B R 3 l i n e w e r e a l p h a b e t i c a l l yd e s i g n a t e d i n o r d e r o f t h e i r si ze s an d a r e s u m m a r i z e d i n T a b l e I I . T h ea p p r o x i m a t e g e n o m e s iz e s o f t h e d e r i v a ti v e s B R 3 - T ( 1 75 0 k b ) a n d B R 3 - G( 1 8 7 0 k b ) a r e 1 5 0 - 2 7 0 k b la r g e r t h a n t h a t o f B R 3 - 3 X ( 16 0 0 k b ) . T h e g e n o m eo f S. citri B R 3 c o n t a i n s m u l t i p l e s p i r o p l a s m a v i r u s S p V l - l i k e s e q u e n c e s .H y b r i d i z a t i o n o f B s s H I I - a n d S a l I - r e s t r i c t e d B R 3 - 3 X , B R 3 - T , a n d B R 3 - Gg e n o m i c D N A s w i th t h e v ir u s S p V 1 - R 8 A 2 D N A r e v e a le d s e v e ra l f r ag m e n t si n e a c h li n e, a n d r e s u l t e d i n v e r y d i f f e re n t p a t t e r n s a m o n g t h e t h r e esp i rop la sm a l ine s (F ig . 1B) .

C o n s t r u c ti o n a n d C o m p a r i s o n o f P h y s i c a l M a p so f t h e G e n o m e s o f S . c i t r i B R 3 L i n e s

T o e x p l o r e t h e r e a s o n f o r t h e l a r g e d i f f e r e n c e s i n r e s t r i c t i o n p a t t e r n s , w em a p p e d t h e g e n o m e s o f t h e s e l in e s b y r e c ip r o c al B s s H I I an d S a l I d o u b l ed i g e s t i o n a n d D N A h y b r i d i z a t i o n , u s i n g 2 0 d i f f e r e n t m a r k e r s . E x c e p t f o rf r a g m e n t s S a l I F a n d H o f B R 3 - 3 X a n d B R 3 - T o r S a l I E a n d H o f B R 3 - G ,which a re lo ca ted w i th in a l a rge B s s H I I f r agme nt , the r e la tive pos i t ions o f al lo t h e r f r a g m e n t s c o u l d b e d e t e r m i n e d . T h e r e s u lt s a ls o al l o w e d th e a ss ig n -m e n t o f t h e 2 0 D N A m a r k e r s t o p o s i t i o n s o n t h e m a p s ( F ig . 2). T h e o r d e r s o ft h e p o s i t io n e d D N A m a r k e r s a s w e l l a s th a t o f m o s t B s s H I I an d S a l Ir e st r ic t io n f r ag m e n t s o f B R 3 - 3 X a n d B R 3 - T w e r e a l m o s t id e n ti ca l. H o w -e v e r, s o m e f ra g m e n t s o f B R 3 - T w e r e l a rg e r t h a n t h e c o r r e s p o n d i n g f r a g -m e n t s i n B R 3 - 3 X , m a k i n g t h e w h o l e g e n o m e o f B R 3 - T ( 17 5 0 k b ) a p p ro x i -m a t e l y 1 5 0 k b l a r g e r t h a n t h a t o f B R 3 - 3 X ( 1 60 0 k b ) .

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ChromosomeAberrations in Spiroplasma c i t r i 27 5Table II. BssHII and SalI Fragments of the G enome of S. citriBR3 Lines

BssH I I B R 3 - 3 X B R 3-T B R 3-G SalI B R 3 - 3 X B R 3 - T B R 3- G(band ) (kb) (kb) (kb) (band ) (kb) (kb) (kb)A a 412 440 510 A a 290 320 390B 310 320 386 B 280 290 270C 258 310 240 C 215 246 260D 170 200 180 D 170 200 224E 130 160 160 E 170 190 190F 125 125 150 F 150 160 165G 120 120 125 G 130 150 160H 75 75 120 H 110 110 130I 83 83 83To tal 1600 1750 1871 To tal 1598 1749 1872

aIn BR3-3X, a band resulting from partial digestion between fragm entsBssHI1A and BssHII Hoften appeared abov e fragment BssHII A, and a band from partial digestion between frag-ments SalI C and SalI I som etimes appeared above fragment SalI A (Fig. 1A).T h e g e n o m e o f B R 3 - G w a s 2 70 k b l a rg e r th a n t h a t o f B R 3 - 3 X . T h e

o r d e r o f t h e m a r k e r s b e t w e e n A 9 o r E 4 6 a n d A 8 o r X 33 , a n d b e t w e e nr e s t r i c t i o n f r a g m e n t s BssHII C a n d BssHII E , w a s o p p o s i t e t o t h a t i nB R 3 - 3 X o r B R 3 - T , s u g g e s t i n g a c h r o m o s o m a l i n v e r s i o n i n t h i s r e g i o n . I na d d i t io n , t h e o v e r l a p p i n g f r a g m e n t s BssHII C a n d SalI B o f B R 3 - G w e r eb o t h a b o u t 1 0 - 20 k b s m a l l e r t h a n t h e c o r r e s p o n d i n g f r a g m e n t s o f B R 3 - 3 X( F i g. 1 C ) , s u g g e s t in g t h a t a d e l e t i o n o f t h a t s iz e o c c u r r e d i n th i s r e g i o n( D e l e t i o n A r e a I ) . S im i l a r ly , t h e o c c u r r e n c e o f a 5 - t o 1 0 - k b d e l e t i o n( D e l e t i o n A r e a I I ) n e a r t h e o t h e r b o r d e r o f t h e l a r g e c h r o m o s o m e in v e rs i onw a s s u g g e s t e d b y t h e o b s e r v a t i o n t h a t f r a g m e n t s BssHII F a n d SalI F o fB R 3 - G w e r e 5 - 1 0 k b s m a l l e r t h a n t h e i r c o u n t e r p a r t s i n B R 3 - 3 X a n d B R 3 - T .

C l o n i n g D N A s M a p p e d t o th e D e le t io n s i n B R 3 - GB y d i ff e r e n ti a l s c re e n i n g , w e i s o l a te d t w o p l a s m i d s c o n t a i n i n g D N A f r a g -m e n t s m a p p e d t o t h e tw o d e l e t i o n a r e a s i n B R 3 - G . A s s h o w n in Fig . 3 A , th ep l a s m i d p E 4 6 , c a r r y i n g a 4 - k b E c o R I f r a g m e n t , r e v e a l e d t w o E c o R I f r a g -m e n t s , o f 4 .0 a n d 1 5.0 k b , in B R 3 - 3 X a n d B R 3 - T b u t o n l y t h e 1 5 .0 -k bf r a g m e n t i n B R 3 - G . I t a ls o r e v e a le d t w o HindIII f r a g m e n t s , o f 8 .0 a n d 1 6 .0k b , in B R 3 - 3 X a n d B R 3 - T , b u t t h e 8 .0 - k b HindIII f r a g m e n t w a s m i s s i n g i nB R 3 - G . T h e s e f i n di n gs s u g g e s t t h a t t h e 4 . 0 -k b E c o R I a n d t h e 8 . 0 - k b HindII If r a g m e n t s o v e r l a p e a c h o t h e r a n d t h a t b o t h w e r e d e l e t e d i n B R 3 - G . B e c a u s et h e p E 4 6 p r o b e r e a c t e d s t r o n g ly w i t h tw o E c o R I a n d t w o HindIII f r a g m e n t sin b o t h B R 3 - 3 X a n d B R 3 - T , s o m e s e q u e n c e s o r t h e w h o l e p r o b e f r a g m e n tm u s t b e d u p l i c a t e d i n t h e g e n o m e . I n B R 3 - 3 X a n d B R 3 - T , t h is p r o b e i sl o c a t e d i n t h e r e g i o n w h e r e t h e 1 0- t o 2 0 - k b d e l e ti o n ( D e l e t i o n A r e a I ) w a s

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Chrom osome Aberrations in Spi rop lasm a c i t ri 277o b s e r v e d i n B R 3 - G ( F ig . 1 C ). T h e f a c t t h a t o n l y o n e BssHII o r SalI f r a g m e n tw a s r e v e a l e d b y th i s p r o b e s u g g e s t s t h a t t h e t w o r e a c t in g EcoRI a n d / o rHindIII f r a g m e n t s w i t h p o s s i b ly d u p l i c a t e d s e q u e n c e s a r e b o t h l o c a t e d i nt h e r e g i o n c o v e r e d b y f ra g m e n t s BssHII C a n d SalI B in B R 3 - 3 X orBssHII Ca n d SalI A i n B R 3 - T ( F i g . 2 ) . T h e o t h e r i s o l a t e d p l a s m i d , p A 8 , r e v e a l e dt h r e e EcoRI f r a g m e n t s a n d t w o HindIII f r a g m e n t s in B R 3 - 3 X a n d B R 3 - T ,b u t o n l y t w o EcoRI f r a g m e n t s a n d a si ng l e HindIII f r a g m e n t o f la r g e r s i ze i nB R 3 - G ( F ig . 3 B ). A s w i t h p E 4 6 , t h i s p r o b e a l so h y b r i d iz e d t o s e v e r a l b a n d s ,i n d ic a t i n g t h a t s o m e s e q u e n c e s o r t h e w h o l e p r o b e D N A w e r e r e p e a t e d int h e g en o m e . U p o n h y b ri di za ti on a g a in s t P F G E - s e p a r a t e d B R 3 g e n o m i c D N A ,t h e p r o b e p A 8 m a p p e d t o th e o t h e r d e l e ti o n re g i on ( D e l e t i o n A r e a I I ) .

M a p p i n g , N u c l e o t i d e S e q u e n c i n g , a n d G e n e t i c O r g a n i z a t io n o f th e D N AS e g m e n t C o r r e s p o n d i n g t o D e l e t io n A r e a I i n B R 3 - G

B y u s in g t h e p l a s m i d p E 4 6 a s a p r o b e f o r f u r t h e r s c r e e n i n g o f B R 3 - 3 Xl i b r a r i e s , t h r e e o t h e r p l a s m i d s - - p S 4 , c a r r y i n g a 4 . 0 - k b Sau3A f r a g m e n t ;p S H 5 , c a r r y i n g a 5 . 0 - k b Sau3A-HindlII f r a g m e n t ; a n d p A 9 , c a r ry i n g a1 .8 - k b EcoRI f r a g m e n t - - w e r e o b t a in e d . T h e i n s er ts o f t h e s e p la s m i d sc o v e r e d t h e w h o l e D e l e t i o n A r e a I ( Fi g. 4 A ) . B y S o u t h e r n h y b r i d iz a t io n s ,t h e l e ft - s id e d e l e t io n b o r d e r ( D e l e t i o n B o r d e r I ) w a s l o c a t e d w i th i n t h e1 .5 - k b Sau3A-HindIII f r a g m e n t , b e c a u s e t h i s p r o b e r e c o g n i z e d a 4 . 2 - k bSau3A b a n d i n B R 3 - G r a t h e r t h a n t h e 4 . 0 - k b Sau3A b a n d i n B R 3 - 3 X a n dB R 3 - T a n d t h e a d j a c e n t 4 .0 -k b EcoRI f r a g m e n t r e v e a le d EcoRI a n d HindIIIb a n d s p r e s e n t i n B R 3 - 3 X a n d B R 3 - T b u t a b s e n t in B R 3 - G . T h e r i g h t- s id ed e l e t i o n b o r d e r ( D e l e t i o n B o r d e r I I ) w a s p o s i t io n e d w i th i n t h e 0 .4 - kbEcoRI-XbaI f r a g m e n t , b e c a u s e t h i s p r o b e r e v e a l e d a 3 . 2 - k b EcoRI f r a g -m e n t i n B R 3 - G r a t h e r t h a n t h e 1 . 8- kb EcoRI f r a g m e n t i n B R 3 - 3 X a n dB R 3 - T a n d t h e a d j a c e n t 2 .6 -k b EcoRI f r a g m e n t r e v e a le d EcoRI a n d HindIIIb a n d s p r e s e n t in B R 3 - 3 X a n d B R 3 - T b u t a b s e n t in B R 3 - G . N u c l e o t id es e q u e n c e s o f t h e f o u r o v e r l a p p in g p l a sm i d s h a v e b e e n d e t e r m i n e d . A sFig. 2. Phy sicalmaps of the genomes of S. citriBR3-3X and i ts derivatives BR3-T and BR3-G.Letters symb olize he restriction fragm ents listed in Table II, and positions of the DN A m arkerslisted in Table I are indicated ab ov e or b elow the restriction m aps . The diamond betweenfragments Sall F and SalIH in BR3-3X and BR3-T, and between SalIH and Sall E in BR3-G,indicates undetermined relative positions o f these SalI fragments within the correspondingBssHII fragment. The two BR3-G deletions, Deletion Area I and Deletion Area II, were basedon the smaller sizes of the corresponding fragments in B R3-G w ith respect to their counterpartsin BR3-3X. The crossed dashed lines indicate the relative positions of the chromosomalinversion in BR3 -G. The sym bolsAA IB and CA2D (preinversion borders) define the BR3-3Xsegments corresponding to D eletion Area 1 and D eletion Area II and their borders beforegenetic events leading to the d eletions and inversion in BR 3-G, and the sym bolsAC and BD(postinversion borders) define the results from exchange oints between the borders o f DeletionArea I and Deletion Area II.

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