CHAPTER 30

Sortin54 of piant Chromosornes

Jarosla-v Doleiel,' Martin A.Hana Sirrrkovà,' liÌí Macas-i'lnlnnLr .r !rf.ii|j.jr, ù. ì!(lZ :rt rrrr ()]. rrì!!. (.r..lj trrpr b,.

.l'lli1,' rr.,,{,I r.r ,,. z: i r6r

( r\.r.iì Il.rN .h cÈrL.rl'.nr 8uùh,rnr{}. L),!Ntr,

fysàl<.' Marie Kutral;ikor i..artd Sergio Lrrcrettil

À phrr Mi t..nrtù. ÌìQg.,r6 r.,l Sb.t S.t.riùr\

.. , f t,.r r ,rt ,rih Àl.L(rrrl

A !r.pl. tr, epÌrnon,l ' \v ' ,r./r \ i. r ltrt,q tl q.1 ,\A. sun|1. tù.l]rarnrl

,,, u " '

A. Srijrpl. lr,ep.ìfution

' , \'., ,,. '... ..

-

I. Introduction

fechniqucs t0r now cvtomcrncwerc origjnatry develop;d lor lhc

l'rtL

, n. l!.. InJ \,nr,.î o, nrit.rr. chfu.n, ..,n(5, ,r'res..r.,rn\rr.r . n,t .r\.e,,.r(, r) nìJrj nJJ

(SJS\tHdì 'ror!'r.dsns

ii, iurt.ìrlrt y\C ,r^ L/ pillrrd pi Ìì fortìiu rì n-orsn

pr,\rrt.ìr .q ^ruÌ

srqJ .ú)'rJi.Ltt nrrttr.r pitlìclrrtsrp ilsfoJUrn-ouroq rou ..nlìr)L sùu..brs.rrluidii toi;uìt:clÌjt ìu.rs.r)llU uo prîrq sr !oLtìÌurtrnnsrfiurosou'o.Ìrl) r\ordùr oÌ torll.]ru t.rrou Y (rrì(rt t) /, r.rl)r1lLr\rtr( :ltrl)lll''tl'l'''ln|''lll'|'l||.'''|j|'|l|'lilqr^losrr to r..ltrrfu .rÌr'(aN6t /, r, srcìiiuÌr1) rtPUu|rùr r.rìl(r r puÌr surun'lur s.(()!oruo.rqr r lsorulÌr l() !ortrtosir.rtl stllU.r.d srsrilIur).1() iclir srqrqinoqtly r.\p rrt'r.ds ),) pL[] .lv qlr.\\ îu u rri îro. 4tnr rs rruÌr srsitrìurl.lrurÌ\rq !rurt)nt.ur 'f.ìs.1 usrLt ir\rrt !.rpror.klù .r.rllo (66art t, r, ìrrsi.l:9af,l '1, r, !ì\oìltnu)) ).tùìtù,1 lur)t|) l)uÌr'(s6()l ,1, r.r Uur[Í].N) l/i rilrrj''''',t t,t att'r I rr'' id/ ,1l.lrr I r\..trrri ..,l "lrtrììor.l.lÌr srql suorr.tif .(r/pur suollÌr]otsur)ll oì .np pin-Uìrrt. sr iE(rpq.lrotrr."()!oukrrqJ qirqr\ ur \ì.ots tuo!)sorro llt. ì-ursn t.rsrî;r\ (taú t) ./, r, rlri.r.ntiurètct(Ì.1 \rr .uro.ri\o ot. p..\tosir iq touu|r lrrll s.urosoruorLl:, t)irLs,it.rrtrurs .,\rq sin;ds tùrt.l ìs)t\ 1.llrosourollti tÌrnIr\rtùr Ì{, UortrUru .r.srft)àrrr.uor s.rr.u.3oli. \\rru Nrìd to lU.udot.^.f irll r|lr.!\ ur.t.lord r.!l()

PiDÌuro sr d.ìs u()rÌù\rt I q.rqr\ur .rùpitrord i(| to uors.rr,\ p.qrfoúr ìj p..nforrur (ló6t) //) r, ritcld.rÌ)sll)o.r prlq efirtiptÌruro.t tr()rj frlrpsr irr.r s.uro\ourorqtr inrf..ord ptir,iqìirq truù'!) rìrtl ut Gaúì ?, r.) tiI.to(] :!6ót ti?ito(l IuI rrìrrrn t)

s .r rsntdìr r Jo ùorÌr(trlord q8rrt ù uluqo o1 przru<uq:rr,is iq uu. sdrì

ìoor pu'r .tqqs i r.r;otoirìrì rrìì suilt{r.nr r()o.r'rttìurq ol i\rìr.rr liurlpi.s:srurrts,t\ rrrtÌo 'i^o s.!"Ètur,\px tfr..\is srrr.i.to uorrItosr iurosotlrc.rq] rol

rclrÌ ì(!)r.Jn rsn rqì (t .tqrìl) l).sr itÌu.nbirt tsoLU u.irl \r)q (a6art .1, r,tizàtoC) sijurlp..s to sdrr r.ro.r przruojll.uis Ìr.r írorp,iÌt ú]rl srtrrcsoúorqr Ìo.sDJt.r trr:ruELt.:,ur uo trsrxl rtrìpitr(,rd

'r'pr,iotdur. u..ìct r\ìrq s(.Ìsis trìu.Ìu

-riidri irrlto !n-rortÌt\/ (|E6ót 't66t 'tù t) t.lrto(] ltrl si,ruirsotlr).[t] lrìÌì!r1o suorsurdsns lo uortìrrpd.r.l q!{ sor.tqord or rnp itùLÌre sll{ irÌtip tEDrur

r{ots u..q sìrq (srrr.urior{.ì .{olt ìuÌrtd) s.trr(\or(lo.rq] lUqd to n-uLÌx)s

trrÌ srsitrur Jrjr;uÌoìrir {oLl ur ssi.rn-ol(l iqt ritnÌàl)ir lrtuil (\iulosouro,q.'nè) sì'rd t.(it.p iN or s.uroU.ì.4 to uorìruorr.IrÌ iq f.rurtdu s ilìr).r; iquno\\ uornìosr t)uìì îurd.lrtrr .u.r_ lrÌrlt rùirtr sr lt (9161 qÌr(.\^ tnìr ùiUu:ìg)s.uro!.ìr nr.p r .rtrr.t i,\nt sèìrads ì!ìrld ruÙì(nL r iitìrJuìorìo.è ìsot\

(f66T .rèì. rJ) s.rto(l "" rlUrLrd rurosoú'o'qtr f"r'(066i U..\ìr.(-t !uìì ìì r(] !ìì^) sJ"|rq't V\C Jttìii.l! itrrosoo!)It.tnuoìt.nitsuo) (af6tîq. l);ud(lpruiuil.krttìrr.ràÌuu.rhrùn.qolu$ Jsui.qsììrts.trrosoulo.rrt. p.r.ìos p vNC s.rÌLturnb.ìr-r:lIrs.di].uìosourorqtriìn_IrsÌnuortìrJosì sìrur..lÀÙ.úù)litr $oLl srsÍtruìr.urosorùo[t] or Uoìlrppìr ut (Zaf,t pr.)uro'qrso8 :SU6t trl,).ìooti316t iollo)s'rorlErri(ìù.urosouro[ttrtr]ùrl.nrlspuÌr

trrl r.(llnu to uorì:rit.| ()J t(!)l lì t.sn ìr iq or t..() Ìd srìq (;ur.lix),irrì sou) sluos- troiq:r Jo u()LÌù.rrrssrt. i lruroliir \\ou ullqÌ..urs^ (zsttl tot.t uoÌr(l Ìart6l'7, /, ourulr'):(16t '7, /, irrD) \ùri(ls trunur.riqnr t|ri.\r\ púrÌ suI rnrt rot

30. sa.ti.g ofI,tanr ctrohroson,ès

Lif ofPlani speci€{ for which Fror.Has B€en Reporrcd

cytohertic Anaivsis of Miroti. Chtofr oso,hes

L)'t oryr ù où . tcùt.rtorL\a/t.ru), rnìt!Li

i.Ò!nxtu |IL nhap,LìJb!ì(t NloniPlìtI fronrpiallsMesopùIll !j!Lofl$ts

i2t1t2()

J21

2)a L (6)

r (ó)

2

a

)

1

2

2l1)I

2

I (r)

N,l

l)c L[LudB],r\(198.1).t).L&L and sch.t |,)N6i

ariiralikov:i ./ dl ftqgS):

AùnùSu har r/ ,l I t99ll\rr'!rrrr ,i/Ltrrlr\ . . cns ,r J, ( ru2 !,,i)

Con,r c/ d1 (l!N7. i98rìl

Schia.zt.hu.rnl /l9orìL.. t al. t.1,)t)ì )I-u.rcticr al (i9r)31. t)ol.t.

lroicTel.vLi rr nr I t9,)a)

Bosclmanls isolalì(lrì

palts (c.9..

F (Dolctel

liDal hi.sh

,e simiLarly

s problen,

)9-i), P ,,rer dl., 1998;

Lg bivarialci. Allho gh

nl DoleZcl.

(PRTNSES)

dn..nìiD!tÈf l-tr. nuhb.Ò nì r. rnLf J\! L \! lh! ,. r xrurn

lM.rc.r\ ù l]1, to,.ri) Atrhouch rr hiìs erÈ.r

'',\n.,h n-,,f,,..,, n.,.^::;;. :jli'i::;:"i,l.i ,1,ii.,'; , .

II. Application

. A curnlll.n t,r^e.d!Lc tnr rhÌnno\orrc ,,r'". ( h.'.,(,er^ .'r ..'i.,,'. .,;;;lì,, urrrr'n n'r n"s - r',n, c,,,'.,..1,,_, 0,, ",,1" I ., .r," "-.,ì, t,:f.'; t;;":t. ; i : : i;'ll j,;J,,:..:ili,li..r,r .o,rr' É Tt,,, c,,,tìr..,. Jur,,,,., L, n\,.,rjrr.,.ec,,,.\r,,rr;.;,ì,;;:.i,:;,.:mu\^, ,,.''.f, .,o.. r,"n, .1n.,,,",ii,.., ,,". r.r,.,,, ". . f ;: . ,;;".,..1..'lrcc\,l',,c.'1.,r,, or L,r\.,, ,r n,$.,..,,,. ,,.."-,,r.,."; ;,.'l-rrcLlcl rufìro\ùnres jrre Èr\cr Iìcludirìf rh,r{'nUl\l\ÚÙ||l''|.'||m',h.U'|r-nl'"'J<.ll..f:|' ''reJ c'io' ro'ú rre ,. r"" '' l'"i' rì ' '

i: :"1 .l'" 'lr 'rrr"'! . r'r fu i\ n'

.h,i re,(,,,' (l'r t{, I'r r|r u'l'n. r.

I ;iill,l i-i;,rii1l iji1lj r;;,;j,ì ;r:.;";; ; ;;

";ri;ll dl,*i;ì,i-ii- [ti6i

sùo]rnlos ìJors puù srrù.aùrìl .g

i'iÎ,ii t ,,ii, i,i;i,,, 'ii-il .-,' 1;i*

,ni,ii,lìiiìir:t :ìiì.Ì!Ì$f;TijiT,rt,fii,iil,j:ffi

,,,.

i|rr|t

st)rrs f

i:i,, ii,i li ;ìi. i, i,,,i:"r,:,'ii;,,irìi i,;

iitI;:;'1;-,1:l,i;"I'r;.i,:ìltlììlJ, ' i' '1;"l': r" r''\

'

, r;'; ì :*: .; ir;; rl

:,:iir,,liiiti r;;;ii_i5tì:tJà1ùIlt rùùtd -v

sIr,:Ireìut\tr.lll ____

I honrF rvith

2 ùnd

I fronìline JlJ(JIC,

Krcm

I ftorìr

:iinTolfarthc

30. sorinÉ ofpl.nt chroDorohcs

1.

r.,-o. Jl-862?) rnd renrxinìnspl,rch.rsc.ì ffolr sjqma.

Horslarcl s Nutfjort Soìutiona. Sot îion A

reagents uscd irlrt not

tfl0 Jng HJlorj.l| ùt MnSOr.H:()t| rrg CrS() jH.O2t ng Znsor rHi)r0 fr! tNrr l,,Nt..()1 .rH,

l0(l tnl rvirh,:ÌcionjTcd H:O arìd srore:rr.t.C

Lj nìi t.n..nl,nLc(Ù lrs()

\tilh dcionizcd Iì)O and srore à1 :1.,(l

7

stecilìcally menrionc.t rvcre

AcljusL \dunrc to t00 lnì

)micalLrdole

i pur

aralog

-1.:16 g Nr:LDt A2.i9 ! FcSO;

\Jt|sr \,,iumc rn.rntn,\|rJlel\ l0iruìJ Hu,,,, ,rL,u,,i, '1i,,. r,i,o\.n,o$, ,,,,. .:',:l .1e{r'rr'u'r^ ' { \,,:'' ,i,,',e.. .. . w , r . . , . J I r, . ,

I I | . rJlrr, . t,.\1, rnJ .,r,t)r\,r..

d. Honrtdnd\ Sto.k Sot,riut (tlx)tJ y CriN(),)r.llljot6g Nllso; 7H-()l.:l g l<N(),0'6 ! NJl.ill;tO.Lj nìt sotùtiùr

^l)5 ùt rrùron B

Adiust lotume t.r 500 ml wirtr .tejonj/ed H,() and store ar.1,C.

c. Hoarldflt's Nutti! t Sot,tio, (tx)

0l) mt \ro.k sotùùr,j nt nnuinrì C.

Adiùst \,,lulnr r. i r nrt $irh Ller(,.,/F,l II U. pr. t..e Lr.. .,,J||ri,|| r \. \\

'oit f,ànuor.p

iìursn S 1ol Hd

ll)l X u.rrr I l?i 0!al).N iì t rl: I

vfd!{N i] Ì!(r 0vr t î .1j!11

's.s.L|urllrd ur u3\r;.rr \u(ÌìPrtu.ruo. truL:I

-rÀllrrlj rP{rtiPtrxr,rcj l

HOTN N I;ursn ! I ol d tnuu iqt tsnlfv O.H pizruoìip rttr.!\ tu 00! or,unìto^ rsnffv

I ^r

or o1) ) ),N ìì..6.fllut) ) vr.t tiN ìillraIlnu, l) ) iLl ìj lrìeil

.r ) jr.r' d r' rr\r; -r 'L I,.. U J r". r I

.r.)|ùft sr.rl t_

rsn arol.q rsnl r^Ìlf\u iqr ,.rEd.,(lqtìÀ\ tur Osa ol iurtor ìsnipv ( t itqPJ iès:tèf.èu uonrrìuo spu.drp pippu runìto^ .ìll) .firt.ptrtru(,t fpV HOUN N I

trurr 3qt lsnltv o;H.pèznror.P qlr,\\ tur 0(ìz ot rlunto,r lsnlpY

'àsn àroJaq lsn uoqntos 3q1 i.ìrd.r,t ( rtqì? L .:rs :p.Pi3uuolllrlrui.rkrr trrtrrt .rìr uo sp sclsp PiptP iunt0^ èqt Èur.rirrs snonurluoJ qlì,{uoqntos luaulnu \.purtirx)H .qr ot uorì1ìtos ì.ols ìriqìiur soqclojdìulìr pfY

uo!1nlos tlt)uqú J I

slonbrlx trlr I ur i).02tu r.urs iuot.Ir ptor..r lo tru 0t uì lÀqliur-soqdo.rdrurìr 3u 9s1l.l i,\tossrc

(It t 0Z) ou tos 4rots ú

suouLtp\ 1iqr.'n rrqdorLLury ;

esn i rol-iq lsnl uorrntos.qr.rrLl.rd Oil p.7ìuoì3p tìlt.\\ ìru 000t ol irlllìo.\ lsnlPY

j 0orlrÌùs [u !1rùooit'\ rl.ors [ù |l

(xt ù Dop t'Si tut!.ttrN s,uuqt;hoH I

i:i:

! !.3

?

;.3 .'!_

:

jds;

E;:-::- -:

:ì:

-i.

:i ri:i

a

:

IÉÉ

.iî'

p9

rl:t.èlOJ'*t^tqt

;lel!tilîl:tclat:i

r:l

E'

E:

ie

10 7.5 using

to 7.5 usiDg

nizcd H,O.

F

í^rù 0l) ú.N ii 1Ì l

{rx u 0t) I l) 'l

961

s.s.qlurJPd ur utÀùj.r'ì suo'ìtrN':ruor Iìrur4

0c rs PnH ÌFr:rtls I)d ft

,4r\ zl

rs

kf 0t

Pl,\] 6

'slonbrlE Itu_s 0

Lrl J,0a lÌ arols s3lrrlrod llrrurs '^oluàr ol rellu urd zt1) ìr qSnorql lail!:l

r'íìiq."r ;."'qi;q ortl pi?rtrorip ltu 0s ul v uú'{.u'rqrlu, ""ru 0s àÀpssi(l

(rn/itu t) uouùlos ì.ols ul:)\(rrllF'lN I

tr èrols s.pllrt.ì lì?urs r^oo'r ol r'rlu Lu?'/ ZZ 0 rr qin(rrql

BuurDs,\q O'H p.zrLoI.P l 0-. ul IdVC ;ru E à\lossrc

(lttr/S(I l (, tto'rrrTos for< l'lv(l e

sl.nìbrt{ì lut 0T ur')o0Z lt.rols ll'N rlut Puìr louritlèoldPJritu d lrt 0ZZ

ppv ,,"1"'iì,,à Lr;;,. ;,,", oì .rls urì1'zz 0 ' q€n.'l r'rlil IJH l"r I

ii"l" si.,t 11.i'1"u'r r"nipv o'H Pézruol'p tlrrN lur 002 or èLunlo^ rrnLpv

'tr tru 911 u' :)o0ariltr4 ùnr 09 r')l

Oitl \ tr.rrr I l'/ 00a

l.)uN i taal)lJ) î i6! I

ll r, .o'ox.ds i sta lì

VI all:NN ìl6tl0s' t 3 agt.0

s3siqlu ud ur 3^l' :ru suorlrìrlrri'uor ll]tlì'l,,111 r\ll"fll

Il

9l9L

0a0i

'E

sr rr lS

nolrÌzlurioÚoH trrnr{ràl^] put uolexlJ .PrqaP*urcn rJ $àr llajPd

l5

l0l1l

to 7.5 rLsing

ìliqùots.

i0 lnin fillerlin 0.5 mì ali

ing lor 60 nir'r rt 20"L rn

30. Soriins.aPlaùr Chromor' e! 11

^diuf volume 1() 2000 n1l $ilh deionircd HrO

9. MrgnesiunÌ Sulf:te Sro.k SolutiorÌ (100 ln'u)Dissolvc 1 23 g o1'MgSOr lH:O in 50 nl of dcionized H'O fillcr through a

0.22,úm fil1cr to rctìove snìîI1 pîrticles. and storc at 4'C

10. Pj\INS l\crction Mix!-inal concontratiors in Parcnthescs rre calculated tor thc volume ''l 50 /'l

Note tlìàt lhi\ 10x L.I DNA polvmcrase bulícr co]rlairs 15 fnM Mgclr'

5 /,1 ll): L'l DNA polv ìtftNc bÙlf'r (l x)j /,1 25 n,V ivlclr (1 ùrrì/)

25111 : n'1/ dCTl d(llP (0'1m'l'/l1 /11 0 I nì't llÚr's'cìn 1l dtr fP (: rù/)I pt 0I nr,U lluorcscein 15 dATP \2 t'M)t1pl Imrvdlll (i1r..V)11 LLt I m'v d,\TP (rl sM)15 41 66 4M PrinÍ '\ (2 {M )

L5 |l1 66 PM Frrnìcr B \2 t'M)0.6 /rl j Lr/&l Tirr DNA lnllmeus' (:r tr/50&ì)

135 4l HrO (5 rll tur èralorxrniì)

11. Srop lluflèr ri,r PtllNS llcrcdonFi al concenrralions arc given ir parcnlhese!'

2921g Na'l 0t'V)I 861 g Narl]DT.\ (0 05 '1/]

A.ìjusi volune to 100 nrl with dcionized H,O AdjLìf final pH 10 8 0 using L N

NaOH bclore a loclaving- Siorc ar'1"C'

12. W.rsh lìuflèr for Ì']l{lNS lr-ci.rionFinal ooncentralions arc given in parenlheses'

1 161 g n'lci' acnì (01'!1)(rsr6 .g NaCl (015 r1'/)

O'5 nl fween 20 (0 0i9'" !/!)

A.ljusl volumc to 100 ml wilh deionized tlro Adjur final pH to 7 5 using 1 N

NaOII beforc autoclaving. Store al '1"C'

13. PCI\ Premix

Final concentradons in parentheses are givcn foÌ the loÌume of 50 fil Notc

that thìs 10x Tdq DNA PolvÍrerasc buffer does nol co tain MP,Cl':'

DTV Z

sv.L PUr résPt (^11) litor^prIn uo qr,{

.r.tri:r àJdadorsolrur è3u:rsèlonH

lsurssa.ord istìrdp.ddrnbàl riìros p!rp. .r4iluoli:r \\oH

'll

L

s

L

slcualel^t rar o pùù rùaqdmbg .i)

pèrììdar.l n s.Luoso(l,o.ftlJ lrlìl r Ìo suorsua.lsns \.oosouìorq] rlottru ruEldlo Surtros pùrì srsltruE ror padoF.p u.iq a^Ìrq paqu.sip srrnpiror.t .q r

s3:rnpcro.rd.^I

u. t )< t srrEnbs (lnd 0Z pur. Lud 0! èzrs rrxl) qseu uol,iN Et(vt{:ù{o1

r.lìrM q.Jlr3sià fm) t?t sz n'ntoÀ .s.rrquìrqJ uorìÌrq.rur lrss-è[urr]lp.is pilrunurr:r ptoq or liìsEq qssu ùrìdo up iurpnì.ur

'Gq8rèq LtI. 0l qlpL.\\ Lur S'rltr-uéì ur .3è) Iur 09/..(|.D JrsÌrlct V

redgd rèìtU uo ùorreurlurab'fè.s Lor (r.lrurÈrp nrl] 8Ì) s.qlrp urad ssqDilìtrad rn uoÌlruurur8 pai\ loI

0q3ràq LtI. ll qrpl{ ur.9l 'rJr,5u.t ú,. E2."":) Iur 000f i,Ir rlsqd V(uorÌDuurît p.as roJ ilprtsctns uiur) a]llr.,l

uortrut^ ro 1 àbrtJ:ror NOt .ìoJ pilrétrc(8€!3T oN;otsr,,,:) rud a spràq Dr sr.u.r.sitod'î.) speiq uorrìrrqrrrJ

uoÌluuf^ Io ruiri]uj.or ^\ot.roI

piìi].t.s(0t€8T o-{iotn)0:) Lud ! tsPìrèq g$ sr.u rsiìo.I,3 é) spciq uoDErqrlpJ

souors uortrr.r puE Surqnl qlrÀ\ r:rtqqnq lunrrrnbV(àqor.l

l/S0ZÌ Vattd r qll\\ Ooztld uorr{tod'3.) rezru.toruoq triìuurpàt\:).! 0 i .! ol piìrsnLDìr irrllrriduì.r 'rtlrq retìr.!\ pilolrJ

ol pàrsnipn .rnr*îdlual lì,or.Ìù,'(ir,rooJ/BuD,,"tt,,,r",,-"iii"%j,;ii

ÍZI

'Ll

1)l

!.Z

-iq itìroqs x'uFrd 3rll .rPd.rd ,(U.uq sruerp.r;ur .qr 13nrrrlui.

(rt 1)!rir !alitln t)(An )

(r.lú io)lr{or 9l)

(rl)

o:tì r.nror.t ilu:Lsr$DùiLùl \/Nd ,rJ t/,a (

fl Iúr Àrt O.\: r.rtrud

^,/ |(

sdlNt) r'l| r l)lljilt\ 4ùL la

lJilù nnlu.r !{, srop r.!nq.qr).'rtnq r .[it.d V\tÌ,./ !0t

zt

d sorllrg oI

.DA 1205/5

gNo.183'10)

gNo. 18318)

t cm heìght).

) cln heìglìr)

:l lì. Sonnrg ÒfPlanl cnramosÒfles -l

frol1l rool iiP\ of young seedlings Prior lo chromosotne ìsolation' nlcristenr cclls

are accumuia!c.l at mctaphasc hy a sequcntial lrcahenl with a DNA s-!nrlcsis

inhìbilor hrdroxyurea ancl a milotic spìn'lle ìdritrilor amìprophos nìetlìvl Rool

tips accun1ulaled at mctapÌìasc are nìldly 1ì:ied $ith Iornaldehvde and intad

chromosomes are relcased ìn1() a l)sìs buffer try Ìneclìanical hl)lÌogeniz'rliorì of

rool tip mc stenls. SusPen!tuns oI intaci chromosomcs are slained b\ DNAn"orochromes. ard tlìeir fl orescencc is arolyzed b) lh\Ì tometer' Chrono

somes can be sorled on a sli.le lor nricroscopic obselvalìon and/or coÌled'd ìrt'r

a lube for PCR and other applicatio s

A. SaDrple Preparation

1. Sccd Cìcmrìmttor

Two ,:lifferc t pro!edures arc used to germiralc seeds of legumcs and cercaìs

In both cascs the gcrminrtion is periomled in the daìk at25'105'C'

d. G4111i1.1tit íf Perlite (Vieia fdlrd' Pistult s'lthJtn)

lmbìtrc tlìe secds for 24 ht ln dcionized HrO lvilh aetaliù (-30 lccdlug' Icnccded t0 PrePare one samPle)

Wel the perlilc $'ith thc Hoagland's nlLlrient solrÌtion rnd put il ìnlo a pl'rs

lic lra,v.

Washthesecdsi deìonized IIzO,laver them onlo the surlace ofperlite co\cr

thcn \lilh a laver of 1-2 cn of \vel perlite

Cover tlìe tra)' \\it1ì îlunrinunr loil and lcal'e thc seeds 1() germinîtc for 2 ll:lays to achicve proPcr lenglh of prnÌaryr roots ( table II )

Renlove ihe sccdlings liom perlite and wash then i deionized II]O

b Geflttifldtion ott d FiItù PaPú (Hordtuù ""lrdrc'

Secde cÚ'Ilc)

Place lelcrd Ìaycrs of Papcr towcls ìnto a glass pcrri dish (]fì cm diarneter)

and top theD ù'ith a lingle shccl ol lìltcr paper'

Moisren the paPcr layers ùìth deionìzed IIrO urlil runofl

Sprea.l thc seeds on the paper surl'ìce (-50 seedlings are Dccded io prcpare

one salnPle)

Cover thc petri dish and lca\e the scecls io gcrmiralc for 2 3 days 1l) àchieve

proPcr lenglb of prhaÍv roors (Table II)

2. Accumul:ìtion of Root Tf {lells nr Metaph.rse

Adìust the lcmperalure of all solutiors to 25" 1 0 5'C prìor to lrse PerÎornì

alÌ incubarions irithc clark in a bìological ircubator al 25'a 05'C Acrate

.ll soluiion!.

lJa

.do.so.r.rrlr .).u]]s.roìrtJ u r.rpun uorsu.(l!ns.ìrll Ìr) {rtnrh.ql iutrur\grtsiu, uotlLr ur/ 0a Ì qSno.rqt r.rurl

lurFr/ z lo Uol]Ìrrìul)]r()r tlurr c ot ì:)otr Ìdv(l;ùLppr iq uors ..lsns ur srúrosoltrortr .r]l ùllìs

s5lt,uV rtpurtút!l toJ fttltlt\S IIVA .r:ì rÌln lr .rruosorn.r'lrl:) I

stlrt toor rrúrr qtLssin.ds ur nr u.\uorrr puc snr)rloqrt ir)ur !r p(trlr.trr strìI s.{unt. iruosoú!)Iltrrs.ìrdsrp o],tp..u iSnpìi Za ìr rtinorqr r.uo piì-ur{s iq lsnrr u(rsrrrdsìrs i(I'àsìr. snìl ut r.lÌrq srsit t(ìg't t0 tur I SurLrrrìno. rtsrp nl.d ssutS E ur tidlrr.sdiìrqs ù Lìlì,$ rllr rrrcr.)rI iurddoq.,iq prlrtosr.ìq oltlr rr:r s.ìrrosoulort.)

.Jr uo uorsuidsns.qt ijols3qnt 3ui.riìsitod Ìr olur qsaur uotiu rrr/-tìc x (É'no.n uoìsui,dsns ìl.rittr{

t èìqPt ùr ùi.\rn- spoìr.d io] rud.r 00!6 rù 3urzrui;'oluoq iq sJúosorro rt. .Ntosl

'r.llnq slsIt t0ut lo tru l 8urunr1uo. eqnr èu3.rjtsilftl trlFr u oturuiqì réJsurJ] PUr (s?r.àds I uo ijurPuidip 'rrrr z t) sru.lsr.r u 1oo.ì :ìsri\q

Jog nr ulru ! rol setllr] rilltl .riÌlrìq sr.r I Lrì sirú.ì 3rl1 qsr^\

III etct!t. ur ua^r.a spor.r.il .l(rt ).! tìr uriqr.Ìrclnrur fuP a.\rtìrxu 3p,\rJàpJìrurot ! otrìr ru.ql .aurrr:llsur.rl iq sloo.r .rìl xrl

O.H pàzìuoìip oruì ru.ql j.rsuìr'Ir puu (u,. t) sdìl roo.r rsr\.llrH

\uoAU.xlsn\ .)trr(\otrro rLl') Jo

rqîrure^osloor:ìqrlxr,tpur'rolErrir.rtrrrrurrrurìrtuor.rrlrr.ÈU (.)-Z ot)OiH pezruorip puE siqn:l']r f) illlt\llfl | rllrN p. I r.ìrEluo. p ol siurtpiisrlrrr!\ riìsìrq rqt.r.Ìsuìr.rr'(,.!Dst^ II .r/,:r/,,.) \') sioids trr.ri. to isr:r ul

II 'tqE.t ur PitrJrpur spoui(l rot in:(tìr.ur pllP rlonDto! ru.rllnrr.u

tÍrlriln !oqdordrlllE rltr{ pi !r icrr I ot s!4trÌtp..s rtrrN r.ìsrat rqì r.tsurrlIt iftut r" p.rsrt spo

urd iri\oii.r rot uorirtos iurì los s.furt;rr)t I .. rl 'r.rni\orfiq ur .tpqnrul().H pr/ruorrp to sin-ueq. tr.r.\is ùr itsno.loir\ !Ìoor.rtl rtsr.\\

II itqìr.L ur pirsrt !p0Ìr.(l lot .IìrqÌrrur p[r rkrlDtosìr.rn,{xorprrt .)rtl ììrllrluo. ir.rl.rl!rtd | ()l siùrtf..s qlrs Ì.ìsr]q .qÌ rilsuù r-t

olH pl)7trlor.p rllr$ p. !r úrr rrls'rtd | (op rour\od 1.rìsr(t rtsrllr ù.do irtl to srt(trt .ql qn_.orqÌ sr(r)r r-ùrlf.is tr)irrtl

(lt ,rtrLu.r) st,.,o., irrrtrr.(l tr) rll""uitr.rlorcl rqr qlrr s;-urlprrs r-F5-

tt

iLioncd

r! pcfi

mctlyl

:d Hrorrniglìt.

ìn Ta-

ì slìarp

30. sorihg ofptant chroDororbcs 15

b. Míthtdlnydr/DApr Stnínínr.lù Bihtídt. A dtfsísAdd MgSOr ltock ro nrlt concentrotìon of l0 mM.Stain thc chrorrrosomc,i ì

r'nn ol 1.5

^.g/ùrr .,.J''^::'ìLl'""

/,', "'l..r'ns

D APt \iotk Lu lrn'rlconcerìrra

ot 20 t".g/ml. or 'Ùtlrn! mjLhr rrnrtrn rr,'rk tn nn.t (urcerlralion

Leavc to equìÌibrate fbr jll Drjr oD jce.FìÌler throuqh a 20 /rm n onlneshI \. rì,in, .he qu,t:,\ ., u.fe .r,,jIJ.,r(r . nuur..c..I.i rr:,.,u.c.,t.

R. l-lo$ C)ro,Ie(r;. qnat)\n dnJ \orrin". v,'vr.//ii.i ut lrr ,,rFr ., J . r tr.rn. r

1

prcpàre lhooreticaÌ Bow

_ compu,e, so,6vare r".ll'.;':1.;;lli;:l,j:l i;ii;"","., ., decuca,ed

Dc,ennire thc,",,,,,,,",1,.1j,::.11:l"ll " ,";;;;,,;,r,.r.,n,o*.,," p."ren.eJ-J .,,t.crinri,jr,. i.ùi\r,llr tì,.. ,o.u.r, .,r.. .. ,,,....,

2. Flou Keryotlprng

':l.il.:ii't'l..;Í:yi:!uni\.'rc''1'|Jhsis the'uson jor,i,sc, rs operalc.o'*, "" "'"r"..i.ìi,"li.l].

I

- -11'J ) Diìrl.$rlh -rrrr '"\\ "uLpur pu\ver: rbrr:si i-ieì.i

'1,ìì "ili'r"Ì :iì:j ("1n'" r' ',. I r\ urr(rirtcir rr murt, L v node

i."l;' "p*'l.i l,i iìi ii:l",ll.:''-'i.i' r) \cr"rirLr Lh€ \c!on.r riNr (argor

I . J,' l''e'L'l \''1 'l)'ì ' \[ " 'rL' r ' '$'-l

rr:,rr Jdrf,.,r " 0 rrr 'r rle' 1""' 'r'' 't '' '1,'' ' r ":r',"rEqlly rhc wafe conranìe

[uìc] S}-.jo. r ard fiIl the lhcarÌì corìlainer rith slcrjte shcath

Adiust shcarh Uui.l pressur!_,ru L psr. xn.t le.rvL rtìc [urd runnrne to filt .ìtp1.,,t1. ',re. r,.o r,,tr,,, .,, ,,, n. Ime,rlInstlll a nozzle (7ll lznì orjtìce) ancl chcck fbr arr bubbles.

d. Insrtrmlnt Alílnn n!

'"ìl;ll ,i:;i"jl;:'":::: ll']. ì ::' \" . '1,\ ,!-o pj,..,2. ,,,,pass -t30/-rr) nnì i" i..'', ,:'. .1.111']

dcrccr"ri r''| r'''''';r' "u'ir*. L'ona

' " " ,'; .. ". i;ì'.,l..il.l"jj " - J ''j J''.' *'À,4 nì '

Sct ùp lhc lrigger sjlnnt 1(

threshotd tcvct r tor wàrd scirÍcr hejghr (FSC Il). an,:Ì sctecr r

RI' .x,:br. ,u, r, . ù! ,lolilì;;";;:,,,:;::;;i::l(,rnr'r rrc :ìn:ìr\s\, pnÌrseie,c,. Bts b, ,r!i ro,'r,, . yr, h-.,J.\dr tì,.$ r..\,.t rj,r,j.,,(J.,.\.

ìsrP ìr tro llìrs:r èLlr a\Es puÌr siruosol orq' 000 0s 000 0z iTrl?uY

v-t'u snsri^ v-l-Ic fo lold-]op u uo EllP 3q1 fEldsld

(Y t'l:t) Ì'rP

.^, 'd tt l fr" Y l l \'li l^ nu' ror'!l\ 'l 'li 1>'r'r" ' n i|'r"' r' '.r"'J.

,J,::"" " j'','i''*' r' ; u r"''|r rd'' r rre' r:' r'^\'J rr" rrrJ^rJrr L r'r'Lr'\

sdunl. iÈq ! ì? Ièpnu

'sucL.p epnl.la ol Ir I'rir pÙP H r)^'l ro lolli roP ìr uo uo'3i]-3il:11;!is

002 i .) .rP.r ^\ofi .lìIrdordcìP ÙE lu 'zrl{qrrls

1I r.l pur' aL'lturs 3qi urd3utt at.lrurs irp

'lPrqLlmb:or ,. '(,u-I'IIJ ^u' ld\ U r rr' rr-oj I rJntl lr'u l' lour/ \r' ui'rr'! ' unìl

'rl \\1r 'Lol u"'rr' "uJ'Ú' ro'É"n

^rrJ ur \J'L rtr' 1r' r"r1 "':'l lu''lr Jr' r' its rrì

'i,1",;il ;ì.: l; ìr..,.' r.r''a'"'"""runu-urrr'J u' ri"ur-'u':( I

sr'(louv ttti!Ú1!g a

tsrp E uo rinsei :tll '^ts PLrÌì s'rurosolÙortll 0001q 0001ìZ 'zIiIUV.v r r;,.,."'r r rr, ",::lì::r ., :l-,:, :l.ìii:J,:ì:"rì"iri"

,,r.,ir.,r,u.,r"qr ....r'r' . ,ro'r' ii,r,rrJrL ,. -,.un,r..,,, a,, ,.,.,.u

,suqip .p,ìr.xi, or H r-ri pu' s 353 ro rorr r.n " " *ttì'j',::ì'j''l:i'

002 '3 i) itìL\\ou 3rELr'lorddìr ìE :zLlrqqs lr tt' 1"t" "td** "q],ffi l

.ql .rrrqlllnbe oÌ (IdVC S rutPruo' rafnq 108-I) qdlÙts iúrutnp rÌ d

èp T'trJ .qr to rùo.rJ ul pi'ìrltl sL r'rlg sstcl puPq (uu tl/fzf !qÌ lrrql srns 'ììrl\sliltt'uH è|nÚ'attti q

p:P3arÌ s| srilatllììnd 3uLr Plr3p puÌ irìip r'sE1 lenp rsnllv'sprlÚor rîqro

rn'n. ,úJ Ur'! irL l t uJ)' rur rur Jrlr"J '' rìr\ (lJÚ '; ) J^" l \

;;"i.';,'; ,.,"".,. Dtr'rLudr'rurur \':u' 1\rrr''uo'r''t u \rrrr

"''i:;. t' :'i ; :'"';' rIour/rior'r"r 'n 'r'\r'u"rir' \r''-rIll ' llru' H ''r " !]]ll l" l l'1".'".

l). JinLUr'irrLlrr' \l' ll'"'' 1"u"'' -'nrrr\r ru \\ 'rrr 'n Jl lu'ur''r''tr"rl-uoLLY

H IIlrull )\IJ t''':^'' rl o

,". HrII rq:,'ur','J III'rrcH ì\I1 '|d 'n r"r^ ' ìot r'r' \ 'l'r\r(l

'Jr t Ìa:Jìod rr{sorul

ris.

\).

r{J_ sÒfiDg ofPtani ctrronosomcs

3. (lhro,ùosollÌc Sortng (rftc, Univrdr.c An,ìtvsjs)

1r-

SNitch on 1he sortìng module and îdiusr tho drop.lÍi!c trequency and ctropclrive amptitude 1() Lrrcak rhe lheam at a.,uitable distaDce from rhc laserilìtercepl poirr (chcck for satetìi1c drops).

AdjLrst lìrc drop drnc phase to obtair slngtc !ide strearrr! (test lno.tc an.l rersort musl be turned on).

Calculatc drop detay and pelforll1 ìls opti i/atìon.Select rhc loìr modc lrd soft c.velope according ro the requircd purn\,.

numbcr ol ohromosomes to bc $rred. an.ì desire.l lotrme fbr ttre sortedfractio.

Run thc \rnìple. and display rhc \i$als on ll dot plol ot FLI puìse wictrh(It t\\)\Lr .r ,|. cAdjuSt rhc FI_l-W a ptìnergaio and widrh o .,cr as needcd 1(] achicvc optinal

resolutìon 0f rhe \vìdlh signal.De1ìne sorlìng regions (nr tlìe FLt W versus FI_t A.lol ptot.Clheck fìrr srabilirv of the break olt poinr and oI the si.tc streams.Sort rhe rcquired runìrer of chronosonìes o.ro a n]icroscope stirtc or into a

pol,r'sr\, rcn e iube as needed.

If reanrl\L. ut .ort.J Jlìrnna.,,I( t î.rr. . -n,tor r{,, .tcp,...||r,! i. r-quìred, sort ar leaf 5 )< l0r chrom,,\,,mes ir r,,l-i0 y I ot LBUl !r . tt,tysryrenetube. fhc soúecl il.aclior can bc aralyzed atler rdding DAt,t to à finatconceotratìon of 2 /.g/ml.

4. Clhrornuomc Identifìcrtion rnrl Puriq ofsortcd Fr:ìctr,ìThe conlcnr of chronìosome pcak! oD fiow kîryot,vpcs aùd rhe purjry of thc

sorled chronro\ome fraclion can bc clliÌnared bl, fluoresccnoe nìicroscotr] orailcr PCIì wilh ohromosomc specìtìc primers.

d. Fhnftye d ùIi.toscolt),

Pjpefte 15 pt ot LBol burrer contafiing 10E (\,^, sucrose onlo a clean m!!ru-scope sticlc, sorr 1000 ctroDìosollrcs 1l) rhe clrop. and air dr!.

Drair ercess lluid and nrount in Veclalhietd anliràde sohli(nr. Blor and \ealwilh rxbbcr cenenr.

Exan ne thc slide \\iih a. cpifluorcscr_rce microscope equjppcl:t $,ith a fìttersel f.r r I)^PT

Lign

jrst

20t)

bris,

h. Pincd ir Siùr DNA LabeliÍsPipcttc 15 /rl of LB0l bufîer conrainnìg 1(ì% (v/v) sLrcrole onto a clcar micro

scotìcslide. sorl 1000 chromosomesro rhc drop. and air dj.yjr an:i!epric box.

r\otc I

tltúrd Z

r:f I

'r .r.lq looÌ morr paseelsÌ.r! seuosorlorqr qrq,\\ rrpun suortrpuo.3ltt uo pLÌD u'.ls erù loo' iltl ul qlè. isEqdErelll Jo uorrrodord iql uo qloqspu.dap tI .ruruodrur érlrlrd lo sr uoÌsu.dsrs arùosoluorqr lo ,{lrtÌnb eqJ

úo4rredèt<I atdures .V

sornpaJord oql Jo slrodsv IÈrrllJD .^

.qdÌl3oloqd pue rprurorq unrprql. qtra urrls'to8 isore.èP /,,S L E uo snàroqd

-orl..tr uru puE àqnt qrìr. ruo{ (td s j,) strnpord dtr.IJo sÌunou! lgnb. iìrluìLu 0l roj uorsu:lrà tùr'u

èqì puE':)oz, tp Llorslrilx3 llllrl a.ÍEd rar u.l ursoqr eqì rot .lerrdor.tduarntnì.cllur1 agt lE n-uLtEiuuìr ulttt I ,Jot6 te orlernlEuep lllur I to Surlsrsrorselr,{r st iq pel\olloj Jof6 rl? ùrlrl z.rol UortnìntÌu.p tertrur:u:).t [rro]r3d

fuerrq rrds puìr ,aàt.ro^ ixrurerd ìtJd lo I?, 0! ppvìIJd ro.l

ft:lrìrp.ulur ueqt èsn ot srtlrosoúorq].ql lJitr plrr ozeaq ro.-J.02 tD iÌotseqnl qr1r. otur sèruosoruorq. 00! lros

.(t,/ 0Z itarìruÌxordd! :q n,r BuD.ros ràllr

eulnlo^ tEuu) ozH piì,'uoì:p.p31r r0 Id 6T,SorurEluor seqnr à:)(t:rpd.rduoltrtaù utDtD )sútaùúod .)

o.ueJssronLJ :)J_I,t puD tdvcl Io nooù^rosqo snoiu-Ètlnrtlrs

'o Ì trs r arlg Irìr p r pue '(JJH ) rtìruù,{3oltllosr rllè]sàron u

, Id v(l .roJ

sÌes r.]ru qtr^\ psddnìb3 .doJsoj]llll riuJ]sèronurd. qlr!\ aprts iqr 3ultulrrl'tuèuÌà. riqqnr qI^\

tìr.s PUìr lol$ uoqnlos àpuluuÌ ptàntrqr.A uI tllnou' puf .prnu ss.]rr. urDr(rèrrnq qsn,r sNIu.t.rll u' apÈLtl (ìtrvld zIt IdvcI qlrl\ aprts urqsrèlunoJ

l]rr.!!Ì dels Surqsr^\ aqìlr.d:lJ ùrur ! ror arnìrr.dlu4 uroor lp rlrqnJ$r pnn rrtrnq qsE^\ (NIà(T aglJo I/ 00T ppv qsrp rrli.l rì ol epls.r{Ì r.jsuer] pr10 r. nq dols rqr îrourèì{

'J.01 r! urur zrot .juqnr r pu! ireJJnq .lots sNlud.ql lo Id 00Ì ppf. ra^or 3q1 i^onr:d

Jo0l t0 u'ur 0I ot Uo'suilxa pun urlu I olllrLtrauur àqt Srotord'è1.ír tuuq aqt rI Jo0t lÈ uftn t puE.JoS!- ln ullu T'J,t6 tE ultrÌ t Jo rsrsuo] s.t.ir lqBrè tnr'\\o ol arlt:J,,fl/ tE utru 0l .J.9sl1ì urlu s lci6 ì0 ![ui I]o stsrsuotr 3lJir tlLrt rqr:uorlrr?r.r:-NItId àql unìI

'èLl'PrÌ iltl rè^o rilolr;lsiitoct oqt àtrqd puÈ lrrrìr{.rn olur rltlr lor]reer SNIììd to trl qZ àl]iLtr.r

'E.rr uaurrèLls.qr.r:^o.prts.ql ol (td SZ) raqltllrq. te.:--:urpr,t ìiq\^

,,I' ràz.lofl ÀeFor't !t

'c.iog

of

30. sortnrg or.Illinr (jb.ohosor)cs

1'.el]C}c]csyrìcl1lol]jz.ldol1.ltclMPr.^l,'.^^

ri," p.n.".r,,,.. a.,.i"t"||'ìse I'cÙnrÙl't;orr

'n., '''. ,. ro,",.' .. ,.',f;-' 'll'; ' ',",, r: ,ù u.Lj l,\ .o DJ ,h

.; i:r; lirììi-:;:,t r',',',4,',ri' t'r,,,;; : ì: ": ;: t';;:'; :;,,.;ì;l:

"1

Itlil iii';;iiii. ir i,,*,i[1.'iii;1, ;,"",,,, * ",:.",,.i:,,iì;t.fi:;f ;iili iì.lfÌl:ii 1::;l_:líil,il:l;Xi::ffffi:lti,lilj.:,;].,ji1:'ncsìrJ:ì,i.ns ,,,,ec.,ed.tr*.,,,,,i,,1i"'..,*a rr";.",,l-q,,. J,,,,n,,,.n.,,"ì,li:,':'"'' "',''"''''ì,, 1'i lr-;l.X' t "'rt""'' ,'"r' ri ,;'r,"1;,",li,lllllì'il. l "" . , ,;,..;'::.:' ,':l'' "' .,''

:: . ; li;:li:iliil,: l;:l:l,.;:::r"^:. ;;,'',j:,r,j;,,ì .' ;;;ii::';.'

..,,,,,,,\,ùj ,.,..,,.1 1 ìr".tur'"s r.n,,.,...,,. i,;.ì,: ,1,n. À',i'.

" :.;;:il;";lì:;: ir ;: t,lliiitll:,ii:;''i; ' ': i; l,'. ì, l-;;lt :;'i)." 1'. " ; ;i;":llll::i l;:: :,::',1 :l'' : ;i;;;ll.ll'ì :ì ::,,;' ""i:o

2.r,refrú,jo,,orchron,osor.er,,r",,,,:,,:"'*""'"''r"r'-";r'.,'.'',,,,.ì,i""'|rlesuro(,'

l he p11r(Lturc n,r chì oD

1,,: ,";; i:,,:;l ;;,it;li::_,": T: ;: ;i: ti-ìì,t"T t;; t;;i i;i;:,,; - .,,rl,l,',,.,,,",,,".,;". 1,i.]ì,ll^ll'" ;'l:l,1,"r,.,., i"i"'""'-';';;;.i;Ì:;l.li;il:.li::,Tì"1,1ìj:i'"i................;';'':r .1í,,""'í.:ll:::

ll;rl'lli"l;;i;l'l;Irril;' ;:: i; l:;rl liill;;,1;ll ;' Î:;l'::"',,,p. r,,. .,.p,i..,11 .,;;;:::i : ., :,, : ne .,h," ;:.;. . Iì,i1,',. ".

;í"j::ì;::Ti*t jr,:#. i:rr..;tiillliiilF :: rntijtiìtxi:ir'' r.di:I-J\ r' .r ,r,, rrr.r;,, -,',,""

:, - rJ\\nìrle,,, *.,r.,r, ,,, ,.'L.l \e.ra,,.i,r *.,n.,,.",,-1111:"'"' .,1",,

'",: " :'i,I,.; '" ""',' r,, ,,. 0,..". j::lj,;1,'] ''. , 0"'^

^ " , ,. .ì:j li,"llì,,1ìB. l lor,l{aryorypine.,,,(J

ct,ro,ho\on,o son,r,g,. rrLr'\ KJn Òt\ trrn-

.,.i:,,1111;*li,:h*."1il:iÍ,:;:tij:"jH.J.,.jj:l,i;l.i:,x.:1,,,,tÌj::ì

)ùl

l\'

r'rl , 1,, r rrL L ,i ,

rìr.t (Jl

piu,rÌlir.r \rrrt.t.

1l )lo!ùrqìJ.rrÌ Jl.ìr,.! .v

'ÌL\:- ------

ùll

)rtJ.

fCIDìt{rl\U

IT

II

; :: ,: ' ,;:1,: ,;l.',',, ,':.,,: , : ,;; ,

, ,ii.' ,..; ,' ;' :-:':I,l " ' j r: j

-,j, t'; ,; ,,,1 : ,t ,l::, , '; :' ,tHiiilif i :ri;iiii:;ili:: îiri,rìir, ìr,:;iìiìli ì,:r; ;i:rrti|,J #'iifr,riìì th-

, ,, :, :'' : ,', , ' ,' , :,t,,,, ' ', ,,,.; ,.

,,

u,irl.)L r_t lìiì.r(i\.J(J ilr.rf,l l,rL ro rL.rrlrirìlìt .rLn,)niLLr(Jr{i. ) I

,'1''', lil", ,,,,,, II ; r';;,i,ji Iì ],,il:,i 'lt,ìli ;,1'il;ìì,.,t' , ''; , ,i

, ,' ; ,,.,,,.t ':

,: ; , ,1,'

":,:::,r;Iu.;r;1,'.,sli[i,r,ii,rrii:riiil.ii,jr'iiilfd;]i'l,,,,,,,, ,,

, ,1.;-'r,,,i, ;i,' ;', i, '', ,,',,,, ,,, "'',,,

,', . ,, I , 1,

, ," r : l;,ì ",t,: -:,:: ;,' ,,:l

i,'i,: : ': ,,ril,j ) .,,' , '', ',;',''",,,,,,,,i

rllr. Sorn,g ol l'lani chrÒùìorìrès

I in I smîll

Polvnrcrîse charn rcaciion $'ith PrùneN 'lcriled lronì chronìosonìe-specihc

DNA \cquenccs is a scnsitjlc tuol (o icleniil)' chronrosomc content ol peak\.on

thc flos kary'(fype rnd to.letecl contrmìnatìon ol sortcd fraclìon bì olher

.rì.olno"rln... wit"' uring P(llì. ncgatile înd po\iLì\'e controls shoLrld be rlw:r\s

lncludctl to check Nhethel the PCR ran properlv \\ìth thc gìlen lrimer pair'

\a[. Instruments

fhc higlì yìcld procedurc tor prcparatiorì of chronosore susPcnsiors descritrcd

hcrc irìvoives tonnalclerrycle fì\rìtion. Our earlicr re!lLlts demonstralcd thal the

firRrion rcsulted in lo$cr resolLÌ1ìon ol l'low klryotvpcs aiìer sLainirg rìth D\^inrcrcalalors such as propidnÌm ìodidc On lhc other lìand, higlì relolution no\\'

rl,\olrDJ\cru. bc'J'r''n.J r< 'rr'ir'U \r'' f)\ \ ll "r'crr' n "r"-' r'

nit't i'. L,' ,l tr<1r"rrr|,1,\ 'o \lrr'lì :r^'rfrolczt r'l ltr'r-rr''"<ìThese flroro!hromes are erciled bi' llV. and thus lhc procedures wcrc dereloped

lor u\c \\,ìth a Beclor DickiÌrsor FACSV antrgc instrllncÌr1 eqtrìpped with ar

tlv kìscr at lhe Iìrl posi(ion For biÌari.llc (DAPI/milhr'ì ìycin) rnallscs' thc

secon.l hser \vas lun;d 10 157.9 nÌì1 FkNlcver. the proccdures describod herc

. .urolirLl,'u:,ll con, r' c.'r 'Ì^\ L\r'rner<r' Jn 'r' r'l' 'l(J rlr'\ "

. "i-, " ..-Ì \ l.\'r'rrl -\rl'"riú-

VII. Results

thal iU DNA

ús lhe \ortrng.

I thn hafPcns

The puritY ol lhc

s.

A. Samplc Pr€paratiÒn

1 CelL (lycle SvÌiclrîonizrrioù lrd N4ctîfhr\e Accurrrrrlntiorr

Ihe pr ocedurc tor ccll cycÌe '1 nchr' 'n zrtiun usrr g hrtltu' \ urtr il'\'ribed hcre

ìs ver) .1tectilc.:tnd nrjtolit rnAice' rJngLnq lronr '15 lo 5i 'rr( r'Lrtrrelv ob-

làinei. The tìllle rhcù the m.lxìmu nunrbcr ot !ooÎ tiP cclls undcrgo nrìrosìs

depends on lhc specìcs and h!l:lro\yurett co ncen 1r at ion. (Table II) frerrflenlof'synchronizcdcellswith.rrrrrprúfh'N-nLLh\L'1"|r 1l 'onccitrJlion\

(-lablc

Il) icàds to the ac!urrulaljon ;f nL leasr -i07ó or rhe cells in metaphase (Fig l)Ailhough lhc tretrlnìent tlone ìs clîeclilc ìn achie\'irg high n]claphallc incliccs'

olemìgìt tÍcatrncrt in icc $rrier iÌnpro!cs chronìosonrc sprerìding and thrrs rc

duccs ìhe occuLLence ot chronosone cì rììps in H rulsarc aid S ccrcot'

2. Prctx, rtion of Clhr otlovrrrre St\pensiorrs

'Ihc cxtenl oî fornralclehlde fiìîLjon crilìcally 'lcternrincs

chronosomc )rield

and llrc qutrìì1y ofsu\pensiorìs ìr gcneral \Veak fi\aliolr rcslìlts in poor prcser!'r

tìor olchronósomc norphology:Most i!(naled chronoso ìes arc danl0gc'l' and

ihe lNpensions cor1lain a largeî ourl ol chronlosolllo debri! lf'onth':olhcr1993).

.rìr ol Surp.ro.ri p.urÈlqo sidilo,úEì ,$ol-l rd,ilojjol NoU ro riììsu.lur r.ulll)s.lonu..\rììrt.' to uorlnqutsrp ur stìnser u(Jrsu.dsns llu)soLUo.[J) Ìo srsÍtEuÌr rql

ltr,rquv t!11t.traùn . r{;-urd,iro i Ùy .\\ot{l \A iFi,v .,,,ro\orì,o.,rt. ) Z

(Z ìrr::t) piz.\lìrÍr rlrlrri;! urjr uorrEullUÌr.srr iu,osou(,rqr uo (slìrrLIiruosorù(rtrp lo uortÌrì'riÀ to nJ.rl .()r) Uorlnlo\.r .rtr lo lr.II..Lt.t pilros purpiììrù'trn.rrsrp i]q irìu lErlt saulos)llro,qr to ù()rtutrurlùipL rcl puìr sluiruuidri.,r . i,. ir.l $,itr .,,rr'., ,r I t5 \r!, I rn.r' ,..,^. i,, ,r,1.,^Dìrtàr ro rIituoJ VNCI r-ursn paIì|r]tlr] iq ioor si.tiro,irrì aot] tErIì:,ro.rU

\.)(1.{ro úr) .\\1)tJ lÌrrr.r.ro.,rll ISuDroS pù. srs,{trùV Jrrràúrorfl ÀotC .g

';-rrrlros pur sriluur: :rulrruolirr\oLt .'oÌ eJqrìms i.'u puI i;otorfirotlÌ pé \ r.sirdlt.À\. \rjrt \.uìosoúr) tltr p.]olorl,.11)lln^ II p\tN rtD.t)) \' to sd| ìoor 0! nì(r.rt p.rrrtosr àq ù|tr liurosouorrtr i0tx ! itirìrurìxor.l.l\/ tu lar\ .l tir:. ,qDl I Ìo sdn ìoor 0r urorj prlulosr rq rir:rs:ìrÌosouù).'rtr,lll x t uurll .rot\ s.r.rds .rJì ùo lturdàp slltrrcsouro.llt.) p.lctosrJo '.qunu all ! .r l.utur pur s.luìrìJ. ruosouìorqr to srrqun! ii,!rt uìÌrìuor.urr'./ r':i..,,r'..r , t., t.t. ,r,r.,r',1.! 'tr j.,r. \,.....r ,r,rrL\ r.tt.Ll

(jq ar,t!'/ ( rl LnlLior n)q,k, ul ,0L: qrr\ (tL.,r lìi7 ùorql,isrirrirÒLlJnl to tÙ.uìÌìùL rrlL rrìrI firrlrLtrf.rrdf l..Lrrtr,/,jr.l ùLìl]. rul.hrl.l\ t.tr,l

'tp

*tÈ

fi,d

à. tÍ.tl\ "-'

&*

*r'*$ s'#

r;v

/L €lí- 4,r,'"'-4*Kr$t(:* la i\) ""y'p"'s

ts..-_a

2.t:10 sor.ins oa Plant ChromoronrA

[i

lre. l o ' i.

Fig. 2 r'rì.orclicll lldv krryrlfcs ùiclcd lor /r'rrlcrn \ttlrn) \')r = L1) Fith l$ri dilltr0nl

cocirì.,.ùlsoL'n,i,liùoI0lr'.nrsonrcl.ak\Th.tocllìcrenlol!xri'rn'ì'l:ì'isr'liuflìciorrlllo$ lÒ discrmi .lc.hnnn.r!r.5 (Al lhis ct b. rchn\'d ar r 'o'

liti'rt ol lrriati{lr ol Li'l(ìr). lhe k yoltpcs s.r. nma.l.d usirg thc Kr\RYa)SÌAR soltùr'' (DoLe'Èl L1r9l)'

prcsenl procedurc contaiù l(r\{ allloorlts or chromaljd! and dchrìs background

îne cocitlc;enr ,.,t variaLjon of chnùosomc p.aks ìanscs fronl 1 s ro 3'J ' Figrires

3. .1. :ìnl:l 5 sho$ lììe resulls of unilarìate anal)'sis or DAI'Ì sllincd cfir!'nosonlcs

nt V. fnhu, P. \d:|,utt. jt'1d H |trl.grtrc Although onlv one or N 1c\\ dìn'ÌosonÈsctìn Lrc re$llcd in lincs \ith shnclrrd karyotvPc. tlìe nlrrìrber ol rcsolvaLrlc

chronosonr-s incretr\cs jn iines $'here karyotvPes lìr\'e beon ch:tugod due 1o onc

or nrore chroÌì1osonrc 1r:Ìnslocalìons

l'. uirdtidt Axdh,sisFigurc 6 showa DAI'I !crsus llliLhraÌnlcin bi\':trialc llo\\'ktrr"-ot\pe\ oblaioed

in t\\o chromosone tranllo ttion ljncs ot v. [ród Thc dislriblrlìon oJ chro'no

some pcaks ovcr the t\\'o .timersìons is !cr.'- lilniied and mosL of then lje or IstraigÉr aiagonrt line. s ggestiDg onl,\' minor diÎicrcnces in the AT:(i(l ratio

arnong the chromosoncs BilarìN1c anal"-sis dìd rol hclp io rcsoh'e chr'trìlosomcs

:, Tt tlt

Rel.lirc l_luores!.D.. ltt.ìr\il)

Fis.3 l:nnrr iri. ftN kr'-voryPri,hrinof nrr!' rlrt lrrlrs s '1clìrnn'$r0s

ir)lr1td fxì'ì l\ro

diri,cnr rinc'.r r,"i,, ta"i \:'i = 2) (A) I iùc qirlì r shtfn ({il{rttPe) kxitol!pe 'nltdrîoùrqr. L cùr b. iisriDrn.r..l. r..Ìidiù! chrfn.oì0s ll r) Lonì

"'nìt'sile JJ'rk' (Bl

Lirc EF rilh r .ec,'ìrtrL.te(l ltiitol!pe ilL six rhrÙn'some\ 'rr bo di!crrnìinil'rì

B

É

/1

"t

mtrer ol

leLy 5 i

tìr flow

:YoLYPing'ì ìralcd

alìre 1lù.È

inìg 10 Úc

a

'.d,iroirrì tìtrnfnk|l Lr qrú\ ,iL) i.l.urt ùL t.lrúLurL,r\Lf iq trLr.ì l9 ! a) \rtr(A,rfIL] I L.Ì. (!tl p.r,0rurrF\rp r! 0rr !.oie{úùLLttrliru. .firoirrì (id,ir rlÌ$)|rpf0ìrsILlr{.urt{v) t.t = ùa) )ri:it)l utr)rn)!t o\iurlì0.r.lrr)ohrn,!rtpiùl(\isrúosroorqr.tosrsitrnr.!rr.xrtrù0.1q.\fui!riIÌ{t)lir,,'r,rLtrit !.3ft

ìrsu4Ù1 irrr.si.url j i^rtÈt.ìl

fl

lqd lof rr uo Irs,\\oprìr.r lros u tursn pèlir.las :urosorrorqr iqÌ to u()ur?trdod V.ìtdrlru\i ilu rxr uE snrrs.rd.r ,trJ / ro ad,\roiipì fJotul,'ls rqÌ ur I irflosou)rqr lrusrrlrur iSrEt rLt.L pilJr.lxi sr ptèri ià,\\ot rroJ.r.qì pur'ùorÌrilosr

.rtr r-urnrP i;rìr q or i^rlrsuis rrour i.re srurosoruorqr.ri;uo-l ùoLsù.dlns ur',,r. .,i,,r. 't. tr r. i. . lr'.t ,tr .u t'r . .1. ',. rr. ' ; ;' :r. !ur,^^ r^ I ,tr

fun;ùrllosIo.Ìur.ql pi^irqJr] .q uutr puoris.r.d !Z or ! ruo.rj ituìî ursirtulSurtros uo ìrtosrr ts.q;rq lot ;urN(J

': rlcr {oU ìr lu un.r sr rtdups ll u.q,\\

t1P!^ uo!)ut \:) .ú

lìort ro5 runxorrrr rrl;1 9

sèìrads s[l] ur n_urcl{ìoirpì NoLJ :itnru^rq to tunrrtodpi)rr(l"t.rlli)lErrPLrPLìesiurJoorlrioJso!.rt.r tloqìr.rf.ùllrtcto.'rsltrl 'Ìt'lllls'srsÍtrur .lrlrt^rlll Sursn p.lxurlurrrsrp lou èri.r iÌrql jìt iurt rqì ur !l puìr I

F.rlr úrtrsrD 1l 0F I ior^(ùonp ! ioonrùuLl. .l ù. rLlr| r trL rn\Fr,',ì

iÌro .diroiirt i.dir ftLNl trLrlnùL! ÌLtLh\ rt(Vl ( =r/Z) ,,'rryi r/,r,/ rr){rù0 lùiLr rLtl

.{ì r0orr r.rrlosr \rùrrrorqr J. \riÌruD iqì rirtr Ir!'Ì:rlLi) sJd,i!,iL,ì tr,,U rr,.r11r0 ì t 5rJ

ft

E

ta

30. sortnrg ofplàri Chroùìoionìcs 25

3lìmiled

Ii!

=

A B

_e._-{s,

"ór-Nli(ltr.in!cin llurre3.erce

Fig.6 ùiraril.flo$ krrv(nv|.\ obtilnod ìlt0r thc rnrN\is.l ùhr.ùrrùnrrs i*nilrd ùfnrw.rrnrslocarror rirÈso vnitt lirbi t.)- = 1).) ]lncJF(Alxrdlir.EF(B).N.t0thxtqillìlh.tx..trnnr,illF.h,onrNon,c5 îllrhrnl\on,6li.!n r nr ri!h1hnc rn(hcrtrng\nrnlLdrlter.n.cs nr A lrCa rrtio.

FLl W vcrsus Fl-l A rcprcscnls onlv rboul2.5% ol the lotal cbromosome popu-lNli(nr i.stcrd of thc lhcorcricrll] crpccrod 1ó.7r1 (]lig.7A).

The puritv ot the sorted lraction deperds on the degree of resolutjorì olchromosome peaks. l1 nray be coùrprolllised bi lhe occurrence of chron1:Ìlicls.chronrosorc Jirgmcnls. tìnd chronrosomc doLrbìets llìal hale the tot.ì1 fiuores'ccnco:jimìltìr 1o lhrì1 ol chronroso.rcs. Il i! jmporlanl 10 !elect rppropriatc soltin.qwnrd(Nsbnvoìd {hcscprrliclcs. Fi!uro7A !ho\v! s(ralcc_v u\ed lodiscrìmìnate

rting and

uring the

A

lihi..eRr.r.è l\ l{- A'.r

Fig. 7 Dor |Ld! ol luorcsc.nc. ruls..kr lersa lluorrsc.rc. tulsc $nrl (,\l ^nalysis.lrhrl)flA!rrÈ\ r!ìtrre(ì nnr vitit lnxt l2Ì = l2) hnr \!i1h I (rn!t.fd (ùrlf L!p t rirotrpe. Rcgnlr

llL.. r.ù\ d,iublrs olrù.Llr.tu.tùfL..hnÌì.frì.s rnd rìtlr.. r'irrhrnl\onì. ùrLh lolì0ddrs rrgnn R2 coùrains L.ùg mtrùnrrl..hrmosù. l. lBl

^rilFn ol .hÌ..ì.nrìr\ Aol,lr(l

nanH.klùL rù11latu \4, 1,1)tuno.nliuìlir.T26!SorLirì!qnrJo\tsust{10r!rchioùhnnrs

t, ; ,,! -r'liiatq .t{

t: ì

30. SorthA ofPlant Ch.or.o$ùr\

'rtng93'n.

iimi

ì00

2'C'tC',tC'2a2'C2'C'

:y.The

ificd topú and

h hain

Fig.9 lf0rtifì.rlior rnd trralrrs orFlrrlr olclrrtrrorfn.2l',rrtrÌ r,,r..l lnì /r,1rar, rrlldr.(2n - l1) rrrnsloc.rnr hnc l2 6J_ \!r,.s.9.1.1..(,.fb.i.slsrvNlrcrl.nrcdrltcrPCR\ithDNA1il !flcd .hr.nrs.nì.s a.d l!ifitrs \tcciiìr 1{r cxch !l 1hc s.!0r /l Lr1Íd,. rlìromo$nes Th.r.rdnls r.î. I)c .ùr.d nirh 50u 0ruùbcr 2) chrnn!\,ùù (ùrc\ mrrkcl 2) trrd 3,i(rl) tlr'. !isùc\ { -ji)0 throù){D.s or.rch lrFc) \u! !s.il r: t),\iliv..Òù1Ì.1 lL.ù.s nìrrktd (r). Ih.spc0rlìciL! ol pnmerr o, rndr\rrltrrl cl,',mor'ùt( Nr. !luvn N sulrcrsùiÍs. \olc lhlr bcsidcs lhc

n,,\irr\e úonr',,1!.,ùttiJ'.ili(Jù |,.du.1fus ohtailr{ onl! rvilh trinrcrs sp0cilìc lor 0hìomo$ne 2

tr\\, (,r,.,t I I r\l

Chronrosomc\ is(rla1c.l accoÌding to the protocol outlined here are mechanicrll) lilrblc and Nilhslarìd t\\o step soúing. \hich mr-v be required in somc cases

lo tic|ìcvc a high purjli, of lhe sorte.lfraction (Lùcretli 1.dl. l993). The exlractedDNA isolhjgh lnolecu lar \\'eight and suilable tol gene mîppi.g rìnd co.str ucli(nl()1 chr.,,ìrosome-specific DNA librarie!. In r. lnbir. so, tcd cl,olrosonc\ wcrcusecl for I'Cìì \\iih sequence specilìc prinrcrs 1o locrlìzc sccrl spccilìc prolcingenes lN,lacrs cr./1. 1993)and.torlhcÍìrsttiùcìrplînls.Locorìrruclaconplelcset of clìromosoùìe specjficDNA librîrics (l\,l.rcrs.1d1., 1996). Ì-lìc use (rfrecon-structed knr)ol)pe! pcnrillcd lorlìng ol rll chr.'nìosorìcs wilhin kar],o1ype an.:l

enabled subcbr o nr osunrì I Scnc localizalio . (llìr..nnosome specìfic DNA librarjescould bc c11cc1i\cl) cnrìclìcd lìrr ììcÌosatellìtes (KoblìZko\':i cr dl, 1998). Thisopcncd î sR\ lbr cllccti!c pr(rducrion oi nìolecuhr markers needed to ircre.rsethc.Ìcnsìly ol gcnclic n1rps. Ihe procedurc lòr isolalion ot large quî.tilic! olinl.lcl chronosoÌrìcs also stin1ulated Ìrrogress in olher area! ot plan t chr(nro!(nrorc\c.rrch. Isolated chromosomes \ere shown \ùìtrble ior \c.ìnnnrg cloclroÌ ll1ì

croscop,! (Schnbed r/ dt, lqq3) for high re$lulion mîppins oi DNA scqucnccsusing nr.!n! h)briclizrtion (Ì-ùchs ?r,r/., li)9.1) and PRINS (KubalÍlo!í.1 dl.,

1997), and for ànaLlsis oî chromosomîl p,oloi.s (Bìn o!ri .,r dl., 1993).

ln addition to lhe abjljl) lo nrodìl_! lhc proco.lLîc 1(] olhcr crops. the fltureof piant ffow cyiogcnclic! $ill dcfcn.ì 1nr rlr. capabilily 1(l .lìscrìnlìnîte and sortspecific chromosonrcs. At prolcn1. lììc usc ol various chronosomiìl focks (e.g.,lmrslocarjon,dcloljon):rptc.rr\1obcthcllroslpoweÍrìIapprorch. Ì heprocedureîor specilìc hbolnrg ol clrorììosolDcs in suspension (l'lìINSBS) deleloped ir r''.

'!!l"r

Il a 8I !rr,,,í) î!,!, ùt rlL.r{ùontr ii{Lsisit|ur.niÌr.ìirr,i.) iraa, ).J.N.rirn,)/,,r ,rr iu,,Fs ,ru,, {,,.trI,i,i, \o, i(, s,rhf,!.,q, ., ,, ,î:i,::iÌT"[Ì"1 1]Ì"11j:li'(41ól) Y'n rtlloúÌ:Atùr't ) 7rt.qr\- r.naÌIrùll.iJ ù {,ìl,i'0ù1,,\\ f,{,,,,). \ \,.o l,,i)Llt ($tt \t ltr\rtr1 ) r.(l[!],irpì tr.0 ilIr,[\(L ur si,roytr!orLtr nm,ltr].to 0tjri.ripp.nrùkJllÌiu.s larrjl) v 1.0.ìvLJ j.tr.rilrs.,\\.qr.ù.N l\ î.sL.fuìrN.\,.!tÌjrrlri$B.1!i (t.tl ./ r,,t/ s.trf)$trDLqr rryd r0 0oiiì' trrr.norrúr.

r. rnprr.ùr l rt! { ùrli (lr r n.rr) L.rìrDossv lN66Ì).l.rjqt(l trr| -f.lài:t.ìa,! r$ùll.<t |!)0ru,fi

, i,,, !,r.t nu,.dniù,', ui \,!n.m, vN. Ì,rÌ, N r,)h,) ,ií.ii:lÍíÍ li'lí.l;ll",.t Dht ,.)!) 7,rr olruor r,) siu.{nuo.trrr rJrrbs*j, u,r rirorr v*., .,,,...*:t ,l:,:rt()rl) lf6al) o:ì rtrr:Ìt0|.(| s i.t\10[t_ H !rtri.ti.t .fl ]l.rLr.trrl\,).0rrllrtrlinùùv

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