- Open Access
Analysis of the leaf methylomes of parents and their hybrids provides new insight into hybrid vigor in Populus deltoides
- Ming Gao†1, 2,
- Qinjun Huang†1, 2,
- Yanguang Chu1, 2,
- Changjun Ding1, 2,
- Bingyu Zhang1, 2 and
- Xiaohua Su1, 2Email author
© Gao et al.; licensee BioMed Central Ltd. 2014
- Published: 20 June 2014
Plants with heterosis/hybrid vigor perform better than their parents in many traits. However, the biological mechanisms underlying heterosis remain unclear. To investigate the significance of DNA methylation to heterosis, a comprehensive analysis of whole-genome DNA methylome profiles of Populus deltoides cl.'55/65' and '10/17' parental lines and their intraspecific F1 hybrids lines was performed using methylated DNA immunoprecipitation (MeDIP) and high-throughput sequencing.
Here, a total of 486.27 million reads were mapped to the reference genome of Populus trichocarpa, with an average unique mapping rate of 57.8%. The parents with similar genetic background had distinct DNA methylation levels. F1 hybrids with hybrid vigor possessed non-additive DNA methylation level (their levels were higher than mid-parent values). The DNA methylation levels in promoter and repetitive sequences and transposable element of better-parent F1 hybrids and parents and lower-parent F1 hybrids were different. Compared with the maternal parent, better-parent F1 hybrids had fewer hypermethylated genes and more hypomethylated ones. Compared with the paternal parent and lower-parent L1, better-parent F1 hybrids had more hypermethylated genes and fewer hypomethylated ones. The differentially methylated genes between better-parent F1 hybrids, the parents and lower-parent F1 hybrids were enriched in the categories metabolic processes, response to stress, binding, and catalytic activity, development, and involved in hormone biosynthesis, signaling pathway.
The methylation patterns of the parents both partially and dynamically passed onto their hybrids, and F1 hybrids has a non-additive mathylation level. A multidimensional process is involved in the formation of heterosis.
- Populus deltoides
- DNA methylation
- hybrid vigor
Heterosis/hybrid vigor is the phenomenon in which progeny are superior to their parents (with distinct genetic backgrounds) in many traits, such as biomass, growth rate, adaptability, fertility, and resistance [1–5]. Since interspecific hybrid tobacco with hybrid vigor was produced in the 1760s by crossing Nicotiana rustuca with N. paniculata , heterosis has often been exploited in crop and tree breeding. However, the genetic basis of heterosis is still far from being understood and is still a controversial subject [1, 7, 8]. Three classic hypotheses, i.e., dominance, overdominance, and epistasis, were proposed as genetic explanations for heterosis. In the dominance hypothesis, the inferior parental alleles in the hybrids are complemented by the superior or dominant alleles from the other parent. In the overdominance hypothesis, heterosis arises from allelic interactions within each of many genetic loci. An alternate model, epistasis, postulates that interactions between different parental genes in hybrids lead to heterosis. Although numerous examples support each of these hypotheses, they only partially explain the genetic basis of heterosis [1, 3, 9].
Genome-wide analyses of heterosis have revealed altered gene expression profiles in F1 hybrids compared with their parents, as well as non-additive patterns of gene expression [2, 10], including studies in maize (Zea mays L.) [11–14], rice (Oryza.sativa L.) [15, 16], Arabidopsis thaliana , wheat (Triticum aestivum L.) , Larix kaempferi (Lamb.) Carr , and Populus tremula . Recently, heterosis was observed in hybrids derived from parents with similar genetic backgrounds. Such parents with highly similar genomic features had distinct epigenomes [21–23], and epi-alleles that arise from epigenetic modification were also identified. Epi-alleles cause allelic variation and altered gene expression activity, which are essential to the architecture of plant heterosis . One type of epigenetic regulation, DNA methylation, primarily serves as an epigenetic silencing mechanism and predominantly occurs in transposons and other repetitive DNA elements [5, 25–29] and has been explored in model plants and crops, such as maize, rice, cotton (Gossypium herbaceum L.), and A. thaliana.
The genus Populus (poplar) includes species that are important for the health of ecosystems and are vital to the timber, paper, and biofuel industries. Poplars are also used as a model woody plant species and models of interest for epigenetic studies [30, 31]. Variations in DNA methylation between genotypes and tissues and in response to drought, as well as the relationship between gene-body DNA methylation and tissue-specific gene expression, have been reported [31–34].
During the last century, many poplar varieties with enhanced growth or adaptability have been generated using inter- or intraspecific hybridization approaches, which take advantage of the presence of heterosis in poplars. Although investigations of the molecular basis of heterosis in poplar have been undertaken via genetic mapping and gene expression profiling, the global patterns of epigenetic modification such as DNA methylation have not been determined, and whether DNA methylation plays a role in the architecture of heterosis is still unclear. In this study, P. deltoides cl.'55/65' was maternal parent which has straight bole, round crown, fast growth, high resistance to Anoplophora glabripennis and strong rooting ability, and P. deltoides cl. '10/17' was paternal parent which fast-growth and high stress resistance. This cross-combination is multigeneration convergent cross. Intraspecific F1 hybrids of P. deltoides with significant hybrid vigor or lower-parental performance were examined. Methylated DNA immunoprecipitation, combined with a high-throughput sequencing (MeDIP-Seq) approach were applied to analyze the genome-wide DNA methylation landscapes in Populus deltoides parental lines and F1 hybrids lines. The results showed that better-parent F1 hybrids have higher methylation levels than the average of the parents, suggesting that non-additive level of DNA methylation is related to heterosis/hybrid vigor. The hypermethylated genes of better-parent F1 hybrids relative to the parents and lower-parent F1 hybrids were enriched in the processes of metabolism and development, which may be highly relevant to heterosis.
Plant materials and growth conditions
Two P. deltoides intraspecific parental lines, P. deltoides cl. '55/65' (Salicaceae, Populus, Section Aigeiros) and P. deltoides cl. '10/17' (Salicaceae, Populus, Section Aigeiros) and their intraspecific hybrids, designated here as H1, H2, H3, L1 and L2, were used in this study. All F1 Hybrids was generated by the same intraspecific cross-combination of P. deltoides cl. '55/65' as maternal parent and P. deltoides cl. '10/17' as paternal parent. P. deltoides cl. '55/65' was primitively generated from the inbred seeds of excellent individual plants in former Yugoslavia and introduced into China in 1981. P. deltoides cl. '10/17' was generated by intraspecific crossing P. deltoides Bartr. cv. 'Shanhaiguanensis' (which was primitively generated from the inbred seeds of excellent individual plants and introduced into China in 1900) with P. deltoides Bartr. cl. 'Harvard' (I-63/51) (which was primitively generated from the inbred seeds of excellent individual plants in Mississippi Delta and introduced into China in 1972).
Hybrids were generated by hand pollination. All seeds were grown in a greenhouse at the Chinese Academy of Forestry (the authority responsible is the Chinese Academy of Forestry, Beijing, China) in January, 2002. One-year-old seedlings were made into cuttings to accelerate cloning, which were planted in the greenhouse in January, 2003 and transplanted to Yuquan mountain nursery (the authority responsible is the Chinese Academy of Forestry, Beijing, China) in May, 2003. No specific permits were required for these locations. The locations are not privately owned in any way, and the field studies did not involve endangered or protected species. A total of 149 F1 hybrids were introduced into Jiaozuo Research Institute of Forestry (Henan province, China) in 2003 and 2004. Of these, 18 F1 hybrids that had good performance in tree height and Diameter at breast height (DBH) were selected over the course of the two-year seedling test. Parents and their 18 F1 hybrids were planted in Xifeng village, Wuzhi Country, Jiaozuo city in Henan province in 2005 and then transplanted to Yangcheng, Wuzhi Country, Jiaozuo city of Henan province (35°8' N, 113°17' E), in 2007. No specific permits were required for these locations. The location is not privately owned in any way, and the field studies did not involve endangered or protected species. This site has an annual average precipitation of 625.4 mm, with an annual average temperature of 15.2ºC (ranging from 14.3ºC to 43.6ºC), an accumulated temperature above 0ºC of 4,633ºC, and a frostless period of 224 days per year. The average relative humidity and annual sunshine duration are 61% and 2,434 hours, respectively. The experimental field had an average soil pH of 6.8 and was irrigated. This trial was designed in randomized complete blocks, with four blocks and eight trees per treatment (planting spacing of 3 m × 5 m). After 5 years of growth, three F1 hybrids (H1, H2, and H3) which exhibited the highest tree heights and largest DBHs and two F1 hybrids (L1 and L2) that showed the lowest tree heights and DBHs were selected.
Since DNA methylation differences among tissues are obvious in Poplar  and leaves are important to plant growth and development, after five years of growth, the leaves at the top of main trunk were collected at the vigorous stage (9:30-10:30 am on August 10, 2011). Three trees (three leaves per tree) per replication were sampled, thus, twelve trees and 36 leaves were sampled for every line. Samples for every parent and F1 hybrid were pooled and stored in liquid nitrogen prior to DNA extraction.
Evaluation of heterosis
Since planting (in 2007), two important economic traits, tree height and DBH were continuously measured. Considering heterosis over higher parent was important for poplar breeding, after five years of growth, heterosis over higher parent was calculated as H = (F1-Ps)/Ps × 100%, where H is the amount of heterosis, F1 is the trait value measured in the hybrid, and Ps is the trait value measured in the higher parent .
Genomic DNA was isolated from each sample using a DNeasy Plant Mini Kit (Qiagen, Courtaboeuf, France). The DNA integrity was verified by agarose gel electrophoresis. The DNA was quantified using a Qubit Fluorometer and a Quant-iT™ dsDNA BRAssay Kit (Life Technologies, USA).
The MeDIP process was almost identical to the method of Pomraning et al . Before carrying out MeDIP, genomic DNA was sheared to 350-450 bp fragments with a Bioruptor (Sonics, Newtown, USA, VC130PB), and the fragments were recovered using a Qiaquick PCR Purification Kit (Qiagen, Courtaboeuf, France). The fragments were end-repaired, phosphorylated, and A-tailed. The fragments were then ligated to Illumina sequencing adapters . The sheared DNA was diluted in 450 µl of TE buffer, denatured in a 100°C heat block for 10 min, and snap-cooled on ice for 5 min. Immunoprecipitation buffer (100 mM Na-Phosphate pH 7.0, 1.4 M NaCl, 0.5% TritonX-100) and 1 µl of 5meC antibody (Diagenode, Liège, Belgium #MAb-5MECYT-100, 1 µg/µl) were added to the DNA solution followed by incubation for 2 h on an orbital rotator at 4°C. Bound DNA was precipitated with sheep anti-mouse IgG Dynabeads (M-280, Invitrogen, California,USA), washed three times with immunoprecipitation buffer for 10 min at room temperature with shaking, resuspended in 250 μl proteinase K digestion buffer (5 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0, 0.05% SDS) with 7 μl of 10 mg/ml proteinase K, and incubated for 3 h on an end-over-end rotator at 50°C to digest the antibodies and release the 5meC-containing DNA. Methylated DNA was extracted by phenol-chloroform extraction followed by ethanol precipitation. The DNA pellets were resuspended in 50 μl TE buffer and stored at -20°C.
The immunoprecipitated DNA was used to generate a DNA colony template library using the Fasteris procedure (Fasteris, Plan-les-Ouates, Switzerland). The DNA samples were quantified using a 2100 Bioanalyzer (Agilent, USA) and a StepOnePlus Real-Time PCR System (ABI, California,USA). Illumina sequencing was performed in a HiSeq-2000 system (Illumina, San Diego, CA, USA).
Bioinformatics processing and statistical analysis
MeDIP-Seq reads were aligned to the Populus trichocarpa v2.2 reference genome (http://www.phytozome.net/poplar.php, February 2012). The alignments were carried out with SOAP aligner (BGI, version 2.01) , allowing up to two mismatches for successful mapping. The mapped rate (the ratio of the number of mapped reads to that of original reads), and the uniquely mapped rate (the ratio of the number of uniquely mapped reads to that of original reads) were calculated. The coverage depth was calculated as the coverage times of specific loci by sequencing reads. The genome coverage was calculated as the proportion of eligible base numbers in the entire genome. In the distribution analysis of the MeDIP-Seq sequencing reads in a chromosome, each chromosome was scanned with windows of 100 kb, the reads coverage depth per window was calculated, and the reads were standardized with the following formula: reads number of specific 100 kb windows * 1,000,000/number of uniquely mapped reads. The methylation coverage of CG/CHG/CHH contexts was calculated as the proportion of CG/CHG/CHH site over certain coverage depth in all CG/CHG/CHH sites from as determined by MeDIP-Seq.
Peak summit coordinates were generated using model-based analysis of ChIP-Seq (MACS; version 1.4.0 beta) . The summit files were then used for further analysis (total peaks number, peak mean length, peak median length, peak total length, and peak covered size in the genome).
To detect differentially methylated gene between the two samples, the peak summits of two samples were merged, and the normalized reads number of each sample the merged region was determined. The false positive reads were removed using a chi-square test. For genes that overlapped with a merged region, if the reads number of sample 2 in this region was more than that of sample 1, then the gene was designated as hypermethylated during the Sample 1 versus Sample 2 comparison, while if the opposite situation occurred, the gene was considered to be hypomethylated.
Gene Ontology (GO) analysis was performed to obtain the functional classifications of differentially methylated genes using the TermFinder tool (http://search.cpan.org/~sherlock/GO-TermFinder-0.86/). P-values were multiple test corrected to reduce false positive rates. GO terms with adjusted P-values of <0.05 were considered to be significant.
The known genes were submitted to the KEGG Automatic Annotation Server (http://www.genome.jp/kegg/pathway.html) for pathway analysis. A hypergeometric test was performed to identify the significantly enriched pathways in differentially methylated genes compared with the whole genome. Pathways with Q-values ≤ 0.05 were considered to be significant.
Heterosis of F1 hybrids
heterosis over better parent %
7.81 ± 0.05
1.26 ± 0.03
12.55 ± 0.06
1.49 ± 0.02
11.09 ± 0.04
0.72 ± 0.01
-5.77 ± 0.03
-20.92 ± 0.03
-7.59 ± 0.05
-21.82 ± 0.05
Mapping of MeDIP-Seq reads to the reference genome
Summary of MeDIP-Seq experimental results
length of sequence reads (bp)
mapped reads b
Percent unique mapped read (%)
Comparison of methylation status among parents and F1 hybrid genomes
Mapping of MeDIP-Seq reads to genes
Methylated peaks analysis
Statistics of peak summits
Peak mean length /bp
Peak coverage in genome
Number of peaks in genome features
Analysis of differentially methylated genes in the parental and F1 hybrid genomes
For all of the differentially methylated genes identified, we performed Gene Ontology (GO) functional category analysis to determine whether these genes were enriched for certain pathway or network (Additional file 2: Figure S2). The results showed that the differentially methylated genes between better-parent hybrid H1 and maternal parent P1 were enriched in 28 biological functional categories, and ten additional enriched functional categories (biological adhesion, cell proliferation, locomotion, reproductive process, extracellular region, extracellular region part, enzyme regulator activity, molecular transducer activity, protein binding, and transcription factor activity) were also found for genes identified in the H1-P2 comparison. Compared with maternal parent P1, better-parent hybrid H2 possessed more hypermethylated genes enriched in 35 functional categories, such as biological adhesion, cell proliferation, protein binding, and transcription factor activity. The differentially methylated genes between H2 and paternal parent P2 were enriched in 33 functional categories (e.g., pigmentation). The differentially methylated genes between better-parent hybrid H3 and maternal parent P1 were enriched in 31 functional categories, and two additional categories (cell proliferation and molecular transducer activity) were found to have enriched differentially methylated genes between H3 and P2. As a whole, the majority of hypermethylated genes between three better-parent hybrids and both parents tended to fall into seven functional categories, including metabolic processes, cellular, response to stress, cell, cell part, binding, and catalytic activity.
Annotations of hypermethylated genes
Start position of the gene in the scaffold
End position of the gene in the scaffold
Predicted GTP-binding protein (ODN superfamily)
Predicted 3-ketosphinganine reductase
Cytochrome P450 CYP4/CYP19/CYP26 subfamilies
15-hydroxyprostaglandin dehydrogenase and related dehydrogenases
Ca2+/calmodulin-dependent protein kinase, EF-Hand protein superfamily
Cytochrome P450 CYP2 subfamily
Serine/threonine protein kinase
Molybdopterin converting factor, small subunit
Leucine-rich repeat protein
Serine/threonine protein kinase
The differentially methylated genes between better- and lower-parent F1 hybrids were also analyzed (Figure 8B). The number of hypermethylated genes of better-parent F1 hybrids versus lower-parent hybrids L1 and L2 were 523 and 132, respectively. For these genes, hypermethylation predominantly occurred in the promoter (171 genes compared with L1 and 27 genes compared with L2) and gene body (296 genes compared with L1 and 80 genes compared with L2), while less hypermethylation occurred in the 5 'UTR (28 genes compared with L1 and 15 genes compared with L2) and the 3' UTR (15 genes compared with L1 and 10 genes compared with L2). A total of 40 hypermethylated genes were found in all three better-parent F1 hybrids compared with lower-parent hybrids L1 and L2, ten of these genes could be annotated (Table 5). The GO functional categories of the 10 genes mainly involve metabolic process (4), primary metabolic process (2), cellular metabolic process (3), signaling (3), small molecule metabolic process (2), anatomical structure development (2), and biological regulation (2). These genes were then submitted to the KEGG Pathway database, yielding pathway information about four genes as follows: (1) POPTR_0012s07360 is calcium-dependent protein kinase gene involved in plant-pathogen interactions (ko04626); (2) POPTR_0015s09720 belongs to cytochrome P450 CYP4/CYP19/CYP26 subfamilies involved in steroid hormone biosynthesis (ko00140); (3) POPTR_0019s09910 encodes a molybdopterin synthase catalytic subunit involved in multiple processes such as metabolism, metabolism of cofactors, vitamin and folate biosynthesis (ko00790), genetic information processing, folding, sorting, and degradation, and the sulfur relay system (ko04122); and (4) POPTR_0008s18420 encodes an erbb2-interacting protein involved in a NOD-like receptor signaling pathway (ko04621).
Several classical hypotheses about heterosis are based on the differences between genomes , and allelic diversity may produce heterosis. However, hybrid vigor can be observed even when parents are genetically very similar . Recent studies have shown that parents with similar genome sequences have distinct epigenomes, which may contribute to heterosis [5, 24]. In Populus, hybrids with heterosis are often obtained by intrasection and interspecific hybridization, whereas hybrids obtained by intersection hybridization always have mid-parent performance, and hybrids with growth vigor are obtained less frequently. In Section Aigeiros, excellent hybrids with heterosis have been produced by intraspecific hybridization; the level of heterosis increases with a decrease in genetic distance between parents and polymerization of excellent genetic composition. Super high yield varieties are often generated by convergent crossing of P. deltoides varieties (strains). In this study, P. deltoides cl. '55/65', was used as the maternal parent and P. deltoides cl. '10/17' was used as the paternal parent. This cross combination is a multigeneration convergent cross, and the level of heterosis is outstanding. Intraspecific F1 hybrids of P. deltoides with significant hybrid vigor or lower-parental performance were examined, providing a unique opportunity to accurately analyze the contribution of DNA methylation to heterosis in trees. This is the first investigation of DNA methylation maps with high resolution in P. deltoides plants and their F1 hybrids at the genome-wide scale using high-throughput sequencing.
A total of 670.55 million reads were generated using MeDIP-Seq, 486.27 million of which could be mapped onto the reference genome of P. trichocarpa, the average of the uniquely mapped rates was 57.8%. The relatively low rate of mapping using genomes of closely related species as a reference suggests that species in different sections within the genus Populus are genetically divergent (P. trichocarpa belongs to sect. Tacamahaca, and P. deltoides belongs to sect. Algeiros). Similar observations were also documented in studies of Populus alba and Populus tremula based on single nucleotide polymorphism (SNP) analysis of the two species . Our dataset of leaf methylomes shows that the parents and F1 hybrids had significant methylation in the CG/CHH/CHG contexts, with CHG and CHH methylation being more consistent, and cytosines in CG context were less methylated than those in the other two contexts. Previous studies have reported that CGs are dominant in methylome, especially in coding regions, while less frequent in general, 5meCHH is more common in repeat regions and short transposable elements [49–51]. In populous, CG and CHG methylation were more consistent within tissues. However, in the two targets with cytosine content < 10%, cytosines in CHH context were methylated more frequently than those in the other two contexts . The two parents had distinct methylomes reflected by different methylation coverage in the CG/CHG/CHH contexts. The methylation coverage of three better-parent F1 hybrids was higher than the average of the parental values (mid-parent value, MPV), indicating that the F1 hybrids had an altered epigenome, and the DNA methylation level was non-additive. Unlike in animal systems, where "Erase and Reset" of cytosine methylation occurs in each generation, in plants, the parental methylation states can be stably inherited by the progeny [52, 53]. However, many plants species often exhibit the remodeling of parental methylation patterns in interspecific hybrids and allopolyploids [54–56]. In these scenarios, DNA methylation partly functions epigenetically and dynamically over generations, thus achieving the control and balance of gene expression under specific circumstances [27, 54, 57].
Early studies proposed that allelic variation is the primary cause of heterosis , but this notion was challenged by the observation that parents with similar genetic backgrounds can also produce hybrids with heterosis, which can arise from the diversity of epialleles. Epi-allelic changes in hybrids occur though changes in siRNA levels, trans-chromosomal methylation (TCM) or trans-chromosomal demethylation (TCdM), which fit the dominance or overdominance hypotheses and indicate that epi-alleles are essential parts of the genetic basis of heterosis. In rice hybrids, DNA methylation at many loci is inherited by non-additive inheritance . Although the two rice hybrids had unequal numbers of non-additively methylated loci, in both hybrids, approximately 75% of such loci had increased methylation levels. The increased DNA methylationwas also reported in reciprocal F1 hybrids between Arabidopsis thaliana Landsberg erecta and C24 . In this study, we found that P. deltoides F1 hybrids with hybrid vigor (H1, H2, and H3) showed higher DNA methylation coverage in three contexts than the MPV. This can partially be explained by the effects of TCM. In this scenario, the better parent derived siRNA molecules associate with both alleles, maintains the methylation state of its own alleles and establishes the de novo methylation of lower parent hypomethylation , resulting in increased methylation levels in the non- or low methylation region. Therefore, the methylation levels of hybrids may exceed MPV. For lower-parent hybrids L1, the fact that DNA methylation coverages in three contexts are lower than parental values can be attributed to the influence of TCdM. The lower parent derived siRNA initially becomes associated across both parental alleles. This association can cause siRNA level to be present at lower levels than the threshold required for the establishment and/or maintenance of methylation, leading to hypomethylation of alleles of the lower parent allele. At the same time, with the loss of methylation, normal siRNA levels cannot be maintained (loss of siRNA), which further reduces the level of DNA methylation, as detected in the lower-parent hybrids L1 with lower methylation levels. Thus, contrasting patterns of methylation between poplar better-parent F1 and lower-parent L1 hybrids may result from an adjustment of methylation levels of the parents, and this difference in methylation may in turn influence and regulate the expression network of target genes, which is beneficial to the establishment of heterosis. Interestingly, one of the hybrids with negative better-parent heterosis (L2) has methylation coverages in three contexts above the midparent value, and the variations in methylation in specific genomic features (such as intron) and in transposable elements and repetitive sequences seem dependent on each genotype. This indicates that the role of DNA methylation in heterosis is complex and multifaceted.
In addition, in some annual herb plant species, distinct epigenomes between parents can give rise to increased DNA methylation levels in the F1 hybrids and contribute to heterosis. For instance, when two rice subspecies, Nipponbare (o. sativa ssp japonica) and 93-11 (o. sativa ssp indica), were used as parents, 82.1 and 70.8% of the different methylation region (DMRs) of the genome of F1 hybrids showed high- or above high-parental DNA methylation levels, respectively . When A. thaliana Landsberg erecta and C24 were used as parental lines, the reciprocal F1 hybrids showed increased DNA methylation levels across the entire genome, especially in the transposable elements .
However, other studies revealed no obviously altered or decreased methylation levels in hybrids compared with their parents. In Arabidopsis thaliana, 97% of the MspI/HpaII recognition sites in the F1 hybrids of a Col-0 and C24 cross retained their levels of methylation . The methylation levels of cotton hybrids were lower than those of the parents, and the demethylation numbers of better-parent hybrids were higher than those of the lower-parent hybrids . This discrepancy may be due to the different approaches used in these two studies versus the present study. The two previous studies used a methylation-sensitive amplified polymorphism assay, which is much less sensitive than MeDIP-Seq and thus could not fully scan all methylation loci and could only partially provide the landscapes of DNA methylation.
The MACS approach can improve the spatial resolution of the aligned data and impart the robustness of the final aligned sequences based on dynamic Poisson distribution . The peak coverage further illustrates that the parents had distinct DNA methylation levels, while F1 hybrids with hybrid vigor possessed elevated DNA methylation levels, and F1 hybrids with negative hybrid vigor possessed declining DNA methylation levels. In the P. deltoides genomes, peak data were found to be more enriched in promoters than in gene bodies, and the CDS showed more enrichment than introns or UTRs in gene bodies. The enrichment levels of various genomic features in the better-parent hybrids, parent and lower-parent hybrid were different. The growth vigor displayed in better-parent hybrids may be attributed to the increased transcriptional inactivation of CG and CHG sites and heterochromatin-mediated gene silencing, which are related to methylated enrichment. Throughout the growth and development of poplar, methylated enrichment may also suppress the expression of a proportion of genes and/or reduce spurious global transcription to enable full transcription or to initiate the expression of other suitable loci, consequently increasing hybrid vigor in the F1 hybrids; this concept deserves further investigation.
The analysis of differentially methylated genes between parents and hybrids has revealed that the hypermethylation levels of better-parent F1 hybrids were between those of the two parents, while the hypermethylation levels of the lower-parent F1 hybrids was lower than lowest value of the parents. This finding suggests that having a methylation level between that of the two parents in F1 hybrids may be more favorable for achieving better-parent heterosis, while deviating from the MPV tends to preclude the establishment of heterosis.
The analysis of GO functional categories showed that the differentially methylated genes between the better-parent F1 hybrids and the parents were enriched in metabolic processes, response to stress, and binding and catalytic activity, which indicates that heterosis in trees may follow a comprehensive process. At the same time, compared with lower-parent F1 hybrids, the hypermethylated genes in the better-parent F1 hybrids were enriched in metabolic and development processes, such as metabolic process, cellular metabolic process, primary metabolic process, small molecule metabolic process, nitrogen compound metabolic process, developmental process, anatomical structure development, and signaling, which implied that differentially methylated genes are involved in heterosis.
Compared with the parents and lower-parent F1 hybrids, the hypermethylated genes in better-parent F1 hybrids were involved in hormone synthesis and response to stress, such as cytochrome P450, participating in the biosynthesis of hormones, defensive compounds and fatty acids, GTP-binding proteins involved in cytoskeleton organization, signal transduction, vesicle trafficking, and stress tolerance. As Ca2 + signal transducers, calcium-dependent protein kinases play an important role in various plant physiological process, including growth, development, defense responses, regulation of reactive oxygen species production, symbiotic interactions, guard cell turgor, osmotic, drought and salt stress, and regulation through hormones such as ABA and GA. In summary, the fact that many differentially methylated genes are involved in diverse biological pathways indicates that the inheritance of heterosis is a multidimensional process.
To date, studies linking epigenetics and heterosis have only been carried out in a few plant species. In this study, we identified genome-wide variations in leaf methylomes between parents and their hybrids in P. deltoides, a perennial forest tree species. The dataset derived from MeDIP-Seq were used to produce DNA methylation maps with high resolution of P. deltoides. cl. '55/65' and P. deltoides cl. '10/17' and their five F1 hybrids. Populus F1 hybrids has a non-additive mathylation level (higher than mid-parent values), which showed that the methylation patterns of the parents partially and dynamically passed onto their hybrids and was remodeled. In addition, the DNA methylomes of better-parent F1 hybrids were significantly different from that of lower-parent F1 hybrids, which indicates that having a methylation level between that of the two parents may be more favorable for the achievement of better-parent heterosis in F1 hybrids, while the deviation from MPV tends to preclude the establishment of heterosis. Compared with the parents and the lower-parent F1 hybrids, the hypermethylated genes in the better-parent F1 hybrids were enriched in the processes of metabolism and development, which may be highly relevant to heterosis.
The publication charges this article were funded by the National Key Technology Research and Development Program for the Twelfth Five-Year-Plan of China (Grant No. 2012BAD01B03) and National Key Technology Research and Development Program for the Eleventh Five-Year-Plan of China (Grant No. 2006BAD01A15).
This article has been published as part of BMC Genetics Volume 15 Supplement 1, 2014: Selected articles from the International Symposium on Quantitative Genetics and Genomics of Woody Plants. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcgenet/supplements/15/S1.
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