Cross-amplification and multiplexing of cpSSRs and nSSRs in two closely related pine species (Pinus sylvestris L. and P. mugo Turra)

Abstract

Background: Simple sequence repeats (SSRs) are widespread molecular markers commonly used in population genetic studies. Nowadays, next-generation sequencing (NGS) methods allow identifying thousands of SSRs in one sequencing run, which greatly facilitates isolation and development of new SSRs. However, their usefulness as molecular markers still must be tested empirically on a number of populations to select SSRs with best parameters for future population genetic research. An alternative approach, cheaper and faster than isolation and characterization of new SSRs, involves cross-amplification of SSRs in closely related species. Aims: Our goal was to develop multiplex PCR protocols that will be useful in population genetic studies of Scots pine (Pinus sylvestris L.) and dwarf mountain pine (P. mugo Turra), and possibly other pine species. Methods: We tested 14 chloroplast (cpSSRs) and 22 nuclear (nSSRs) microsatellite markers originally designed for Japanese black pine (P. thunbergii Parl.), P. sylvestris and loblolly pine (P. taeda L.) in four populations of P. sylvestris and P. mugo across different locations in Europe. We designed six multiplex PCRs, which were subsequently screened for their ability to provide repeatable and high quality amplification products using capillary electrophoresis. Results: The transfer rate in our study was similar in both pine species, and it was very high for cpSSRs (93% and 86% for P. sylvestris and P. mugo, respectively) and moderate for nSSRs (59% for both species). We managed to design five well-performing multiplex reactions out of six initially tested. Most of the tested loci were polymorphic. Moreover, the allelic patterns detected at some cpSSRs were species-specific. Conclusions: We provide a set of five multiplexes which can be used in genetic studies of both P. sylvestris and P. mugo. Chloroplast marker PCP30277 is a good candidate for a cheap species diagnostic marker suitable for tracking interspecific gene flow between hybridizing species of P. sylvestris and P. mugo.

Functional characteristics of EST-SSR markers available for Scots pine

“…On the other hand, detection of redundant markers, especially ones reversely oriented relative to one another within the same gene region, by conventional methods can be problematic. E.g., among the redundant markers we have identified,4 EST-SSR pairs were used by research teams within the same study: PtTX2146/PtSIFG_3145 [71], lw_isotig00080/lw_isotig00081 [72], psyl16/psyl17 [73][74][75][76][77][78], psyl18/psyl19 [73,79].…”

Evolutionary targets of gene expression divergence in a complex of closely related pine species

“…However, they are genetically similar, and previous studies showed little differentiation between the taxa at karyotype (Bogunić et al, 2011), mitochondrial and plastid genomes (Heuertz et al, 2010; Dzialuk et al, 2017; Zaborowska et al, 2019; Sokołowska et al, 2021), nuclear loci (Monteleone et al, 2006; Wachowiak et al, 2013; Zaborowska et al, 2021), and candidate genes that showed low (<0.1%) net genetic divergence between the taxa (Wachowiak et al, 2013). So far, only singular species diagnostic markers have been found at mitochondrial, plastid, and nuclear regions (Wachowiak et al, 2000; Kormut'ák et al, 2005; Żukowska & Wachowiak, 2017; Zaborowska et al, 2019). This close genetic similarity between the species is usually explained by relatively recent speciation in presence of gene flow (Christensen, 1987; Monteleone et al, 2006; Jasińska et al, 2010) and segregation of ancestral variation (Wachowiak et al, 2011, 2013).…”