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      Widespread natural variation of DNA methylation within angiosperms

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          Abstract

          Background

          DNA methylation is an important feature of plant epigenomes, involved in the formation of heterochromatin and affecting gene expression. Extensive variation of DNA methylation patterns within a species has been uncovered from studies of natural variation. However, the extent to which DNA methylation varies between flowering plant species is still unclear. To understand the variation in genomic patterning of DNA methylation across flowering plant species, we compared single base resolution DNA methylomes of 34 diverse angiosperm species.

          Results

          By analyzing whole-genome bisulfite sequencing data in a phylogenetic context, it becomes clear that there is extensive variation throughout angiosperms in gene body DNA methylation, euchromatic silencing of transposons and repeats, as well as silencing of heterochromatic transposons. The Brassicaceae have reduced CHG methylation levels and also reduced or loss of CG gene body methylation. The Poaceae are characterized by a lack or reduction of heterochromatic CHH methylation and enrichment of CHH methylation in genic regions. Furthermore, low levels of CHH methylation are observed in a number of species, especially in clonally propagated species.

          Conclusions

          These results reveal the extent of variation in DNA methylation in angiosperms and show that DNA methylation patterns are broadly a reflection of the evolutionary and life histories of plant species.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13059-016-1059-0) contains supplementary material, which is available to authorized users.

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          The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.

          The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.
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            The tomato genome sequence provides insights into fleshy fruit evolution

            Introductory Paragraph Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera 1 and includes annual and perennial plants from diverse habitats. We present a high quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, S. pimpinellifolium 2 , and compare them to each other and to potato (S. tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show >8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato, small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness. Main Text The genome of the inbred tomato cultivar ‘Heinz 1706’ was sequenced and assembled using a combination of Sanger and “next generation” technologies (Supplementary Section 1). The predicted genome size is ~900 Mb, consistent with prior estimates 3 , of which 760 Mb were assembled in 91 scaffolds aligned to the 12 tomato chromosomes, with most gaps restricted to pericentromeric regions (Fig. 1A; Supplementary Fig. 1). Base accuracy is approximately one substitution error per 29.4 kb and one indel error per 6.4 kb. The scaffolds were linked with two BAC-based physical maps and anchored/oriented using a high-density genetic map, introgression line mapping and BAC fluorescence in situ hybridisation (FISH). The genome of S. pimpinellifolium (accession LA1589) was sequenced and assembled de novo using Illumina short reads, yielding a 739 Mb draft genome (Supplementary Section 3). Estimated divergence between the wild and domesticated genomes is 0.6% (5.4M SNPs distributed along the chromosomes (Fig. 1A, Supplementary Fig. 1)). Tomato chromosomes consist of pericentric heterochromatin and distal euchromatin, with repeats concentrated within and around centromeres, in chromomeres and at telomeres (Fig. 1A, Supplementary Fig. 1). Substantially higher densities of recombination, genes and transcripts are observed in euchromatin, while chloroplast insertions (Supplementary Sections 1.22-1.23) and conserved miRNA genes (Supplementary Section 2.9) are more evenly distributed throughout the genome. The genome is highly syntenic with those of other economically important Solanaceae (Fig. 1B). Compared to the genomes of Arabidopsis 4 and sorghum 5 , tomato has fewer high-copy, full-length LTR retrotransposons with older average insertion ages (2.8 versus 0.8 mya) and fewer high-frequency k-mers (Supplementary Section 2.10). This supports previous findings that the tomato genome is unusual among angiosperms by being largely comprised of low-copy DNA 6,7 . The pipeline used to annotate the tomato and potato 8 genomes is described in Supplementary Section 2. It predicted 34,727 and 35,004 protein-coding genes, respectively. Of these, 30,855 and 32,988, respectively, are supported by RNA-Seq data, and 31,741 and 32,056, respectively, show high similarity to Arabidopsis genes (Supplementary section 2.1). Chromosomal organisation of genes, transcripts, repeats and sRNAs is very similar in the two species (Supplementary Figures 2-4). The protein coding genes of tomato, potato, Arabidopsis, rice and grape were clustered into 23,208 gene groups (≥2 members), of which 8,615 are common to all five genomes, 1,727 are confined to eudicots (tomato, potato, grape and Arabidopsis), and 727 are confined to plants with fleshy fruits (tomato, potato and grape) (Supplementary Section 5.1, Supplementary Fig. 5). Relative expression of all tomato genes was determined by replicated strand-specific Illumina RNA-Seq of root, leaf, flower (2 stages) and fruit (6 stages) in addition to leaf and fruit (3 stages) of S. pimpinellifolium (Supplementary Table 1). sRNA sequencing data supported the prediction of 96 conserved miRNA genes in tomato and 120 in potato, a number consistent with other plant species (Fig. 1A, Supplementary Figures 1 and 3, Supplementary Section 2.9). Among the 34 miRNA families identified, 10 are highly conserved in plants and similarly represented in the two species, whereas other, less conserved families are more abundant in potato. Several miRNAs, predicted to target TIR-NBS-LRR genes, appeared to be preferentially or exclusively expressed in potato (Supplementary Section 2.9). Supplementary section 4 deals with comparative genomic studies. Sequence alignment of 71 Mb of euchromatic tomato genomic DNA to their potato 8 counterparts revealed 8.7% nucleotide divergence (Supplementary Section 4.1). Intergenic and repeat-rich heterochromatic sequences showed more than 30% nucleotide divergence, consistent with the high sequence diversity in these regions among potato genotypes 8 . Alignment of tomato-potato orthologous regions confirmed 9 large inversions known from cytological or genetic studies and several smaller ones (Fig. 1C). The exact number of small inversions is difficult to determine due to the lack of orientation of most potato scaffolds. 18,320 clearly orthologous tomato-potato gene pairs were identified. Of these, 138 (0.75%) had significantly higher than average non-synonymous (Ka) versus synonymous (Ks) nucleotide substitution rate ratios (ω), suggesting diversifying selection, whereas 147 (0.80%) had significantly lower than average ω, suggesting purifying selection (Supplementary Table 2). The proportions of high and low ω between sorghum and maize (Zea mays) are 0.70% and 1.19%, respectively, after 11.9 Myr of divergence 9 , suggesting that diversifying selection may have been stronger in tomato-potato. The highest densities of low-ω genes are found in collinear blocks with average Ks >1.5, tracing to a genome triplication shared with grape (see below) (Fig. 1C, Supplementary Fig. 6, Supplementary Table 3). These genes, which have been preserved in paleo-duplicated locations for more than 100 Myr 10,11 are more constrained than ‘average’ genes and are enriched for transcription factors and genes otherwise related to gene regulation (Supplementary Tables 3-4). Sequence comparison of 32,955 annotated genes in tomato and S. pimpinellifolium revealed 6,659 identical genes and 3,730 with only synonymous changes. A total of 22,888 genes had non-synonymous changes, including gains and losses of stop codons with potential consequences for gene function (Supplementary Tables 5-7). Several pericentric regions, predicted to contain genes, are absent or polymorphic in the broader S. pimpinellifolium germplasm (Supplementary Table 8, Supplementary Fig. 7). Within cultivated germplasm, particularly among the small-fruited cherry tomatoes, several chromosomal segments are more closely related to S. pimpinellifolium than to ‘Heinz 1706’ (Supplementary Figures 8-9), supporting previous observations on recent admixture of these gene pools due to breeding 12 . ‘Heinz 1706’ itself has been reported to carry introgressions from S. pimpinellifolium 13 , traces of which are detectable on chromosomes 4, 9, 11 and 12 (Supplementary Table 9). Comparison of the tomato and grape genomes supports the hypothesis that a whole-genome triplication affecting the rosid lineage occurred in a common eudicot ancestor 11 (Fig. 2B). The distribution of Ks between corresponding gene pairs in duplicated blocks suggests that one polyploidisation in the solanaceous lineage preceded the rosid-asterid (tomato-grape) divergence (Supplementary Fig. 10). Comparison to the grape genome also reveals a more recent triplication in tomato and potato. While few individual tomato/potato genes remain triplicated (Supplementary Tables 10-11), 73% of tomato gene models are in blocks that are orthologous to one grape region, collectively covering 84% of the grape gene space. Among these grape genomic regions, 22.5% have one orthologous region in tomato, 39.9% have two, and 21.6% have three, indicating that a whole genome triplication occurred in the Solanum lineage, followed by widespread gene loss. This triplication, also evident in potato (Supplementary Fig. 11) is estimated at 71 (+/-19.4) mya based on Ks of paralogous genes (Supplementary Fig. 10), and therefore predates the ~7.3 mya tomato-potato divergence. Based on alignments to single grape genome segments, the tomato genome can be partitioned into three non-overlapping ‘subgenomes’ (Fig. 2A). The number of euasterid lineages that have experienced the recent triplication remains unclear and awaits complete euasterid I and II genome sequences. Ks distributions show that euasterids I and II, and indeed the rosid-asterid lineages, all diverged from common ancestry at or near the pan-eudicot triplication (Fig. 2B), suggesting that this event may have contributed to formation of major eudicot lineages in a short period of several million years 14 , partially explaining the explosive radiation of angiosperm plants on earth 15 . Supplementary section 5 reports on the analysis of specific gene families. Fleshy fruits (Supplementary Fig. 12) are an important means of attracting vertebrate frugivores for seed dispersal 16 . Combined orthology and synteny analyses suggest that both genome triplications added new gene family members that mediate important fruit-specific functions (Fig. 3). These include transcription factors and enzymes necessary for ethylene biosynthesis (RIN, CNR, ACS) and perception (LeETR3/NR, LeETR4) 17 , red light photoreceptors influencing fruit quality (PHYB1/PHYB2) and ethylene- and light-regulated genes mediating lycopene biosynthesis (PSY1/PSY2). Several cytochrome P450 subfamilies associated with toxic alkaloid biosynthesis show contraction or complete loss in tomato and the extant genes show negligible expression in ripe fruits (Supplementary Section 5.4). Fruit texture has profound agronomic and sensory importance and is controlled in part by cell wall structure and composition 18 . More than 50 genes showing differential expression during fruit development and ripening encode proteins involved in modification of wall architecture (Fig. 4A and Supplementary Section 5.7). For example, a family of xyloglucan endotransglucosylase-/hydrolases (XTHs) has expanded both in the recent whole genome triplication and through tandem duplication. One of the triplicated members, SlXTH10, shows differential loss between tomato and potato (Fig. 4A, Supplementary Table 12), suggesting genetically driven specialisation in the remodelling of fruit cell walls. Similar to soybean and potato and in contrast to Arabidopsis, tomato sRNAs map preferentially to euchromatin (Supplementary Fig. 2). sRNAs from tomato flowers and fruits 19 map to 8,416 gene promoters. Differential expression of sRNAs during fruit development is apparent for 2,687 promoters, including those of cell wall-related genes (Fig. 4B) and occurs preferentially at key developmental transitions (e.g. flower to fruit, fruit growth to fruit ripening, Supplementary Section 2.8). The genome sequences of tomato, S. pimpinellifolium and potato provide a starting point for comparing gene family evolution and sub-functionalization in the Solanaceae. A striking example is the SELF PRUNING (SP) gene family, which includes the homolog of Arabidopsis FT, encoding the mobile flowering hormone florigen 20 and its antagonist SP, encoding the ortholog of TFL1. Nearly a century ago, a spontaneous mutation in SP spawned the “determinate” varieties that now dominate the tomato mechanical harvesting industry 21 . The genome sequence has revealed that the SP family has expanded in the Solanum lineage compared to Arabidopsis, driven by the Solanum triplication and tandem duplication (Supplementary Fig. 13). In potato, SP3D and SP6A control flowering and tuberisation, respectively 22 , whereas SP3D in tomato, known as SINGLE FLOWER TRUSS, similarly controls flowering, but also drives heterosis for fruit yield in an epistatic relationship with SP 23,24,25 . Interestingly, SP6A in S. lycopersicum is inactivated by a premature stop codon, but remains functionally intact in S. pimpinellifolium. Thus, allelic variation in a subset of SP family genes has played a major role in the generation of both shared and species-specific variation in Solanaceous agricultural traits. The genome sequences of tomato and S. pimpinellifolium also provide a basis for understanding the bottlenecks that have narrowed tomato genetic diversity: the domestication of S. pimpinellifolium in the Americas, the export of a small number of accessions to Europe in the 16th Century, and the intensive breeding that followed. Charles Rick pioneered the use of trait introgression from wild tomato relatives to increase genetic diversity of cultivated tomatoes 26 . Introgression lines exist for seven wild tomato species, including S. pimpinellifolium, in the background of cultivated tomato. The genome sequences presented here and the availability of millions of SNPs will allow breeders to revisit this rich trait reservoir and identify domestication genes, providing biological knowledge and empowering biodiversity-based breeding. Methods Summary A total of 21 Gb of Roche/454 Titanium shotgun and matepair reads and 3.3 Gb of Sanger paired-end reads, including ~200,000 BAC and fosmid end sequence pairs, were generated from the ‘Heinz 1706’ inbred line (Supplementary Sections 1.1-1.7), assembled using both Newbler and CABOG and integrated into a single assembly (Supplementary Sections 1.17-1.18). The scaffolds were anchored using two BAC-based physical maps, one high density genetic map, overgo hybridization and genome-wide BAC FISH (Supplementary Sections 1.8-1.16 and 1.19). Over 99.9% of BAC/fosmid end pairs mapped consistently on the assembly and over 98% of EST sequences could be aligned to the assembly (Supplementary Section 1.20). Chloroplast genome insertions in the nuclear genome were validated using a matepair method and the flanking regions were identified (Supplementary Sections 1.22-1.24). Annotation was carried out using a pipeline based on EuGene that integrates de novo gene prediction, RNA-Seq alignment and rich function annotation (Supplementary Section 2). To facilitate interspecies comparison, the potato genome was re-annotated using the same pipeline. LTR retrotransposons were detected de novo with the LTR-STRUC program and dated by the sequence divergence between left and right solo LTR (Supplementary Section 2.10). The genome of S. pimpinellifolium was sequenced to 40x depth using Illumina paired end reads and assembled using ABySS (Supplementary Section 3). The tomato and potato genomes were aligned using LASTZ (Supplementary Section 4.1). Identification of triplicated regions was done using BLASTP, in-house generated scripts and three way comparisons between tomato, potato and S. pimpinellifolium using MCscan (Supplementary Sections 4.2-4.4). Specific gene families/groups (genes for ascorbate, carotenoid and jasmonate biosynthesis, cytochrome P450s, genes controlling cell wall architecture, hormonal and transcriptional regulators, resistance genes) were subjected to expert curation/analysis, (Supplementary Section 5). PHYML and MEGA were used to reconstruct phylogenetic trees and MCSCAN was used to infer gene collinearity (Supplementary Section 5.2). Supplementary Material 1 2 3 4
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              The Sorghum bicolor genome and the diversification of grasses.

              Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the approximately 730-megabase Sorghum bicolor (L.) Moench genome, placing approximately 98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the approximately 75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization approximately 70 million years ago, most duplicated gene sets lost one member before the sorghum-rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum's drought tolerance.
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                Author and article information

                Contributors
                (706) 542-1887 , schmitz@uga.edu
                Journal
                Genome Biol
                Genome Biol
                Genome Biology
                BioMed Central (London )
                1474-7596
                1474-760X
                27 September 2016
                27 September 2016
                2016
                : 17
                : 194
                Affiliations
                [1 ]Department of Genetics, University of Georgia, 120 East Green Street, Athens, GA 30602 USA
                [2 ]Institute of Bioinformatics, University of Georgia, Athens, GA 30602 USA
                [3 ]Department of Plant Biology, University of Georgia, Athens, GA 30602 USA
                [4 ]Center for Applied Genetic Technologies, University of Georgia, Athens, GA 30602 USA
                [5 ]Department of Plant Biology, Microbial and Plant Genomics Institute, University of Minnesota, Saint Paul, MN 55108 USA
                [6 ]Plant and Wildlife Science Department, Brigham Young University, Provo, UT 84602 USA
                [7 ]National Root Crops Research Institute (NRCRI), Umudike, Km 8 Ikot Ekpene Road, PMB 7006, Umuahia, 440001 Nigeria
                [8 ]HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806 USA
                [9 ]Department of Energy Joint Genome Institute, Walnut Creek, CA USA
                Article
                1059
                10.1186/s13059-016-1059-0
                5037628
                27671052
                fd11ee03-04f5-4c11-96bf-54fb2607bf00
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 2 June 2016
                : 9 September 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000152, Division of Molecular and Cellular Biosciences;
                Award ID: MCB – 1339194
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000154, Division of Integrative Organismal Systems;
                Award ID: IOS – 1402183
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000875, Pew Charitable Trusts;
                Funded by: FundRef http://dx.doi.org/10.13039/100006132, Office of Science;
                Award ID: DE-AC02-05CH11231
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100007699, University of Georgia;
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                Genetics
                Genetics

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