Effects of Population Bottlenecks on Immunity Genes in
NZ's Threatened Birds.
Allan Wilson Centre Summer Studentship 2012-2013
Student: Chris Harris
Supervised by: Professor Ian Jamieson, Dr Catherine Grueber and Dr Tania King
Introduction
Genetic diversity among individuals of a population has fundamental implications for conservation
efforts. Decreased genetic diversity within a population leads to inbreeding which often leads to a
decrease in fitness. If genetic variability is low then it is likely that few or none of the individuals
have the characteristics to cope with changes in the environment. Such a population could be
suddenly wiped out, potentially threatening an entire ecosystem. Such a disaster could be averted by
human intervention, for example by maximising out-breeding. To this end, it is first necessary to
determine the amount and nature of genetic diversity present. This project aims to determine the
genetic diversity in immunity genes of various endangered New Zealand native birds.
The Toll-like receptors (TLRs) are membrane bound proteins of the innate immune system (Iwasaki
and Medzhitov, 2010). They detect distinctive features of invasive pathogenic microbes and are
essential for activating the adaptive immune system. The TLRs are encoded by a multi-gene family
comprised of 10 – 15 genes with large differences between and within vertebrate groups. However,
they are relatively highly conserved within species due to their crucial functional role (Barreiro et al.
2009). The genetic variation at TLR genes has been directly related to a variety of pathogenic
responses in vertebrates (Alcaide and Edwards, 2011). Comparing the variability of these TLR
genes with neutral genetic diversity (e.g. microsatellites) in native New Zealand birds will provide a
significant insight into the viability of these populations.
My contribution to the project was to amplify and sequence TLR genes TLR1LA, TLR1LB, TLR3,
TLR4, TLR5, TLR7 and TLR21 across 24 unrelated Takahe individuals and TLR1LB, TLR5 and
TLR15 across at least 18 unrelated Kokako individuals. Each of these genes plays a special role in
the avian immune system. TLR1LA and TLR1LB bind to lipoproteins in the cell wall of bacteria,
fungi and parasites (Uematsu and Akira, 2008). TLR4 targets gram-negative bacteria (Hoshino et al.,
1
1999). TLR5 detects flagellated bacteria. TLR3 and TLR7 detect viral RNA. There are indications
of an association between TLR21 and detection of microbial DNA (Brownlie et al., 2009) and
TLR15 and components of both gram-negative and gram-positive bacteria (Nerren et al., 2009).
Methods
The extra-cellular domains of the TLR genes were amplified using polymerase chain reaction
(PCR) primers designed by Alcaide and Edwards (2011) and CEG unpublished primers (see tables
1 and 2). Once the sequences underwent PCR, they were ran in a 1% agarose 1 X TAE gel stained
with SYBR safe DNA gel stain (Invitrogen) at approximately 100 volts for 10 minutes. The size of
the TLR product ranged from 800 to 1200 base pairs. If the desired bands in the gel were
considered too weak, the number of cycles in the PCR was increased from 35 to 40 cycles and/or
the volume of the PCR was increased from 15 μl to 30 μl. Sometimes gels were combined to
increase the DNA yield.
Following PCR and gel electrophoresis, the bands were excised and purified by using the Dneasy
Ultra-Sep gel purification kit (Omega Bio-Trek) to obtain a 50 μl eluted product. Purified PCR
products were then concentrated to a volume of approximately 8 μl using the Eppendorf
concentrator (at 40 °C for 20 minutes). Purified and concentrated PCR products were sequenced
first in the forward direction and then in the backward direction if a clearer sequence was required.
The PCR products were sent to Macrogen for sequencing. If sequence quality was low then the
PCR product was sequenced by an ABI 3730xl Genetic Analyser (Genetic Analysis Services at
Otago). Correct amplification was confirmed by BLAST search.
Sequences were then edited, aligned and analysed using Sequencher v5.0 (Gene Codes Corporation).
Results
PCR products for at least 18 individuals for 8 TLR loci in Takahe and Kokako were successfully
sequenced. In Takahe this revealed one amino acid change in TLR4. The variability among
individuals was therefore very low in Takahe.
Conclusions
Due to the extremely low genetic variability of TLRs in the Takahe, this population is at severe risk
of being decimated by disease. This should be taken into account in any conservation effort for
Takahe. Further study is ongoing for Kokako.
2
Acknowledgements
I would like to thank Professor Ian Jamieson, Dr Catherine Grueber and Dr Tania King for their
time, professionalism, wisdom and confidence in me during the project. I learned a lot in the
laboratory and I am very thankful for this. Thanks go to the Allan Wilson Centre for providing the
funding.
TABLE 1
LOCI
TAKAHE PRIMERS, forward (F) and reverse (R)
TAKAHE PCR annealing temperature (°C)
TLR1LA
F = av TLR1LAF, R = avTLR1LAR
58
TLR1LB
F = avTLR1LBF, R = avTLR1LBR
56
TLR3
F = avTLR3F, R = avTLR1LBR
55
TLR4
F = avTLR4F, R = AplTLR4R
58
TLR5
F = OleTLR5F, R = PpoTLR5R
56
TLR7
F = avTLR7F, R = avTLR7R
54
TLR21
F = PpoTLR21F, R = PpoTLR21R
57
TABLE 2
KOKAKO PCR annealing temperature
(°C)
LOCI
KOKAKO PRIMERS, forward (F) and reverse (R)
TLR1LB
F = PcaTLR1LBF, R = PcaTLR1LBR
56
TLR5
F = avTLR5F, R = avTLR5R
50
TLR15
F = finchTLR15F, R = avTLR15R
50
References
Alcaide, M. and S. V. Edwards (2011). "Molecular Evolution of the Toll-Like Receptor Multigene
Family in Birds." Molecular Biology and Evolution 28(5): 1703-1715
Barreiro LB, Ben-Ali M, Quach H, et al. 18 co-authors (2009). Evolutionary dynamics of human
Toll-like receptors and their different contributions to host defense. PLoS Genet. 5:e1000562.
Brownlie R, Zhu J, Allan B, Mutwiri GK, Babiuk LA, Potter A, Griebel P (2009). Chicken TLR21
acts as a functional homologue to mammalian TLR9 in the recognition of CpG
oligodeoxynucleotides. Mol Immunol. 46:3163–3170.
3
Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K., and Akira, S.
(1999). TLR4-deficient mice are hyporesponsive to LPS: evidence for TLR4 as the Lps gene
product. J. Immunol. 162, 3749–3752.
Iwasaki A, Medzhitov R (2010). Regulation of adaptive immunity by the innate immune system.
Science 327:291–295.
Nerren JR, He H, Genovese KJ, Kogut MH (2009). mRNA expression of the avian-specific toll-like
receptor 15 is induced by multiple species of bacteria. J Immunol. 182:135.58.
Uematsu S, Akira S (2008). Toll-like receptors (TLRs) and their ligands. Handb Exp Pharmacol.
1:1–20.
4