Beyond genes: How the extended genotype of plants facilitates adaptation
Adaptation to environmental change is required for species to persist, however rapid environmental change may exceed the limits of traditional genetic adaptation leading to widespread decline. Recent work has highlighted the ‘extended genotype’ as additional factors influencing adaptive phenotypes. This project will examine DNA methylation and polyploidization as both a cause and consequence of the adaptation process using natural populations of the model cereal Brachypodium distachyon. The project will determine the architecture of these features and how their variability impacts adaptive traits such as flowering time. From the functional role of the extended genotype we will be able to predict and select genetic responses to environment.
With a rapidly changing environment, the ability for plants to quickly adapt to new environmental challenges is being tested. Genetic adaptation through varying life history strategies has long been thought the major driver of adaptation to the environment, however the rate of environmental change may be too great for genetic adaptation to compensate. Beyond this, populations consisting of low genetic diversity are seen, and persist, in nature. Recent insights indicate ‘extended genotype’ features -including DNA methylation variation, repetitive DNA elements, and polyploidization- may act as factors inducing rapid environmental adaptation regardless of the genetic variation found in the population. This project aims to identify the causal effects of extended genotype variation in the model cereal Brachypodium and how it is associated with adaptive traits such as growth rate and seasonal flowering time. Specific genes targeted by extended genotype factors will be identified across 300 low diversity lines in four controlled growing conditions using whole genome bisulfite sequencing. My expertise with extended genotype profiling, along with the novel resources for assaying natural populations will lead to breakthroughs in understanding the specific role that the extended genotype plays in rapid plant environmental adaptation.
All successful organisms require the ability to adapt to their environment. This is especially true for plants, as their sessile nature requires them to adapt insitu to the
many seasonal environmental factors they experience using the heritable genetic and potentially epigenetic information they contain. Heritable information is readily
observed as allelic variation between genes.When such features are adaptive, they control traits that provide greater fitness in a specific environment. Adaptation can be limited by standing genetic variation, so barring other mechanisms; the genetic makeup of the population (diversity and recombination) we could define the ‘adaptability’ of a population. Some plants and many crops are highly inbreeding leading to populations with little genetic variation. These inbred populations can establish and spread, and often display heritable phenotypic variation in adaptive traits such as flowering time . In this case, and likely for all plants, there may be additional heritable factors beyond genetic variation influencing phenotype and adaptability.
Figure 1. (A) Examples of extended genotype factors. (B) Map of populations collected from Canberra to Adelaide. (C) Examples of B. distachyon life history strategies for flowering time (images from the Amasino lab). Examples are matched to example locations in Australia. (D) Example of differentially methylated regions (black boxes) in a hypothetical genome comparison. Red indicates high DNA methylation, blue indicates depletion. Regions are mapped to genes and nearby transposons (below).
These factors beyond genetic variation may be considered part of an organism’s ‘extended genotype’(Fig 1A). The extended genotype can include a variety of items including epigenetic modifications such as DNA methylation  that may display variation independent of the underlying genetic content of an individual. These chromatin modifications can result in epialleles. The position of heterochromatin  and repetitive transposable DNA elements within the genome can also provide unique regulatory environments impacting gene expression and DNA methylation [4-6]. Indeed variation in repeat content has been observed and is associated with genetic variation in trans in specific populations . Another component of the extended genotype is polyploidy, the duplication/hybridization of entire sets of chromosomes in the genome . Variation in ploidy can allow for unique gene regulation and gene sub-functionalization to occur  and has been shown to be associated with species segregation in natural environments [10-12]. The major question of this project is to understand how extended genotype factors influence growing season adaptability across simulated natural environments.
The model cereal, Brachypodium distachyon provides a unique plant system to answer this question. With natural populations found throughout the world and Australia (Fig 1B) of diploid (2n=10), allotetraploid (2n=20), and allohexaploid (2n=30) individuals it provides a unique opportunity to examine the impact of polyploidization on adaptation . It is also well suited for genomic analyses with a small diploid genome  closely related to other agricultural species such as barley and wheat . To understand the mechanisms underlying adaptation to growing season variation, I will identify genetic loci that are regulated by extended genotype activity and associate with adaptive traits such as growth rate and flowering time (Fig 1B-D). Plants (300) selected across a transect from Canberra to Adelaide will be grown under four contrasting experimental conditions simulating diurnal and seasonal light quality and temperature of typical winter or spring growing conditions where these populations are found in Australia. The resulting quantitative trait loci for seasonal flowering time will be finely dissected with future downstream analysis (see 5. Feasibility and Benefit). The functional pathways identified can be used to develop (epi)allelic selection criteria for increased adaptability of plants further marginalized by climate change.
This project will answer the questions: Ia. Does DNA methylation variation directly associate with the adaptive phenotypic variation of seasonal flowering time? Ib. Is this more prominent within local inbred mapping set where genetic diversity is low or among a diverse global mapping set with high genetic diversity? II. How has polyploidization impacted DNA methylation and influenced adaptive traits?
A key aspect of this project is the unique set of populations that are available for study. Brachypodium distachyon populations across southern Australia live in a range of environments from open fields to woodland areas shaded by a canopy. Beyond these differences in light quality (Figure 2A), differences in water availability from river banks and floodways to higher plains as well as variations in plant density are also apparent. The combination of these factors can be used to define the growing season, in which plants must select a life history strategy (overwintering vs. rapid cycling) that is optimal for their location. Two unique diploid subspecies, an allotetraploid (four genome copies resulting from the hybridization of the two diploid subspecies), and an allohexaploid are known to exist in natural populations. Populations may consist of multiple genotypes and ploidy levels or be more inbred with limited genetic diversity at a single ploidy level. Hundreds of individuals have been collected across these environmental gradients and have been genotyped by sequencing. This allows target mapping populations with high and low genetic diversity to be selected for these studies. This project will build upon resources developed in the ARC Centre of Excellence for Plant Energy Biology.
Aim I: Demonstrate that phenotypic changes caused by DNA methylation variation
DNA methylation can provide a mechanism of epigenetic information that allows for heritable variation outside of mendelian genetic variation. Recent advances by Lister, a close collaborator (and coCI in CoE Plant Energy Biology), have allowed DNA methylation profiling on a genome-wide scale . A major goal of epigenetic research is to demonstrate that epigenetic variation can have phenotypic consequences. To date, studies have been able to associate DNA methylation variation to gene expression changes [17,6] however this has not been successfully associated to whole plant phenotypes. Nor has it been shown to have function or relevance in natural environments. However, recent examples of environmental factors influencing epigenetic signals have been reported and have been shown to act across generations . Research under this aim will expand my expertise identifying DNA methylation variation across individuals to show that phenotypic changes within adaptive traits can be directly caused or act through DNA methylation variation independent of genetic variation.
Figure 2. (A)Location of known Brachypodium populations across southeastern australia (red dots). Color gradient indicates solar radiation variation in the driest quarter (ala.org.au) which defines growing season (winter or spring). (B,C) Example of fine-scale light quality in growth chambers. LED lighting can simulate diurnal spectral variation (sunrise and sunet) which reproduces outdoor seasonal signals and regulates flowering . Imaging systems in chambers provide detailed analysis of growth rate and flowering time.
To examine the impact of DNA methylation as a source of adaptive variation, 300 diploid (2n=10) Bracypodium distachyon individuals from a low diversity inbred mapping set, and 300 total individuals from high diversity mapping set spanning the native and introduced range across Australia and global populations will be examined (Figure 1B). These samples will be selected from the large-scale Brachypodium genotyping projects that are ongoing in the Borevitz lab. Seeds will be planted in specialized growth chambers programmed to simulate open fields under spring or winter conditions. These chambers have been developed in the Borevitz lab to provide fine temporal-scale control of light quality (up to 10 LED spectral bands), temperature and moisture conditions beyond traditional growth chambers (Figure 2B-C). Plants will be grown under two plantings mimicking winter and spring growing seasons . All lines will be grown to measure growth rate (photosynthetic area/time) and flowering time through high-throughput imaging technologies developed between the Borevitz lab and the High Resolution Plant Phenomics Centre in Canberra. Low-coverage (~2-5X) whole genome methylC-seq  will be performed on the 1200 total samples spanning the two growing seasons and 2 replicate blocks of related genotypes. Sequencing data obtained by MethylC-seq will be used to identify common differentially methylated regions (DMRs) within the two mapping sets of lines as well as across environments. The resulting data will be used to perform genome-wide association studies (GWAS) using both available typical genetic variants (SNPs) and the identified common DMRs for associations with traits across environments (Figure 3). Quantitative Trait Loci (QTL) and epiQTL, associated with seasonal growth rate and flowering time may be jointly controlled by SNPs or epialleles that are independent of each other (Figure 3A-C) or those displaying similar associations between both genetic and methylation markers (Figure 3D). Associations found only by DMRs are likely to be unique epigenetic regulators of the tested phenotype. Results from both environments will be examined jointly to identify (epi)GxE interactions as candidate adaptive loci.
The unique mapping sets of Brachypodium distachyon available for this project, as well as the genotyping data already in hand, allow these experiments to be started immediately. A challenge of this aim will be to develop analysis methods to identify and classify common epigenetic variation from ~2-5X genomic coverage each for thousands of samples. This will allow phenotypic associations among SNPs and DMRs from large samples to be tested. As this has not been done previously, bioinformatic challenges will exist for read mapping, the calling of methylation variants, and the ability to associate them with observed phenotypes. However, extensions of high sample multiplexing used in Genotype-by-Sequencing (GBS) can be used for MethylC-seq analysis. Computational methods including Hidden Markov Models  will jointly look across samples and along the chromosome to detect common DMRs. This work will be performed in collaboration with Ryan Lister at the University of Western Australia who has experience in the emerging molecular technologies described in this proposal. The high sample coverage methods developed in this aim can be applied to other experiments within this proposal as well as be adapted for other experimental systems used by the wider community.
Figure 3: Hypothetical example of genome-wide association approaches. Associations (blue) are plotted across chromosomes for a traditional GWAS with SNP markers (top) and DNA methylation variants (bottom). (A, B) Example of genetic associations with no methylation variation. (C) Example of methylation association acting independently of underlying genetic association. (D) Example of association by both SNP and DNA methylation markers.
Aim II: Investigate epigenomic variation of homeologs within polyploids
The creation of a polyploid genome is a strong ‘genomic shock’ creating a new chromosomal load in a
single generation. Further, the new reproductive barrier and subsequent inbreeding that may arise can limit survival. Most animal polyploids that occur spontaneously are not maintained in natural populations . However, plant polyploids that do adapt to this new genomic context are frequently observed in nature, for example wheat, and often display valuable phenotypic traits in relationship to environmental adaptation . Although polyploidy is common, it is largely unknown what mechanisms have lead to their success once created and/or if there are specific targets of selection leading to stable polyploids. In Arabidopsis thaliana, specific genes have been identified that are associated with the prevalence of polyploidization 
indicating that some individuals may contain alleles that promote the creation of new polyploids. This aim will examine the extended genotype landscape of natural polyploid populations independently derived within Brachypodium distachyon to determine if there are patterns of conserved variation between polyploids and their progenitors.
DNA methylation will be profiled using MethylC-seq  on 100 individuals from an inbred allotetraploid population and 200 diverse samples of allotetraploid (2n=20) and alloheaploid (2n=30) individuals from across the growing range. Plants will also be grown across simulated spring and winter environments within the climate chambers in the Borevitz lab. Adaptive phenotypes will be measured as described in Aim I. This data will be combined with the diploid data from Aim I to conduct an initial analysis of genotypic differences across ploidy levels to identify genetic loci that are associated with adaptive phenotypes across these environments. Although these aims utilize different genetic material, known loci that are involved in flowering time may be confirmed across both aims I and II.
Differences in DNA methylation may occur at individual genetic loci as DMRs or on a larger scale of chromosomal shifts in heterochromatin patterns. Indeed, Brachypodium polyploids have displayed nucleolar dominance based on cytogenetic studies  indicating the possibility of sub-genome selection within polyploid individuals. The sequencing data from diploid samples as described in Aim I will be utilized to perform genome-wide association using ploidy level as a phenotype analogous to recent extended genotype studies in Arabidopsis [7,24]. Beyond this, the number of novel transposon insertion events will be classified as an additional phenotype by examining unmapped sequencing reads with signatures of known transposable element sequences. Patterns of DNA methylation variation will be identified across ploidy levels as well as within populations of the same ploidy level. If methylation variants are discovered that are conserved across independent polyploid lines, it may indicate epigenetic regulation that has been selected in these successful polyploid populations. It is also possible that there are novel methylation variants both across and between populations, which may indicate unique regulatory variation within polyploid lines. These variants may provide a source of variation independent of mendelian genetic variation which could lead to increased adaptability to environments previously inaccessible to their diploid progenitors. The genes targeted by methylation variation will be classified and investigated in future experiments, beyond the scope of this proposal, as possible loci impacting the actions, or consequences, of polyploidization.
The results of this project will be translatable to economically important relatives of Brachypodium distachyon such as wheat and barley. The ability to identify previously unidentified extended genotype factors promoting adaptability to environmental variation both within and across growing seasons may allow these crop systems to ‘cope’ better when environmental and/or seasonal conditions change. This project will establish a paradigm for how any rapidly colonizing population can adapt to new environments. The factors impacting rapid adaptation ability will be extremely valuable for the prediction of endangered and foundation species that are more likely to be successful across new environments, or with increased environmental variability due to climate change.
A major outcome of this research will be a more complete picture of how epigenetic modifications, and the extended genotype play a role in the adaptive ability of plant systems across environments. This knowledge can be applied by selecting and/or engineering germplasm that contains genetic and epigenetic variants that promote adaptive ability for new or unstable growing environments. This proposal lays the foundation to fine-map loci and modify the extended genotype of individuals. Recent advances in genome editing technology now allow for targeted mutagenesis at loci of interest within plant systems  as well as genomic reduction of polyploids . (Epi)genetic loci that are identified by this project can be used for targeted genomic-editing to develop new alleles in plants collected from natural populations. With many cutting-edge technologies and novel protocols to be developed, it will take time beyond the scope of this proposal to develop functional validation to determine a causal link between extended genotype factors and phenotypes in the field. However as methods are validated, and tools developed, the results of Aim I and II will provide years of functional follow up work.
1. Platt A, et al. The scale of population structure in Arabidopsis thaliana. PLoS Genet. 2010 Feb 12;6(2):e1000843.
2. Cedar H, Bergman Y. Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet. 2009 May;10(5):295-304.
3. Grewal SI, Jia S. Heterochromatin revisited. Nat Rev Genet. 2007 Jan;8(1):35-46.
4. Slotkin RK, Martienssen R. Transposable elements and the epigenetic regulation of the genome. Nat Rev Genet. 2007 Apr;8(4):272-85.
5. Eichten SR, et al. Spreading of heterochromatin is limited to specific families of maize retrotransposons. PLoS Genet. 2012;8(12):e1003127.
6. Eichten SR, et al. Epigenetic and Genetic Influences on DNA Methylation Variation in Maize Populations. Plant Cell. 2013 Aug
7. Long Q, et al. Massive genomic variation and strong selection in Arabidopsis thaliana lines from Sweden. Nat Genet. 2013 Aug;45(8):884-90.
8. Parisod C, et al. Evolutionary consequences of autopolyploidy. New Phytol. 2010 Apr;186(1):5-17.
9. Soltis DE, et al. What we still don’t know about polyploidy. Taxon 2010 59: 1387-1403
10. Duchoslav M, et al. Complex distribution patterns, ecology and coexistence of ploidy levels of Allium oleraceum (Alliaceae) in the Czech Republic. Ann Bot. 2010 May;105(5):719-35.
11. Ramsey J. Polyploidy and ecological adaptation in wild yarrow. PNAS. 2011 Apr 26;108(17):7096-101
12. Manzaneda AJ, et al. Environmental aridity is associated with cytotype segregation and polyploidy occurrence in Brachypodium distachyon (Poaceae). New Phytol. 2012 Feb;193(3):797-805.
13. Catalán P, et al. Evolution and taxonomic split of the model grass Brachypodium distachyon. Ann Bot. 2012 Feb;109(2):385-405.
14. International Brachypodium Initiative. Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature. 2010 Feb 11;463(7282):763-8.
15. Mur LA, et al. Exploiting the Brachypodium Tool Box in cereal and grass research. New Phytol. 2011 Jul;191(2):334-47.
16. Lister R, et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell. 2008 May 2;133(3):523-36.
17. Schmitz RJ, et al. Patterns of population epigenomic diversity. Nature. 2013 Mar 14;495(7440):193-8.
18. Zhong SH, et al. Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis. Proc Natl Acad Sci U S A. 2013 May 28;110(22):9171-6.
19. Li Y, et al. Association mapping of local climate-sensitive quantitative trait loci in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2010 Dec 7;107(49):21199-204.
20. Emerson JJ, et al. Natural selection shapes genome-wide patterns of copy-number polymorphism in Drosophila melanogaster. Science, 2008. 320 (5883) 1629-1631.
21. Otto SP, Whitton J. Polyploid incidence and evolution. Annu Rev Genet. 2000;34:401-437.
22. Hollister JD, et al. Genetic adaptation associated with genome-doubling in autotetraploid Arabidopsis arenosa. PLoS Genet.2012;8(12):e1003093.
23. Idziak D, Hasterok R. Cytogenetic evidence of nucleolar dominance in allotetraploid species of Brachypodium. Genome. 2008 May;51(5):387-91.
24. Chao DY, et al. Polyploids exhibit higher potassium uptake and salinity tolerance in Arabidopsis. Science. 2013 Aug 9;341(6146):658-9
25. Li JF, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013 Aug 8;31(8):688-91.
26. Li Y, et al. Genetic Variation for Life History Sensitivity to Seasonal Warming in Arabidopsis thaliana. Genetics, 2013. 113.157628
27. Eichten SR, Borevitz JO. Methylation’s Mark on Inheritance. Nature News & Views, 2013. 10/1038/nature11960