Identification of F₁ Hybrid Progenies in Mango Based on Fluorescent SSR Markers

Round 1

Reviewer 1 Report

Please start the abstract with a general information about mango.

Write the purpose of the study in the abstract.

Include more numerical data than the study results in the abstract section.

In the abstract and in the article, write the full explanation for the first use of abbreviations (eg SSR: simple sequence repeat)

 

LOGIN

“Add a reference in the introductory section up to the 31st and 37th lines. It may be more accurate to use FAO data.

Add reference to the second paragraph of the introduction. Most of the information is written without reference.

The aim of the study should be written more clearly in the last paragraph of the introduction.

MATERIAL and METHODS

In the material and method section, the maternal and paternal parents of 65 hybrid individuals should be written (for example Jinhuang as maternal etc…)

General comments;

Increase the literature used in the study.

Examples of other species using SSR markers can be added in the introduction.

Expected and observed heterozygosity data can be added if available.

Author Response

1. We have rivised the abstract section, including: added the general information about mango, added the purpose of the study, and completed more data and full explanation. The details see abstract.
2. In the introduction section, we added six references from 2 to 7, and added the aim of the study in the last paragraph of the introduction. We did not use the FAO data, because the FAO statistics data include mangoes, mangosteen and guava, not mangoes alone, So we use the official mango data of Ministry of Agriculture and Rural Affairs. Examples of other species using SSR markers were added in the introduction, the details see reference
3. In the material and method section, we added the maternal and paternal parents of 65 hybrid individuals, and the methods of expected heterozygosity and observed heterozygosity.
4. In the results section, we added the data of expected heterozygosity and observed heterozygosity.

Reviewer 2 Report

This paper is about using multiple SSR markers to verify/distinguish Mongo hybrids. High throughput fluorescent multiplex M13-tailed PCR was utilized. 

There is a need to improve the manuscript in the following aspects: 

1.  A clear and accurate description of how the PCR was conducted. Based on the descriptions in section 2.4 (lanes 114-127), it is not clear if both forward and reverse PCR primers had the M13 tail sequence; the sentence 'the forward primers were labeled with ...fluorescent dye at the 5'-end of the upstream primer' is confusing; also the protocol seems to suggest only two primers were used for each reaction. Was a universal M13 primer labeled with fluorescent dyes included in the PCR? If this 3rd is not used, then what is the purpose of adding an M13 tail to primers? 

2. The authors tested 93 SSR primers and finally selected 8 SSR primers for this research project. It is observed that 7 SSR loci have only 2 alleles and only one SSR loci (ES83) has 3 alleles. Authors need to explain why not using more polymorphic markers for more effective identification of true hybrids, such as AAxBB, ABxCD and ABxCC markers (lanes 293-310). 

3. It would be good to have some morphological verification of the hybrid status of tree saplings.  

Author Response

1. We have revised the material and methods section, the details are as follows: PCR amplification was performed in a 20 µL reaction volume containing 2 µL DNA (20 ng/µL), 10 µL 2×Taq PCR Mix, 0.1µL forward primer(10 pmol/µL), 0.4µL tail primer, 0.5 µL reverse primer (10 pmol/µL), and 7µL nuclease free water. The M13 tail (sequence, TGTAAAACGACGGCCAGT) was added to the 5’end of the each forward primer pair, with an annealing temperature of 53 ℃. The tail was alternatively labeled with the following four dyes: PET(red),NED(yellow), HEX(green) and FAM(blue).
2. In this study, we developed SSR markers based on transcriptome data, 218 primer pairs showed stable amplification for seven mango genotypes (Among of which, include Jinhuang and Renong No.1), 93 of the primer pairs yielded polymorphic products. In this study, First 34 SSR primer pairs with different amplification bands between ‘Jinhuang’ and ‘Renong No.1’were selected for hybrid identification. We use these 34 SSR Pairs to amplification two parents and six progenies, 26 primer pairs were discarded because of their low level of polymorphism between parents and its six progenies or other shortcomings identified during the preliminary amplification tests. Finally, in this study, eight SSRs pairs were selected for the whole population identification. In addition, it may be that we have not screened enough primers. So far, we didn’t found more effective polymorphic markers (AAxBB, ABxCD and ABxCC) markers to identify true hybrids.
3. Next, it is necessary for us to develop more effective SSR markers based on reference genome.
4. We have carried out morphological verification, and the relevant data has been published in the Acta Horticulturae Sinica. So the data of morphological verification of hybrid offspring population has not been included in this paper.

Round 2

Reviewer 1 Report

Ms is ready for publication