A Fusarium verticillioides MAT1-2 Strain near Isogenic to the Sequenced FGSC7600 Strain for Producing Homozygous Multigene Mutants

Round 1

Reviewer 1 Report

In this study, the authors generated a Fusarium verticillioides MAT1-2 strain near isogenic to the sequenced wild-type MAT1-1 strain, FGSC7600, which could be an imprtant genetic tool for the analysis of multigene mutants in this fungus. The study is of great interest, and the manuscript is well written.  My major concern is that why did the authors choose a Sk^K isolate rather than a SK^S isolate as the parental strain?

Minor concerns：

1 In Fig. 3, the data should be presented in a better way and pictures with higher resolution are required.  

2 Please highlight the details of Fig. 4. 

3 Fig. 5 needs to be reorganized and Fig. 5C could be moved to the supplemental data.

Author Response

Reviewer 1 and 3 question regarding use of FGSC7603:

FGSC7603 (Sk^K), was chosen because of its history as a lab strain that has repeatedly been used successfully in past Fusarium verticillioides gene function studies. In practice this will reduce ascospore production from 8 to 4 spores per ascus. Theoretically, there should be few changes in the progeny ratio results for the vast majority of genes. Those closely linked to the mating-type and killer locus will show skewing, but with the availability of the full genome this should be obvious to researchers using the new strain FGSC27326.

 

Reviewer 1:

Detail Comments:

• Figure 3: The resolution of the revised figure has been significantly improved. We will be happy to work with the editorial staff if additional improvement is needed, but we believe the important information is presented in the revised version.
• Detail added in Figure 4 legend.
• Figure 5 improved as suggested but left organization intact.

Reviewer 2 Report

The paper entitled ‘A Fusarium verticillioides MAT1-2 strain near isogenic to the sequenced FGSC7600 strain for producing homozygous multi-gene mutants’ is devoted to the development of genetic tool for improved analysis of multigene mutants in Fusarium verticilloides. I believe that the manuscript is timely and could be interesting for the readers. Generally, it could be accepted for publication in JoF, but, from my point of view, several things should be taken into account.

1. Introduction. The authors should more comprehensively describe the dangers and significance of Fusarium verticillioides, as well as Fusarium fungi and their mycotoxins at all. Also, more information about the current or performed studies devoted to obtaining and analysis of Fusarium and/or other ascomycetes multigene mutants is needed.

2. I do not see any information if the obtained sequences were deposited in any open database.

3. In ‘Discussion’ and ‘Conclusions’, the authors should add more information about practical value of the obtained results and methods they developed.

4. More references in the list and more detailed discussion are needed.

Line 28: ‘Fusarium verticillioides’ should be among the Keywords

Lines 34-36: I do not think this sentence is appropriate in the paper

Lines 155-157 (Figure 5): What DNA marker was used? Also, the PCR products lengths shoud be mentioned.

Figure 5, C: Were these primers developed in this study or used before?

 

Author Response

Reviewer 2:

Major points:

• Added additional background information on the pathosystem to the introduction as suggested.
• Generational genome sequences have been deposited at NCBI as described in added text in section 4.2.
• Added statements as suggested in both the Discussion and in the Conclusion sections.
• More references were added for additional support as requested.

 

Minor points:

• Added Fusarium verticillioides to the key words as suggested.
• We choose to leave the statement as this is our focus.
• We have noted the requested information in the legend of Fig. 5.
• These primers were new to this study and that is noted in Fig. 5 legend.

 

Reviewer 3 Report

 

How/why was the female parent FGSC7603 selected? Simply out of convenience, availability, particular genetic traits, no other necessary spore killer variants in your possession? Were there other candidates in your possession considered to be used?

Could you entertain the reader with a contemplation in the discussion section about what results could be expected in 8^th generation backcross? Would FGSC7603 genotype percentage go down another percent or you reached the point where necessary non-discardable genes from 7603 will be retained permanently? Would your final product be even more near isogenic or is 7 generations optimal in terms of resources used and result obtained?

 

Line 155: Description of figure 5. Its not necessary, but I would consider it an improvement if you would write the name of the marker/ladder used. Readers get curious about what size fragments are there.

Line 242: “Work bench versions 11.0” – version?

Line 273: Author contributions are missing. As well as other “statements”. (at least I think that usually “not applicable” should be written.)

Line 278: References seem to be arranged alphabetically. First time I see this in MDPI. Double check if they should not be arranged in the order they appear in the text.

Author Response

Reviewer 1 and 3 question regarding use of FGSC7603:

FGSC7603 (Sk^K), was chosen because of its history as a lab strain that has repeatedly been used successfully in past Fusarium verticillioides gene function studies. In practice this will reduce ascospore production from 8 to 4 spores per ascus. Theoretically, there should be few changes in the progeny ratio results for the vast majority of genes. Those closely linked to the mating-type and killer locus will show skewing, but with the availability of the full genome this should be obvious to researchers using the new strain FGSC27326.

 

Reviewer 3:

Major points:

• Why FGSC7603 was chosen is addressed above.
• We have a statement in the discussion regarding the use of the final near isogenic MAT1-2 strain in further crosses to generate multimutant progeny.

Minor points:

• Added marker to the legend.
• “versions” changed to “version” L242.
• Added Authors contributions.
• Reformatted bibliography.