Low Mutation Load in a Supergene Underpinning Alternative Male Mating Strategies in Ruff (Calidris pugnax)

A paradox in evolutionary biology is how supergenes can maintain high fitness despite reduced effective population size, the suppression of recombination, and the expected accumulation of mutational load. The ruff supergene involves 2 rare inversion haplotypes (satellite and faeder). These are recessive lethals but with dominant effects on male mating strategies, plumage, and body size. Sequence divergence to the wild-type (independent) haplotype indicates that the inversion could be as old as 4 million years. Here, we have constructed a highly contiguous genome assembly of the inversion region for both the independent and satellite haplotypes. Based on the new data, we estimate that the recombination event(s) creating the satellite haplotype occurred only about 70,000 yr ago. Contrary to expectations for supergenes, we find no substantial expansion of repeats and only a modest mutation load on the satellite and faeder haplotypes despite high sequence divergence to the non-inverted haplotype (1.46%). The essential centromere protein N (CENPN) gene is disrupted by the inversion and is as well conserved on the inversion haplotypes as on the noninversion haplotype. These results suggest that the inversion may be much younger than previously thought. The low mutation load, despite recessive lethality, may be explained by the introgression of the inversion from a now extinct lineage

Molecular identification of colour pattern genes in birds

Birds display a spectacular range of plumage pigmentation. The purpose of this thesis was to elucidate genetic mechanisms that contribute to pattern formation on individual feathers and the body. In study I and II, we investigated two barring patterns in chicken. We show that in the Fayoumi breed autosomal barring is associated with a 1Mb un-recombined region on chromosome 11, which contains the MC1R gene. Our functional analysis strongly suggests that autosomal barring is primarily caused by activating MC1R mutations and that other loci contribute to the appearance of the pattern. In study II, we demonstrate that sex-linked barring is created by a combination of cis-regulatory and missense mutations in the CDKN2A/ ARF gene. We demonstrate that the up-regulation of CDKN2A expression is caused by non-coding mutation(s) and is resulting in a dilute barring pattern. Functional testing revealed that the two missense mutations in ARF hamper its function and restrict the diluting effect of the non-coding mutations. Only the combination of both regulatory and missense mutations generates clear barring pattern as observed e.g. in the Barred Plymouth Rock. In study III and IV, we investigated the genetic mechanisms driving pigment pattern variation in the ruff (Philomachus pugnax). We first identified a 4.5 Mb inversion to be associated with the two male reproductive morphs called satellite and faeder. These morphs differ substantially in behavior, reproductive strategy, body size and plumage appearance between each other as well as from the third, more prevalent morph, the independent. The inversion disrupts the CENPN gene making this genomic re-arrangement homozygous lethal. We identified a large set of variants; among them four missense mutations in MC1R associated with the Satellite allele. In study IV, we explored whether these MC1R mutations are contributing to the light display plumage of the satellite morph. Our data shows that MC1R is up-regulated in all coloured satellite feathers and that this is due to a higher expression of the Independent allele. Evaluation of MC1R signaling in cell culture models subsequently revealed that the mutations alter receptor properties such as cAMP production, sensitization and surface expression but also suggests that transfection assays using mammalian cells might not reveal the complex function MC1R is most likely having in avian melanocytes

Mutations in \u3ci\u3eDMRT3\u3c/i\u3e Affect Locomotion in Horses and Spinal Circuit Function in Mice

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left–right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favorable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation

Identification de gènes de dessin de plumage chez les oiseaux

Le plumage des oiseaux montre une diversité de coloration spectaculaire. L’objectif de cette thèse est d’élucider les mécanismes génétiques contribuant à cette diversité en étudiant plus particulièrement la formation de dessins sur la plume et sur le corps. Les études I et II sont consacrées à deux dessins de barrure chez le poulet : la barrure autosomale et la barrure liée au sexe. En réalisant un croisement en retour à partir de la race Fayoumi, nous avons pu démontrer que la barrure autosomale est associée à une région du chromosome 11 et porte le gène de pigmentation MC1R. L’analyse fonctionnelle met en évidence qu’une mutation faux-sens, et non une modification de régulation, a le rôle d’une mutation causale. Dans l’étude II, nous démontrons que la barrure liée au sexe est créée par la combinaison de mutations de régulation en cis et de mutations faux-sens dans le gène CDKN2A. L’activation de l’expression de CDKN2A par deux mutations non-codantes détermine un phénotype de barrure très dilué. Des tests fonctionnels in vitro révèlent que les deux mutations faux-sens identifiées dans le même gène restreignent sa fonction et diminuent l’effet de dilution des mutations non-codantes. Seule la combinaison des mutations de régulation et d’une des mutation faux-sens, produit la barrure bien nette observée dans les races de poule actuelles. Les études III et IV précisent les mécanismes contrôlant la variation des dessins de plumage chez les oiseaux sauvages. Le chevalier combattant Philomachus pugnax présente trois formes reproductives chez le mâle, dénommées ‘indépendant’, ‘satellite’ et ‘faeder’. Nous avons d’abord identifié une inversion de 4.5 Mb chez les mâles satellite et faeder, qui diffèrent nettement entre eux, comme de l’indépendant, par leur comportement, leur stratégie reproductive et l’aspect de leur plumage. Un examen plus précis de la structure de l’inversion a montré que les points de cassure interrompaient le gène CENPN ce qui rend l’inversion létale à l’état homozygote. Nous avons identifié un grand nombre de variants contribuant assez probablement au phénotype et avons découvert quatre SNPs dans l’allèle MC1R du mâle satellite. L’étude IV a consisté à analyser la contribution de ces mutations de MC1R au plumage clair du mâle satellite pendant la reproduction. Nos données montrent que MC1R est activé dans les plumes colorées du mâle satellite en raison d’une plus forte expression de l’allèle indépendant. L’étude de la signalisation de MC1R en culture cellulaire a ensuite révélé que les mutations altèrent les propriétés du récepteur, mais suggère aussi que l’évaluation fonctionnelle du MC1R aviaire en cellules mammaliennes ne rend pas complètement compte du rôle complexe que MC1R joue dans les mélanocytes aviaires.Birds display a spectacular range of plumage pigmentation. The purpose of this thesis was to elucidate genetic mechanisms that contribute to pattern formation on individual feathers and the body. In study I and II, we investigated two barring patterns in chicken. We show that in the Fayoumi breed autosomal barring is associated with a 1Mb un-recombined region on chromosome 11, which contains the MC1R gene. Our functional analysis strongly suggests that autosomal barring is primarily caused by activating MC1R mutations and that other loci contribute to the appearance of the pattern. In study II, we demonstrate that sex-linked barring is created by a combination of cisregulatory and missense mutations in the CDKN2A/ ARF gene. We demonstrate that the upregulation of CDKN2A expression is caused by non-coding mutation(s) and is resulting in a dilute barring pattern. Functional testing revealed that the two missense mutations in ARF hamper its function and restrict the diluting effect of the non-coding mutations. Only the combination of both regulatory and missense mutations generates clear barring pattern as observed e.g. in the Barred Plymouth Rock. In study III and IV, we investigated the genetic mechanisms driving pigment pattern variation in the ruff (Philomachus pugnax). We first identified a 4.5 Mb inversion to be associated with the two male reproductive morphs called satellite and faeder. These morphs differ substantially in behavior, reproductive strategy, body size and plumage appearance between each other as well as from the third, more prevalent morph, the independent. The inversion disrupts the CENPN gene making this genomic re-arrangement homozygous lethal. We identified a large set of variants; among them four missense mutations in MC1R associated with the Satellite allele. In study IV, we explored whether these MC1R mutations are contributing to the light display plumage of the satellite morph. Our data shows that MC1R is up-regulated in all coloured satellite feathers and that this is due to a higher expression of the Independent allele. Evaluation of MC1R signaling in cell culture models subsequently revealed that the mutations alter receptor properties such as cAMP production, sensitization and surface expression but also suggests that transfection assays using mammalian cells might not reveal the complex function MC1R is most likely having in avian melanocytes

Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

Conclusion: By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species

Identification de gènes de dessin de plumage chez les oiseaux

Birds display a spectacular range of plumage pigmentation. The purpose of this thesis was to elucidate genetic mechanisms that contribute to pattern formation on individual feathers and the body. In study I and II, we investigated two barring patterns in chicken. We show that in the Fayoumi breed autosomal barring is associated with a 1Mb un-recombined region on chromosome 11, which contains the MC1R gene. Our functional analysis strongly suggests that autosomal barring is primarily caused by activating MC1R mutations and that other loci contribute to the appearance of the pattern. In study II, we demonstrate that sex-linked barring is created by a combination of cisregulatory and missense mutations in the CDKN2A/ ARF gene. We demonstrate that the upregulation of CDKN2A expression is caused by non-coding mutation(s) and is resulting in a dilute barring pattern. Functional testing revealed that the two missense mutations in ARF hamper its function and restrict the diluting effect of the non-coding mutations. Only the combination of both regulatory and missense mutations generates clear barring pattern as observed e.g. in the Barred Plymouth Rock. In study III and IV, we investigated the genetic mechanisms driving pigment pattern variation in the ruff (Philomachus pugnax). We first identified a 4.5 Mb inversion to be associated with the two male reproductive morphs called satellite and faeder. These morphs differ substantially in behavior, reproductive strategy, body size and plumage appearance between each other as well as from the third, more prevalent morph, the independent. The inversion disrupts the CENPN gene making this genomic re-arrangement homozygous lethal. We identified a large set of variants; among them four missense mutations in MC1R associated with the Satellite allele. In study IV, we explored whether these MC1R mutations are contributing to the light display plumage of the satellite morph. Our data shows that MC1R is up-regulated in all coloured satellite feathers and that this is due to a higher expression of the Independent allele. Evaluation of MC1R signaling in cell culture models subsequently revealed that the mutations alter receptor properties such as cAMP production, sensitization and surface expression but also suggests that transfection assays using mammalian cells might not reveal the complex function MC1R is most likely having in avian melanocytes.Le plumage des oiseaux montre une diversité de coloration spectaculaire. L’objectif de cette thèse est d’élucider les mécanismes génétiques contribuant à cette diversité en étudiant plus particulièrement la formation de dessins sur la plume et sur le corps. Les études I et II sont consacrées à deux dessins de barrure chez le poulet : la barrure autosomale et la barrure liée au sexe. En réalisant un croisement en retour à partir de la race Fayoumi, nous avons pu démontrer que la barrure autosomale est associée à une région du chromosome 11 et porte le gène de pigmentation MC1R. L’analyse fonctionnelle met en évidence qu’une mutation faux-sens, et non une modification de régulation, a le rôle d’une mutation causale. Dans l’étude II, nous démontrons que la barrure liée au sexe est créée par la combinaison de mutations de régulation en cis et de mutations faux-sens dans le gène CDKN2A. L’activation de l’expression de CDKN2A par deux mutations non-codantes détermine un phénotype de barrure très dilué. Des tests fonctionnels in vitro révèlent que les deux mutations faux-sens identifiées dans le même gène restreignent sa fonction et diminuent l’effet de dilution des mutations non-codantes. Seule la combinaison des mutations de régulation et d’une des mutation faux-sens, produit la barrure bien nette observée dans les races de poule actuelles. Les études III et IV précisent les mécanismes contrôlant la variation des dessins de plumage chez les oiseaux sauvages. Le chevalier combattant Philomachus pugnax présente trois formes reproductives chez le mâle, dénommées ‘indépendant’, ‘satellite’ et ‘faeder’. Nous avons d’abord identifié une inversion de 4.5 Mb chez les mâles satellite et faeder, qui diffèrent nettement entre eux, comme de l’indépendant, par leur comportement, leur stratégie reproductive et l’aspect de leur plumage. Un examen plus précis de la structure de l’inversion a montré que les points de cassure interrompaient le gène CENPN ce qui rend l’inversion létale à l’état homozygote. Nous avons identifié un grand nombre de variants contribuant assez probablement au phénotype et avons découvert quatre SNPs dans l’allèle MC1R du mâle satellite. L’étude IV a consisté à analyser la contribution de ces mutations de MC1R au plumage clair du mâle satellite pendant la reproduction. Nos données montrent que MC1R est activé dans les plumes colorées du mâle satellite en raison d’une plus forte expression de l’allèle indépendant. L’étude de la signalisation de MC1R en culture cellulaire a ensuite révélé que les mutations altèrent les propriétés du récepteur, mais suggère aussi que l’évaluation fonctionnelle du MC1R aviaire en cellules mammaliennes ne rend pas complètement compte du rôle complexe que MC1R joue dans les mélanocytes aviaires

The feather pattern autosomal barring in chicken is strongly associated with segregation at the MC1R locus

Color patterns within individual feathers are common in birds but little is known about the genetic mechanisms causing such patterns. Here, we investigate the genetic basis for autosomal barring in chicken, a horizontal striping pattern on individual feathers. Using an informative backcross, we demonstrate that the MC1R locus is strongly associated with this phenotype. A deletion at SOX10, underlying the dark brown phenotype on its own, affects the manifestation of the barring pattern. The coding variant L133Q in MC1R is the most likely causal mutation for autosomal barring in this pedigree. Furthermore, a genetic screen across six different breeds showing different patterning phenotypes revealed that the most striking shared characteristics among these breeds were that they all carried the MC1R alleles Birchen or brown. Our data suggest that the presence of activating MC1R mutations enhancing pigment synthesis is an important mechanism underlying pigmentation patterns on individual feathers in chicken. We propose that MC1R and its antagonist ASIP play a critical role for determining within-feather pigmentation patterns in birds by acting as activator and inhibitor possibly in a Turing reaction-diffusion model

Towards understanding the molecular basis of pigment pattern formation in birds

Towards understanding the molecular basis of pigment pattern formation in birds. 20. Meeting of the European Society for Pigment Cell Research (ESPCR – 2017

The barred beauty- how a tumour suppressor locus can delight your eyes

The barred beauty- how a tumour suppressor locus can delight your eyes. Avian Model Systems