Development of Retrotransposon-Based Molecular Markers for Characterization of Persea americana (Avocado) Cultivars and Horticultural Races

Round 1

Reviewer 1 Report

1) Normally we standardize the annealing temperature through gradient PCR which has  not been done and it varies from one primer to another. 

2) Gel images are not satisfactory  

3) In my view, 10 ng genomic DNA has given a satisfactory result during iPBS amplification, kindly recheck

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 1, thanks for your comments, see the answer below.

1) Normally we standardize the annealing temperature through gradient PCR which has not been done and it varies from one primer to another.

We agree with the reviewer, as standardization of annealing temperature between primers facilitates PCR implementation. Indeed, in the case of new designed primers for IRAP implementation, we standardize their melting temperature (~62ºC) at the primer-design step. In this case, annealing temperature was also optimized by gradient PCR (Figure 4). However, in the case of PBS primers, the annealing temperature was taken as described by the original publication, as it has been previously validated by Kalendar and coworkers. In this case, annealing temperature has not changed, in order to maintain, as much as possible, the most reproducible conditions between different works involving iPBS. Honestly, we have tried to optimize annealing temperature of different PBS primers in the past, when working with other plant species, and the annealing temperatures from the original authors have always yielded best results.

2) Gel images are not satisfactory 

We have replaced al figures by their respective high-quality images. We have also increased image sizes when possible, in order to improve quality of gels.

3) In my view, 10 ng genomic DNA has given a satisfactory result during iPBS amplification, kindly recheck

We have checked three DNA amounts for iPBS. Among them, 2 ng yielded the most reproducible results (most discrete bands, and technical replicates consistency). As the reviewer suggests, also 10 ng could be used, but some bands (>1.8 Kb) amplified with lower efficiency (Figure 1). It is probably caused by higher amount of PCR inhibitors included in PCR reactions. Thus, we selected 2 ng as the optimum DNA amount, which was maintained in the rest of the work.

 4) Proposed changes, included in the attached PDF, are considered as follows:

Line 17: “per hectare” has been omitted

Headings: “Agronomy 2021” has been changed by “Agronomy 2022”

Line 82: “is the case” has been change by “in the case”

Line 95: “whose” by “which”

Several lines of PDF: all appearances of “P. Americana” have been changed by “P. americana”

Line 217: “seems” by “did”

Line 218: “to” has been omitted

Line 244: “they” has been included

Line 323: “the presence in the avocado genome of multiple copies of the potential LTRs identified in this work.” Has been changed by “the presence of multiple copies of the potential LTRs”.

Line 377: “Mussa” by “Musa”

Line 395: “Regarding” by “Respect”

Line 423: “Since” by “which”

Line 616: “Guajava” by “guajava”

Line 620: “Tabacum” by “tabacum”

Line 627: “Allium Ledebourianum” by “Allium ledebourianum”. In addition, all species scientific names have been checked.

Line 633 “Spp.” By “spp.”, all appearances in the document have been corrected.

 

 

 

 

 

Reviewer 2 Report

 

Summary:

The avocado plant is economically significant. There are 3 main cultivars of the avocado plant, with the “Hass” cultivar being the most dominant. However, the dominant cultivar is saline sensitive. There have been a recommendation to exploit the traits from other cultivars, for instance, West Indian, which happens to be saline resistant as well as pest resistant. To be able to do so smoothly, the characterization of avocado cultivars, including the identification of their molecular markers, is essential.

There have been quite many attempts to identify molecular markers in avocado plants previously using RFLP, AFLP, RAPD, SSRs, or SNPs: “classic methods”. However, the authors of the current study assert that there has been only one work investigating avocado’s molecular marker identification using a retrotransposon-based technique so far and that technique is quite different from what they have been using in this work (ISTR vs iPBS and IRAP), potentially yielding novel sets of molecular markers.

In the current work, IRAP primers were made based on LTRs obtained through iPBS. The congruency in the result between iPBS and IRAP technique was assessed. While the resulting phylogenetic tree did not perfectly match, there is a significant degree of similarities in the results obtained using the two techniques, implying reliability. More importantly, the authors successfully demonstrate the usability of the iPBS technique in identifying molecular markers in avocados. iPBS, which can work even when there is no reference genome available, are especially useful in identifying the molecular markers in other, less-known cultivars of Avocado whose reference genomes are yet to be determined. 

 

In terms of delivery, some atypical diction and grammar (& formatting inconsistencies) are found and thus may reduce future manuscript readers’ comprehension speed; the overall meanings still could be deduced if extra efforts are exerted.  

 

Some suggestions:

Line 19: “… in demand”, instead of “… on demand” ?

Line 21: iPBS and IRAP -> perhaps better use the full form in the abstract.

Line 43: “However, the reproductive biology of P. americana, despite being mainly asynchronous (i.e. female flowers bloom first than male ones)…” -> favors cross fertilization precisely because their flowerings are asynchronous (?)  

Line 76: “… involving different classic methods such as Restriction Fragment Length Polymorphism (RFLP)…”

Line 82: “… as is the case for other agro-82 nomically-important plant species [41,42].”

Line 88: remove comma.

Line 95:  “… transcription step, which recognize a Primer Binding Site (PBS) sequence placed near to the 5’ LTR of the retrotransposon [53].”

Line 108: ‘private exploitation’ -> private collection?

Line 145: I wonder, was it really 10 hours? Not too long?

Line 168: “…direct forward and reverse…” ?

Line 235: “A260/A280” ?

Line 237: “… , whose which failed to …”

Line 294-296: I am curious, I know you just followed recommendation from Kalendar et al., but could you briefly explain how did you shortlist 8 potential LTRs from 12 LTR clusters? Why the other 4 potential LTRs were not used?

Line 349: ‘On the other hand, …’ perhaps should be changed to ‘Moreover, …’ or ‘Furthermore, …’ because the content of this sentence supports the content of the previous sentence, I think.

Line 435: “In resume…” -> “In short…” ?

 

Author Response

Dear reviewer 2, thanks for your suggestions see the answer below.

Line 19: “… in demand”, instead of “… on demand”?

Has been changed as suggested

Line 21: iPBS and IRAP -> perhaps better use the full form in the abstract.

The full form has been included.

Line 43: “However, the reproductive biology of P. americana, despite being mainly asynchronous (i.e. female flowers bloom first than male ones)…” -> favors cross fertilization precisely because their flowerings are asynchronous (?) 

We agree with the reviewer. The sentence has been changed to correct the meaning.

Line 76: “… involving different classic methods such as Restriction Fragment Length Polymorphism (RFLP)…”

Done

Line 82: “… as is the case for other agro-82 nomically-important plant species [41,42].”

Done

Line 88: remove comma.

Done

Line 95:  “… transcription step, which recognize a Primer Binding Site (PBS) sequence placed near to the 5’ LTR of the retrotransposon [53].”

Done

Line 108: ‘private exploitation’ -> private collection?

Done

Line 145: I wonder, was it really 10 hours? Not too long?

Yes, agarose gel electrophoresis was run for 10 hours (120V), when 20 cm length gels were used. The EF time has been previously optimized in our lab, when we have implemented iPBS in other plant species. Indeed, we have run EFs until 16 hours in some cases. The explanation is a better separation of the higher molecular weight fragments, without lost of resolution.

Line 168: “…direct forward and reverse…” ?

Done

Line 235: “A260/A280” ?

Done

Line 237: “… , whose which failed to …”

Done

Line 294-296: I am curious, I know you just followed recommendation from Kalendar et al., but could you briefly explain how did you shortlist 8 potential LTRs from 12 LTR clusters? Why the other 4 potential LTRs were not used?

A longer explanation is stated at the methods section (173 – 180). Also, the complete process for LTR identification has been included as Supplementary Information (Fig. S1).

The following sentence has been included: “However, potential LTRs were not clearly identified from LTR clusters 5, 8, 9 and 10, as the expected conserved region flanked by 5’-TG and CA-3’ dinucleotides, was not found as expected.”

Line 349: ‘On the other hand, …’ perhaps should be changed to ‘Moreover, …’ or ‘Furthermore, …’ because the content of this sentence supports the content of the previous sentence, I think.

Done

Line 435: “In resume…” -> “In short…” ?

Done

 

Reviewer 3 Report

This paper is well written and adds to options available for genetic analysis of avocado cultivars. My comments to the authors are below.

Introduction
If available, the authors should indicate genome size and number of chromosomes in P. americana.
The authors should also demonstrate the advantage of iPBS or IRAP over other molecular markers, especially NGS markers such as SNPs (This can be done in the discussion section).
Line 53/54: Rewrite the ending of the sentence 'have been started to be exploited'
Line 88: Remove comma between 'these strategies'.
Line 95: Change 'whose recognize' to 'which recognize'

Materials and Methods
Line 129: Change 'contained' to 'containing'
Line 147: Rephrase ending of the statement.

Results
Line 231: Change 'increase' to 'increased'
Line 244: Rephrase 'since produce clear band patterns with high repeatability'
Line 244: Change 'The rest six primers were excluded' to 'Six primers were excluded since high amount.....'
Figure 3b: In the description add a note indicating what the letters in brackets represent.

Discussion
Line 376: change to 'agronomic interest such as'
Line 406: spelling of techniques

Author Response

Dear reviewer 3, thanks for your comments and suggestions see the answer below.

This paper is well written and adds to options available for genetic analysis of avocado cultivars. My comments to the authors are below. Introduction

If available, the authors should indicate genome size and number of chromosomes in P. americana.

The next sentence has been modified, just before the most suitable references, in the discussion:

Line 385: “Recently, draft versions P. americana cv. Drymifolia (Mexican horticultural race) and Hass (GxM hybrid) genomes have been published [77]”

Has been changed by:

“The haploid genome size for the Hass cultivar approximately ranges between 1.33 -1.63 Gb [40], and draft versions of P. americana cv. Drymifolia (Mexican horticultural race) and Hass (GxM hybrid) genomes have been published, confirming the presence of 12 chromosomes [77]”

The authors should also demonstrate the advantage of iPBS or IRAP over other molecular markers, especially NGS markers such as SNPs (This can be done in the discussion section).

The next sentence has been modified in the discussion section:

Line 381: The clear advantage of iPBS strategy is that prior knowledge of nucleotide sequences from target species is not required, since a relatively small set of "universal" primers can be used for analysis of any eukaryotic organism [46,49].

Has been changed by:

The clear advantage of iPBS strategy is that prior knowledge of nucleotide sequences from target species is not required, since a relatively small set of "universal" primers can be used for analysis of any eukaryotic organism [46,49]. Moreover, co-dominant markers as microsatellites or SNPs, requires allele dosage determination, which is especially difficult for partially heterozygous genotypes in polyploid species. This limitation is usually overcome after transformation of co-dominant genotypes into dominant ones, thus providing essentially the same binary information as iPBS or IRAP. Therefore, another important advantage arises when iPBS and IRAP are implemented for analysis of polyploid species, as dominant multi-locus genotypes are directly obtained, which can be immediately analyzed with most available bioinformatic tools [46,49]. Finally, their set-up simplicity, low cost, and scarce laboratory resources needed, make iPBS and IRAP very attractive tools for population genetic analysis in the field of agronomy.

Line 53/54: Rewrite the ending of the sentence 'have been started to be exploited'

This sentence has been modified as follows: “are being exploited by pharmaceutical industry”

Line 88: Remove comma between 'these strategies'.

Comma has been removed

Line 95: Change 'whose recognize' to 'which recognize'
Done

Materials and Methods
Line 129: Change 'contained' to 'containing'

Done

Line 147: Rephrase ending of the statement.

The sentence: “To visualize DNA fragments, gels were immersed in 1X GelRed (Biotium, USA) for 1.5-2 h and exposed to ultraviolet light with the aim of the image capture system ChemiDoc XRS+ (BioRad, USA).”

Has been changed by:

Gels were immersed in 1X GelRed (Biotium, USA) for 1.5-2 h, and exposed to ultraviolet light in a ChemiDoc XRS+ (BioRad, USA) to visualize DNA fragmets.

Results
Line 231: Change 'increase' to 'increased'

Done

Line 244: Rephrase 'since produce clear band patterns with high repeatability'

This sentence has been modified as follows: “since they produce clear band patterns with high repeatability”

Line 244: Change 'The rest six primers were excluded' to 'Six primers were excluded since high amount.....'

Done

Figure 3b: In the description add a note indicating what the letters in brackets represent.

Done

Discussion
Line 376: change to 'agronomic interest such as'

Done

Line 406: spelling of techniques

In this case, “techniques” has been changed by “tools”