Dealing with paralogy in RADseq data: in silico detection and single nucleotide polymorphism validation in Robinia pseudoacacia L.

Abstract

The RADseq technology allows researchers to efficiently develop thousands of polymorphic loci across multiple individuals with little or no prior information on the genome. However, many questions remain about the biases inherent to this technology. Notably, sequence misalignments arising from paralogy may affect the development of single nucleotide polymorphism (SNP) markers and the estimation of genetic diversity. We evaluated the impact of putative paralog loci on genetic diversity estimation during the development of SNPs from a RADseq dataset for the nonmodel tree species Robinia pseudoacacia L. We sequenced nine genotypes and analyzed the frequency of putative paralogous RAD loci as a function of both the depth of coverage and the mismatch threshold allowed between loci. Putative paralogy was detected in a very variable number of loci, from 1% to more than 20%, with the depth of coverage having a major influence on the result. Putative paralogy artificially increased the observed degree of polymorphism and resulting estimates of diversity. The choice of the depth of coverage also affected diversity estimation and SNP validation: A low threshold decreased the chances of detecting minor alleles while a high threshold increased allelic dropout. SNP validation was better for the low threshold (4×) than for the high threshold (18×) we tested. Using the strategy developed here, we were able to validate more than 80% of the SNPs tested by means of individual genotyping, resulting in a readily usable set of 330 SNPs, suitable for use in population genetics applications.

Comparative phylogeography of trans‐Andean freshwater fishes based on genome‐wide nuclear and mitochondrial markers

“…Note that the temporal divergence of our target fish orders (Gymnotiformes, Characiformes and Siluriformes) is well over 100 my old (Arcila et al, ; Betancur‐R et al., ). There is also a potential for paralogous locus assembly (Mastretta‐Yanes et al, ; Verdu et al, ), a problem that is exacerbated in fishes due to multiple rounds of whole genome duplications (Hughes et al, ). By contrast, data missingness is not likely to pose a large effect on our analyses of sequence capture data given that we successfully assembled ~92% of the ~one thousand target loci across both populations and species.…”

A few north Appalachian populations are the source of European black locust

“…SNPs have recently been developed on black locust (Verdu et al, ) using the double‐digest RAD approach. Nine samples (from six North American trees, two European trees, and one Iranian tree) were digested with Eco RI/ Mse I and subsequent libraries were sequenced using Illumina technology.…”

“…Two genotyping experiments were performed using these SNPs: (a) All collected samples (initial dataset) were genotyped using 113 SNPs, which were selected according to the procedure presented in Bouteiller et al (); (b) after clone removal (see Section 3), 163 individuals were subsampled randomly within the populations in each range to maintain the sampling design between ranges (additional dataset: 69 individuals from the USA and 96 from Europe) and genotyped using a total of 251 SNPs. These additional SNPs were among the SNPs developed by Verdu et al (), which had first been discarded as we had initially chosen to prioritize the more polymorphic SNPs. Four additional multiplexes of SNPs (138 SNPs in total) were thus designed using the MassArray Assay Editor 4.0.1.4 software (see Verdu et al, for more details regarding the procedure).…”

“…These additional SNPs were among the SNPs developed by Verdu et al (), which had first been discarded as we had initially chosen to prioritize the more polymorphic SNPs. Four additional multiplexes of SNPs (138 SNPs in total) were thus designed using the MassArray Assay Editor 4.0.1.4 software (see Verdu et al, for more details regarding the procedure). SNP genotyping was performed using the Sequenom MassARRAY System (Agena Bioscience, San Diego, USA) at the Bordeaux Genome Transcriptome Facility (https://pgtb.cgfb.u-bordeaux.fr/en), and using the iPLEX Gold chemistry genotyping kit according to the manufacturer's instructions.…”

“…Modern molecular and statistical tools used in population genetics have proved to be useful for finding the geographic origins of invasive species, complementing or providing a solution to the lack of available historical knowledge (Besnard et al, ; Chown et al, ; Cristescu, ; Hoos, Whitman Miller, Ruiz, Vrijenhoek, & Geller, ). Using SNP markers developed for the black locust (Verdu et al, ), we investigated its introduction history and genetic diversity in its native range and European invasive range, in particular by answering the following questions: (a) Can we identify the native population sources of European black locust? (b) What is the genetic differentiation within and between ranges?…”