Watch Out for a Second SNP: Focus on Multi-Nucleotide Variants in Coding Regions and Rescued Stop-Gained

Most single-nucleotide polymorphisms (SNPs) are located in non-coding regions, but the fraction usually studied is harbored in protein-coding regions because potential impacts on proteins are relatively easy to predict by popular tools such as the Variant Effect Predictor. These tools annotate variants independently without considering the potential effect of grouped or haplotypic variations, often called “multi-nucleotide variants” (MNVs). Here, we used a large RNA-seq dataset to survey MNVs, comprising 382 chicken samples originating from 11 populations analyzed in the companion paper in which 9.5M SNPs— including 3.3M SNPs with reliable genotypes—were detected. We focused our study on in-codon MNVs and evaluate their potential mis-annotation. Using GATK HaplotypeCaller read-based phasing results, we identified 2,965 MNVs observed in at least five individuals located in 1,792 genes. We found 41.1% of them showing a novel impact when compared to the effect of their constituent SNPs analyzed separately. The biggest impact variation flux concerns the originally annotated stop-gained consequences, for which around 95% were rescued; this flux is followed by the missense consequences for which 37% were reannotated with a different amino acid. We then present in more depth the rescued stop-gained MNVs and give an illustration in the SLC27A4 gene. As previously shown in human datasets, our results in chicken demonstrate the value of haplotype-aware variant annotation, and the interest to consider MNVs in the coding region, particularly when searching for severe functional consequence such as stop-gained variants.

Genetic mapping of sex and self-incompatibility determinants in the androdioecious plant Phillyrea angustifolia

The diversity of mating and sexual systems in Angiosperms is spectacular, but the factors driving their evolution remain poorly understood. In plants of the Oleaceae family, an unusual self-incompatibility (SI) system has been discovered recently, whereby only two distinct homomorphic SI specificities segregate stably. To understand the role of this peculiar SI system in preventing or promoting the diversity of sexual phenotypes observed across the family, an essential first step is to characterize the genetic architecture of these two traits. Here, we developed a high-density genetic map of the androdioecious shrub P. angustifolia based on a F1 cross between a hermaphrodite and a male parent with distinct SI genotypes. Using a double restriction-site associated digestion (ddRAD) sequencing approach, we obtained reliable genotypes for 196 offspring and their two parents at 10,388 markers. The resulting map comprises 23 linkage groups totaling 1,855.13 cM on the sex-averaged map. We found strong signals of association for the sex and SI phenotypes, that were each associated with a unique set of markers on linkage group 12 and 18 respectively, demonstrating inheritance of these traits as single, independent, mendelian factors. The P. angustifolia linkage map shows robust synteny to the olive tree genome overall. Two of the six markers strictly associated with SI in P. angustifolia have strong similarity with a recently identified 741kb chromosomal region fully linked to the SI phenotype on chromosome 18 of the olive tree genome, providing strong cross-validation support. The SI locus stands out as being markedly more rearranged, while the sex locus has remained relatively more collinear. This P. angustifolia linkage map will be a useful resource to investigate the various ways by which the sex and SI determination systems have co-evolved in the broader phylogenetic context of the Oleaceae family.

Noninvasive population assessment of moose (Alces alces) by SNP genotyping of fecal pellets

Noninvasive genetic studies of wild animals enable the recovery of information infeasible to obtain using other means. However, the low quantity and quality of noninvasively collected DNA often challenge the retrieval of reliable genotypes, which may cause biases in downstream analyses. In this study, we optimized SNP (single nucleotide polymorphism) genotyping of fecal samples from moose (Alces alces) with the main purpose of exploring the potential of using noninvasively retrieved genotypes for individual- and sex identification. Fecal pellets were collected during the late winter of 2016 on the Swedish island of Öland in the Baltic Sea and DNA was extracted and genotyped using 86 autosomal, six sex-specific and five species diagnostic SNPs. The SNP error rate of the quality filtered dataset was 0.06 and the probability of identity for siblings below 0.001. Following a thorough quality filtering process, 182 reliable genotypes were obtained, corresponding to 100 unique individuals (37 males, 63 females), with an estimated male proportion of 37% (± 9%). The population size, estimated using two different capture-mark-recapture approaches, was found to be in the range of 115–156 individuals (95% CI). Furthermore, moose on Öland showed significantly lower heterozygosity levels (zHexp = −5.51, N = 69, pHexp = 3.56·10−8, zHobs = −3.58, N = 69, pHobs = 3.38·10−4) and appeared genetically differentiated from moose on the Swedish mainland. Thus, we show that quality controlled noninvasively derived SNP genotypes can be highly informative for individual and population monitoring in a large ungulate.

Validating the use of non-invasively sourced DNA for population genetic studies using pedigree data

Non-invasive genetic sampling has provided valuable ecological data for many species – data which may have been unobtainable using invasive sampling methods. However, DNA obtained non-invasively may be prone to increased levels of amplification failure and genotyping error. Utilizing genotype data from 32 pedigreed koalas, this study aimed to validate the reliability of final consensus genotypes obtained using DNA isolated from koala scats. Pedigree analysis, duplicate genotyping, analysis of mismatched loci and tests for null alleles were used to look for evidence of errors. All genetically confirmed parent–offspring relationships were found to follow Mendelian rules of inheritance. Duplicate genotypes matched in all cases and there was no evidence of null alleles. Related individuals always had different 12-marker genotypes having a minimum of three unique loci (in one full sibling pair), a mode of seven unique loci and a maximum of 11 unique loci. This study demonstrates the capacity of DNA recovered from koala scats to provide reliable genotypes that can unequivocally discriminate individuals and infer parentage, provided data are missing from no more than two loci. Validating data obtained using non-invasive sampling is an important step, allowing potential problems to be identified at an early stage.

Conservation genetics of the capercaillie in Poland – estimating the size of the Tatra National Park population by the genotyping of non-invasive samples

Knowledge about population size is of high importance for conservationists. We used non-invasively collected samples and microsatellite genotyping to estimate the size of the Tatra National Park population of the endangered capercaillie Tetrao urogallus. This population is one of the most important strongholds of the species in Poland. In 2016 over 150 samples (faeces and feathers) of the capercaillie were collected throughout area of the Tatra National Park. Then, DNA was extracted and genetic profiles were evaluated, using nine microsatellite markers. We obtained 81 reliable genotypes. Among them, 34 unique genotypes were found, corresponding to Minimum Number of individuals Alive in the investigated population. Application of capture-recapture models in the R package Capwire indicated, that the area was inhabited by approx. 54 birds, whereas regression model suggested presence of 36–64 individuals. Previous field surveys suggested that the number of birds in the Tatra National Park is about 50. Hence, we assumed that genetic tagging of non-invasive samples performs well in estimating the abundance of the capercaillie in the investigated population.

Piles of scats for piles of DNA: deriving DNA of lizards from their faeces

Non-invasive genetic sampling using scats has a well established role in conservation biology, but has rarely been applied to reptiles. Using scats from captive and wild Egernia stokesii (Squamata, Scincidae) we evaluated two storage and six DNA-extraction methods and the reliability of subsequent genotype and sequence data. Accurate genotype and sequence data were obtained from frozen and dried captive lizard scat DNA extracted using a QIAamp® DNA Stool Mini Kit and a modified Gentra® Puregene® method, but success rates were reduced for wild lizard scats. Wild E. stokesii eat more plants than their captive counterparts, possibly resulting in scat DNA extracts containing plant compounds that inhibit PCR-amplifications. Notably, reliable genotypes and sequences were obtained from wild E. stokesii scat DNA extracted using a Qiagen DNeasy® Plant Mini Kit, a method designed to remove plant inhibitory compounds. Results highlight the opportunity for using scat-derived DNA in lizard studies, particularly for species that deposit scats in piles.