RUFF GENOMICS · Genomics of reproductive strategies in ruffs

Genomics of reproductive strategies in ruffs

Summary of Research The coordinator, Dr. Clemens Küpper (CK), has successfully completed his Marie Curie Intra-European Fellow at University of Sheffield on 31 March 2015. His project “Genomics of reproductive strategies in ruffs” has had two main objectives. First, to identify the genomic regions that harbour gene variants that differ in the three male mating phenotypes (Objective 1). For this, he employed a combination of linkage mapping, genome wide association mapping and whole genome sequencing of captive and wild ruffs. The second objective was to identify and test candidate genes responsible for the differences in male phenotypes through fine mapping (Objective 2). Objective 1 The coordinator managed to obtain more samples of wild and captive ruffs than initially planned. He started new research collaborations with collaborators in The Netherlands (Theunis Piersma and Yvonne Verkuil, University of Groningen kindly provided samples of unrelated faeders and satellites) and went to Belarus where he sampled ruffs during the spring migration with help of new collaborators Natalia Karliova and Pavel Pinchuk, Turov Ornithological Station. CK also visited the named collaborator Dr. David Lank at Simon-Fraser-University, Burnaby to obtain further samples of the captive ruff population that was the source of the linkage mapping and got to know the pedigree set up and new insight into the reproductive biology of ruffs. Mapping of traits to the genome location requires a precise genome map. This genome map also provides guidance for the subsequent fine mapping based on the hit of the associated markers. Many draft genomes that have been produced using short length reads only are of inferior quality (with only short contigs being assembled) which subsequently makes it harder to fine map the traits of interest. In order to get better insights into the sequencing process and to obtain a high quality genome map the coordinator became involved with the genome sequencing of the ruff and prepared and submitted samples for Illumina HiSeq (six paired end and mate pair libraries of various insert sizes ranging from 200 bp to 5kbp) and one library for Pacific Biosciences (Pac Bio) Sequencing. He also prepared RNA samples for subsequent gene annotation. The resulting genome assembly is of high quality totalling 1.165 Gbp with an N50 scaffold length of 880 kbp consisting of 41k contigs (mean: 28 kbp). A large numbers of captive and wild samples were then genotyped using restriction site associated DNA sequencing (RAD sequencing) libraries to create a) a high density genetic marker map (42 unrelated individuals) for a genome wide association study and b) a low density marker map for linkage mapping (300 individuals). Results of both approaches pointed towards a single genomic region as source of variation for the mating types. This was confirmed when association studies were repeated with the data of 17 whole genome sequenced ruffs that were also generated as part of the project. Objective 2 Whole genome sequencing and linkage mapping suggested that the two alternative mating strategies of ruffs share an inverted chromosomal segment. We managed to find the exact breakpoints and concluded that the inversion is spanning about 1/5 of the entire chromosome. The inversion contains more than 100 genes of which about 80% differ between morphs. There is a heterogenous pattern of differentiation between morphs within the inversion with some regions more similar between faeders and satellites and some regions more similar between satellite and independent ruffs. However, recombination between inversion haplotypes is restricted because the inversion is lethal in its homozygous state. Several strong candidate genes that are involved hormone metabolism including steroid synthesis are located in the inversion and we identified not only morph specific gene copies but also new phenotypic differences that link these genes to the different morphs in ruffs and may explain observed differences in appearance and aggression behaviour. Societal Implications of the project The project provided a striking example how cutting edge DNA technology can help to identify the genetic basis of complex behavioural phenotypes in non-model organisms and will no doubt open a large number of fruitful new research avenues. We newly discovered that a large chromosomal inversion is responsible for modulating mating behaviours and identified several candidate genes in this inversion that should be investigated in more depth in the future. Being project coordinator helped CK to re-integrate into European science community after a short parental leave period since returning from the US in 2012. He built up essential bioinformatics skills, first by attending courses in Python and UNIX and then analysing the data himself. The coordinator also attended career workshops provided by University of Sheffield about (1) preparation of grant proposal and (2) efficient teaching. To obtain more teaching experience the coordinator prepared and delivered guest lectures to Sheffield undergraduates in 2013 and 2014 and co-organised two workshops for postgraduates/postdocs. He also obtained more experience by supervising two masters students and one research technician that assisted with the sample preparation at University of Sheffield. CK collaborated with another Marie Curie fellow (Dr. Maaike de Jong, University of Bristol) and helped her with her own RAD sequencing analysis. All these newly acquired skills have improved the academic profile of the coordinator. He has successfully passed the postdoctoral stage and has now taken up a position as Assistant Professor. Keeping with the spirit of European mobility and knowledge transfer he moved to a different European country and is now at the Institute of Zoology, University of Graz, Austria. In his new post he intends to apply the newly learned skills acquired to his teaching and research. Importantly, he will stay connected with his colleagues at University of Sheffield and continue to collaborate on the ruff project.

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