Objective Pinus tabuliformis is an important ecological and timber tree species in northern China, possessing high economic and ecological value. This study conducted parentage identification, investigated the physiological and molecular mechanisms of cold tolerance, and performed seedling evaluation on its hybrid progeny. The research aims to provide a scientific basis for genetic improvement and germplasm innovation in Pinus species.

Method Taking hybrid progeny of P. tabuliformis as the research subject, this study performed parentage identification and genetic analysis using molecular markers at two levels: organellar (chloroplast and mitochondrial) and nuclear (SSR) genomes. Systematic comparisons were made with parental P. tabuliformis and another high-quality pine species, P. sylvestris var. mongolica, in terms of growth traits, physiological and biochemical indicators, as well as transcriptomic responses under low-temperature stress. Finally, a multi-trait comprehensive evaluation and selection of superior individuals were conducted using the fuzzy membership function method.

Result (1) Analysis of chloroplast and mitochondrial DNA sequences confirmed that the paternal parent of the hybrid progeny was P. tabuliformis and the maternal parent was P. densiflora var. zhangwuensis. Both genetic distance and cluster analysis indicated that the hybrid progeny were most closely related to P. tabuliformis and grouped within the same clade, suggesting that their genetic characteristics were primarily inherited from P. tabuliformis. (2)Compared with P. tabuliformis and P. sylvestris var. mongolica, the 3-year-old hybrid seedlings exhibited greater average height and ground diameter, indicating superior growth performance. (3) During natural overwintering, low-temperature stress significantly affected the physiological metabolism of the three pine species. The hybrid progeny exhibited more active photosynthetic pigment metabolism, the greatest increase in malondialdehyde accumulation, and rapid and efficient responses in soluble sugar and soluble protein levels. Both peroxidase and superoxide dismutase activities remained at relatively high levels. Overall, the hybrid progeny demonstrated superior physiological regulation capacity compared to P. tabuliformis and P. sylvestris var. mongolica, indicating stronger adaptability to low temperatures. (4) Gene expression analysis among the three pine species revealed that the hybrid progeny had the highest number of differentially expressed genes under low-temperature stress, with the most pronounced changes in transcription factors and protein kinases. Four key transcription factors (PtZFP30、PtNAC83、PtMYB230和PtNF_YB3) were identified. (5) Using the fuzzy membership function method for comprehensive evaluation of growth and physiological traits, 25 superior individuals were selected from 150 hybrid progeny. Their average membership degree (0.578–0.458) was significantly higher than that of P. tabuliformis (0.457) and P. sylvestris var. mongolica (0.455).

Conclusion This study systematically elucidated the advantages of P. tabuliformis hybrid progeny in terms of growth, physiology, and molecular mechanisms, established a comprehensive multi-index evaluation and selection system, and selected 25 superior individuals. It not only provided empirical evidence for understanding the mechanisms of heterosis in forest trees but also layed a material and theoretical foundation for the breeding of new pine varieties.