This study has shown that in order to identify and confidently remove unreliable markers in heterozygote deficient populations, additional techniques are required beyond those currently applied to microsatellite data. The currently used tests such as examination of HWE, heterozygosity, amplification failure, allele frequency distributions and linkage equilibrium are not sufficient to identify reliability in primer sets. This may particularly be the case for invertebrate species, many of which commonly display departures from HWE and heterozygote deficiencies [14, 19]. The results show that the majority of the loci examined (6/7) displayed heterozygote deficiency (Additional file 2) and H. ulvae therefore exhibits similar characteristics to many of the marine and freshwater invertebrates (as detailed in Additional file 1). The technique presented here has shown that after primer redesign, Hulv-06 and Hulv-07 cannot be reliably genotyped due to the discrepancies between each primer set. Therefore it is concluded that these loci, along with designed primers, should not be used in future studies. Similarly, the technique has also provided increased confidence in the remaining loci (Hulv-01, Hulv-03, Hulv-04 and Hulv-05), which all showed similar genotype outputs regardless of the primer set implemented. For this reason, the technique described here presents a useful method for helping to assess the reliability of designed primers in heterozygote-deficient populations.
Previous published studies have suggested many causes for heterozygote deficiency in invertebrates, including selection, inbreeding, mutation and null alleles, SINEs, poor primer design and amplification error [14, 19]. However, attempts to ascertain the exact source of the heterozygote deficiency have often had limited success [21, 24], and while it is not the purpose of this study to identify the source of the heterozygote deficiency, the technique and results obtained can be used to shed light into the cause of the heterozygosity in the species.
The first factor to consider is selection. While some microsatellite loci have been shown to be affected by selection [37, 38], the majority are still considered to be selectively neutral . This study took care not to incorporate tri-nucleotide repeats, which have been shown to occur in coding regions of the genome and thus are more likely to be subject to selection . Therefore, while it cannot be categorically ruled out, selection is not likely to be the cause of the observed deficiencies. Similarly, given what is known about the life stages of the target organism, inbreeding seems unlikely. In species with limited dispersal or direct development, it is simple to imagine inbreeding as a dominant force [41, 42], which can be detrimental to species fitness [43, 44]. However, given that Hydrobia ulvae has a dispersal phase of up to four weeks , a good potential for mixing of progeny by tides and currents, and the likelihood of widespread settlement of larvae within the estuary, it seems highly improbable that inbreeding is occurring. Another alternative potential cause is the Wahlund effect, which is a heterozygote deficiency due to the accidental pooling of discrete sub-populations. Indeed, by disregarding sub-structuring within populations one would expect to see a common deficiency across the majority of the loci as observed in this study [14, 42]. However, it is unlikely that samples taken from a homogeneous area of less than 1 m2, as done here, could contain many different sub-populations . Nonetheless, in order to be certain, additional genotypic analysis would be required on individuals from identified cohorts and known local spatial localities .
Perhaps the most interesting factor to consider with regard to the technique presented here is the presence of null alleles and their potential effect upon levels of heterozygosity. Null alleles represent base-pair mutations in the primer regions which cause primer binding to weaken and/or fail, resulting in a failure to amplify certain alleles . As a result, the presence of null alleles in data sets has been commonly suggested as a contributor to heterozygote deficiency [46, 47] (Additional file 1). While microsatellite regions are often highly polymorphic due to a high rate of mutation through replication slippage and proof-reading events , the flanking regions surrounding microsatellite repeat regions are generally considered to be more conserved. However, given the very high levels of polymorphism shown in microsatellite loci examined for H. ulvae (Additional file 2), it is possible that the sequence flanking the repeat regions may also exhibit increased levels of mutation which would certainly reduce the effectiveness of primer binding and result in an abundance of null alleles. Microsatellite loci in humans have been estimated to have mutation rates of about 10-4 . However, microsatellite mutation is known to vary between different taxa [50, 51], and while little is known specifically about the mutation rate in marine molluscs, several studies, including this current study, have shown high polymorphism in microsatellite loci in marine invertebrates [14, 20, 52], suggesting that mutation rates may be high. For this reason further investigation in marine invertebrates is required (i.e. genetic sequencing) to determine whether mutations in the flanking regions introduce errors into genotype data and consequently influence levels of heterozygosity.
Despite this, there are several reasons to suggest that mutation and null alleles are not the explanation for the overall heterozygote deficiency observed in Hydrobia ulvae. First, given their nature one would typically expect null alleles to occur at a minority of loci and not across the majority of loci as seen in this study [14, 19, 39]. Secondly, results from the double-primer technique show similar heterozygote deficiencies and null allele frequencies in both primer sets A and B in all loci with the exception of Hulv-06 and Hulv-07 (Additional file 4). If nulls were the explanation for the heterozygote deficiency then we would not expect both primer sets to be equally affected.
Mutation and null alleles do however, present one possible explanation for the poor match between primer sets for Hulv-06 and Hulv-07, particularly as the predicted null allele frequencies at these loci were shown by the software MICROCHECKER to be much higher than for all the other loci examined (Additional file 4). Indeed, when coupled with the low amplification rate of individuals shown by Hulv-06_A and Hulv-07_A primers (83.3 and 70.8%, respectively), there is evidence to suggest mutation in the flanking regions and null alleles. Alternatively, the mismatch between primer sets A and B for Hulv-06 and Hulv-07 could be due to similarity in the flanking regions of different loci. In the study by Meglecz et al.  on lepidopterans (butterflies and moths), high flanking sequence similarity was observed at a number of loci, which led to difficulties in designing effective primer sets. Likewise, in a study on the marine gastropod Littorina saxatilis, anomalous large alleles were identified that may signify the presence of flanking similarity in marine invertebrates . While no evidence has been found in the present study for large alleles above 500 bp as described by Sokolov et al. , it is entirely possible that flanking region similarity could introduce errors into genotyped data and thus disrupt the levels of heterozygosity observed. However, while further investigation into the matter is required, the fact that very little sequence similarity was identified in this study suggests that it is not a dominant factor.
While the precise nature of the overall heterozygote deficiency still remains unclear, it is concluded that mutation and null alleles are the likely explanation for the unreliability observed at two of the loci examined (Hulv-06 and Hulv-07). Drawing definitive conclusions on the presence of null alleles, the cause of heterozygote deficiency and primer reliability, has been limited in this study due to constraints on primer redesign imposed by the restricted length of flanking regions. However, when redesigning primers, attempts were made to make B-primers as distinct from A-primers as possible, particularly at the 3' end of the primer, as this region is known to affect product amplification and polymerase extension [34, 36, 35]. Given that the occurrence of restricted flanking regions in microsatellite studies has not been commonly reported, and that information regarding the length of flanking regions in different species is scarce, it is difficult to estimate the likelihood of primer redesign complications in other species; nevertheless, it is believed that the technique presented here provides an important contribution to the field, as it offers a practical alternative to merely assuming primer reliability in heterozygote-deficient populations. Most importantly, this study has highlighted the need for increased caution in assessing primer reliability using only standard assessments, especially in populations commonly exhibiting heterozygote deficiency and deviations from HWE. Moreover, this study has illustrated that additional testing of designed primers in heterozygote deficient populations can identify potentially unreliable markers, amplification errors and genotyping failures, which are commonly associated with microsatellite studies [12, 39]. For these reasons, the methodology presented will be of potential interest/application to future studies on invertebrate and non-invertebrate species alike. Indeed, the technique will be of value in all microsatellite studies that seek increased confidence in genotype assignments. While doubling expenditure on primers may be seen as a disadvantage, the benefits make the technique a sound investment when one considers the high cost of running unreliable primers in large-scale population analysis. The technique will also be of particular use in studies where small primer sets are used due to difficulties in microsatellite isolation. In these cases, it is imperative that all primers consistently amplify a reliable product, given the low number being implemented. Similarly, the genotype data validation provided by this technique will also enable increased confidence in species where multiple banding patterns and anomalous large alleles are often noted .
In addition to the technique proposed, five novel Hydrobia ulvae polymorphic loci (Hulv-01, Hulv-02, Hulv-03, Hulv-04 & Hulv-05) (Additional file 2) are presented for further use in population studies. Given their level of polymorphism, these microsatellites, and the primers described, will provide valuable tools in the study of genetic mixing and population differentiation in Hydrobia ulvae.