# Comparative Chloroplast Genomes ofSorghum Species: Sequence Divergence and Phylogenetic Relationships

Authors: Yun Song; Yan Chen; Jizhou Lv; Jin Xu; Shuifang Zhu; MingFu Li
Journal: BioMed Research International
 (2019)

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## Abstract

Sorghum comprises 31 species that exhibit considerable morphological and ecological diversity. The phylogenetic relationships among Sorghum species still remain unresolved due to lower information on the traditional DNA markers, which provides a limited resolution for identifyingSorghum species. In this study, we sequenced the complete chloroplast genomes ofSorghum sudanense andS. propinquum and analyzed the published chloroplast genomes ofS. bicolor andS. timorenseto retrieve valuable chloroplast molecular resources forSorghum. The chloroplast genomes ranged in length from 140,629 to 140,755 bp, and their gene contents, gene orders, and GC contents were similar to those for other Poaceae species but were slightly different in the number of SSRs. Comparative analyses among the four chloroplast genomes revealed 651 variable sites, 137 indels, and nine small inversions. Four highly divergent DNA regions (rps16-trnQ,trnG-trnM,rbcL-psaI, andrps15-ndhF), which were suitable for phylogenetic and species identification, were detected in theSorghum chloroplast genomes. A phylogenetic analysis strongly supported thatSorghum is a monophyletic group in the tribe Andropogoneae. Overall, the genomic resources in this study could provide potential molecular markers for phylogeny and species identification inSorghum.


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## Body

## 1. Introduction

Sorghum bicolor (L.) Moench, sorghum, is the fifth in both production and planted area of cereal crops worldwide. It is extensively cultivated in marginal rainfall areas of the tropics and subtropics. The wild species of sorghum represent a potentially diverse source of germplasm for sorghum breeding programs. Sorghum comprises 31 species that exhibit considerable morphological and ecological diversity [1–3]. The genusSorghum has been taxonomically classified into five subgenera or sections:Chaetosorghum,Heterosorghum,Parasorghum, Stiposorghum, andSorghum[3]. Phylogenies based on a sequence analysis suggest that theSorghum subgenera or section designations may not correspond to evolutionary relationships [1, 4, 5]. The phylogenetic relationships within subgenera or sections ofSorghum are not clear, and little is known about the phylogenetic relationships among the species.To determine the phylogenetic relationships ofSorghum, molecular markers, including chloroplast genome regions (such asndhF,psbZ-trnG,trnY-trnD,trnY-psbM, andtrnT-trnL), and multiple nuclear genes (ITS, Pepc4, and GBSSI) have been analyzed [4–9]. However, many relationships within the genus remain unresolved because these markers are of low diversity and only provide a limited resolution for identifying closely related taxa. The development of more effective genetic resources is necessary to infer phylogenic relationships and to identify the species ofSorghum.In recent years, an increasing number of researchers have focused on the chloroplast genome to develop genetic markers for phylogeny and DNA barcoding. In general, chloroplast genomes are in the range of 120-160 kb in length and encode 120 to 130 genes [10]. The chloroplast genome has a conserved quadripartite structure that consists of a large single-copy region (LSC) and a small single-copy region (SSC), which are separated by a pair of inverted repeats (IRs). Moreover, chloroplast genomes are inherited uniparentally (maternally in most angiosperms plants) at a slower evolutionary rate of change compared to nuclear genomes. For these reasons, the chloroplast genome is a potentially useful tool for phylogenetic studies, population genetics, phylogeography, and species identification. Mutations in the chloroplast genome are clustered as mutation hotspots, and this mutational dynamic has resulted in highly variable regions in the genome [11]. Those variable regions are used for phylogeny and species identification [12, 13].In this study, we sequenced the complete chloroplast genomes ofS. sudanense andS. propinquum which belong to subgenera ofSorghum and compared the resulting sequences with the published chloroplast genome ofS. bicolor [14] andS. timorense (GenBank accession number: KF998272). The objective was to compare the chloroplast genomic structure and sequence variation within the genusSorghum to retrieve valuable chloroplast molecular markers for species identification and to clarify the phylogenetic relationship of the tribe Andropogonodae.


## 2. Materials and Methods

### 2.1. Plant Materials, DNA Extraction, and Sequencing

The plant materials ofS. sudanense andS. propinquum were provided by the National Grass Germplasm Bank of China. Fresh leaves from each species were immediately dried with silica gel prior to DNA extraction. The total genomic DNA was isolated from each individual plant using the mCTAB extraction protocol [15] and was purified using the Wizard DNA CleanUp System (Promega, Madison, WI, USA). The total DNA quantity was evaluated by the value of the ratio of absorbance measurements at 260 nm and 280 nm (A260/A280) using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), whereas a visual assessment of the DNA size and integrity was performed using gel electrophoresis. We identified the materials using the ITS sequences. The ITS sequencing methods followed Ng'uni et al. [6] and the ITS sequences were submitted to GenBank (accession numbers: MK514589 and MK514590).The chloroplast genomes ofS. sudanense andS. propinquum were sequenced using the long-range PCR method reported by Donget al. [10]. The PCR protocol was as follows: preheating at 98°C for 2 min, 40 cycles at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 5 min, followed by a final extension at 72°C for 10 min. PCR amplification was performed in an Applied Biosystems Veriti™ 96-Well Thermal Cycler (Model #: 9902, made in Singapore).PCR products were randomly fragmented into 400–600 bp using an ultrasonicator. An Illumina paired-end DNA library with a 500 bp insert size was constructed using a NEBNext® Ultra™DNA Library Prep Kit following the manufacturer’s instructions. The library was sequenced by the Illumina Hiseq X Ten platform double terminal sequencing method.


### 2.2. Assembly and Annotation

The paired-end reads were qualitatively assessed and were assembled using SPAdes 3.6.1 [16]. Chloroplast genome sequence contigs were selected from the initial assembly by performing a BLAST search using theSorghum bicolor chloroplast genome sequence as a reference (GenBank accession number: EF115542). The selected contigs were assembled with Sequencher 5.4.5 (http://www.genecodes.com). The gaps and ambiguous sequences were manually adjusted after Sanger sequencing. PCR amplification and Sanger sequencing were performed to verify the four junction regions between the IRs and the LSC/SSC [17]. The chloroplast genome annotation was performed with Plann [18] using theSorghum bicolorreference sequence from GenBank. The chloroplast genome map was drawn using Genome Vx software [19].


### 2.3. Simple Sequence Repeat (SSR) Analysis

Perl script MISA (http://pgrc.ipk-gatersleben.de/misa/) was used to detect the chloroplast simple sequence repeats in four chloroplast genome sequences ofSorghum. Its parameters were set as follows: the minimum numbers of repeats for mononucleotide, dinucleotides, trinucleotides, tetranucleotides, pentanucleotide, and hexanucleotides were 10, 5, 4, 3, 3, and 3, respectively. At the same time, the SSR of the IR, LSC, SSC, and coding regions, introns, and intergenic regions that correspond to different regions were analyzed.


### 2.4. Variation Analyses

All sequencedSorghum chloroplast genomes were aligned using MIFFT v7 [20]. SNPs and the microstructure (indels and inversions) were checked in the fourSorghum chloroplast genomes. The SNPs were calculated using MEGA 6.0 [21]. Based on the aligned sequence matrix, the indel events were checked manually and were further divided into two categories: microsatellite-related indels (SSR-indel) and non-microsatellite-related indels (NR-indel).Using theS. sudanense chloroplast genome sequence as the standard reference, the size, location, and evolutionary direction of the microstructure events were counted. The proposed secondary structures of the inverted regions were analyzed using mfold software [22].


### 2.5. Molecular Marker Development

A sliding window analysis was conducted to generate nucleotide diversity of the chloroplast genome using DnaSP v5.10 software [23]. The step size was set to 100 bp with a 600 bp window length.


### 2.6. Phylogenetic Reconstruction

To investigate the phylogenetic position ofSorghum, we used 41 complete chloroplast genomes (Table S1). Among them, 36 were from Andropogonodae, and five other species from different tribes (Garnotia tenella, Centotheca lappacea,Chasmanthium laxum,Gynerium sagittatum, andPseudolasiacis leptolomoides) were used as the outgroups. Sequence alignments were carried out using MIFFT v7 [20] and then were adjusted manually using Se-Al 2.0. [24].Phylogenetic analyses were conducted using the maximum likelihood (ML) and the Bayesian inference (BI) methods. The ML analysis was conducted using RAxML version 8.0.20 with 500 bootstrap replicates. The GTRGAMMA model was used in all of the ML analyses as is suggested in the RAxML manual.MrBayes 3.2.2 [25] was used to perform a Bayesian inference analysis. The Markov chain Monte Carlo (MCMC) analysis was run for 2 × 5,000,000 generations. The average standard deviation of split frequencies remained below 0.01 after the fifty percent burn-in. The remaining trees were used to build a 50% majority-rule consensus tree.


## 2.1. Plant Materials, DNA Extraction, and Sequencing

The plant materials ofS. sudanense andS. propinquum were provided by the National Grass Germplasm Bank of China. Fresh leaves from each species were immediately dried with silica gel prior to DNA extraction. The total genomic DNA was isolated from each individual plant using the mCTAB extraction protocol [15] and was purified using the Wizard DNA CleanUp System (Promega, Madison, WI, USA). The total DNA quantity was evaluated by the value of the ratio of absorbance measurements at 260 nm and 280 nm (A260/A280) using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), whereas a visual assessment of the DNA size and integrity was performed using gel electrophoresis. We identified the materials using the ITS sequences. The ITS sequencing methods followed Ng'uni et al. [6] and the ITS sequences were submitted to GenBank (accession numbers: MK514589 and MK514590).The chloroplast genomes ofS. sudanense andS. propinquum were sequenced using the long-range PCR method reported by Donget al. [10]. The PCR protocol was as follows: preheating at 98°C for 2 min, 40 cycles at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 5 min, followed by a final extension at 72°C for 10 min. PCR amplification was performed in an Applied Biosystems Veriti™ 96-Well Thermal Cycler (Model #: 9902, made in Singapore).PCR products were randomly fragmented into 400–600 bp using an ultrasonicator. An Illumina paired-end DNA library with a 500 bp insert size was constructed using a NEBNext® Ultra™DNA Library Prep Kit following the manufacturer’s instructions. The library was sequenced by the Illumina Hiseq X Ten platform double terminal sequencing method.


## 2.2. Assembly and Annotation

The paired-end reads were qualitatively assessed and were assembled using SPAdes 3.6.1 [16]. Chloroplast genome sequence contigs were selected from the initial assembly by performing a BLAST search using theSorghum bicolor chloroplast genome sequence as a reference (GenBank accession number: EF115542). The selected contigs were assembled with Sequencher 5.4.5 (http://www.genecodes.com). The gaps and ambiguous sequences were manually adjusted after Sanger sequencing. PCR amplification and Sanger sequencing were performed to verify the four junction regions between the IRs and the LSC/SSC [17]. The chloroplast genome annotation was performed with Plann [18] using theSorghum bicolorreference sequence from GenBank. The chloroplast genome map was drawn using Genome Vx software [19].


## 2.3. Simple Sequence Repeat (SSR) Analysis

Perl script MISA (http://pgrc.ipk-gatersleben.de/misa/) was used to detect the chloroplast simple sequence repeats in four chloroplast genome sequences ofSorghum. Its parameters were set as follows: the minimum numbers of repeats for mononucleotide, dinucleotides, trinucleotides, tetranucleotides, pentanucleotide, and hexanucleotides were 10, 5, 4, 3, 3, and 3, respectively. At the same time, the SSR of the IR, LSC, SSC, and coding regions, introns, and intergenic regions that correspond to different regions were analyzed.


## 2.4. Variation Analyses

All sequencedSorghum chloroplast genomes were aligned using MIFFT v7 [20]. SNPs and the microstructure (indels and inversions) were checked in the fourSorghum chloroplast genomes. The SNPs were calculated using MEGA 6.0 [21]. Based on the aligned sequence matrix, the indel events were checked manually and were further divided into two categories: microsatellite-related indels (SSR-indel) and non-microsatellite-related indels (NR-indel).Using theS. sudanense chloroplast genome sequence as the standard reference, the size, location, and evolutionary direction of the microstructure events were counted. The proposed secondary structures of the inverted regions were analyzed using mfold software [22].


## 2.5. Molecular Marker Development

A sliding window analysis was conducted to generate nucleotide diversity of the chloroplast genome using DnaSP v5.10 software [23]. The step size was set to 100 bp with a 600 bp window length.


## 2.6. Phylogenetic Reconstruction

To investigate the phylogenetic position ofSorghum, we used 41 complete chloroplast genomes (Table S1). Among them, 36 were from Andropogonodae, and five other species from different tribes (Garnotia tenella, Centotheca lappacea,Chasmanthium laxum,Gynerium sagittatum, andPseudolasiacis leptolomoides) were used as the outgroups. Sequence alignments were carried out using MIFFT v7 [20] and then were adjusted manually using Se-Al 2.0. [24].Phylogenetic analyses were conducted using the maximum likelihood (ML) and the Bayesian inference (BI) methods. The ML analysis was conducted using RAxML version 8.0.20 with 500 bootstrap replicates. The GTRGAMMA model was used in all of the ML analyses as is suggested in the RAxML manual.MrBayes 3.2.2 [25] was used to perform a Bayesian inference analysis. The Markov chain Monte Carlo (MCMC) analysis was run for 2 × 5,000,000 generations. The average standard deviation of split frequencies remained below 0.01 after the fifty percent burn-in. The remaining trees were used to build a 50% majority-rule consensus tree.


## 3. Results and Discussion

### 3.1. Features ofSorghum Chloroplast Genomes

The plastomes of the four species contain no significant differences in their contents of genes and introns, the gene order in the four genomes is identical, and the sizes of LSC, SSC, and IR regions are very similar. The overall GC content of the chloroplast genome is 38.5%, which is consistent with reported Poaceae species [12, 26].A total of 110 unique genes were identified in theSorghum chloroplast genome, including 77 protein-coding genes, 29 tRNA genes, and 4 ribosomal RNA genes (Figure 1, Table 1 and Table S2). Notably, seven protein-coding genes (rps15,rps12,rps7,ndhB,rpl23,rpl2, andrps19) eight tRNA genes (trnA-UGC,trnH-GUG,trnI-CAU,trnI-GAU,trnL-CAA,trnN-GUU,trnR-ACG, andtrnV-GAC), and all of the rRNA genes are duplicated in the IR regions, which is common in most Poaceae genomes. In theSorghum chloroplast genome, there were 18 intron-containing genes. Among them, ten protein-coding genes (petB,petD,atpF,ndhB,ndhA,rpoC1,rps12,rps16,rpl16, andrpl2) and six tRNA genes have a single intron and two genes (clpP andycf3) that contained two introns. Therps12 is a trans-splicing gene, with the 5′ end located in the LSC region and the duplicated 3′ end located in the IR region. ThematKwas located within the intron oftrnK-UUU.Table 1
Details of the complete chloroplast genomes of the fourSorghumspecies.
Genome features S. sudanense S. propinquum S. bicolor S. timorense Size (bp) 140755 140642 140754 140629 LSC length (bp) 82686 82572 82685 82587 IR length (bp) 22783 22782 22783 22752 SSC length (bp) 12503 12506 12503 12538 Total genes 110 110 110 110 Protein coding genes 77 77 77 77 tRNA genes 29 29 29 29 rRNA genes 4 4 4 4 Overall GC content (%) 38.5 38.5 38.5 38.5 Accession number in GenBank MH926028 MH926027 EF115542 KF998272Figure 1
Map of the Sorghum chloroplast genome. The genes inside and outside the circle are transcribed in the clockwise and counterclockwise directions, respectively. Genes belonging to different functional groups are shown in different colors. Thick lines indicate the extent of the inverted repeats (IRa and IRb) that separate the genomes into small single-copy (SSC) and large single-copy (LSC) regions.


### 3.2. Simple Sequence Repeats

Simple sequence repeats (SSRs) are a type of 1–6 nucleotide unit tandem repeat sequence that is frequently observed in chloroplast genomes. These are important molecular markers for plant population genetics, evolution, and ecological studies because of their high diversity in copy numbers within species due to slipped strand mispairing during DNA replication on a single DNA strand [27, 28].There were 38, 41, 41, and 45 simple sequence repeats in the chloroplast genomes ofS. timorense,S. bicolor,S. sudanense, andS. propinquum, respectively (Figure 2, Table S3). The mononucleotide SSRs were the richest, with a proportion of 60.61%, followed by dinucleotide SSRs (14.55%), tetranucleotide SSRs (19.39%), and trinucleotide SSRs (4.24%). One hexanucleotide SSR was found inSorghum sudanense andSorghum bicolor. Pentanucleotide was not detected in the Sorghum chloroplast genomes. The majority of SSRs in all species were A/T mononucleotides. Chloroplast genome SSRs were composed of adenine or thymine repeats and rarely contained tandem guanine (G) or cytosine (C) repeats. The majority of SSRs were located in the LSC region (71.52). Furthermore, most of the SSRs were found in space regions (73.33%), followed by exon regions (16.97%) and intron regions (9.70%). SSRs in the chloroplast genome have been shown to be extremely useful for resolving genetic diversity between closely related taxa and, hence, increase the power of interspecific studies [29, 30], possibly in combination with other informative nuclear genome SSRs.Figure 2
Analyses of simple sequence repeat (SSR) in the four Sorghum chloroplast genomes. (a) Number of different SSRs types detected by MISA; (b) number of SSRs in LSC, SSC, and IR regions; (c) number of SSRs in spacer, exon, and intron; (d) frequency of identified SSR motifs in the different repeat classes.
(a) (b) (c) (d)


### 3.3. Numbers and Pattern of SNP Mutations

In total, 651 single nucleotide substitutions (SNP) were detected in the fourSorghum chloroplast genomes, 518 of which were found in the LSC region, 18 in the IR region, and 97 in the SSC region. The number of SNP among the fourSorghumspecies was found to be 3 to 631.S. timorenseexhibits higher divergence than other three species.S. sudanenseandS. bicolor show the lowest sequence divergence.The pattern of SNP mutation is shown in Figure3. There were 345 transitions (Ts) and 306 transversions (Tv) and the Tv to Ts ratio was 1:0.89, which indicated a bias in favor of transitions. The most frequently occurring mutations were from A to G and from T to C substitutions (179), while from C to G and from G to C exhibited the lowest frequency (30). Despite the higher A+T contents in chloroplast genomes, AT to TA transversions among the four types of transversions were found to occur significantly less frequently (Figure 3). It is clear that there is a bias in the chloroplast genomes [31].Figure 3
The patterns of nucleotide substitutions among the four Sorghum chloroplast genomes. The patterns were divided into six types as indicated by the six non-strand-specific base-substitution types (i.e., numbers of considered G to A and C to T sites for each respective set of associated mutation types).


### 3.4. Indels

There were 137 indels in the chloroplast genome, which was identified among the fourSorghum chloroplast genomes (Tables S4 and S5), including 43 indels that are caused by SSR variations (SSR-indels) and 94 non-SSR-related indels (NR-indels). The majority of SSR-indels were related to A/T types SSRs (39 times). Only one dinucleotide SSR indel was identified, which is located inndhF-rpl32. All of the SSR-indels were found in the noncoding regions of the LSC/SSC section.The size of NR-indels ranged from 1 to 165 bp, with one bp long indel and 5 bp long indels being the most common (TableS5 and Figure 4). The largest one, found inrpoC with 165 bp length, was a deletion in theS. sudanense. The second longest, which was found inrps16-trnQwith 152 bp length, was an insertion inS. timorense. Finally, 46 insertion indels and 42 deletion indels were specific toS. timorense, one insertion indel and two deletion indels were specific toS. sudanense, and one insertion inrpoC1 intron was specific toS. propinquum.Most of the NR-indels were located in noncoding regions (81.91% in space and 15.96% in introns).Figure 4
NR-indels identified in the chloroplast genomes of the four Sorghum chloroplast genomes . (a) Numbers of individual NR-indels shown by sequence length; (b) relative frequency of NR-indel occurrence in LSC, SSC, and IR regions.
(a) (b) (c)Indels were another important class of genetic variation compared with nucleotide substitutions. Several molecular processes are known to create indels. Polymerase slippage processes during DNA replication or repair can result in the addition or deletion of short spans of sequence that repeat at one side of the region flanking the indel [32], which mainly created SSR-indel type. SSR-indels in chloroplast genome were primarily found in AT-regions and often involve long stretches of repeats of a single nucleotide [33]. In theSorghum chloroplast genome, most of the SSR-indels (90.70%) were A/T types. Hairpins or the stem-loop secondary structure and intramolecular recombination are thought to cause the majority of NR-indel mutations [33]. Different types of indels also show varying amounts of homoplasy. SSR-indels seem to be more prone to homoplasy between different species [28, 34]. In this study, NR-indels were often less homoplasious (Table S5). An increasing number of studies have shown that indel characters can be extremely useful for inferring relationships among more closely related taxa [30, 35, 36].


### 3.5. Small Inversions

Nine small inversions of 2 to 6 bp were identified in theSorghum chloroplast genomes (Table 2). Eight inversions occurred in the LSC region, and one occurred in the SSC region. Most of the small inversions are in intergenic spacer regions, with only two exceptions. One is a 4 bp inversion within the coding region ofccsA, and the other is a 4 bp inversion in therpl16 intron. All of the inversions and their inverted repeating flanking sequences can form stem-loop structures. The franking repeats are from 3 to 20 bp in length. All inversions occurred inS. timorense except the inversion in ccsA, which occurred inS. sudanense.Table 2
The location, direction, and length of nine small inversions in the fourSorghumchloroplast genomes.
Length of inversions (bp) Direction of the small inversions Location Length of inversion Length of inverted repeat S. sudanense S. propinquum S. bicolor S. timorense rps16-trnQ 2 12 no no no yes trnT-trnE 2 6 no no no yes trnT-trnE 2 5 no no no yes psbM-petN 6 14 no no no yes rbcL-rpl32 6 8 no no no yes petA-psbJ 6 14 no no no yes rpl33-rps18 2 3 no no no yes rpl16 intron 4 8 no no no yes ccsA 4 20 no yes yes yesMany small inversions may have been generated by parallel or back mutation events during chloroplast genome evolution [37, 38]. However, recent studies suggest that, at least in some groups, some small inversions are valuable for a phylogenetic relationship [34]. All of the small inversions in the fourSorghum chloroplast genomes had phylogenetic information.


### 3.6. Divergent Hotspots

Divergent hotspots in the chloroplast genomes between different species at the genus level have provided abundant informative loci for systematic plant and DNA barcoding research [11, 39, 40]. Furthermore, a sliding window analysis using DnaSP detected highly variable regions in theSorghumchloroplast genome. Nucleotide diversity values within 800 bp varied from 0 to 0.01167, and the average value of PI was 0.00965. The IR regions exhibited lower variability than the LSC and SSC regions (Figure 5). There were four mutational hotspots that showed remarkably higher PI values (>0.01), including three intergenic regions (rps16-trnQ,trnG-trnM, andrbcL-psaI) in the LSC and one intergenic region (rps15-ndhF) in the SSC from the chloroplast genomes.Figure 5
Sliding window analysis of the Sorghum chloroplast genomes. X-axis: position of the midpoint of a window; Y-axis: nucleotide diversity of each window.Rps16-trnQ are highly variable in most plant groups and have been used in previous phylogenetic studies [11, 41–43]. In Veroniceae,trnG-trnM was also identified as a highly variable locus [44]. K Saltonstall [45] provided a set of primers to amplify therbcL-psaI region in the grass. TherbcL-psaIhas been used for phylogeographic inference ofPhragmites australis [46].rps15-ndhF combined with five other chloroplast markers has been used to successfully resolve relationships and investigate the biogeography in woody bamboos (Poaceae: Bambusoideae) [47]. These four mutation “hotspot” regions could provide adequate genetic information forSorghum species identification and phylogeny analysis.


### 3.7. Phylogenetic Analysis

Chloroplast genome sequences have been successfully used for the reconstruction of phylogenetic relationships among plant lineages [48–51]. Phylogenetic analyses of plant species using a small number of loci might frequently be insufficient to resolve evolutionary relationships, particularly at low taxonomic levels [52, 53]. Much of the previous phylogenetic work based on whole chloroplast genomes has been used to resolve difficult phylogenetic relationships among closely related species [40, 54].To understand the evolution of Andropogoneae, an improved resolution of phylogenetic relationships has been achieved using the fully sequenced chloroplast genome sequences of 38 Andropogoneae species (Figures 6 andS1). The maximum likelihood (ML) and Bayesian inference (BI) trees exhibited similar phylogenetic topologies. The phylogenetic analyses supported the monophyly of Andropogoneae with strong bootstrap support (BS) of 100% and posterior probabilities (PP) of 1.0 and contributed to clarifying intergeneric relationships (Figures 6 and S1).Arthraxonwas well resolved as the first-branching lineage (BS=100; PP=1.0). The short branch lengths in some nodes of the tree suggested the rapid radiation evolutionary history in these clades. Skendzic et al. [55] used ITS andtrnL–F to investigate the phylogeny of Andropogoneae; the result showed that most of Clayton and Renvoize’s [56] subtribes are not monophyletic. Using the chloroplast genome dataset, this study inferred the clear relationship of Andropogoneae, and this result is consistent with Skendzic et al. ’s.Sorghum was a monophyletic sister toPseudosorghumandMiscanthus (BS=100, PP=1.0). The fourSorghumspecies were grouped into two groups.S. sudanense, S. bicolor, andS. propinquumformed a group.S. sudanense, S. bicolor, andS. propinquumbelong to the subgenusSorghumwhich contain ten species. The phylogeny of subgenusSorghum was unclear because of the low divergence among those species. Several studies used chloroplast markers (ndhF,psbZ-trnG,trnY-trnD,trnY-psbM, andtrnT-trnL) and nuclear markers (ITS, Pepc4, and GBSSI) to infer the phylogeny ofSorghum[4–6, 8]. Those results supported thatS. sudanense, S. bicolor, andS. propinquumformed a group.S. sudanenseis believed to be segregate from a natural hybrid betweenS. bicolor andS. arundinaceum[57]. This is consistent with the present results, which placeS. sudanense in close relationship withS. bicolor with 100% support (Figure 6).Figure 6
Phylogenetic relationships of the Andropogoneae species constructed from the complete chloroplast genome sequences using maximum likelihood (ML) and Bayesian inference (BI). ML topology shown with ML bootstrap support value (BP)/Bayesian posterior probability (PP) given at each node. Nodes with 100 BP/1.0 PP are not marked.Therefore, it is crucial to use more species to better understand Andropogoneae andSorghum phylogeny and evolution. This study provides a basis for the future phylogenesis of Andropogoneae species.


## 3.1. Features ofSorghum Chloroplast Genomes

The plastomes of the four species contain no significant differences in their contents of genes and introns, the gene order in the four genomes is identical, and the sizes of LSC, SSC, and IR regions are very similar. The overall GC content of the chloroplast genome is 38.5%, which is consistent with reported Poaceae species [12, 26].A total of 110 unique genes were identified in theSorghum chloroplast genome, including 77 protein-coding genes, 29 tRNA genes, and 4 ribosomal RNA genes (Figure 1, Table 1 and Table S2). Notably, seven protein-coding genes (rps15,rps12,rps7,ndhB,rpl23,rpl2, andrps19) eight tRNA genes (trnA-UGC,trnH-GUG,trnI-CAU,trnI-GAU,trnL-CAA,trnN-GUU,trnR-ACG, andtrnV-GAC), and all of the rRNA genes are duplicated in the IR regions, which is common in most Poaceae genomes. In theSorghum chloroplast genome, there were 18 intron-containing genes. Among them, ten protein-coding genes (petB,petD,atpF,ndhB,ndhA,rpoC1,rps12,rps16,rpl16, andrpl2) and six tRNA genes have a single intron and two genes (clpP andycf3) that contained two introns. Therps12 is a trans-splicing gene, with the 5′ end located in the LSC region and the duplicated 3′ end located in the IR region. ThematKwas located within the intron oftrnK-UUU.Table 1
Details of the complete chloroplast genomes of the fourSorghumspecies.
Genome features S. sudanense S. propinquum S. bicolor S. timorense Size (bp) 140755 140642 140754 140629 LSC length (bp) 82686 82572 82685 82587 IR length (bp) 22783 22782 22783 22752 SSC length (bp) 12503 12506 12503 12538 Total genes 110 110 110 110 Protein coding genes 77 77 77 77 tRNA genes 29 29 29 29 rRNA genes 4 4 4 4 Overall GC content (%) 38.5 38.5 38.5 38.5 Accession number in GenBank MH926028 MH926027 EF115542 KF998272Figure 1
Map of the Sorghum chloroplast genome. The genes inside and outside the circle are transcribed in the clockwise and counterclockwise directions, respectively. Genes belonging to different functional groups are shown in different colors. Thick lines indicate the extent of the inverted repeats (IRa and IRb) that separate the genomes into small single-copy (SSC) and large single-copy (LSC) regions.


## 3.2. Simple Sequence Repeats

Simple sequence repeats (SSRs) are a type of 1–6 nucleotide unit tandem repeat sequence that is frequently observed in chloroplast genomes. These are important molecular markers for plant population genetics, evolution, and ecological studies because of their high diversity in copy numbers within species due to slipped strand mispairing during DNA replication on a single DNA strand [27, 28].There were 38, 41, 41, and 45 simple sequence repeats in the chloroplast genomes ofS. timorense,S. bicolor,S. sudanense, andS. propinquum, respectively (Figure 2, Table S3). The mononucleotide SSRs were the richest, with a proportion of 60.61%, followed by dinucleotide SSRs (14.55%), tetranucleotide SSRs (19.39%), and trinucleotide SSRs (4.24%). One hexanucleotide SSR was found inSorghum sudanense andSorghum bicolor. Pentanucleotide was not detected in the Sorghum chloroplast genomes. The majority of SSRs in all species were A/T mononucleotides. Chloroplast genome SSRs were composed of adenine or thymine repeats and rarely contained tandem guanine (G) or cytosine (C) repeats. The majority of SSRs were located in the LSC region (71.52). Furthermore, most of the SSRs were found in space regions (73.33%), followed by exon regions (16.97%) and intron regions (9.70%). SSRs in the chloroplast genome have been shown to be extremely useful for resolving genetic diversity between closely related taxa and, hence, increase the power of interspecific studies [29, 30], possibly in combination with other informative nuclear genome SSRs.Figure 2
Analyses of simple sequence repeat (SSR) in the four Sorghum chloroplast genomes. (a) Number of different SSRs types detected by MISA; (b) number of SSRs in LSC, SSC, and IR regions; (c) number of SSRs in spacer, exon, and intron; (d) frequency of identified SSR motifs in the different repeat classes.
(a) (b) (c) (d)


## 3.3. Numbers and Pattern of SNP Mutations

In total, 651 single nucleotide substitutions (SNP) were detected in the fourSorghum chloroplast genomes, 518 of which were found in the LSC region, 18 in the IR region, and 97 in the SSC region. The number of SNP among the fourSorghumspecies was found to be 3 to 631.S. timorenseexhibits higher divergence than other three species.S. sudanenseandS. bicolor show the lowest sequence divergence.The pattern of SNP mutation is shown in Figure3. There were 345 transitions (Ts) and 306 transversions (Tv) and the Tv to Ts ratio was 1:0.89, which indicated a bias in favor of transitions. The most frequently occurring mutations were from A to G and from T to C substitutions (179), while from C to G and from G to C exhibited the lowest frequency (30). Despite the higher A+T contents in chloroplast genomes, AT to TA transversions among the four types of transversions were found to occur significantly less frequently (Figure 3). It is clear that there is a bias in the chloroplast genomes [31].Figure 3
The patterns of nucleotide substitutions among the four Sorghum chloroplast genomes. The patterns were divided into six types as indicated by the six non-strand-specific base-substitution types (i.e., numbers of considered G to A and C to T sites for each respective set of associated mutation types).


## 3.4. Indels

There were 137 indels in the chloroplast genome, which was identified among the fourSorghum chloroplast genomes (Tables S4 and S5), including 43 indels that are caused by SSR variations (SSR-indels) and 94 non-SSR-related indels (NR-indels). The majority of SSR-indels were related to A/T types SSRs (39 times). Only one dinucleotide SSR indel was identified, which is located inndhF-rpl32. All of the SSR-indels were found in the noncoding regions of the LSC/SSC section.The size of NR-indels ranged from 1 to 165 bp, with one bp long indel and 5 bp long indels being the most common (TableS5 and Figure 4). The largest one, found inrpoC with 165 bp length, was a deletion in theS. sudanense. The second longest, which was found inrps16-trnQwith 152 bp length, was an insertion inS. timorense. Finally, 46 insertion indels and 42 deletion indels were specific toS. timorense, one insertion indel and two deletion indels were specific toS. sudanense, and one insertion inrpoC1 intron was specific toS. propinquum.Most of the NR-indels were located in noncoding regions (81.91% in space and 15.96% in introns).Figure 4
NR-indels identified in the chloroplast genomes of the four Sorghum chloroplast genomes . (a) Numbers of individual NR-indels shown by sequence length; (b) relative frequency of NR-indel occurrence in LSC, SSC, and IR regions.
(a) (b) (c)Indels were another important class of genetic variation compared with nucleotide substitutions. Several molecular processes are known to create indels. Polymerase slippage processes during DNA replication or repair can result in the addition or deletion of short spans of sequence that repeat at one side of the region flanking the indel [32], which mainly created SSR-indel type. SSR-indels in chloroplast genome were primarily found in AT-regions and often involve long stretches of repeats of a single nucleotide [33]. In theSorghum chloroplast genome, most of the SSR-indels (90.70%) were A/T types. Hairpins or the stem-loop secondary structure and intramolecular recombination are thought to cause the majority of NR-indel mutations [33]. Different types of indels also show varying amounts of homoplasy. SSR-indels seem to be more prone to homoplasy between different species [28, 34]. In this study, NR-indels were often less homoplasious (Table S5). An increasing number of studies have shown that indel characters can be extremely useful for inferring relationships among more closely related taxa [30, 35, 36].


## 3.5. Small Inversions

Nine small inversions of 2 to 6 bp were identified in theSorghum chloroplast genomes (Table 2). Eight inversions occurred in the LSC region, and one occurred in the SSC region. Most of the small inversions are in intergenic spacer regions, with only two exceptions. One is a 4 bp inversion within the coding region ofccsA, and the other is a 4 bp inversion in therpl16 intron. All of the inversions and their inverted repeating flanking sequences can form stem-loop structures. The franking repeats are from 3 to 20 bp in length. All inversions occurred inS. timorense except the inversion in ccsA, which occurred inS. sudanense.Table 2
The location, direction, and length of nine small inversions in the fourSorghumchloroplast genomes.
Length of inversions (bp) Direction of the small inversions Location Length of inversion Length of inverted repeat S. sudanense S. propinquum S. bicolor S. timorense rps16-trnQ 2 12 no no no yes trnT-trnE 2 6 no no no yes trnT-trnE 2 5 no no no yes psbM-petN 6 14 no no no yes rbcL-rpl32 6 8 no no no yes petA-psbJ 6 14 no no no yes rpl33-rps18 2 3 no no no yes rpl16 intron 4 8 no no no yes ccsA 4 20 no yes yes yesMany small inversions may have been generated by parallel or back mutation events during chloroplast genome evolution [37, 38]. However, recent studies suggest that, at least in some groups, some small inversions are valuable for a phylogenetic relationship [34]. All of the small inversions in the fourSorghum chloroplast genomes had phylogenetic information.


## 3.6. Divergent Hotspots

Divergent hotspots in the chloroplast genomes between different species at the genus level have provided abundant informative loci for systematic plant and DNA barcoding research [11, 39, 40]. Furthermore, a sliding window analysis using DnaSP detected highly variable regions in theSorghumchloroplast genome. Nucleotide diversity values within 800 bp varied from 0 to 0.01167, and the average value of PI was 0.00965. The IR regions exhibited lower variability than the LSC and SSC regions (Figure 5). There were four mutational hotspots that showed remarkably higher PI values (>0.01), including three intergenic regions (rps16-trnQ,trnG-trnM, andrbcL-psaI) in the LSC and one intergenic region (rps15-ndhF) in the SSC from the chloroplast genomes.Figure 5
Sliding window analysis of the Sorghum chloroplast genomes. X-axis: position of the midpoint of a window; Y-axis: nucleotide diversity of each window.Rps16-trnQ are highly variable in most plant groups and have been used in previous phylogenetic studies [11, 41–43]. In Veroniceae,trnG-trnM was also identified as a highly variable locus [44]. K Saltonstall [45] provided a set of primers to amplify therbcL-psaI region in the grass. TherbcL-psaIhas been used for phylogeographic inference ofPhragmites australis [46].rps15-ndhF combined with five other chloroplast markers has been used to successfully resolve relationships and investigate the biogeography in woody bamboos (Poaceae: Bambusoideae) [47]. These four mutation “hotspot” regions could provide adequate genetic information forSorghum species identification and phylogeny analysis.


## 3.7. Phylogenetic Analysis

Chloroplast genome sequences have been successfully used for the reconstruction of phylogenetic relationships among plant lineages [48–51]. Phylogenetic analyses of plant species using a small number of loci might frequently be insufficient to resolve evolutionary relationships, particularly at low taxonomic levels [52, 53]. Much of the previous phylogenetic work based on whole chloroplast genomes has been used to resolve difficult phylogenetic relationships among closely related species [40, 54].To understand the evolution of Andropogoneae, an improved resolution of phylogenetic relationships has been achieved using the fully sequenced chloroplast genome sequences of 38 Andropogoneae species (Figures 6 andS1). The maximum likelihood (ML) and Bayesian inference (BI) trees exhibited similar phylogenetic topologies. The phylogenetic analyses supported the monophyly of Andropogoneae with strong bootstrap support (BS) of 100% and posterior probabilities (PP) of 1.0 and contributed to clarifying intergeneric relationships (Figures 6 and S1).Arthraxonwas well resolved as the first-branching lineage (BS=100; PP=1.0). The short branch lengths in some nodes of the tree suggested the rapid radiation evolutionary history in these clades. Skendzic et al. [55] used ITS andtrnL–F to investigate the phylogeny of Andropogoneae; the result showed that most of Clayton and Renvoize’s [56] subtribes are not monophyletic. Using the chloroplast genome dataset, this study inferred the clear relationship of Andropogoneae, and this result is consistent with Skendzic et al. ’s.Sorghum was a monophyletic sister toPseudosorghumandMiscanthus (BS=100, PP=1.0). The fourSorghumspecies were grouped into two groups.S. sudanense, S. bicolor, andS. propinquumformed a group.S. sudanense, S. bicolor, andS. propinquumbelong to the subgenusSorghumwhich contain ten species. The phylogeny of subgenusSorghum was unclear because of the low divergence among those species. Several studies used chloroplast markers (ndhF,psbZ-trnG,trnY-trnD,trnY-psbM, andtrnT-trnL) and nuclear markers (ITS, Pepc4, and GBSSI) to infer the phylogeny ofSorghum[4–6, 8]. Those results supported thatS. sudanense, S. bicolor, andS. propinquumformed a group.S. sudanenseis believed to be segregate from a natural hybrid betweenS. bicolor andS. arundinaceum[57]. This is consistent with the present results, which placeS. sudanense in close relationship withS. bicolor with 100% support (Figure 6).Figure 6
Phylogenetic relationships of the Andropogoneae species constructed from the complete chloroplast genome sequences using maximum likelihood (ML) and Bayesian inference (BI). ML topology shown with ML bootstrap support value (BP)/Bayesian posterior probability (PP) given at each node. Nodes with 100 BP/1.0 PP are not marked.Therefore, it is crucial to use more species to better understand Andropogoneae andSorghum phylogeny and evolution. This study provides a basis for the future phylogenesis of Andropogoneae species.



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