Introduction

The Amorphophallus (Araceae) genus contains more than 170 species, mainly distributed throughout Asia and Africa. Twenty-six species were found in Sichuan, Chongqing, Yunan, Guizhou and Hubei Provinces in China1. Because the bulbs of Amorphophallus are rich in glucomannan, they have been widely used in food, medicine, the chemical industry and so on2. In general, the Amorphophallus genus produces starch and glucomannan, depending on the species. Much research has focused on in vitro propagation systems, due to the accumulation of pathogens from normal asexual reproduction, to increase the yield of Amorphophallus3,4. The nucleotide sequences (ITS1) and plastid sequences (rbcL and matK) revealed a new subgeneric delineation by large-scale phylogenetic analysis of Amorphophallus5.

The genome size of Amorphophallus is quite large, approximately 20 times larger than the rice genome6. Furthermore, large variation exists in the genomic sequences of Amorphophallus species. Therefore, sequencing the whole genome of Amorphophallus species is very difficult. Complete sequencing of chloroplast (cp) genomes is much easier to achieve in Amorphophallus species. The plant chloroplast is a key plastid involved in photosynthesis and carbon fixation7. Chloroplast genomes are more conserved than nuclear genomes and contain four important regions: a large single-copy (LSC) region, a small single-copy (SSC) region and a pair of inverted repeats (IRA, IRB)8. The cp genome contains important information and genetic markers for phylogenetic and taxonomic analyses between plant species and individuals9,10,11 because of the low rates of polymorphisms, indels and SNPs in cps. More than 800 cp genomes have been sequenced and deposited in the NCBI. The first cp genome was discovered in Zea mays12, and a complete sequence was determined in Nicotiana tabacum and Marchantia polymorpha13,14. A circular cp genome of Aquilaria sinensis was found to be 159,565 bp long and contained 82 protein-coding genes. Zhang et al. reported sequences for five Epimedium species cp genomes, which provided valuable genetic information for accurately identifying species and assisted in the utilization of Epimedium plants15. These complete cp genome sequences have been widely used in the development of molecular markers for phylogenetic research16,17. Because of the ability for intracellular gene transfer and the conservation, diversity, and genetic basis of chloroplasts, transgene development has allowed for the engineering of high-value agricultural or biomedical products18. With the advent of high-throughput sequencing technology, it has become both standard practice and inexpensive to obtain cp genome sequences.

In this study, for the first time, we sequenced the complete cp genomes of four major Amorphophallus species using high-throughput sequencing technology and the Illumina HiSeq2500 platform. This study had four aims: (1) determine the size range and structure of four Amorphophallus species cp genomes; (2) compare the variations of simple sequence repeats (SSRs) among four major Amorphophallus cp genomes; (3) examine the indels and SNPs among four major Amorphophallus cp genomes; (4) confirm the phylogenetic relationship among four Amorphophallus species, as well as other species, using the complete cp genomes. These results will provide valuable and basic sequence information for taxonomic study and the development of molecular markers for further species identification of Amorphophallus. After the completion of the whole cp genome sequence, it is possible to build a database of the species. Based on the differences in the gene sequences of the four cp genomes, a DNA barcode can easily be developed to allow for the building of an efficient platform for postgenomics species research, such as subsequent gene excavation and functional verification of DNA sequence information.

Results and Discussions

Organization of four chloroplast genomes

Approximately 2G of data for each cp genome was obtained with a 300 bp read length. Gap closing was based on the sequence of the complete cp genome from Colocasia esculenta (NC_016753)19. The chloroplast genome sequences of the four genomes ranged from 162,853 bp (A. bulbifer) to 167,424 bp (A. konjac) (Fig. 1, Table 1). The same typical quadripartite structure was displayed in the four cp genomes. Two IR regions (25,379-26,120 bp) were separated by an LSC region (90,467-92,660 bp) and an SSC region (21,628-22,839 bp) (Table 1). The IRB region was 39 bp longer than the IRA region in the A. konjac cp genome. The IR/LSC and IR/SSC borders of the four Amorphophallus cp genomes were compared (Fig. S1). The variation of the IR/LSC and IR/SSC borders was considered to be the primary mechanism causing the length differences of angiosperm cp genomes20. The GC content ranged from 35.39% to 35.90% for the four cp genomes (Table 1). These four Amorphophallus cp genome data were deposited in GenBank.

Figure 1
figure 1

Gene maps of the four Amorphophallus cp genomes. (A) A. albus, (B) A. bulbifer, (C) A. konjac, (D) A. muelleri. The annotation of the genome was performed using DOGMA. The genes that are drawn on the outside of the circle are transcribed clockwise, while those inside are transcribed counter clockwise. Genes belonging to different functional groups are color coded. Small single copy (SSC), large single copy (LSC), and inverted repeats (IRa, IRb) are indicated.

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Table 1 Summary of the sequencing data for the four Amorphophallus species.
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Divergence hotspots in four chloroplast genomes

The A. albus cp genome contains 113 genes, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The A. bulbifer cp genome contains 111 genes, including 78 protein-coding genes, 29 tRNA genes and 4 rRNA genes. Both the A. konjac and A. muelleri cp genomes contain 112 genes, comprising 79 protein-coding genes, 29 tRNA genes and 4 rRNA genes. All of the features are shown in Table 1 and annotated in Fig. 1. All these genes play different roles in the chloroplast, and the classification is shown in Table 2.

Table 2 List of genes encoded by the four Amorphophallus chloroplast genomes.
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The estimated deletion of some genes was detected in some Amorphophallus cp genomes (Table 3). The ycf1 gene was present in three cp genomes but not in the A. bulbifer cp genome. Another gene named trnL-CAA appeared in the A. albus and A. konjac cp genomes. The trnG-GCC gene was lost in the A. konjac cp genome. The accD gene was found only in the A. muelleri cp genome, and psbE was missing only in the A. konjac cp genome. The rpl2 and rpl23 genes were annotated in the IRA and IRB regions of the four cp genomes, but they were only found in the IRA region and were lost in the IRB region in the A. albus cp genome.

Table 3 Summary of genes estimate deletion in the four Amorphophallus cp genomes.
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In addition to some genes being deleted, variations in the copy numbers and intron numbers of some genes were also found in the four cp genomes. Eight protein-coding genes, four rRNA genes, nine tRNA genes and two putative genes were present in two copies. Moreover, trnT-GGU was found to have two copies only in the A. bulbifer and A. muelleri cp genomes. In addition, three copies of the rps12 gene were found. Fifteen genes contained introns, including four tRNA genes, ten protein-coding genes and one putative gene. The psbF and ycf2 genes containing one intron were only found in the A. muelleri cp genome. There were no introns in the clpP gene in the A. bulbifer and A. muelleri cp genomes, but there were two introns in this gene in the A. albus and A. konjac cp genomes, while ycf3 and infA had two introns in each of the four cp genomes. All of the above divergences are shown in Table 2. The development of molecular markers for the identification of Amorphophallus species was much easier based on the divergence hotspot regions of the four Amorphophallus cp genomes.

COG analysis

COG (clusters of orthologous groups of proteins) and KOG (eukaryotic ortholog groups) are based on the relationship between orthologous genes in the NCBI annotation system for prokaryotes and eukaryotes21, respectively. Homologous genes from different species can be divided into different ortholog clusters combining evolutionary relationships. There are 4,873 categories in COG and 4,852 in KOG. Genes that are orthologs have the same function, and the functional annotation can be inferred to other members of the same COG/KOG clusters. All of the genes from the four cp genomes were classified into six categories: energy production and conversion; translation, ribosomal structure and biogenesis; posttranslational modification, protein turnover and chaperones; transcription; carbohydrate transport and metabolism; and lipid metabolism. The number of genes classified under each function in the four Amorphophallus genomes is shown in Fig. S2.

SSR polymorphisms and SNP/Indel analysis

SSRs are important molecular markers for plant evolutionary and ecological studies15, and they are widely present in the cp genome. With MISA analysis, 134–164 SSRs were detected in the four cp genomes (Table 4). Among these SSRs, mono-, di-, tri-, tetra-, and hexanucleotides were detected. The mononucleotide SSRs were most common, with 70.15% of the SSRs observed in A. bulbifer. In addition, most of the mononucleotide SSRs were composed of A and T repeat units, and the majority of the dinucleotides were composed of AT and TA. The cp SSRs are normally composed of short polyA or polyT repeats22. Higher contents of A/T and AT/TA repeats in cp SSRs were also detected in the Metasequoia glyptostroboides cp genome23. Hexanucleotide repeat unit SSRs were in the A. muelleri cp genome only at a portion of 2.08%. In short, the cp SSRs represented rich variation and were absolutely useful for polymorphism analysis in the Amorphophallus species.

Table 4 Simple sequence repeats (SSRs) in the four Amorphophallus species cp genomes.
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Using the A. albus cp genome as the reference sequence, we compared the SNP/indel loci of the four cp genomes. SNP markers were detected in 65 protein-coding genes in A. bulbifer, A. konjac and A. muelleri cp genomes. Eleven genes were in the SSC region, and 54 genes were in the LSC region, indicating that the protein-coding genes in the IR region were more conserved. These 65 genes were divided into four categories according to their different functions in plant chloroplasts, including photosynthetic apparatus, photosynthetic metabolism, gene expression, and other genes. Nine hundred sixty-nine and 943 SNP markers were detected between A. albus and A. bulbifer in protein-coding genes and noncoding regions, respectively. One hundred and four and 176 SNP markers were detected between A. albus and A. konjac in protein-coding genes and noncoding regions, respectively. Nine hundred and seventy-eight and 926 SNP markers were detected between A. albus and A. muelleri in protein-coding genes and noncoding regions, respectively. The SNPs in the A. konjac cp genome were significantly fewer than those in the A. bulbifer and A. muelleri cp genomes. One hundred and fifty-nine SNP sites were found in Oryza. sativa and Oryza. nivara chloroplast genomes24, 591 SNP markers were detected between the Solanum tuberosum and S. bulbocastanum plastomes25, and 464 were detected between the plastomes of P. ginseng and P. notoginseng26.

All of the SNPs were classified into two types, including synonymous (S) and nonsynonymous (N) (Table 5, Fig. 2). For the 969 and 978 SNP markers in the gene coding regions of the A. bulbifer and A. muelleri cp genomes, respectively, 696 and 708 belonged to the nonsynonymous type, and 273 and 270 belonged to the synonymous type. Synonymous and nonsynonymous SNP makers from the gene coding genes shared very similar numbers in these two cp genomes. There were 32 synonymous SNPs and 72 nonsynonymous SNPs in the protein coding regions of the A. konjac cp genome. Forty-eight nonsynonymous and 47 synonymous SNP sites were detected in the Machilus cp genome, implying that a substitution constraint mechanism existed27. Genes ycf3, rpoC1 and clpP were detected with SNP markers in their introns. Six, 1 and 6 SNP markers were found in one intron from rpoC1; 6, 1 and 5 SNP markers were found in one intron from ycf3; 23, 7 and 25 SNP markers were found in two introns from clpP in the A. bulbifer, A. konjac and A. muelleri cp genomes, respectively. ClpP and ycf1 were the variation hotspots for SNPs and indels, and they were usually used for investigating sequence variation in seed plants28,29.

Table 5 Comparisons of mutation changes, number of synonymous (S) and nonsynonymous (N) substitutions per gene of protein coding cp genes among A. bulbifer, A. konjac and A. muelleri.
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Figure 2
figure 2

SNPs statistics of A. bulbifer, A. konjac and A. muelleri cp genomes. The Amorphophallus albus cp genome was used as the reference sequence for SNPs analyses for the other three cp genomes. SNPs belonging to different type groups are color coded. (A) Number of SNPs in the A. bulbifer cp genome sequence. (B) The number of SNPs in the A. konjac cp genome sequence. (C) The number of SNPs in the A. muelleri cp genome sequence.

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cpSSR and SNP markers will be useful in testing maternal inheritance of the cp genome, identifying species differentiation and even in breeding programs30. cpSSRs have been demonstrated to be useful in gene flow studies to estimate seed and pollen contribution31 and in phylogeographic analyses32.

Twenty-two protein-coding genes from three Amorphophallus cp genomes contained indels (Table 6). Only two coding genes were detected to contain indels in the A. konjac cp genome; one indel was in rps15, and two indels existed in ycf1. The indel numbers of each coding gene from the A. bulbifer and A. muelleri cp genomes are shown in Fig. 3A,B. The gene ycf1 was a hotspot for indel variation, and almost half of the number of indels existed in this gene (Fig. 3). Such mutational loci in cp genomes showed highly variable regions in the genomes.

Table 6 Comparisons of InDels of protein coding cp genes among A. bulbifer, A. konjac and A. muelleri.
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Figure 3
figure 3

InDels statistics of A. bulbifer and A. muelleri cp genomes. The Amorphophallus albus cp genome was used as the reference sequence for InDels analyses for the other three cp genomes. InDels belonging to different coding genes are color coded. Only three InDels were detected in the A. konjac cp genome, so the statistics results are shown in the main text. (A) The number of InDels of each coding gene in the A. bulbifer cp genome sequence, (B) The number of InDels of each coding gene in the A. muelleri cp genome sequence.

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ndhK regression in four Amorphophallus species cp genomes

The ndhK gene was a new gene represented in the four Amorphophallus species cp genomes. It was 744 bp in length in the A. albus and A. konjac cp genomes, and 741 bp in length in the A. bulbifer and A. muelleri cp genomes. The gene ndhK is present in a novel protein complex of the thylakoid membrane and shows homology to a mitochondrial gene that encodes a subunit of the NADH-ubiquinone oxidoreductase of the mitochondria33. ndhK was reported as a gene encoding a subunit of PSII, but later, this protein was classified as a subunit of NADH dehydrogenase, and the gene has been renamed ndhK34,35. In many plants, such as Glycine max, Epimedium acuminatum, Psilotum nudum, Machilus yunnanensis, Actinidia chinensis, Veronica persica, and Aquilaria sinensis (Lour.) Gilg., ndhK was lost from their cp genomes15,18,27,36,37,38,39. ndhK has been found in the Paramecium aurelia mitochondrial (mt) genome40. The presence of this gene in the mt genome raises interesting questions concerning its evolutionary origin. The gene ndhK may play a crucial role in photosynthesis in four Amorphophallus species, and its presence in the cp genomes can be used as a marker for distinguishing them from other family species.

Phylogenetic analysis

Phylogenetic analysis of Amorphophallus species has been reported using different aspects, such as several chloroplast genes41, two chloroplast genes, leafy intron sequences42, plastid DNA markers and fingerprinting43. These studies simply demonstrated Amorphophallus sample species relationships and did not include the four Amorphophallus species that are the major commercial cultivation species used in our study. In addition, whole chloroplast sequences were much more accurate than individual gene sequences for phylogenetic analysis. In the present study, complete cp genomes sequences of four Amorphophallus species and other plants (Table S1) were used to perform phylogenetic analyses (Fig. 4). The clade of the four species of Amorphophallus was grouped with other Araceae species as expected. A. albus and A. konjac were clustered into one clade, and A. bulbifer and A. muelleri were clustered into another clade. These results showed that A. albus and A. konjac had a close relationship, and A. bulbifer and A. muelleri were closely related. The matK and rbcL genes were also used for phylogenetic analysis among the Amorphophallus genus (Figs S3 and S4). Both of the phylogenetic trees indicated that the Amorphophallus species were grouped into three major clades named Africa, southeast Asia, and Continental Asia. The Continental Asia clade covered the taxa distributed from India to China and Thailand, which were subdivided into two subclades, Continental Asia I and II. The four Amorphophallus species in our study were all derived from the Chinese mainland; A. albus and A. konjac were grouped as Continental Asia I, and A. bulbifer and A. muelleri were grouped as Continental Asia II. The first two species came from the central region of China, and the other two species were collected from the southern region of China near Burma. The matK and rbcL genes well supported clades in consensus trees and the resolution of ingroup relationships within Amorphophallus44. All the results suggested that the relationship in Amorphophallus was consistent with the biogeographical distribution. A. konjac and A. bulbifer were also classified in two different clades by Sedayu42. A. albus and A. konjac have the same chromosome number (2N = 2X = 26), while A. bulbifer and A. muelleri are triploid (3N = 3X = 39). The propagation coefficient of A. albus and A. konjac did not exceed single digits, while the propagation coefficient in A. bulbifer and A. muelleri increased significantly because of aerial bulbs growing in the stems. The aerial bulbs diminish the need for sexual reproduction and lead to a significantly increased reproductive capacity. In many cases, the evolutionary process is closely linked with the reproduction system of the species. A. muelleri and A. bulbifer reproduce, thus far, through apomictic processes. The corm of A. bulbifer is light red, and that of A. muelleri is light yellow. These phenotypes also demonstrated the relationship among the four Amorphophallus species. The sequenced cp genomes of the four Amorphophallus species provide a large amount of genetic information for phylogenetic analysis and taxonomic study.

Figure 4
figure 4

Phylogenetic tree based on 30 complete cp genome sequences.

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Conclusion

We sequenced the chloroplast genomes of four Amorphophallus plants: A. albus, A. bulbifer, A. konjac, and A. muelleri. We annotated the four cp genomes and analyzed the structural divergence among the four cp genomes; moreover, we identified the SSR loci and SNPs in protein-coding genes. These SSRs and SNPs could be selected for use in developing markers and in phylogenetic analysis. Comparing the cp genomes among some plants suggested that psbG regressed in the A. albus, A. konjac, A. bulbifer and A. muelleri cp genomes. We also detected that some genes and introns were lost, in addition to copy differences of some genes among the four cp genomes. The results of SNP detection demonstrated that very few of the SNPs were identified between the A. albus and A. konjac cp genomes; on the contrary, a large number of SNPs between A. bulbifer and A. muelleri were identified when the A. albus cp genome was used as the reference sequence. Interestingly, the SNPs were almost the same in the A. bulbifer and A. muelleri cp genomes. The indel results were very similar between A. albus and A. konjac because only three indels were detected in the A. konjac cp genome. In addition, phylogenetic analysis using complete cp genome sequences showed that A. albus and A. konjac were in a clade and A. bulbifer and A. muelleri were in a different clade. The clustering analysis results verified the results of the SNP data. All the data will be very helpful in further research on Amorphophallus plants and chloroplasts and in expanding our understanding of the evolutionary history of the Amorphophallus cp genomes. All of these divergences in the four cp genomes were significant for taxonomic and evolutionary studies, as well as for genetic engineering developments in the future.

Methods

Plant material preparation and sequencing

Fresh young leaves of A. albus, A. bulbifer, A. konjac and A. muelleri were collected from live individuals at the greenhouse of Wuhan University in China. Five micrograms of cp DNA was isolated from leaves and sheared into 300 bp DNA fragments using a Covaris M220 (Covaris, United States). NEB Next ® UltraTM DNA Library Prep Kit for Illumina (NEB, United States) was used to build the library after DNA fragmentation. The genomic DNA of four species was sequenced on a single HiSeq2500 flow cell lane (Illumina Inc.) by the Chinese National Human Genome Center (http://www.chgc.sh.cn/), Shanghai, China.

Plant cp genome assembly and annotation

Trimmomatic v 0.3245 was used for raw data processing, and the resulting clean data were used for assembly and post analysis. Fastqc v0.10.046 was used to evaluate the quality of the data visually. Velvet v1.2.0747 was used to assemble the clean data, and the complete chloroplast genome sequence was obtained after gap closing. DOGMA48 was used to annotate the cp genomes and predict the rRNA/tRNA of A. albus, A bulbifer, A. konjac, and A. muelleri. COGs (clusters of orthologous groups of proteins) were analyzed through rpsblast v2.2.30+49. The circular cp genome maps were drawn using the OrganellarGenomeDRAW program50.

Mutation events analysis

To compare the mutations among the four complete cp genomes, MISA and MUMMER 3.23 software was used for SSR and SNP/indel analyses, respectively. The A. albus cp genome was used as a reference sequence for SNP/indel analyses. Definition of microsatellites (unit size/minimum number of repeats): (1/10) (2/5) (3/4) (4/4) (5/4) (6/4).

Phylogenetic analysis

We selected twenty-six cp genomes (Table S1), representing the nine families, for phylogenetic analysis. The matK and rbcL genes were used for phylogenetic analysis among the Amorphophallus genus, and the selected species are shown in Tables S2 and S3. MEGA 6.06 software was used for building the evolutionary tree. The analysis was carried out based on the complete cp DNA sequences.