Molecular Characterization and Marker Development of the HMW-GS Gene from Thinopyrum elongatum for Improving Wheat Quality
Round 1
Reviewer 1 Report
Astragalus sinicus is an important winter-growing cover crop. It is widely utilized, not only as a cover crop for its benefits in fertilizing the soil but also as a landscape ground cover plant for the sea of flowers. Anthocyanins are involved in the pigmentation of plants in leaves, and flowers, which is a crucial characteristic trait for Astragalus sinicus. Chalcone isomerase (CHI) is a key enzyme in the synthesis of anthocyanins. However, the research on the CHI gene of Astragalus sinicus remains unexplored. In this study, the sequence of CHI in Astragalus sinicus (AsiCHI) was cloned by rapid amplification of cDNA ends (RACE) technology. The expression profiles of the AsiCHI gene in different tissues of Astragalus sinicus were subsequently analyzed by qRT-PCR. Furthermore, the function of the AsiCHI is identified by the performance of ectopic expression in Arabidopsis thaliana. The results showed that the full-length cDNA of the AsiCHI gene (GeneBank: OQ870547) was 972 bp in length and contained an open reading frame of 660 bp. The encoded protein contains 219 amino acids with a molecular weight of 24.14 kDa and a theoretical isoelectric point of 5.11. The sequence alignment and phylogenetic analysis illustrated the high similarity between the AsiCHI protein and CHI proteins of other Astragalus species. In addition, the highest expression level of AsiCHI was observed in leaves and showed a positive correlation with anthocyanin content. The functional analysis further revealed that the overexpression of AsiCHI enhanced the anthocyanidin accumulation in the transgenic lines. This study provided a better understanding of AsiCHI and elucidated its role in anthocyanin production.
The storage protein such as gluten determine the quality of cereal’s flour. In current MS, authors have described, a molecular marker to identify Glu-1Ey subunit gene in Th. elongata alien introgression lines.
Overall the results provide important clues to improve the flour texture and quality using specific markers for breeding. The study could be considered for publications after careful revision.
Some comments:
1. First explain Th. Elongate in abstract and introduction well and provide its relevance to wheat Triticum aestivum.
2. In Figure 1. Please show specific molecular weight sizes of bands of Glu-1E and other proteins.
3. Figure 2B. The sequence font could be increased to make it clearly visible.
4. In abstract please include significance values and other statistical data, avg, sd/se, etc. Also, do the same in conclusions section.
5. Figure 4 font size and resolution needs to be improved.
6. Author needs to include other molecular marker based analysis studies used for various crop improvement and discuss in introduction and discussion sections. For example, Multiplex molecular marker-assisted analysis of significant pathogens of cotton (Gossypium sp.), 2022; Biocatalysis and Agricultural Biotechnology https://doi.org/10.1016/j.bcab.2022.102557 (Cotton); Assessment of genetic diversity and volatile content of commercially grown banana (Musa spp.) cultivars, Hinge et al., Scientific Reports, 2022; https://doi.org/10.1038/s41598-022-11992-1 (Banana); Microsatellite and RAPD analysis of grape (Vitis spp.) accessions and identification of duplicates/misnomers in germplasm collection, Upadhyay et al., 2010 Indian J Hortic Volume 67 Pages 8-15; Microsatellite analysis to differentiate clones of Thompson seedless grapevine, Upadhyay et al., 2010, Ind Journal of Horticulture, Volume 67 Issue 2 Pages 260-263
7. Table 2. I think it should be “net gluten content” instead of ‘wet’; please rectify this.
Language needs editing.
Author Response
Dear Reviewer:
Thank you very much for your valuable comments on our manuscript. We revised the manuscript according to your suggestion. The point to point responses is also submitted as a attchment file.
Thank you again for your support.
Best regards,
Hongxiang
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript ijms-2467580, entitled “Molecular characterization and marker development of HMW-GS gene from Thinopyrum elongata for improving wheat quality” submitted by Dai et al. reported and discussed the results of a genetic and qualitative study on Thinopyrum elongata in order to obtain information useful for the food use of this species or for common wheat breeding.
Considering the importance of this kind of study to improve knowledge on cereals to improve wheat and flour quality, I believe that the manuscript is of potential interest to readers of “International Journal of Molecular Sciences” and falls within its scope.
In general, the experimental activity was carried out following strict scientific logic and according to widely used methods which have made it possible to obtain reliable results.
In particular, the experimental design considered four varieties of Th. Elongate and one common spring variety.
Title: it is ok
Abstract: well written, briefly summarises the major aspects of the study.
Keyword: should be revised. Avoid to repeat the words already mentioned in the title.
Introduction: It is ok. It presents a well description of the state of the art introducing well the role of the proposed study in the framework of current knowledge on the subject.
Results and discussion: in general, are clear and well-written. I suggest to improve image quality and dimensions; figure 4 in particular.
Materials and Methods: Are clear and well detailed. The biological materials of Th. elongata are species or varieties? In my opinion they are varieties. Revise the MS according. Make the numerous acronyms explicit the first time they are mentioned. In “Determination of Wheat Quality” provide references to the methods.
Author Response
Dear Reviewer:
Thank you very much for your valuable comments on our manuscript. We revised the manuscript according to your suggestion.
Thank you again for your support.
Best regards,
Hongxiang
Author Response File: Author Response.pdf
Reviewer 3 Report
The present document describes the impact of inserting genes of Thinopyrum elongata to increase the quality of bred wheat.
The introduction is complete with good references, only in two sentences it will be interesting to include at least one (see place in the attached document)
The results are well presented, clear and easily understandable, there is also a good discussion.
Conclusion ok
There are a few minor changes to be made, please see attached file
The material and method is complete.
Comments for author File: Comments.pdf
Author Response
Dear Reviewer:
Thank you very much for your valuable comments on our manuscript. We checked the attached file and revised the manuscript according to your suggestion.
Thank you again for your support.
Best regards,
Hongxiang
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Authors have addressed majority of the comments.
English needs attention prior to publication.
