Microsatellite Loci Identified by Cross-Species Amplifications in the Globally Vulnerable Relict Gull, Larus relictus

Fifty-two Relict gull muscle samples were taken from the natural dead chicks from 52 different nest sites in Hongjian Nur Nature Reserve in China (38°13’ N - 39°27’N, 109°42’ N - 110°54‘E) in 2016 and were stored in Absolute Ethyl Alcohol. Genomic DNA was isolated from the samples using a standard phenol: chloroform based extraction technique.

We tested the cross-amplification of 90 microsatellite primer pairs developed for other closely species within the Charadriiformes: nine on Saunders’s Gull Larus saundersi (Jiang et al., 2011), 22 on Ivory Gull Pagophila eburnea (Yannic et al., 2011), seven on Red-billed Gull Larus novaehollandiae scopulinus (Given et al., 2002), six on Herring Gull Larus argentatus (Gregory and Quinn, 2005), five on American Herring Gull Larus smithsonianus (Crochet et al., 2003), seven on Black-legged Kittiwake Rissa tridactyla (Verkuil et al., 2009), 31 on Little Terns Sternula albifrons (Noreikiene et al., 2012) and three on Roseate Tern Sterna dougallii (Szczys et al., 2005) for variability in Relict Gull.

PCR amplification was run in 25 μL volume containing 2.5 μL 10×PCR Buffer, 0.5 μL dNTP mix (10 mmol/µL), 2 μL MgCl2 (25 mmol/µL), 0.2 μL Taq polymerase (5U/µL), 0.5 μL of each primer, 17.8 μL H2O and about 50 ng DNA. PCR reactions were carried out in one cycling profile (an initial denaturation of 3 min at 95 °C, followed by 10 cycles of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C, and 20 cycles of 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C, and 6 min of final elongation at 72 °C. PCR products were analyzed by Songon Biotech, Shanghai, China, on ABI PRISM 3730XL Genetic Analyzer for polymorphism testing with GeneMapper v3.7 for the size range reading.

We tested for deviations from linkage disequilibrium (LD), and Hardy–Weinberg equilibrium (HWE) by Markov chain method using the following parameters: dememorization = 10000, number of batches = 20, and number of iterations/batch = 5000 in Genepop 4.3. Bonferroni correction (α=0.05) was applied to the two above tests. Null alleles were checked in Micro-Checker 2.2.3. The observed and expected heterozygosity, and polymorphic information content were calculated with Cervus 3.0.

Out of a total of 90 microsatellites combination tested, 56 (62%) positive amplifications, of which 11 loci were polymorphic. None of the markers was observed departing from HWE and no null alleles were found in the 11 loci. The polymorphism loci had 2-10 alleles per locus and with PIC ranging from 0.373 to 0.775 (Table I). The observed heterozygosities ranged from 0.481 to 0.827.

The genetic variability estimated by microsatellite markers significantly outnumbered the genetic variability assessed from the two mtDNA and one nuclear gene that has been previously used (Yang et al., 2015). However, this particular set of microsatellite loci are still relatively invariant compared to many microsatellite loci examined in other species (e.g. Jiang et al., 2011; Yannic et al., 2011) and possibly at other loci in this species. One possibility is that the populations are small with relatively low level of gene flow. As revealed by mitochondrial and nuclear sequence data, the sampled population indeed appears to have low variation compared with that in closely related gull (Yang et al., 2015). And that only about 20% (11 out of 56) of tested primers were variable in our study is also consistent with this explanation.

Significant LD was found only between Locus RBG29 and LARZAP11. LD creates pseudo-replication for analysis where locus is assumed to be independent. In this scenario, one of the linked loci should be excluded to avoid increased Type I error (Selkoe and Toonen, 2006). However, for many ecological questions, application of linked loci could be beneficial. For example, LD can help understanding the patterns of gene exchange and history of changes in population size (e.g. Tishkoff et al., 1996). Therefore, these microsatellite markers could be useful for inferring population patterns and processes, and analyzing kinship among individuals in Relict Gull.

Table I.- Characteristics of eleven microsatellite loci in Relict gull. Initial species, number of alleles (Na), size range, observed (Ho) and expected (He) heterozygosities, polymorphic information content (PIC) and Hardy-Weinberg exact test (P-val) are shown.

1All of the 11 loci were successfully amplified in the 52 samples.

In conclusion, 11 polymorphic microsatellite loci in Relict gull were identified by cross-species amplifications. The characteristics of these loci provide useful information for further studies on genetic diversity, population structure, conservation status as well as kinship among individuals in this species.

This work was funded by the National Key Research and Development Program of China (No. 2016YFC0500400) and the National Nature Science Foundation of China (No. D41371106). We thank Qiang Wang, Qingxiong Wang, Yong Lyu and Shuanzhu Gong for the assistance of field work, and Mingju E for the help with laboratory work.

Authors have declared no conflict of interest.