Male-Specific Sequence in Populus simonii Provides Insights into Gender Determination of Poplar

Round 1

Reviewer 1 Report

- Explain the primer design briefly.

- As a suggestion, I recommend add former published sequences to the sequence alignment (Fig. 2).

- It was best that you use a cloning-based sequencing method instead of PCR-based sequencing. As you know, the PCR-based sequencing method may be detected false SNPs.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Line 2: Mistake in headline. Change “provide” to “provides”.

Abstract: What is environmental pollution by seeds? I recommend to change the phrase. Do not use word pollution for seeds. It is a big problem causing allergies, fires and others difficulties, but not pollution.

Line 34: Again, environmental problems. Are they really problems for the environment? For people -yes, but not for the environment.

Line 85: How old are they? (adult poplars).

Line 90. Table 1. Why the order of poplars in the table and at the picture with PCR products is different?

Lines 95-98: Please correct this sentence: “A total of 25 μL PCR amplification system for gel electrophoresis containing 12.5 μL Premix TaqTM (RR902, Takara, San Jose, CA, USA), 1 μL (10 μM) forward(5′-CACAACCTAAGCAATAGTTGGCA-3′) primer, 1 μL (10 μM) reverse(5′- ATGTCTTTGAGCTTTGGTGCTG-3′) primer, 3 μL (10ng·μL-1) template DNA, and 7.5 μL ddH2O.” Where is a verb? Why system is for gel electrophoresis but nut for PCR? Please explain and correct.

Line 101: What is “The PCR amplification for Sanger”. Was that termination reaction or regular PCR. If PCR, what did you amplified and which primers did you use? If you did termination reaction for sequencing – it is not PCR. Please may clear two PCRs.

Line 106: Change “Mega7” to “Mega 7”. Please add the reference to the software.

Line 128: Why the length of the band is approximately 400 bp? What was an expected size? Did you sequence those bands to prove PCR specifity?

Line 156: Figure 1. There are no positive and negative controls of PCR. Should be presented in such type of work.

Line 163: Why did you take only 7 individuals out of 19 for sequencing?

SNP detection. Working with hybrids and performing PCR it is better to clone PCR products in E. coli and then check several colonies by sequencing. You may find both haplotypes in hybrid species. Due to your PCR worked both on hybrid and non-hydride poplars it is better to clone PCR products obtained from hybrids. Detected SNPs should be additionally proved by screening of cloned PCR products obtained from hybrids.

You showed, that sex-specific sequence is located at the chromosome 19, but you could not find it in the genome assembly? Why? The chromosome 19 of P. simonii is presented in the gene bank. Did you try to do nucleotide BLAST or alignment with low percent of similarity with chromosome 19?

Mistakes should be corrected.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

This study by Ziyue et al., is a very short and simple story, but it provides some useful information for gender determination of Populus species, including Tacamahaca and Aigeiros, which will be benefit for controlling seed hair pollution.

I am curious about how the author identified the male-specific sequence; However, I can’t find the reference No. 17, which probably is a patent. That will be better If the author can introduce a little bit more about how the male-specific sequence was identified.

The manuscript is okay to read. The figures’ quality looks fine, However, there are a few small formatting errors. The author please check it carefully throughout the manuscript.

Such as line 58 “[11].Whereas” a space should be there.

Line 198“P. deltoides :3420,3415 and T20 ;”

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

All my comments were considered and corrections were included to the updated version.