Abstract: To understand the pattern of codon preference used in the chloroplast genome of Pinus densata, the factors that influence the formation of codon preference were explored. Methods such as neutral mapping analysis, ENC-plot, PR2-plot analysis, and correspondence analysis were performed using CodonW and CUSP online software. The results showed that the average GC content of chloroplast codons in P. densata was 39.84%, and the contents of GC1and GC2were 48.81% and 40.70%, respectively, which were higher than GC3(30.52%), indicating that their chloroplast genome codon endings preferred to end with A and U. The mean value of effective codon number ENC was 48.77, indicating a weak codon preference. Results of the neutral plot analysis, ENC-plot analysis, and PR2-plot bias analysis showed that selection pressure was the main factor influencing the codon preference of P. densata, followed by a combination of factors such as mutations. Further correspondence analysis revealed that axis 1 accounted for 21.18% of the overall variation and was the main influencing factor, which was not correlated with ENC and GC3, but was significantly correlated with CAI. The results of these analyses indicated that natural selection was the main influencing factor on chloroplast codon preference in P. densata, and was also influenced by other factors; 15 codons were identified as optimal codons, showing a strong preference for NNA and NNU codons with high representation. This study provided basic information for future genetic engineering of P. densata chloroplasts, codon optimization, improving the expression level of exogenous genes and the yield and quality of alpine pine seeds.

Key words: Pinus densata, chloroplast genome, codon preference, optimal codon