Evaluation of two approaches to genotyping major histocompatibility complex class I in a passerine-CE-SSCP and 454 pyrosequencing.
Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE-SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE-SSCP.
Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Květná 8, 60365 Brno, Czech Republic. promerova@seznam.cz
Marta
M
Promerová
Wiesław
W
Babik
Josef
J
Bryja
Tomáš
T
Albrecht
Michał
M
Stuglik
Jacek
J
Radwan
D000818:Animals; D030161:Avian Proteins / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005802:Genes, MHC Class II; D005838:Genotype; D008969:Molecular Sequence Data; D046109:Passeriformes / Q000145:classification / Q000235:genetics*; D010802:Phylogeny; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA / Q000379:methods*
D030161:Avian Proteins