Autosomal and Z-linked microsatellite markers enhanced for cross-species utility and assessed in a range of birds, including species of conservation concern

Abstract

Microsatellite markers were designed to be of utility for genotyping multiple species of birds, including those of conservation concern, hence saving resources and enabling species/genome comparisons. We used the proven approach of Dawson et al. (Mol Ecol Resour 10:475–494, 2010) and assessed markers in multiple species, including nine species of conservation interest. We ensured both primer sequences matched multiple species (13 loci) or designed primer sets from expressed sequence tags (2 loci). Eleven primer sets were 100 % identical to the zebra finch (Taeniopygia guttata) and a second passerine species and/or the chicken (Gallus gallus). All 15 loci were polymorphic when assessed in a non-source species (Gouldian finch, Erythrura gouldiae) suggesting utility in multiple species. Four of the five Z-linked loci were assessed in at least nine additional species each (including ratites). All were variable in multiple species, demonstrating cross-species utility and potential for identifying Z chromosome rearrangements.

Introduction

In order to create enhanced microsatellite markers of high cross-species utility we followed the approach of Dawson et al. (2010). We designed primer sets from alignments of multiple species/expressed sequence tags and characterised these in non-source species. Those markers identified as Z-linked based on sequence homology to the zebra finch and genotyping were tested in additional bird species. Z markers with wide cross-species amplification are particularly useful because species-specific allele sizes allow the identification of the parental species of a hybrid individual (e.g. Lifjeld et al. 2010), whereas the more variable Z markers allow the study of cross-species chromosomal rearrangements (e.g. Backström et al. 2006).

Methods

We followed the approach of Dawson et al. (2010) to enhance the markers for high cross-species utility. We created consensus zebra finch–passerine, zebra finch–chicken or zebra finch–passerine–chicken sequences from homologous microsatellite sequences using MEGA3 (for details see Supplementary File 1). Primer sets were designed from these multi-species consensus sequences using PRIMER3 v0.4.0 using a maximum of one degenerate base per primer set.

All markers were assessed in a non-source species, the near-threatened Gouldian finch (Erythrura gouldiae). Since this species was not used in the design of the markers, successful amplification and polymorphism would suggest the marker will be of utility in many species. We tested known sexes to diagnose if the markers were Z-linked or autosomal (Table 1; up to 20 female and 20 male; sexed using the marker Z-002A, Dawson 2007). Four of the Z markers were genotyped in 10–23 additional species (including nine species of conservation concern) and the saltwater crocodile Crocodylus porosus (Table 2; 1–40 individuals per species).

Table 1 Fifteen microsatellite markers of enhanced cross-species utility and characterised in estrildid finches

Table 2 Four Z-linked bird markers enhanced for cross-species utility and assessed in a wide range of bird species (including nine of conservation concern) and the saltwater crocodile (Crocodylus porosus)

Genomic DNA was extracted from blood using an ammonium acetate protocol. Each fluorescent PCR contained approximately 10 ng genomic DNA, in 2-µl volumes using QIAGEN Multiplex PCR Master Mix or 10-µl volumes with 2.5 mM MgCl₂ and BIOLINE Taq DNA polymerase and buffer. PCR amplification was performed using a DNA Engine Tetrad thermal cycler. PCR amplification conditions were 94 °C for 15 min (QIAGEN) or 3 min (Bioline); then 35 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s; followed by one cycle of 72 °C for 10 min. PCR products were loaded on a 48-capillary ABI 3730 DNA Analyzer and genotypes assigned using GeneMapper software (Applied Biosystems). Heterozygosities were calculated using cervus and deviation from Hardy–Weinberg equilibrium (HWE) calculated using genepop. Z-linked markers were assessed for HWE when typed in a minimum of 10 individuals and using males only.

Results

Eleven primer sets were 100 % identical to zebra finch and a second bird species and two sets mismatched at 1–4 bases (ApCo104-ZFC and Ase12-ZFS; Supplementary File 1). Two other sets were designed from zebra finch Expressed Sequence Tags (ESTs) (ZEST09-005 and ZEST09-018; Table 1).

Based on their high multispecies sequence homology and proven utility in multiple non-source species (Table 1, 2), these markers are expected to be of utility for studying many species, including those of conservation concern, especially passerines (see Dawson et al. 2010). Only one locus (Ase12) deviated from HWE, possibly due to null alleles (Table 1). A selection of these markers are being used in parentage studies for multiple species (DAD unpublished data).

For the five loci homologous to the zebra finch Z chromosome, females were always homozygous (hemizygous), confirming their Z-linked nature in other species, and suggesting no W-linked homologues amplified (Table 1). Four of the Z-linked sequences were highly conserved between genetically distant species (zebra finch-chicken; Z-013, Z-037, Z-040 and Ase50) and, as expected, amplified across a wide range of species, including ratites and saltwater crocodile (81–100 %; Table 2, Supplementary Table 1). These were variable in multiple species demonstrating potential for cross-utility and identifying chromosomal rearrangements between species. Allele sizes in some orders were invariant within a species, yet varied in size between species, suggesting potential for identifying species and hybrids (Table 2).