Analysis of the variation and genetic stability of chloroplast genome of Pinus taeda

Chloroplasts are utilized in genetic studies due to their compact genomes and simple structure. While their inheritance is predominantly maternal in most plants, it is paternal in many gymnosperms. This study investigated intraspecific variation and inheritance patterns of the chloroplast genome in loblolly pine (Pinus taeda). We sequenced chloroplast DNA from 54 individuals in the second generation core breeding population and performed genome-wide SNP discovery via a custom Python pipeline. From the 81 initially identified SNP loci, a final set of 32 high-frequency loci (variation frequency > 5%) was selected for validation, comprising 14 in intergenic spacer regions and 18 within gene regions. Despite overall conservation, we identified a hypervariable region in the ycf1 gene, which contained 8 SNP loci and accounted for 44.4% of all coding region SNPs. We then designed 23 primer pairs to amplify the regions containing these candidate variants across a 9 × 5 tester design, encompassing a total of 75 individuals (45 progeny and 30 parents). Phylogenetic and haplotype analyses based on these SNPs revealed no maternal haplotype sharing between any of the eight maternal parents and their progeny, and showed that progeny consistently clustered with specific paternal parents, such as W03 and N4. Furthermore, analysis of SNP profiles allowed reconstruction of paternal lineages and detection of patterns indicative of pollen contamination. Collectively, these results validate both the paternal inheritance of the chloroplast genome in P. taeda and the potential utility of chloroplast markers in conifer breeding.