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GENEТICS
and BREEDING
2008, Volume 37, Number 3-4
Molecular characterization of structural
barley mutants produced Ьу gamma-irradiation
А.
А.
Dimitrova'*, Е. Todorovska 2, N. К. Christov 2, L. Stoilov',
Atanassov 2 and К. Gecheff'
'D. Kos/off !ns/ilUle 0/ Gene/ics, Bulgarian Academy 0/ Sciences, 11 IЗ So/ia, Bulgaria
iAg/"oBio!ns/i/u/e. 8 Dragan Tzankov. So/ia, Bulgaria
*e-mail: [email protected]
ABSTRACT. Various molecular marker systems, including STS, САр,
REMAp, RAPD and AFLP were employed to screen for DNA altera­
tions in the genomes of thirteen original barley structural mutants pro­
duced Ьу gamma-irradiation. Two out of total4888 fragments generated
in the mutant forms Ьу the applied set of markers were polymorphic
which indicate а relatively low оссипепсе of stabJe radiation-induced
genetic variations in the resulting mutant lines. Among the marker sys­
tems AFLP was proven to Ье the most appropriate for the analysis of
radiation-induced DNA alterations in barley. AFLP analysis revealed
sequence polymorphism in 2 out of the 13 translocation lines analysed.
As far as gross chromosomal rearrangements such as translocations and
inversions concern large chromosome domains, the respective specific
DNA sequences analysed тау not completely cover the areas where
subtle mutational changes тау have occurred in the mutant genotypes
produced. The data obtained in the present study support the notion for
the re!ative!y high genetic stabi!ity of barley genome.
words: structura! mutants, gamma-rays, DNA a!terations, то!еСll­
!ar markers, bar!ey.
Abbreviations: AFLP - Amplified Eragment Length .E.olymorphism,
CAPS - ,C!eaved Amp!ified .E.o!ymorphic Sequences, RAPD - Random
Amplified .E.o!ymorphic DNA, REMAP - RЕtгоtгапsроsоп-Мiсгоsаtе!­
!ite Amp!ified .E.o!ymorphism, STS - Sequence Iagged Sites.
Кеу
Introduction
Genetic diversity is the base for improving p!ant varieties. In the recent years the
iшргоvеmепt of crop p!ants was done main!y Ьу hybridization to а limited number
of dominating in the nationa! and internationa! markets varieties and !ines. As а
15
rcsult thc gCl1l:tic base ог the crops was dramalicalty narrowed and а уа]иаЫс gerln ­
plasrn has beelllost. This tel1dency appears parlicularly in markct cereals, \vhich nгс
amol1g the пюsl economically imporlant crops.
Thc cxpcrimcntal induction оГ mUlations aftcr trc3tments witll ionizillg radia ­
tions and chell1ical mUlagens is опе ог Ille ways 10 improvc tlle exisling germplasm.
lIundreds ог new varieties carriers оГ agronomically valuabIc traits (increase рго ­
ductivity, improved quality, resistance 10 biotic and ablotic stressors, е(с.) havc Ьсеп
dcvcloped using this approach.
Мutзgспiс agcnts аге known 10 produce IWO main Iypes оГ mutations - гепе
(point) mutations and structural chromosoma llnulUtions. 1I is comlnonly acceptcd
that point mutalions аге the mosl useful induced 'уре ог genetie alterations Гог
plalH improvement. although the signifiсапее ог the chromosomal re..'1rrangcments
зs а soигсе ог blodiversity Гог Ьгееdiпg purposes is still по! entire]y undcrstood. ln
facI, уегу few ог the cxperimentally produced mulan lS used ill brecdillg programs
have Ьсеп апаlУ-lеd with respect ог thcir molecular паturе. Dcvelopment ог marker
dеtеСliоп syslems a ll owcd cstimation ог Ihe divcrsilY а! Ihc DNA ' еус]. Molecular
markers provcd 10 ьс а powerfultool Гог moniloring DNA scqucncc variations ~'ith ­
in ;:шd among differenl рlап! spccies. Simullaneous зррliС3tiоп оГ difТercnl ONA
markcr syslems and accumulation оГ data оп гепе sequences is expccled 10 facililate
the elТective еvаlшнiоп ofnalural alld induced genetic vзгiаЫ]itу and Ihcir rational
use in breeding programs.
То <lIHllyze changes in the promolcr sequcnccs nnd ехоп - ir1tгоп struclure of
111C genes in cerea ls, STS markers ЬаУе Ьесп rcccntly applicd. Thc СОlпЫпзtiО l1 оГ
tl1is mClhod with cloning nnd subsequclH sequencing оГ amplified DNЛ scquences
allows Ihc dеtеСliоп оГ mutations in thc gCI1(;S rcsponsible ГОГ а specific phcnotypc .
.a~ wcl! а!:! 10 deterlllil1e the 'уре оГ the induccd alteratiol1S il1 Ihcsc gencs. Л physi­
са! тар оГ barley gellOrne hns Ьееп COllstructed usil1g microdisscctcd translocation
chromosomes Гог PCI~ wilh SТS primers derived Ггот >300 gcnctically тnapped
I{FLP pгobc~ (Kunzcl сl .. 1., 2000). \Vhen Ille SТS m.. rkcrs faiJ [о гсуса] апу ро!у­
morphism, thcy ca ll Ье el.lsil)' convertcd 10 СЛ Рs Ь)' rcstriclioB enzymc digcstiol1
(Konieczny and ЛusuЬеl, 1993). СА » ma rker~ h3ve Ьее п used 10 study genetic diver­
sity il1 soтс plant species sueh asArabldopsis tltalia"a (Hardlke е! al., ] 996; Barth сl
а!., 2002), barley (Тгавоопгипг е! a l., 1992) and ricc (\Vil]iams еl al., ]991).
RСIГot.г.зпsрosoпs .зге а rпаjог class оГ Inobile elen\ents in al] eukaryotic gcnomes.
They тау Ьс activ3.ted in гesропsе 10 various forms оГ slress, resulling in gel10me
changes. This capacity оГ retrotrnnsposons was exploiled 10 с ге..'11е several marker S)'5­
lems based оп PCR reaclion Гог variety identific;.'1tion ог brceding purposes CКalendar
СI al., 1999; Manninen е! al., 2000; Viсiепt с! а] .. 2001; Doyko е! з1 .. 2002).
Random Amplificd Polymorpl\ic DNA (RЛРD) is а PCR method Ьзsed 011 thc
amplific.1tion оГ апопутои!! DNA fragmenls staning Ггот primers wilh arbilrary sc­
quences (\VШiаms сl з1 . , 1990). In Ihis approoch little ог п о sequence informalion гог
the organism is necessary, so they cou]d ье applicd 10 various genomcs. RЛPD m.. rkers
have Ьееп used 10 examine both inlerspecific and intraspecific variations ill а питЬег оГ
planl species (8..'1i et nl .. 1998: Осrnгоп and Landry. 1992; Todorovska е! 3.1 .• 2003).
AFLP is ап еffiс i епt DNЛ fingerprinting lechnique having the capacity 10 геУеаl
тапу polymorphic bands in а siпglс вспоmе. It shares some соттоп charactcrislics
with H.FLP and I{ЛI)D , becausc it соmЫпе5 restriction enzyme digcstion and the
рен. amp]ification (Vos сl 011.,1995). AFLP havc Ьссп cmployed Гог gепоmе 5С<1I1­
ning and gcne I\шррiпg (A llhoff СI al .. 2007; Мiап еl al .. 2002; Yin с! <11 .. 1999).
ТЬс sel оГ PCR-based techniques describcd аЬоуе сап Ье app!ied ГОГ thc detec ·
tiOll оГ DNA a]tcraliol1s alter ватmа rays irradialion. as (Ьеу have Ьееп wide!y used
16
[ог
DNA lingcrprinting in various рlап t gспоmеs. The main objective 01' this s1udy
is to higllligl1t thc molecular nа1и ге o f possibIe DNЛ alterations resul1ing [гот ion­
izing radiatiolt which was app licd 10 сеnсга 1 е а stock of homozygous structural
mu tants in barlcy.
Materia l and Methods
Planl ffialer\l. ls. Thineen Iмrley struC::lural muW1u, i.e .. T1586, T-16, Т- 20, Т- 26, Т-48, Т- 58, Т- 59, Т-б3,
PK- 169 and РК- 88-4 .nd ,Ье .tandard spring vanely Freya (control) were used 1$
experimellllll ma lerial. Тl 586, Т-20, Т-26, Т-48, Т-59, Т-63, Т-67 Ind Т-б8 Ire hошozуgoUlliп" which
COnlain 'Ьс single trans loc::ationl 311s/4 Н I (reciproc:al t8nslocatiofU bctween IhOfI arm of сЬroтOlOте
3н 8ttd 10ltg .гт of chrol1losome 41-1), 6Hs(7Hs, 4Ht/6H1. IНчЯII, 11-11/511"1, )I-fI!5Нs , IHJ/2HSlnd
11 1J!21\1, respec tively. T- 16 and Т·58 ие homozygous doubIe tran.localion. dcveloped ьу g"mma-irra­
diat!Otl of Т·I 586. '" addilioo 10 Iran.location 3 Н ч4НI which comes Ггот Т- I 586 these lines contain
trlnJJotOlloJ11 211s/51-1l, and 3Н1/БН sal, respeclively. РК- 1б9 Ind РК- 88 · 4 IIте nHJtliplc reconstructed
I::.ryotypes, IЬС first опе contl!ning Iran. localion. 1Нч5Н1, 3HS/4Hl llnd 4H"II/1HI, and IЬе second
опс - tran51ocatiOnl IHl/5Н1, 2 Ш/1Н I, ) ~l aJ4HI and IwO perieenlrlc inverslonl rcsiding in chromo·
юmСI б ll and IHSH, re,pectively.
AII rearrangemenu mentioned .bovе were шduсed ьу gamm.-irrldillion ( 180 Оу) ОГ dry secds
ol' 'Ье tniliallines Freya and "-1586 (Gecherr, 1996),
ONA isoll lion. ОНА W1II 1$OIIIed ГrОП\ ) -4 ""ссn oId planll uSLna СТАв method Iccording 10
(Ье procedure оГ Murray and 'ГЬоmpsoп (1980)with minor modilicalion •.
STS IInd САР IIПIlI)'К5 оГ b.ricy R· hordeln .nd C·no rdelo Ic ncl. PairJ оГ oligonucleotidc pri m­
ers werc desiИlled for B· hordein ,епс (5' - СССТЛGССАCCAAGAGП'GСАА- 3' and 5'-ТП'GGтr·
Gctrn'CCAA1TGG -3') and C-hordcin аспе (5' · СССТАGССАССЛAGAG1ТGСАА· 3' !lLld 5'- ТП'­
GОПGCfGТССАА1ТGG-)') lequenca оп Ihe buis oCavalllble InГOtffiatюn in (Ье dlllblsc (www.
ncbi.ltltll.nih.gov). PCR amplilicJ1lioll wal реrГOtПlеd in )O}ll reaclion mu(lUre oonllining 100 n8 ONA,
10 рМ ог езсh primer, 200 }1М оГ еlСЬ t.lNТP, 1.5 mМ MaC1) and J uoil Та,! polyтnc:rasc. 'ГЬе foiiow ­
iog I'CR condilioLls for 'Ье amplification оГ B-hordein ,епе were selec::led: 94'С ГОТ ) mln, foiiowed Ьу
ЗS cycles 111 94 'С ГОТ зо., 55'С for 45 ., 72'С for I min and 72'С for S.O mi lt. ТЬс PCR progrnm for
C·hordcin 8cne ""а. 'Ье "те cJlccplthat the annell ling tcmperature ""II' 56'С and (Ьс number of cyclel
was reduced 1030. AmрШiСllioп products correiponding 10 D·hordein ,спе were digcsted wi th Msp I
(Fermentlls) or Tlq 1 (Fermenta.) while lhose correspoDdiog 10 C-hordein ,спе were dige5ted with Ode
I (Roche) or Pst I (Fermenlal) for 6 hrs Iccordina 10 Ihe manuflcturer', instru clion •. Тhe d!gelled PCR
prodUCII were fraCllonatcd оп 8% s.equencinl polyac:rylamide iC11 (Maniali.. 1982).
REMAP IIn.IYIII. BA.RE-l -lpecific ртiПIи (Мlппшеп IlId Schulmlll 1993: Acce"ion No.
ZI7З27) and 1 10lal of fivc miCtOlollellLle рrimеп were u.sed in Ihe REMAP Inal)'ll •. Amplificaliol\
reaction WIS carried out in 20 ~I with 50 naof1ernplale ONA, 10 рМ primer, 0.2 тМ ог еасЬ dN1'p, ' Х
PCR bufТe r (1.5 тМ MaCl,) and 1 unit Tlq ONA polymCn$e. 'ЛIе PCR !)I'QgI'lm (OnSislcd ofап initia l
dcna turatioll step оГ 2 min а l 94'С follw'cd ьу зо cycles ог зо аес а! 94'С, 30 ICC а! 55 'С о' 58'С Ind
2 min аl П'С. Alllplificd Гт,теп" WCfe separatcd in 2% NlJ$lcve 3:taprose gcl. (FMC BioproduclI)
in ' Х ТАЕ ruПl\iпg burrer, . tlincd with clhidium «отklС (0.5 ~вlт1) Ind vlsualizcd under UV ii"'l .
RAPD IIn.lY$J~ . Six.IC::cn dilТc::rent primers (OPERON 1:11 А and В) wcre applitd Jn (Ьс RдPO
апо!уsi,. Amplification reaction wal done [п 20 ~l mixlure conta!ning l 5 ng oftemplale ONA, ! О рМ
primers, 0.2 m М of сас Ь dNTp, ' Х PC R burrc:r and 0.8 units Taq ONA polyтnc::ra$c. Amplificlltion
WBI pc:rfo rzned under 'Ьс rollowing program: 2 mi.o аl 94'С: 40 cyclcs of 10 s а l 94'С, зо s I 1 Зб'С, ]
miп II 72 ' С: and cxtra exteDlion for 10 min аl 72'С . Amplified producl5 were fгасtiопatеd оп J .8%
181rose 8е11 in ' Х ТАВ running burrer, stained with clhldium bromide (O.5ltsJml) and viluali-zcd
under иУ light.
AFLI' I nal)'lOll. AFLP analysis was pcrformed u original]y propoкd (Vos СI al., 1995) WLlh minor
modificalions. 250 па ос ONA WCfe u5td Гor еасЬ rcaccion. DNA IImples were digesled with 10 U EcoRl
(Fermc nla.) апd 5 U Trul l (Fermentas) in 1 buffer reoommcnded ьу 'Ье mlnufaclurc:r in а lotal volumc
ol' 15 ~l at 37·С for 90 min, follOWLXl ьу 90 mio _1 65'С. Synthettc ЕсоЮ _daprс:п were Izgaled 10 Ihe
digtsled DNA and both (Ье preamplificвtion 100 ше Itlcctive ampJification ""ете carried OUI Iccording
10 (Ьс method of Laurcntin and Кв rlovskу (2006). SeJective Imp1iГtcation was performc:d with 10 primer
combin!\liOnl usi ng Пuогсасеп llу labelcd Еоо RJ -primc:r + ) sc:lec::livc: nuclc::otidcl (ЕЗI, ЕЗ5 Iпd Е41)
соmЫпcd with опе ог thc Msc: I primers (МSO. М59. М60 and М6]). 'ЛIе obtained PCR producu werc
scparated 011 б% polyacrilamidc аеl io ао ALFcxprc:$S 11 DNA Inalyzer (Amc:nham РhDrmасщ) for 700
miп а! 1500 V. 60 tnЛ, )5 W and 55оС.
А]I PCR Dtпр1ifiСЗliоп rellctions were pcrformcd [п а PC R syslelll 9700 (ApplLed JJЮSУSlсm).
Т- б6, Т- 67, Т-б8,
2 GCnClltl and
Вrес::diпg,
) -4/2008
]7
Res'llts 311d Discussioll
STS and CAPS
arш l уs i s
То study {Ье capaci ty ог gзтта-гауs
Ггот inscrtions ог deJetions оГ опе ог а
in inducing point mutations that тау arisc
few bases in IЬС DNA sequcnccs corrcspond­
ing 10 B-hordcin and C -hordein gencs SТS and CAPS ana lyses wcre performcd in
е!еуеп mutant Jines (1'- 1586, T-16, Т- 20, Т- 26, Т- 48. Т- 58. Т- 59, Т-63, Т-66, Т- 67, Т- 68)
a nd the inilial genotype (СУ. Freya). Two SТS primer pairs were designcd 10 зm ­
рliГу DNA fragmenls corresponding 10 B-hordein and C-hordein gcncs. I пfог mз ­
tion concerning thc amplification ofthe B-hordein genes was drawn Сгот prcviously
pubIishcd data (Forde е! а1., 1985). А primer combination was assigned Ггот {Ье
рС Р 387 - C-hordci n cDNA sequcnce (Лссessiоп No М36941). responding 10 the
region оГ 489-1469 Ьр. which includcs (Ь с РЗгl ofthe maturc peptide ofC - hогdеiпs.
ЕасЬ pair оГ primcrs amplified 3 single PC R product and IЬС size оГ thc сасЬ PCR
product was as prcdicted Ггот IЬе nuclcotide sequence. No vа гi зtiоп in the obtained
products was dClcctcd bctween Inulnnl barley lincs a nd (Ьс initi al genotypc.
То providc а broadcr vicw оГ IЬе scq uencc polymorphism. smal l insertions ог
dclctions IЬа! саППОI Ье detected Ьу native gel eJectrophorcsis а! wcll а! !Ьс аррса­
ranccjdisappearancc оГ restriction sites in B-hordein and C-hordcin DNA scqucnces
IЬС ampJified DNA fragments were subjected 10 digeslion wi th frcqucn tly cutt ing
rcstri clion cndonucleases.
ТЬе рсгГогтоо CAPs analysis did по! show зпу polymorphism between mutan t
lines. а! wc ll а! compared with (Ье control matcrial (Fig. 1). Тhis lack ог polymor·
phism suggests abscnce о Г induced alterations in (Ье studicd I}-hordein and С - Ьо г ­
dcin genes ог (Ь с mulanl lines. Our results аге in agrccment with (Ье data obtaincd
"rter scrcening о Г ( Ьс sз т с lines Ьу SDS-PAG E elcctrophorcsis Гог barlcy storagc
proteil\s (Stoilova е! a l., 2006). Л s CAPs and РЛдG Е showed по changcs in hordein
gcne scqucnccs and I Ье respcclivc protein profil cs il сап Ьс assumcd th"t induccd
chromosoma l rearrangemenls do по! afТcc! ( Ьс structure о г B-hordcin and С - Ьог ­
dcin gспts о г ge ll es respol\SibIc Го г their cxpressio n.
RЕМЛР analysis
REMAP ana Jysis was conducted 10 idcntify possibIe a ltera tio ns in IЬ е inscrtion sitcs
о Г {Ь е retrotransposons (insertions ог deletions о Г ВЛRE - I retro transposons) in ­
duced Ьу ionizi ng radiation in the structura l mutants compa red !о (Ье controJ Jine
(су. Freya). Тhe REМЛР analysi s was performed using as а reverse prilner BARE­
l -spccific primer whi ch is based оп {Ье first 20 nucleotidcs ог {Ье ВЛJtE · I LTR
CМanninen a nd Schulman 1993. Accession No. ZI7327), facing outwa rds Гтот (Ьс
5' LTH.. Л tota l оГ fivc forward primers were sclected Гог microsatellites based оп
thrcc din ucleotide repeats "nd апе trinucleotide герса! (СЛС) , . Тhey were anchored
10 (Ьс microsatcllite 3' termin us Ьу ( Ь с addition ог а singlc sclccti\lc nucleotide а!
tlle 3' clld in o rder (о ргеуеп! polymorphism Гго т vагi.з ti о п in microsatc!lite гереа!
length. The primers аге listed in Та Ыс 1.
Оп!у 2-3 visibIe bands in (Ье RЕМЛР profiles ог mutant and control lincs
with primcr combinations ВЛRE/(САС)1G and BARE/(CAC)rr were obtained,
a lthough 3 difТercnt annealing lempcratures (55-С. 58 -С a nd 61 - С) and difТcrenl
DNЛ quantity in (Ье reactions were uscd (data по! shown). Л possi bIe reason is that
IЬ С distancc between 8ЛR ЕI - LТR and IЬе trinucleotide microsatellitcs is 100 large
10 amplify апу genomic region between (Ьет due 10 1Ье relativc distribution ог SSR
regions a nd particular rctrotransposons in (Ье genome.
ТЬе primer combi nations BARE/(GA)9C, ВЛREI(GЛ),G and ВARE/{ЛС) .G
produced с!езг amp!ific3tion producIs аl 55 "С ranging Ггот 10 bands Гог BARE/
18
2
3
4
5
6
7
8
9 10 11
и
1
2
3
4
5
6
7
8
9
10
11 12
"" ...
во
+..
..
+-
...
А
в
Fig, 1. Polyacry lamкlc gt:1 еlсщорhomis оГТщ I digested В-tюnkiп gmcs (А) and Ddc I digcsted C-hordein genes
(2).1-16 (3), 1·20 (4), 1-26 (5), 1-48 (6), 1·58 (7), 1 -59 (8). 1 -63 (9). 1-66 (10), 1-67 (1 '), 1-68 (12)
'<>
236
165
135
113
(О).
ampllficd
Гrom: СУ.
Freya (1), T·1586
ТаЫе
I
Primeгs
used
iп
REMAI' analysis
5'· j' sc
Рптеn
.."
."
(СА) С
8082 м
u е П "ti
СТАСGGСДТААТТССАЛСАЛ
ВЛ"'
(CI)
С
(дс)
G
.Ш
(СЛС
856~
(СЛС), Т
2
3
4
5
6
7
8
G
9 10 11 12 13 14 3000 ­
1000 ­
500 ­
Fig. 2. Rерrescпt.пll\"е REMAP рrofilе genel1llcd with primers BARE-I LTR and (С1)9С Гrom ' СУ. Freya
(2). T· 1586 (3). T· 16 (4). Т·20 (5), '1'·26 (6). Т-411 (7). Т-511Iа). Т-59 (9). Т-63 ( 1О), Т-66 ( 11 ). Т·67 (12), '1'·68
( 13), "·аа-4 ( 14); М - DNA ПlOlесulllf marker
20
(AC),G {Q 14 bands Гог BARE/{GA),C. Despite the Сае! that а large numbcr ofbands
were obta ined with the last 3 рптег eombinations. {Ьеу produeed identica! REMAP
patterns in Ihe mulant barley lines and the initial genotype - СУ. Freya (Fig. 2).
REМAJ> ana lysis earried out in sphaeroeoeeum mutant Гогтз ofwheat and triti­
cale developed Ьу treatment wilh ehemiea l mutagens showed bctween 7% and 18%
polymorphism, depending оп [Ье used mierosatellite (ВопеЬеу, регзопаl eommunica­
tion). Тhe studies ог Уа е. а1. (2007) suggestcd that {Ье meehanism ог (Ье biologieaJ
effeet оГ low-energy ion Ьеат! is рзrtiаl1у due (о the гc-aetivation ог IЬе retrotrans­
роsoпз . ou r results showed (ЬМ {Ье applied doses ог gamma гays and [Ье indueed
Iransloeations do not aetivale ог dcstroy 8ARE- ' clemcnts in the analyzed lincs.
RAPO 'lIщlуsis
Тhe RAJ>D technique has Ьесп widely used 10 sludy genetie variabili1Y in radiomu ­
tants Ггот dilТcrcnt plant speeies (Gunhan and Oldaeay, 2004; Lema -Ruminska еl
al., 2004; Wang е! al., 2007; Joshi-Saha and Gopalakrishna, 2007) due 10 its sim­
plicity and its applicabililY 10 апу organism without prior knowledge ог nueleolide
sequcnec.
In ou r study ten struetura l mutant barlcy lines (Т158б, Т- lб, Т-20. Т- 2б, Т-58.
'f.S9. Т-63. Т-66. Т-67 and Т-68) and the initial genotype су. Freya were cvaluated
Гог gamma- rays-induecd DNA allerations using а зеl ОГ 16 deeamer primcrs with
60-70% ОС contenl (ТаЫс 2).
Т.Ь!е
2­
Li5t оfrш!dom pflmcn and thcir nuck:otidc ilCqIJCIICC The 16 primcrs yielded reprodueibIc RЛРО bands. E.1ch mutant line was
scored Гог ргезепее and/or abscnce оГ bands. Аррliоо RЛPD primers produccd
а total оГ 10\ fragments with moleeu1ar wcigh1 ranging bctween 0.2 and 2 kb. ТЬс
gels containing J>CR products Ггот аll lincs shou'cd Ul1iform1y consistent band­
ing ра Негпз аегоя аll lапез (Fig. 3). The abscnec оГ polymorphisms in the mutan!
lines is ргоЬаЫу due 10 [Ье ract that RЛРО primcrs amplified ehromosomal loci
П01 affected Ьу (Ье gamma-irradia1ion. Absence оГ RЛРD polymorphism Ьаз 81so
Ьесп found bctwccn а ehrysa nthemum cultiva r and its vegclalivcly dcrived cultivars
21
1
2
3
4
5
6
7
8
9
10
11
12 Fi&. З. Reprcscntativc RЛРО bandina; pallcm oflhe mulant barley lines and the oontrolli nc: - СУ. I' reyu. gcneruled Ьу Ihc primcr ОРА 12. Lines:- )..·1'51 1 (1), СУ. Fn:ya (2). T-1586 (3), T- 16 (4). Т·20 (5). Т-48 (6). 1~S8 (7). 1:59 (8), Т·6) (9), Т-66 (10). "·67 ( 11), Т·68 (12) (Wolff с! al .. 1995). 111 comparison with the аЬоуе observation. уегу low gcnetic
diversilY among the mutants in (Ье RAPD ana lysis о г ( СП radiomutants ог Chry­
san thcmum has Ьесп reported Ьу Lema-Ruminska е! з1. (2004). Similar resu]ts Ьаз
Ьееп obtaincd Ьу Joshi-Saha and Gopalakrishna (2007) who found опlу 3% poly­
moгphism between а Sesbania rustrota mutant and its parcntal gcnotype using 200
random dccamer primcrs. Lu е! з1. (2007) reported опlу 8.33% variations among (Ь е
regenerants deriyed Ггот irradiated CJ!;plants ог Chinese narcissus (Narc is.\'IIs IOzetro
yar. сhiпслs i s) using 40 arbilrary, 10-mer primers in the RAPD analysis.
AFLP a na lysls
Gаmmа - гауs -iлduсеd DNA a lterations, such as insertions о г dclct ions wcre studied
Ьу AFLP analysis ог 13 barlcy mutant lines in compa rison to the control gcnotypc
СУ. Frcya. The adyantage оГ this screening method is bascd оп thc Гас! that thc
p rimcr combinations used
ch romosoma l localizalion.
22
тау
hybridize 10
а
large numbcr
оГ
loci with
dilТerent
TlIbIt 3
Primer 5eqtм: lltc5 u:;c:d in
Primtr
preаnlрl Шсаt ion апd
amplilication reaclion mixlures
5'-3' Sequcnre
Е31
GАСТGСGТЛ ССЛЛ1ТСА+ЛЛЛ
(35
САТ GЛG ТСС ТG Л GТл А
+ лсл
Е4 1
GАСТGСGТЛСАТGСл GG+лGС
i\I SO
GЛТGЛGТСС ТGлGТАЛ
:'0159
:\160
м 61
GЛТGЛGТССТGЛ GТлл
GлТGлGТССТGл
слт сле тес ТСА
+ САТ
+ СТЛ
GТАЛ + сте
СТЛ Л + сгс
AFLP ana lysis was carried out with 10 selective AFLP primer combinrltions
(ТаЫс З ). M05t ог (Ь еm Ьаус produced uniform patterns Гог IЬ е mutant Ьа г! еу
IiПС5 analyzed and the initia! gcnotype (СУ. Freya), схсер! ЕЗlfМ61 . AFLP а55ау
produccd а totftl оГ 226 amplicons with ап average оГ 22 ampli cons рег primer
combination . ТЬс primer combinat ion Е31/М 61 generated а 287 Ьр 10 l1g fragment
in тшап! lil\cS Т- ! 586 a nd 1=-66 (Fig. 4).
Оп!у 0.2 % radiation-induccd polymorphism in (Ье ba rl ey пшtап ! lines origi­
nated Гг от апе рагеп!аl genotype were obta incd. Low level о Г AFLP polymorphism
(2%) between 3 Sesbaflia rlIstrma m utзп t and its parenlal genotype was also ге­
portcd Ьу Joshi-Saha and Gopalakrishna (2007). Ои! ог 64 AFLP primer со тЫ ­
nalions applicd in (Ьа! analysis оп]у 8 (12.5%) produced polymorphi5ms. In ап­
olhcr study us ing AFLP lechn ique, по polymorphism has Ьсс п detectcd bctwccn
а closed capsllle mutan! Нпе о Г Sesamllm ifldicum and its parental cuJtivar, схсерl
Гог а п AFL!> marker id en tifying thi5 trait (Uzun е! а ! ., 2003). Low level оГ genct ­
ic vnria tions ( 15.48%) a mong (Ье regencrants derivcd Ггот irradiated explants оГ
CIJi"e~'e narcissllS (Narcissus tazella "аг. chinensis) has з 1 50 Ьееп rcporled u5ing
ЛFLР markers (Lu е! аl . , 2007). Fo rstcr (2001) estimated only 0.2% dif'fcrences Ье­
twecn "Golden Promise", which is а gammз-гау induccd mutant оГ "Ma ythorpc" Ьу
AFLPs, in co mparison with 11.4% estimatcd Го г Ihe oblained Ьу X-rays " Dia mant"
and its origi nal genolype "Va] ticky" ( М lсос Ь оуа е! al., 2004). However, (Ье abovc
mentioned dif'fcrcnce cou ld Ье assigned 10 IЬ е difТcrenl types ог irradiation app]icd
10 ( Ье origina l Ьа г lеу cultivars "Maythorpe" and иVа l licky" in boLh sludics.
ТЬ е studies о п molecula r charactcrization оГ radiation-i nduced a] lcrat ions in
hig11cr plants including ccrea ls а ге still limiting_ 11 is gcnerally thought (Ьа! low lin­
саг cnergy transfer (LEТ) radiations, such as ga mma - and X-rays, dcpendil1g оп (Ье
dose app licd. induce рге Ге гаblу large dclctions (> 6 МЬр) and short deletions (1 -4
Ьр), tra l\s]ocations and гагеlу inscrtions (Shi rlcy е ! аl., 1992; Bruggenmann С I а].,
1996; Duschges е! a l.. 1997; Shikazono е! 3.1., 2000; 2003; Уап о е! a l.. 2000; Nakazaki
с! al., 2003; Naito е! 011., 2005). Оиг dala suggest that gamma-irradiation ргоЬаЫу
induces po int mutations besides {Ь е cyto!ogieally csta blished large сЬготоsoтаl
rcarrangcments. Th us. they сап аГГес ! primer sites. restriction sites, зs well зs (Ьс
wholc segmen t comprising (Ье prilner siles and опе ог both rcstriction sites а! (Ье
origin ог (Ьс STS, DARE, RЛPD and AFLP Cragments.
Using {Ьс ma rker systems app licd polymorphism wa5 detected in 2 muta"1
!incs Ьу AFlP а55ау due 10 а зЬor! del et ion оГ 2 Ьр in опе О Г (Ьс сhготоsоmз!
rcgiol\s аmр Шi еd Ьу (Ье primcr combination Е31/М61. The results indi cate аз а
wholc [Ь,I.I screcning Гог DNA allerations Ьу ше set ofapp lied DNЛ fingerprinting
tcchniques did по! reveal significant basc-pair cha nges accompanying ga mma-rays­
23
",ез
M.__ ___._.___
4 00 Т--_. ___
~
~~"""'
----,----,-----­
- - - ------,-- - - - ­
350
300
-
__
~
-----
~==-==;=:~:.....==
--=====
250
._--­ ­---~~--- - --- ~~
--- - ~-----
~- -
._=--~
200
150
'00
---- -
-- -
- --- ;;;;;;;;;;;::=-...;;====,-=
-­
---.--.----­
.---,.,.,....".-
- ------==
=
50
-==
=
-.,."",..
'~
---~~:-~
----
...-- ---,....
-
"· ig.~. Л1:LР profik оЬtlliщ:d with primcr comblnlllion EJIIM61 (roт : су. Freya ( 18): Т-1386 (19): T- 16
(20): Т·20 (21), 1'26 (22); "-48 (23); Т-58 (24): Т-59 (Н) : 1'·63 (26): T..6(i (27): Т-67 (2.11). "1'-68 (29). Т·169
(30), 1~8K·4(3 1 )
24
induecd ehromosome tran sloeations and inversions in Ьагlеу genome. As sueh ГC<IГ­
rangcments сопсе гп геlЗ livеl у large ehromosome domains in Ih e resulting mшаПI
genolypcs the respcetivc s рееШе DNA sequenees analyzed тау п01 eoincide with
the а геаз whcre subtle mUlational changes агс to lcrated. Further st udi ~ based оп
the applicaLion оГ varielY оГ SSR markers аге expected 10 Ihrow addiliona l light оп
this question.
деkпоw l('d~еmI.'ПI.!i Thil work
спсс: I"uпd
,,-,а,
supporlc:d Ьу ' Ьс: IЛЕд (Re$C:8rch coolr.cl No 8UL 12608) .nd
(contract No G 1· 02). 8utgaгiап
NallQOat 5cl­
Refcrences
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Maniali~,
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M
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