ORMANBAKANUGIMINISTRY OF FORESTRY

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onCaNiZaS YON rOUrrESi SEKRETERYAS ISECRETENAT OF THE ORGANIT]NG COMITTEE

Ankara. Ekim / October 1993

K r z r L g A M ( P i N U S B R U T T A T E N . ) ' D A I Z O E N Z i M

ANALiZLERIYLE ORfJ iN AYIRIMT

PROVENANCE SEPARATION

ANALYSTS IN PINUS

BY USING ISOENZYME

B R U T I A T E N .

t 2 1Zeki YAEYA0c[0' P. Ahnei AYAZ- ltusa GEI{Q^

I fi.T.ii. 0rnan Fak., 0rnan lttih. Bttl., Trabzon-TcR[iYE

K.T.t i . Pen-Edebiyat I 'ak., Biyoloj i Bci l . , Trabzon-i iR[ iYE

6ZET : Bu ga l rgma, as l i agag t i i r l e r im izden b i r i o lan

k rz r l gam (P inus b ru t i a Ten . ) ' r n k i i g t i k a lan la rdak i ( seg i l en

tohum megcere le r i nde ) gene t i k va ryasyon la r rn r bu lup o r -

taya g rka rmak , daha son ra muh teme len yap t l acak o lan o r i j i n

a lan la r rn r s r -n l - r l and r rma ga l rgma la r rna baz o lug tu rmak ve

bu baz i izer ine i lave or i j in ler in sapLanmasr amacry la

yap r lmrg t r r . Deneme le rde fa rk l r o r i j i n l e rden e lde ed i l en

karrgrk tohum c i rnek ler i ve bu tohumlarrn kuru endosperm-- l

l e r i ( - ) k u l l a n r l m r g t r r . D e n e m e y e a l r n a n p o p u l a s y o n l a r

a ras rnda , LAP (Leuc ine aminopep t idase ) enz im s i s tem iy le ya -

p r l a n a n a l i z l e r d e g e n e t i k f a r k l r l r k l a r r n o l d u g u s a p t a n -

mrg t r r . Boy lece e lek t ro fo re t i k yon te rn le r l e ( i zoenz im

a n a L i z l e r i i l e ) k : - z : - l g a m d a s e g i l e n t o h u m m e g c e r e l e r i n i n

( o r i j i n l e r i n ) gene t i k s t r r i k t i i r l e r i n in ag rk lanab i l ece ! i

o r taya konmug tu r .

ABSTRACT : This study was

a r c h i t e c t u r e i n s o m e s e l e c t e d

T e n . . F o r e a c h s e e d s t a n d s ,

dr ied endosperms were used

m a d e t o a s s e s s t h e g e n e t i c

s e e d s t a n d s o f P i n u s b r u t i a

m i x e d s e e d s a m p l e s a n d t h e i r

i n I a b o r a t o r y a n a l y s i s , a n d

2_some genet ic d i f fe rences were de termined be tween the

popu la t ions by the use o f e lec t rophore t ic methods . LAP

( L e u c i n e a m i n o p e p t i d a s e ) e n z l r m e s y s t e m w a s u s e d i n t h e s e

a n a l y s i s . R e s u l t s s h o w e d t h a t . i s o e n z y m e a n a l y s i s c a n b e

s a f e l y u s e d t o c e r t i f i c a t e s e e d s t a n d s

T e n .

o f P i n u s b r u t i a

ctRis

Is1ah gal rgmalarr -nr -n b i r programa baqlanmasr ve bu

p ro l ram la r rn da sa l l r k l r b i r gek i l de haz r r l anmasr ge rek i r .

Aks i takdi rde i ler ide or taya grkabi lecek hata lar r p€ l rEr

kaybr yanrnda zaman kaybrna da sebep o labi lecekt i r . Bu ne-

denle or i j in denemeler in in mut laka genet ik b i r baza dayan-

d r r r lmas r ge rekmek ted i r .

Is lah gal rgmalarrnda i1k agama cograf ik varyasyon ga-

l rgma la r r o lma l rd r r . Bu ga l rgma la r o r i j i n deneme le r i ne baz

o lug tu racak t r r . Ay r r ca i l g i l i t i i r i i n doga l yay r l rg a lan r ,

kag ade t ve hang i o r i j i n l e r l e t ems i l ed i lmes i ge rek t i l i de

o r taya g rka r r lmrg o lacak t r r . Co l ra f i k va ryasyon ga l rgma la -

11 tamamlandrk t .an son ra o r i j i n deneme le r i bag la t r lma l rd r r .

Orman agac r popu lasyon la r rn rn gene t i k va ryasyon la r r

hakk rnda b i l g i ed inmek ig in son y : - l l a rda i zoenz im ana l i z -

l e r i n d e n y a r a r l a n r l m a k t a d : - r ( L , 2 , 3 , . . . . 1 0 ) . O r m a n c r l r k t a

y r l l a rdan be r i t ohumla r " t ohum ka l i t e be lges i " i l e sak lan -

mag ve ku l l an rm ye r le r i ne g6nder i lm ig , t ohum kayna l rn rn

gene t i k yap r -s rn r o r taya koyan b i r be lge ku l l an r lmamrg t : - r .

Ancak bug t i n b i1 'ok imyasa l -gene t i k yon temle r le popu las l ' on Ia -

r r n g e n e t i k y a p r l a r r e m i n v e h r z l r b i r g e k i l d e b e l i r l e n e -

b i l m e k t e d i r ( 1 ) . Y i n e i z o e n z i m a n a l i z l e r i y l e d o g a l p o p u -

l a s y o n l a r r n a k r a b a l r k a n a l i z l e r i y a p r l a b i l d i g i g i b i , k l o -

\)

na l me lez leme ana l i z l e r i i I e me lez lemen in bagar rs r da de -

ne t l eneb i lmek ted i r (21 .

i zoenz im ana l i z l e r i uzun y r I l a r t rp ta , b i yok imyada ve

b iyo lo j i de fa rk l r p rob lem le r in goz r im t inde kuL lan r ld rk tan

sonra , son y r l l a rda ta r tm ve o rmanc r l r k kes im inde de ku l -

l a n r l m a y a b a g l a n m r g t r r . o r m a n c r l r k t a , o z e l l i k l e g e n e t i k -

r s lah ga l rgma la r rnda , t aksonomj -de ve tohum t i r e t im i g ib i

a lan la rda ku l l an rm ye r i bu lmug tu r ( 3 ) .

t z o e n z i m l e r , a y n l - e n z i m t i p i n i n g e g i t l i v a r y a n t l a r r d r r .

Bun la r madde deg ig im inde ay r r - , f onks i yon l .a ra sah ip t i r l e r .

t zoenz im le r b i t k i se l doku la rdan ( tohum, tomurcuk v .b . ) i -

zoLe ed i l d i k ten son ra , po rc i z legmig o r tam la rda (h id ro l i ze

o lmug pa ta t ,es n igas tas r v .b . ) e lek t r i kse l b i r a landa k i i t l e

ve y t i k l e r i ne gc i re geq i t , l i uzak l r k la rda ha reke t ede r le r . Bu

elekt roforet ik ayrr - Imadan sonra enzimfere uygun b i r gek i l -

de boyama yapr l r r ve sonugta renk l i bant lar ( z imogram) o-

Iugu r . Deger lend i rme le r bu ban t l a ra gc i re yapa lmak tad r r .

i zoenz im le r f i zyo lo j i k ve mor fo lo j i k oze l l i k l e r g ib i

p r imer feno t i p i k c i ze l l i k l e rd i r (3 ) ve gen le r t a ra f rndan

k o n t r o l e d i l i r . G e n e L l i k l e g e v r e s e l f a k t o r l e r d e n e t k i l e n -

med ik le r i i g i n , gene t i k va ryasyon la r r enz im le r o lug tu r rnak -

t a d r r . D o l a y r s r y l a g e n e t i k y a p r y a i l i g k i n d u y a r l r b i l g i -

l e r i ge rd i k le r i i g i n , geno t i p i k ana l i z l e rde g t i ven l i b i r

g e k i l d e k u l l a n r l a b i l m e k t e d i r .

Bu ga l r -gmanr -n amac l , "Orman Agaq la r r ve Tohumla r r I s -

Iah A rag t r rma Mt id t i r l t i g i i " nce seg i l en k rz r l gam tohum megce-

r e l e r i a r a s r n d a g e n o t i p i k f a r k l r l r k l a r o l u p o l m a d r l r n r o r -

taya koymak t r r . Seg i l en 3 tohum megceres ine a i t ka rma

t o h u r n c j r n e k l e r i i I e g a l r g m a l a r a b a g l a n m l g o l u p d a h a s o n -

r a k r y r l l a r d a q a l r g m a n r n g e n i g l e t i l m e s i p l a n l a n m a k t a d r r .

IDetay l r anal iz ler sonucunda, co l raf ik varyasyonlar gene-

t i k ana l i z l e re dayandr r r l acak , mevcu t ve yen i t esp i t ed i -

l ecek tohum megcere re r i bu ana l i z sonug la r rna go re yen i -

den s rn r r l and r r r l acak ve k rz r l gam ig in b i r o r i j i n ha r i -

t as r d r i zen lenecek t i r .

MATERYAI, VE YdNTEM

Bu on ga r rgmada , p inus b ru t i a Ten . ' n rn t i g l oka r popu-

lasyonundan (o r i j i n ) t ohu rn la r ku l ran r lmrg t r r . A l rnan to -

humlar "orman ASaglarr ve Tohumlarr rsrah Aragtrrma Mridi ir-

l i ig i i " taraf rndan sagranmrgtr r . Deneme materyal in in top lan-

d r g r m e g c e r e l e r e i r i g k i n g e n e l b i l g i l e r T a b r o L ' d e v e r i l -

m ig t , i r .

Laboratuvar anal iz ler inde her or i j inden ayrr_ ayrr kar-

ma tohum c j rnek ler i kurranr lmrg ve enzim s is temin in anar j -z i

hapro id b i r doku oran endospermde gerqeklegt i r i lmigt i r .

Ho rno jen i zasyon tamponu o la rak T r i s -HC1 (0 .97 g T r i s -Hc l +

1 0 0 m l d e s t i l e s u , p H 7 . 3 ) k u l l a n r l m r g t r r .

P inus b ru t i a t ohumra r r , d iqe r kon i fe r t ohumra r r g ib i ,

anatomik bakrmdan r ig fark l r tabakadan o lugur : Endosperm

(n, ) , embr i l 'o ve tohum kabusu. Bu gal rgmad.a Lamamen endo-

spe rm ku l l an r lmrg t r r . Endosperm sadece ana aqac r -n

geno t ip in i t ag : -mak tad r r . ga l rgma la r Ba r te l s ( l - ) t a ra f rndan

kul lanr lan yontemrer le s i i rd i i r i i lmt ig t r i r . Aragtr r r lan enzim

s is tem i LAP o lup , ay r rma s i s tem i o la rak ta Tc* ku l l an r r -

m r g t r r .

LAP ie in

- J e r t a m p o n u o l a r a k T C + d e s t i r e s u ( 5 0 m l T C , p H 7 . 0 +

1 0 0 m l D e s t i l e s u ) v e 1 6 . 5 g h i d r o l i z e o l m u g p a t a t e s

n igas tas r + 3 g sakka roz ka r rg rmr ku l l an r lmrg t r r .

5T a b l o 1 : A r a g t r r m a d a K u l l a n r l a n T o h u m M a t e r y a l i n i n

E l d e e d i l d i q i T o h u m M e g c e r e l e r i .

T a b l e 1 : D e t a i l s o f S t u d i e d S e e d S t a n d s .

M e g .

Stand

N o . M e g . B u l . Y e r

N o . S t a n d P l a c e

Yt ikse 1t i EnLem

Al t i t u te Lac i tu te

( m )

B o y I a m

Long i tu te

1 aI J

i l R= J

f , 3

S rndrrgr -Se1'dan

Kumluca-KumIuca

Amasya

3 9 ' l - 2 ' 0 0 "

3 6 ' 2 6 ' 2 0 "

4 0 ' 5 0 ' 4 3 "

2 B ' l 2 ' 0 0 "

3 0 0 1 5 ' 5 5 "

3 6 ' , 2 1 ' L 9 "

4 0 0

250

4 3 0

geki l 1 : E lekt rof roze Cihazrnrn gemat ik Gc5r i in i igr i .

F igure 1 : The Schemat ic Apperance of E lect rophores is

Inst rument .

z l -mogram

S t a r t

r -> o rnek ye r leg t i rmeI r - ) n i g a s t a j e l

t l i : T r lc id /an ' rv :cus )

LElek t rod tamponu oLa rak i se TC ( 18 g / I T r i s -HC l + 10

g / I s i t r i k a s i t m o n o h i d r a t , p H 7 . 0 1 k u l l a n r l m r g t r r .

Homjen izas l zon sonucu e lde ed i l en doku eks t rak t l a r r

( i zoenz im le r ) wha tman no : l - k romotog ra f i kaq rd rna emd i r i l e -

rek h id roL i ze n igas ta j e l i konan e rek t ro fo rez c ihaz rna ak -

ta r r lmrg t r r (gek i l l - ) . Daha son ra c ihaza e rek t r i k ak rmr

ve r i lm ig ve o lugan e lek t r i kse l a landa i zoenz im le r 4 -5 saa t

s i i rey le kogturu lmugtur . Bu s i i re sonunda, je I c ihazdan

ar rnmrg ve a l t rndan l - -2 mm ka l rn r r k ta b i r d i l im kes i l e rek

boyama kabrna konmugtur . 6nce c in tamponda (Tr is-Mareic

ac id , pH 5 .4 ) L0 dk . bek le t i lm ig t i r . Bunun a rd rndan c in

tampon kabtan alrnmrg ve yerine boyama tamponu konmugtur.

i zoenz im le r i n go r i i n i i r ha le ge t i r i Lmes i i g in , b i yok imyasar

boyama y6ntemler inden faydalanr lmrgt r r . Boyama tamponu

o la rak 25 m l T r i s -Ma le i c ac id + 25 mI des t i l e su + 20 mg

L-Leuc in + 20 mg Fas t B lack K sa l t ka r rg r -mr - ku l ran r lmrg t r r .

B o y a m a g o z e l t i s i n i n i l a v e s i n i n a r d r n d a n j e I d i l i m i ,

i . i ze r i nde enz im band la r r ( z imogram) gc in in r i r ha le ge lene ka -

da r yak laq rk 35 Co 'de ka ran l r k b i r o r tamda tu t ,u lmug tu r .

Daha son ra p ro toko r d r i zen lenmig ve bundan ya ra r l an r la rak

g e n f r e k a n s l a r r h e s a p l a n m r g t r r .

soNug vE oNERiLER

K r z r l g a m t o h u m e n d o s p e r m l e r i n d e a r a g t r r r l a n L A p z i m o g -

r a m r - s a d e c e i k i i z o e n z i m z o n u n d a n o l u g m a k t a d r r . B u z o n r a r

L A P - A v e L A P - B r o k u s l a r r o l a r a k i s i m l e n d i r i l i r . y a p r l a n b u

o n c a l r s m a d a , b o v a m a s o r ' - l c ' t 3 l r : ' i r L i r - h r l : g e 1 . : r L A p - A 7 - -

n u n d a h e r h a n g i b i r v a r y a s y o n g o z r e n e m e m i g t i r . A n c a k L A P - B

Izonrrnda t ig ar ler t ip i (LAp-BL, LAp-82 ve LAp-83| gozren-

m i g t i r . B u a l r e r r e r i n ( i z o e n z i m a L r e r l e r i ) f r e k a n s r a r r h e -

s a p l a n a r a k T a b I o 2 ' d e v e r i l m i g t i r .

Bu tab lodan da gc i r i i l eb i l eceg i g ib i , denemede ku l l an r -

l an popurasyon la r a ras rnda gene t i k f a rk r r l r k ra r mevcuL tu r .

Ni tek im LAP-B3 a l le l i Amasya popurasyonund.a yokken,

Kumluca -Kumluca popu lasyonunda 0 .500 . Seydan popu lasyonun-

da 0 .066 o ran rnda tems i l ed i rmek ted i r . Ayn r gek i l de LAP-B I

a l l e l i A m a s y a o r i j i n i n d e 0 . 9 0 0 o r a n r n d a t e m s i r e d i r i r k e n ,

Kumluca o r i j i n i nde 0 .366 o ran rnda tems i l ed i rmek ted i r .

LAP-82 a l l e l i Amasya o r i j i n i nde 0 .100 o ran : -nda bu lunu rken ,

K u m l u c a o r i j i n i n d e 0 . 1 6 6 , S e y d a n o r i j i n i n d e 0 . 5 6 6 o r a n r n -

da tems i r ed i rmek ted i r . Endosperm c j rnek re r i nde be r i r l enen

Tab lo 2 : Be l i r l enen Enz im-Gen

A l le l F rekans la r r .

Tab le 2 : A1 le l F reguenc ies fn

Lokus la r rna t l i gk in

De te rm ined Enzyme Loc i .

ORTJiNLER

( ORIGINS )

BAL

B1 B t B 3

Amasya

Kumluca

Seydan

0 . 9 0 0

0 . 3 3 3

0 . 3 6 6

0 . 1 0 0

0 . 1 _ 6 6

0 . 5 6 6

0 . 0 0 0

0 . 5 0 0

0 . 0 6 6

gen Lokus la r rndak i

t e r i l m i q t i r .

a 1 1 e 1 l e r g e k i L 2 ' d e g e m a t i k o l a r a k g o s -

Ic i ze t renecek oLu rsa , LAp-B gen l okusunda i i g a r l e l be -

l i r r e n m i g o l u p , h e r b i r a l r e l d e c i z e l r i k r e 8 3 a r r e r i n d e g e -

ne t i k f a rk l r - I r k d i kka t i gekmek ted i r . N i tek i rn 83 a l l e r i A_masya o r i j i n i nde tems i l ed i lmezken , d j -ge r o r i j i n l e rde

fa rk r r o ran la rda bu runmak tad r r . i nce renen popu lasyonra r rn

bu lunduk la r r ye r l e r d i kka te a l rnd rg rnd ,a , gene t i k o la rak

o r taya g rkan va ryasyon la r rn , ds r rnda cog ra f r k va ryasyon la r

o rduqu da kes in l i k kazanmak tad . r r . Ancak bu ga l rgma b i r dnga l : -gmadr r . Daha gok ma te rya l ku l ran r ra rak a ras t r rma la r rn

devam et t i r i lmesi gerekrnektedi r .

sonug o l . a rak d i yeb i r i r i z k i , e rek t ro fo re t i k yc in temre r

sayesinde gen envanter i yaparmasr , buna gore populasyonla-

gek i l 2 : Endosperm le rde Ber i r renen Gen Lokus la r rndak i

A l Ie l l e r i n gemat i k O la rak Gc is te r i lmes i .

F igu re 2 : A D iag ramat i c r l r us t ra t i on o f A l l e l s rn

Determined Gen Loci .

LOKUSLAR( L O C r )

O R I J I N L E R ( O R I G I N S )

Amasya Kumluca Seydan

LAP-A I I I I I r I I

I1

LAP-B I

r r n b i r b i r i n d e n a y r r l m a s r d o l a y r s r y r a a q a g l a n d r r m a g a r r g -

m a l a r r n d a g e n e t i k y a p r s r b i l r n e n m a t e r y a r k u l l a n : - m r m t i m -

k r i n h a l e g e l m e k t e d i r . B u n e d e n l - e d o Q a l o r m a n a g a c r t t i r l e -

: i - - . t : : - i - - - . - . , . ]< : - : xa1-bet ; re , ;en J ,= f i env . l .n te r l - ga i rgmalar rna

b a g l a n m a l r v e o r i j i n h a r i t a l a r r h a z r r r a n a r a k o r i j i n d e n e -

I Ir 1

I r2 Z

r5

r3

m e l e r i y l e tohum mesceres] - s e q i m l e r i b u

otur tu lmaf rdr r

B u q a l r g m a l a r s r r a s r n d a t e s p i t e d i l e c e k g e n e t i k

q e g i t l i l i k i n - s i t u v e e x - s i t u c i n l e m l e r i y l e k o r u m a a L t r n a

a l r n m a l r d r r . A k s i h a l d e T i i r k i y e o r m a n c r l r g i n r n g e l e c e q i n d e

c j n e m l i b i r y e r t e g k i l e d e c e k , h r z l . r g e l i g e n d o l a I o r m a n a -

gac l t i . i r i imt iz , bug i in ig in zeng in b i r gen havuzuna sah ip o l -

d u q u m u z k r z r l q a m d a , s o n r a k i y r l l a r d a y a p : . l a c a k a g a q l a n -

d r r m a g a l r g m a l a r r - n r - n g e l e c e g i t e h l i k e a l t r n a g i r e c e k t i r .

G e l e c e k n e s i l l e r i e i n m e v c u t G E N E T I K M I R A S m u t l a k a

k o r u n m a l r d r r .

q

b a z a

KAYNAKqA

B e r g m a n n , E . , I 9 7 L : G e n e t i s c h e U n t e r s u c h u n g e n b e i

P i c e a a b i e s m i t H i l f e d e r i s o e n z y m I d e n d i f i z i e r u n g .

A l I g . F o r s t - u . J . Z L g . L 4 2 . J g . 1 1 , 2 7 8 - 2 8 0 .

B a r t e l s , H . , L 9 7 L : I s o e n z y m e u n d i h r e B e d e u t u n g f r i r

F o r s t p f l a n z e n z i i c h t u n g u n d - g e n e t i k . A l l g e n . F o r s t z t s c h r .

2 6 , 5 0 - 5 2 .

B e r g m a n n , F . u n d M i i l l e r - S t a r c k , G . , l - 9 8 9 : N a c h w e i s v o n

I s o e n z y m e n b e i W a l d b a u m a r t e n . F o r s t l i c h e F a k r i l t a t d e r

U n i . G o t t i n g e n .

B e r g m a n n , E . , L 9 7 3 a : G e n e t i s c h e U n t e r z u c h u n g e n b e i

P i c e a a b i e s m i t H i l f e d e r I s o e n z y m - I d e n d i f i z i e r u n g .

u_)

( 2 )

( 3 )

( 4 )

T h e o r e t i c a l

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