First draft genome assembly of the Argane tree ( Argania spinosa).

Background: The Argane tree ( Argania spinosa L. Skeels) is an endemic tree of southwestern Morocco that plays an important socioeconomic and ecologic role for a dense human population in an arid zone. Several studies confirmed the importance of this species as a food and feed source and as a resource for both pharmaceutical and cosmetic compounds. Unfortunately, the argane tree ecosystem is facing significant threats from environmental changes (global warming, over-population) and over-exploitation. Limited research has been conducted, however, on argane tree genetics and genomics, which hinders its conservation and genetic improvement.
Methods: Here, we present a draft genome assembly of A. spinosa. A reliable reference genome of  A. spinosa was created using a hybrid  de novo assembly approach combining short and long sequencing reads.
Results: In total, 144 Gb Illumina HiSeq reads and 7.2 Gb PacBio reads were produced and assembled. The final draft genome comprises 75 327 scaffolds totaling 671 Mb with an N50 of 49 916 kb. The draft assembly is close to the genome size estimated by k-mers distribution and covers 89% of complete and 4.3 % of partial Arabidopsis orthologous groups in BUSCO.
Conclusion: The A. spinosa genome will be useful for assessing biodiversity leading to efficient conservation of this endangered endemic tree. Furthermore, the genome may enable genome-assisted cultivar breeding, and provide a better understanding of important metabolic pathways and their underlying genes for both cosmetic and pharmacological purposes. Copyright:

Introduction

Argania spinosa (L. Skeels) is a tree endemic to the Middle West of Morocco and occupying arid and semi-arid regions totaling up to around 900,000 ha [1]. The argane tree forest was recognized as biosphere reserve (Arganeraie Biosphere Reserve) by UNESCO in 1998 [2]. It is the only unique member of the tropical Sapotaceae family in Morocco [3]. In addition to its ecological role in preventing soil erosion and desertification, the argane tree has great cultural and socio-economic importance. The oil extracted from the seed is considered the most expensive edible oil in the world with great cosmetic value and therapeutic potential [4– 6]. Argane oil represents a significant source of dietary fatty acids, while the Argane fruit is used as livestock feed by the local population [7– 9]. Phytochemical composition of Argane fruits reveals different classes of bioactive compounds, including essential oils, fatty acids, triacylglycerols, flavonoids and their acylglycosyl derivatives, monophenols, phenolic acids, cinnamic acids, saponins, triterpenes, phytosterols, ubiquinone, melatonin, new aminophenols, and vitamin E. Argane oil contains high levels of antioxidant compounds. The long-chain fatty acids in Argane oil are primarily represented by unsaturated oleic acid, then linoleic acid, palmitic acid and stearic acid [10].

The distribution area of the Argane forest decreased drastically during the 18th century. Furthermore, about 44 % of the forest was again lost between 1970 and 2007. While there are multiple causes, desertification and overgrazing form the main pressures on the Argane forest [11, 12]. Therefore, the management and conservation of the remaining genetic resources of Argane forest are urgent priorities. In recent decades, several studies have been conducted to evaluate the genetic diversity of the Argane tree using morphological [13, 14], chemical [10, 15], biochemical [6, 16] and standard molecular marker techniques, all with the aim of describing the genetic diversity of Argane trees and addressing ecological and conservation issues [17– 25]. The karyotype of A. spinosa (L.) is constituted of ten pairs of chromosomes (2n =2x =20) [3]. Until now, no reference genome was available of the A. spinosa species. Here, we present the Argane tree genome assembled from short and long DNA reads using a hybrid assembly strategy.

Methods and results

Plant material

The Argane tree ( Argania spinosa, taxid 85883, Sapotaceae, family, order Ericales), named Argane AMGHAR, to be sequenced was selected for its biological and ecological characteristics ( Figure 1). This was a 9-year-old shrub, with weeping form (geotropic, unlike the erect have) with only one main trunk 3 m in height. The ripe fruits has a rounded shape. The plant had semi-evergreen dwarf leaves. The shrub is native to the valley of the plain of Sous, an arid climate with an annual average rainfall of around 220 mm, located between the hills of the Anti-Atlas towards the South East, the Western High Atlas towards the North-West and the Atlantic Ocean towards the West (9°32′ 00″N, 30°24′ 00″W; Altitude: 126 m).

Figure 1.

Information on the Argania.spinosa individual named # Argane Amghar whose DNA has been sequenced.

( A) Map of Sous region with Argania species distribution. ( B) Picture of the tree # Argane Amghar. (Photograph taken by A. El Mousadik).

DNA sequencing and data description

Genomic DNA was extracted from lyophilized leaf tissues of a single tree ( Argane AMGHAR) using the Plant DNeasy mini kit (Qiagen, USA). The Argane tree genome was shotgun-sequenced using both Pacbio TM (Menlo Park, CA, USA) and Illumina TM (San Diego, CA, USA) sequencing technologies, generating 7.6 Gb and 144 Gb of data, respectively. Paired-end libraries with average insert sizes of 600 bp were constructed with Nextera TM DNA Library Prep Kit for Illumina (New England BiolabsTM, New Brunswick, MA, USA). These libraries were sequenced on an Illumina HiSeq XTen platform using the PE-150 module and yielded 957,451,810 reads ( Table 1). These data were trimmed of adapters, yielding a clean set of 936,053,040 reads, representing 236× genome coverage, assuming a genome size of 573 Mb as estimated by the k-mer frequency analysis (described below). Raw reads were deposited at the NCBI Sequence Read Archive (SRA) under accession numbers: SRX3207155 and SRX3207156, corresponding to two independent runs from the same plant DNA sample. In addition, single-molecule long reads from the PacBio RS II platform (Pacific Biosciences, USA) were used to assist the subsequent de novo genome assembly using Illumina. Genomic sequencing libraries were constructed using the PacBio DNA template preparation kit 2.0 (Pacific Biosciences of California, Inc., Menlo Park, CA) for SMRT sequencing on the PacBio RS II machine (Pacific Biosciences of California, Inc.) according to the manufacturer’s instructions, with a size range of 2-15 kb. The constructed libraries were sequenced on six SMRT cells on a PacBio RSII sequencer. The sequences of the 6 SMRT cell runs were deposited at the NCBI SRA under accession numbers: SRX1898029/ SRX1898030/ SRX1898031/ SRX1898032/ SRX1898033/ SRX1898034. The sequencing runs produced about 7.6 Gb, consisting of 6,705,437 reads with an average read length of 2.5 kb and representing about 13× genome coverage, again assuming a genome size of 573 Mb ( Table 1).

Table 1.

Summary of reads generated from genome sequencing and used in the assembly.

Library technology	Raw data		Trimmed data
	Number of reads	Number of bases	Number of reads	Number of bases
Illumina HiSeq X Ten	957,451,810	144,575,223,310	936,053,040	135,539,587,270
PacBio RS II	6,705,437	7,649,825,228	1,910,887	7,078,584,472

In silico genome size estimation

Trimmed reads from the Illumina platform were subjected to k-mer frequency distribution analysis with JELLYFISH v2.1.4 software [26, 27]. Analysis parameters were set at - k 21 and 25, and the final result was plotted as a frequency graph ( Figure 2). Two distinctive modes were observed from the distribution curve: the higher peak at a depth of 44 and reflecting the high heterozygosity of the Argane genome; the lower peak provided a peak depth of 87 for the estimation of the genome size [28]. Based on the total number of k-mers obtained, the Argane genome size was calculated to be approximately 573 Mb and 615 Mb, for 21- and 25-mers respectively, using the following formula: total number of k-mer / Peak depth. The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % and the duplicated fraction of the genome is estimated to be 3,19% ( Figure 2). The estimated genome size seems to be credible compared to the ones of four other Sapotaceae family members. In fact, according to the Plant DNA c-values Database, the genome sizes of these four species ranged from 273 Mb in Mimusops elengi L. (c = 0.28 pg) to 2,513 Mb in Isonandra villosa L. (c = 2.57 pg). The other two species are Planchonella eerwah (c = 0.54 pg, 528 Mb) and Madhuca longifolia (c = 0.99 pg, 968 Mb).

Figure 2.

Distribution of 21 and 25-mers using Jellyfish with PE data Argane whole genome sequencing.

Genome assembly and evaluation

Prior to assembly, Illumina raw reads and PacBio CCS reads were trimmed for adaptor removal using bbduk.sh from BBmap suite ( https://github.com/BioInfoTools/BBMap). Short and long reads were assembled following a hybrid approach using MaSuRCA assembler v.3.2.2 [29]. The initial assembly consists of 671,690,540 bp composed of 82,183 contigs with the largest size being 422,848 bp and an N50 of 43,654 bp. The very few contigs (8) with length less than 200 bp were filtered out and the remaining contigs were scaffolded into 75,327 scaffolds totaling 670,096,797 bp; the N50 reached 49,916 bp and the assembly accounted for 2,982,868 Ns with 445.14 Ns per 100 kb ( Table 2). The scaffolding was done using initial contigs and implemented in MaSuRCA v3.2.4 assembler script using Celera Assembler v8.3. The GC content was estimated to be 33%. The assembly was screened by VecScreen to look for and remove remaining vector contamination. Based on the VecSreen report, contigs containing mitochondrial/chloroplast were also removed. Trimmed PE reads were mapped on the final assembly using CLC genomics (v11.0, CLCbio, Arhus, Denmark) with 0.8 in length and 0.9 in sequence similarity. In total, 94% of the reads were mapped against the Argane genome. The 6% reads that were unmapped may result from the stringency of mapping criteria used.

Table 2.

Statistics of the Argania spinosa genome assembly.

	Number	Total size (bp)	N50 (bp)	Largest (bp)
Contigs	82,183	671,690,540	43,654	422,848
Scaffolds ≥ 200 bp	75,327	670,096,797	49,916	422,848

N50 size defined as the value N such that at least 50% of the genome is covered by scaffolds of size N or larger.

The difference between the genome size estimation and assembly size may be due to the use of parameters excluding extremely high frequency k-mers. They often represent organelle sequences, eventual contaminants inflating the genome size estimation [30], or the high-frequency of repetitive regions found in plant genomes. Furthermore, the genome is highly heterozygous and different allelic regions would inflate assembly size. To assess the completeness of the final assembly, a Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 software approach was used with “embryophyta_odb9” lineage-specific orthologous groups [31]. Thus from a total of 1,440 BUSCO genes, 1291 genes (89%) were complete (1179 in single copy and 112 duplicated), 62 genes (4.3%) were represented partially while 87 genes (6%) were missing” from the assembly.

Conclusions

This draft genome assembly is a first step towards a global and integrative omics strategy for exhaustive characterization of the Argane tree. In particular, future work will focus on structurally annotating the genome using predictive tools and transcriptome analysis. Other future work will focus on functional gene annotation, finding evidence for genome duplication and comparative genome evolution. A reliable annotation is highly dependent on transcriptomic data, analysis and research. Sequencing of different parts and developmental stages of the Argane transcriptome is ongoing. The metabolome, and analysis of Argane oil biosynthesis, as well as the tree’s microbiome should also be analyzed. To this end, and in order to coordinate the strong interests of the Plant Genomics community for this precious tree, the International Argane Genome Consortium (IAGC) and a resource website has been created ( www.arganome.org).

Data availability

All of the A. spinosa datasets can be retrieved under BioProject accession number PRJNA294096: http://identifiers.org/bioproject:PRJNA294096. The raw reads are available at NCBI Sequence Reads Archive under accession number SRP077839: http://identifiers.org/insdc.sra:SRP077839. The complete genome sequence assembly project has been deposited at GenBank under accession number QLOD00000000: http://identifiers.org/ncbigi/GI:1408199612. Data can also be retrieved via the International Argane Genome Consortium (IAGC) website: http://www.arganome.org.

Author information

Slimane Khayi and Nour Elhouda Azza are co-first authors; Rachid Mentag and Hassan Ghazal contributed equally as supervisors.

The authors have revised the manuscript and have addressed all the concerns.

It is hoped that the future updates to the draft genome will include the annotation of repeat regions (TE, retrotransposons, etc) and gene predictions and annotations.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Genome sequencing and assembly, Comparative genomics.

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.

The authors addressed my concerns with their revisions.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Whole genome shotgun assembly, pan-genome analysis

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.

In the article “First draft genome assembly of the Argane tree ( Argania spinosa)”, the authors present the draft assembly of the Argane tree using a combination of Illumina and PacBio reads. An argument for the importance of the Argane tree  to the agriculture and economy of the endemic region is given. The assertion that “the genome may enable genome-assisted cultivar breeding, and provide a better understanding of important metabolic pathways and their underlying genes for both cosmetic and pharmacological purposes” seems reasonable based on the use of previous genome sequences for important agricultural resources. This article does not claim much beyond providing a reasonable draft genome for A. spinosa. The primary evidence for reasonable quality is three-fold: a k-mer analysis of genome size using Jellyfish, 94% of Illumina reads mapping back to the assembly, and a BUSCO analysis showing 89% full length and 4.3% partial length gene matches for these single copy genes. This is probably sufficient to claim reasonable quality and the usefulness of the genome for downstream research given the contiguity shown by the N50 contig size.

The authors do miss an opportunity to look more closely at the haplotype separation that may be occurring as they speculate possibly leading to a somewhat larger genome size than indicated by Jellyfish. I’m not sure why the number of BUSCO genes with more than one full or partial length match was not given – perhaps there were none? The implication that heterozygosity is uniform “The double peak of  k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58%” seems unfounded without more evidence nor is it clear that is the model resulting in this estimate.

Lastly the grammar would benefit from editing. For example, “A reliable annotation is highly dependent on transcriptomic research, and sequencing of Argane transcriptome analysis of different parts and developmental stages or the plant is ongoing.” Might be better as “A reliable annotation is highly dependent on transcriptomic data, analysis and research. Sequencing of different parts and developmental stages of the Argane transcriptome is ongoing.”.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Whole genome shotgun assembly, pan-genome analysis

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.

Answers to Reviewer #2

1- The authors do miss an opportunity to look more closely at the haplotype separation that may be occurring as they speculate possibly leading to a somewhat larger genome size than indicated by Jellyfish. I’m not sure why the number of BUSCO genes with more than one full or partial length match was not given – perhaps there were none? The implication that heterozygosity is uniform “The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58%” seems unfounded without more evidence nor is it clear that is the model resulting in this estimate.

Answer. We agree with the reviewer. The manuscript has been corrected: 2- Lastly the grammar would benefit from editing. For example, “A reliable annotation is highly dependent on transcriptomic research, and sequencing of Argane transcriptome analysis of different parts and developmental stages or the plant is ongoing.” Might be better as “A reliable annotation is highly dependent on transcriptomic data, analysis and research. Sequencing of different parts and developmental stages of the Argane transcriptome is ongoing.”.

“..which showed that the assembly contained 89% (1271 genes) of complete and 4.3% (62 genes) of partial sequences that were Arabidopsis orthologs.” has been replaced by “. Thus, from a total of 1,440 BUSCO genes, 1291 genes (89%) were complete (1179 in single copy and 112 duplicated), 62 genes (4.3%) were represented partially while 87 genes (6%) were missing” from the assembly.

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

" A reliable annotation is highly dependent on transcriptomic research, and sequencing of Argane transcriptome analysis of different parts and developmental stages or the plant is ongoing" has been replaced by " A reliable annotation is highly dependent on transcriptomic data, analysis and research. Sequencing of different parts and developmental stages of the Argane transcriptome is ongoing.”

In the current manuscript, the authors present the first draft genome assembly for the argan tree Argania spinosa, a tree with significant ecological as well as commercial importance.

The authors have used a hybrid assembly approach, combing data from both Illumina short-reads and PacBio long-reads technology. Use of such hybrid approaches is welcome, as they can combine the  advantages offered by different technological platforms.

Based on a k-mer analysis, the authors also provide a reasonable estimate of the genome-size and the level of heterozygosity. These are important metrics to report, as they can guide the methods to be used for genome assembly, as well as for interpretation of the assembled genome sequence. The draft genome has been satisfactorily assembled, using state of the art computational methods, and it contains 89% of the predicted core genes as defined in the BUSCO software.

It is hoped that in their future studies, the authors will follow up with more improvements to the draft genome, including gene annotations using transcriptomic data.

Overall, manuscript provides valuable information and sequence resources that will be useful to the scientific community.  I have only 1 major and 6 minor comments/suggestions that I think the authors should be able to address.

Major reservation:

1. Both Illumina as well as PacBio reads have been processed and trimmed before being used as an input to the MaSuRCA software. However, the instructions for the MaSuRCA software explicitly ask for using any third-party tools, or performing any trimming, cleaning or error correction on Illumina reads, as it will most likely lead to a deteriorated assembly  (See https://github.com/alekseyzimin/masurca#overview). It is therefore strongly suggested that the authors try an assembly with the raw Illumina reads directly, as it might lead to a better assembly. If such an analysis was already performed but the assembly was worse than the current version, please provide this information in the manuscript.

Minor comment/suggestion:

1. For PacBio data, please specify the form of processed reads that were input to the MaSuRCA software: were they the raw PacBio reads, or the Circular Consensus Sequence  (CCS) reads?

2. The authors report that the genomic DNA was size-selected for fragments above 2kb. What was the approximate maximum size of input DNA for PacBio library preparation. Also, what was the maximum size of PacBio reads (e.g after the CCS step) ?

3. The raw coverage obtained from Illumina data is reported as 160X. But as per my calculations, based on number of trimmed bases in table 1 and a estimated genome size of 573 Mb, I obtain 135,539,587,270 / 573,000,000 =  236X. Please correct or explain further.

4. In addition to the observed heterozygosity, the repeat content of the genome could be an important reason for a fragmented assembly. The k-mer analysis can also be used to estimate the % of genome that is comprised of repeats. It will be great if this estimate is provided.

5. For the BUSCO analysis, the authors report using the Arabidopsis lineage-specific orthologous groups. However, from the BUSCO manual, the only valid value for the lineage parameter seems to be “embryophyta_odb9”, which internally uses Arabidopsis hmms for Augustus-based gene predictions, but the member proteins are form multiple species. If this is the lineage parameter the authors used, please specifiy it as “embryophyta_odb9” rather than Arabidopsis, or provide more details on how the Arabidopsis BUSCO groups were determined.

6. The genome sequences of the organelles mitochondria and chloroplast are also very important in investigating the biology of any species, and also serve as important resource for evolutionary and phylogenetic studies. Therefore, it will be great if instead of completely removing the organellar genomes, the authors provide them as separate set of sequences, in separate files and ideally as separate GenBank entries.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

NA

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.

1. Both Illumina as well as PacBio reads have been processed and trimmed before being used as an input to the MaSuRCA software. However, the instructions for the MaSuRCA software explicitly ask for  using any third-party tools, or performing any trimming, cleaning or error correction on Illumina reads, as it will most likely lead to a deteriorated assembly  (See https://github.com/alekseyzimin/masurca#overview). It is therefore strongly suggested that the authors try an assembly with the raw Illumina reads directly, as it might lead to a better assembly. If such an analysis was already performed but the assembly was worse than the current version, please provide this information in the manuscript.

Response:

Both illumina and PacBio reads have been trimmed only for adaptor removal.  No quality trimming was performed. (The manuscript has been corrected):

“Quality-filtered reads from the Illumina” has been replaced by “ Trimmed reads from the Illumina”

“These data was trimmed of adapters and low-quality sequences,…” has been replaced by “These data was trimmed of adapters,…”

“Prior to assembly, Illumina and PacBio raw reads were trimmed for quality and adaptor removal using bbduk.sh” has been replaced by   Prior to assembly, Illumina raw reads and PacBio CCS reads were trimmed for adaptor removal using

Minor comments/suggestions:

1. For PacBio data, please specify the form of processed reads that were input to the MaSuRCA software: were they the raw PacBio reads, or the Circular Consensus Sequence  (CCS) reads? Response:

CCS PacBio reads were processed within MaSuRCA software

2. The authors report that the genomic DNA was size-selected for fragments above 2kb. What was the approximate maximum size of input DNA for PacBio library preparation. Also, what was the maximum size of PacBio reads (e.g after the CCS step) ?

Response:

DNA extracted from the lyophilized leaves was divided into HMW and LMW fractions, and each was used separately to make PacBio libraries. The best PacBio SMRT cell yielded 4G of data on 163,000 spots, with an average read size of 24,468 bases.

3. The raw coverage obtained from Illumina data is reported as 160X. But as per my calculations, based on number of trimmed bases in table 1 and an estimated genome size of 573 Mb, I obtain 135,539,587,270 / 573,000,000 = 236X. Please correct or explain further.

Response:

We agree with the reviewer. The manuscript have been corrected:“These data was trimmed of adapters and low-quality sequences, yielding a clean set of 936,053,040 reads, representing 160× genome coverage,” has now been replaced by “These data was trimmed of adapters and low-quality sequences, yielding a clean set of 936,053,040 reads, representing 236x genome coverage,”

4. In addition to the observed heterozygosity, the repeat content of the genome could be an important reason for a fragmented assembly. The k-mer analysis can also be used to estimate the % of genome that is comprised of repeats. It will be great if this estimate is provided.

Response:

Based on the 21-mer distribution analysis, the duplicated fraction of the genome is estimated to be 3,19%. The revised version of the manuscript has been corrected:

“The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % ( Figure 2).” Has been replaced by “The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % and the duplicated fraction of the genome is estimated to be 3,19% ( Figure 2).”

5. For the BUSCO analysis, the authors report using the Arabidopsis lineage-specific orthologous groups. However, from the BUSCO manual, the only valid value for the lineage parameter seems to be “embryophyta_odb9”, which internally uses Arabidopsis hmms for Augustus-based gene predictions, but the member proteins are form multiple species. If this is the lineage parameter the authors used, please specifiy it as “embryophyta_odb9” rather than Arabidopsis, or provide more details on how the Arabidopsis BUSCO groups were determined.

Response:

We agree. The lineage parameter used is “embryophyta_odb9”. (The manuscript has been corrected):

“To assess the completeness of the final assembly, a Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 software approach was used with Arabidopsis lineage-specific orthologous groups 31,” has been replaced by “To assess the completeness of the final assembly, a ”

6. The genome sequences of the organelles mitochondria and chloroplast are also very important in investigating the biology of any species, and also serve as important resource for evolutionary and phylogenetic studies. Therefore, it will be great if instead of completely removing the organellar genomes, the authors provide them as separate set of sequences, in separate files and ideally as separate GenBank entries.

Response:

We agree with the reviewer. The work is in progress and we plant to publish the organellar genomes soon in a separate manuscript.

Answers to Reviewer #1

Major reservation:

1. Both Illumina as well as PacBio reads have been processed and trimmed before being used as an input to the MaSuRCA software. However, the instructions for the MaSuRCA software explicitly ask for   using any third-party tools, or performing any trimming, cleaning or error correction on Illumina reads, as it will most likely lead to a deteriorated assembly  (See https://github.com/alekseyzimin/masurca#overview). It is therefore strongly suggested that the authors try an assembly with the raw Illumina reads directly, as it might lead to a better assembly. If such an analysis was already performed but the assembly was worse than the current version, please provide this information in the manuscript.

Answer.We agree with the reviewer. Both illumina and PacBio reads have been trimmed only for adaptor removal. No quality trimming was performed. The manuscript has been corrected:

Minor comment/suggestion:

“Quality-filtered reads from the Illumina” has been replaced by “Trimmed reads from the Illumina”

“These data was trimmed of adapters and low-quality sequences,…” has been replaced by “These data was trimmed of adapters,…”

“Prior to assembly, Illumina and PacBio raw reads were trimmed for quality and adaptor removal using bbduk.sh” has beenreplaced by “Prior to assembly, Illumina raw reads and PacBio CCS reads were trimmed for adaptor removal using bbduk.sh”

1- For PacBio data, please specify the form of processed reads that were input to the MaSuRCA software: were they the raw PacBio reads, or the Circular Consensus Sequence  (CCS) reads?

Answer. CCS PacBio reads were processed within MaSuRCA software. The manuscript has been corrected:

“Prior to assembly, Short and long reads were…” has been replaced by “Prior to assembly, Illumina raw reads and PacBio CCS reads were …”

2- The authors report that the genomic DNA was size-selected for fragments above 2kb. What was the approximate maximum size of input DNA for PacBio library preparation. Also, what was the maximum size of PacBio reads (e.g after the CCS step) ?

Answer.DNA extracted from the lyophilized leaves was divided into HMW and LMW fractions, and each was used separately to make PacBio libraries. The best PacBio SMRT cell yielded 4G of data on 163,000 spots, with an average read size of 24,468 bases.

3- The raw coverage obtained from Illumina data is reported as 160X. But as per my calculations, based on number of trimmed bases in table 1 and a estimated genome size of 573 Mb, I obtain 135,539,587,270 / 573,000,000 = 236X. Please correct or explain further.

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

“….yielding a clean set of 936,053,040 reads, representing 160× genome coverage,” has now been replaced by “…yielding a clean set of 936,053,040 reads, representing 236x genome coverage,”

4- In addition to the observed heterozygosity, the repeat content of the genome could be an important reason for a fragmented assembly. The k-mer analysis can also be used to estimate the % of genome that is comprised of repeats. It will be great if this estimate is provided.

Answer.Based on the 21-mer distribution analysis, the duplicated fraction of the genome is estimated to be 3,19%. The revised version of the manuscript has been corrected:

“The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % ( Figure 2).” Has been replaced by “The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % and the duplicated fraction of the genome is estimated to be 3,19% ( Figure 2).”

5- For the BUSCO analysis, the authors report using the Arabidopsis lineage-specific orthologous groups. However, from the BUSCO manual, the only valid value for the lineage parameter seems to be “embryophyta_odb9”, which internally uses Arabidopsis hmms for Augustus-based gene predictions, but the member proteins are form multiple species. If this is the lineage parameter the authors used, please specifiy it as “embryophyta_odb9” rather than Arabidopsis, or provide more details on how the Arabidopsis BUSCO groups were determined.

Answer. We agree with the reviewer. The lineage parameter used was “embryophyta_odb9”. The manuscript has been corrected:

“To assess the completeness of the final assembly, a  Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 software approach was used with Arabidopsis lineage-specific orthologous groups...” has been replaced by “To assess the completeness of the final assembly, a  Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 software approach was used with “embryophyta_odb9” lineage-specific orthologous groups...”

6- The genome sequences of the organelles mitochondria and chloroplast are also very important in investigating the biology of any species, and also serve as important resource for evolutionary and phylogenetic studies. Therefore, it will be great if instead of completely removing the organellar genomes, the authors provide them as separate set of sequences, in separate files and ideally as separate GenBank entries.

Answer.We agree with the reviewer. The work is in progress and we plan to publish the organellar genomes soon in separate manuscripts.