Month: May 2017

On a clear, windy day in winter 2016 (Avignon is (in)famous for its strong northerly Mistral wind), we finally got the results of the SNP genotyping of Spanish and Italian populations of Aleppo pine, performed in collaboration with our long time partners, INIA-CIFOR in Madrid, Spain and CNR-IBBR in Florence, Italy.

Reference assembly had gone smoothly, individual samples had produced a wealth of good-quality Illumina reads. We expected clean, straightforward data analyses, as we already had good results for silver fir, European larch and Atlas cedar (this is part of the ANR-funded FLAG project).

But then, a strange signal showed up in the data. We had two groups of samples: adult trees and seedlings, and when we looked at their polymorphism patterns, they looked very different:

only few polymorphic sites were in common, and often the two groups were fixed for different alleles (each column in the picture is a SNP site, each colour (blue, light blue, gray) one of the three possible genotypes of a bi-allelic SNP). The whole thing just did not make any sense.

The two groups were from different plates (one full plate for adults, two half-plates for the seedlings), so with my postdoc Hadrien Lalagüe we looked for possible differences in DNA quality, sequencing batch, anything technical that would explain such a result. No pattern appeared. We were desperate for a solution for the problem, lest we accepted to toss the whole data set.

Then few weeks later we looked again at the picture. It seemed like the two groups belonged to two different, closely related species, not even to different populations.
Oh, wait a minute… did we say ‘two different species’? Perhaps, if they look like two different species, it is because they are two different species!

Can you hear that kind of ‘falsifiable hypothesis’ stuff opening its way to our brains? Popper’s spook was hovering over us.

Then we had a wider look at our data set. What other species could half of the samples belong to?

One DNA plate of maritime pine had been shipped to the sequencing lab together with the Aleppo pine plates. We held our suspect number one.

What was the most likely mistake? Nobody could possibly mistake two half plates for one full plate, right? So the only possible swap was between plate A and plate B.

What would be the expected polymorphism pattern? Because plate B was genotyped against an internal reference sequence, nothing wrong would appear in its data, no matter what the species was. On the contrary, plates A, C1 and C2 would now be a mix of two pine species – exactly the kind of mix that would produce a pattern of partially shared, partially non-overlapping polymorphisms.

The hypothesis held. Popper’s spook was smiling, but he expected us to do more: to put our hypothesis to experimental test.

If our hypothesis was correct (plate A contained maritime pine, not Aleppo, and plate B had Aleppo, not maritime pine), then a read-mapping with plates B, C1, C2 would yield the expected nice pattern of shared SNPs.

And ta-dah! No differences in polymorphism patterns when plate A was replaced with plate B in the read-mapping (plate A, mapped against itself, was fine, of course).

We did a further check on the absolute number of polymorphisms that appeared within different groups of plates (more polymorphisms expected when mixing two different species), and showed that any group including plate A (now maritime pine) has systematically more SNPs than any group made from the remaining plates (now all Aleppo pine). So we re-ran our SNP analyses and all turned out to be fine.

Popper’s spook was now looking very happily at us.

There are few lessons to draw from this story.

First: even though we forest scientists are used to make statistical inferences, as opposed to testing hypotheses, the good old hypotetico-deductive method is still alive and kicking. It is a vital piece of our toolkit.

Second: look at your data with your eyes. The most likely hypothesis will probably pop up.

Third: never throw out an apparently bad data set too quickly. Maybe it is just begging you to look at it again, and from a different angle.

Opening of a PhD position to study the population genomics and conservation of three Alpine grouse species

A fully-funded 3-year PhD position is available for an enthusiastic student with a background in conservation, population or evolutionary genetics, to analyse patterns of genomic variation in three Alpine grouse species for conservation and management purposes.

The student will spend approximately half their time in the two collaborating research groups, those of Giorgio Bertorelle (University of Ferrara, Italy – UNIFE), and Heidi C. Hauffe (Fondazione Edmund Mach, Trento, Italy – FEM). Barbara Crestanello in the Hauffe group, and expert in tetraonid conservation genetics and genomics, will act as an additional supervisor.

The student will register at UNIFE, and academic training include seminars and courses, as well as participation in national and international conferences.

Brief project description:
This project will focus on three charismatic alpine bird species of conservation and management concern for which data on mtDNA and STR markers for more than 200 individuals per species are already available from Trentino and surrounding regions. The main goals of the project are to a) type SNP markers for a subset of the above samples using GBS; b) compare the power of mtDNA/STRs and SNPs to reconstruct demographic history; c) identify if and how different ecological niches, reproductive systems, and hunting pressures affect the genomic variation; d) translate the results into efficient management and conservation strategies; e) use available technologies to develop SNP sets that can be used for future cost-effective conservation genomic investigations.

Informal enquiries for further details of the aims of the project should be sent to barbara.crestanello@fmach.it, heidi.hauffe@fmach.it, or giorgio.bertorelle@unife.it.

The position is for candidates with a degree equivalent to an Italian “Magistrale” degree (Master), and in an appropriate subject (e.g. Biology, Biotechnology, Mathematics). A keen interest in data analysis as applied to conservation, and preferably, at least 6 months’ experience in a basic molecular biology laboratory, are requested. Good English skills are necessary, but knowledge of Italian is not essential (although it obviously helps for living in Italy!).

The formal online application form will be available around mid June at the site www.unife.it/studenti/dottorato/concorsi. However, interested candidates are welcome to send already to giorgio.bertorelle@unife.it an application letter, stating the applicant’s motivation for the position, experience and skills related to the requirements listed above, a full CV, and contact information (including email addresses) for 2 potential referees. Please send your application file as a single pdf.

Ferrara is an ancient Medieval and Renaissance town located in North-Eastern Italy, 50 km North of Bologna and 100 km south of Venice.  Far from being shrouded in the past, Ferrara is a cyclist- and pedestrian-friendly sustainable town where young people can experience a high quality of life, take advantage of well-maintained infrastructures, and pleasantly blend in. More information can be found at http://www.unife.it/international/student-life

THE INDIANA UNIVERSITY LIBRARY in Bloomington, IN is a stocky, ten-storey building where windows are a rarity.

In the dusk and dust of the tenth floor, one day in summer 2004, I was lucky enough to find the full series of the Proceedings of the Carlsberg Laboratories. I was looking for a rather unusual reference:

Winge O., 1923 On sex chromosomes, sex determination and preponderance of females in some dioecious plants. C. R. Trav. Lab. Carlsberg 15: 1–26.

When I eventually found it, it felt like Indiana Jones grasping the long-sought talisman.

Why on earth would I need such an esoteric paper?

Few weeks earlier, as a post-doc in Lynda Delph’s lab (a very happy time indeed), I was doing linkage mapping with AFLP markers (sounds like the Stone Age, doesn’t it?) in Silene latifolia. The data, produced by Michele Arntz, were nice and tidy, and the map was coming out beautifully. Statistical support was excellent – actually, I’ve never seen such enormous LOD-scores, after or before then – so I was 100% confident in the map.

But something was wrong. ALL the literature on the species, which happens to be dioecious (i.e. it has separate sexes; the species, not the literature), claimed that, as in humans, the two sex chromosomes recombine only at one end (details vary, but all articles agreed on the one-end-only crossing overs).

And I had two nice recombinant blocks, one on each side of the non-recombining region. Oops. Ouch.

We went through all the analyses again, checked the data, the samples, re-ran the mapping with subsets of the mapping population. No way. The data resisted our efforts to force them to conform to the literary, consensus evidence.

It was time to ask for expert advice. So we called the best expert we could reach, a scientist with a very long experience in all matters plant sex chromosomes (let us treat this scientist as an anonymous referee, and let us not reveal her identity). She listened to us carefully, then after a pause she said: “but that’s… HERETIC!”.

I still consider this sentence as the best professional compliment I have ever received. But this did not push us forward an inch. We still had a blatant contradiction between our data and what science expected them to say, and nobody to explain why. Then Lynda, with her typical matter-of-factly, rigorous approach to science, decided we should go through the literature. ALL the literature on the subject.

Everybody quoted the 1923 Winge paper and several papers by Westergaard, published in the 40’s and in the 50’s (Westergaard (1946), Hereditas 32: 419-443; Westergaard (1948), Hereditas 34: 257-279; Westergaard (1958), Advances in Genetics 9: 217-281). All more recent literature quoted those papers as saying that “Silene latifolia sex chromosomes recombine at one end only”. Clearly, if there was a confrontation to be had, it was between our data and those papers.

Hence my travel in space and time to the last floor of IU library (Westergaard’s papers were easier to obtain).

The reading of those papers revealed the complex and surprising truth. Winge did not say a word about chiasmata patterns. Westergaard had carefully characterised the chiasmata on the sex chromosomes in the 1946 paper, but some data were hard to interpret, and so he chose to describe only “unusually favourable [mitotic] plates” (what we would call today “cherrypicking”, even though I acknowledge his great honesty in declaring it). Plus, he dismissed some chiasmata observed in the supposed “differential” arms as being “rare”. And went on describing differential and homologous arms of X and Y chromosomes.

Then, fatally, he depicted in Figure 5 a summary showing X and Y chromosomes with only one homologous arm, as in the figure above, the upper pane of which is based on his one.  Westergaard, by all means, was a rigorous scientist, but he probably oversimplified his results in that drawing, and there we go: starting with this figure (and if you do not read the paper), you are dead sure that only one side recombines. Yet there was no contradiction between the data in the paper and our data. Unfortunately, following authors likely “quoted” only that figure from the 1946 paper. The result: against evidence, only one arm recombined now. Period.

This is serious. Such things should never happen in science. Certainly, most scientists read most of the papers they quote most of the time (for sure, compared to many politicians and opinion makers, we are diamond-grade examples of intellectual rigour, to be honest); but this story tells me that one must be suspicious when everybody quotes a very old, often hard to access, article (I bet very few people have read many of the Sewall Wright’s papers they quote, for instance; of the few who have read them, most have not understood a line of them; I belong to the latter category).

So, this is my take-home message from this story: read carefully all the papers you quote. If you cannot read them, then quote some other, more recent paper (e.g. a review), not the original message. In this case, the Chinese whispers chain must be explicit, if it has to exist at all (which I’d rather prefer not, anyway). Let us not convert science into a matter of ipse dixit. Let us stick to facts.

P.S. we actually published our linkage map, with sex chromosomes nicely recombining at both ends, here and here.

European beech (Fagus sylvatica L.) is a keystone tree species in European forests, making up 14-18% of total forest cover and with a range spanning from Greece to Sweden. Beech is the focus of multiple, long-lasting and intensive international research programs in ecology and population genetics. Despite this, little is known about the genetic bases of the adaptation of this species to environmental variation, no genomic reference is available and patterns of genomic diversity are virtually unexplored. Based on genome sequencing and re-sequencing data, obtained on natural populations and provenance tests throughout Europe, we propose to study patterns of adaptive diversity in coding and promoter regions, to: (a) determine patterns of genomic diversity determined by adaptive processes at multiple geographical scales, from stand to region to range; (b) estimate intensity of selection, through a combination of analytical and modelling approaches; (c) model and predict the ability of European beech to cope with climate change through adaptation.

For more information, please contact Ivan Scotti (ivan.scotti[at]inra.fr) and visit the BEECHGENOMES project post.

Genome sequence variation in European beech (Fagus sylvatica L.): analysing adaptation and adaptability in an ecologically and economically major European forest tree species challenged by climate change.

The European beech (Fagus sylvatica L.) is a major keystone forest species, covering more than fifteen percent of European forests, and is a commercially important timber species. It is the focus of intensive, high-impact science in ecology, forestry, genetics and tree physiology. Notwithstanding its importance, genomic resources and a solid knowledge of the genomic bases of adaptation are lacking for the species.

The BEECHGENOMES project (2017-2020), funded by the France Génomique call and led by INRA-URFM (Ivan Scotti), will tackle two topics: (1) producing a reference genome sequence for European beech; (2) obtaining high density variant maps, through genotyping-by-sequencing, from a large sample (>2000 trees) collected throughout Europe; (3) identifying patterns of local adaptation at multiple scales, from stand to landscape to region to range. The BEECHGENOMES project has tight links to a former program (FLAG) and to a current H2020-funded program (GENTREE), both also led by INRA-URFM. The program will be carried out by a consortium of fourteen research teams from six countries.

The MAP below shows the wide choice of sampling sites for intensively studied sites (blue), regional transects (yellow, purple), latitudinal transect (red) and provenances in the provenance tests (green)

We are seeking candidates for a Ph. D. thesis on the project – see post

Contact: Ivan Scotti, ivan.scotti[at]inra.fr.