Development of new SNP and INDEL loci for the valuable African timber species Lophira alata

The timber of the species Lophira alata (azobe) is very popular for outdoor constructions, which favours its overexploitation and illegal logging. We sampled individuals from Liberia, Ivory Coast, Ghana, Nigeria, Cameroon, Gabon, Congo Brazzaville and Republic Democratic of Congo to discover new nuclear and plastidial SNP and INDEL loci through restriction associated DNA sequencing (RADSeq) and low coverage MiSeq genome sequencing. From an initial set of 397 loci, a final set of 126 loci was selected for timber tracking purposes.

Lophira alata is a tropical pioneer timber species typical from secondary humid forests. Its distribution ranges from Sierra Leone in Western Africa to Congo in Central Africa. The timber is highly valuable because of its resistance against insects and moulding, which enables it's use in outdoor constructions. Despite a minimum cutting diameter of at least 60 cm in all producer countries, this species is heavily exploited and classified as vulnerable in the IUCN red list.
The genus Lophira has been poorly studied until now and only consist of two species: L. alata typical from humid forest habitats while the species L. lanceolata is mostly found in savannahs. However, when both species are growing in sympatry, species identification is difficult (Biwole et al. 2012). Genetic studies are limited to the development and use of SSRs markers (Ewédjè et al. 2020;Pineiro et al. 2015), which aimed at studying the genetic structure and hybridisation between L. alata and L. lanceolata. With 10 SSRs, the presence of a cryptic species in L. alata was suggested in West Gabon (Ewédjè et al 2020). Thus, little information is available on the diversity and differentiation within both species.
After the entry into force of timber regulations (among others European Timber Regulation and US Lacey Act) requiring that the species identity and the country of origin are declared and correct, tracking tools to control the claims on species and origin received increasing attention (Dormontt et al. 2015). Although many genetic markers have been successfully developed on tropical and temperate species, the low DNA quality obtained from timber strongly hinders the use of SSRs markers in timber tracking (Blanc-Jolivet and Liesebach 2015). By contrast, SNP loci usually provide good results and sets of SNP loci for origin identification have already been developed for other African species (Jardine et al. 2016;Blanc-Jolivet et al. 2017, 2018.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s1268 6-020-01173 -5) contains supplementary material, which is available to authorized users. Cambium or leaf samples from 103 individual trees stemming from West and Central Africa (Table 1) were dried in silicagel in the field. DNA was extracted according to Dumolin et al. (1995). Three and nine samples were selected for respectively, restriction site associated DNA sequencing [RADSeq, (Miller et al. 2007)] and low coverage Illumina MiSeq genome sequencing (Straub et al. 2012). The use of both NGS methods allowed the SNP and INDEL discovery in the nuclear and in the plastidial genomes. A MassARRAY ® iPLEX™ design [Assay Design Suite v2.0 (Agena Biosci-ence™, San Diego, USA)] included loci which showed polymorphism among sequenced individuals and for which the sequences did not show the presence of another SNP locus in a 50 bp neighbourhood. 397 loci were run in 12 multiplexes on a MassARRAY ® iPLEX™ platform (Agena Biosci-ence™, San Diego, USA) using the iPLEX™ GOLD chemistry (Supplementary material S1) on 94 samples (Table 1).
In order to select loci showing a differentiation over the species distribution range, we grouped all individuals according to their country of origin. We estimated, when applicable, average diversity, Gregorius' differentiation (Gregorius 1987) and correlation among genetic and geographic distance for each locus (Supplementary material S2). Because most African species show a strong genetic split between Western and Central African populations, we also estimated differentiation parameters within Western African and Central African countries to catch loci showing local genetic differentiation. On the three datasets, we selected the loci with the highest genetic differentiation, the highest genetic-geographic distance correlation, while loci with very low calling rates were excluded (GDA-NT, unpublished). 126 selected loci were included in four MassARRAY ® iPLEX™ multiplexes (Supplementary material S3), from which 75, 20 and 28 SNP loci were located in the nuclear, chloroplastic and mitochondrial genome respectively. Two chloroplastic and one mitochondrial INDELs were also included. Theoretical performance for the verification of the country of origin reached 86% (self-assignment test with leave-one-Out procedure using the Bayesian criteria of Rannala and Mountain 1997) when we splited the Nigeria and Cameroon individuals according to a STRU CTU RE analysis with two putative genetic groups (Pritchard et al. 2000). Indeed, the genetic boarder between West and Central Africa seems to be located between Nigeria and Cameroon where both groups are present in sympatry.
Our new set of 126 loci including both nuclear and plastidial loci will be of great importance for further population genetics and biogeographic studies as well as timber origin tracking in Lophira alata.