Comparative analysis of the complete chloroplast genomes of thirteen Bougainvillea cultivars from South China with implications for their genome structures and phylogenetic relationships

Abstract

Bougainvillea spp., belonging to the Nyctaginaceae family, have high economic and horticultural value in South China. Despite the high similarity in terms of leaf appearance and hybridization among Bougainvillea species, especially Bougainvillea × buttiana, their phylogenetic relationships are very complicated and controversial. In this study, we sequenced, assembled and analyzed thirteen complete chloroplast genomes of Bougainvillea cultivars from South China, including ten B. × buttiana cultivars and three other Bougainvillea cultivars, and identified their phylogenetic relationships within the Bougainvillea genus and other species of the Nyctaginaceae family for the first time. These 13 chloroplast genomes had typical quadripartite structures, comprising a large single-copy (LSC) region (85,169-85,695 bp), a small single-copy (SSC) region (18,050-21,789 bp), and a pair of inverted-repeat (IR) regions (25,377-25,426 bp). These genomes each contained 112 different genes, including 79 protein-coding genes, 29 tRNAs and 4 rRNAs. The gene content, codon usage, simple sequence repeats (SSRs), and long repeats were essentially conserved among these 13 genomes. Single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were detected among these 13 genomes. Four divergent regions, namely, trnH-GUG_psbA, trnS-GCU_trnG-UCC-exon1, trnS-GGA_rps4, and ccsA_ndhD, were identified from the comparative analysis of 16 Bougainvillea cultivar genomes. Among the 46 chloroplast genomes of the Nyctaginaceae family, nine genes, namely, rps12, rbcL, ndhF, rpoB, rpoC2, ndhI, psbT, ycf2, and ycf3, were found to be under positive selection at the amino acid site level. Phylogenetic relationships within the Bougainvillea genus and other species of the Nyctaginaceae family based on complete chloroplast genomes and protein-coding genes revealed that the Bougainvillea genus was a sister to the Belemia genus with strong support and that 35 Bougainvillea individuals were divided into 4 strongly supported clades, namely, Clades Ⅰ, Ⅱ, Ⅲ and Ⅳ. Clade Ⅰ included 6 individuals, which contained 2 cultivars, namely, B. × buttiana 'Gautama's Red' and B. spectabilis 'Flame'. Clades Ⅱ only contained Bougainvillea spinosa. Clade Ⅲ comprised 7 individuals of wild species. Clade Ⅳ included 21 individuals and contained 11 cultivars, namely, B. × buttiana 'Mahara', B. × buttiana 'California Gold', B. × buttiana 'Double Salmon', B. × buttiana 'Double Yellow', B. × buttiana 'Los Banos Beauty', B. × buttiana 'Big Chitra', B. × buttiana 'San Diego Red', B. × buttiana 'Barbara Karst', B. glabra 'White Stripe', B. spectabilis 'Splendens' and B. × buttiana 'Miss Manila' sp. 1. In conclusion, this study not only provided valuable genome resources but also helped to identify Bougainvillea cultivars and understand the chloroplast genome evolution of the Nyctaginaceae family.

Fig 1. Morphologies among 13 cultivars of the Bougainvillea genus.

A, Bougainvillea×buttiana ‘Mahara’; B, B.×buttiana ‘Gautama’s Red’; C, B. ×buttiana ‘California Gold’; D, B.×buttiana ‘Double Salmon’; E, B.×buttiana ‘Double Yellow’; F, B. ×buttiana ‘Big Chitra’; G, B.×buttiana ‘Los Banos Beauty’; H, Bougainvillea glabra ‘White Stripe’; I, Bougainvillea spectabilis ‘Flame’; J, B. spectabilis ‘Splendens’; K, B. × buttiana ‘Barbara Karst’; L, B. × buttiana ‘San Diego Red’; M and N, B. × buttiana ‘Miss Manila’ sp. 1. armed with simple or no thorns, derived from B. × buttiana ‘Miss Manila’.

Fig 2. Chloroplast genome map of B. × buttiana ‘Mahara’ (the outermost three rings) and CGView comparison of 13 complete chloroplast genomes of Bougainvillea cultivars (the inner rings with different colors).

Genes shown on the outside of the outermost first ring are transcribed counter-clockwise, and those on the inside are transcribed clockwise. The second ring with the darker gray color corresponds to the GC content, whereas the third ring with the lighter gray color corresponds to the AT content of the B. × buttiana ‘Mahara’ chloroplast genome generated by OGDRAW. The gray arrowheads indicate the directions of the genes. LSC, large single -copy region; IR, inverted repeat; SSC, small single-copy region. The innermost first black ring indicates the chloroplast genome size of B. × buttiana ‘Mahara’. The innermost second and third rings indicate deviations in the GC content and GC skew, respectively, in the chloroplast genome of C. barbatus: GC skew + indicates G > C, and GC skew − indicates G < C. CGView comparison of the 13 complete chloroplast genomes of Bougainvillea cultivars displayed from the innermost 4th colored ring to the outer 16th ring: B. × buttiana ‘Mahara’, B. × buttiana ‘Gautama’s Red’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘Los Banos Beauty’, B. glabra ‘White Stripe’, B. spectabilis ‘Flame’, B. spectabilis ‘Splendens’, B.× buttiana ‘Barbara Karst’, B.× buttiana ‘San Diego Red’, and B. × buttiana ‘Miss Manila’ sp. 1, respectively. Chloroplast genome similar and highly divergent locations are represented by continuous and interrupted track lines, respectively.

Fig 3. Distribution of SSRs in the 13 newly sequenced Bougainvillea chloroplast genomes.

(A) Numbers of different SSR types detected in the 13 chloroplast genomes. (B) Frequencies of SSRs in the LSC, IR and SSC regions. (C) Frequencies of identified SSR motifs in different repeat class types.

Fig 4. Analysis of long repeat sequences in the 13 newly sequenced Bougainvillea chloroplast genomes.

(A) Total numbers of four long repeat types. (B) Length distribution of long repeats in each sequenced chloroplast genome.

Fig 5. Codon contents of all protein-coding genes of 13 newly sequenced complete chloroplast genomes of the Bougainvillea cultivars.

(A) Codon content and codon usage of the 20 amino acids and stop codons of all protein-coding genes. Each histogram from left to right is shown for B. × buttiana ‘Mahara’, B. × buttiana ‘Gautama’s Red’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘Los Banos Beauty’, B. glabra ‘White Stripe’, B. spectabilis ‘Flame’, B. spectabilis ‘Splendens’, B.× buttiana ‘Barbara Karst’, B.× buttiana ‘San Diego Red’, and B. × buttiana ‘Miss Manila’ sp. 1, respectively. (B) Heatmap analysis of the codon distribution of all protein-coding genes in the 13 newly sequenced chloroplast genomes.

Fig 6. Comparison of the borders of the LSC, SSC, and IR regions among the 16 Bougainvillea chloroplast genomes.

The 13 newly sequenced Bougainvillea chloroplast genomes identified in this study are shown in blue.

Fig 7. Complete chloroplast genome comparison of the 16 Bougainvillea chloroplast genomes using B. × buttiana ‘Mahara’ as a reference.

The gray arrows and thick black lines above the alignment indicate gene orientation. Purple bars represent exons, sky-blue bars represent untranslated regions (UTRs), red bars represent non-coding sequences (CNS), gray bars represent mRNAs, and white regions represent sequence differences among the analyzed chloroplast genomes. The y-axis represents the identity percentage ranging from 50% to 100%. The 13 sequenced Bougainvillea chloroplast genomes in this study are shown in bold.

Fig 8. Comparisons of nucleotide diversity (Pi) values among 16 complete chloroplast genomes of Bougainvillea cultivars.

(A) Protein-coding genes. Protein-coding genes with Pi values > 0.0038 are labeled with gene names. (B) Intergenic regions. Intergenic regions with Pi values > 0.0113 are labeled with intergenic region names.

Fig 9. Phylogenetic relationships of Nyctaginaceae species based on chloroplast genomes sequences reconstructed using maximum likelihood (ML) and Bayesian inference (BI) methods.

(A) ML tree. (B) BI tree. The 13 newly sequenced Bougainvillea chloroplast genomes identified in this study are shown in bold.