- Methodology
- Open Access
Universal endogenous gene controls for bisulphite conversion in analysis of plant DNA methylation
- Jing Wang1,
- Chongnan Wang1,
- Yan Long1,
- Clare Hopkins2,
- Smita Kurup2,
- Kede Liu1,
- Graham J King3Email author and
- Jinling Meng1Email author
https://doi.org/10.1186/1746-4811-7-39
© Wang et al; licensee BioMed Central Ltd. 2011
- Received: 25 August 2011
- Accepted: 2 December 2011
- Published: 2 December 2011
Abstract
Accurate analysis of DNA methylation by bisulphite sequencing depends on the complete conversion of all cytosines into uracil. Until now there has been no standard or universal gene identified as an endogenous control to monitor the conversion frequency in plants. Here, we report the development of PCR based assays for one nuclear gene IND (INDEHISCENT) and two mitochondrial genes, NAD (NICOTINAMIDE ADENINE DINUCLEOTIDE) and ATP1 (ATPase SUBUNIT 1). We demonstrated their efficacy as bisulphite conversion controls in Brassica and other plant taxa. The target regions amplified by four primer pairs were found to be consistently free from DNA methylation. Primer pairs for IND.a and NAD were effective within Brassica species, whereas two primer pairs for ATP1 provided reliable controls across a representative range of dicot and monocot angiosperm species. These primer sets may therefore be adopted as controls in plant methylation analysis for a wide range of studies.
Keywords
- DNA methylation
- plants
- bisulphite
Background
Methylation of cytosine plays an important role in epigenetic gene regulation in vertebrates and higher plants [1]. In contrast to animals, where methylated cytosine residues are primarily observed within the symmetrical CpG dinucleotide, plants display cytosine methylation in any DNA context, including symmetric CG and CHG (where H = A, T or C) and asymmetric CHH [2]. Over the past few decades, four major approaches have been used for distinguishing the epigenetic mark 5-methylcytosine (5mC) from unmethylated cytosine. These include methods based on isochizimer restriction endonucleases, bisulphite conversion of DNA, immunoprecipitation and mass spectrometry [3, 4]. Bisulphite conversion of DNA, originally developed by Frommer et al. [5], involves treatment of DNA with sodium bisulphite, where under optimized conditions unmethylated cytosine is converted to uracil, whilst methylated cytosine (both 5mC and 5-hydroxymethylcytosine) remains unchanged. DNA sequence changes resulting from bisulphite conversion can then be detected by a variety of methods, including PCR amplification, followed by DNA sequencing where in the original uracil residues are reported as thymine. The primary advantage of this technique is that it provides base-pair resolution of methylation patterns, which is particularly useful in plants for distinguishing between the different cytosine sequence contexts [6]. Following a number of substantial improvements based on the original protocol, bisulphite sequencing is now accepted as the gold standard for detecting changes in DNA methylation [3]. The combination of bisulphite conversion and next-generation high-throughput sequencing has recently provided powerful tools for revealing DNA methylation patterns on a genome-wide scale [7–10].
Although bisulphite-based methods are reasonably accurate and reproducible in comparison with other methods, successful detection is dependent on the complete bisulphite conversion of all unmethylated cytosine into uracil [11]. Incomplete conversion complicates downstream data analysis, especially in plants where larger and more complex genomes are likely to contain a high level of 5mC. False-positive 5-methylcytosines (cytosine read as 5-methylcytosine) are common, since it is often difficult to determine whether an unconverted cytosine represents true methylation or incomplete treatment. Both incomplete DNA denaturation prior to bisulphite treatment and reannealing during treatment can lead to incomplete bisulphite conversion, since bisulphite converts single-stranded but not double-stranded DNA [5]. As a result, repeated denaturation cycles during the bisulphite treatment are required to ensure complete conversion [4], which is now a standard feature of protocols recommended for commercial bisulphite conversion kits. However, it is still necessary to include some form of control to monitor bisulphite conversion for each sample assayed. The completion of bisulphite conversion can be tested by monitoring exogenously spiked DNA controls, or retention of endogenous non-target sequence cytosine dinucleotides [4]. Theoretically, any DNA sample with a known consistent methylation pattern could be used as a control. In mammalian genomes, unamplified, nearly methylation-free genomic DNA from specific cell lines has been used as the template to optimize and test conditions for genome-wide bisulphite conversion, PCR amplification and subsequent library construction [8]. In Arabidopsis, specific unmethylated genes and chloroplast DNA have been used for establishing the degree of conversion [9, 12, 13]. Plant mitochondrial DNA is another potential control for monitoring conversion, since mitochondrial genomes are free of methylated cytosines and can be isolated with nuclear DNA from all organs and tissues [14]. However, to date the full sequence of mitochondrial genomes has only been established for a small number of plant species.
In this study, we first identified a nuclear endogenous gene IND.a, present in Brassica 'A' genomes, which remains unmethylated in different organs and tissues. We then designed primer pairs for two mitochondrial genes, ATP1 and NAD. Two primer pairs for ATP1 were effective across all dicotyledonous and monocotyledonous species tested, and are therefore valuable as universal controls for DNA methylation analysis of target genes or whole genome analysis in plants.
Results and Discussion
Diagram of primer design and amplification of IND.a_A3. (A) The alignment of IND.a_A3 region among B. rapa, B. oleracea and B. napus. The blue open box showed primer region. (B-C) PCR products from IND.a_A3 were abundant in genomic DNA and bisulphite treated DNA from floral bud of B. napus and B. rapa, however, extremely low in B. oleracea. TapidorDH and Westar10 are cultivars of B. napus, Chiifu-401 and 3H-120 of B. rapa, and Alboglabra Bailey is a B. oleracea.
In order to develop an assay that would be applicable to all Brassica species, we next considered candidate genes within the mitochondrial genome. The orthologues of Arabidopsis NAD and ATP1 were chosen as suitable targets for developing the control assay due to their potential conservation across plant taxa, and the key role they play in energy production and storage. Two primer pairs for ATP1 and one for NAD were designed based on a region within the genes conserved amongst different species (Additional file 1). Bisulphite sequencing of eight clones for each treatment indicated complete conversion of cytosine to uracil within the target region of NAD for all Brassica species. This indicated that, as expected, the gene is free of methylated cytosine. However, it was not possible to amplify this target region of NAD from other species. In contrast, although some sequence polymorphism was present in different families of plants (data not shown), the PCR products generated using two primer pairs from the ATP1 gene were of identical size (227 bp for ATP1-1 and 252 bp for ATP1-2) in all species tested. Following bisulphite sequencing of eight clones from each treatment, ATP1 genes were also found to be universally unmethylated. This result is consistent with the known lack of 5-methylcytosine within mitochondrial genomes [14]. However, the transfer and incorporation of regions of mitochondrial and plastidic genomes within the nuclear chromosomes is relatively common amongst flowering plants [21]. In Arabidopsis, the mitochondrial ATP1 gene has been found in the nuclear genome where it is methylated at a low level [13, 22]. Detailed analysis of the regions flanked by the two primers ATP1-1 and ATP1-2 indicates that these are free of methylated DNA [13]. Although the methylation status of nuclear ATP1 sequences in other plant species is unknown, it appears that the PCR products we generated here are clearly free of 5-methylcytosine, irrespective of their organellar or nuclear origin.
Schematic representations of restriction endonuclease loci near the target region of ATP1-2 in different species.
DNA methylation profiles of Bra.ATS1 and BnaA.ATS1. (A) Scheme of Bra.ATS1 located on chromosome A1 of B. rapa. (B) Bisulphite sequencing of two promoter regions, P1 and P2, was performed on DNA collected from floral buds from Westar 10 and Chiifu-401. W represents Westar10 and C represents Chiifu-401. 1-10 designate 10 random clones.
Sodium bisulphite conversion frequencies of four treatments
Treatment | Number of clones | Conversion frequency (%) | ||||
---|---|---|---|---|---|---|
CG | CHG | CHH | Total | t test (P < 0.01) | ||
One round treatment | 15 | 97.57 | 98.79 | 98.18 | 98.18 | A |
One round treatment + purification | 15 | 96.36 | 96.97 | 98.99 | 98.06 | A |
Two round treatment | 15 | 99.80 | 99.39 | 99.80 | 99.64 | B |
Two round treatment + purification | 15 | 99.39 | 100.00 | 99.80 | 99.76 | B |
Analysis for PCR bias of different primer sets
Primer set | Percentage (%) of PCR products from genomic DNA in TapidorDH/Value of bias | Average value of bias | Percentage (%) of PCR products from genomic DNA in Nipponbare/Value of bias | Average value of bias | ||||||
---|---|---|---|---|---|---|---|---|---|---|
80 a | 60 a | 40 a | 20 a | 80 a | 60 a | 40 a | 20 a | |||
ATP1-1 | 95/4.75 | 90/6.00 | 85/8.50 | 75/12.00 | 7.81 | 95/4.75 | 65/1.24 | 60/2.25 | 45/3.27 | 2.88 |
ATP1-2 | 90/2.25 | 80/2.67 | 65/2.79 | 30/1.71 | 2.36 | 85/1.42 | 70/1.56 | 60/2.25 | 40/2.67 | 1.98 |
IND.a_A3 | 95/4.75 | 85/3.78 | 85/8.50 | 40/2.67 | 4.93 | / | / | / | / | / |
NAD | 95/4.75 | 90/6.00 | 85/8.50 | 70/9.33 | 7.15 | / | / | / | / | / |
Conclusion
Description of six primer sets tested and the resultant PCR products
Primer pair | Forward primer sequence (5'→3') | Reverse primer sequence (5'→3') | Tm (°C) | Expected size (bp) | No. of cytosine in target region |
---|---|---|---|---|---|
IND.a-A3 | GGAGGAGGAGAGGAAGYAGAAGAA | CCTRRCACCATCCTCTTCAATATCC | 58, 58 | 239 | 43 |
ATP1-1 | TGAAYGAGATTYAAGYTGGGGAAATGGT | CCCTCTTCCATCAATARRTACTCCCA | 50, 56 | 227 | 64 |
ATP1-2 | TAGTAAAYAGGYGGTGGYATATYGA | CTCTRTTTCCAAACARATTTRTCCATC | 50, 56 | 252 | 15 |
NAD | AGTTTYTGYTAGAYGAGAAATAAGGA | CCTACTCACTCRRACAATRCTCT | 50, 56 | 276 | 24 |
ATS1-P1 | AGGTTYAGGGTTTTGGTAGTGAGAAGGGA | TCCATRACAATCCTAACAACAATTATCA | 51, 54 | 305 | 54-58 |
ATS1-P2 | TGGAGGAGYAGAGGYGAAGYTTGA | ACCAARACCCRCCACAACACATRCCT | 55, 64 | 227 | 24-28 |
Materials and methods
Plant materials
Plant materials used in this research
Family | Genus &Species | Accession | Tissue | Environment/Location | Development stage | Gene detection |
---|---|---|---|---|---|---|
Brassicaceae | Brassica rapa | Chiifu-401 | Bud | Field (HAU) | Budding | IND.a, ATP1, NAD |
Silique (4 cm) | Field (HAU) | Silique setting | IND.a, ATP1, NAD | |||
3H-120 | Leaf | Field (HAU) | Seedling | ATP1, NAD | ||
Brassica oleracea | Alboglabra Bailey | Bud | Field (HAU) | Budding | ATP1, NAD | |
Silique (4 cm) | Field (HAU) | Silique setting | ATP1, NAD | |||
Brassica nigra | Giebra | Leaf | Field (HAU) | Seedling | ATP1, NAD | |
Brassica napus | TapidorDH | Bud | Field (HAU) | Budding | IND.a, ATP1, NAD | |
Silique (4 cm) | Field (HAU) | Silique setting | IND.a, ATP1, NAD | |||
Leaf _2 | CE RRes | Seedling | IND.a, ATP1, NAD | |||
Leaf _6 | CE RRes | Seedling | IND.a, ATP1, NAD | |||
Leaf_9 | CE RRes | Seedling | IND.a, ATP1, NAD | |||
Leaf_14 | CE RRes | Seedling | IND.a, ATP1, NAD | |||
Westar10 | Bud | Field (HAU) | Budding | IND.a, ATP1, NAD | ||
Silique (4 cm) | Field(HAU) | Silique setting | IND.a, ATP1, NAD | |||
HC1 | Leaf | Field (HAU) | Budding | ATP1, NAD | ||
CH-21 | Leaf | Field (HAU) | Budding | ATP1, NAD | ||
Brassica juncea | Hn2 | Leaf | Field (HAU) | Budding | ATP1, NAD | |
Brassica carinata | NC3 | Leaf | Field (HAU) | Seedling | ATP1, NAD | |
Malvaceae | Gossypium hirsutum | Lizhongmian-1 | Leaf | Field (HAU) | Seedling | ATP1, |
Gossypium herbaceum | Licaomian-1 | Leaf | Field (HAU) | Seedling | ATP1, | |
Fabaceae | Glycine max | Zhongdou30 | Leaf | Field (OSR) | Flowering | ATP1, |
Rutaceae | Citrus unshiu Marcow | Guoqing No.1 | Leaf | Field (HAU) | Fruit setting | ATP1, |
Citrus grandis Osbeck | HB pummel | Leaf | Field (HAU) | Fruit setting | ATP1, | |
Citrus sinensis Osbeck | Newhall Navel orange | Leaf | Field (HAU) | Fruit setting | ATP1, | |
Citrus limon Burm.f | Eureka lemon | Leaf | Field (HAU) | Fruit setting | ATP1, | |
Citrus reticulata Blanco | 'Egan No.1' | Leaf | Field (HAU) | Fruit setting | ATP1, | |
Solanaceae | Solanum lycopersicum | Micro Tom | Leaf | Glasshouse (HAU) | Flowering | ATP1, |
Solanum lycopersicum | M82 | Leaf | Glasshouse (HAU) | Flowering | ATP1, | |
Nicotiana tabacum | Li yancao-1 | Leaf | Field (HAU) | Seedling | ATP1, | |
Poaceae | Triticum aestivum | Huahui8 | Leaf | Field (HAU) | Seedling | ATP1, |
Zea mays | Mo17 | Leaf | Field (HAU) | Booting | ATP1, | |
Oryza sativa ssp. japonica | Nipponbare | Leaf | Field (HAU) | Seedling | ATP1, | |
Oryza sativa ssp. indica | Zhenshan97 | Leaf | Field (HAU) | Seedling | ATP1, | |
Oryza rufipogon | Griff. | Leaf | Field (HAU) | Seedling | ATP1, |
Bisulphite sequencing
Collection of plant material, storage and DNA extraction followed the DNeasy Plant Mini Kit (Qiagen) handbook. 450-750 ng genomic DNA was subjected to two successive treatments of sodium bisulphite conversion using the EpiTect Bisulphite kit (Qiagen) according to the manufacturer's instructions. The reaction was then purified once more using the PCR purification kit (Qiagen). Forward (F) and reverse (R) primers for bisulphite sequencing PCR were designed using Kismeth http://katahdin.mssm.edu/kismeth based on the reference sequences in GenBank (Additional file 1).The bisulphite-treated DNA was amplified using Maxima™ Hot start Taq DNA polymerase (Fermentas). The thermal cycling program was 95°C for 4 min followed by 35 cycles of 95°C for 30 s, annealing for 30 s, and extension at 72°C for 45 s, ending with a 10 min extension at 72°C. PCR products were cloned into the pMD18-T vector (TaKaRa), and 8-15 individual clones were sequenced. Percentage methylation (% C) was calculated as 100 ×C/(C + T). DNA cytosine methylation in the CG, CHG, and CHH contexts was analyzed and displayed using CyMATE [27].
PCR bias analysis
Un-converted genomic DNA, either from TapidorDH leaf 9 (dicotyledon) or Nipponbare seedling leaf (monocotyledon), was diluted to the same concentration as bisulphite treated DNA. Four sets of samples were prepared with 80:20, 60:40, 40:60, 20:80 ratios of genomic to bisulphite treated DNA. PCR products generated from these templates with different primer combinations were cloned into the pMD18-T vector (TaKaRa). Twenty individual clones from each primer set were selected randomly for SSCP (single strand conformation polymorphism) analysis to discriminate different products from genomic DNA and bisulphate treated DNA. A value for the bias (b) was calculated as b = [y(100 - x)]/[x(100 - y)]; y is the percent of PCR products from genomic DNA and x is the percent of genomic DNA in mixed sample [25].
Declarations
Acknowledgements
We thank Zhibiao Ye, Sibin Yu, Wenwu Guo, Jianbin Yan, Xianhong Ge, Kaisheng Li (Huazhong Agricultural University, Wuhan, China) and Haifeng Chen (Oilseed Crop Research Institute Chinese Academy of Agricultural Science, Wuhan, China) for providing the samples. This work was supported by the 111 Project (B07041) and Postdoctoral Science Foundation (20100480915) in China. Rothamsted Research receives grant-aided funds from the UK Biotechnology and Biological Sciences Research Council.
Authors’ Affiliations
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