Blog

Analysis of codon base composition

The contents of T₃, C₃, A₃, G₃, GC₁, GC₂, GC₃, and GC_all in cp coding gene codons of 49 Dendrobium species were analyzed in this study (Figure S1, Table S3). The results revealed an asymmetric expression of the four nucleotides in these codons. Specifically, the third position of codons showed a higher frequency of A/T usage compared to G/C.The nucleotide expression frequencies at the third codon position were as follows: T₃ (45.72%−47.79%), A₃ (40.92%−43.96%), G₃ (16.68%−19.13%), and C₃ (16.64%−19.51%). Analysis of GC content across the coding regions showed values of 42.14%−48.65% for GC₁, 37.37%−41.08% for GC₂, and 28.81%−32.14% for GC₃, all of which were below 50%. This indicates an overall preference for codons rich in A/T nucleotides. Notably, the GC content exhibited the trend GC₁ > GC₂ > GC₃, highlighting a stronger preference for codons ending with A/T in the cp coding genes of Dendrobium species.

GC content is a critical indicator of genomic characteristics. we analyzed the correlation among GC₁, GC₂, GC₃, and GC_all (Fig. 1, Table S4). The results revealed that GC_all was significantly correlated with GC₁, GC₂, and GC₃ (P < 0.01). Furthermore, GC₁ and GC₂ were significantly correlated (P < 0.05), indicating that the GC composition at the first and second codon positions may influence each other. In contrast, GC₃ showed no correlation with GC₁ or GC₂, suggesting that the GC composition at the third position is independent of the first and second positions.

RSCU analysis

RSCU is an important parameter for measuring codon preference. RSCU > 1 indicates a strong preference for that codon, categorizing as a high-frequency codon. Conversely, RSCU < 1 signifies a weak preference, classifying as a low-frequency codon. Excluding the initiation codon (AUG) and the termination codons (UGG, UAG, and UAA), there were 30 codons with RSCU > 1, representing 46.88% of the total number of codons (Fig. 2 and Table S5). Notably, 29 codons ended with A or U bases, accounting for 96.67% of the high-frequency codons. Among these, AGA, which encodes Arginine, exhibited the highest RSCU value, followed by GCU, which encodes Alanine. Additionally, 30 low-frequency codons with RSCU < 1 and 28 ended with G/C, accounting for 93.33%. These results indicated that the high-frequency codons tend to end with A/U, while the low-frequency codons are more likely to end with G/C.

Factors affecting CUB in 49 Dendrobium species

PR2-plot analysis

A PR2-plot analysis was conducted to analyze the relationship between the third base of codons (A₃/T₃ and G₃/C₃) in CDS of cp genomes from 49 Dendrobium species. As shown in Fig. 3, cp coding genes in the four regions were unevenly distributed, with the majority located far from the center. This deviation suggests that CUB in these genes may be primarily governed by natural selection.

Neutrality plot analysis

To further explore the influence of natural selection and mutation pressure on CUB, neutrality plot analysis was conducted using GC₁₂ and GC₃ as lateral and vertical axes, respectively. The results showed that GC₁₂ was distributed from 29.01% to 53.24%, while GC₃ was distributed from 20.39% to 46.86% in the CDS of the cp genome (Fig. 4). Notably, few genes were distributed along the diagonal, indicating a weak correlation among the nucleotide compositions at GC₁₂ and GC₃. Furthermore, the regression coefficient (slope) ranged from −0.038 to 0.368.

ENC-plot analysis

An ENC-plot analysis for GC₃ was performed to investigate the factors influencing CUB in CDS of Dendrobium cp genome. When the CUB is influenced only by mutation pressure, the cp genes will fall on the ENC expected curve. If the cp genes fall below the ENC expected curve, then the CUB is mainly driven by other factors such as natural selection, in addition to mutation pressure [38]. As shown in Fig. 5, the Majority of the cp genes in the 49 Dendrobium species were far from the expected ENC curve, indicating that natural selection may be the primary factor driving CUB in the cp genome of Dendrobium, while mutation pressure plays a secondary role.

The calculated ENC ratios [(ENCexp-ENCobs)/ENCexp] range from −0.15 to 0.35 (Table 1), representing the difference between ENCobs values and ENCexp values. Approximately 73% of the genes have an ENC ratio greater than 0, which is consistent with the results of the ENC-plot analysis.

Optimal codons of Dendrobium

Eleven to nineteen optimal codons were identified across 49 Dendrobium species, with most optimal codons ending with A/U. Notably, D. polyanthum and D. devonianum shared the same optimal codons: GCA, GAA, UUU, GGA, CAU, UUA, AAU, CCU, AGA, CGA, AGU, GUA, and GUU. Similarly, D. nobile, D. linawianum, D. flexicaule, and D. officinale shared the same optimal codons: GCA, GAA, UUU, GGA, GGU, CAU, AUU, AAA, UUA, CCU, CAA, CGA, CGU, AGU, ACU, and UAU (Figure S2 and Table S6).

Phylogenetic analysis

Phylogenetic trees of 49 Dendrobium cp genomes were constructed based on the cp genomics and CDS using the ML method, with C. hookerianum and C. lowianum as outgroups. The resulting phylogenetic topologies revealed that the Dendrobium species was divided into three distinct groups (Group A, Group B, and Group C), and some species showed overlap between the cp genomics and CDS phylogenetic trees (Fig. 6). Discrepancies were also observed between molecular biological evidence and traditional plant morphological classifications within individual clusters. Species in sct. Chrysotoxae is found across Groups A, B, and C, challenging traditional morphological classifications and suggesting that of sct. Chrysotoxae species may have maternal sources from sct. Dendrobium, sct. Holochrysa, and sct. Chrysotoxae. Similarly, D. hrysocrepis from sct. Holochrysa appeared in Group A, distinctly separated from Group B, indicating possible maternal lineage derived from sct. Dendrobium. D. hercoglossum as the sole representative from sct. Breviflores is more closely related to sct. Dendrobium. However, it is difficult to define the phylogenetic relationship between sct. Breviflores and sct. Dendrobium.

Additionally, PLS-DA was conducted to evaluate the relationship between CUB and phylogeny based on RSCU values of Dendrobium species. The PLS-DA showed that Groups A and C were each divided into two subgroups, consistent with the phylogenetic trees derived from the cp genomics and CDS (Figure S3). In contrast, Group B could not form a distinct cluster. The species were dispersed into Groups A and C. These findings suggest that RSCU values are valuable tools for revealing the evolutionary relationships among Dendrobium species.