Plastid genome of <i>Passiflora tripartita</i> var. <i>mollissima</i> (poro-poro) from Hu&aacute;nuco, Peru

Flavio Aliaga; Mario Zapata-Cruz; Silvia Ana Valverde-Zavaleta

doi:10.12688/f1000research.138150.3

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Genome Note

Revised

Plastid genome of Passiflora tripartita var. mollissima (poro-poro) from Huánuco, Peru

[version 3; peer review: 2 approved]

Flavio Aliaga ^1-3, Mario Zapata-Cruz⁴, Silvia Ana Valverde-Zavaleta⁴

PUBLISHED 19 Feb 2024

Author details Author details

¹ Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru
² Capítulo de Ingeniería Agronómica, Consejo Departamental de La Libertad (CDLL), Colegio de Ingenieros del Perú (CIP), Trujillo, 13008, Peru
³ Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, 13011, Peru
⁴ Plant Science Laboratory (PSL), Trujillo, 13009, Peru

Flavio Aliaga
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Mario Zapata-Cruz
Roles: Conceptualization, Investigation, Methodology, Project Administration, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Silvia Ana Valverde-Zavaleta
Roles: Conceptualization, Investigation, Methodology, Project Administration, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Genomics and Genetics gateway.

Abstract

Passiflora tripartita var. mollissima, known locally as poro-poro, is an important native fruit used in traditional Peruvian medicine with relevant agro-industrial and pharmaceutical potential for its antioxidant capacity for human health. However, to date, only a few genetic data are available, which limits exploring its genetic diversity and developing new genetic studies for its improvement. We report the poro-poro plastid genome to expand the knowledge of its molecular markers, evolutionary studies, molecular pathways, and conservation genetics. The complete chloroplast (cp) genome is 163,451 bp in length with a typical quadripartite structure, containing a large single-copy region of 85,525 bp and a small single-copy region of 13,518 bp, separated by a pair of inverted repeat regions (IR) of 32,204 bp, and the overall GC content was 36.87%. This cp genome contains 128 genes (110 genes were unique and 18 genes were found duplicated in each IR region), including 84 protein-coding genes, 36 transfer RNA-coding genes, eight ribosomal RNA-coding genes, and 13 genes with introns (11 genes with one intron and two genes with two introns). The inverted repeat region boundaries among species were similar in organization, gene order, and content, with a few revisions. The phylogenetic tree reconstructed based on single-copy orthologous genes and maximum likelihood analysis demonstrates poro-poro is most closely related to Passiflora menispermifolia and Passiflora oerstedii. In summary, our study constitutes a valuable resource for studying molecular evolution, phylogenetics, and domestication. It also provides a powerful foundation for conservation genetics research and plant breeding programs. To our knowledge, this is the first report on the plastid genome of Passiflora tripartita var. mollissima from Peru.

Keywords

Plastid genome, Passifloraceae, Passiflora tripartita var. mollissima, poro-poro, native fruit, Huánuco, Peru

Corresponding author: Flavio Aliaga

Competing interests: No competing interests were disclosed.

Grant information: This research was funded by Plant Science Laboratory E.I.R.L. (Sach’a Ruru grant: RIC-2022-101).

Copyright: © 2024 Aliaga F et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Aliaga F, Zapata-Cruz M and Valverde-Zavaleta SA. Plastid genome of Passiflora tripartita var. mollissima (poro-poro) from Huánuco, Peru [version 3; peer review: 2 approved]. F1000Research 2024, 12:795 (https://doi.org/10.12688/f1000research.138150.3) First published: 07 Jul 2023, 12:795 (https://doi.org/10.12688/f1000research.138150.1) Latest published: 19 Feb 2024, 12:795 (https://doi.org/10.12688/f1000research.138150.3)

Revised Amendments from Version 2

We update the circular genome map ensuring a more concise and comprehensible representation.

See the authors' detailed response to the review by Abdullah Abdullah
See the authors' detailed response to the review by Rahul G Shelke

Introduction

Passiflora tripartita var. mollissima (Kunth) Holms-Niels. & P.M. Jørg (ITIS, 2022) previously known as Passiflora mollissima (Kunth) Bailey (Primot et al., 2005), is a semi-perennial fruit plant (Mayorga et al., 2020). It is a diploid species with a small number of chromosomes (2n = 18) (Coppens D’Eeckenbrugge, 2001), which is placed in the section Elkea of supersection Tacsonia of subgenus Passiflora belonging to the Passifloraceae family (Segura et al., 2005; Ocampo & Coppens d’Eeckenbrugge, 2017). Poro-poro is a native fruit of the Andean region (Ocampo & Coppens d’Eeckenbrugge, 2017). It grows in the Peruvian highlands in the departments of Ancash, Junín, Moquegua, Huancavelica, and Huánuco at altitudes of 1,000–4,000 m.a.s.l. (Tapia & Fries, 2007; Ríos-García, 2017). It is widely used in traditional medicine (Ríos-García, 2017) and is considered one of the best Passiflora species based on its organoleptic characteristics (Primot et al., 2005). This fruit provides a source of vitamins (A, B3, and C) and minerals (magnesium, potassium, phosphorus, sodium, chlorine, iron, calcium, sulfur, zinc, copper, selenium, cobalt, and nickel) (Leterme et al., 2006; Chaparro-Rojas et al., 2014). In addition, it has an elevated antioxidant activity and high content of carotenoids (118.8 mg β-carotene), phenols (460.1 mg gallic acid), and flavonoids (1907.6 mg catechin/100 g) (Leterme et al., 2006; Chaparro-Rojas et al., 2014). Specifically, the high concentration of flavan-3-ols (a group of bioactive compounds) has been associated with beneficial effects on human health, such as cardiovascular protection, neurodegenerative diseases, and as an anti-cancer, anti-microbial, and anti-parasitic agent (Giambanelli et al., 2020; Luo et al., 2022).

Plastome sequences from over 4000 species (Zhou et al., 2021) are small in size with high copy numbers and conserved sequences, enabling a significant understanding of plant molecular evolution, structural variations, and evolutionary relationships of plant diversity (Daniell et al., 2016; Dobrogojski et al., 2020). The plastid genome has a quadripartite structure: a large single-copy (LSC) of 80–90 kilobase pairs (kb), a small single-copy (SSC) of 16–27 kb, and two sets of inverted repeats (IRa and IRb) of 20–28 kb, with 110–130 unique genes, including protein-coding genes, transfer RNA (tRNA), and ribosomal RNA (rRNA) (Ozeki et al., 1989; Wang & Lanfear, 2019). In recent years, declining genome sequencing costs resulted in more than 780 complete plant genomes of different species becoming available (Marks et al., 2021; Sun et al., 2022). Recently, some Passiflora plastid genomes such as Passiflora edulis (Cauz-Santos et al., 2017), Passiflora xishuangbannaensis (Hao & Wu, 2021), Passiflora caerulea (Niu et al., 2021), Passiflora serrulata (Mou et al., 2021), Passiflora foetida (Hopley et al., 2021), and Passiflora arbelaezii (Shrestha et al., 2019), became publicly available. However, despite the scarcity of genomic information on underutilized crops (Gioppato et al., 2019), we have only begun to investigate the genomics of plants of great importance for plant breeding programs. The purpose of this research was to obtain the poro-poro plastid genome, which constitutes a valuable resource for studying the molecular evolution, phylogenetics, and domestication of species with beneficial characteristics for human health. In the present study, we report the first plastid genome sequence submitted for an isolate of Passiflora tripartita var. mollissima, and important native fruit of Peru.

Methods

Plant materials

In November 2022, the fresh leaves of Passiflora tripartita var. mollissima were collected from Raccha Cedrón locality of Quisqui District, Huánuco Province from Peru (9°53′37″S, 76°26′02″W, altitude 2,945 m.a.s.l.). A herbarium voucher specimen (USM<PER>:MHN331530) was deposited in the Herbario San Marcos (USM) of the Museo de Historia Natural (MHN) at the Universidad Nacional Mayor de San Marcos (UNMSM) (see the Extended data, Aliaga et al., 2023a).

DNA extraction

Total genomic DNA was extracted from approximately 100 mg fresh leaves (from voucher number USM<PER>:MHN331530) according to Doyle’s (1991) method with slight modifications. The DNA isolation buffer consisted of buffer cetyl-trimethyl ammonium bromide (CTAB) 3% (30g/L CTAB, 100 mM Tris-HCl pH 8.0, 10nM EDTA, 1.4 M NaCl, 0,2% 2-mercaptoethanol), 70% ethanol, chloroform-isoamyl alcohol (24:1), 10 mM ammonium acetate, isopropanol, TE buffer (10 mM Tris-H, 1 mM EDTA), and RNAase A (10 ug/ml). Genomic DNA quality was assessed using a fluorometry-based Qubit (Thermo Fisher Scientific, USA, catalog number: Q33238) coupled to a Broad Range Assay kit (Thermo Fisher Scientific, USA, catalog number: Q33230). High-quality DNA (230/260 and 260/280 ratios >1.8) were normalized (20 ng/μL) to examine its integrity using 1% (w/v) agarose gel electrophoresis (see the Extended data, Aliaga et al., 2023b) with the following equipment: Horizontal gel system (Fisher Scientific, Denmark, catalog number: 11833293, 150mm (length), 100 mm (width)), Transilluminator (Fisher Scientific, Spain, catalog number: 12864008), and digital camera (Canon, Spain, catalog number: 2955C002); Reagents: TAE buffer (40 mM Tris, 20mM NaAc, 1mM EDTA, pH 7.2), loading buffer 6X (Promega, USA, catalog number: G1881, 0.4% orange G, 0.03% bromophenol blue, 0.03% xylene cyanol FF, 15% Ficoll^® 400, 10mM Tris-HCl pH 7.5 and 50mM EDTA pH 8.0) and Ethidium bromide (Promega, USA, catalog number H5041, 10 mg/ml), and 1 Kb Plus DNA Ladder (ThermoFisher, USA, catalog number: 10787018).

Genome sequencing, assembly, and annotation

Qualified DNA was fragmented, and the TruSeq Nano DNA kit (Illumina, San Diego, CA, USA, catalog number: FC-121-4001) was used to construct an Illumina paired-end (PE) library. PE sequencing (2 × 150 bp) was performed using the Illumina NovaSeq 6000 platform (Modi et al., 2021) (Illumina, San Diego, Ca, USA, catalog number: 20012850) (Macrogen, Inc., Seoul, Republic of Korea). The quality control of reads was carried out using the FastQC (Wingett & Andrews, 2018) program. All adapters were removed using the Cutadapt (Martin, 2011) program. After that, PE reads (2 × 150 bp) were evaluated for quality using QUAST (Gurevich et al., 2013) analysis, and subsequent steps used clean data. Then, clean reads obtained were assembled into a circular contig using NOVOPlasty v.4.3 (Dierckxsens et al., 2017), with P. edulis (NC_034285) as the reference (Cauz-Santos et al., 2017). Assembled genome was annotated using CpGAVAS2 (Shi et al., 2019), an integrated plastome sequence annotator and GeSeq (Tillich et al., 2017). Transfer RNAs were also checked with ARAGORN v.1.2.38 (Laslett and Canback, 2004), Chloë v.0.1.0 (https://github.com/ian-small/chloe) and tRNAscan-SE v2.0 (Chan et al., 2019) incorporated in GeSeq using default settings. A circular genome map was constructed using OGDRAW v.1.3.1 (Greiner et al., 2019). Finally, the completed sequences were submitted to the NCBI GenBank under the accession number OQ910395 (GenBank, 2023).

Phylogenetic analysis

We used 26 complete plastome sequences to infer the phylogenetic relationships among Passiflora species, and Vitis vinifera was used as an outgroup (see the Extended data, Aliaga et al., 2023c). Single-copy orthologous genes were identified using the Orthofinder version 2.2.6 pipeline (Emms & Kelly, 2019). For each gene family, the nucleotide sequences were aligned using the L-INS-i algorithm in MAFFT v7.453 (Katoh & Standley, 2013). A phylogenetic tree based on maximum likelihood (ML) was constructed using RAxML v8.2.12 (Stamatakis, 2014) with the GTRCAT model. A phylogenetic ML tree was reconstructed and edited using MEGA 11 (Tamura et al., 2021) with 1000 replicates.

Results

Plastome of Passiflora tripartiva var. mollissima

The plastid genome sequences of P. tripartita var. mollissima (poro-poro) (Figure 1) was 163,451 bp in length, with an average coverage depth of 100 ×, with a typical quadripartite structure consisting of a large single-copy (LSC) region of 85,525 bp (52.32% in total) and a small single-copy (SSC) region of 13,518 bp (8.27%), separated by a pair of inverted repeat regions (IRs) of 32,204 bp (19.70%). The poro-poro plastome is 12,045 bp longer than that of one of the most economically important species, passion fruit (P. edulis) (Cauz-Santos et al., 2017), and is only 7,117 bp longer than that of the longest Passiflora plastome reported, i.e., P. arbelaezii (Shrestha et al., 2019). The plastome sequence of poro-poro has a similar quadripartite architecture to other plants (Ohyama et al., 1986; Shinozaki et al., 1986; Nguyen et al., 2021). However, the LSC region is 4,150 bp longer than that of P. xishuangbannaensis but is 98bp, 195 bp, and 1,927 bp shorter than that of P. caerulea, P. edulis, and P. arbelaezii, respectivety. The SSC region is 121 bp, 140 bp, 359 bp, and 754 bp longer than that of P. caerulea, P. edulis, P. xishuangbannaensis, and P. arbelaezii, respectively. The IRs regions are 6,024 bp, 6,050 bp, and 11,600 longer than that of P. caerulea, P. edulis, and P. xishuangbannaensis, respectively; however, it is 2,972 bp shorter than that of P. arbelaezii (Cauz-Santos et al., 2017; Shrestha et al., 2019; Hao & Wu, 2021; Niu et al., 2021). The plastome structure of the P. tripartita var. mollissima consisted of A = 30.79%, T(U) = 32.34%, C = 18.67% and G = 18.20%. The overall AT content of the plastid genome was 63.13%, whereas the overall GC content was 36.87% as similar to that of other reported chloroplast genomes from the same family, such as 36.90% in P. arbelaezii (Shrestha et al., 2019), 37% in P. edulis and P. serrulata (Cauz-Santos et al., 2017; Mou et al., 2021), 37.03% in P. caerulea (Niu et al., 2021), and 37.1% in P. xishuangbannaensis (Hao & Wu, 2021).

Figure 1. Plastid genome of Passiflora tripartita var. mollissima.

The thick lines indicate the IR1 and IR2 regions, which separate the large single-copy (LSC) and small single-copy (SSC) regions. Genes marked inside the circle are transcribed clockwise, and genes marked outside the circle are transcribed counterclockwise. Genes are color-coded based on their function, shown at the bottom left. The inner circle indicates the inverted boundaries and guanine and cytosine (GC) content.

Poro-poro plastid genome annotation identified 128 genes, of which 110 were unique, and 18 were duplicated in the inverted repeat (IR) region. The plastome contained 84 protein-coding genes, 36 transfer RNA (tRNA)-coding genes, eight ribosomal RNA (rRNA)-coding genes, and 13 genes with introns (11 genes with one intron and two genes with two introns), as shown in Table 1. The poro-poro plastid genome contained 110 unique genes, of which there were 28 tRNA genes, four rRNA genes, and 78 protein-coding genes. The latter comprised 19 ribosomal subunit genes (nine large subunits and 10 small subunit), four DNA-directed RNA polymerase genes, 46 genes were involved in photosynthesis (11 encoded subunits of the NADH oxidoreductase, seven for photosystem I, 15 for photosystem II, six for the cytochrome b6/f complex, six for different subunits of ATP synthase, and one for the large chain of ribulose biphosphate carboxylase), eight genes were involved in different functions, and one gene was of unknown function (Table 2).

Table 1. Plastid genome features of the P. tripartita var. mollissima.

Features	Poro-poro¹
Genome size (bp)	163,451
^aLSC length (bp)	85,525
^bSSC length (bp)	13,518
^cIR length (bp)	32,204
Total GC content (%)	36.87
^dA content (%)	30.79
^eT(U) content (%)	32.34
^fG content (%)	18.20
^gC content (%)	18.67
Total number of genes	128
Protein-coding genes	84
^hrRNA coding genes	8
ⁱtRNA coding genes	36
Genes duplicated in IR regions	18
Total introns	13
Single introns (gene)	11
Double introns (gene)	2

1 Poro-poro is the common name of Passiflora tripartita var. mollissima in Peru.

a LSC: a large single-copy.

b SSC: a small single-copy.

c IR: inverted repeat.

d A: adenine.

e T(U): thymine (uracil).

f G: guanine.

g C: cytosine.

h rRNA: ribosomal RNA.

i tRNA: transfer RNA.

Table 2. Genes present in the plastid genome of P. tripartita var. mollissima.

Category	Group of genes	Gene names
Photosynthesis	Subunits of photosystem I	psaA, psaB, psaC, psaI, psaJ, ycf3*, ycf4*
	Subunits of photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
	Subunits of cytochrome b/f complex	petA, petB, petD, petG, petL, petN*
	Subunits of ATP synthase	atpA, atpB, atpE, atpF, atpH, atpI
	Subunits of NADH dehydrogenase	ndhA, ndhB** (X2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
	Large subunit of RUBISCO	rbcL
Self-replication	Large subunits of ribosome	rpl2* (X2), rpl14, rpl16, rpl20, rpl22, rpl23* (X2), rpl32, rpl33, rpl36
	Small subunits of ribosome	rps2, rps3, rps4, rps8, rps11, rps12** (X2)^a, rps14, rps15, rps18, rps19 (X2)
	DNA-dependent RNA polymerase	rpoA, rpoB, rpoC1, rpoC2*
	Ribosomal RNAs	rrn4.5 (X2), rrn5 (X2), rrn16 (X2), rrn23 (X2)
	Transfer RNAs	trnA-UGC* (X2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnH-GUG, trnI-CAU (X2), trnI-GAU* (X2), trnK-UUU, trnL-CAA* (X2), trnL-UAA, trnL-UAG, trnM-CAU* (X2), trnN-GUU (X2), trnP-UGG, trnQ-UUG, trnR-ACG (X2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC (X2), trnV-UAC, trnW-CCA, trnY-GUA*
Other genes	Maturase	matK
	Protease	clpP
	Envelope membrane protein	cemA
	Acetyl-CoA carboxylase	accD
	C-type cytochrome synthesis gene	ccsA
	Translation initiation factor	InfA
	Component of TIC complex	ycf1, ycf2
Genes of unknown	Proteins of unknown function	ycf15 (X2)

* Gene contains one intron.

** Gene contains two introns.

a Gene divided into two independent transcription units.

In the plastid genome, 13 genes contained introns distributed as follows: the LSC, SSC, and IRs regions contained seven genes (petD, rpl16, rpoC1, trnK-UUU, trnL-UAA, trnV-UAC, and ycf3), one gene (ndhA), and five genes (ndhB, rpl2, rps12, trnA-UGC, and trnI-GAU) respectively. Similarly, these genes included six protein-coding genes, each with a single intron (petD, ndhA, ndhB, rpoC1, rpl2, and rpl16); five tRNA genes, each with a single intron (trnA-UGC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC); and two protein-coding genes with two introns (ycf3 and rps12). Except for 18 genes that were duplicated in the IR region (ndhB, rps19, rpl2, rpl23, rps12, ycf15, rrn4.5, rrn5, rrn16, rrn23, trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnM-CAU, trnN-GUU, trnR-ACG, and trnV-GAC) all genes contained a single copy, as shown in Table 2. The ycf1 sequence encodes a protein essential for plant viability and a vital component of the translocon on the inner chloroplast membrane (TIC) complex (Kikuchi et al., 2013), and ycf2 is a component of the ATPase motor protein associated with the TIC complex (Kikuchi et al., 2018).

Contraction and expansion of the IR boundary

In this study, the IR boundary analysis of four Passiflora species revealed that the structure and sequences of four junctions, JLB (junction between LSC and IRB), JSB (junction between SSC and IRB), JSA (junction between SSC and IRA), and JLA (junction between LSC and IRA), between the two inverted repeats (IRa and IRb) and the two single-copy regions (LSC and SSC) of P. tripartita var. mollissima, P. oerstedii (147 073 bp; Genbank accession: NC_038124), P. foetida (162 266 bp; Genbank accession: NC_043825), and P. edulis (151 406 bp; Genbank accession: NC_034285) were similar (Figure 2). The genes of rps3, rps19, rpl2, rps15, ycf1, ndhF, ndhH, and psbA were located mainly near the IR/LSC and IR/SSC boundaries of the plastome for these four species of Passiflora. In the same order that was described, rps3 is entirely located in the LSC region, at distances of 206 bp, 264 bp, 159 bp, and 206 bp, respectively, from the JLB boundary. For rps19, which is in both IR regions, the nucleotide distance from the JLB boundary varies from 128 – 210 bp. In P. oerstedii, both copies of the rps2 gene are in the IR región, and the ndhH gene is located in the SSC region.

Figure 2. Comparison of IR/SC boundary regions of four Passiflora species.

Boxes represent the nearby border genes. Gaps between the ends of boundaries and adjacent genes, as well as the sizes of gene segments positioned within a boundary, are depicted. The junction sites of LSC/IRb, IRb/SSC, SSC/IRa, and IRa/LSC are denoted as JLB, JSB, JSA, and JLA, respectively.

The rps15 gene crossed the SSC/IRb boundary, expanding 243 bp and 17 bp in P. tripartita var. mollissima, respectively. The rps15 gene is located 182 bp away from the SSC/IRa boundary in P. foetida and is located at the end of the SSC region, expanding 81 bp and 20 bp in P. oerstedii and P. edulis, respectively. In all species compared, the ndhF gene is located 234 bp away from the SSC/IRa boundary in P. tripartita var. mollissima, and is located 29 bp, 14 bp, and 219 bp away from the SSC/IRb boundary in P. oerstedii, P. foetida, and P. edulis. Furthermore, the ycf1 gene in P. oerstedii, P. foetida, and P. edulis is located 266 – 481 bp away from the SSC/IRa boundary, except for P. tripartita var. mollissima, which was not present in JSA.

The infA gene, which codes for translation initiation factor 1, is present in P. tripartita var. mollissima, but it is absent from the P. foetida, P. oerstedii, and P. edulis cp genomes. Furthermore, trnG-UCC and ycf68 are unique genes in P. foetida and P. edulis, respectively. The plastome of P. tripartita var. mollissima contained seven genes (ycf1, ycf2, ycf15, rpl20, rpl22, accD, infA) that were lost or non-functional genes in P. edulis; and compared to P. foetida, P. oersteddi, and P. edulis, the trnfM-CAU gene was not found.

Phylogenetic reconstruction

To identify the evolutionary position of Passiflora tripartita var. mollissima in the Passifloraceae family, phylogenetic relationships based on the OrthoFinder clustering method were used to avoid erroneous rearrangements in phylogenetic tree reconstruction and provides a more reliable evolutionary analysis (Gabaldón, 2005; Zhang et al., 2012). The phylogenetic tree was constructed based on single-copy orthologous genes (Emms & Kelly, 2019) and maximum likelihood analysis with the complete annotated protein sequences of 27 plastid genomes, of which 26 were from Passiflora species. One species, Vitis vinifera, was chosen as the outgroup.

Maximum likelihood (ML) bootstrap values ranged from 38%–92% for seven of the 25 nodes. All nodes except the indicated ones (seven nodes) exhibited bootstrap support (BS) values of 100%. These Passiflora species were divided into four groups: subgenus Passiflora (P. nitida, P. quadrangularis, P. cincinnata, P. caerulea, P. edulis, P. laurifolia, P. vitifolia, P. serratifolia, P. serrulata, P. ligularis, P. serratodigitata, P. actinia, P. menispermifolia and P. oerstedii), subgenus Tetrapathea (P. tetrandra), subgenus Decaloba (P. microstipula, P. xishuangbannaensis, P. biflora, P. lutea, P. jatunsachensis, P. suberosa and P. tenuiloba), and subgenus Deidamoides (P. contracta and P. arbelaezii). The relationships between the four subgenera of Passiflora species (Passiflora, Tetrapathea, Decaloba, and Deidamoides) were congruent and strongly supported by the same patterns as previously reported (Cauz-Santos et al., 2020; Pacheco et al., 2020). These results resolved Passiflora tripartita var. mollissima belonging to the subgenus Passiflora, which was closely related to P. menispermifolia and P. oerstedii with 100% BS, and was sister to P. tetrandra (subgenus Tetrapathea), P. biflora (subgenus Decaloba), and P. contracta (subgenus Deidamoides), as shown in the cladogram (Figure 3).

Figure 3. Phylogenetic tree of 27 plastid genomes using maximum likelihood analysis based on single-copy orthologous protein.

Bootstrap values on the branches were calculated from 1000 replicates.

Data availability

Underlying data

Nucleotide: Passiflora tripartita var. mollissima chloroplast, complete genome. Accession number: OQ910395. https://identifiers.org/nucleotide:OQ910395 (GenBank, 2023).

Extended data

Figshare: Herbarium specimen voucher of Passiflora tripartita var. mollissima (Kunth) Holms-Niels. & P.M. Jørg (USM:MHN331530). https://doi.org/10.6084/m9.figshare.23556654 (Aliaga et al., 2023a).

Figshare: Gel imagen of DNA isolate from poro-poro sample. https://doi.org/10.6084/m9.figshare.23560755 (Aliaga et al., 2023b).

Figshare: Details of the plastid genome sequences used for phylogenetic analysis. https://doi.org/10.6084/m9.figshare.23556834 (Aliaga et al., 2023c).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgements

We thank the Universidad Privada del Norte (UPN) for funding the APC. We thank the Servicio Nacional Forestal y de Fauna Silvestre (SERFOR) for authorized this research project (AUT-IFL-2022-058). We thank the Consejo Departamental de La Libertad (CDLL) - Colegio de Ingenieros del Perú (CIP) for the support and promotion of this research at the regional and national level. We thank Prof. Dr. Esteban Hopp (Universidad de Buenos Aires) for their careful reading of the manuscript and their constructive remaks. We thank MSc. Rocío Natalia González Guerra(Macrogen, Inc. and Macrogen Spain) for her support and guidance in the NGS sequencing of this plant species. We thank Petr Sklenář and Filip Kolar for their help in the sample collection. We thank curator Julio C. Torres–Martinez (Museo de Historia Natural, Universidad Nacional Mayor de San Marcos) for the taxonomy identification and deposit of the plant specimen. We thank Dr. Rajest Mahato and Dr. Giusseppe D’Auria for the recommendations and bioinformatics support. We thank Mr. Julián Vasquez-Arriaga for administrative support.

An earlier version of this article can be found on Preprints.org (doi: https://doi.org/10.20944/preprints202306.0463.v2).

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Version 3

VERSION 3 PUBLISHED 07 Jul 2023

Author details Author details

Mario Zapata-Cruz
Roles: Conceptualization, Investigation, Methodology, Project Administration, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This research was funded by Plant Science Laboratory E.I.R.L. (Sach’a Ruru grant: RIC-2022-101).

Article Versions (3)

version 3

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Published: 19 Feb 2024, 12:795

https://doi.org/10.12688/f1000research.138150.3

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https://doi.org/10.12688/f1000research.138150.2

version 1

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https://doi.org/10.12688/f1000research.138150.1

© 2024 Aliaga F et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Aliaga F, Zapata-Cruz M and Valverde-Zavaleta SA. Plastid genome of Passiflora tripartita var. mollissima (poro-poro) from Huánuco, Peru [version 3; peer review: 2 approved] F1000Research 2024, 12:795 (https://doi.org/10.12688/f1000research.138150.3)

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Reviewer Report 22 Feb 2024

Rahul G Shelke, Independent Researcher, Amravati, India

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https://doi.org/10.5256/f1000research.162741.r248377

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Reviewer Report 14 Feb 2024

Rahul G Shelke, Independent Researcher, Amravati, India

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https://doi.org/10.5256/f1000research.160734.r236402

I have thoroughly reviewed the manuscript and would like to bring to your attention a minor issue in Fig. 1.
Upon careful examination, I have observed that certain genes, including rrn23, rrn23-fragment, infA, rpoA, etc., bear double annotations in the figure. I appreciate the authors' efforts in incorporating the suggested changes throughout the manuscript. However, I recommend addressing this specific matter to enhance clarity for readers.
To rectify this, I propose that the extra annotations be removed from the GenBank file before generating the diagram. This correction will streamline the information presented in Fig. 1, ensuring a more concise and comprehensible representation for new readers.
I believe implementing this adjustment will further enhance the overall quality of the manuscript.

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Organelle genome sequencing, Transcriptome assembling, Genetic diversity, Barcoding and DNA markers etc

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Author Response 13 Apr 2024

Flavio Aliaga, Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, 13011, Peru

13 Apr 2024

Author Response

The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us ... Continue reading The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

In the previous version (V.2), we did not update the circular map (Fig. 1). In this new version, all the genes correctly mentioned in Table 2 are observed in Figure 1. Likewise, we clarify that the gene rps12 is divided into two independent transcription units (Table 2).

We appreciate you greatly for your immense support in reviewing this manuscript.
The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

In the previous version (V.2), we did not update the circular map (Fig. 1). In this new version, all the genes correctly mentioned in Table 2 are observed in Figure 1. Likewise, we clarify that the gene rps12 is divided into two independent transcription units (Table 2).

We appreciate you greatly for your immense support in reviewing this manuscript.
Competing Interests: No competing interests were disclosed. Close
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Author Response 13 Apr 2024

Flavio Aliaga, Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, 13011, Peru

13 Apr 2024

Author Response

The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us ... Continue reading The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

In the previous version (V.2), we did not update the circular map (Fig. 1). In this new version, all the genes correctly mentioned in Table 2 are observed in Figure 1. Likewise, we clarify that the gene rps12 is divided into two independent transcription units (Table 2).

We appreciate you greatly for your immense support in reviewing this manuscript.
The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

In the previous version (V.2), we did not update the circular map (Fig. 1). In this new version, all the genes correctly mentioned in Table 2 are observed in Figure 1. Likewise, we clarify that the gene rps12 is divided into two independent transcription units (Table 2).

We appreciate you greatly for your immense support in reviewing this manuscript.
Competing Interests: No competing interests were disclosed. Close
Report a concern

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Reviewer Report 19 Jan 2024

Abdullah Abdullah, Alpha Genomics Private Limited, Islamabad, Quaid-i-Azam University, Islamabad, Islamabad Capital Territory, Pakistan

Approved

https://doi.org/10.5256/f1000research.160734.r236401

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Reviewer Report 04 Sep 2023

Abdullah Abdullah, Alpha Genomics Private Limited, Islamabad, Quaid-i-Azam University, Islamabad, Islamabad Capital Territory, Pakistan

Approved with Reservations

https://doi.org/10.5256/f1000research.151329.r191281

Authors need to describe how the current genome will be helpful. I found many species of the genus Passiflora in the NCBI. So, authors need clear points about the need for the existing genome. In addition, all the genomes have similar features so the current genome is helpful for the study of evolution and phylogenetics.

The authors mentioned the number of reported genomes up to 800. This statement is not correct and is based on earlier observations. Now the number has increased significantly.

I found authors are not entirely aware of the utilization of some software. For example, authors mention fastqc for trimming while fastqc checks the quality of reads.

The authors used many programs for annotations. I will suggest authors use GeSeq along with Chole, Aragorn and tRNAScan on GeSeq servers for annotation of transfer RNA.

The authors need to give some information on how the current genome is similar to or differs from other reported genomes of Passiflora.

Are the rationale for sequencing the genome and the species significance clearly described?

Partly
Are the protocols appropriate and is the work technically sound?

Partly
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Partly
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: I can assess all aspects of the manuscript and published up to 18 articles in the same field.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 23 Jan 2024

Flavio Aliaga, Capítulo de Ingeniería Agronómica, Consejo Departamental de La Libertad (CDLL), Colegio de Ingenieros del Perú (CIP), Trujillo, 13008, Peru

23 Jan 2024

Author Response
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad ... Continue reading
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru

1. Authors need to describe how the current genome will be helpful. I found many species of the genus Passiflora in the NCBI. So, authors need clear points about the need for the existing genome.

Answer: We would like to thank reviewer 2 for the insightful response. We have considered your comment because the poro-poro plastid genome constitutes a valuable resource for studying the molecular evolution, phylogenetics, and domestication of species with beneficial characteristics for human health.

2. In addition, all the genomes have similar features so the current genome is helpful for the study of evolution and phylogenetics.

Answer: We are grateful to reviewer 2 for the constructive and valuable comments. Thank you.

3. The authors mentioned the number of reported genomes up to 800. This statement is not correct and is based on earlier observations. Now the number has increased significantly.

Answer. We agree with the reviewer. The manuscript has been corrected:

" Plastome sequences from over 4000 species…”.

4. I found authors are not entirely aware of the utilization of some software. For example, authors mention fastqc for trimming while fastqc checks the quality of reads.

Answer. It has been revised, thank you.

5. The authors used many programs for annotations. I will suggest authors use GeSeq along with Chole, Aragorn and tRNAScan on GeSeq servers for annotation of transfer RNA.

Answer. It has been revised, thank you.

6. The authors need to give some information on how the current genome is similar to or differs from other reported genomes of Passiflora

Answer. Thank you for your comment. We have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru

1. Authors need to describe how the current genome will be helpful. I found many species of the genus Passiflora in the NCBI. So, authors need clear points about the need for the existing genome.

Answer: We would like to thank reviewer 2 for the insightful response. We have considered your comment because the poro-poro plastid genome constitutes a valuable resource for studying the molecular evolution, phylogenetics, and domestication of species with beneficial characteristics for human health.

2. In addition, all the genomes have similar features so the current genome is helpful for the study of evolution and phylogenetics.

Answer: We are grateful to reviewer 2 for the constructive and valuable comments. Thank you.

3. The authors mentioned the number of reported genomes up to 800. This statement is not correct and is based on earlier observations. Now the number has increased significantly.

Answer. We agree with the reviewer. The manuscript has been corrected:

" Plastome sequences from over 4000 species…”.

4. I found authors are not entirely aware of the utilization of some software. For example, authors mention fastqc for trimming while fastqc checks the quality of reads.

Answer. It has been revised, thank you.

5. The authors used many programs for annotations. I will suggest authors use GeSeq along with Chole, Aragorn and tRNAScan on GeSeq servers for annotation of transfer RNA.

Answer. It has been revised, thank you.

6. The authors need to give some information on how the current genome is similar to or differs from other reported genomes of Passiflora

Answer. Thank you for your comment. We have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 23 Jan 2024

Flavio Aliaga, Capítulo de Ingeniería Agronómica, Consejo Departamental de La Libertad (CDLL), Colegio de Ingenieros del Perú (CIP), Trujillo, 13008, Peru

23 Jan 2024

Author Response
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad ... Continue reading
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru

1. Authors need to describe how the current genome will be helpful. I found many species of the genus Passiflora in the NCBI. So, authors need clear points about the need for the existing genome.

Answer: We would like to thank reviewer 2 for the insightful response. We have considered your comment because the poro-poro plastid genome constitutes a valuable resource for studying the molecular evolution, phylogenetics, and domestication of species with beneficial characteristics for human health.

2. In addition, all the genomes have similar features so the current genome is helpful for the study of evolution and phylogenetics.

Answer: We are grateful to reviewer 2 for the constructive and valuable comments. Thank you.

3. The authors mentioned the number of reported genomes up to 800. This statement is not correct and is based on earlier observations. Now the number has increased significantly.

Answer. We agree with the reviewer. The manuscript has been corrected:

" Plastome sequences from over 4000 species…”.

4. I found authors are not entirely aware of the utilization of some software. For example, authors mention fastqc for trimming while fastqc checks the quality of reads.

Answer. It has been revised, thank you.

5. The authors used many programs for annotations. I will suggest authors use GeSeq along with Chole, Aragorn and tRNAScan on GeSeq servers for annotation of transfer RNA.

Answer. It has been revised, thank you.

6. The authors need to give some information on how the current genome is similar to or differs from other reported genomes of Passiflora

Answer. Thank you for your comment. We have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru

1. Authors need to describe how the current genome will be helpful. I found many species of the genus Passiflora in the NCBI. So, authors need clear points about the need for the existing genome.

Answer: We would like to thank reviewer 2 for the insightful response. We have considered your comment because the poro-poro plastid genome constitutes a valuable resource for studying the molecular evolution, phylogenetics, and domestication of species with beneficial characteristics for human health.

2. In addition, all the genomes have similar features so the current genome is helpful for the study of evolution and phylogenetics.

Answer: We are grateful to reviewer 2 for the constructive and valuable comments. Thank you.

3. The authors mentioned the number of reported genomes up to 800. This statement is not correct and is based on earlier observations. Now the number has increased significantly.

Answer. We agree with the reviewer. The manuscript has been corrected:

" Plastome sequences from over 4000 species…”.

4. I found authors are not entirely aware of the utilization of some software. For example, authors mention fastqc for trimming while fastqc checks the quality of reads.

Answer. It has been revised, thank you.

5. The authors used many programs for annotations. I will suggest authors use GeSeq along with Chole, Aragorn and tRNAScan on GeSeq servers for annotation of transfer RNA.

Answer. It has been revised, thank you.

6. The authors need to give some information on how the current genome is similar to or differs from other reported genomes of Passiflora

Answer. Thank you for your comment. We have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.
Competing Interests: No competing interests were disclosed. Close
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Views

Reviewer Report 30 Aug 2023

Rahul G Shelke, Independent Researcher, Amravati, India

Approved with Reservations

https://doi.org/10.5256/f1000research.151329.r191279

I extend my appreciation to the authors for their commendable efforts in extensively exploring the plastid genome of Passiflora tripartita var. mollissima, demonstrating remarkable depth and breadth in their approach. The manuscript substantially enhances our comprehension of this species, emphasizing its plastid genome, genetic diversity, and evolutionary relationships. This newfound insight carries far-reaching implications, fostering the conservation and sustainable utilization of this species. Consequently, the manuscript's significance is markedly elevated, making a noteworthy contribution to the scientific community's understanding of genetic resource stewardship.

Here is a concise summary of the study's key points:

Implications and Novelty: The findings of the study hold significant implications. The genetic insights uncovered have the potential to catalyze further investigations in areas such as molecular evolution, conservation genetics, and the development of plant breeding programs. Notably, the manuscript's distinction as the first documentation of the plastid genome of Passiflora tripartita var. mollissima from Peru underscores its novel contribution to the scientific community.
Methodological Rigor: The authors' selection of the Illumina Novaseq 6000 platform for DNA sequencing is well-suited to the research objectives. The subsequent analyses and methodologies applied exhibit robustness and alignment with the research goals.

Major comments:

While the genes in Table 2 have been organized into categories, it is apparent that certain instances lack gene information or are unclear in their presentation. This calls for a restructuring of the table to ensure a heightened level of clarity and coherence.
In Table 2, the authors have indicated the absence of a duplicated copy of the rrn4.5 gene. It is advised that the authors review the annotation of this gene. Typically, rRNA genes are located within the IR regions and therefore exist in duplicated copies. However, in Figure 1 of the chloroplast genome, the rrn4.5 gene is depicted in a duplicated copy. I recommend that the authors rectify any discrepancies pertaining to the rrn4.5 gene both in the table and within the text.
The authors have provided comprehensive insights into the expansion and contraction of the LSC, SSC, and IR regions in the compared species. To further enhance reader comprehension, I recommend that the authors consider incorporating the corresponding IRSCOPE figure within the manuscript. This addition would allow readers to access detailed information more effectively and contribute to a clearer understanding of the discussed concepts.
Kindly provide information regarding the genes that are missing or subject to gene loss within the compared species.
In the abstract, the authors have included the statement: “In summary, our study provides the basis for developing new molecular markers that constitutes a valuable resource for studying molecular evolution and domestication.” However, upon reviewing the manuscript, it is evident that the authors have not conducted any SSR, tandem repeat, or DNA diversity analysis, nor have they proposed any markers. Given this situation, I recommend that the authors consider removing the phrase “developing new molecular markers” from the abstract to accurately reflect the scope of their study.

Minor comments:

The inclusion of specific methodology details such as "Total genomic DNA was extracted from fresh leaves (herbarium voucher: USM:MHN331531)" and "The DNA was sequenced using Illumina Novaseq 6000 platform" in the abstract might be better suited for the methodology section rather than the abstract itself. Focusing the abstract on the core findings and implications of the study could enhance its conciseness and clarity.
Add the full botanical name of Passiflora tripartita in the phylogeny tree to ensure consistency.
Gene names should be in italics.
It is recommended to include the coverage of the genome assembly within the main text of the manuscript.

The authors adeptly tackle gaps in our understanding of this species' genetics, comparative genomics, and evolutionary relationships. By thoughtfully engaging with reviewer feedback and subsequently refining their manuscript, the authors stand poised to elevate the impact and significance of their research within the realm of plant genomics and beyond. This process will significantly enhance the accuracy and reliability of gene classification and annotation, thereby elevating the overall quality of the manuscript.

Are the rationale for sequencing the genome and the species significance clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Yes
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Organelle genome sequencing, Transcriptome assembling, Genetic diversity, Barcoding and DNA markers etc

CITE

Report a concern

Author Response 23 Jan 2024

Flavio Aliaga, Capítulo de Ingeniería Agronómica, Consejo Departamental de La Libertad (CDLL), Colegio de Ingenieros del Perú (CIP), Trujillo, 13008, Peru

23 Jan 2024

Author Response
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad ... Continue reading
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru

The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

Answers to Reviewer #1

Major comments:

1. While the genes in Table 2 have been organized into categories, it is apparent that certain instances lack gene information or are unclear in their presentation. This calls for a restructuring of the table to ensure a heightened level of clarity and coherence.

Answer: We agree with the reviewer. The manuscript has been revised, thanks.

2. In Table 2, the authors have indicated the absence of a duplicated copy of the rrn4.5 gene. It is advised that the authors review the annotation of this gene. Typically, rRNA genes are located within the IR regions and therefore exist in duplicated copies. However, in Fiure 1 of the chloroplast genome, the rrn4.5 gene is depicted in a duplicated copy. I recommend that the authors rectify any discrepancies pertaining to the rrn4.5 gene both in the table and within the text.

Answer: We would like to thank the reviewer for highlighting this important comment. In fact, we checked and updated the duplicated copy of the rrn4.5 gene. Thank you.

3. The authors have provided comprehensive insights into the expansion and contraction of the LSC, SSC, and IR regions in the compared species. To further enhance reader comprehension, I recommend that the authors consider incorporating the corresponding IRSCOPE figure within the manuscript. This addition would allow readers to access detailed information more effectively and contribute to a clearer understanding of the discussed concepts.

Answer: Thank you for your comment. According to your suggestions, we have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.

4. Kindly provide information regarding the genes that are missing or subject to gene loss within the compared species.

Answer: Thank you for your reminding. Done.

5. In the abstract, the authors have included the statement: “In summary, our study provides the basis for developing new molecular markers that constitutes a valuable resource for studying molecular evolution and domestication.” However, upon reviewing the manuscript, it is evident that the authors have not conducted any SSR, tandem repeat, or DNA diversity analysis, nor have they proposed any markers. Given this situation, I recommend that the authors consider removing the phrase “developing new molecular markers” from the abstract to accurately reflect the scope of their study.

Answer. We agree with the reviewer. The manuscript has been corrected:

“In summary, our study constitutes a valuable resource for studying molecular evolution, phylogenetics, and domestication…”

Minor comments:

1. The inclusion of specific methodology details such as "Total genomic DNA was extracted from fresh leaves (herbarium voucher: USM:MHN331531)" and "The DNA was sequenced using Illumina Novaseq 6000 platform" in the abstract might be better suited for the methodology section rather than the abstract itself. Focusing the abstract on the core findings and implications of the study could enhance its conciseness and clarity.

Answer. Yes, we agree with the reviewer. Done.

2. Add the full botanical name of Passiflora tripartita in the phylogeny tree to ensure consistency.

Answer. Done.

3. Gene names should be in italics. It is recommended to include the coverage of the genome assembly within the main text of the manuscript.

Answer. Done.

4. The authors adeptly tackle gaps in our understanding of this species' genetics, comparative genomics, and evolutionary relationships. By thoughtfully engaging with reviewer feedback and subsequently refining their manuscript, the authors stand poised to elevate the impact and significance of their research within the realm of plant genomics and beyond. This process will significantly enhance the accuracy and reliability of gene classification and annotation, thereby elevating the overall quality of the manuscript.

Answer: We thank reviewer 1 for all constructive comments that led to substantial improvement of the manuscript. Thank you; it is much appreciated.
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru

The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

Answers to Reviewer #1

Major comments:

1. While the genes in Table 2 have been organized into categories, it is apparent that certain instances lack gene information or are unclear in their presentation. This calls for a restructuring of the table to ensure a heightened level of clarity and coherence.

Answer: We agree with the reviewer. The manuscript has been revised, thanks.

2. In Table 2, the authors have indicated the absence of a duplicated copy of the rrn4.5 gene. It is advised that the authors review the annotation of this gene. Typically, rRNA genes are located within the IR regions and therefore exist in duplicated copies. However, in Fiure 1 of the chloroplast genome, the rrn4.5 gene is depicted in a duplicated copy. I recommend that the authors rectify any discrepancies pertaining to the rrn4.5 gene both in the table and within the text.

Answer: We would like to thank the reviewer for highlighting this important comment. In fact, we checked and updated the duplicated copy of the rrn4.5 gene. Thank you.

3. The authors have provided comprehensive insights into the expansion and contraction of the LSC, SSC, and IR regions in the compared species. To further enhance reader comprehension, I recommend that the authors consider incorporating the corresponding IRSCOPE figure within the manuscript. This addition would allow readers to access detailed information more effectively and contribute to a clearer understanding of the discussed concepts.

Answer: Thank you for your comment. According to your suggestions, we have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.

4. Kindly provide information regarding the genes that are missing or subject to gene loss within the compared species.

Answer: Thank you for your reminding. Done.

5. In the abstract, the authors have included the statement: “In summary, our study provides the basis for developing new molecular markers that constitutes a valuable resource for studying molecular evolution and domestication.” However, upon reviewing the manuscript, it is evident that the authors have not conducted any SSR, tandem repeat, or DNA diversity analysis, nor have they proposed any markers. Given this situation, I recommend that the authors consider removing the phrase “developing new molecular markers” from the abstract to accurately reflect the scope of their study.

Answer. We agree with the reviewer. The manuscript has been corrected:

“In summary, our study constitutes a valuable resource for studying molecular evolution, phylogenetics, and domestication…”

Minor comments:

1. The inclusion of specific methodology details such as "Total genomic DNA was extracted from fresh leaves (herbarium voucher: USM:MHN331531)" and "The DNA was sequenced using Illumina Novaseq 6000 platform" in the abstract might be better suited for the methodology section rather than the abstract itself. Focusing the abstract on the core findings and implications of the study could enhance its conciseness and clarity.

Answer. Yes, we agree with the reviewer. Done.

2. Add the full botanical name of Passiflora tripartita in the phylogeny tree to ensure consistency.

Answer. Done.

3. Gene names should be in italics. It is recommended to include the coverage of the genome assembly within the main text of the manuscript.

Answer. Done.

4. The authors adeptly tackle gaps in our understanding of this species' genetics, comparative genomics, and evolutionary relationships. By thoughtfully engaging with reviewer feedback and subsequently refining their manuscript, the authors stand poised to elevate the impact and significance of their research within the realm of plant genomics and beyond. This process will significantly enhance the accuracy and reliability of gene classification and annotation, thereby elevating the overall quality of the manuscript.

Answer: We thank reviewer 1 for all constructive comments that led to substantial improvement of the manuscript. Thank you; it is much appreciated.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 23 Jan 2024

Flavio Aliaga, Capítulo de Ingeniería Agronómica, Consejo Departamental de La Libertad (CDLL), Colegio de Ingenieros del Perú (CIP), Trujillo, 13008, Peru

23 Jan 2024

Author Response
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad ... Continue reading
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru

The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

Answers to Reviewer #1

Major comments:

1. While the genes in Table 2 have been organized into categories, it is apparent that certain instances lack gene information or are unclear in their presentation. This calls for a restructuring of the table to ensure a heightened level of clarity and coherence.

Answer: We agree with the reviewer. The manuscript has been revised, thanks.

2. In Table 2, the authors have indicated the absence of a duplicated copy of the rrn4.5 gene. It is advised that the authors review the annotation of this gene. Typically, rRNA genes are located within the IR regions and therefore exist in duplicated copies. However, in Fiure 1 of the chloroplast genome, the rrn4.5 gene is depicted in a duplicated copy. I recommend that the authors rectify any discrepancies pertaining to the rrn4.5 gene both in the table and within the text.

Answer: We would like to thank the reviewer for highlighting this important comment. In fact, we checked and updated the duplicated copy of the rrn4.5 gene. Thank you.

3. The authors have provided comprehensive insights into the expansion and contraction of the LSC, SSC, and IR regions in the compared species. To further enhance reader comprehension, I recommend that the authors consider incorporating the corresponding IRSCOPE figure within the manuscript. This addition would allow readers to access detailed information more effectively and contribute to a clearer understanding of the discussed concepts.

Answer: Thank you for your comment. According to your suggestions, we have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.

4. Kindly provide information regarding the genes that are missing or subject to gene loss within the compared species.

Answer: Thank you for your reminding. Done.

5. In the abstract, the authors have included the statement: “In summary, our study provides the basis for developing new molecular markers that constitutes a valuable resource for studying molecular evolution and domestication.” However, upon reviewing the manuscript, it is evident that the authors have not conducted any SSR, tandem repeat, or DNA diversity analysis, nor have they proposed any markers. Given this situation, I recommend that the authors consider removing the phrase “developing new molecular markers” from the abstract to accurately reflect the scope of their study.

Answer. We agree with the reviewer. The manuscript has been corrected:

“In summary, our study constitutes a valuable resource for studying molecular evolution, phylogenetics, and domestication…”

Minor comments:

1. The inclusion of specific methodology details such as "Total genomic DNA was extracted from fresh leaves (herbarium voucher: USM:MHN331531)" and "The DNA was sequenced using Illumina Novaseq 6000 platform" in the abstract might be better suited for the methodology section rather than the abstract itself. Focusing the abstract on the core findings and implications of the study could enhance its conciseness and clarity.

Answer. Yes, we agree with the reviewer. Done.

2. Add the full botanical name of Passiflora tripartita in the phylogeny tree to ensure consistency.

Answer. Done.

3. Gene names should be in italics. It is recommended to include the coverage of the genome assembly within the main text of the manuscript.

Answer. Done.

4. The authors adeptly tackle gaps in our understanding of this species' genetics, comparative genomics, and evolutionary relationships. By thoughtfully engaging with reviewer feedback and subsequently refining their manuscript, the authors stand poised to elevate the impact and significance of their research within the realm of plant genomics and beyond. This process will significantly enhance the accuracy and reliability of gene classification and annotation, thereby elevating the overall quality of the manuscript.

Answer: We thank reviewer 1 for all constructive comments that led to substantial improvement of the manuscript. Thank you; it is much appreciated.
Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru

The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

Answers to Reviewer #1

Major comments:

1. While the genes in Table 2 have been organized into categories, it is apparent that certain instances lack gene information or are unclear in their presentation. This calls for a restructuring of the table to ensure a heightened level of clarity and coherence.

Answer: We agree with the reviewer. The manuscript has been revised, thanks.

2. In Table 2, the authors have indicated the absence of a duplicated copy of the rrn4.5 gene. It is advised that the authors review the annotation of this gene. Typically, rRNA genes are located within the IR regions and therefore exist in duplicated copies. However, in Fiure 1 of the chloroplast genome, the rrn4.5 gene is depicted in a duplicated copy. I recommend that the authors rectify any discrepancies pertaining to the rrn4.5 gene both in the table and within the text.

Answer: We would like to thank the reviewer for highlighting this important comment. In fact, we checked and updated the duplicated copy of the rrn4.5 gene. Thank you.

3. The authors have provided comprehensive insights into the expansion and contraction of the LSC, SSC, and IR regions in the compared species. To further enhance reader comprehension, I recommend that the authors consider incorporating the corresponding IRSCOPE figure within the manuscript. This addition would allow readers to access detailed information more effectively and contribute to a clearer understanding of the discussed concepts.

Answer: Thank you for your comment. According to your suggestions, we have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.

4. Kindly provide information regarding the genes that are missing or subject to gene loss within the compared species.

Answer: Thank you for your reminding. Done.

5. In the abstract, the authors have included the statement: “In summary, our study provides the basis for developing new molecular markers that constitutes a valuable resource for studying molecular evolution and domestication.” However, upon reviewing the manuscript, it is evident that the authors have not conducted any SSR, tandem repeat, or DNA diversity analysis, nor have they proposed any markers. Given this situation, I recommend that the authors consider removing the phrase “developing new molecular markers” from the abstract to accurately reflect the scope of their study.

Answer. We agree with the reviewer. The manuscript has been corrected:

“In summary, our study constitutes a valuable resource for studying molecular evolution, phylogenetics, and domestication…”

Minor comments:

1. The inclusion of specific methodology details such as "Total genomic DNA was extracted from fresh leaves (herbarium voucher: USM:MHN331531)" and "The DNA was sequenced using Illumina Novaseq 6000 platform" in the abstract might be better suited for the methodology section rather than the abstract itself. Focusing the abstract on the core findings and implications of the study could enhance its conciseness and clarity.

Answer. Yes, we agree with the reviewer. Done.

2. Add the full botanical name of Passiflora tripartita in the phylogeny tree to ensure consistency.

Answer. Done.

3. Gene names should be in italics. It is recommended to include the coverage of the genome assembly within the main text of the manuscript.

Answer. Done.

4. The authors adeptly tackle gaps in our understanding of this species' genetics, comparative genomics, and evolutionary relationships. By thoughtfully engaging with reviewer feedback and subsequently refining their manuscript, the authors stand poised to elevate the impact and significance of their research within the realm of plant genomics and beyond. This process will significantly enhance the accuracy and reliability of gene classification and annotation, thereby elevating the overall quality of the manuscript.

Answer: We thank reviewer 1 for all constructive comments that led to substantial improvement of the manuscript. Thank you; it is much appreciated.
Competing Interests: No competing interests were disclosed. Close
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Version 3

VERSION 3 PUBLISHED 07 Jul 2023

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Version 3 (revision) 19 Feb 24	read
Version 2 (revision) 09 Jan 24	read	read
Version 1 07 Jul 23	read	read

Rahul G Shelke, Independent Researcher, Amravati, India
Abdullah Abdullah, Quaid-i-Azam University, Islamabad, Pakistan

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22 Feb 2024 | for Version 3

Rahul G Shelke, Independent Researcher, Amravati, India

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Approved

Since the authors attentively considered every recommendation, I have nothing else to add.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Organelle genome sequencing, Transcriptome assembling, Genetic diversity, Barcoding and DNA markers etc

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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14 Feb 2024 | for Version 2

Rahul G Shelke, Independent Researcher, Amravati, India

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Approved

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Organelle genome sequencing, Transcriptome assembling, Genetic diversity, Barcoding and DNA markers etc

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Author Response

13 Apr 2024

Flavio Aliaga, Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, 13011, Peru

The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

In the previous version (V.2), we did not update the circular map (Fig. 1). In this new version, all the genes correctly mentioned in Table 2 are observed in Figure 1. Likewise, we clarify that the gene rps12 is divided into two independent transcription units (Table 2).

We appreciate you greatly for your immense support in reviewing this manuscript.

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No competing interests were disclosed.

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19 Jan 2024 | for Version 2

Abdullah Abdullah, Alpha Genomics Private Limited, Islamabad, Quaid-i-Azam University, Islamabad, Islamabad Capital Territory, Pakistan

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Approved

The article can be accepted.

Competing Interests

No competing interests were disclosed.

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I can assess all aspects of the manuscript and published up to 18 articles in the same field.

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04 Sep 2023 | for Version 1

Abdullah Abdullah, Alpha Genomics Private Limited, Islamabad, Quaid-i-Azam University, Islamabad, Islamabad Capital Territory, Pakistan

20 Views Cite this report Responses(1)

Approved With Reservations

Are the rationale for sequencing the genome and the species significance clearly described?

Partly
Are the protocols appropriate and is the work technically sound?

Partly
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Partly
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

I can assess all aspects of the manuscript and published up to 18 articles in the same field.

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Author Response

23 Jan 2024

Flavio Aliaga, Capítulo de Ingeniería Agronómica, Consejo Departamental de La Libertad (CDLL), Colegio de Ingenieros del Perú (CIP), Trujillo, 13008, Peru

" Plastome sequences from over 4000 species…”.

4. I found authors are not entirely aware of the utilization of some software. For example, authors mention fastqc for trimming while fastqc checks the quality of reads.

Answer. It has been revised, thank you.

5. The authors used many programs for annotations. I will suggest authors use GeSeq along with Chole, Aragorn and tRNAScan on GeSeq servers for annotation of transfer RNA.

Answer. It has been revised, thank you.

6. The authors need to give some information on how the current genome is similar to or differs from other reported genomes of Passiflora

Answer. Thank you for your comment. We have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.

View more View less

Competing Interests

No competing interests were disclosed.

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Reviewer Report

37 Views

30 Aug 2023 | for Version 1

Rahul G Shelke, Independent Researcher, Amravati, India

37 Views Cite this report Responses(1)

Approved With Reservations

Implications and Novelty: The findings of the study hold significant implications. The genetic insights uncovered have the potential to catalyze further investigations in areas such as molecular evolution, conservation genetics, and the development of plant breeding programs. Notably, the manuscript's distinction as the first documentation of the plastid genome of Passiflora tripartita var. mollissima from Peru underscores its novel contribution to the scientific community.
Methodological Rigor: The authors' selection of the Illumina Novaseq 6000 platform for DNA sequencing is well-suited to the research objectives. The subsequent analyses and methodologies applied exhibit robustness and alignment with the research goals.

Major comments:

While the genes in Table 2 have been organized into categories, it is apparent that certain instances lack gene information or are unclear in their presentation. This calls for a restructuring of the table to ensure a heightened level of clarity and coherence.
In Table 2, the authors have indicated the absence of a duplicated copy of the rrn4.5 gene. It is advised that the authors review the annotation of this gene. Typically, rRNA genes are located within the IR regions and therefore exist in duplicated copies. However, in Figure 1 of the chloroplast genome, the rrn4.5 gene is depicted in a duplicated copy. I recommend that the authors rectify any discrepancies pertaining to the rrn4.5 gene both in the table and within the text.
The authors have provided comprehensive insights into the expansion and contraction of the LSC, SSC, and IR regions in the compared species. To further enhance reader comprehension, I recommend that the authors consider incorporating the corresponding IRSCOPE figure within the manuscript. This addition would allow readers to access detailed information more effectively and contribute to a clearer understanding of the discussed concepts.
Kindly provide information regarding the genes that are missing or subject to gene loss within the compared species.
In the abstract, the authors have included the statement: “In summary, our study provides the basis for developing new molecular markers that constitutes a valuable resource for studying molecular evolution and domestication.” However, upon reviewing the manuscript, it is evident that the authors have not conducted any SSR, tandem repeat, or DNA diversity analysis, nor have they proposed any markers. Given this situation, I recommend that the authors consider removing the phrase “developing new molecular markers” from the abstract to accurately reflect the scope of their study.

Minor comments:

The inclusion of specific methodology details such as "Total genomic DNA was extracted from fresh leaves (herbarium voucher: USM:MHN331531)" and "The DNA was sequenced using Illumina Novaseq 6000 platform" in the abstract might be better suited for the methodology section rather than the abstract itself. Focusing the abstract on the core findings and implications of the study could enhance its conciseness and clarity.
Add the full botanical name of Passiflora tripartita in the phylogeny tree to ensure consistency.
Gene names should be in italics.
It is recommended to include the coverage of the genome assembly within the main text of the manuscript.

Are the rationale for sequencing the genome and the species significance clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Yes
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Organelle genome sequencing, Transcriptome assembling, Genetic diversity, Barcoding and DNA markers etc

Respond to this report

Responses (1)

Author Response

23 Jan 2024

Flavio Aliaga, Capítulo de Ingeniería Agronómica, Consejo Departamental de La Libertad (CDLL), Colegio de Ingenieros del Perú (CIP), Trujillo, 13008, Peru

Corresponding Author: Flavio Aliaga
Grupo de Investigación en Ecología Evolutiva, Protección de Cultivos, Remediación Ambiental, y Biotecnología (EPROBIO), Universidad Privada del Norte, Trujillo, Peru.
Dirección de Investigación, Innovación y Responsabilidad Social, Universidad Privada del Norte, Trujillo, 13011, Peru

The authors thank Dr. Rahul G. Shelke for taking the time and effort necessary to review the manuscript. We sincerely appreciate all the valuable comments and suggestions that helped us improve the quality of the manuscript.

Answers to Reviewer #1

Major comments:

1. While the genes in Table 2 have been organized into categories, it is apparent that certain instances lack gene information or are unclear in their presentation. This calls for a restructuring of the table to ensure a heightened level of clarity and coherence.

Answer: We agree with the reviewer. The manuscript has been revised, thanks.

2. In Table 2, the authors have indicated the absence of a duplicated copy of the rrn4.5 gene. It is advised that the authors review the annotation of this gene. Typically, rRNA genes are located within the IR regions and therefore exist in duplicated copies. However, in Fiure 1 of the chloroplast genome, the rrn4.5 gene is depicted in a duplicated copy. I recommend that the authors rectify any discrepancies pertaining to the rrn4.5 gene both in the table and within the text.

Answer: We would like to thank the reviewer for highlighting this important comment. In fact, we checked and updated the duplicated copy of the rrn4.5 gene. Thank you.

3. The authors have provided comprehensive insights into the expansion and contraction of the LSC, SSC, and IR regions in the compared species. To further enhance reader comprehension, I recommend that the authors consider incorporating the corresponding IRSCOPE figure within the manuscript. This addition would allow readers to access detailed information more effectively and contribute to a clearer understanding of the discussed concepts.

Answer: Thank you for your comment. According to your suggestions, we have added the figure 2 titled “Comparison of IR/SC boundary regions of four Passiflora species”.

4. Kindly provide information regarding the genes that are missing or subject to gene loss within the compared species.

Answer: Thank you for your reminding. Done.

5. In the abstract, the authors have included the statement: “In summary, our study provides the basis for developing new molecular markers that constitutes a valuable resource for studying molecular evolution and domestication.” However, upon reviewing the manuscript, it is evident that the authors have not conducted any SSR, tandem repeat, or DNA diversity analysis, nor have they proposed any markers. Given this situation, I recommend that the authors consider removing the phrase “developing new molecular markers” from the abstract to accurately reflect the scope of their study.

Answer. We agree with the reviewer. The manuscript has been corrected:

“In summary, our study constitutes a valuable resource for studying molecular evolution, phylogenetics, and domestication…”

Minor comments:

1. The inclusion of specific methodology details such as "Total genomic DNA was extracted from fresh leaves (herbarium voucher: USM:MHN331531)" and "The DNA was sequenced using Illumina Novaseq 6000 platform" in the abstract might be better suited for the methodology section rather than the abstract itself. Focusing the abstract on the core findings and implications of the study could enhance its conciseness and clarity.

Answer. Yes, we agree with the reviewer. Done.

2. Add the full botanical name of Passiflora tripartita in the phylogeny tree to ensure consistency.

Answer. Done.

3. Gene names should be in italics. It is recommended to include the coverage of the genome assembly within the main text of the manuscript.

Answer. Done.

4. The authors adeptly tackle gaps in our understanding of this species' genetics, comparative genomics, and evolutionary relationships. By thoughtfully engaging with reviewer feedback and subsequently refining their manuscript, the authors stand poised to elevate the impact and significance of their research within the realm of plant genomics and beyond. This process will significantly enhance the accuracy and reliability of gene classification and annotation, thereby elevating the overall quality of the manuscript.

Answer: We thank reviewer 1 for all constructive comments that led to substantial improvement of the manuscript. Thank you; it is much appreciated.

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Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Plastid genome of Passiflora tripartita var. mollissima (poro-poro) from Huánuco, Peru

Abstract

Keywords

Revised Amendments from Version 2

Introduction

Methods

Plant materials

DNA extraction

Genome sequencing, assembly, and annotation

Phylogenetic analysis

Results

Plastome of Passiflora tripartiva var. mollissima

Figure 1. Plastid genome of Passiflora tripartita var. mollissima.

Table 1. Plastid genome features of the P. tripartita var. mollissima.

Table 2. Genes present in the plastid genome of P. tripartita var. mollissima.

Contraction and expansion of the IR boundary

Figure 2. Comparison of IR/SC boundary regions of four Passiflora species.

Phylogenetic reconstruction

Figure 3. Phylogenetic tree of 27 plastid genomes using maximum likelihood analysis based on single-copy orthologous protein.

Data availability

Underlying data

Extended data

Acknowledgements

References

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