A method for genotype validation and primer assessment in heterozygote-deficient species, as demonstrated in the prosobranch mollusc Hydrobia ulvae

BACKGROUND: In studies where microsatellite markers are employed, it is essential that the primers designed will reliably and consistently amplify target loci. In populations conforming to Hardy-Weinberg equilibrium (HWE), screening for unreliable markers often relies on the identification of heterozygote deficiencies and subsequent departures from HWE. However, since many populations naturally deviate from HWE, such as many marine invertebrates, it can be difficult to distinguish heterozygote deficiencies resulting from unreliable markers from natural processes. Thus, studies of populations that are suspected to deviate from HWE naturally would benefit from a method to validate genotype data-sets and test the reliability of the designed primers. Levels of heterozygosity are reported for the prosobranch mollusc Hydrobia ulvae (Pennant) together with a method of genotype validation and primer assessment that utilises two primer sets for each locus. Microsatellite loci presented are the first described for the species Hydrobia ulvae; the five loci presented will be of value in further study of populations of H. ulvae. RESULTS: We have developed a novel method of testing primer reliability in naturally heterozygote deficient populations. After the design of an initial primer set, genotyping in 48 Hydrobia ulvae specimens using a single primer set (Primer set_A) revealed heterozygote deficiency in six of the seven loci examined. Redesign of six of the primer pairs (Primer set_B), re-genotyping of the successful individuals from Primer set_A using Primer set_B, and comparison of genotypes between the two primer sets, enabled the identification of two loci (Hulv-06 & Hulv-07) that showed a high degree of discrepancy between primer sets A and B (0% & only 25% alleles matching, respectively), suggesting unreliability in these primers. The discrepancies included changes from heterozygotes to homozygotes or vice versa, and some individuals who also displayed new alleles of unexpected sizes. Of the other four loci examined (Hulv-01, Hulv-03, Hulv-04, & Hulv-05), all showed more than 95% agreement between primer sets. Hulv-01, Hulv-03, & Hulv-05 displayed similar levels of heterozygosity with both primer sets suggesting that these loci are indeed heterozygote deficient, while Hulv-08 showed no deficiency in either primer set. CONCLUSION: The simple method described to identify unreliable markers will prove a useful technique for many population studies, and also emphasises the dangers in using a single primer set and assuming marker reliability in populations shown to naturally deviate from HWE

A new marker based on the avian spindlin gene that is able to sex most birds, including species problematic to sex with CHD markers

We have developed a new marker (Z43B) that can be successfully used to identify the sex of most birds (69%), including species difficult or impossible to sex with other markers. We utilized the zebra finch Taeniopygia guttata EST microsatellite sequence (CK309496) which displays sequence homology to the 5′ untranslated region (UTR) of the avian spindlin gene. This gene is known to be present on the Z and W chromosomes. To maximize cross-species utility, the primer set was designed from a consensus sequence created from homologs of CK309496 that were isolated from multiple distantly related species. Both the forward and reverse primer sequences were 100% identical to 14 avian species, including the Z chromosome of eight species and the chicken Gallus gallus W chromosome, as well as the saltwater crocodile Crocodylus porosus. The Z43B primer set was assessed by genotyping individuals of known sex belonging to 61 non-ratite species and a single ratite. The Z and W amplicons differed in size making it possible to distinguish between males (ZZ) and females (ZW) for the majority (69%) of non-ratite species tested, comprising 10 orders of birds. We predict that this marker will be useful for obtaining sex-typing data for ca 6,869 species of birds (69% of non-ratites but not galliforms). A wide range of species could be sex-typed including passerines, shorebirds, eagles, falcons, bee-eaters, cranes, shags, parrots, penguins, ducks, and a ratite species, the brown kiwi, Apteryx australis. Those species sexed include species impossible or problematic to sex-type with other markers (magpie, albatross, petrel, eagle, falcon, crane, and penguin species)

Conserved microsatellite markers of high cross-species utility for flying, ground and tree squirrels

Many squirrel species around the world are threatened by forest loss and fragmentation. To facilitate studies of squirrel biodiversity, particularly of flying squirrels in Southeast Asia, we identified Hylopetes, Menetes, Glaucomys and Sciurus squirrel microsatellite sequences with homologs in a second squirrel species (Spermophilus tridecemlineatus), designed 40 consensus markers and tested three squirrel species. When tested in four individuals per species, 26 markers were variable in Hylopetes phayrei, 25 markers in H. lepidus and 25 markers in Menetes berdmorei. Eleven markers were selected from 14 that were polymorphic in all three species. Cross-species utility was confirmed for these 11 markers in seven additional squirrel species, including: the flying squirrels H. phayrei, H. lepidus, H. spadiceus and Petaurista petaurista; a ground squirrel, M. berdmorei; and the tree squirrels, Callosciurus caniceps and C. finlaysoni. The other markers that were variable in one or multiple species are also useful for those specific species

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

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

Identification of 24 new microsatellite loci in the sweat bee Lasioglossum malachurum (Hymenoptera: Halictidae)

OBJECTIVE: The objective here is to identify highly polymorphic microsatellite loci for the Palaearctic sweat bee Lasioglossum malachurum. Sweat bees (Hymenoptera: Halictidae) are widespread pollinators that exhibit an unusually large range of social behaviours from non-social, where each female nests alone, to eusocial, where a single queen reproduces while the other members of the colony help to rear her offspring. They thus represent excellent models for understanding social evolution. RESULTS: 24 new microsatellite loci were successfully optimized. When amplified across 23-40 unrelated females, the number of alleles per locus ranged from 3 to 17 and the observed heterozygosities 0.45 to 0.95. Only one locus showed evidence of significant deviation from Hardy-Weinberg equilibrium. No evidence of linkage disequilibrium was found. These 24 loci will enable researchers to gain greater understanding of colony relationships within this species, an important model for the study of eusociality. Furthermore, 22 of the same loci were also successfully amplified in L. calceatum, suggesting that these loci may be useful for investigating the ecology and evolution of sweat bees in general

A multiplex marker set for microsatellite typing and sexing of sooty terns Onychoprion fuscatus

OBJECTIVES: Seabirds have suffered dramatic population declines in recent decades with one such species being the sooty tern Onychoprion fuscatus. An urgent call to re-assess their conservation status has been made given that some populations, such as the one on Ascension Island, South Atlantic, have declined by over 80% in three generations. Little is known about their population genetics, which would aid conservation management through understanding ecological processes and vulnerability to environmental change. We developed a multiplex microsatellite marker set for sooty terns including sex-typing markers to assist population genetics studies. RESULTS: Fifty microsatellite loci were isolated and tested in 23 individuals from Ascension Island. Thirty-one were polymorphic and displayed between 4 and 20 alleles. Three loci were Z-linked and two autosomal loci deviated from Hardy-Weinberg equilibrium. The remaining 26 autosomal loci together with three sex-typing makers were optimised in seven polymerase chain reaction plexes. These 26 highly polymorphic markers will be useful for understanding genetic structure of the Ascension Island population and the species as a whole. Combining these with recently developed microsatellite markers isolated from Indian Ocean birds will allow for assessment of global population structure and genetic diversity

The characterisation of microsatellite markers reveals tetraploidy in the Greater Water Parsnip, Sium latifolium (Apiaceae).

BACKGROUND: The Greater Water Parsnip, Sium latifolium (Apiaceae), is a marginal aquatic perennial currently endangered in England and consequently the focus of a number of conservation translocation projects. Microsatellite markers were developed for S. latifolium to facilitate comparison of genetic diversity and composition between natural and introduced populations. RESULTS: We selected 65 S. latifolium microsatellite (MiSeq) sequences and designed primer pairs for these. Primer sets were tested in 32 individuals. We found 15 polymorphic loci that amplified consistently. For the selected 15 loci, the number of alleles per locus ranged from 8 to 17. For all loci, S. latifolium individuals displayed up to four alleles indicating polyploidy in this species. CONCLUSIONS: These are the first microsatellite loci developed for S. latifolium and each individual displayed 1-4 alleles per locus, suggesting polyploidy in this species. These markers provide a valuable resource in evaluating the population genetic composition of this endangered species and thus will be useful for guiding conservation and future translocations of the species

Widespread gene flow between oceans in a pelagic seabird species complex

Global-scale gene flow is an important concern in conservation biology as it has the potential to either increase or decrease genetic diversity in species and populations. Although many studies focus on the gene flow between different populations of a single species, the potential for gene flow and introgression between species is understudied, particularly in seabirds. The only well studied example of a mixed-species, hybridising population of petrels exists on Round Island, in the Indian Ocean. Previous research assumed that Round Island represents a point of secondary contact between Atlantic (Pterodroma arminjoniana) and Pacific species (P. neglecta and P. heraldica). This study uses microsatellite genotyping and tracking data to address the possibility of between-species hybridisation occurring outside the Indian Ocean. Dispersal and gene flow spanning three oceans was demonstrated between the species in this complex. Analysis of migration rates estimated using BAYESASS revealed unidirectional movement of petrels from the Atlantic and Pacific into the Indian Ocean. Conversely, STRUCTURE analysis revealed gene-flow between species of the Atlantic and Pacific Oceans, with potential three-way hybrids occurring outside the Indian Ocean. Additionally, geolocation tracking of Round Island petrels revealed two individuals travelling to the Atlantic and Pacific. These results suggest that inter-specific hybrids in Pterodroma petrels are more common than was previously assumed. This study is the first of its kind to investigate gene flow between populations of closely related Procellariform species on a global scale, demonstrating the need for consideration of widespread migration and hybridisation in the conservation of threatened seabirds