Review Reports - Investigation of Watermelon Collection for Mutations Affecting Male Sterility

Round 1

Reviewer 1 Report

This research investigates a group of melon varieties to search for mutations that affect male sterility. Basic research idea: The research idea focuses on identifying new genetic sources of male sterility in melons (Citrullus lanatus) by examining a wide range of genetic patterns. Male sterility is an important system and is used to develop new hybrid varieties and produce their seeds at a lower and more competitive cost. The experiment was collection of 150 watermelon genotypes that were examined over a period of nine years . Researchers discovered two spontaneous mutations in the watermelon collection. The second mutation, found in the ‘Concurrent’ cultivar, showed a monogenic recessive genetic pattern for male sterility. Although the new mutation in ‘Concurrent’ is accompanied by female sterility and reduced pollen production, this mutation can be maintained and propagated by self-pollination of fertile plants that are presumably heterozygous. The fruits of fertile plants of the ‘Concurrent’ variety are characterized by good sensory qualities (pleasant taste, high sweetness, juiciness, melting granular texture). This makes the variety suitable for hybridization. The researchers used a probabilistic method to determine the minimum number of melon plants required (4,492 plants) to detect at least one mutation with a probability level of P3 – 0.95. This provides a strategic framework for future breeding programs. But authors should discuss these weaknesses.
- The new mutation in the ‘Concurrent’ variety not only affects male sterility, but is also accompanied by female sterility and changes in leaf shape, and the male flowers produce little pollen.
- No direct offspring were obtained from the mutant plants themselves, indicating female sterility and premature cessation of growth.
- The study needs for further genetic analysis.

- Table 2 did not clarify the significance tests and comparison between means.

Author Response

Our answers are in the attached file.

Author Response File: Author Response.docx

Reviewer 2 Report

Major revision.

The manuscript entitled “Investigation of watermelon collection for mutations affecting male sterility” reports a long-term screening of a watermelon germplasm collection to identify mutations associated with male sterility. The topic is relevant to hybrid breeding and seed production, and the authors present observations accumulated over nine years, which adds value to the study. The identification of naturally occurring sterility-related mutations is potentially useful for both basic research and breeding programs. However, the manuscript is largely descriptive and lacks sufficient genetic, developmental, and statistical analyses to support some of its conclusions. It needs to be revised according to the following comments:

The segregation ratio (25% mutant : 75% normal) suggests Mendelian inheritance, but no formal genetic analysis or statistical validation is provided.
Most results are presented qualitatively. Key traits such as pollen production, flower abortion rate, and fertility are not supported by quantitative measurements.
- Please provide numerical data (means ± SD/SE) and indicate the number of biological replicates.
- Statistical methods should be clearly described and applied where appropriate.
The study identifies two mutations affecting floral development and fertility, yet the genetic basis of these mutations remains largely unexplored.
- Please clarify whether this ratio was statistically tested (e.g., χ² test).
The Materials and Methods section lacks sufficient detail to ensure reproducibility.
- How were male sterility and partial fertility assessed (visual inspection, pollen staining, seed set)?
- How many plants per genotype were evaluated each year?
- Were environmental effects across years considered or controlled?
The description of floral abnormalities would benefit greatly from clear, high-quality figures.
- Please include detailed images of normal vs. mutant male and female flowers, pollen morphology, and leaf phenotypes.
- Figure legends should be more descriptive and self-explanatory.

Author Response

Our answers are in the attached file.

Author Response File: Author Response.docx

Reviewer 3 Report

In the current manuscript, the authors identified two watermelon variants that confer male sterility. Although the Asar variant was determined to be not heritable, for the variant from the Concurrent variety, the identified trait of interest was shown to segregate 3:1, indicative of the putative mutation being located at a Mendelian recessive allele. The phenotype of the variant was characterized, along with statically determined occurrence of the putative mutation.

My suggestions:

Although the plants of interest studied in this paper have been meticulously phenotyped and characterized, no efforts were put in to genotype the lines of interest. This is fine given the scope of the study. However, to help the readers avoid being confused, it would be helpful for the authors to minorly adjust the wording for several instances, e.g. in the title, the word “mutations” would be largely ungrounded given that only one line of interest was shown to pass heritable male sterility traits down to segregation, hence it may be more appropriate to use the word “lines”, or similar, here; also, at line 185, the word “genotype” seemed a bit inappropriate given that no genotyping data were shown or cited in the paper.
Related, on line 182, “Screening a watermelon collection for natural mutants”: it would be more suitable to change “mutants” to “putative mutants”, or something similar; in fact, the fact that the trait on the Asar variant was not heritable would possibly mean that it was not due to a mutation.

It would be helpful if the authors could elaborate on the 150 lines tested, particularly the Asar and Concurrent varieties, a little bit of background for which would be greatly helpful to readers not familiar with the plants.
Figure 1 legend: it would be helpful to expand the words to be more descriptive. Also, it would be helpful to indicate which panels are for Asar, and which are for Concurrent.
Figure 3: please add more descriptive language to help the readers understand the contents.

Author Response

Our answers are in the attached file.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I am satified with the submission.