*2.3. Development of Selected CAPS Markers and Further Validation of Their Taxon Specificity*

From potentially taxon-specific SNPs (Table S3), some SNPs were selected for the development of cleaved amplified polymorphic site (CAPS) markers which fulfill the following criteria: (i) a SNP is located in a recognition site of a restriction enzyme; (ii) a SNP allele of the target taxon is part of the recognition site sequence, i.e., amplicon of the target taxon will be cut by the restriction enzyme whereas the others will not; (iii) a SNP is not located close to one of the gene ends (design of flanking primers is possible within the gene).

Specific primers were designed in the flanking regions of selected SNPs considering that the amplicon size should not exceed 200 bp because mtDNA extracted from processed wood products may be highly degraded. Moreover, primer sequences should match the respective genic region in all 13 species included in the genic alignments (see above) with a perfect match to Fagales species (as far as possible) and with a perfect match or at most one to two mismatches to the other sequences.

Selected CAPS markers were then pre-validated using DNA samples of different deciduous tree and conifer species. As an example, Figure 2 presents the pre-validation of the potential *Fagus*-specific CAPS marker 3\_Fagus\_*ccmFc* (marker description in Table 1). Only the five *Fagus* individuals from five different *Fagus* species included in the pre-validation provided the pattern of the digested PCR product with two fragments of 108 and 45 bp each, whereas all individuals from the other genera of deciduous trees or conifers showed the band of the non-digested PCR product of 153 bp (Figure 2).

**Figure 2.** *Cont*.

**Figure 2.** Pre-validation of the *Fagus*-specific mitochondrial CAPS marker 3\_Fagus\_*ccmFc* (marker description in Table 1) with DNA samples of *F. sylvatica*, and four other *Fagus* species as well as 41 non-*Fagus* species comprising 35 species of deciduous trees, 5 conifer species, and *Ginkgo biloba*. After digestion, PCR fragments were separated on a 1.5% agarose gel. Related primer sequences are provided in Table S4. Extended validation of CAPS markers was performed with much more individuals (see main text, Section 4.10, and Table S5).


**Table 1.** Mitochondrial CAPS markers specific \* for genus *Fagus*, family Fagaceae, or order Fagales.

The name of the gene where the SNP is included is part of the marker name, e.g., "*matR*". SNP positions are related to the nucleotide position in the DNA sequence of the respective gene in *Populus tremula* (GenBank KT337313.1) [44] used as reference. The "target" is the taxon given in column "taxon specificity". Related primer sequences are provided in Table S4. \*, The term "taxon specificity" in this context means the specificity of the related marker compared to all individuals of different deciduous tree species and conifer species included in the marker development (see above) and validation in this study (Table S5 and Section 4.10).

Only CAPS markers that successfully passed the pre-validation were subjected to extended validation as detailed below (see also Section 4.10). Table 1 summarizes the features of four successfully validated mitochondrial CAPS markers, and Figure 3 compares the related digestion patterns of these markers in one individual each of *F. sylvatica* (Fagaceae, Fagales), *Quercus robur* (Fagaceae, Fagales), *Betula pendula* (Betulacea, Fagales), and *Populus tremula* (Salicaceae, Malpighiales) as a non-Fagales species.

**Figure 3.** Representative restriction patterns of the CAPS markers (Table 1) analyzed in individuals of three Fagales and one non-Fagales species each. After digestion, PCR fragments were separated on a 1.5% agarose gel.

In the entire validation (pre-validation and extended validation), each CAPS marker (Table 1; Figure 3) was successfully validated with around 100 individuals of 59–63 tree species that belong to 14–15 families (including four Fagales families) in nine to ten orders in total (see also 4.10). All species within the entire validation, the numbers of individuals per species, and details about the origin of the individuals are summarized in Table S5. The species list contains—among others—species that are known to be potentially included in wood composite products.

Besides, the two *Fagus*-specific CAPS markers—3\_Fagus\_*matR* and 3\_Fagus\_*ccmFc* (Table 1)—were successfully validated in 25 additional *F. sylvatica*, five additional *F. orientalis*, four additional *F. grandifolia* and *F. engleriana* each, and two additional *F. crenata* individuals (Table S5). Although these two *Fagus-*specific markers were originally selected based on potentially *F. sylvatica*-specific SNPs (identified in the genic alignments described above including *F. sylvatica* as the only *Fagus* species), they turned out to be specific for the genus *Fagus* in the validation.
