Methylation sensitive-sequence related amplified polymorphism (MS-SRAP) marker system and its application to de novo methylation detection in Brassica napus

DNA methylation is one of the most important epigenetic processes that participates in the organization of chromatin structure and plays an important role in regulating gene expression. Sequence-related amplified polymorphism (SRAP) is a simple but an efficient gene amplification marker system for both mapping and gene tagging in plants. Combined methylation sensitive restriction enzyme digested genomic DNA with SRAP and methylation sensitive-sequence related amplified polymorphism (MSSRAP) marker system was developed to investigate de novo DNA methylation in the plant genome. To investigate the efficiency of this new marker system, DNA samples of 5 different tissues from 3 individuals of Brassica napus W10 (DH line) were employed for de novo DNA methylation analysis. Results indicated that approximately 0.2% reproducible polymorphic fragments were found among the five different tissues. Sequencing results demonstrated that some of these polymorphic fragments were matched well with the chloroplast encoding genes, photosynthetic related genes and the genes encoding protein with unknown functions in Genbank. Therefore, MS-SRAP marker system is a simple but efficient system for detecting gene de novo methylation.


INTRODUCTION
Epigenetics can be defined as a study of heritable changes that modulate regional chromatin organization without altering the corresponding DNA sequence.Methylation of the C5 position of cytosine residue which (an asymmertric site, where H is A, C or T) sequences is *Corresponding author.E-mail: Limaoteng426@163.com or zhchy@mail.hzau.edu.cn.
the most common covalent modification in higher plants.The presence of 5-methylcytosine in genomic DNA is the result of enzymatic activity of the 5C DNA methyltransferases, that is, maintenance and de novo methyltransferases.It is thought that one of the mechanisms of de novo DNA methylation is guided by small interfering RNA (siRNA) (Chan et al., 2004).In contrast to de novo DNA methylation, plants have also developed a glycosylasebased mechanism for removing DNA methylation (Kinoshita et al., 2004;Gong et al., 2002).This epigenetic process participates in the organization of chromatin structure and plays an important role in regulating gene expression during normal plant growth (Finnegan et al., 1996;Richards, 1997).
A methylated promoter is usually associated with a close configuration and is no longer accessible to transcription factors, resulting in disabling of the activity of the promoter; while un-methylated promoter displays an open chromatin structure and is accessible to transcription factors and other co-activators to initiate gene transcription.However, recent reports indicate that a DNA de novo methylation could spread from the coding region to the other regions such as the promoter regions both in plants and mammals, and this can also lead to an inhibition of gene transcriptin (Hisano et al., 2003;Singal and vanWert, 2001;Thomas et al., 1996).Therefore, studies on the status of DNA methylation level in the genome, especially de novo DNA methylation, become more and more meaningful for investigating the gene spatial expression pattern during development.
Some techniques have been developed for analyzing global and gene specific DNA methylation.For a global analysis of methylated hot-spots or CpG islands in the genome, several methods such as methylation sensitivesequence related amplified polymorphism (MS-AFLP) (Yamamoto et al., 2001) and CpG island microarray (Gitan et al., 2002;Shi et al., 2003) have been created.But each system has its own advantages and disadvantages.For example, MS-AFLP, that is, methylation restriction enzyme is used to digest genomic DNA followed by polymerase chain reaction (PCR) amplification, and is now widely used for genomic DNA methylation detection due to its high multiplexing ratio.However, the disadvantage of this method is not only on the complexity but also on less gene fragments amplified.CpG island microarray is a high throughput technology, but considerably expensive, which prevents it to be widely used.Therefore, a strong demand for cheap and convenient marker system is required for better uncovering the de novo DNA methylation or demethylation genome widely.
Recently, a new marker system, sequence-related amplified polymorphism (SRAP), was developed by combining two different arbitrary primers containing AATT with CCGG core sequence, respectively (Li and Quiros, 2001).This rationale is based on the fact that exons are normally GC-rich regions, whereas AT-rich regions are normally found on promoters or introns.Approximately, 45% of the gel-isolated bands matched with known functional genes in the database of GenBank after genomic DNA was employed with PCR using this marker system and with gel separation (Li and Quiros, 2001).
In the present study, a new marker system, MS-SRAP, was developed which combined a digestion of genomic DNA using methylation sensitive restriction enzyme with the SRAP marker system.Tissue specific de novo methylation/de-methylation of few functional genes was revealed based on this maker system.

Plant material and DNA extraction
Seeds of a DH line, Brassica napus cv W10, derived from Westar Zhang et al. 3341 were planted in the greenhouse and used for sampling different tissues in this experiment only because an efficient gene transformation system has been developed(unpublished data).At the flowering time, tissues with the same age were sampled from different parts, that is, young leaves (YL), mature leaves (ML), small buds (SB), mature buds (MB) and bagged flowers (F) of three individuals and used for DNA extraction, respectively.Total DNA was isolated following the method of Horn and Rafalski (1992).DNA concentrations were determined with a Beckman DU640 UV/V spectrophotometer.

Arbitrary primers
Arbitrary primers were designed according to the concept of Li and Quiros (2001) and Hu and Vick (2003) (Table 1).Each of the arbitrary primers containing a core region of AATT or CCGG sequence, 3 selective nucleotides at the 3' end and the filler sequences at the 5' end, was synthesized by Sangon Company (Shanghai, China).

PCR amplifications
Five micro gram total genomic DNA from the 5 different tissues were digested with 10 U HpaII restriction enzyme in a 20 µl reaction volume overnight at 37°C.After incubation, the digested products were dissolved using water to a final concentration of 50 ng/µl for PCR amplification, respectively.PCR reaction was carried out in a 15 µl volume containing 2 µl of 50 ng/µl digested DNA sample, 50 ng of each forward and reverse arbitrary primers, 200 µM of each dNTP, 1.2 U Taq polymerase (Promega, Madison, WI, USA) and 1×•Taq polymerase buffer supplied with the enzyme.The PCR reactions were done as described by Hu and Vick (2003).The amplicons were separated by denaturing acrylamide gels and detected by silver staining.

Sequencing analysis
Each polymorphic band reflecting different methylation status at the same locus among the five different tissues were excised from the acrylamide gel and transferred into a 1.5 ml tube containing 20 µl sterile ddH2O, respectively.After boiling for 5 min in the water, the recovered DNA was dissolved and then 2 µl of them were used as the PCR templates for re-amplification with the corresponding primer pair, respectively.The re-amplified products with the expected size were inserted into pEGMT-easy vector (Promega) and sequenced by Gene Company Limited (Shanghai, China).

Polymorphism
In the present study, 170 different primer combinations were performed for PCR amplification among the five different tissues.On an average, each PCR reaction can generate as much as 50 scorable fragments after separation on polyacrylamide gel.Approximately, 0.2% polymorphic fragments, which can be fully reproducible when same DNA samples were employed in independent experiment, were found among the five different tissues in our experiment.Considering methylation of the C5 position of cytosine residue which can be by methylation Fortunately, as revealed by Li and Quiros (2001) that at least 40% of polymorphic fragments are representing a coding sequence, it is also likely that about 40% of these 18 polymorphic fragments are coding sequences.Additionally, DNA methylation can spread from promoter region to the coding region or from coding region to the promoter region, and then de novo genes' methylation among these polymorphic fragments is possibly associated with the silencing of gene expression.Thereafter, it may provide an opportunity to investigate the biological roles that gene de novo methylation played during development.For example, the important case detected in this experiment may imply that silencing of genes expression via epigenetic modification is common and is so much essential for embryo development; by  contrast, DNA de novo de-methylation events occurred in the reproductive tissues relative to the vegetative tissues that may reflect that the initiation of some genes' expression is possibly very essential for promoting plant to transit from vegetative to reproductive stage.

Sequencing of polymorphic fragments
Eighteen polymorphic bands from amplification with 15 different primer combinations were isolated and inserted into TA-clone vector.Previous studies showed that some sequences homologous to plastid encoded genes were found to be methylated in amyloplast and resulted in a reduced expression ability (Macherel et al., 1985).Except for the case of Chlamydomonas reinhardtii, methylated DNA is not usually found in chloroplasts (Sager and Lane, 1972).However, whether the expression of the methylated gene homologous to chloroplast-encoded genes also have been silenced or reduced in this study, it remains unclear.Among them, rpoB, encoding one of the subunit of plastid encoded RNA polymerase (RNAPs), is one of the most interesting genes.The plastid genome is transcribed by two distinct RNAPs, the plastid encoded RNAP (PEP) and nucleus-encoded RNAP (NEP).Some plastid genes transcribed exclusively by PEP; some by NEP only, and the remaining parts transcribed by both of them (Eberhard et al., 2002;Cahoon et al., 2004).It is thought that NEP expressed and played a role at the early stage of plastid development, while PEP expressed at the mature stage.However, the mechanism of the dynamic relationship between the activities of PEP and NEP remains unknown.
The different methylation of rpoB gene discovered in this experiment from different tissues may provide a clue for further investigating the dynamic relationship between the activities of PEP and NEP.In addition to plastid encoded genes, photosynthetic related gene such as Protochlorophyllide reductase B gene, and the other genes responsible for encoding unknown protein were revealed as well by using this maker system.In conclusion, MS-SRAP marker system is reliable for genome-wide de novo gene methylation/de-methylation investigation.

Figure 1 .
Figure 1.Two major types of de novo DNA methylation were found by using MS-SRAP marker system.A, Leaf specific type de novo DNA methylation revealed with primer pair me1 and em4.The arrow showed the polymorphic bands presented in young and mature leave tissues of B. napus; B, Flower specific type de novo DNA methylation revealed with me10 and em12.The arrow showed the polymorphic band only presented in flower tissue of B. napus.YL represents young leaves; ML represents mature leaves; SB represents small buds; MB represents mature buds and F means bagged flowers.

Table 1 .
Arbitrary primers used for MS-SRAP PCR amplification in this experiment.

Table 2 .
Characteristics of the polymorphic bands amplified from five tissues of B. napus at the flowering stage using 15 primer combinations.