DNA Methylation Detection of Oil Palm (Elaeis guineensis Jacq ...

JOURNAL of BIOTECHNOLOGY RESEARCH in TROPICAL REGION, Vol. 1, Oct. 2008 (Special Edition)

ISSN: 1979-9756

DNA Methylation Detection of Oil Palm (Elaeis guineensis Jacq) Somatic Embryo by Randomly Amplified DNA Fingerprinting with Methylation–Sensitive (RAF-SM) and RP-HPLC Nesti F. Sianipar1*, Gustaaf A. Wattimena2, Maggy Thenawijaya Soehartono3, Hajrial Aswidinnoor2, Nurita Toruan-Mathius4,Gale Ginting5 1

Biology Departement, Faculty of Science and Mathematics, University of Pelita Harapan, Jalan MH. Thamrin Boulevard 1100, Lippo Karawaci, Tangerang 15811, Banten 2 Agronomy Departement, Bogor Agriculture University, Campus of IPB-Darmaga, Bogor 3 Food Technology Faculty, Bogor Agriculture University, Campus of IPB-Darmaga, Bogor 4 SEAMEO Biotrop, Jalan Raya Tajur Km 6 PO. Box 116, Bogor 5 Oil Palm Research Center of Indonesia, PO. Box. 1103 Medan 20001, Jalan Brigjen. Katamso 51 Kp. Baru, Medan 20158

Abstract Oil palm is the most important vegetable oil producing plant in Indonesia. A mass production of oil palm transplants to support new oil palm estate can be conducted through tissue culture. However, the primary problem of tissue culture generated transplants is the increase reproductive abnormalities postulated due to the epigenetic changes during somatic embryo (SE) formation. The objective of this research was to study the relationship of cytosine methylation of genomic DNA and abnormality in several SE growth stages and normal mother plant. Epigenetic characterization was conducted by Reverse Phase-High Performance Liquid Chromathography (RP-HPLC) and RAF-methylation sensitive technique. The genomic DNA digestion with MspI and HpaII by RAF-SM technique could detect the location of cytosine methylation. HpaII cuts the mCCGG, but if both the C’s were methylated, the sequence will not be cut. MspI cuts the genomic DNA sequence when the cytosine experiencing an internal methylation (CmCGG). The RAF-SM technique could detect methylation location of internal, external and fully methylated at 124-457 bp of genomic DNA. The RP-HPLC analysis showed very little changes of cytosine (0.25–2.72%) in clone 638. However, the cytosine methylation changes of normal and abnormal SE were due to hypomethylation in clone 638. Abnormality of SE at cotyledon stage was due to location change of cytosine methylation in the genomic DNA sequence. Keywords: somatic-embryo abnormality, oil-palm, DNA methylation, RAF-sensitive methylation, RPHPLC.

*Correspondence to: Nesti F. Sianipar e-mail: [email protected]

INTRODUCTION DNA methylation involves the addition of methyl group to the carbon-5 position of the cytosine ring, which is catalysed by DNA methyltransferase (Singal & Ginder, 1999). DNA methylation has been reported to be expressed in a number of biological processes such as regulation of imprinted genes, X chromosome inactivation and tumor suppressor gene silencing in cancerous cells or carcinogenesis (Gonzalgo & Jones, 1997; Zhang, 2002), wich lead to abnormality. In DNA genome, methylation usually occurs in the CpG island, a CG rich region, upstream of the promoter region. The development of in vitro clonal propagation of oil palm by somatic embryogenesis has resulted in the identification of mantled fruit phenotype among adult regenerants (Rival et al., 1997). This abnormality affects the formation of floral organs in both male and female flowers and has been observed on an average of 5% of regenerants (Jaligot et al., 2000). The observation of the variable expression of mantled phenotype suggested that the underlying cause might be epigenetic. Alterations in DNA methylation patterns have been widely found in plant displaying phenotypic defect, in the case of aberrant floral morphogenesis (Finnegan et al., 2000) and somaclonal variants (Kaeppler et al., 2000). Shah & Ahmed-Parveez (1995) showed that level of 5-methylcytocine, measured by high pressure liquid chromatography, were significantly higher in abnormal compared to normal regenerants. Several other studies have reported that hypomethylation occured at a higher frequency in the genome of abnormal regenerants compared to their normal counter parts (Jaligot et al., 2000).

DNA Methylation Detection of Oil Palm (Elaeis guineensis Jacq) Somatic Embryo by Randomly Amplified DNA Fingerprinting with Methylation–Sensitive (RAF-SM) and RP-HPLC

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ISSN: 1979-9756


Recently, several methods to detect DNA methylation have been developed. Detection of DNA methylation in eukariotic organism tissues depends on the application of bisulfites or Isoschizomers. Isoschizomers share the same recognition sites but show differential sensitivity to the DNA methylation (McClelland et al., 1994). Polymorphic DNA fragments can be generated after digesting methylated genomic DNA with isoschizomers. In general, any method capable of displaying polymorphism of digested DNA fragments can be used to detect DNA methylation (Reyna-Lopez et al., 1997; Xiong et al., 1999). In this study, the methylation levels of the whole genomes from abnormal cotyledon somatic embryo and leaf tissue of normal mother plant were detected and analyzed by Randomly Amplified DNA Fingerprinting-Methylation Sensitive (RAF-MS) with RPHPLC. This work investigated the extent of methylation from quantitative level using RP-HPLC technique. RAF-MS technique is a powerful tool to detect base methylation in the genome. In this study, the technique was used to investigate changes in methylation status of normal and abnormal tissue culture clone of oil palm. The RAF-MS technique was preferred as it provides high resolution of amplified fragment length polymorphism, while the use of isoschizomeric enzymes (HpaII/MspI) allows the evaluation status within the corresponding target site (CCGG). The analysis of global DNA methylation is defined as the relation between methylated deoxycytosines and total deoxycytosines of the genome and expressed as a percentage, can be performed by HPCE system (Fraga et al., 2002) or HPLC system (Johnston et al., 2005). The objectives of this research were to study the relationship of cytosine methylation of genomic DNA and abnormality at several SE growth stages and plantlets by RAF-MS and RP-HPLC techniques.

MATERIALS AND METHODS Plant material: Normal mother plant and somatic embryo of oil palm MK638 obtained through in vitro somatic embryogenesis were sampled at Oil Palm Research Center of Indonesia (PPKS), Medan. DNA extraction: DNA for RAF-SM analysis was extracted from young leaves of normal mother plant and somatic embryo of oil palm using a modified method of Doyle & Doyle (1990). RAF-MS assay: The RAF-MS technique was performed according to the general steps as described by Waldron et al. (2002) with certain modification to incorporate the use of methylation-sensitive restriction enzymes. This modified assay involved the use of the isoschizomers enzymes, HpaII/MspI. Each reaction volume (10 µl) cointained 1x PCR buffer (10 mM Tris pH 8.0, 10 mM KCl, 5 mM MgCl2), 20 µM dNTPs, 1.5 units AmpliTaqI Stoffel Fagment DNA polymerase, 1 µCi α-labelled 33PdATP, 5 µM single oligonucleotides (Operon Technologies Inc. A kits) (Table 1) and 50 ng of genomic DNA template. PCR was performed with a hot start (85oC), an initial denaturation at 94oC for 5 min, followed by 30 amplification cycles of : 94oC for 30 and 60 sec each at 57, 56, 55, 54 and 53oC, and final extension step at 72oC for 5 min. Eight primers were initially tested. To find DNA fragments, DNA analyzer were used. GeneScanTM-500LIZ was used as size standard. Two microlitres aliquots from the PCR result was used. Into the sample, 0.2 µl GeneScanTM-500LIZ and 7.8 µl HiDi formamide were added. The sample was then denatured at 95oC for 5 min and cooled on ice. These samples were the run on a 3130 DNA Analyser (Applied Biosystems).

Table 1. Primer sequences from Operon Technologies Inc. Primer

1

AB-16

5’-CCCGGATGGT-3’

2

AE-11

5’-AAGACCGGGA-3’

3

AO-12

5’-TCCCGGTCTC-3

4

AP-20

5’-CCCGGATACA-3’

5

BB-18

5’-CAACCGGTCT-3’

6

W-15

5-ACACCGGAAC-3’

Analysis of methylation levels by RP-HPLC: Absolute methylation levels were measured by reversed-phase high-performance liquid chromatography (RP-HPLC) with enzymatic hydrolysis to deSianipar, et.al.

Primer sequence

No.

(5’–3’)

grade the total genomic DNA from mother plant and in vitro somatic embryo. In the enzymatic method, 1-10 µg of DNA was dissolved in water, heated for 2 min in boiling water and cooled rapidly on ice. 2


Five microlitres of 10 mM ZnSO4 and 10 µl of nuclease S1 were added to each sample and the reaction was incubated at 37oC for 16 h. A 10 µl portion of 0.5 M Tris pH 8.3 and 10 µl bacterial alkaline phosphatase were added and the mixture was incubated at 37oC for an additional 2 h before centrifugation to remove suspended proteins. Sample aliquots of 100 µl was injected onto a supercoil LC-18S column (150x4.6 mm; Supelco) and separated with a Hewlett Packard automated HPLC instrument. Elution was carried out in mobile phase of 0.05 M NH4H2PO4, 8% methanol 4.2 at flow rate of 1 ml/min at room temperature. Identification of specific nucleoside peaks was obtained by running standard samples of cytidine, 5-methylcytosine and a nucleosides test mixture. Each sample was separated at least twice. The bases were detected at 254 nm and the percentage of 5-methylcytosine (5 mC) was calculated as (5 mC/[5 mC+C]x100.

ISSN: 1979-9756

RESULTS AND DISCUSSION Detection of cytosine methylation sites by RAF – MS technique: Using the RAF-MS technique, we detected and analyzed the methylation levels of the whole genomes from abnormal SE cotyledon and leaf tissue of normal mother plant. The result was obtained by digestion with HpaII and MspI and amplification using 6 primers. Out of 6 primers, 4 primers can amplify (AB-16, AE-11, AO-12 and AP20 primers). The result showed that methylation patterns which occur between a normal mother plant and abnormal somatic embryo cotyledon was different. With primer AB-16 (5’-CCCGGATGGT-3’), the result showed a different methylation pattern between normal mother plant and abnormal SE cotyledon. In the normal plants the cytosine methylation sites were at 124 and 378 bp, while in the abnormal SE were at 138, 375 and 385 bp. In an abnormal SE cotyledon some external methylations were also notified at 375 and 385 bp site and full-methylation (mCmCGG) at 138 bp site (Table 2 and Fig. 1).

Table 2. The result of cytosine methylation site detection on oil palm DNA genome by RAF-MS technique on clone MK 638. Primer AB-16 (5’-CCCGG ATGGT-3’)

Sample

Full (uncut)

HpaII cut

MspI cut

Explanation

Band (bp) 124

EG 2K-

+ -

+ -

+ -

Full methylation (mCmCGG) Mutation

138

EG 2K-

+

+

+

Primer not complementing Full methylation (mCmCGG); mutation

375

EG 2K-

+

+

-

Primer not complementing Mutation and external metylation (mCCGG)

378

EG 2K-

+ -

+ -

+ -

Full metylation (mCmCGG) Mutation

385

EG 2K-

+

+

-

Primer not complementing Mutation and external metylation (mCCGG)

Band (bp) 168

EG 2K-

+ -

+ -

+ -


446

EG 2K-

+ -

+ -

-

External methylation (mCCGG) Mutation

457

EG 2K-

+ +

-

+ +

Internal methylation (mCCGG) Internal methylation (CmCGG)

Band (bp) 195

EG 2K-

+ -

+ -

+ -


290

EG 2K-

+ -

+ -

+ -


Primer AE-11 (5’-AAGAC CGGGA-3’)

Primer AO-12 (5’-TCCCG GTCTC-3’)


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Primer AP-20 (5’-CCCGG ATACA-3’) Band (bp) 235

EG 2K-

+

+

-

Primer not complementing External methylation (mCCGG), Mutation

Fig. 1. Fluorescence detection technique of RAF with primer AB-16 (5’-CCCGGATGGT-3’). Labeled by FAM (6-canboxyfluorecein) on oil palm plant clone MK638. The normal mother plant DNA genome was uncut (R1-EG), uncut abnormal SE cotyledon DNA genome (R1-2K-), normal mother plant DNA genome cut by HpaII enzyme (HR1-EG), abnormal SE cotyledon DNA genome cut by HpaII enzyme (HR1-2K-), normal mother plant DNA genome cut by MspI enzyme (MR1EG), abnormal SE cotyledon DNA genome cut by MspI enzyme (MR1-2K-).

Application of primer AE-11 (5’AAGACCGGGA-3’) on mother plant genomic DNA identified a full methylation located at 168 bp site and internal methylations at 446 and 457 bp sites. On abnormal SE cotyledon, internal methylation was also identified at 457 bp site. Primer AO12 (5’-TCCCGGTCTC-3’) on normal mother plant DNA gave full methylation at 195 and 290 bp sites, while on DNA fragment of abnormal SE cotyledon identified point mutations at those sites. Primer AP20 (5’-CCCGGATACA-3’) identified external methylation and point mutation at 235 bp site of abnormal SE cotyledon DNA (Table 2). Methylation patterns obtained after MspI and HpaII digestion on normal mother plant and abnormal SE cotyledon DNA were classified into full methylation, internal methylation (hemimethylation) and external methylation. The RAF-MS technique with AB-16, AE-11, AO-12 and AP-20 (Table 2) could detect all those methylation paterns on either normal mother plant or abnormal SE cotyledon genomic DNA. DNA methylation occurs at specific sites. Bird (1995) mentioned that eukaryotic genome is usually heterogenously methylated at a certain site, while the rest of the domain remains unmethylated. Most of methyl groups are found at CG. DNA of higher Sianipar, et.al.

eukaryote is methylated at C (carbon) of some cytosines. The proportion of modified cytosine (methylcytosine/5-mC) was more than 30% in several plant species (Matassi et al., 1992). MspI and HpaII as the digestion enzymes on genomic DNA to detect cytosine methylation sites have been used on several plant, such as rice, apple, Arabidopsis and Pisum. The method has been used to identify CpG methylation by some recognizing enzyme on CCGG sites of rice genome (Xiong et al., 1999), to characterize methylation change related to micropropagation on banana (PerazaEcheverria et al., 2001) and to detect somaclonal variation of oil palm (Matthes et al., 2001). The MspI enzyme cuts only the C5mCGG, but not the 5mCCGG (McCleland et al., 1994). Belluci et al. (2002) mention that internal metylation can be detected by MspI enzyme as it cannot cut methylated cytosine (C5mCGG). Quantification of cytosine methylation by RPHPLC technique: On clone MK638, the different of methylcytosine contents between normal and abnormal globular was 0.64%, normal and abnormal SE cotyledon was 0.25%, and plantlet and normal mother plant was 2.72%. The results showed that the change of methylcytosine content between nor 4


mal and abnormal globular SE cotyledon decreased by 0.64% and at normal and abnormal cotyledon stage decreased by 0.25%. The change of methylcytosine contents between normal and abnormal SE cotyledon, and between plantlet and normal mother plant were due to hypomethylation (Fig. 2). Rival et al. (2004) found that hypomethylation occured on fast growing calli (100% abnormal regenerants), compared to compact calli (5% abnormal regenerants) at the same clonal line. The result of methylcitosine (5-mC) quantification on clone MK638 showed that methylcitosine content change in the range of 0.25-2.72%. Kubis et al. (2003) stated that the difference of cytosine methylation level between calli and its parent or explant source was about 2.2% and it was insignificant. Methylation level was lower on plants from mantled fruit clone than parents. This research showed that clone MK638, the different of cytosine methylation content between normal and abnormal globular SE, plantlet and normal mother plant was insignificant. Abnormality on somatic embryo at globular stage of cotyledon was due to the change of genomic DNA sequence or mutation. The change of metylcytosine was due to metylation decrease or hypomethylation. Kakutani et al. (1996) found that hypometylation, decreases in metylcytosin content, on mutant Arabidopsis thaliana. DNA hypometylation increases mutation rate because of the present of transposition element or increasing recombination rate of genome restructuring. Wolffe et al. (1999) stated that methylcytosine residue in genomic DNA plays an important role in gene expression.

typic abnormalities were mainly due to accumulated mutation. Finnegan et al. (1996) mentioned that aberration development was postulated as a part of hypomethylation of global genome inducted genetically or epigenetically in a single sequence of plant or animal. In conclusion that primer AB-16, AE-11, AO-12 and AP-12 could detect cytosine methylation at different sites between abnormal SE cotyledon and normal mother plant. RAF technique could detect the site of abnormal SE cotyledon and normal mother plant. Internal methylation, external methylation, and full methylation were detected at 124-57 bp sites on either abnormal SE cotyledon or normal mother plant. The difference of cytosine methylation content between normal and abnormal globular SE, normal and abnormal SE cotyledon, and plantlet and normal mother plant were very small (0.25–2.72%). The changes of methylcytosine contents in abnormal and normal SE, plantlet and normal mother plant were due to hypomethylation.

ACKNOWLEDGEMENTS The authors would like to thank the Oil Palm Research Center of Indonesia (PPKS) for providing mother plant and somatic embryo samples. We also would like to thank Dr. Rudy Lukman for his permission to work with the RAF-MS technique in the laboratory of the Biotechnology Department at PT. BISI International Tbk.

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60 50 Normal Globular

40 Percentage of 5mC

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Abnormal Globular

30

Normal Cotyledon Abnormal Cotyledon

20 Plantlet

10

Mother

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