Theor Appl Genel (19961 'J2: 532-540 iD Spri ngt'r· Verlag 1006

D. M. Spooner' J. Tivang . J. Nienhuis· J. T. Miller D. S. Douches' A. Contreras-M.

Comparison of four molecular markers in measuring relationships among the wild potato relatives Solanum section Etuberosum (subgenus Potatoe)

Abstract We evaluated chl()ropla~t DNA (cpONA). j~o­zymes. si ngle to low-copy nuclcu Ol\ ,\., (RFLPs). and fan­dom amplified polymorphic ONA~ iRAPDq In lerms of concord,wce fOf genet ic d istanc,,' of I) accessions each of SO/i/lllilil I:'IlIbeml/I/li anu S. pa/lillre; (jnd 4 acces~ions

(Jr' S, ffmWI(/f~!UI/!lm. The"e sell-compatible. diploid (2n,"24), ;}nd morphol (lie' ica II y vel'} 'i imi lar laX :,i comti tute •111 ,pec ie." in SI! /Will III "ec l. Er /I/lI! 1"11 S filII. a gro 1I p 0 f non­mber-beanng pecies c1o\,'ly reLI1l'd to S%moJi ~ecl. Pe­

liJlU (the potato and i Is wild I'el uti Ye.' 1. Genellc di \lallce and mullid[mentional ~c;}lil1g resulh show general concor­d'.IJlce of isozymes, RFLPs and RAPDs between all three laxa: cpDNA sho\~~ S. ('/uhemsulI! and S. f!ulllsire to be more ,~lJni lar 10 each other than to S.jCr!lwu!r :;UII fllrl, Inter, ~peci fic samp ling v,lriance shows a gradation or re,ol ution fran' alloL) IlIC (low) 10 RAPD to RFLP (hIgh): whIle lJltra­,speci fic cornr~ll'i~ons gI'aded from RFLP'i (low) to RA PD~

(high: Lick of ~lIfficienl all07yme vJnablillY \'.'ithm spe­cies precluded compari'ion, 1'01" aliozYI1le..;), Experi mental error was low in RFLPs and RAPD~.

Key words Genetic di-ralH::e PotatO' RAPD RFLP, So/al/II/II ~ection ElldJl!m \ 11111

Communicated by G, E. H;II,t

D, rvl. Spouner (8 I ' J, T, iVl iller Vegclabk Crops Research Unit. USDA, Agric'ultural Rcseml'h Sn· vil'e, Deparllnenl of ]-[OrtICU lture, U ni ver"ly "r'\\'lscon'lil. J ~75 Lin­den Drive. M,ldl">n WI. 5:171\0-150[1, USA

J, Ti\',m~ 'J NlcnllHI' Departmetlt (11 H"rtluL!llIre. lllli\n'lly (11 WI",'ulhin. I~75 Linden Drive. M .ull'l)ll. \\11. 5' 706-15LJ(). US A

I) S DOlll'he, DcpJI't menl of' Crnp ~l1d SOil Sl' ,once,. 'v1 iell Ig~1I St~lc UIHVcr"t)'. [,1St Lllbillg, M I. 4~~'~-1 32.~, L;SA

A CUIllrcr",-M, In'tlilito de Prl1dUl"l'ilJIl }- S;lIlldad Vc:gt'tal. Lrlnn'llbd Austral de Chile. C",lil:l No ,~6 7, V,ildl vi,1. Chi Ie'

.\ dme, M~' l'Itl't"ar}- \(l re""rI!aclll,ill}- ,IllU a ",liLible dJld, h,,"'evcr. Ih<' llSDA nellha guar~lll('c" Ilur wmr,lllb Ihe ,Ianuaru 11[ lhe pro­dUCl, anu th" I" ': ul tll<, Ilalll~ hJ' US DA i mph"" n" approval 01 tile jJ""ilucl to til,' eXc'lll' 1\)1) ot miler" Ihdl Illd)' <-\!>o he 'illlt,lhlc

Introduction

SOIUIIIIII1 L. section Eluhcrosulli (JUl.) A, Chi Id I~ COIl]­

pri ,ed or morphol ogicftll y very., i rni lar ~reci~,. Accord1 ng to Hawl,<.:s ( 1990), the grou p contilins five specie~: S. Inc \', IrlOil' Ph iI. (with lwo 'lIo~pecic,). S. I!fllhe/'IJ.\II1n LIIHJI.. S.

/t'rill/lli!c:illlllmi Phil.. S. [lo/II.I/rc Schltdl., and S, .1/1/7(/11­

dllllJlIl F. Meigen. The Jailer ha.' been cOIl,idered 10 be rare or ex tl nct by Correl I ( 19(2). Hawkes ( J990 l. and Brucltn (1966). The late,t revi,ion of section t'lli he (() 1/1111 (ContIT­ras and Spooner 1000 in pre ~,,! recognize.' Ihrei' "pee ie'i. S, 1!11I/N.'rtJSIII7I (1I1d tid 1I1g S. ,I 1.1!1i1lit/ili 11111 1- S. fe mal/de ~;it­

Ittl/II. and S. palus/J e (includiJl!:, S, hrcl'ide//.I l. SO/ill/tlllI fJU"

/llI"ll'f and S. ('ltIhfmSII/IJ occur in ,outhern Chile with S, pO/llslrc generally funher ~oulh and in adpcent southern ,Argentina, while S, .f{'monde:;iwlt/III is endemic to Ivlnsa­tien;\ hland III the Juan Fern,indez Arclllpdago. o.'i{) km w..::,t of continenlal Chile- (Fig. I J,

All member~ of ~eCll()n Ef/l/)C/"(!I'111iI lack tubers, 'llil otherwi se ure very si mi lar morphological Iy to ll1embcr~

or ~ecI ion Pell!/il DUlllon, A II or the specie~ W',' 'l: If-col11­patlble and set abundant seed ltl natu reo Despltc strong crOS'ii ng barrier~ bel ween members of ~ection £luhcro.1 11111

:lI1d section PelO!iI, fen i Ie ,exU:JI hybrid~ l'all be obtai ned bet~een qions Y1<! bndglllg spene, and polyploidlzatioll \ Ehlen reldt and H;jnllelll:J1l 1984: Hermsen ;jnd 'Iuylor 1979: Hermsen et al. 1981),

Ba,ed 011 morphological and cros,i Ilg dat<,,- Con..: II ( 1962) and Hawke, ( 1990) class! fied 5, ouhcm.1 filii, S. fu­IWl1de~i{/lIl.lIlI. and S. po/u I'lre inlO SO/(I II 1.111/ sect ion Pe!olo.

senes £1/1/7<:1"11.1'0 JUL. A chloropla~l DNA krDNA) clad­i,t ic allaly~j'i (Spooner et ell. 1993) eWllli ned I accessiOIl each or these three 'ipecies in :J wider examination of reLt­tiollship~ \vl1hin SO/UIIIIIII, C\'J1110/lllllldm Sendtn" and L1­

copen icol1 Mil.. 'fbi, unaly~i, \upponed an al ternate cl a~­sdicallon (Child 1990) thaI removed ,cries E!ubem,1'(! from ~tioll P('!o/li and recogn ized it at the higher ,ecllonal rank, Th i rty- ri YC ~y napomorphie, (~harcd deri ved charaClerS) uni ted ;\11 Ihree specie,. Three ~ynapolllol'pllles 1I ni led S. {w/usl!'e ancl 5, ('1II!JcmsU/lI, while 5 fel"l!(llIde:iwuml

533

was established as a sister cl ade, dIstinguished by thr.:.: uutapomorphies (derived characters supporting only 1 ac­ces ion). No diff renee wa~ detected between S. polusl!'e

and S. eluherosum. An allozyme study. evaluating 32 acceSSIOns, show-:J

close relationships between all lhree species, with S. po­

ll/SIre and S. eluberosurn more closely related than either to S.fernande:;;arlul/1 (Spooner el al. 1992). The study fur­ther showed lower within-population diversity hr all three species compared to Ihe diversity ob\erved between pop­ulalions.

Classification and organization of genOlypes into ,,)'s­tematic r lationships is based on a wide array of morpho­logical, biochemical. and molecular descriptors. Regard­less of the type of characters chosen for analysis. all are assumed to have a genetic basis and to be appropriate. No charaet r can be viewed in a vacuum, and ](5 true value can only be evaluated in compari~on to independent dalllstts (Crawford 1990). To measure genetic relationships accu­nJlely. Ihe ideal de~cflpt()r would provide an unbiased estimate of totul genome varialion and be ~ufficienlly

abundant to minimize errors due to ~ampling variance. III the sludy presented here we exp~l1lded on a pri\H'

cpON A study (Spooner et al. 199:1) by churaClerizi ng more acce~si()I's of S. !Jall/,I'I!"e, S. 1!I1I/!I!fO.\·lIIl1, Jnd S. fernande­~i([!llIm. We use dat<.l from ;j prior alloz)'me study (Spooner ct al. 1992) and use si ngle to low-copy nuclear ON A probes to generate nuclear restriction fragment length pOlymor­phisms (RFLPs). and random amplified polymorphic DNAs (RAPDs). These are four very diffcl-ent marker s)'strl11s, Chioropiasl DNA is usually maternally inherited a, a single linkage bl()ck oj functional genes. hozymes are codornil1ant secondary fUllctional gene products. Sin­gIe-copy genom i<; RFLP" generall y arc non-funel iooal eo­do'nin'lI11 ONA sequences. while RAPDs generally are non-functional domin;lJlt DNA .,equenees. Our objective was to evaluate the COl1cord<.lnce of these four m<.lrker systems ill terms of genel ic d i"lance among these lhree species.

Materials and methods

\Ve analY'c'd 15 ac.:c\sion' eal'h of 5 IJall.lslrI:' and 5. clllbl:'mSIIIII.

and .. aCC~,~LOJh of S (clllllI/de:;lIlIum (Table I). Most 01 these ac­ceS~LOn~ came Irom recenl gnn! pla~m-l·otleclingexpeditions [0 Ar­gemina (Spooner and Clau,,~n Il),:/,) and Chilo:: (Spoono::r et ..d_ 1991). J.nd were evaluated ny SpOlln~'r et:11 (I ',192) using ulh.'lymes_ Ac­('e~~ions and vouchers '-Ire deposited at the National Re\earch Sup­purt Progrllm-6INRSP-6 (Bamberg and Marlin 19')3; Bamberg and Spooner 1994) formerly called the Inter-Regional Potato Introduc­tion Project. JR - JJ The accessions occur In 24 generalized areas (Fig. I). and we chose them to represent the maximum geographic dlslrr­hutlon availahle from Iht:: cotlectlon. Compkte Jocallty data can he t)bt,ttn~'d from Conln;r,., .md Spooner (1996). The Jow nllmher:. of S j~'''''I~~l'/{h'-:/{1I11l1// aee~"ion, ex,"lnJned repres~nt the true rarity of thi, 'peei,·.

Tahle I EX,Il111l1ed pupulalwn, of SO/OIll"/1 fI'rIlUIIJ,'c'(lI)WIJ (lrn). S fl"ii<'!"{)1'II1II (ell)). unu 5 Ih,hl.\Ir(' (pbl (SCC llg. I jor 111ilp loc;ili­tle\). Specie, nUl11 her, LOrre,p()]\J 10 Spouller ct al. (1992) except I'or th" addition of 1m x.y

"ipeeie,. Map StlJdv~ PI' number localllY"

trn I I :1,1 320270 C. Skol/l'bcrg \ II

fro 2 I a.c.I.t -7:1.(,~ C Ocholl 130(j~

frn x I " _~66752 G. AIIJffWIl1 1561) 1m y I il .'i60755 C" Sf< etb _~ 2 ,IJ·.I 558o:;X(, SCo "325 lOth 4 3 a,c.l.I 55:5285 SCo 432-+ etb 5 .1 a~L'J.I 55l:nO.1 S 4</73 etb 6 4 «.j .<;StU04 .s 4</7.:/ tlb 7 5 a_I 55S2S7 SCI) 432fJ etb R 7 a.e .1".1 5582Xl:-: S Cn 4328 ern 9 8 a,l 55R290 S Cn 4331 lOth 10 9 a,cX.1 88311 Co 1322 eth II fO (IX .1.1 245924 D. COffell ('1-13 eth 12 fO '<:u·.f.i 2459.19 D ("fTeff CI34 db!3 JO a,i 498412 A MI)/lloido \ II

elb 14 I(J O1.e,1.1 55:':2')4 S Co 4338 .ctb 1.'\ J() a,l·X.t 5.'\8295 S Co 4340 etb 16 f f a,cJ.1 5.'\83 I I S 44f)() etll 17 J3 ",c,f.i 558297" S CIl 4349 ph IX (, a.e J,I 558233 5 CI) 432'1 pI:; J':/ 12 a,eJ,1 5582RO S4484 pl,20 14 a.e.l".\ 245763 D. Corr-ell C /4 pl,21 15 <I,e.l.1 558241 S Cli ./3':12 pl,22 16 :t.e. 1.1 5.58276 S 4469 pis 23 17 a.c. f.i 245764 D Correll Cl5 pts ~4 t8 il,eJ.i 558246 S Co 4.i'lS ph 25 t9 a,e.t.1 55816 5('14510 ph 26 20 a,cJ.1 558257­ S Co ';406 pl,17 21 J.C.1.1 47340 I Diel1l ,1'.11

pts 28 n <.t.l 5S81XIl S C/4539 ph 29 2.' a,i 558257 S 4450 ph _~() 24 a,e.I.1 55X25~ S 4-151 pI., 31 ~,e.Li 21 N22N EBS 338 pI, 32 :I.e. 1'.1 4'-18415 H IJnk!r<'l' 8/-8

., See Fi" I h a=RAPD. c=cpDNA, f=nRFLf'. i=allu7.Yl11e , USDA plant Introduction l\un1be" (see Bamhng ~l1d M;lrl']1 l<)lJ3)

CI=Andrea Clausen. Co=Andr6 ('ontreru'.I: IlS=ErwlI\ B~ur Sor­Wilen! Gene-bank, Germany, S=Da\> IU Sj)(l(lner " This colt~L'1i'.ln wa, ,em 1<\ th~ U.S Potato Station by thl' Emlll BJur Sorti rllCI11 Gcneb~l1k without collecto[ or loe~lity (bl~

f H. Bnicher-s loe'liity data (Chi Ie, Quetrihu~) i, COl1fu'lIlg Thf Pe­nln'ula Quetrihue t, In the range of 'i. {mlllsll'/' tn Argentina. <t.' are th ree ~ep,1 rate locall1 ie~ spe II eu Qu et rah uC In Chde: g M iSidentilteu 111 Bamberg ~nd IvIJrttll ( 1',19_, J a, 5' /)"/,,.\'11'"

DNA "ulation ..lnd r~>lriction ,ile comparison. erONA. RFLP,

DNA ''', obt,llned from 5 to 10 g uf bulked Ire,h le,lf ll';"'e 01- 5 plan" p~r <tee~S'lon from 2-molllh old plan!.,_ Preparation, 111 101:11

DNA followed the procedur<:' of Doy!.c :lnd D\lyle (19'i)71 unu wer~

pUrified ovcr (,(I graUH'nt, Restriction endonuele;1Sc digestion.,. agaro~e ~el ~Iectrophore"i';. ~\Ild the llnid,ncClional tr;Hl\l'er uf DNA fr<lgmetll~ from agarose geb to nylon filter:. (Blolrans'M . leN for cpDNA. Zeta-Probe'" . Blo-Rad for RFLPs) followed (he melholb of Palmer (19::$6). For cpDNA vanallon, 2 Jig 01 each DNA sample wa, digesled WJth 14 cIlUOl1l1clt::.Jse, (Am!. BlIInH I. Bg/l. BSINr. Clar. DI'lI!. EroRL EUJRV, Hincll. Hil/dll!. Hphl. Ned, S.I'II. Xlwl). For RFLP vJlmttion, .'\ I-lf, 01 each DNA ,ample was uigested separately WIth 4 ,'ndonllclease, (Dml. £,,)Rl. [loRY. Nil/dlll) f1ccurding to

534

36'

34'

RIO NEGRO

, ­

- - _.­ -,42"

ZAPAlJ\

$olanLJm femandazianum

72" 70°

VII

o v

, -9'.:< ' : 10. \ <

VIII

~\rH06 ;:AJ.t., • " , ,1.'...... ~~J

':'''l).\_)~.~, ~ ..NEUQUEN' -" IX :.' I

(J

'" Q

74"

o

c

'" OJ

u

o

CONCEPTION

40°

38"':

Fig. 1 DI,trl bUliOlb nl' the 34 nopul;1tlons of 5010/11,111 jenJuilde:l­WUWl IIria/l,~I~s), S, ew!Jer(l,I'!i,iI (s'Ilwres), and 5 pu/listre (circlt!s) exalmneJ ill Ihl' stuJy, Jiv](JeJ into 24 geographIc area, (,ee Table 1)

m,lnufaClul c)" IllstruClio~, (BR L). Hybridizatioll prolocoh 1'01­lo\ved those oj Glil~lwt ta~1 0 and Spoo~er ( I994),

Chloropl~q ON A

Nylon fi Itn, were probed W II h 12 P,-Il an d 2 Sail clones. reprcsell!­ing the entire cpDNA genOI'''' of Peill/Jiu ncept for the ~mC\1I sin­gL.".copy reglo~ (SyhlllU anJ GOlllleb 1986), fi ve c1onc" of N'cOli,,­/Iii covenng the ,mall single-copy reglo~ (Olmstead and P,;Illler [992) were used to cOl1lpkte the cOverage for the entire cpDNA mole­cule (approxi m~kly IS5 kb long)

Nuclear RFLPs

A senOl1llC Ilhr~ry 01 lotal DNA from So/al1l<ln I,lwrt'ju JUl, and Buk, wa, used a., described by J-1v$ab ~nd Spl.'oner (1992). A tVla] or 25 unlrmpped pro!:>cs I P4 1. PS2. P93. PI () I. P 132, P 140, P 14g, PISS, P 161. P204. P209. P263. P279. P-'07, P352, P-'613, P374, P392. P3n, P470. P47-'. P5A2. P6 :l. P778. PS72) was used tor RFLP ~nalysi"

To minimizc the collection of potentially "ynonymous data. the en­zyme yielding the greatest number of clearly disccrnabJe band, per probe was visually scored (Giannattasio and Spooner 1994).

RA PO primer sekc!lnn and ,corln)"

We e val uaterl ~n arbl trilry ,et of 8 acccss i () n, I hJt reprc,~nt Iwo 01 the thrc'e' specie, (or p0Iymorphl,ln, with 96 Operon technology (AI~med,l, Cali t,) H)-mer primer" 'J wenty,one of th~,e pnmcrs (Opcron Teeh nvlogJes. A 1m eda, Call j oml;1) were se leeted 0 n the ba SI, of c Ie ~rI Y dl scern ab Ie pol ymorphl C banrk opa4, 0r;1 5, op,11 5. op~ 16, opa 18. opd.'. ope I, opd, "piS, "rr7, vpli,- opr9, opr 12, opr 13. op,3, ops7, ors'), op, II. \l[J~ [7. O[J' 19. Dptl, Fr~gmenh rant:lng from 0,3 kb to'2 ~b werc scor~d vi\u~lly f(H pres~nce and c;,hsence,

RAPD <ul1pllflcOlion

RA PD re~Cll()nS were pertormed In an Idaho Technolvgy Air Ther­rnnl Cycld'-". Model I G05. rro~nllnmed tor 40 cycles, The cycling prO!o('ol for the fir"! 2 cycles wa.S denatur~lIon al () I °C for 60 " an­nea ling at .17°C for 7 ~. e Iongatj on ;.it 7rc for 7() s. The ~ uhscqllent 3::; ('yel es \vere Id en tIcal except for J dendtu ral ion tjme of I s Ido~

Santo' et al. 1994), Reac1)()n, were performed In thin-walled gla,,-capillary tubes

Th~ r~aCllon hurter" '" compo,ed (Jf 50 rnM TRIS. pH 8.5. 2 mil1 MgCI 1. 20 mM KCI. SOU ,-,)"/n11 BSA. 2.5'-, Ficoll 400, 002'10 xyle~e cylal1ol, Reaction conc~ntrati()n, wer~ I 00 ~M Dj\;]P,. 2 ng/ill DNA ternpl~le, 0.4 IlM RAPD prj Il1n. and 0 G units Tuq DN A P(l Iymera~e.

RAPD profiles were r~so IveJ by el ec tmph, lrr,Sl, (5 VIc Ill) fur :I h In 1.5% U Ilrapure'-" (BR L) ~garo.sc gcl~. Gel, were ~talned with <"IhldlUIll bromide and photographed unJer UV Ilglll (270 nm) with Polaroid 667 film,

Chloroplast DNA ~n~IYSI,

The cpDNA data was ex~n)nled with PA UP. vcr, inn 3 I (Swoftord 1(93), The tree wa, unrooteu, The dala were nnalyzed by Wagner parsi mony, which gives equal weight 10 ,He galli' alld sile losse~,

The most parsi monlOu,s tr~es wer~ ,ought USI ng ~n exh~lI~lIve ~earl'h

strutegy,

Allo7ym~. RFLP. and RAPD ~n~lyqs

The gen ellC dl,tances {G D) ~ mong ~cce"ion s b~sed on a IIOLY me, RFLP, and RA PD dM~ w~re calculated using N~I', ] (Nci I ')72). the coefficient of Nei-Li (Nei and Li 1979). and the complement to the ,llI1ple matching coeffi ~l("'t (Sneath and Sokal 1913 \ Th" Sl mple malchlng c0el'l'Icient lI1easures ~-,socI~tjon, among an aC('esslon', hand scores that ~re "all ke In .stale," and IncIud e, the concordanl prescnc~ II I) or ahscnc~ 1(0) of f)l()Jendar f)larker b~nds (Dudley 1994: Tiv~ng el al 1994) These gL'ne!lC di,tances arc ~xpr~,,~d ii'

GD(xy) = I[(x;c y)+ I(x;cy)], wlll'le I( x;cy) represent, the ,urn of all discordant observations wllile Ii X =y) IS the ~UI1l of nil concordanl ohservation, between aece.sSlOns nnu y, The rc,ulllng genetic di,­lanc~ malf\ces gel1er~ted by allv~ymes 02x32), RFLP, CSx25) ~nd

RAPD.s (-'4x34) were reduced 1o IWV dimensions u'lng Km,ko, monotoniC mullidlmcnslon~1scaling (MDS) an,dy>ls wuh Syst:u S.2 (Wilkin,on 1992)

To compare lhe sampling v~ri~n(e of g~netic fel~lI()nshlp' based on 'll1ozyme. RFLP. ~nd RAPO data. we drew 1000 bootstrap s~m­plb, ..:ach of Size II [11=4.6, 7.9, 12 (stopped for allozymes). 16,20. 26, 33,43 (for RFLP,l. 55, 70,90. I 16, I '·8, 178 (fur RAPD,) I. in­

dependently fmm each v!' lhe three II10kcuiar marker data,eb (TI­v~ng ~t ~I 1994). The GD between ~I] pairs ot genvtype~ wa' c~J­clliated fol' eJch boot,lrap ,ample The vari~nce amon~ the 1000 bootstrap samp!.:s for each pair of g~nOlYres was ,tandaf,-Ji..;~d to the coe ffinellt 0 f vari at ion (C V) by d iv Idi n:'. the sta ndard dev I al j on by the bvotslrap .sam ple mean. Natu fa I log trans fornl allons were II sed to Ilneafl(e' the relatlon,hlp between menn C:V, and the ,ample Size, Regression ~n~lvsls w~s used to evaluak slopes and Intercepts for the allozyme, RfLP ~nd RAPD ualasets,

Pallwise gellgraphlc distance, between ull 24 generalized gco­graph icare", w ~s obt a ined by (;on vert ing 1~t1tude lind lung lluJe d~ ta

535

to dIstance. Concordance among marker system, iJnd geogr~,phic di, ­lance were evaluated with the Spearman-r~nk correlatIon ,tali'llc", using JMP software ver. ion 3,fl2 (SAS InstIt1H,·. I()')4) ,

Table 2 Chl"roplJst f)i\'A IWrII ,eel. t/l.dWI'OSWII

No, Enzym~ Region

restriclion ~ite I1lUlatlon~ within S%­

Size (kb) Specie~"

Experiu1ental errors

Duplication of 2 accessions (pis 21 ;.lIld ph ~O) IV,1S included in the RFLP dataset; similarly, J accessions (elb 5, ph 18. ph 22) were in­cluded in the RAPD datas l. We scored both d~la~et~ blindly. Error was c. timaled as the failure of bands (either RFLP or RAPD) to be scored identically over duplicated accessions

Results and discussion

Chloroplast DNA cladistic <lnalysis

Cladistic analysis transforms ataxon by character ~tate d<ltu matrix into a tree of relationships based on minimil.ing character state changes in the tree, but directIon is given only with use of an outgroup. Analysis of the entire chlo­roplast genome wi th 14 re~lrict ion en7ymes and 14 clones revealed the presence of eight restriction variants among the 25 accessions exnm ined. Til rC'e of the~e v<.lriants were shared by more than nile taxon, the other five were un iq lie to S.jernai1dez;orwltI (Table 2). Four ident ic<.l), 9-step, most parStrnonlOlJ' tree~ (consistency index of 0.67, excluding autapomorphi~s) were generated with Wagner parsimony. A ~trict consenSl1~ tree o~· :l:ese four tree~ was complete] y unresol ved, Accessions etb 4, etb 16, and pIs 21 shared one or two restrictionite variants (elb 4) with S.fernande:i­{I/W/11. This result was in concordance with the resu Its of Spooner et ai, ( J993). II ltll the detection of two additIonal autapolllorph ies for S. !erf!cmd('zialil/lrI, and the di ~covery

of the tllree shared I ,:Slnct ion si te variants in some acces­~ions of S, eluoerosum <lnu S. poll/sl(, with S./emal1de:i­{JfWIfi. The cpONA data were not included 1I1 the sampling variance an(lly~~ (rig. 2) because of insufficient numbers of marker.'- and are not appropnate for phenetic analysis (Fig. 3).

AlJozyme, RFLP, RAPD datasets

The allozyme study (If Spooner et aL (1992) evaJuated seven enzyme systems, encodin~ J 2 enzyme loci, and dis­tinguished 19 presu mpI ive alleles. Fi ve enzyme sy"tems were polymorphic, encoding 5 enl,ym..: 'oei. resulting in 12 ,)0;) morphlc alleles. A total 01''25 RFLP probes were eval­uate,; using anyone of the four enzyme,,: J7 probe-enzyme combinations were polymorphic resulting in 50 polymor­pl)l(: band:-.. A lOud of 21 RAPD prllners were evaiuated. I'l:~ulling in 179 polymorphic band'>. The RFLl' Clnd RAPD dala are available from lhe corn.~~pondi ng aUlhor.

r:x perime ntaJ ern) r S

The failure of duplicated genotypes to be scored identi­cally constitutes experiment(j] error. This error i~ con-

I BsINI P3 2.0 + 1.6 = 3 6 Inti :2 ') Drol PIO 2.8 + 0.5 =.'L1 foo 2 :\ Oml P6 15 +:\.5= 185 etb 4. fnd 2 -4 Oml T.wn 10 -' 0 + 2.2 -= :> 2 fJld 2 5 EcoRV PIS/PlY 1.0 = 0.7 , 03 clb 4. pls:!1 <'> Hillel I P3 2.7 + (l.g = .1.5 Illd :2 7 Hphl 1'37 J.7 + (l.l = 1.8 lilo 2 8 Neil T37/TJS/T39 90+ J I = 10.1 elb 4. cth 16.

pb :2 J, fud 2

" See Table I ror definilion of abbreviations

b wilhin species

R2 = 0,999

parameter estimates estimate Prob>ltl

Common In1ercept 290 RFLP 291 0.001 RAPD 288 0.000 Common Slope ·0.509 "'5

5 10 20 30 40 60 80 100 200 50 70 90 150

Sample siz.e, bands (narurallog scale)

Fig. 2a. b Linear n':;ression~ of l'Oetfl<:lenb of ",mallon 00 boo\­,tr'lp sample size for allolyme~, RFLP,. <loll RAPD" The upper anJ lo"'er quarltle are Il1dlUlt~d by !>(Ir,1

founded wi th the germ plasm sump) ing error in this ~tudy,

since 5 ind Ividual plant~ per accession wa." bulked iI1to one DNA sample, but it can be minimized by bulking momy 1Il­

di viduals, The mean experimeotal errors observed in thi ~

ex perimel1t were 3.93'% and 1.38% for RFLPs and RAPOs, respectively. These percentages are within the r:ll1ge of thos..: reponed for Brassica by dos Santos el a!. ( 1994).

100 90 80 70 60 50

0)40

~ 30 a> 52 ~ 20 ::>

-S'""* 10c o 'ffi

20

10

a among species

R2 = 0.999

• Allozymes

" RFL Ps

-- RAPDs

paramster estimate:s

Commof'l Intercepl allozyme RHP RAPD Common Slope

. estimate

130 146 122 125

-OS 11

0.000 0.000 0.00'

n,

---

536

Fig.3a-c Multidil11entional scaling IMDSj plots of genetic distances u.>IIlS a <lIIOlymes. b RFLP,. and c Ri\PD,

0

-1

c 0

-U5 c OJ E '0 -0 § as (f)

0

-1

e03­d .&.p?! e04' AlJozyme

,16 eOSb-P3? ,06 e07.Ap31 e08el4 -p20e09 J

p21p18,

ell

't--- pn~19t.&. p24p23 J ,26 p25dO 0 0

p30fIll .&.p28ell ~19 el3 ell

~? !..

!OI

I I I

b AFLPeO) 0 pl9 ~

e17 ~e04 p32 Aof31 p18.&. P17~P20p21

pl\"";/ '4 '&'p30ell e08 p1J p14 p22 ~."

el5 ell ... el4 p26

Q f02

I

e33 0c el3 RAPD el~elO 0

pi)elb ~I08 116 e. 4 30\ pn pl0, p26

~31 ~1~~_-P28e060c:rJ1j el7 e09 p32- I... p24

.&. ,-p13pl9Ap31 'p27

0-'

,010 pl8 0104

-1

t02.)10'tOl

loy

II

-1 0 1 2 First dimension

Correlation s among genetic di stance estllnalOrs uses allelic ralios) and GO (discarding allelic ratios). Be­eau~e of a 97,9% fixation rate. alkl ie informal ion with ali\>­

Ctn [elations JJllong the three gl:netil: distJI1Ce estinwtors zymes has limited impact on genetic relatiomhip esti mates, (Nei's J. Nei-Li. G'» ~ere evaluated within the three marker The RFLP and RAPD d,tta were collected using bulked tis­systems (<lIl07ymes, RFLP~, RAPDs): they ranged from sue: thus the aile Iic frequency wa~ not obtJi ned, 1n Iight of 0.98010 0.99Y. The lowest correlation (0.986) among esti­ the high correlations among the e~lim<llOrs. we elected to lise mators WilS observed wilhin allozyme.... between Nei'~ I (lhal GO for all subsequent comparisons due to its ~implicity,

537

Table 3 Geographic and genetic distances Wilhll1 and among specics

Comparative stali~tics

Sect. Elllberrw/l/1

Among ~pecie,t> pis - etb pls - I'rn db - frn

Within "pee ies pis etb 1ftl

" Total comparisons h See Table 1 for definition.' ,

Only olle accession avail~l:>lc

lvlean Mean genetic distance geographil' distance All<lljmcs RFLP RAPD (kill)

/I DI,tance II oISI,II1l~e /I DI'tane'"

Totitl 490 0.29); .~OO o JIO 59.' () 350

3% 225 ().307 143 11.441 24U U.463 I 115 30 0.450 II IJ )+2 00 OAS2 9S1 30 0.62':) II 11-\93 04 U 444

241 IU5 0.168 7S 0,124 lOS U,1U1'> 225 105 0.281 55 1),10] 12U O,2IiO

() I 0.000 U NA' f, {J 074

Sampl!lIg van;mce for tsozymes, RFLPs, and RAPO ....

Sampli ng variance il~"oci3led wi lh genetic rei ati ollsh ips occur when discrepa ncies are detected between a random subset of molecular marker bands and all available bands (Ti vang et a1. 1994). Sampl ing van ance among species showed <I gradation of intercepts (resol 1Ilion) from allo­zyme (low) to RAPO to RFLP (high: Fig, 2a). All of till: marker systems can be observed to share a common slope, Jndica! ing that the rate of informat ion addit ion per band will remain conqanl for each system. Despile the greater resolution of RFLP~ ill detnmining interspecific relation­,hips. RAPDs yielded a beller estimate (9.6(/(, CY) because of their greater numbers.

Sampling vari.lIlce withlll species likewise indicated a gradation of resolUI ion from RFLPs (low) 10 RAPDs (high: Fig. 2b). Both marker systems likewise share a COrnlT'on slope. Becau"e of the lower number of polymorphic bands observed for the within-species comparisons (mean of 24 bands for RFLP, WII,I S. elll/)I!r(),I'u/11 and S, palllS/re. anL! a mean of 70 bands for RAPDs for al I three species), the pre­cision of these comparisons was greatly compromised. This problem had the gl'~'ate"t impact on dllozymes, which had only a mean of 4 polymorphic bands within each of the three spell .:s. Becau~~' of Ihis low level of polymor­phism, the Jilozymes cou Id not be inel ude<J in the- regres­sion am lysi s.

Even though RAPDs appear to be the mo~t efficienl system 1'\)1' Il1traspeci flc analysi s in section £liIberostll11, the I<lck of polymorph ic bands suggests that the phenet ic relationshIps within each ~pecies ns displayed in Fig. 3 are unreliable. Using the regresSion paramelers for RAPO..... we eSlimaled thaI a mean or 650 polymorphic bands are required for an arbitrary preCision ofa 10% CY. Thi~ mean.., th;,1 an addltiollal55 eq'J;1I1y polymorphic primer<; must be localed.

The ,creening results indicated that an add:[ional 200 pruners musl be evaluated \0 discover these 55 primer"

Multid imensional seal ing (M DS) and geographic and genet ic di slanc~,

MDS is a mliltiv~lriate procedure thaI IS related 10 factor and principle component anal ysi s, It ha, the advantage over these latter proced ures in lhat iI frcquen tly accounts for most of the variation in two dimensions (Nienhuis el al. 1993: WlIkinson 1992), The percentage of variance in the GO matrices explained by the first two dimensions of MOS (Fig. 3) accounted for98.8%, 98.9%. and 95,9% of the var­illnce associated with Ihe GD matrices for Ihe alloz.yme. RJ:LP. and RAPO datasets. respe<':lively. Each of th(' MOS (Fig. 3) plots separate S, fJolu )Ire. S, ('1IJhe roSIJIli. and S. jernande;.iQl'lum into three groups dnd appear to ha ve a si m­dar re13tive positioning among them. However, the <\110­zyme data show considerable overlap bet ween S. !W!tIS/ re and S, elubero,HlIII

Quanlificat ion of vi 'iual observat ions b<lsed on the IY1 OS analysis (Table 3) confirmed Ihe general trend of greater distance between species than within specie.." The pooled average distance between species is 0.459±O.077 ilmong dll marker systems, 0nly allozyme observations ex<.:eeued v:·t Iues greater than one standard deviation of the mean, This is in concordance with the I,Hge sampling variance obt<.lined with allo/ymes and SllppOrlS the vi~llal observa­lion of approximately equal nuclear genomIc dlslance among lhe lhree species. A less apparellt visual clue. quan­tified in Table 3. showed that a consistently greater mean distance among the marker sy.... tcrns i" observed wilhin S. r:fuberosum (0.257) than within S. pail/SIre (D, 134). Sol{{­l1um .!frllWldfzi(/nui/l showed the smallesl mean eli stance among acces"ions. but this could be an Clnifact or the few

538

Table 4 Spearman rank correlation statLstics among gcogr>JphlC. al­lozyme. RFLP. and RAPD diswnccs

Geography AII07ymes RFLP RAPD

Geography I Allozymcs 0263 I RFLP 0.527 0.410 I RAPD 0.436 0.494 0.750

accessions available. A preliminary conclusIon >.uggests that S. elllbcrosulI1 has the greatest diver~ity of all three species,

Concordan;.;-; of dawsets

Concordance of the three marker systems was evaluated lISll1g Spearman-rank correlatIons. On Iy i:lcce~sions in­eluded in all of the three marker systelTI~ were considered (Table 1). The selection of a non-par;ometric correlntion of pairwise i:lssociation wa~ just ified by our inlerest in the pre~ervaljon 01 relati ve L'Jn!-;,,;, not magnitude of distance. Geographic distan('e wa~ included only to maximize the information content of our stud:. The concordance of re­sults from isozymes, RFLPs. and RAPOs as displayed in the MDS plots (hg. 3) shows the ability of these markers 10 measure gelktic distance in S. elubero.~um. S..fernalldc­"lanum, and S. pa/Hslre. The concordance among molecu­lar marker systems shows an increasing correlation be­tween Isozymes to RFLP~ to RAPDs (Table 4). This trend is in agreement with the sampling variance increasing wilh greater numbers of bands and the discrimination abil ity among the marker syst~ms. A pOSItive correlation among alllhree methods to geography was observed; i.e.. Ihere is a grea te r siIII iIari ty amon g close Iy spaced access ion s th an :lmong distantly located accessions. The data do nOI allow more re lined invc:~tigations into possible cI inal pal1erns be­cause of the lack 01 frequency data within acccs~ions.

Sy~tematic relat ionshlps witb: 11 sect ion E/Ilherosilm

So!anum ji:rnandczionulII is t'lldernic to Masatierra '~I and (Cnntreras and Spoon..:r 1996). This island, 650 km west 01 continental Chtle, I~ 4 million years old (Sluessy el aI. 1984) and forms pan of the hl3n I~ernandez Archipelago. Isozyme data suggests Ihat S. fernandcGianum is 1.1 mil­lion ye:u s old (Spooner ,'1 ;;1. 11)92), and distributional data ~tTgest that S. j'ernllnde;;iwJlu/l is derived from S. P011l5­trl!. S. clIlberosl-llII. or their ancestor.

A crONA c;adislic Ilnalysis (Spooner el al. 1993), us-IIg appropriate outgroups for senes Elubcnl'i/lm placed S.

ell/bem.lum and S. palu,llre on one clade (supported by 3 ~ynapomorphie~) and S. jcnwndczia/'luIIJ on another clade (dislingulshed by three uutapomorphiesl but provided no conclusion ... or pro~2,~nilOr denvalive relationships. Our ne\',,' cpONA data simp:) point OUI previou~ly undiscov­

ered poJymorphisms in S. pa!uslrc and S. e/llberosu/1/ while failing to resolve their illlerspecific relationships. ['he low levels of' diversity of cpONA compared to that of nuclear genes was expected (Wolfe et a!. 1987).

Our isozyme, RFLP, and RAPO results. unlike the cpONA d<Jta. show approximately an equal distance be­tween all three species. The interspecific relationships of Ihese species, therefore, remallls unresolved wilh these dJLa <.IS we II. The discordance betw;,;.:n the more distant re­lill ionship of S. fernandez/mil/III relat ive to S. eluberosum aod S. pail/Sire by cpDNA data, and their approximately equal sepal;ltion wi th the other nuclear III :trkers is unre­sol ved. A possible explc.mation is an ancestral "chloroplast capture" phenomenon \Vhere S. eJubero.\um and S. polus­Ire could [HIVe hybndized with backcrossing to one or the other specie~ to share ch loropl ast type.s. This phenomenon has been documenled in other plant groups (Ricseberg and Wendel 1993).

The greater variability in S. e/llberos/l1/I (0.257) Ihan in S. [Iall/Slre (0.134) sugge,~ls Ihallhe former is ancestral and has had time 10 accumulate marc variability,

Conclusion

Our study i~ in agreement with most intraspecific ~tudies

Ihat have compared marker systems ei ther statist ically (Al­drich and Ooebley 1992; dos Santo~ et al. 1994; Beer et al. 1993: Gerdes nnd Tracy 1994; Hallden et al. 1994: Helin el al. 1994; Messmer et al. 1992; Smilh and Smith 1992; Thorman et <.11. 1994) or by other methods (Chase et al. 1991 : Havey and Muehnenbauer \989; Pri nce e! ill. 1992: Wang et aJ. 1992; Wang and Tanblcy 1989; Yang and Qui­ros 1993) in.showing a general concordance of results at low taxonomic levels. Tilorman el 1.11. (1994) investigated ~ix cultivated Brassico species and one species of Rllpha­llUS, They showed good RFLP/RAPD intraspecific concor­dance. but poor interspecific concordance. Our ~Iudy. in contrast. showed good interspecific RFLP/RAPD Concor­dance and inferred good intraspecific concordnnce. Other sludies claiming good concordance of RAPDs and various other datasets are those of Halward et al. (1992) van Coppe­nolle et al. (1993). Wi Ik ie et al. (1993). and Will iams i:lnd 51. Clair (J 993). Our intraspecific concordance is tcnta­tive,I1Ilwever, since relatively few RFLP and RAPO mark­ers yielded low levels of precision.

Questions regarding the reliabilily of RAPDs ha\ l' been raised by 5 mith et al. (1994), Thorman et al. (1994), and Wi II iams i:lnd St. Clair ( 1993 l. These studies used RAPD fra!, P.1ents as probes to show non-homology of some com­igrating RAPOs from differenl specl,:s. suggesting the need for similar homology studies with other taxa. Addi­tionally. Smith et al. (1994) showed fragment absences (0

be low-re'wJution presences, and tIle RA PO react ion to be dependent on the synthesis of RAPO products from unre­lated loci. Our ~tudy does no! provide ex peri melllal data for homology, neither by Inbelling fra~menls as probes nor by inherilance studies investigating the allelic nalure of

bands. The concordance of our independent RFLP and RAPD resulls (Fig. 3. Table 4) sUflgests that most of the RAPDs have biological relevance Ifl elucidating genetic distances among species in sect. Eiuberusum.

The sampling variance of RAPDs in comparison to that of RFLPs in our study is greater in interspecific compari­sons (Fig. 2a) than in intraspecific comparisons (Fig. 2b). These results suggest that the magnitude of non-homology (noise) increases with increasing t3X0l10m ie levels. De­spite this noise. the MDS plots of RFLl's and RAPOs (Fig. 3b,c) show similar associCltions among species, sug­gesting that the noise levels in this study haw reduced im­

• pact on biologically relevant results. Addlt tonal support for the limited impact of noise is confi rmed by the rela­tively low experimental error for RFLPs and RAPOs. In contrast to interspe,cific comparisons, tntraspecific com­parisons show greater noise wilh RFLPs. This may he caused simply by the lower fesolution ofthi s marker within these genetically similar species.

Our study showed the applicabIlity of RAPDs for bOlh intn.l- and interspecific studies in So/anulll section Etube­I"U.I'UIJl. These species. however, ;,Ife three morphologically very similar diploid lllbreeders. The applicability of RAPDs and concordance of these three marker systems h<. \ e not been tested at different levels of genet ie di ver­gence and taxonomy. in various breedin8 systems. and at dl ffl'rent ploidy levels of sect. P<::Io/(/.

Acknowledgments We Ihank B. Karus for technical assistance. K. Hosaka tor RFLP probes: R G Ollnsteud. J. D Palmer. and K, J, Sytsma for cpDNA probes. Andreu Clausen ror cullaboratlof\ for col­lecting S, palu,I'lre in Argenlina, amJ R. Whllku.' und O. S Smith for (omment., on an earlier \'er'10n of the manuscript

ThiS work wa., SUpp()'t~d by d NR I Competitive Grants Pro­gram/USDA Award Number 94-:17300-0297 and by USDA, ARS germ plasm collecting grilnts to f\rgenlina and ChJie 10 DaVid M Spooner; and IBPGR grant 1193- 105 to J. NienhUIS.

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