MSAP甲基化文献
Plant Science161(2001)359–367
Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive ampli?cation
polymorphism(MSAP)
Santy Peraza-Echeverria,Virginia Aurora Herrera-Valencia,Andrew James-Kay* Unidad de Biotecnolog?′a,Centro de In6estigacio′n Cient?′?ca de Yucata′n,Calle43No.130,Col.Chuburna′de Hidalgo,C.P.97200,Me′rida,
Yucata′n,Mexico
Received1November2000;received in revised form9April2001;accepted12April2001
Abstract
The extent of DNA methylation polymorphisms was evaluated in micropropagated banana(Musa AAA cv.‘Grand Naine’) derived from either the vegetative apex of the sucker or the?oral apex of the male in?orescence using the methylation-sensitive ampli?cation polymorphism(MSAP)technique.In all,465fragments,each representing a recognition site cleaved by either or both of the isoschizomers were ampli?ed using eight combinations of primers.A total of107sites(23%)were found to be methylated at cytosine in the genome of micropropagated banana plants.In plants micropropagated from the male in?orescence explant14(3%)DNA methylation events were polymorphic,while plants micropropagated from the sucker explant produced8 (1.7%)polymorphisms.No DNA methylation polymorphisms were detected in conventionally propagated banana plants.These results demonstrated the usefulness of MSAP to detect DNA methylation events in micropropagated banana plants and indicate that DNA methylation polymorphisms are associated with micropropagation.?2001Elsevier Science Ireland Ltd.All rights reserved.
Keywords:DNA methylation;Banana;Micropropagation;MSAP
https://www.sodocs.net/doc/3d9496998.html,/locate/plantsci
1.Introduction
Plant tissue culture can induce a variety of genetic
and epigenetic changes in regenerants,phenomenon
known as somaclonal variation.Changes in DNA
methylation has been hypothesized as an underlying
mechanism of tissue culture induced mutagenesis
due to the high frequency of quantitative pheno-
typic variation,the activation of transposable ele-
ments,heterochromatin-induced chromosome-
breakage events,and the sequence changes due to
deamination of5-methylcytosine to thymine[1–3].
Methylation of DNA in plants has been associated
with regulation of gene expression,genome defense,
cell differentiation,chromatin inactivation and ge-
nomic imprinting.Higher plants are predominately
methylated at the dinucleotide CpG and trinucle-
otide CpXpG in the form of5-methylcytosine[4].
Variation in DNA methylation patterns in regen-
erants has been described in rice[5],maize[6],
potato[7],tomato[8]and more recently,oil palm
[9].In the case of oil palm propagated via somatic
embryos,DNA hypomethylation has been found to
be associated with a well characterized phenotypic
somaclonal variant.
In micropropagated bananas somaclonal varia-
tion detectable at the level of the phenotype has
been reported to vary between1and50%[10].This
variation is one of the major drawbacks that has
limited the expansion of the tissue culture tech-
nology.Various factors such as genotype,origin
of shoots in vitro(adventitious or axillary buds),
number of subcultures,the choice of explant *Corresponding author.Tel.:+52-99-9813923;fax:+52-99-
9813900.
E-mail addresses:santy@cicy.mx(S.Peraza-Echeverria),vir-
gihv@cicy.mx(V.A.Herrera-Valencia),andyj007@cicy.mx(A.
James-Kay).
0168-9452/01/$-see front matter?2001Elsevier Science Ireland Ltd.All rights reserved.
PII:S0168-9452(01)00421-6
S.Peraza-Eche6erria et al./Plant Science161(2001)359–367 360
and the degree of dedifferentiation of the tissues in culture have all been shown to in?uence both the quantity and the type of somaclonal variation in micropropagated bananas[10–15].Somaclonal variants have been observed mainly in plant stature,in?orescence morphology and leaf abnor-malities[16].For commercial propagation of Cavendish bananas the vegetative apex of the sucker is most frequently used as an explant, however it is possible to obtain reversion of?oral to vegetative apices in vitro[17,18].Perceived ad-vantages for the use of the in?orescence as an explant include:decontamination is easier as there is no contact with soil;in the case of suspected chimeric clones it is possible to retain?delity of the fruit bunch characteristics by propagation from the male?oral apex;a lower incidence of virus infec-tion in regenerants[19].
In contrast to the wealth of data available on banana somaclonal variation at the phenotypic level,the possiblity of an epigenetic basis of this variation remains unknown.In order to initiate epigenetic studies of the level of DNA methylation in banana micropropagation,the methylation-sen-sitive ampli?cation polymorphism(MSAP)tech-nique was used,which is a relatively new modi?cation of the ampli?cation fragment length polymorphism(AFLP)technique,?rst developed to determine DNA methylation events in dimor-phic fungi[20]and later adapted for the detection of cytosine methylation in the rice genome[21].The MSAP technique utilizes the restriction isoschizomer pair Msp I and Hpa II(instead of Mse I as in the original protocol[22])whose ability to cleave at the sequence5%-CCGG-3%is affected by the methylation state of the external or internal cytosine residues.Hpa II is inactive if one or both cytosines are fully methylated(both strands methy-lated)but cleaves the hemimethylated sequence (only one DNA strand methylated),whereas Msp I cleaves C m CGG but not m CCGG[23].In this study we report the occurrence of DNA methyla-tion changes in micropropagated bananas from sucker and male in?orescence explants.
2.Materials and methods
2.1.Plant material
All plant material was obtained from a commer-cial plantation of Musa AAA cv.‘Grand Naine’near the town of Teapa,Tabasco state,Mexico. This plantation was derived from plants propa-gated solely by conventional means.Five corms were obtained from?ve individual plants and they were used for conventional propagation;these were cut in two so that each half had an axillary bud,and potted into sterilised compost(60%local soil and 40%peat).Approximately3weeks after planting shoots with leaves had emerged.Ten regenerants from one male in?orescence explant and10regen-erants from one sucker explant were obtained by tissue culture;these explants came from the same healthy mother plant,which was randomly selected in the?eld.
2.2.Tissue culture
Due to the differences in the nature of the two types of explants decontamination and induction procedures differed.A modi?cation of the protocol of Sandoval et al.[24]was used for the sucker.The sword sucker was reduced in size to a5cm explant, consisting of the meristem and the leaf bases, surface sterilised in a solution of commercial hypochlorite(6%Cl)for20min,and further reduced in size to2cm in length in sterile condi-tions.The explant was then immersed in a commer-cial hypochlorite(0.6%Cl)solution for10min and rinsed three times with sterile water.Further size reduction resulted in an explant of ca.1cm in length containing the vegetative apex.In the case of the male in?orescence,a modi?ed protocol based on that of Cronauer and Krikorian[17]was fol-lowed.The male in?orescence was dissected to obtain a conical explant of approximately4cm in length,immersed under sterile conditions in70% ethanol for5min and then reduced in size to obtain an apex of6–8mm in length. Micropropagation was carried out using Mu-rashige and Skoog medium[25]with3%sucrose and10mg l?1ascorbic acid;prior to autoclaving the pH was adjusted to5.8and2.5%gelrite added for semisolid medium.This medium was aug-mented for the induction phase with22.19m M 6-benzylaminopurine(BAP)and4.92m M indole-butyric acid(IBA)and for the shoot multiplication phase with10.0m M BAP and1m M indoleacetic acid(IAA).For induction the sucker explant was cultured in a tube on semi-solid induc-tion medium at a constant28°C for30days in darkness.The male in?orescence explant was?rst placed in liquid induction MS medium as
S.Peraza-Eche6erria et al./Plant Science161(2001)359–367361
indicated by Swamy and Sahijram[18]to mini-mize oxidation of phenolic compounds and cul-tured at a constant28°C in darkness for30days. After this time the?oral explant was transferred to a semi-solid induction medium as described above.Before moving to the multiplication medium both explant types were cut longitudi-nally taking care that wounded tissue was in con-tact with the medium as recommended by Sandoval et al.[24].For shoot multiplication both types of explants were cultured at28°C for 10days in total darkness and then further cul-tured at28°C for10days in photoperiods of16h light/8h darkness(Osram Daylight?uorescent tubes,35–40m E per m?2per s?1)subcultures were carried out every20days.After?ve subcul-tures regenerants were transferred to a plant growth regulator free medium for rooting, weaned in a peat/perlite compost and then trans-ferred to the greenhouse for acclimatization. These plants have now been planted in the?eld for future studies.
2.3.DNA extraction and MSAP assay
Genomic DNA was extracted from fully devel-oped leaves of both conventionally propagated and acclimatized micropropagated plants(6weeks post de-?asking)using the Nucleon Phy-topure kit(Amersham).
To detect MSAP,two digestion reactions were set up at the same time for each genomic DNA sample.In the?rst reaction200ng of the ge-nomic DNA was digested with5U of Eco R I (Gibco BRL)plus5U of Msp I(Gibco BRL)in a?nal volume of50m l containing50mM Tris–HCl(pH8.0),10mM MgCl2and50mM NaCl for5h at37°C.The second digestion reaction was carried out as above however Hpa II(Gibco BRL)was used instead of Msp I.The digestion reactions were then ligated to the adapters by adding10m l of ligation mixture containing1×T4DNA ligase buffer(Gibco BRL),10U T4 DNA ligase(Gibco BRL),5pmol Eco R I adapter and50pmol Msp I-Hpa II adapter (Table1).The ligation reaction was incubated at 23°C for5h.The digestion and ligation reactions were stopped by incubating at65°C for10min. The preampli?cation reaction was performed by using5m l of the above ligation product with 75ng Eco R I primer with one selective base (Eco RI+1),75ng Msp I-Hpa II primer with no selective base(Msp I-Hpa II+0),1×PCR buffer(Gibco BRL),1U Taq polymerase(Gibco BRL),1.5mM MgCl2and0.4mM dNTP’s in a ?nal volume of50m l.Reaction conditions were 20cycles of94°C for1min,56°C for1min and 72°C for2min.
Selective ampli?cation was conducted in vol-umes of20m l.For selective ampli?cation the preampli?ed mixtures were diluted1:25from their original volume with TE.A volume of5m l of these diluted samples was mixed with30ng of Eco R I with two selective bases,10ng of Msp I-Hpa II primer with two or three selective bases end-labelled with0.8m Ci[a32P]ATP,1×PCR buffer,0.5U Taq DNA polymerase and1.5mM MgCl2.Fragments present in these mixtures were ampli?ed for1cycle of94°C for30s,65°C for 30s and72°C for1min.In the following12 cycles the annealing temperature of65°C was lowered by0.7°C each cycle,followed by24cy-cles with the following program:94°C for30s, 56°C for1min and72°C for2min with a?nal extension at72°C for5min.All ampli?cation reactions were conducted in a Perkin Elmer9600 thermocycler(Perkin Elmer).A full list of adapters and primers used in this study is given in Table1.
Table1
Sequences of adapters and primers used for MSAP analysis Eco R I adapter5%-CTCGTAGACTGCGTACC-3%
3%-CATCTGACGCATGGTTAA-5%
Eco R I+1primer5%-GACTGCGTACCAATTCA-3%
E+AG
Eco R I+2primers
E+AT
E+AC
5%-CGACTCAGGACTCAT-3% Msp I-Hpa II adapter
3%-TGAGTCCTGAGTAGCAG-5% Msp I-Hpa II+0primer5%-GATGAGTCCTGAGTCGG-3% Msp I-Hpa II+2primers MH+CT
MH+GT
MH+ATG
Msp I-Hpa II+3primers
MH+CAT
MH+CTC
MH+CTA
S.Peraza-Eche6erria et al./Plant Science161(2001)359–367
362
Fig.1.MSAP electrophoresis patterns in micropropagated banana plants:(A)MSAP pattern of regenerants from the male in?orescence(1–10)and sucker explants(11–20)of one mother plant using the primer combination MH-ATG/E-AG;M and H refers to digestio′n with Eco R I+Msp I and Eco R I+Hpa II,respectively;CP refers to conventionally propagated plants DNA pool.L=50bp ladder(Gibco BRL);(B)Enlarged view of the left boxed area in(A)indicates DNA methylation polymorphisms in regenerants from the male in?orescence(see arrows);(C)Enlarged view of the right boxed area in(A)indicates DNA methylation polymorphisms in regenerants from the sucker(see arrow).
2.4.MSAP electrophoresis and autoradiography The MSAP products were mixed with an equal volume of tracking dye(98%formamide,10mM EDTA pH8.0,0.1%bromophenol blue and0.1% xylene cyanol)denatured at95°C for3min and immediately cooled on ice.Aliquots(5m l)of each reaction were electrophoresed on6%denaturing polyacrylamide gel(30cm×50cm×0.4mm) containing8M urea and1×TBE.Gels were run at60W for2h,dried and exposed to Kodak BioMax X-ray?lm for2–4days before being developed.The size of the fragments was calculated using a50bp ladder(Gibco BRL).2.5.Data analysis
A DNA methylation event was detected when bands present in the autoradiographs from the reaction Eco R I+Msp I(M)were absent from the reaction Eco R I+Hpa II(H).This indicated that the internal cytosine was methylated(5%-Cm-CGG-3%).The contrary situation,where a band was present in H but absent in M,indicated that the external cytosine of one DNA strand was methylated(5%-m CCGG-3%).This is regarded as the hemimethylated state[23].A DNA methylation event was considered to be a polymorphism when at least one plant differed from the others in the
S.Peraza-Eche6erria et al./Plant Science161(2001)359–367363 Table2
Total number of bands and number of DNA methylation events detected by MSAP in leaf tissue of micropropagated plants(MP) and conventionally propagated plants(CP)
Number of bands
Oligonucleotide combination Number of methylation events
MP CP MP CP
64651915
E-AG/MH-ATG
7162
E-AC/MH-ATG2017
E-AT/MH-ATG486
6
57
E-AG/MH-CTC55
5183
4742
E-AG/MH-CAT1615
7663
E-AC/MH-GT138
E-AG/MH-GT58601513
5052
E-AT/MH-CA107
107(23%)
46545684(18.4%)
Total
methylation pattern,for example,a band was present in both M and H in one plant but was only found in M in other plants.When,in one or some samples,no bands were detected in both digestions this was not considered as a polymor-phism due to the fact that it is not possible to discard the presence of an AFLP event.Events in which there was an obvious difference in band intensity were also considered as DNA methyla-tion polymorphisms.
The proportion of polymorphisms(P)was esti-mated by division of the number of DNA methy-lation polymorphisms by the total number of bands.To determine if differences were statisti-cally signi?cant between treatments,standard er-rors were calculated using the equation of Sokal and Rohlf[26]:SE(P)= (P(1?P)/n),where: SE,standard error;P,proportion of polymor-phisms;n,total number of bands.To obtain a 95%con?dence interval the normal approximation of the binomial distribution was used:P9t(h) SE(P),where t,normal distribution value;h,prob-ability of type I error.
3.Results
In this analysis eight primer combinations pro-duced a total of456bands from leaf tissue of conventionally propagated banana plants and465 bands from the equivalent leaf tissue of mi-cropropagated banana plants.Of these fragments, 84bands(18.4%)from conventionally propagated plants were considered to have been produced by DNA methylation events whilst107bands(23%) from the micropropagated plants were considered to have been produced by DNA methylation events(Table2).Fig.1A shows representative DNA methylation events at the cytosine of the dinucleotide CpG cleaved by Msp I(present band) but not cleaved by Hpa II(absent band).Fig.2 shows a representative MSAP pattern for conven-tionally propagated banana plants.
DNA methylation polymorphisms were detected in?ve of the primer combinations among mi-cropropagated plants(Table3);for example,using the primer combination MH-ATG/E-AG(Fig. 1B)the regenerants1and2from the male in?ores-cence explant are demethylated in two sites(see arrows),but methylated in the remainder of the micropropagated plants(but not the DNA pool of 10conventionally propagated plants).With the same primer combination in the regenerant num-ber15,a demethylated site of almost200bp was detected,but this site is methylated in the rest of the regenerants derived from both explant types and also in the conventionally propagated plants (Fig.1C).A total of14reproducible DNA methy-lation polymorphisms were detected in regenerants from the male in?orescence and eight in regener-ants from the sucker explant(Table3);these dif-ferences were not statistically signi?cant(Table4). Conventionally propagated plants did not show any DNA methylation polymorphisms using the same primer combinations tested in the regener-ants(Table3).This result suggests that the DNA methylation polymorphisms were associated with the tissue culture process.
S.Peraza-Eche6erria et al./Plant Science161(2001)359–367
364
Fig.2.MSAP electrophoresis pattern in conventionally prop-
agated plants using the primer combination MH-ATG/E-AG;
M and H refers to digestion with Eco R I+Msp I and Eco R
I+Hpa II,respectively.L=50bp ladder(Gibco BRL).
requirement for prior genome information other
than the approximate genome size.Other advan-
tages are the high number of methylation events
detected using a relatively small number of primer
combinations and the additional ability to clone
and characterize novel methylated sequences.We
therefore consider MSAP to be a suitable technique
to evaluate epigenetic changes at the level of DNA
methylation which may be associated with the
phenomenon of somaclonal variation.
The prime objective of this study was to evaluate
the occurrence of DNA methylation changes in
micropropagated banana plants.The results
demonstrated that the micropropagation process
may be responsible for DNA methylation pattern
alterations in plants derived from both explant
types since conventionally-propagated plants do
not show DNA methylation polymorphisms at
least with the eight primer combinations tested.
Some polymorphisms such as those found in a
sucker-derived regenerant(Fig.1C)suggest that
sequences such as transposons could be activated
by the tissue culture process,as for example those
reported by Brettell and Dennis[28]who found a
correlation between AC element activation and AC
element demethylation in regenerants of Zea mays.
On the other hand,this kind of polymorphism
could be related to the ectopic expression of genes
such has recently been demonstrated to occur in
Arabidopsis thaliana plants carrying the methy-
transferase METI-antisense transgene and inwhich
there was found to be ectopic expression of?oral
homeotic genes in leaves[29].
The hypermethylation found in micropropagated
banana plants(Table2),in comparison to conven-
tionally propagated plants,has similarities with the
situation found in rice seedling(hypermethylated)
and rice?ag leaves as reported by Xiong et al.[21].
It is thus likely that this is a tissue-speci?c and
developmentally regulated phenomenon.However
during conventional propagation of banana this
situation probably never occurs since no functional
leaves of comparable size to those of6-week old
micropropagated banana plants are produced.It
has been reported that the leaf anatomy and mor-
phology of young ex vitro banana plants is different
to that of?eld grown plants[30]hence differences
in methylation may be a re?ection of the different
developmental processes involved;for a review see
Finnegan et al.[4,31].An alternative explanation is
that the hypermethylation common to all regener-
ants,is induced by elements of tissue culture per se 4.Discussion
The present study has demonstrated the sensitiv-
ity of the MSAP technique in the detection of DNA
methylation events in the genome of micropropa-
gated banana plants.This conclusion is in agree-
ment with the results reported in the rice genome
by Xiong et al.[21],who detected195(16.3%)DNA
methylation events among a total of1076ampli?ed
bands with the use of16primers,and more recently
Ashikawa[27]reported similar results also in the
rice genome.To our knowledge this is the?rst
report of the use of the MSAP technique to evalu-
ate DNA methylation changes in plants derived
from tissue culture.Previous analysis of the methy-
lation status of in vitro-cultured plants have most
frequently utilized restriction fragment length poly-
morphism(RFLP)[1,5,6,8].However,the latter
technique has several disadvantages such as the
high number of probes required,the lower amount
of information produced and the necessity for prior
sequence information.MSAP is based on the
AFLP technology[22]and therefore there is no
S .Peraza -Eche 6erria et al ./Plant Science 161(2001)359–367365
Table 3
Polymorphisms in cytosine methylation detected with MSAP in leaf tissue of micropropagated banana plants derived from in ?orescence (n =10)and sucker explants (n =10)and banana plants propagated conventionally from corms (n =10)Conventionally propagated plants
Oligonucleotide Sucker-derived regenerants
In ?orescence-derived regenerants combination 410E-AG /MH-ATG 01E-AC /MH-ATG 300E-AT /MH-ATG 030E-AG /MH-CTC 400E-AG /MH-CAT 03E-AC /MH-GT 2001E-AG /MH-GT 000E-AT /MH-CA 00
14
8
Total
Table 4
Standard errors and 95%con ?dence intervals calculated for DNA methylation polymorphisms in banana plants micropropagated from male in ?orescence and sucker explants 95%con ?dence
Explant origin of
Estimated proportion Percentage of
Standard error interval (10?3)micropropagated plants polymorphisms (%)(P )(10?3)3[14.5–45.6]Male in ?orescence 7.9130.10 1.7
[5.4–29]
6.02
17.20
Sucker
dence is required to fully assess the effect of the explant source on the extent of DNA methylation polymorphisms,using a more extensive number of primer combinations.In addition,other plant methylation sites such as the trinucleotide CXG could be evaluated.The question as to whether DNA methylation changes are associated with banana ‘off-types ’will require molecular character-ization of the polymorphisms and a phenotypic evaluation of the regenerants in the ?eld,this work is currently in progress.Acknowledgements
This work was ?nanced in part under Interna-tional Atomic Energy Agency Research Contract No.9639,Belgium.S.Peraza-Echeverria and V.Herrera-Valencia acknowledge CONACyT (Mex-ico)for the scholarships 126280and 129567respec-tively,which enabled them to participate in this work.We thank Dr Georgina Reyna Lo ′pez for technical assessment.We are also thankful to Dr Octavio Mart ?′nez de la Vega for statistical assess-ment,and to Dr William Cress for his critical review of this manuscript.
and is not developmentally related.For example,plant growth regulators in particular auxins (used in this study)have been shown to induce methyla-tion changes in plant tissue cultures [32–34].
In some samples the ampli ?ed fragments cleaved by both enzymes differed in band intensity (Fig.1B),such differences may infer variation in the methylation level among the cells in the target tissue as suggested by Xiong et al.[21].At the global level differences in band intensity were clearly seen at some sites among both micropropagated and con-ventionally propagated plants (Fig.1A and Fig.2)and may indicate the presence of repeated elements.Repeated elements are known to be preferentially methylated in higher plants [35]and have also been found to be subject to tissue-culture induced methy-lation [8].In this regard it is of interest to observe the intense bands cleaved by both enzymes above 750bp in Fig.2;this site is demethylated in conventionally propagated plants whereas the cor-responding site in most of the micropropagated plants (Fig.1A)is methylated.
Although,in this study,no signi ?cant statistical differences were found between the regenerants of both explant types,we consider that further evi-
S.Peraza-Eche6erria et al./Plant Science161(2001)359–367 366
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