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Journal of Molecular Evolution

, Volume 77, Issue 1–2, pp 8–21 | Cite as

Characteristics of MHC Class I Genes in House Sparrows Passer domesticus as Revealed by Long cDNA Transcripts and Amplicon Sequencing

  • Maria Karlsson
  • Helena WesterdahlEmail author
Original Article

Abstract

In birds the major histocompatibility complex (MHC) organization differs both among and within orders; chickens Gallus gallus of the order Galliformes have a simple arrangement, while many songbirds of the order Passeriformes have a more complex arrangement with larger numbers of MHC class I and II genes. Chicken MHC genes are found at two independent loci, classical MHC-B and non-classical MHC-Y, whereas non-classical MHC genes are yet to be verified in passerines. Here we characterize MHC class I transcripts (α1 to α3 domain) and perform amplicon sequencing using a next-generation sequencing technique on exon 3 from house sparrow Passer domesticus (a passerine) families. Then we use phylogenetic, selection, and segregation analyses to gain a better understanding of the MHC class I organization. Trees based on the α1 and α2 domain revealed a distinct cluster with short terminal branches for transcripts with a 6-bp deletion. Interestingly, this cluster was not seen in the tree based on the α3 domain. 21 exon 3 sequences were verified in a single individual and the average numbers within an individual were nine and five for sequences with and without a 6-bp deletion, respectively. All individuals had exon 3 sequences with and without a 6-bp deletion. The sequences with a 6-bp deletion have many characteristics in common with non-classical MHC, e.g., highly conserved amino acid positions were substituted compared with the other alleles, low nucleotide diversity and just a single site was subject to positive selection. However, these alleles also have characteristics that suggest they could be classical, e.g., complete linkage and absence of a distinct cluster in a tree based on the α3 domain. Thus, we cannot determine for certain whether or not the alleles with a 6-bp deletion are non-classical based on our present data. Further analyses on segregation patterns of these alleles in combination with dating the 6-bp deletion through MHC characterization across the genus Passer may solve this matter in the future.

Keywords

MHC class I House sparrow Amplicon 454-sequencing cDNA Linkage 

Notes

Acknowledgments

We would like to thank Terry Burke for letting us use DNA from the sparrows on Lundy and the Landmark trust and the Lundy Company for letting us work on Lundy. We are grateful to all the staff on Lundy especially Kevin Welsh, Patricia, and Roger Fursdon. Thanks to Ian Cleasby for assistance in the field. We are grateful to Mimi Lannefors and Anna Drews for help in the laboratory and to Emily O’Connor, Kristin Scherman, and Maria Strandh for valuable comments on earlier versions of the manuscript. The project was funded by the Swedish Research Council, VR (621-2206-2876) to Helena Westerdahl and Stiftelsen Lunds Djurskyddsfond to Maria Karlsson.

Supplementary material

239_2013_9575_MOESM1_ESM.pdf (22 kb)
Supplementary Fig. 1. Alignment of house sparrow MHC-I amino acid sequences (Pado-UA), covering the α2 region, retrieved from 454-amplicon sequencing, species specific nomenclature and GenBank accession numbers are used for all sequences (Pado-UA*200-260 (novel from the present study), Pado-UA*324 JN609623, Pado-UA*326 JN609626, Pado-UA*312 JN609636, Pado-UA*309 JN609635, Pado-UA*322 JN609643, Pado-UA*317 JN609640 (found previously and also in the present study) in comparison with the house sparrow transcripts (Pado-UA*230-233, *240-243, this study), great reed warbler (Acar cN3, AJ005503; cN15, AJ005505) and chicken (MHC-B, Gaga, HQ141386; MHC-Y NM_001030675). Amino acid sequences are numbered according to full-length chicken MHC-I. Pado-UA*240-242 have no deletions, Pado-UA*243 has a 3 bp deletion (site 149) and Pado-UA*230-233 have 6 bp deletions (site 246-247). Identity with sequence Pado-UA*240 is indicated with dots, codons corresponding to the PBR according to human HLA (Björkman et al. 1987) with (P); sites subject to negative selection (N) and to positive selection (+) according to HyPhy. HyPhy was run for 38 454-sequences with a 6 bp deletion (with 6 bp deletion), for 20 454-sequences without a 6 bp deletion (without 6 bp deletion) and for the 58 sequences combined (all 454-sequences) (PDF 21 kb)
239_2013_9575_MOESM2_ESM.pdf (18 kb)
Supplementary Fig. 2. Phylogenetic reconstruction of house sparrow (Pado-UA*230-233 and Pado-UA*240-243) and great reed warbler (Acar cN3, AJ005503; cN15, AJ005505) exon 2, 3 and 4 MHC-I nucleotide sequences, with chicken sequences (MHC-B, Gaga, HQ141386; MHC-Y, NM_001030675) as outgroups, using Neighbor-joining (Jukes-Cantor, bootstrap (bt) values based on 1000 replicates). (a) Phylogenetic reconstruction based on nucleotides in exon 2, (b) phylogenetic reconstruction based on nucleotides in exon 3, (c) phylogenetic reconstruction based on nucleotides in exon 4, (d) phylogenetic reconstruction based on synonymous substitutions in exon 2, (e) phylogenetic reconstruction based on synonymous substitutions in exon 3, (f) phylogenetic reconstruction based on synonymous substitutions in exon 4, (g) phylogenetic reconstruction based on nonsynonymous substitutions in exon 2, (h) phylogenetic reconstruction based on nonsynonymous substitutions in exon 3 and (i) phylogenetic reconstruction based on nonsynonymous substitutions in exon 4. The house sparrow sequences with a 6 bp deletion form a significantly supported monophyletic cluster in all trees based on exons 2 and 3 (a bt = 99,b bt = 100,d bt = 77,e bt = 82,g bt = 99,and h bt = 99), but not in trees based on exon 4 (c, f, i) (PDF 18 kb)
239_2013_9575_MOESM3_ESM.jpg (247 kb)
Supplementary Fig. 3. Inheritance patterns of house sparrow exon 3 sequences (in this paragraph called alleles, with (‘short’) and without (‘long’) the 6bp deletion) in three house sparrow families with 2-4 chicks. Alleles are retrieved from 454-amplicon sequencing, species specific nomenclature and GenBank accession numbers are used for all sequences (Pado-UA*200-260 (novel from the present study), Pado-UA*324 JN609623, Pado-UA*326 JN609626, Pado-UA*312 JN609636, Pado-UA*309 JN609635, Pado-UA*322 JN609643, Pado-UA*317 JN609640 (found previously and also in the present study). Male alleles are indicated with blue, and the two male haplotypes are indicated light and dark blue. Female alleles are indicated with red and the two female haplotypes are pink and red. When alleles are found in both the male and female in-pair haplotypes cannot be inferred and these alleles are non-informative in considering inheritance (indicated green). In clutch 3 the female has three ‘short’ alleles, one allele is red and is inherited in all chicks except in Chick2, Chick2 inherits two pink alleles. This female furthermore has three ‘long’ alleles, one is red and is inherited in all four chicks except in Chick2, Chick2 again inherits two pink alleles. In clutch 3 the male has eight ‘short’ alleles, four are dark blue and they are inherited in all four chicks. This male has six ‘long’ alleles, three are dark blue and are inherited in all four chicks. This male also has four ‘short’ light blue alleles and three ‘long’ light blue alleles, these alleles are not found in any of the four chicks. We find the same pattern of inheritance independent of whether we study inheritance of ‘long’ or ‘short’ alleles, this finding indicates that the ‘long’ and ‘short’ alleles are linked, hence found in the same chromosomal region. Three individuals with 454-read numbers lower than 120 for the ‘short’ alleles were added in this analysis (number of reads from amplification with the ‘short’ primer combination; 64 in Chick2 Clutch3, 65 inChick2 Clutch2 and 92 in the male in Clutch1) (JPG 248 kb)
239_2013_9575_MOESM4_ESM.docx (14 kb)
Supplementary Table 1. Verified MHC-I exon 2-4 cDNA sequences, covering the α1, α2 and α3 regions, in individual 1 and 2 (Ind1 and Ind2), respectively (with a 6 bp deletion, Pado-UA*230-233; without a 6 bp deletion, Pado-UA*240-343) (DOCX 14 kb)

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© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of BiologyLund UniversityLundSweden

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