Final Report Summary - SLMHC (Sheltered load at the MHC)
The aim of this project was to characterise the deleterious mutations (known as 'sheltered load') that are linked to the immune genes of the major histocompatibility complex (MHC). As the evolutionary genetic model we study a small freshwater fish, the guppy Poecilia reticulata. The Marie Curie fellow has interrupted her Marie Curie contract because she has been offered a permanent position. Her stay at the University of Hull has thus lasted less than 9 months instead of 24 months as initially planned. However, the first steps of the project have been realised and will provide interesting results. The fellow plans to carry on the project in her new institution (CNRS, National Museum of Natural History, Paris, France) in collaboration with Dr van Oosterhout and Dr Mark McMullan, a postdoc in the team of Dr van Oosterhout, now located in the University of East Anglia (United Kingdom).
The main findings during nine months of research are:
(1) We built a bacterial artificial chromosome (BAC) library from 200 flash-frozen guppies to make sure we will have enough deoxyribonucleic acid (DNA). We screened this BAC library using MHC class IIB primers and detect four positive clones containing the gene of interest (class II MHC).
(2) We developed a set of new primers and detect 10 positive clones for MHC class I.
(3) We have sequenced these clones and are currently performing the bioinformatics to analyse the nucleotide variation at the National Museum of Natural History, Paris, France.
(4) We also analysed the sheltered load at the MHC by performing a study using Sanger sequencing of MHC class IIB genes. We thus investigate circa 1000 base pairs (bp) of MHC class II DAB genes in four wild populations of the guppy Poecilia reticulate. We detected a group of 27 sequences that are virtually identical (2.3 ± 0.6 bp mean pairwise difference) and show no signal of selection (dN / dS = 0.06).
(5) We detected an average of 2.96 (± 1.29 S.D.) sequences per guppy and show that some individuals carry more than two DAB genes.
(6) We found evidence of several gene conversion events which may have homogenised sequence variation and reduced the visible copy number variation (CNV). Indeed, we would have missed 40.9 % of the haplotype variation if we had only sequenced exon 2, but which was revealed by distinct polymorphisms in the intron. We argue that gene conversion could result in a systematic underestimation of the CNV in studies of the MHC and other multigene families.
(7) We screened microsatellite DNA to reconstruct the populations' demography and employ computer simulations to show that balancing selection can maintain the observed number of sequences in a population with CNV ranging from one to three DAB loci. We found that parasite-mediated selection alone cannot explain the presence of so many apparently functionally-equivalent sequences.
(8) We also examined and rejected other hypotheses that might explain this observation, including asymmetric overdominance, gene conversion, neofunctionalisation and the duplication-degeneration-complementation (DDC) models.
(9) We proposed that balancing selection operates outside the PBR, for example on linked immune genes or on the 'sheltered load' that is thought to be associated to the MHC (cf. ABC evolution).
The main findings during nine months of research are:
(1) We built a bacterial artificial chromosome (BAC) library from 200 flash-frozen guppies to make sure we will have enough deoxyribonucleic acid (DNA). We screened this BAC library using MHC class IIB primers and detect four positive clones containing the gene of interest (class II MHC).
(2) We developed a set of new primers and detect 10 positive clones for MHC class I.
(3) We have sequenced these clones and are currently performing the bioinformatics to analyse the nucleotide variation at the National Museum of Natural History, Paris, France.
(4) We also analysed the sheltered load at the MHC by performing a study using Sanger sequencing of MHC class IIB genes. We thus investigate circa 1000 base pairs (bp) of MHC class II DAB genes in four wild populations of the guppy Poecilia reticulate. We detected a group of 27 sequences that are virtually identical (2.3 ± 0.6 bp mean pairwise difference) and show no signal of selection (dN / dS = 0.06).
(5) We detected an average of 2.96 (± 1.29 S.D.) sequences per guppy and show that some individuals carry more than two DAB genes.
(6) We found evidence of several gene conversion events which may have homogenised sequence variation and reduced the visible copy number variation (CNV). Indeed, we would have missed 40.9 % of the haplotype variation if we had only sequenced exon 2, but which was revealed by distinct polymorphisms in the intron. We argue that gene conversion could result in a systematic underestimation of the CNV in studies of the MHC and other multigene families.
(7) We screened microsatellite DNA to reconstruct the populations' demography and employ computer simulations to show that balancing selection can maintain the observed number of sequences in a population with CNV ranging from one to three DAB loci. We found that parasite-mediated selection alone cannot explain the presence of so many apparently functionally-equivalent sequences.
(8) We also examined and rejected other hypotheses that might explain this observation, including asymmetric overdominance, gene conversion, neofunctionalisation and the duplication-degeneration-complementation (DDC) models.
(9) We proposed that balancing selection operates outside the PBR, for example on linked immune genes or on the 'sheltered load' that is thought to be associated to the MHC (cf. ABC evolution).