Open pollination is widely used in forest tree breeding programs, often resulting in progeny with unknown paternal origin. The main objective of this study was to investigate paternal relationships and infer relatedness among Pinus pinaster Ait. individuals using an optimized multiplex amplicon sequencing approach targeting 69 polymorphic SNP loci. To achieve this objective, a custom bioinformatic pipeline was implemented in R for the processing of multiplex paired-end MiSeq sequencing data obtained from the DNA of 137 individual trees.

The specific workflow included read quality assessment, filtering of low-quality reads based on Phred score, ambiguous nucleotide content and minimum sequence length, followed by automated assignment of reads to locus-specific primer pairs using IUPAC-compatible matching with a maximum of one mismatch. Reverse-read information was processed in the appropriate orientation and combined with forward-read information to support genotype calling for each sample and locus. Reads matching the expected primer and control regions were extracted and used to determine the nucleotide state at the target polymorphic position. The pipeline accounted for homozygous and heterozygous profiles, as well as INDEL-type variation when present, generating per-sample genotype calls in an allele-pair format. Matched reads were additionally exported as locus-specific FASTA files to support traceability, alignment inspection and validation of ambiguous or unexpected amplification patterns. The final output consisted of a multi-locus SNP genotype matrix compiling the genetic profiles obtained for the analysed individuals. This matrix was subsequently subjected to parentage analysis using dedicated software, resulting in the successful assignment of 36 out of 49 offspring to candidate pollen donors within the studied population. In addition, the SNP profiles enabled the verification and reassignment of clone identities for 17 trees with missing or inconsistent records.

Together, the assignment of offspring to candidate pollen donors, the inference of genetic relationships within the breeding population to support pedigree reconstruction and inbreeding avoidance, and the verification of germplasm identity demonstrate the value of integrating a SNP marker panel, multiplex sequencing, bioinformatic processing and parentage analysis in the management of the Portuguese maritime pine breeding program.