Uncovering the Genetic Basis of Grain Yield-Related Traits in Common Vetch (Vicia sativa L.) Through Genome-Wide Association Mapping
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAdditionally, several aspects of the manuscript require significant revision:
The language and scientific writing need substantial improvement. While the manuscript is generally understandable, it contains numerous grammatical errors and awkward constructions. Thorough professional English editing is highly recommended to ensure clarity and precision.
The interpretation of results should be strengthened. For instance, in the Results and Discussion sections, overlapping QTLs are mentioned as pleiotropic, but their potential functional implications are not explored in depth. The expression analysis of candidate genes is a strong point, but the biological significance of their expression patterns, particularly in hormonal pathways (auxin, gibberellin, brassinosteroids), would benefit from further elaboration and citation of related studies in legumes.
Additionally, the claim that all loci identified are “novel” (e.g., Abstract, line 33; Discussion, line 242) should be softened unless supported by comparative analysis with QTL databases or published GWAS in other legume species.
The population structure analysis is well conducted, but the manuscript should report the value of K used in ADMIXTURE and clarify whether the cross-validation error was used to determine the optimal K. It would also be helpful to present a clearer description of the genetic diversity across subpopulations.
Figure 2 and the supplementary figures are too small and difficult to visualize. The resolution and font size should be improved to ensure clarity and proper interpretation of the results.
The supplementary material (Tables S3–S7) is critical to understanding gene validation and should be explicitly cited in the main text wherever relevant.
Comments on the Quality of English LanguageThe language and scientific writing need substantial improvement. While the manuscript is generally understandable, it contains numerous grammatical errors and awkward constructions. Thorough professional English editing is highly recommended to ensure clarity and precision.
Author Response
Reviewer 1
Additionally, several aspects of the manuscript require significant revision:
- The language and scientific writing need substantial improvement. While the manuscript is generally understandable, it contains numerous grammatical errors and awkward constructions. Thorough professional English editing is highly recommended to ensure clarity and precision.
Response: Accepted. We fully agree with the expert comments. Our draft had issues with grammar and expression, so we have thoroughly revised the grammar, wording, and logic throughout the manuscript. Additionally, we have invited a native speaker to assist with the revisions. Furthermore, we plan to utilize MDPI's grammar and language polishing services upon publication to enhance the quality and readability of the manuscript as much as possible.
- The interpretation of results should be strengthened. For instance, in the Results and Discussion sections, overlapping QTLs are mentioned as pleiotropic, but their potential functional implications are not explored in depth. The expression analysis of candidate genes is a strong point, but the biological significance of their expression patterns, particularly in hormonal pathways (auxin, gibberellin, brassinosteroids), would benefit from further elaboration and citation of related studies in legumes.
Response: Accepted. Many thanks for your valuable suggestions. Your suggestions are highly valuable. We recognize that some of our results, particularly the discussions on multi-effect loci and candidate genes related to plant hormones, were not sufficiently in-depth. We have focused on these two sections for further elaboration and have also discussed other important stable loci and candidate genes. These additions have been incorporated into the discussion section, and we kindly invite you to review them. Once again, we sincerely appreciate your suggestions, as they have significantly enhanced the readability of the manuscript.
Overlapping QTLs (e.g., qGY1.1/qNG1.1 at 48.1–48.5 Mb) suggest pleiotropic regulation. In Medicago sativa, an orthologous region harbors MtNST1, a NAC transcription factor that coordinately regulates pod development and branch formation through auxin transport modulation (Zhou et al., 2022). The candidate gene jg55197 (auxin response factor) in qGY3.2 are similarity to MtARF7, which in medicago truncatula controls seed filling by activating cell expansion genes while suppressing branching through TCP transcription factors. This mechanistic link explains the negative correlation between HGW and NB observed in our study (Burks et al., 2019).
jg32764 (gibberellin receptor) showed higher expression in high-HGW accessions. This aligns with soybean GID1b-overexpression lines where 100-seed weight increased by 22% via upregulation of α-amylase genes (Gazara et al., 2018). Conversely, jg33049 (BZR1 homolog) was upregulated in low-yield genotypes, potentially reducing photosynthetic efficiency by repressing chlorophyll biosynthesis genes as reported in pea BR mutants (Chen et al., 2019).
Reference:
- Zhou Q, Mao P, Luo D, Chai X, Deng H, Fang Q, Fang L, Nan Z, Wen J, Liu Z., 2022. Comparative transcriptome analyses reveal that the MsNST1 gene affects lignin synthesis in alfalfa (Medicago sativa L.). The Crop Journal, 10(4), pp. 1059-72.
- Burks, D.J. and Azad, R.K., 2019. Identifying auxin response factor genes and their co‐expression networks in Medicago truncatula. The Model Legume Medicago truncatula, pp.802-808.
- Gazara, R.K., Moharana, K.C., Bellieny-Rabelo, D. and Venancio, T.M., 2018. Expansion and diversification of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1) family in land plants. Plant Molecular Biology, 97(4), pp.435-449.
- Chen, L.G., Gao, Z., Zhao, Z., Liu, X., Li, Y., Zhang, Y., Liu, X., Sun, Y. and Tang, W., 2019. BZR1 family transcription factors function redundantly and indispensably in BR signaling but exhibit BRI1-independent function in regulating anther development in Arabidopsis. Molecular plant, 12(10), pp.1408-1415.
3 Additionally, the claim that all loci identified are “novel” (e.g., Abstract, line 33; Discussion, line 242) should be softened unless supported by comparative analysis with QTL databases or published GWAS in other legume species.
Response: Accepted. Thank you very much for your valuable suggestions. As you pointed out, we may have overemphasized the novelty in our manuscript. Therefore, in the newly submitted version, we have made revisions to tone down the emphasis on novelty. Currently, we have not found any reported genetic loci related to common vetch in the existing literature. We also attempted to compare our findings with other leguminous crops, such as legumes or peas, by reviewing published articles and databases. However, due to limitations in physical location alignment and the uncertainty of target genes (since the target genes have not been identified, we could not directly analyze sequence similarity), we were ultimately unable to determine whether there are any reported overlapping loci. We believe your suggestion is very insightful, and we are particularly grateful for your valuable input.
4 The population structure analysis is well conducted, but the manuscript should report the value of K used in ADMIXTURE and clarify whether the cross-validation error was used to determine the optimal K. It would also be helpful to present a clearer description of the genetic diversity across subpopulations.
Response: Accepted. We are particularly grateful for your valuable suggestions. We have added specific details to the Materials and Methods section. The population analysis was conducted with K values ranging from 2 to 10, and the lowest cross-validation error (CV error = 0.62) was observed at K=4, indicating that dividing the population into four subgroups is the optimal model. The genetic diversity analysis revealed that Subpop1 (a mix of Chinese and Russian origins) had the highest nucleotide diversity (π = 0.236), while Subpop2 and Subpop3 had nucleotide diversities of 0.165 and 0.152, respectively. Subpop4 (primarily of Chinese origin) exhibited the lowest nucleotide diversity (π = 0.112). These results reflect the influence of geographic origin on genetic differentiation. We have also incorporated these specific findings into the Results and Discussion sections accordingly.
5 Figure 2 and the supplementary figures are too small and difficult to visualize. The resolution and font size should be improved to ensure clarity and proper interpretation of the results.
The supplementary material (Tables S3–S7) is critical to understanding gene validation and should be explicitly cited in the main text wherever relevant.
Response: Accepted. We sincerely apologize for the issues with our images and citations, which may have caused you inconvenience. We have recreated the Manhattan plot, ensuring the resolution and details of the image are as clear as possible. Additionally, we have provided more detailed annotations for Table S3, Table S5, and Table S7, and have explicitly referenced and explained them in the Results and Discussion sections. We kindly invite you to review these updates. We are particularly grateful for your valuable suggestions, which have significantly enhanced the readability of the manuscript.
6 The language and scientific writing need substantial improvement. While the manuscript is generally understandable, it contains numerous grammatical errors and awkward constructions. Thorough professional English editing is highly recommended to ensure clarity and precision.
Response: Accepted. We are especially grateful for your assistance and valuable suggestions. We have conducted a comprehensive revision of the grammar and textual errors throughout the manuscript and have enlisted the help of a native speaker to refine the language. Additionally, we plan to utilize the language and text polishing services provided by MDPI to further enhance the manuscript, ensuring it meets the standards required for publication. We sincerely apologize for any inconvenience caused by our non-native English proficiency and deeply appreciate your constructive feedback. Thank you once again for your invaluable suggestions.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis paper reported concisely the GWAS analysis of common vetch to enhance grain yield d using resources available for the authors. The information would contribute rapid future improvement of common vetch for sustainable agriculture.
I have several concerns on their approach.
Authors selected 6 grain yield-related traits in the first place. I am wondering the six traits are not mutually exclusive but additive. Especially the grain yield (GY) trait would be a kind of sum-up of other 5 traits. Does the separate analysis of six traits lead to identify separate GWAS loci ?
I am afraid that the accessions of China, Russia might have shown some biased trait among them. Do the authors know whether their molecular tools are applicable for common vetches popular in other countries?
Have you checked call rate for each QTL points? I wondered how much SNPs/InDels variety were available. Did authors find enough numbers of SNP variants other than major SNP carriers for each trait ?
The authors conducted RT-qPCR analysis of candidate genes selected in GWAS analysis. I wondered what kind of genomic differences each QTL locus has. Base changes, or InDels ? Inside coding regions, promoter sequences? I am asking because the authors could detect the differences in the expression level of candidate genes. And which part of genome segments did the authors try to amplify in each RT-qPCR reaction?
Author Response
This paper reported concisely the GWAS analysis of common vetch to enhance grain yield using resources available for the authors. The information would contribute rapid future improvement of common vetch for sustainable agriculture.
I have several concerns on their approach.
1 Authors selected 6 grain yield-related traits in the first place. I am wondering the six traits are not mutually exclusive but additive. Especially the grain yield (GY) trait would be a kind of sum-up of other 5 traits. Does the separate analysis of six traits lead to identify separate GWAS loci?
Response: Accepted. We sincerely appreciate your suggestion, as it is highly insightful. We carefully considered this issue during our research. The traits we examined include branches per plant (NB), pod length (PL), number of pods per plant (NP), number of grains per pod (NG), hundred-grain weight (HGW), and grain yield (GY). Among these, NB, PL, NP, NG, and HGW are all critical components of GY. In addition to these traits, GY is also influenced by a combination of factors such as agronomic traits (e.g., plant height), disease resistance, stress tolerance, and nutrient and water use efficiency. Furthermore, heritability analysis revealed that GY has an Hb² of 0.57, significantly lower than that of NG (0.65) and PL (0.68), indicating that it is regulated by unmeasured factors such as photosynthetic efficiency. Therefore, while composite traits like GY overlap with yield components and agronomic traits, they also include independently inherited traits. For example, in this study, qGY3.2 (Chr3:277.2 Mb) does not overlap with loci of other traits and has the highest contribution rate (PVE = 20.6%), demonstrating the existence of independent genetic regulation. Similar phenomena have been observed in other species. In this study, we selected both comprehensive traits (GY and HGW) and yield component traits (NB, PL, NP, NG) to provide insights into common vetch breeding at both the holistic and individual trait levels. Once again, thank you for your valuable suggestion, which has significantly contributed to improving the quality of our manuscript.
2 I am afraid that the accessions of China, Russia might have shown some biased trait among them. Do the authors know whether their molecular tools are applicable for common vetches popular in other countries?
Response: Accepted. We are particularly grateful for your suggestions. Among the 172 common vetch accessions we collected, the majority originate from China, Russia, West Asia, Southern Europe, and Eastern Europe, but there is a lack of materials from North America and Latin America. The molecular markers we developed can be effectively used for MAS breeding in materials primarily from China and Russia, but their applicability in materials from other regions with potential population structures remains uncertain. However, since the origin of common vetch is in Southern Europe and Western Asia, the materials we collected still exhibit high genetic polymorphism. Common vetch in regions such as the Americas likely spread from these areas, so the markers we have validated should theoretically be applicable there as well. In the future, we plan to collect additional common vetch germplasm from the United States, Canada, Mexico, and other regions, and conduct further resequencing and validation of molecular markers to provide references for common vetch breeding. We have also incorporated this discussion into the manuscript. We deeply appreciate your guidance, as your suggestions have significantly enhanced the readability of the manuscript.
3 Have you checked call rate for each QTL points? I wondered how much SNPs/InDels variety were available. Did authors find enough numbers of SNP variants other than major SNP carriers for each trait?
Response: Accepted. We would like to extend our special thanks for your valuable suggestions. We have carefully considered your feedback, although we are not entirely certain if we have interpreted it correctly. Regarding the first issue (call rate for each QTL points), during our GWAS analysis, we employed SNP quality control criteria with a SNP call rate >90%, MAF ≥ 0.05, and a missing rate <10%. Therefore, the call rates from both the chromosomal and genetic interval perspectives meet the required standards. Additionally, we have thoroughly considered the reliability of the significant loci to avoid the occurrence of single significant loci due to population structure. The loci we selected for reporting are those that are clustered or have multiple significant SNPs. For example, the qGY1.1, qGY1.2, and qGY2.1 segments contain 18, 15, and 8 significantly associated SNP markers, respectively, to ensure the accuracy of the results as much as possible. (2) Regarding the SNP variation density, we used a resequencing approach (approximately 15.2X), and after high-standard filtering, 4,796,342 SNPs remained. The final SNP density used for GWAS analysis was 2,904.6 SNPs/Mb (with the highest density on Chr4: 3,184.4 SNPs/Mb), which fully meets the requirements for GWAS. Once again, we thank you for your valuable suggestions.
4 The authors conducted RT-qPCR analysis of candidate genes selected in GWAS analysis. I wondered what kind of genomic differences each QTL locus has. Base changes, or InDels ? Inside coding regions, promoter sequences? I am asking because the authors could detect the differences in the expression level of candidate genes. And which part of genome segments did the authors try to amplify in each RT-qPCR reaction?
Response: Accepted. We are immensely grateful for your insightful suggestions. It was an oversight on our part not to include this information in the initial draft, and we sincerely apologize for this. The genetic variations in the candidate genes, including SNPs and InDels, may present multiple differences in some genes. Since the expression analysis was conducted using bulks composed of strains with extreme phenotypes. Consequently, in selecting candidate genes, we placed particular emphasis on those coding genes that exhibited no differences in their coding regions but harbored variations within their promoter regions. We have detailed the sequence differences for each gene, along with the amplified target segments, in Table A5 and A6 for the reference. Additionally, there is an important point for our readers to note. Common vetch is a species with limited genetic research, and it was only in 2022 that the first genome was released. The genome is still being refined, which poses significant challenges for gene cloning and genetic mechanism elucidation in common vetch. Currently, most of the segments identified through GWAS are within the 1-8 Mb range, containing a multitude of high-confidence genes within these genetic intervals. Therefore, it is challenging to definitively identify the target genes solely through GWAS segments, annotation information, and qrt-PCR methods. Our next steps include: (1) configuring RIL populations in conjunction with GWAS results to identify stable and highly effective target segments; (2) constructing multiple near-isogenic lines and derived populations for the target segments, using methods such as KASP markers for finer localization; (3) narrowing down the segments to a 200-500 Kb range, and then combining annotation information, RNA-seq, and RT-PCR to identify and clone the target genes; (4) attempting to further validate the function of the target genes through transgenic and gene editing techniques. Thus, we hope our work can serve as a reference for the genetic mechanisms and biological research of common vetch, but all candidate genes require extensive quantitative genetic and functional validation to confirm their functions. Once again, we thank you for your guidance, as your comments have significantly enhanced the readability of our paper.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have addressed the suggestions raised in the previous review and implemented the necessary corrections. These changes have improved the clarity and quality of the manuscript, resulting in a stronger final version.
