Paper: ”Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens”

Since I love author blog posts about papers, I thought I’d write a little about papers I’ve contributed too. So far, they’re not that many, but maybe it can be a habit.

Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens” was published in BMC Genomics in 2012. The title says it very well: the paper looks at differential expression and DNA methylation of a subset of genes in the hypothalamus of Red Junglefowl and domestic White Leghorn chickens. My contribution was during my MSc project in the group. Previously (Lindqvist & al 2007; Nätt & al 2009) Daniel Nätt, Pelle Jensen and others found a transgenerational effect of unpredictable light stress on domestic chickens. After that, and being interested in chicken domestication, a DNA methylation comparison of wild and domestic seems like a natural thing to do. And it turns out Red Junglefowl and White Leghorns differ in expression of a bunch of genes and in methylation of certain promoters (where promoter is operationally defined as a region around the start of the gene model). And when looking at two generations, the contrasts are correlated between parent and offspring. There is some heritable basis of the differences in gene expression and  DNA methylation.

In Red Junglefowl, ancestor of domestic chickens, gene expression and methylation profiles in thalamus/hypothalamus differed substantially from that of a domesticated egg laying breed. Expression as well as methylation differences were largely maintained in the offspring, demonstrating reliable inheritance of epigenetic variation.

What I did was methylation sensitive high resolution melting. HRM is a typing method based on real time PCR. After PCR you often make a melting curve by ramping up the temperature, denaturing the PCR product. The melting characteristics depend on the sequence, so you can use melting to check that you get the expected PCR product, and it turns out that the difference can be big enough to type SNPs. And if you can type SNPs, you can analyse DNA methylation. So we treat the DNA with bisulfite, which deaminates cytosines to uracil unless they are protected by methylation, and get a converted sequence where an unmethylated C is like a C>T SNP. We set up standard curves with a mixture of whole-genome amplified and in vitro methylated DNA and measured the degree of methylation.

That is averaging over the population of DNA molecules in the sample; I’ve been wondering how HRM performs when the CpGs in the amplicon have heterogenous methylation differences. We’ve used HRM for genotyping as well, and it works, but we’ve switched to pyrosequencing, which gives cleaner results and where the assay design is much easier to get right the first time. I don’t know whether the same applies for methylation analysis with pyro.


My favourite part of the paper is figure 4b (licence: cc:by 2.0) which shows methylation analysis in the advanced intercross of Red Junglefowl and White Leghorns, which immediately leads to, as mentioned in the paper, the thought of DNA methylation QTL mapping.


Nätt, D., Rubin, C. J., Wright, D., Johnsson, M., Beltéky, J., Andersson, L., & Jensen, P. (2012). Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens. BMC genomics, 13(1), 59.

Lindqvist C, Janczak AM, Nätt D, Baranowska I, Lindqvist N, et al. (2007) Transmission of Stress-Induced Learning Impairment and Associated Brain Gene Expression from Parents to Offspring in Chickens. PLoS ONE 2(4): e364. doi:10.1371/journal.pone.0000364

Nätt D, Lindqvist N, Stranneheim H, Lundeberg J, Torjesen PA, et al. (2009) Inheritance of Acquired Behaviour Adaptations and Brain Gene Expression in Chickens. PLoS ONE 4(7): e6405. doi:10.1371/journal.pone.0006405

Journal club of one: ”Short copy number variations potentially associated with tonic immobility response in newly hatched chicks”

(‘Journal club of one’ will be quick notes on papers, probably mostly about my favourite topics — genetics and the noble chicken.)

Abe, Nagao & Inoue-Murayama (2013), recently published this paper in PLOS ONE about copy number variants and tonic immobility in two kinds of domestic chicken. This obviously interests me for several reasons: I’m working on the genetic basis of some traits in the chicken; tonic immobility is a fun and strange behaviour — how it works and if it has any adaptive importance is pretty much unknown, but it is a classic from the chicken literature — and the authors use QTL regions derived directly from the F2 generation of cross that I’m working on — we’ve published one paper so far on the F8 generation.

Results: They use arrays and qPCR to search for copy number variants in three regions on chromosome one in two breeds (White Leghorn and Nagoya, a Japanese breed). After quite a bit of filtering they end up with a few variants that differ between the breeds. The breeds also differ in their tonic immobility behaviour with Leghorns going into tonic immobility after three attempts on average and lying still for 75 s and Nagoya taking 4.5 attempts and lying for 100 s on average. But the copy number variants were not associated with tonic immobility attempts or duration within breeds, so there is not really any evidence that they affect tonic immobility behaviour.


Apart from the issue that the regions (more than 60 Mb) will contain lots of other variants, we do not know whether these regions affect tonic immobility behaviour in these breeds in the first place. The intercross that the QTL come from is a wild by domestic Red Junglefowl x White Leghorn cross, and while Nagoya seem a very interesting breed that is distant from White Leghorn they are not junglefowl. When it comes to the Leghorn side of the experiments, I wouldn’t be surprised White Leghorn bred on a Swedish research institute and a Japanese research institute differed quite a bit. The breed differences in tonic immobility is not necessarily due to the genetic variants identified in this particular cross, especially since behaviour is probably very polygenic, and an F2 QTL study by necessity only scratches the surface.

In the discussion the authors bring up power: There were 71 Nagoya and 39 White Leghorn individuals and the experiment might be unable to reliably detect associations within the breeds. That does seem likely, but making a good informed guess about the expected effect is not so easy. A hint could come from looking at the effect sizes in the QTL study, but there is no guarantee that genetic background will not affect them. I don’t know really what this calculation comes from: ”Sample sizes would need to be increased more than 20-fold over the current study design” — maybe 11 tested copy number variants times two breeds? To me, that seems both overly optimistic, because it assumes that the entire breed difference would be due to these three QTL on chromosome 1, and overly pessimistic, since it assumes that the three QTL would fractionate into 11 variants.

Finally, with all diversity in the chicken, there’s certainly a place both for within and between population studies of various chickens with all kinds of genomic! Comparing breeds with different selection histories should be very interesting for distinguishing early ‘domestication QTL’ from ‘productivity QTL’ selected under modern chicken breeding. And I wish somebody would figure out a little more about how tonic immobility works.


Abe H, Nagao K, Inoue-Murayama M (2013) Short Copy Number Variations Potentially Associated with Tonic Immobility Responses in Newly Hatched Chicks. PLoS ONE 8(11): e80205. doi:10.1371/journal.pone.0080205

From my halftime seminar

A couple of weeks ago I presented my halftime seminar at IFM Biology, Linköping university. The halftime at our department isn’t a particularly dramatic event, but it means that after you’ve been going for two and a half years (since a typical Swedish PhD programme is four years plus 20% teaching to a total of five years), you get to talk about what you’ve been up to and discuss it with an invited opponent. I talked about combining genetic mapping and gene expression to search for quantitative trait genes for chicken domestication traits, and the work done so far particularly with relative comb mass. To give my esteemed readers an overview of what my project is about, here come a few of my slides about the mapping work — it is described in detail in Johnsson & al (2012). Yes, it does feel very good to write that — shout-outs to all the coauthors! This is part what I said on the seminar, part digression more suited for the blog format. Enjoy!

Slide04(Photo: Dominic Wright)

The common theme of my PhD project is genetic mapping and genetical genomics in an experimental intercross of wild and domestic chickens. The photo shows some of them as chicks. Since plumage colour is one of the things that segregate in this cross, their feathers actually make a very nice illustration of what is going on. We’re interested in traits that differ between wild and domestic chickens, so we use a cross based on a Red Jungefowl male and three domestic White Leghorn females. Their offspring have been mated with each other for several generations, giving rise to what is called an advanced intercross line. Genetic variants that cause differences between White Leghorn and Red Jungefowl chickens will segregate among the birds of the cross, and are mixed by recombination at meiosis. Some of the birds have the Red Junglefowl variant and some have the White Leghorn variant at a given part of their genome. By measuring traits that vary in the cross, and genotyping the birds for a map of genetic markers, we can find chromosomal chunks that are associated with particular traits, i.e. regions of the genome where we’re reasonably confident harbour a variant affecting the trait. These chromosomal chunks tend to be rather large, though, and contain several genes. My job is to use gene expression measurements from the cross to help zero in on the right genes.

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