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Article

The First Complete Chloroplast Genome Sequence of Mortiño (Vaccinium floribundum) and Comparative Analyses with Other Vaccinium Species

by
Karla E. Rojas López
†,
Carolina E. Armijos
†,
Manuela Parra
and
María de Lourdes Torres
*
Laboratorio de Biotecnología de Plantas, Universidad San Francisco de Quito (USFQ), Diego de Robles y Vía Interoceánica, Quito 170901, Ecuador
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2023, 9(3), 302; https://doi.org/10.3390/horticulturae9030302
Submission received: 30 January 2023 / Revised: 14 February 2023 / Accepted: 21 February 2023 / Published: 23 February 2023
(This article belongs to the Special Issue Advances in Berry Crops Production, Genomics and Breeding)
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Abstract

:
Vaccinium floribundum, commonly known as mortiño, is a native high Andean wild species of cultural and economic importance. Genomic resources for V. floribundum are scarce, and a clear phylogenetic and evolutionary history for this species has yet to be elucidated. This study aimed to assemble the complete chloroplast genome sequence of this species and perform an in-depth comparative analysis with other Vaccinium species. The chloroplast genome of V. floribundum was obtained using Oxford Nanopore Technology (ONT). The de novo assembly of the chloroplast genome of V. floribundum resulted in a 187,966 bp sequence, which contained 134 genes (84 Protein Coding Genes (PCGs), 42 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes). The comparative analysis of the V. floribundum chloroplast genome with other nine chloroplast genomes of the Vaccinium species suggested that a contraction/expansion event of the inverted repeat (IR) regions could have occurred, causing the relocation of psbA and rpl32 genes. Additionally, a possible loss of function of the ndhF gene was found. For the phylogenetic analysis based on 87 genes, the chloroplast genome of 19 species (including V. floribundum) was used and revealed that V. myrtillus could be a sister group of V. floribundum. Altogether, our findings provide insights into the plastome characteristics and the phylogeny of V. floribundum. This study describes the complete chloroplast genome sequence of V. floribundum as the first genomic resource available for an Andean species native to Ecuador.

1. Introduction

The Vaccinieae, a tribe belonging to the Ericaceae family, is a unique and megadiverse group in its floral and vegetative morphology. Vaccinium is the only genus within this tribe that occurs in temperate and tropical zones, including the Northern Hemisphere, Southeast Asia, Malaysia, and the South American tropics (from Mexico to northern Argentina) [1,2,3,4]. This genus has more than 450–500 species [5], many of them identified as popular berry crops (e.g., blueberry, V. angustifolium; cranberry, V. macrocarpon Aiton; huckleberry, V. ovatum Pursh) [6,7].
Vaccinium floribundum Kunth—also known as Andean blueberry or mortiño—is a wild deciduous perennial and terrestrial shrub distributed across the high Andes from Venezuela to Bolivia, between 1600 and 4500 m above sea level (masl) [8,9,10,11,12,13]. This shrub can grow up to 2–3.5 m and has an inflorescence formed by 6–10 pink flowers bearing round blue to nearly black edible berries [8,14,15]. This species contains a unique combination of bioactive compounds, nutrients, and fiber, which play an essential role in its characterization as a functional food [15]. Moreover, different studies have established that its berries have a high content of anthocyanins, phenolics, flavonoids, polyphenols, and vitamin C, which make this species attractive because of its medicinal properties, mainly as an antioxidant, anti-inflammatory, antitumor, and antidiabetic [8,9,11,14,16,17,18]. In Ecuador, mortiño is used as the main ingredient in the production of marmalades, ice cream, wine, and the traditional “Dia de los Muertos” (Day of the Souls) drink, called “colada morada” [1,9,13,14,19,20,21,22]. This wild berry species has not been domesticated. Therefore, the berries consumed at local markets are harvested directly from the “paramo”, a high-altitude tundra-like ecosystem characterized by dominant non-arboreal vegetation, high ultraviolet (UV) irradiation, low temperatures (8 to 17 °C), and high humidity. Local indigenous communities harvest mortiño fruits from August to November, representing an important source of income for them [15,18,19,20,24].
The paramo is an ecosystem of great importance for many countries, as it provides one of the primary sources of freshwater [9,25]. However, in recent years, this ecosystem has been under pressure from anthropogenic activities such as burning and grazing, causing widespread habitat loss and fragmentation [23]. Mortiño plays a significant environmental and ecological role in this ecosystem, because it is one of the first species to grow after deforestation and human-made fires, due to its high regenerative capacity [17,22].
Previous studies have characterized the genetic diversity and population structure of mortiño in the Andean Highlands of Ecuador [10,13]. Unfortunately, at the moment, there is no genomic information that would otherwise help elucidate the phylogeny of this species, better understand the status of its populations, and thus contribute to the conservation efforts and sustainable use of this interesting species of the Andean region.
One of the genomic resources that can provide helpful information for a plant species’ genetic characterization is its chloroplast genome (hereinafter referred to as cp genome). This organelle is the metabolic center of plants and plays an essential role in their physiology, development, and evolution [26]. The plastid genome of higher plants is a circular molecule of double-stranded DNA, ranging from 120 to 180 kb in size and containing about 130 genes, depending on the plant species, which mainly codify enzymes involved in photosynthesis [7]. It has a quadripartite structure with two inverted repeats (IRa and IRb, 20–28 kb in length) separated by two regions of unique DNA: large (LSC, 80–90 kb in length) and small (SSC, 16–27 kb in length) single copy regions [7,26,27,28]. These regions are used as “super-barcodes” in phylogenic reconstructions and evolutionary history inference, as they provide sequence-based polymorphisms [4] and have unique genetic properties (i.e., uniparental inheritance, well-preserved gene arrangements, small size, haploidy, and non-recombinant nature) [4,7,26].
To date, 16 cp genome sequences from Vaccinium species have been published: V. macrocarpon (2), V. duclouxii, V. microcarpum, V. fragile, V. oldhamii, V. bracteatum, V. vitis-idaea, V. uliginosum, V. japonicum, V. corymbosum (3), V. virgatum, V. angustifolium, and V. myrtillus (Table S1) [4,6,7,27,29,30,31,32,33,34]. Most of these are reported as plastome assemblies, without any information on comparative analyses [4]. Yet, these types of studies are important, as they can reveal interesting structural variations, such as IR or gene loss, insertions/deletions (InDels), inversions, substitutions, genome rearrangements, and translocations associated with environmental adaptations [7,35]. Only two investigations have performed comparative analyses that could clarify the Vaccinium cp genome evolution [4,7]. The first of these studies [7] found that there were variations in the intergenic spacer lengths, and sequence divergence in noncoding regions between V. bracteatum, V. vitis-idaea, V. uliginosum, V. macrocarpon, and V. oldhamii. The second study [4] revealed an inverted SSC region and insertions/deletions around the IR borders between the cp genome sequence of V. macrocarpon and that of V. corymbosum, V. corymbosum hybrids, V. virgatum, V. angustifolium, and V. myrtillus. The Vaccinium cp genomes described in [4] were highly conserved in terms of structure and sequence.
New findings on the genetics of mortiño could aid in determining the phylogeny and evolutionary history of this unique species of the Andean paramo [22]. The objectives of this study were (a) to provide the complete cp genome sequence of V. floribundum as a valuable genomic resource to assist future studies of mortiño, and (b) to create a phylogenetic tree that resolves V. floribundum’s genetic relationship with other Vaccinium species.

2. Materials and Methods

2.1. Plant Material

A total of 50 young leaves were collected from a single V. floribundum individual from Lloa, Pichincha province, in Ecuador (S 0°11.55122′ W 78°35.216′), under the MAE-DNB-CM-2016-0046-M-0002 collection permit. Leaves were stored in silica gel during their transportation to the USFQ Plant Biotechnology Laboratory. After arrival, they were stored at −20 °C overnight and then at −80 °C until DNA extraction.

2.2. Genomic DNA Extraction, Library Preparation, and Sequencing

High-molecular-weight genomic DNA (HMW-gDNA) was extracted from young leaf tissue using the protocol described by Oxford Nanopore Technologies (ONT) Community “High molecular weight gDNA extraction from Arabidopsis leaves (Arabidopsis thaliana L Er)”, based on Carlson Lysis Buffer followed by purification using the QIAGEN Genomic-tip 100/G. After this step, the Circulomics Short Read Eliminator Kit was used to size-select extracted DNA before library preparation (removing reads < 10 kb). DNA quantity, quality, and integrity were assessed using a Qubit® (Life Technologies, Darmstadt, Germany) fluorometer with a 1 × dsDNA BR assay kit, Nanodrop 2000 (Thermo ScientificTM, Waltham, MA, USA), and an agarose gel (1%), respectively. Libraries were then constructed according to the ONT ligation library protocol (SQK-LSK109) with some modifications: input DNA was 2 μg instead of 1 μg, and in the adapter ligation step, the incubation time was 20 min instead of 10 min. These modifications were made as a troubleshooting step to improve the amount of genomic data generated in the sequencing experiments (according to suggestions made by the ONT Community). Long-read sequencing was then carried out in 2 R.9.4.1 flow cells in the MinION sequencer. Basecalling was performed using MinKNOW, with Albacore v2.0.2 (Albacore, RRID:SCR_015897).

2.3. V. floribundum cp Genome Assembly and Annotation

Adapters from long reads were removed using Porechop v0.2.4 (https://github.com/rrwick/Porechop, accessed on 1 November 2022) with default parameters. Afterward, bases with quality < 9 were trimmed on both sides of reads and filtered based on a size > 1000 bp using Nanofilt v2.8.0 [36]. To facilitate cp genome assembly, cp reads were extracted by aligning them to a reference dataset of 7 “Reference Sequences” (complete, integrated, non-redundant, thoroughly annotated set of reference sequences) of Vaccinium cp genomes from NCBI (accessed on 10 November 2022): V. oldhamii (NC_042713.1), V. bracteatum (LC521967.1), V. uliginosum (LC521968.1), V. japonicum (MW006668.1), V. vitis-idaea (LC521969.1), V. corymbosum Sharpblue Blueberry (SB) (MZ328079.1), and V. macrocarpon (NC019616.1), using BlasR v.5.3.5 with default parameters [37].
To assemble the cp genome of V. floribundum, different assembly tools were used: Flye v. 2.9.1 with parameters “flye --nano-raw reads.fasta --out-dir out_nano --threads 4—asm-coverage 50” [38], Canu v.2.2 with parameters “minReadLength = 1000, minOverlapLength = 500, genomeSize = 180 k, correctedErrorRate = 0.016” [39], and ptGaul v.1 with default parameters, using V. macrocarpon (NC019616.1) as a reference [40]. All genome assemblies were then polished with Apollo v.2.0 (3 total polishing rounds) using the default parameters [41].
Assembly graphs for each assembler (except for Canu, which did not generate an assembly graph) were then evaluated using Bandage v.0.9.0 [42] to visualize genome structures (Figures S1 and S2). After this step, the assembly with a distinctive quadripartite structure (ptGAUL) was selected for further analysis. In an additional step, to verify the evenness of the coverage in every site of the V. floribundum cp genome, all raw reads extracted with BlasR were mapped to the ptGAUL assembled genome using Samtools coverage [43] and plotted with an in-house script (Figure S3 and Material S1). For the annotation step, an online tool was employed: GeSeq v.2.03 using Chloë [44]. Then, manual curation of the annotated genome was performed using Geneious bioinformatics software version 2022.2.2 [45]. We used the R package seqinr v.4.2-23 from R software [46] for relative codon usage and amino acid frequency analyses (Table S2). Finally, the architecture of the V. floribundum cp genome was visualized employing OGDRAW v.1.3.1 [47].

2.4. SSR Analysis

MISA-web v.2.1 software [48] was used to identify microsatellites in the V. floribundum cp genome, with a minimum number of repetitions set at 10 for mononucleotide repeats, 5 for dinucleotide repeats, 4 for trinucleotide repeats, and 3 for tetra-, penta-, and hexa-nucleotide repeats, following the same parameters described by [4].

2.5. Comparison of Vaccinium cp Genomes

Eleven Vaccinium cp genome sequences found on the NCBI (accessed on 15 December 2022) as “Reference Sequences” were used to perform the comparative analyses: V. japonicum (MW006668.1), V. macrocarpon (NC019616.1), V. bracteatum (LC521967.1), V. uliginosum (LC521968.1), V. vitis-idaea (LC521969.1), V. angustifolium (NC068713.1), V. myrtillus (NC068715.1), V. corymbosum (NC068711.1), V. virgatum (NC068712.1), V. oldhamii (NC042713.1), and V. corymbosum SB (MZ328079.1).
To determine the similarity of V. floribundum cp genome and the 11 aforementioned Vaccinium cp genomes, comparative analyses of pseudogenes content, percentage of identity, gene content, and gene content by region were conducted (Tables S3–S6, respectively). However, for genome structure comparisons (performed in mVISTA [49] and IRscope [50] software), two Vaccinium cp genomes (V. oldhamii (NC042713.1) and V. corymbosum SB (MZ328079.1)) were left out. The reason for this was that 10 was the maximum number of species (9 Vaccinium cp genomes and V. floribundum cp genome) able to be compared in these programs. Moreover, we decided to exclude these two species because V. corymbosum SB is a variety of V. corymbosum already included (NC068711.1), and V. oldhamii was the most distant species to V. floribundum (in a preliminary phylogenetic tree analysis; Figure S4).
The mVISTA program [49] was used with the Shuffle-LAGAN model (http://genome.lbl.gov/vista/mvista/ accessed on 27 December 2022). IRScope [50] was then used to evaluate the expansion and contraction of junction sites within the V. floribundum cp genome and the previously mentioned Vaccinium species. Progressive Mauve alignment v.2.4.0 with default parameters [51] was used to examine local and large-scale rearrangements. The percentage of identity analysis was performed using the Multiple Alignment Using Fast Fourier Transform (MAFFT) with the following parameters: “maft --adjustdirection input > output” [52].

2.6. Phylogenetic Analyses

To infer the phylogeny of V. floribundum, the complete cp genome sequences of 16 Vaccinium species available on NCBI were retrieved (Reference sequences and Unverified sequences) (accessed on 15 December 2022): V. japonicum (MW006668.1), V. macrocarpon (NC019616.1), V. microcarpum (MK715444.1), V. corymbosum SB (MZ328079.1), V. fragile (MK816301.1), V. duclouxii (MK816300.1), V. mandarinorum (MW8011356.1), V. bracteatum (LC521967.1), V. oldhamii (NC042713.1), V. carlessii (MW801354.1), V. uliginosum (LC521968.1), V. vitis-idaea (LC521969.1), V. angustifolium (NC068713.1), V. myrtillus (NC068715.1), V. corymbosum (NC068711.1), and V. virgatum (NC068712.1). In this analysis we included unverified sequences (which are not thoroughly annotated) in addition to the set of 11 “Reference sequences” previously mentioned (Section 2.5), because in this analysis we are comparing gene sequences rather than genome features, and the inclusion of all these species could give us a better resolution in the phylogenetic tree reconstruction. Two Actinidia species cp genomes (NC053769.1, NC051888.1) were used as an outgroup to root the tree. To reconstruct the phylogenetic tree, we first identified 87 genes (Material S2) shared among all the Vaccinium species using PhyloHerb v1.1.2 [53], and then we used a maximum likelihood (ML) approach employing RAxML v8.2.11 software with 1000 patterns and bootstrap replicates under the GTR + GAMMA nucleotide substitution model of evolution [54]. The resulting tree was edited with FigTree v.1.4.4 [55].

3. Results

3.1. Vaccinium floribundum cp Genome Sequencing and Assembly

A total of ~925 Mb (186,686 raw reads) were obtained for the V. floribundum cp genome using ONT after the filtering step using BlasR v.5.3.5. This represented an average coverage of 2188X per site. The evenness of the assembled genome was verified with the graph generated using the Samtools coverage output file and an in-house script (see Methods Section 2.3) (Figure S3), indicating a suitable de novo assembly result. The complete cp sequence of V. floribundum was deposited in the GeneBank with the accession number OQ331035. After de novo assembly of the V. floribundum cp genome, its total length was 187,966 bp, which exhibited a typical quadripartite structure consisting of a pair of IR regions of 38,421 bp separated by a LSC region of 107,283 bp and SSC region of 3841 bp (Figure 1 and Table 1). The total GC content (%) of the cp genome was 36.8%, where IR regions exhibited the highest GC content (38.3%), followed by the LSC (35.9%) and SSC regions (29.5%) (Table 1).
In V. floribundum cp genome, a total of 134 genes were found: 84 PCGs, 42 tRNA genes, and 8 rRNA genes (Table 1 and Table 2). Of those 134 genes, 20 were duplicated because of their presence in the IRa/b regions (10 PCGs, 4 rRNA, and 6 tRNA genes), and one was duplicated, but present in the LSC (trnfM-CAU); 25 presented introns (14 were PCGs and 11 tRNA genes). Seven genes were found to be putative pseudogenes (accD, clpP1, infA, ycf2, ycf1, ycf68, and ndhF) (Table 2 and Table S3).

3.2. Relative Synonymous Codon Usage (RSCU), Amino Acid Frequency, and Simple Sequence Repeats (SSR) Identification in V. floribundum cp Genome

The most abundant amino acid was Leucine (Leu) (count = 6341), followed by Serine (Ser) (count = 6029) (Table S3). Isoleucine (Ile) was the amino acid present with the least number of counts (count = 5244). Moreover, for Tryptophan (Trp) and Methionine (Met) amino acids, only one codon was recognized. Thirty-one codons revealed a RSCU < 1, and 33 codons were detected to be used more frequently than the expected usage at equilibrium (RSCU > 1). Codons Arg (AGA), Gly (GGA), Gln (CAA), Ser (TCT), and Asn (AAT) presented the highest RSCU values (Figure 2, Table S2).
Ninety-two SSRs were identified in the V. floribundum cp genome. Among these, dinucleotide (in green) and tetranucleotide (in blue) repeats were the most abundant (33), followed by mononucleotide (in yellow) (12), hexanucleotide (in red) (9) and trinucleotide (in orange) (5) (Figure 3).

3.3. Comparison of Sequence Divergence between the cp Genome of V. floribundum and Other Vaccinium Species

To determine the level of divergence between the cp genome of V. floribundum and the other nine Vaccinium species, V. japonicum (MW006668.1), V. macrocarpon (NC019616.1), V. bracteatum (LC521967.1), V. uliginosum (LC521968.1), V. vitis-idaea (LC521969.1), V. angustifolium (NC_068713.1), V. myrtillus (NC_068715.1), V. corymbosum (NC_068711.1), V. virgatum (NC_068712.1), mVISTA analysis was performed (Figure 4). The alignment resulting from this analysis showed a sequence identity of 70% between all Vaccinium species and V. floribundum. The IR regions exhibited less similarity across all the cp genome sequences for all Vaccinium species, including V. floribundum, as is shown by the average pairwise identity for these regions (72.25%) (Table S4). Notwithstanding, the LSC and SSC regions presented a high pairwise identity for all species (97% LSC; 93.5% SSC) (Table S4).
A colinear analysis was performed to further explore and compare V. floribundum cp genome with the other Vaccinium species included in this study (Figure 5). Although the Mauve alignment result showed that there were no major rearrangements in the cp genomes of the 10 Vaccinium species (including V. floribundum), there was a local colonial block (LCB) (shown in blue) relocated to the SSC region in the cp genome of V. floribundum (Figure 5).

3.4. Contraction and Expansion Analysis of the V. floribundum cp Genome Borders versus Other Vaccinium Species

Cp genome structures such as the gene content and order were contrasted between V. floribundum and the other nine Vaccinium species using a gene boundary junction graph (Figure 6). The V. floribundum cp genome (187,966 bp) was the fourth largest cp genome after V. virgatum (195,878 bp), V. myrtillus (191,744 bp), and V. corymbosum (191,378 bp). The LSC of the V. floribundum cp genome (107,283 bp) had 324 bp less compared to the largest LSC found in V. angustifolium (107,607 bp). The SSC region of the V. floribundum cp genome was the largest (3841 bp) compared to the other Vaccinium species. In addition, the V. floribundum IRa/b region’s length was 38,421 bp.
As can be seen in Figure 6, we found that the trnV-GAC gene was located near the junction (61–62 bp) of the LSC and IRb regions in all Vaccinium species cp genomes, excluding the V. floribundum cp genome. In the latter, this gene was 927 bp away from the LSC/IRb border. Another gene that was found to have a different distance to the LSC/IRb border was the trnH-GUG gene. This gene was found to be 1165–1176 bp away from the LSC/IRb border in the other Vaccinium species cp genomes, and in the V. floribundum cp genome it was only 310 bp away from this border. Similarly, the ndhF gene maintained its position in the SSC region in the 10 Vaccinium cp genomes analyzed, and had a wide variation in distance between its location within this region and the IRb/SSC (70–1158 bp) and SSC/IRa (87–700 bp) borders. In the case of the V. floribundum cp genome, the ndhF gene was 624 bp away from the IRb/SSC border and 2837 bp away from the SSC/IRa border. Additionally, the ndhF gene in the V. floribundum cp genome is depicted as a small fragment due to its possible pseudogenization.
Another interesting finding was that the V. floribundum cp genome had only one copy of the rpl32 gene, whereas the other Vaccinium species had two copies (Table S5). This finding coincided with the Mauve result (blue block) (Figure 5), suggesting a rpl32 gene rearrangement in the cp genome of V. floribundum. The last gene found to be in a different arrangement in the V. floribundum cp genome compared to the other Vaccinium species cp genomes was the psbA gene. This gene was 38 bp away from the IRa/LSC border in the V. floribundum cp genome, whereas its location in the other Vaccinium species cp genomes was within the IRa/LSC regions (Figure 6).

3.5. Phylogenetic Analysis

In order to resolve the phylogenetic relationship of V. floribundum, a maximum likelihood (ML) tree was constructed using 87 genes (Material S2) from 17 Vaccinium species cp genomes (including V. floribundum) and two outgroup species (from the Actidinia genus) (Figure 7). The resulting tree was highly resolved, as all nodes had 100% bootstrap support (BS), except for the clade (91%) comprising the sections Oxycoccus (100%), Vitis-idaea (92%), Pyxothamnus, and Myrtillus (78%). Only one member of the Oxycoccoides section was identified (V. japonicum), two for the Oxycoccus section (V. macrocarpon and V. microcarpum), one for the Vitis-idaea section (V. vitis-idaea), one for the Pyxothamnus section (V. floribundum), one for the Myrtillus section (V. myrtillus), four for the Cyanococcus section (V. angustifolium, V. virgatum, V.corymbosum, V. corymbosum SB), one for the Vaccinium section (V. uliginosum), two for the Eococcus section (V. mandarinorum and V. ducloxii), one for the Baccula-Nigra section (V. fragile), one for the Bracteata section (V. bracteatum), and one for the Ciliata section (V. oldhamii). As can be seen on the tree, V. floribundum was grouped within the same clade as V. myrtillus (section Myrtillus), and both species were closely related to V. vitis-idaea (section Vitis-idaea).

4. Discussion

In this study, we described for the first time the cp genome sequence of V. floribundum using ONT and compared it with the cp genomes of other Vaccinium species. Based on the comparative analyses, we found that V. floribundum was more closely related to V. myrtillus and that its cp genome exhibited an expansion/contraction event of its IR region’s boundaries. This information can be used for the future study of this unique and greatly importance Andean species.
The length of the V. floribundum cp genome (~188 kb) was within the range reported for higher plant species (107–218 kb) [56]. Interestingly, its IR regions (38,421 bp) were longer compared to the typical size of Ericales (34,232 bp) [57]. Its SSC region was the longest (3841 bp) in relation to other Vaccinium cp genomes (2997 bp in V. angustifolium and 3518 bp in V. vitis-idaea) [7,27,33,35].
In terms of gene content, cp genomes of higher plants usually have a total of 110–130 unique genes, mostly encoding enzymes and proteins involved in photosynthesis, transcription, and translation [56,58]. We found that the V. floribundum cp genome exhibited 113 unique genes and 134 genes in total: 84 PCGs, 8 rRNA genes, and 42 tRNA genes. The gene content found for the V. floribundum cp genome was similar to that found for the re-annotated cp genomes of the Vaccinium species analyzed in this study (Tables S5 and S6). We identified the presence of one more gene (trnI-AAU) in V. bracteatum, V. uliginosum, V. vitis-idaea, V. floribundum, V. angustifolium, V. myrtillus, V. corymbosum, and V. virgatum compared to the study in [4] (Tables S5 and S6). Six pseudogenes were identified in the V. floribundum cp genome, which included (Table S3) accD, clP1, infA, ycf1, ycf2, and ycf68, similar to what has been described in other Ericales cp genomes [4,27,59,60]. The ycf15 gene was mentioned in [4] as a pseudogene in V. angustifolium, V. myrtillus, V. corymbosum, and V. virgatum. However, in our study it was absent in the re-annotated cp genomes of V. bracteatum, V. uliginosum, V. vitis-idaea, V. japonicum, V. corymbosum SB, V. oldhamii, and V. macrocarpon, including V. floribundum (Tables S5 and S6). This could suggest a parallel loss of this gene during the evolution of Vaccinium, similar to what was observed in the Papaveraceae family [61].
The V. floribundum cp genome contained one more presumed pseudogene (ndhF) compared to the rest of the analyzed Vaccinium species cp genomes. This gene was truncated and presented in frame stop codons. Pseudogenization of the ndhF gene has been previously reported in Orchids [62] and Amaryllidaceae [63]. ndh genes encode for the NADH dehydrogenase-like complex (NDH-1) that regulates photosynthetic electron transport and is relevant in environments where light availability is rapidly changing [64]. Plants with either high or low light disposal have been reported to lose ndh gene functions [63,65,66]. Interestingly, the paramo, the ecosystem where mortiño grows, is characterized by intense UV radiation [67]. This environmental condition (high UV radiation) could be linked to the loss of function of the ndhF gene in V. floribundum. In one study [68], environmental characteristics such as air temperature, humidity, and photosynthetic active radiation (PAR) were evaluated in the paramo to determine the physiological response of Polylepis cuadrijuga. The authors found that, on clear days, this ecosystem had a high PAR of 600 µmoles/m2s, which is within the photoinhibition threshold for C3 plants [68]. Over long and continuous exposure to these light conditions, the photosystem activity suffers irreversible damage [68]. Based on those findings, other plants living in the paramo, such as mortiño, could be subjected to similar PAR values, which could alter their photosynthetic competence. Hence, this provides a plausible explanation for the pseudogenization of the V. floribundum ndhF gene. In addition, the paramo is considered a biodiversity hotspot, where unique adaptations to extreme environmental conditions evolved in several endemic and native species [69,70,71,72,73]; therefore, the gain and loss of function of certain genes (such as ndhF gene) could have taken place in plant species as a response to this fast evolving ecosystem. Another explanation for this finding is that the ndhF function could have been transferred to the nuclear genome, as reported in other studies [62,63,74]. To verify whether this has occurred in the cp genome of V. floribundum, it would be necessary to perform a genome-wide analysis.
V. floribundum had a similar cp genome structure as found in other Ericaceae. The trnV and trnH genes were placed at the LSC/IRb and IRa/LSC junctions (respectively) (Figure 6); likewise, the ndhF gene was present in the SSC region (Figure 6) [57]. Nonetheless, the psbA gene in the V. floribundum cp genome was only located in the LSC region (Figure 6), in contrast to other Ericales, where it is present within the IRa/LSC boundary. Similarly, the V. floribundum cp genome had the rpl32 gene placed only in the SSC region, while in other Ericaceae, it is placed inside the IRa/b regions [57]. Therefore, in relation to other Vaccinium species cp genomes, the V. floribundum cp genome could have undergone a possible expansion/contraction in its IR regions, shifting its IR/SSC-IR/LSC boundaries. This has been reported previously in cp genomes of different genera, such as Litsea [75], Punica [76], Paphiopedilum [77], and Stemona [78]. Additionally, the expansion/contraction of the IR regions could justify the difference in plastome sizes within the Vaccinium genus and contribute to the different gene arrangements of their cp genomes [75,76,77,78,79].
In the present study, we performed a maximum likelihood (ML) tree using 87 cp genes due to the high level of divergence in the IR regions within the Vaccinium cp genomes (Figure 6). In contrast to the phylogenetic analysis of [4], we included three new species to the Vaccinium tree (V. floribundum, V. mandarinorum, and V. carlesii). The expanded taxon sampling enabled a different reconstruction of the evolutionary relationships for Vaccinium species compared to what was done in [4]. For example, in the case of V. japonicum, this species appears to be closely related to the rest of Vaccinium ancestors, with a bootstrap support of 100%. Additionally, V. angustifolium seems to have diverged earlier within the Cyanococcus clade. Overall, the rest of the branches agree with the genetic relationships reported between sections in other phylogenetic studies where whole cp genomes have been used [4,80].
Although each node of the phylogenetic tree in this study (Figure 7) was well supported, showing a bootstrap value above 70% [81], V. floribundum was identified as a sister taxon to V. myrtillus, with the lowest bootstrap support (78%). According to [5], who used nrITS and matK molecular markers to infer the evolutionary relationship of 93 species from the Vaccinieae tribe, including V. floribundum, V. floribundum clustered under the section Pyxothamnus (with bootstrap support of 62%) and was closely related to V. consanguineum and V. ovatum. In this context, it is not yet possible to clearly resolve V. floribundum’s phylogenetic relationships within the Vaccinium genus as they are not strongly supported. A more accurate and reliable phylogenetic history of this species could be achieved by including more wild Vaccinium species from around the world. Two other Vaccinium species, V. distichum, and V. crenatum [22], have been recorded in Ecuador, with no available cp genome that would be interesting to analyze.

5. Conclusions

The V. floribundum cp genome was successfully sequenced and assembled. This demonstrates that ONT is sufficient to obtain a continuous and high-quality cp genome assembly. The V. floribundum cp genome shared a similar structure and gene content with other Vaccinium species’ cp genomes. However, some interesting differences were found in this study, such as psbA and rpl32 rearrangement in the LSC and SSC regions, respectively, and ndhF possible pseudogenization. Moreover, through the reconstruction of a phylogenetic tree using 87 cp genes among 17 Vaccinium species, we found that V. myrtillus could be the closest relative to V. floribundum, despite having previously shown different evolutionary relationships when using only nrITS and matK molecular markers [5]. To have a better resolution of the genetic relationships between V. floribundum and other Vaccinium species, it would be important to expand the sampling of taxa from the different sections of Vaccinium. Overall, our results revealed for the first time the complete cp genome sequence of V. floribundum, an economically and culturally important species of the Ecuadorian paramo, and an ecologically relevant species of the Andean Highlands. This genetic resource could help to better understand the unique biology of mortiño, a plant adapted to an extreme environment in one of the most rapidly evolving hotspots on Earth [69], and provide a tool for further studies of the Vaccinium genus.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9030302/s1, Figure S1: Bandage graph result from Flye genome assembly of V. floribundum cp genome. Figure S2: Bandage graph result from ptGAUL genome assembly of V. floribundum cp genome. Figure S3: Depth coverage of V. floribundum cp genome by base position. Figure S4. Preliminary phylogenetic tree constructed with 87 genes of 13 Vaccinium species cp genomes including V. floribundum cp genome using RAxML. Table S1: Summary of features from 16 characterized Vaccinium complete cp genomes. Table S2: Amino Acid Codons Relative Synonymous Codon Usage (RSCU) and amino acid frequency (Aafreq) in the cp genome of V. floribundum. Table S3: List of identified pseudogenes in 12 Vaccinium cp genomes including V. floribundum cp genome. Table S4: Percentage of identity between 12 Vaccinium plastomes* obtained from a sequence alignment using Multiple Alignment Using Fast Fourier Transform (MAFFT). Table S5: List of identified genes in 12 Vaccinium cp genomes including V. floribundum cp genome. Table S6: Summary of features of 12 Vaccinium cp genomes including V. floribundum cp genome from NCBI and their description in the present study based on each region (LSC, SSC, and IR regions). Material S1: In-house script to create the V. floribundum cp genome coverage graph. Material S2: List of genes used to create the phylogenetic tree of Vaccinium.

Author Contributions

K.E.R.L.: methodology, software, validation, formal analysis, investigation, data curation, writing—original draft, funding acquisition. C.E.A.: methodology, software, validation, formal analysis, investigation, data curation, writing—original draft. M.P.: formal analysis, investigation, data curation, writing—original draft. M.d.L.T.: conceptualization, supervision, investigation, project administration, funding acquisition, writing—review & editing. K.E.R.L. and C.E.A. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Universidad San Francisco de Quito grant number 16819. The APC was funded by Universidad San Francisco de Quito.

Data Availability Statement

The V. floribundum cp genome sequence was submitted to the NCBI GeneBank under the accession number OQ331035. The NCBI GeneBank accession number of the rest of the Vaccinium species used in the present study are listed as follows: V. japonicum (MW006668.1), V. macrocarpon (NC019616.1), V. microcarpum (MK715444.1), V. corymbosum SB (MZ328079.1), V. fragile (MK816301.1), V. duclouxii (MK816300.1), V. mandarinorum (MW8011356.1), V. bracteatum (LC521967.1), V. oldhamii (NC_042713.1), V. carlessii (MW801354.1), V. uliginosum (LC521968.1), V. vitis-idaea (LC521969.1), V. angustifolium (NC_068713.1), V. myrtillus (NC_068715.1), V. corymbosum (NC_068711.1), and V. virgatum (NC_068712.1). The NCBI GeneBank accession numbers of the outgroup sequences used for the phylogenetic analysis are Actidinia rubus (NC053769.1) and Actidinia fulvicoma (NC051888.1).

Acknowledgments

We would like to thank the Plant Biotechnology Laboratory COCIBA; USFQ, for their support during the execution of this project, with special thanks to Milton Gordillo and Sebastian Jordan for their technical assistance. We thank Pamela Vega and Andres Villavicencio for their assistance on the field trip. Sample collection was performed in compliance with the research permit MAE-DNB-CM-2016-0046-M-0002 granted by the Ministry of Environment, Ecuador.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Cp genome map of V. floribundum. On the bottom left, the genes corresponding to the different functional groups are depicted with distinctive colors. Genes that are inside the circle are transcribed in a clockwise direction. Genes outside the circle are transcribed in a counterclockwise direction. The light grey line inside the circle represents the AT content, and the dark grey area illustrates the GC content. IRa/IRb, LSC, and SSC regions are shown by black lines surrounding the dark grey area.
Figure 1. Cp genome map of V. floribundum. On the bottom left, the genes corresponding to the different functional groups are depicted with distinctive colors. Genes that are inside the circle are transcribed in a clockwise direction. Genes outside the circle are transcribed in a counterclockwise direction. The light grey line inside the circle represents the AT content, and the dark grey area illustrates the GC content. IRa/IRb, LSC, and SSC regions are shown by black lines surrounding the dark grey area.
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Figure 2. Codon usage analysis and amino acid frequencies (%) in V. floribundum cp genome. (A) Relative synonymous codon usage (RSCU). (B) Amino acid frequencies (aafreq %). The x-axis shows codons that are represented by different colors.
Figure 2. Codon usage analysis and amino acid frequencies (%) in V. floribundum cp genome. (A) Relative synonymous codon usage (RSCU). (B) Amino acid frequencies (aafreq %). The x-axis shows codons that are represented by different colors.
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Figure 3. Microsatellite frequency classified by microsatellite motifs found in the cp genome of V. floribundum.
Figure 3. Microsatellite frequency classified by microsatellite motifs found in the cp genome of V. floribundum.
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Figure 4. mVISTA cp genome alignments of V. floribundum and the other nine Vaccinium species. The x-axis corresponds to the coordinates of the V. floribundum cp genome used as a reference. The y-axis (horizontal bars) represents the percent identity (50–100%) across regions in each of the analyzed Vaccinium species. Dissimilar regions are in white. Annotated genes appear at the top.
Figure 4. mVISTA cp genome alignments of V. floribundum and the other nine Vaccinium species. The x-axis corresponds to the coordinates of the V. floribundum cp genome used as a reference. The y-axis (horizontal bars) represents the percent identity (50–100%) across regions in each of the analyzed Vaccinium species. Dissimilar regions are in white. Annotated genes appear at the top.
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Figure 5. Colinear analysis using progressive Mauve of V. floribundum and other nine Vaccinium species cp genomes. The LCBs are depicted as shaded colored boxes connected by a line. The numbers on top of the x-axis indicate the coordinates in bp in the analyzed genomes. LSC, IRb, and SSC regions are presented by blue arrows in the V. floribundum cp genome.
Figure 5. Colinear analysis using progressive Mauve of V. floribundum and other nine Vaccinium species cp genomes. The LCBs are depicted as shaded colored boxes connected by a line. The numbers on top of the x-axis indicate the coordinates in bp in the analyzed genomes. LSC, IRb, and SSC regions are presented by blue arrows in the V. floribundum cp genome.
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Figure 6. Gene border junction graph of the LSC, SSC, and IR regions in V. floribundum cp genome and the other nine Vaccinium species obtained with IRScope. The numbers with the arrows represent the distance in bp between each junction. Note that the distances are not drawn to scale and that the cp genomes follow the typical quadripartite structure with one LSC, one SSC, and two IR regions.
Figure 6. Gene border junction graph of the LSC, SSC, and IR regions in V. floribundum cp genome and the other nine Vaccinium species obtained with IRScope. The numbers with the arrows represent the distance in bp between each junction. Note that the distances are not drawn to scale and that the cp genomes follow the typical quadripartite structure with one LSC, one SSC, and two IR regions.
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Figure 7. Phylogenetic tree constructed with 87 genes of 17 Vaccinium species cp genomes including the V. floribundum cp genome using RAxML. Branch labels indicate the BS values (%). The scale bar represents nucleotide substitutions per site. Two sequences of Actinidia genus (A. rubus and A. fulvicoma) were used as an outgroup to root the tree. Sections of Vaccinium are represented by different colors.
Figure 7. Phylogenetic tree constructed with 87 genes of 17 Vaccinium species cp genomes including the V. floribundum cp genome using RAxML. Branch labels indicate the BS values (%). The scale bar represents nucleotide substitutions per site. Two sequences of Actinidia genus (A. rubus and A. fulvicoma) were used as an outgroup to root the tree. Sections of Vaccinium are represented by different colors.
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Table
Table 1. Assembly and annotation statistics of the cp genome of V. floribundum.
Table 1. Assembly and annotation statistics of the cp genome of V. floribundum.
Whole cp Genome Sequence
Length (bp)187,966
GC content (%)36.8
Protein Coding Genes (PCGs) 184
Unique Genes113
Genes 1134
Transfer RNA (tRNA) genes 142
Ribosomal RNA (rRNA) genes 18
LSC region
Length (bp)107,283
PCGs 162
Genes 192
tRNA genes 130
rRNA genes 10
GC content (%)35.9
IR regions
Length (bp)38,421
PCGs 110
Genes 120
tRNA genes 16
rRNA genes 14
GC content (%)38.3
SSC region
Length (bp)3841
PCGs 12
Genes 12
tRNA genes 10
tRNA genes 10
GC content (%)29.5
1 Including all copies.
Table
Table 2. Genes present in V. floribundum cp genome.
Table 2. Genes present in V. floribundum cp genome.
Gene CategoryGene GroupGene Name
PhotosynthesisSubunits of ATP synthaseatpA, atpB, atpE, atpF2, atpH, atpI
Subunits of NADH-dehydrogenasendhA2, 3, 5, ndhB2, ndhC, ndhD3, 5, ndhE3, 5, ndhF1, ndhG3, 5, ndhH3, 5, ndhI3, 5, ndhJ, ndhK
Subunits of cytochrome b/f complexpetA, petB2, petD2, petG, petL, petN
Subunits of photosystem IpsaA, psaB, psaC3, 5, psaI, psaJ
Subunits of photosystem IIpsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbT, psbZ
Subunit of rubiscorbcL
rRNArrn163, 5, rrn232, 3, 5, rrn4.53, 5, rrn53, 5
tRNAtrnA-UGC2, 3, 5, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU3, 4, trnG-GCC, trnG-UCC2, trnH-GUG3, 5, trnI-AAU, trnI-CAU, trnI-GAU2, 3, 5, trnK-UUU2, trnL-CAA, trnL-UAA2, trnL-UAG3, 5, trnM-CAU, trnN-GUU3, 5, trnP-GGG, trnP-UGG, trnQ-UUG, trnR-ACG3, 5, trnR-UCU, trnS-CGA2, trnS-GCA2, trnS-GCU, trnS-GGA, trnS-UGA, trnT-CGU2, trnT-GGU, trnT-UGU, trnV-GAC, trnV-UAC2, trnW-CCA, trnY-GUA
Self-replicationThe large subunit of the ribosome (LSU)rpl14, rpl162, rpl2, rpl20, rpl22, rpl23, rpl32, rpl33, rpl36
The small subunit of the ribosome (SSU)rpoA, rpoB, rpoC12, rpoC2
DNA-dependent RNA polymeraserps11, rps12, rps14, rps153, 5, rps162, 3, 5, rps18, rps19, rps2, rps3, rps4, rps7, rps8
Other genesc-type cytochrome synthesisccsA3, 5
Envelop membrane proteincemA
UnknownHypothetical cp reading framepbfI (psbN), ycf2, ycf3 (pafI) 2, ycf4 (pafII)
1 Putative pseudogenes included for comparison purposes with other Vaccinium species; 2 genes with introns; 3 genes with two copies; 4 genes duplicated not present in IR; 5 genes duplicated in IR.
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López, K.E.R.; Armijos, C.E.; Parra, M.; Torres, M.d.L. The First Complete Chloroplast Genome Sequence of Mortiño (Vaccinium floribundum) and Comparative Analyses with Other Vaccinium Species. Horticulturae 2023, 9, 302. https://doi.org/10.3390/horticulturae9030302

AMA Style

López KER, Armijos CE, Parra M, Torres MdL. The First Complete Chloroplast Genome Sequence of Mortiño (Vaccinium floribundum) and Comparative Analyses with Other Vaccinium Species. Horticulturae. 2023; 9(3):302. https://doi.org/10.3390/horticulturae9030302

Chicago/Turabian Style

López, Karla E. Rojas, Carolina E. Armijos, Manuela Parra, and María de Lourdes Torres. 2023. "The First Complete Chloroplast Genome Sequence of Mortiño (Vaccinium floribundum) and Comparative Analyses with Other Vaccinium Species" Horticulturae 9, no. 3: 302. https://doi.org/10.3390/horticulturae9030302

APA Style

López, K. E. R., Armijos, C. E., Parra, M., & Torres, M. d. L. (2023). The First Complete Chloroplast Genome Sequence of Mortiño (Vaccinium floribundum) and Comparative Analyses with Other Vaccinium Species. Horticulturae, 9(3), 302. https://doi.org/10.3390/horticulturae9030302

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