Complete Chloroplast Genome of Nicotiana otophora and its Comparison with Related Species

Abstract

Nicotiana otophora is a wild parental species of Nicotiana tabacum, an interspecific hybrid of Nicotiana tomentosiformis and Nicotiana sylvestris. However, N. otophora is least understood as an alternative paternal donor. Here, we compared the fully assembled chloroplast (cp) genome of N. otophora and with those of closely related species. The analysis showed a cp genome size of 156,073 bp and exhibited a typical quadripartite structure, which contains a pair of inverted repeats separated by small and large single copies, containing 163 representative genes, with 165 microsatellites distributed unevenly throughout the genome. Comparative analysis of a gene with known function across Nicotiana species revealed 76 protein-coding sequences, 20 tRNA sequences, and 3 rRNA sequence shared between the cp genomes. The analysis revealed that N. otophora is a sister species to N. tomentosiformis within the Nicotiana genus, and Atropha belladonna and Datura stramonium are their closest relatives. These findings provide a valuable analysis of the complete N. otophora cp genome, which can identify species, elucidate taxonomy, and reconstruct the phylogeny of genus Nicotiana.

Keywords: Nicotiana; SSRs; cp genome; phylogeny; repeat analysis; sequence divergence.

Figure 1

Gene map of the N. otophora chloroplast genome. Genes drawn inside the circle are transcribed clockwise, and those outside are counterclockwise. Genes belonging to different functional groups are color-coded. The darker gray in the inner circle corresponds to GC content, and the lighter gray corresponds to AT content.

Figure 2

Amino acid frequencies of the N. otophora cp protein coding sequences. The frequencies of amino acids were calculated for all 110 protein-coding genes from the start to the stop codon.

Figure 3

Analysis of repeated sequences in five Nicotiana chloroplast genomes. (A), Total of three repeat types; (B), Frequency of the palindromic repeat by length; (C), frequency of the direct repeat by length; and (D), Frequency of tandem repeat by length.

Figure 4

Analysis of simple sequence repeat (SSR) in the five Nicotiana chloroplast genomes. (A) Number different SSRs types detected in five genomes and (B) Frequency of identified SSR motifs in different repeat class types.

Figure 5

Venn diagram illustrating the proportion of genes in five Nicotiana cp genomes. (A) Number of protein coding genes shared by five Nicotiana cp genomes. (B) Number of unique genes identified in each cp genome.

Figure 6

Visualization alignment of five chloroplast genome sequences. VISTA-based identity plot showing sequence identity among five Nicotiana species using N. otophora as a reference. The thick black line shows the inverted repeats (IRs) in the chloroplast genomes.

Figure 7

Sliding window analysis of N. otophora with four Nicotiana cp genomes. (A) Analysis of LSC regions, (B) Analysis of SSC regions, and (C) Analysis of IR regions. (window length: 600 bp, step size: 200 bp). X-axis, position of the midpoint of a window; Y-axis, nucleotide diversity of each window.

Figure 8

Phylogenetic relationship of N. otophora with related species based on 75 protein-coding genes shared by all cp genomes. Tree constructed by maximum likelihood (A), maximum parsimony and Bayesian inference (B) with Citrus aurantifolia and Citrus sinensis as outgroups.