Chloroplast genome of four Amorphophallus species: genomic features,comparative analysis, and phylogenetic relationships among Amorphophallus species

Abstract

Background: The genus Amorphophallus (Araceae) contains approximately 250 species, most of which have high ecological and economic significance. The chloroplast genome data and the comprehensive analysis of the chloroplast genome structure of Amorphophallus is limited. In this study, four chloroplast genomes of Amorphophallus were sequenced and assembled. For the first time, comparative analyses of chloroplast genomes were conducted on the 13 Amorphophallus species in conjunction with nine published sequences.

Results: The Amorphophallus chloroplast genomes exhibited typical quadripartite structures with lengths ranging from 164,417 to 177,076 bp. These structures consisted of a large single copy (LSC, 90,705 - 98,561 bp), a small single copy (SSC, 14,172 - 21,575 bp), and a pair of inverted repeats (IRs, 26,225 - 35,204 bp). The genomes contain 108 - 113 unique genes, including 76 - 79 protein-coding genes, 28 - 29 tRNA genes, and 4 rRNA genes. The molecular structure, gene order, content, codon usage, long repeats, and simple sequence repeats (SSRs) within Amorphophallus were generally conserved. However, several variations in intron loss and gene expansion on the IR-SSC boundary regions were found among these 13 genomes. Four mutational hotspot regions, including trnM-atpE, atpB, atpB-rbcL and ycf1 were identified. They could identify and phylogeny future species in the genus Amorphophallus. Positive selection was found for rpl36, ccsA, rpl16, rps4, rps8, rps11, rps12, rps14, clpP, rps3, ycf1, rpl20, rps2, rps18, rps19, atpA, atpF, rpl14, rpoA, rpoC1, rpoC2 and rps15 based on the analyses of Ka/Ks ratios. Phylogenetic inferences based on the complete chloroplast genomes revealed a sister relationship between Amorphophallus and Caladieae. All Amorphophallus species formed a monophyletic evolutionary clade and were divided into three groups, including CA-II, SEA, and CA-I. Amorphophallus albus, A. krausei, A. kachinensis and A. konjac were clustered into the CA-II clade, A. paeoniifolius and A. titanum were clustered into the SEA clade, A. muelleri 'zhuyajin1', Amorphophallus sp, A. coaetaneus, A. tonkinensis and A. yunnanensis were clustered into CA- I clade.

Conclusions: The genome structure and gene content of Amorphophallus chloroplast genomes are consistent across various species. In this study, the structural variation and comparative genome of chloroplast genomes of Amorphophallus were comprehensively analyzed for the first time. The results provide important genetic information for species classification, identification, molecular breeding, and evolutionary exploration of the genus Amorphophallus.

Keywords: Amorphophallus; Chloroplast genome; Genome comparison; Phylogenetic analysis.

Fig. 1

Species reference image of Amorphophallus. A A. krausei; B A. albus Yunnan; C-G A. muelleri ‘zhuyajin1’, C petiole detail, D leaf detail, E flower bud details before flowering, F and G inflorescence; H–L Amorphophallus sp, H petiole detail, I leaf detail, J flower bud details before flowering, K and L inflorescence

Fig. 2

Chloroplast genome maps of Amorphophallus with annotated genes. Genes within the circle are clockwise, while those beyond the circle are counterclockwise. Different colors indicate functional gene groups. The darker and lighter shades of gray in the inner circle represent the content of GC and AT, respectively

Fig. 3

Heat map analysis for relative synonymous codon usage (RSCU) values of all protein-coding genes of 13 complete chloroplast genomes in Amorphophallus. Red and blue indicates higher and lower RSCU values, respectively. The species in bold are sequenced in this study

Fig. 4

Analysis of repeats and SRRs in seven complete chloroplast genomes of the Amorphophallus. A Different types of repeats in each chloroplast genome. B Numbers of tandem repeats more than 30 bp long in each chloroplast genome. C Numbers of palindromic repeats more than 30 bp long in each chloroplast genome. D Numbers of forward repeats more than 30 bp long in each chloroplast genome. E Total numbers and different types of SSRs detected in each chloroplast genome. Mono: mononucleotide, Di: dinucleotide, Tri: trinucleotides, Tetra: tetranucleotide, Penta: pentanucleotide, Hexa: hexanucleotide. The species in bold are sequenced in this study

Fig. 5

Comparison of the LSC, SSC, and IR boundaries among 13 chloroplast genomes. The light blue, orange, and light green blocks indicate the LSC, IR, and SSC regions. JLB: junction of the LSC and the IRb; JSB: junction of the IRb and the SSC; JSA: connection of the IRa and the SSC; JLA: connection of the SSC and the IRb. The species in bold are sequenced in this study

Fig. 6

Comparison of the chloroplast genome sequences of 13 Amorphophallus species. A Sequence variation analysis generated with mVISTA. Gray arrows indicated the position and direction of each gene. Purple, blue, pink, and gray bars represent exons, untranslated regions (UTRs), non-coding sequences (CNS), and mRNA, respectively. The scales on the Y-axis represent the average percent identity of sequence similarity ranging from 50 to 100%. B Collinear block analyses of Amorphophallus genome. The white, black, green and colours blue represent protein-coding genes, tRNA genes, intron containing tRNA genes, and rRNA genes, respectively. The species in bold are sequenced in this study

Fig. 7

Sliding window analysis for the nucleotide diversity (Pi) of the whole chloroplast genomes for Amorphophallus species. Window length and step size are 600 bp and 200 bp, respectively. The y-axis represents the nucleotide diversity of each window; the X-axis represents the position of the window’s midpoint. The species in bold are sequenced in this study

Fig. 8

Comparison of non-synonymous (Ka)/synonymous (Ks) substitution ratios among 13 species of Amorphophallus. The species in bold are sequenced in this study

Fig. 9

Phylogenetic trees were constructed using the maximum likelihood (ML) based on the complete chloroplast genomes of 54 Araceae species. The numbers above the nodes indicate support values. The species in bold are sequenced in this study