Comprehensive Evaluation of Agronomic Traits and Nutritional Composition in Summer-Sown Vegetable Soybean Varieties from Shanghai, China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

My opinion on the manuscript is in the attached file.

Comments for author File: Comments.pdf

Author Response

We greatly appreciate the reviewer’s positive and constructive comments on our manuscript. We have carefully addressed all concerns raised and revised the manuscript accordingly. The point-by-point responses are provided below.

 

Comments 1: The chosen research methods allow achieving the set aim in the manuscript. The methods are not clearly described. The analyses for proteins, fats, amino acids, sugars and isoflavones are described very schematically. It is not mentioned under what conditions the contents of individual sugars and starch were determined. The model of the high-performance liquid chromatograph, the columns used for the studies of saponins and isoflavones, as well as the conditions under which the analyses were carried out are not described. The obtained results are presented in 5 tables and 6 figures.

Response 1: Thank you for pointing this out, and we agree with this comment. Therefore, we have comprehensively supplemented and improved the experimental methods section. The determination methods for crude protein, crude fat, free amino acids, soluble sugars (the sum of sucrose, glucose, and fructose), starch, vitamin C, saponins, and isoflavones have been fully refined and elaborated. All relevant revisions have been highlighted in red in Section 2.3 of the revised manuscript.

 

Comments 2: In keywords: Why are isoflavones included in the keywords, but the other studied components are not included, such as saponins. Aren't they included in the nutritional quality?

Response 2: Thank you very much for this valuable comment. We initially selected isoflavones as a keyword because isoflavones are core characteristic functional components of soybean and the primary research focus of this study. To make the keywords more comprehensive and consistent with the complete research content, we have now supplemented saponins into the revised keywords list in red in the revised manuscript.

 

Comments 3: Why is a “landrace” included, is this so important?

Response 3: Landrace was included as a keyword because all experimental materials in this study are landraces with typical genetic backgrounds and regional characteristics. Including “landrace” can more accurately reflect the material characteristics and research scope of this study, highlighting the features of germplasm resources.

 

Comments 4: In introduction Page 2 line 42 – It is written “R6 growth stage”, What does R6 mean?

Response 4: Thank you for pointing this out. The R6 growth stage refers to 35–39 days after flowering, when at least one pod at the nodes of the top four fully expanded leaves on the main stem contains green seeds that completely fill the pod cavity. This stage is also the optimal harvest stage for vegetable soybean. A more detailed definition can be found in Reference 6.

 

Comments 5: The introduction does not provide data on the nutritional composition of the other soybean varieties included in the study. This missing part should be supplemented by incorporating findings from other authors regarding the chemical composition of soybean seeds.

Response 5: Thank you very much for this valuable comment, and we fully agree with it. In the revised manuscript, we have supplemented relevant findings from previous studies on the contents of key nutritional components in different soybean varieties, including soluble sugars, free amino acids, isoflavones, saponins, crude protein and crude fat, in the Introduction section, with the revised parts highlighted in red (P2, Line 45-51).

 

Comments 6: In materials and methods Page 3 line 108 – It is written “2.3. Analysis for protein, free amino acids, fatty acids, isoflavnoes”, but fatty acids have not been studied, but are written in the subtitle. In addition, there is a spelling mistake in the spelling of isoflavones.

Response 6: Thank you for pointing this out, and we have corrected the spelling error of “isoflavnoes” into “isoflavones”. Meanwhile, considering that fatty acids were not analyzed in our study, we have removed this item from the subtitle. The updated subtitle is “2.3. Analysis for protein, free amino acids and isoflavones” (P3). Relevant revisions are highlighted in red in the revised paper.

 

Comments 7: Page 3 line 109-111 and 115-116 - It is written “Nutritional composition was also analyzed, focusing on concentrations of crude protein, crude fat, free amino acid, saponins, Vitmin C, isoflavones, starch, and total sugar (sum of sucrose, glucose, and fructose) [20].” and “Soluble sugar and starch contents were determined by the anthrone sulfuric acid colorimetric method [22].”. In the first sentence for determining sugars, reference 20 is listed, and in the next sentence, reference 22. Please specify which reference you used?

Response 7: Thank you for pointing this out. We apologize for the unclear citations. Reference [20] serves as the general reference for the determination of crude protein, crude fat, free amino acids, saponins, vitamin C, isoflavones, starch, and total sugar. Separate references are used for the detection of each individual component. We have rearranged the citations and adjusted paragraph divisions for better clarity, with all changes marked in red (P3-P4).

 

Comments 8: A colorimetric method was applied, but the wavelengths at which the measurements were conducted are not specified. The same applies to the methods for proteins and fats - at the beginning it is 20, and then it is 1.

Response 8: Thank you for pointing this out. We have added the specific measuring wavelength of the colorimetric method in the text. In addition, we have standardized the citations for protein and fat determination methods to avoid confusion caused by inconsistent reference numbers. All changes are highlighted in red in the revised manuscript (P3-P4).

 

Comments 9: In results Page 5 line 154 – 163 - The text is in a smaller font. Page 8 line 235-236 – It is written “In contrast, crude fat exhibited greater phenotypic plasticity, ranging from 8.85% to 15.08%, with a mean of 11.66±1.67% FW and a CV of 14.31%.”, but in Table 4 it is written” Crude fat (% DW) - 8.85; 5.08; 11.66; 1.67, 14.31”. The presentation of the analytical results lacks consistency in terms of the measurement basis used for the reported values. It remains unclear which basis should be considered valid — FW or DW — as the manuscript does not provide sufficient justification for the chosen approach.

Response 9: Thanks for pointing out these issues. We have corrected the abnormal font size on Page 7 (Lines 277–237). The conflicting labels of FW and DW were unintentional errors. All crude fat data in the main text and Table 4 are calculated on a dry weight (DW) basis. We have revised the wrong “FW” to “DW” in the corresponding sentence and added unified notes for the measurement basis of nutritional indicators (P11, Line 333). All changes are marked in red.

 

Comments 10: Furthermore, the rationale for expressing only the fat content on a dry‑matter basis, while all other constituents are reported relative to fresh material, is not explained, creating ambiguity in the interpretation and comparability of the data.

Response 10: Thanks for pointing out the inconsistency in the reporting basis for our analytical data. This was an oversight in our presentation, and we sincerely apologize for the confusion. We chose to report protein and fat contents on a dry weight (DW) basis for the following reasons. These two components are the major storage nutrients in vegetable soybean seeds, and their accumulation is largely determined by genotypic characteristics. However, moisture content in fresh samples can vary considerably due to differences in maturity, harvest time, and growing conditions. Expressing protein and fat on a DW basis effectively eliminates the confounding effect of water variation, thereby allowing for more accurate reflection of the intrinsic differences among varieties in their capacity to accumulate these macronutrients. In contrast, other quality attributes, such as vitamin C and soluble sugars, are more closely associated with fresh-eating taste, postharvest physiological activity, and direct consumer intake. Therefore, we retained the fresh weight (FW) basis for these parameters, which better represents their actual levels in the vegetable as consumed. To ensure clarity, we have now explicitly indicated the measurement basis (DW or FW) for every parameter in all relevant tables and in the main text of the revised manuscript (P11-P12).

 

Comments 11: Page 8–Figure 4-On the ordinate, write Contents.

Response 11: Thank you for your comment. We have updated the ordinate label of Figure 4 to “Contents” in accordance with your suggestion. The change is marked in red.

 

Comments 12: Page 9 – line 259 – It is written “to 6.01 mg/g”, but in table 4 the max Total  free amino acids (mg/g) are 6.00 mg/g.

Response 12: Thank you for pointing out this numerical inconsistency. We have checked the data carefully. The maximum content of total free amino acids is 6.00 mg/g as shown in Table 4. We have revised “6.01 mg/g” in the text to “6.00 mg/g” to unify all data. This correction is highlighted in red in the revised manuscript (P12, Line 351).

 

Comments 13: Page 10 line 301 – Table 5 – It written “Table 5. Variation of isoflavone and composition among 30 soybean varieties.”, but should be “Table 5. Variation of isoflavone and composition among 30 soybean varieties (μg/g DW).”. No units of measurement are specified for this component. In addition to these inconsistencies, the description of the analytical procedures lacks essential detail regarding the reporting of individual components. No units of measurement are specified for this component, which hinders the clarity and reproducibility of the results and raises concerns about the interpretability of the presented data.

Response 13: Thanks for your comments. We have added the unit (μg/g DW) to the title of Table 5 (P13). Meanwhile, we supplemented the details of analytical methods and unified the unit labels for all detected components in the manuscript and tables (P3-P4). All revisions are marked in red.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses an interesting topic and provides a valuable dataset regarding the agronomic performance, nutritional composition, and isoflavone profiles of Shanghai-native vegetable soybean landraces. The study may contribute to future breeding programs and germplasm utilization strategies. However, several points require clarification and further discussion before the manuscript can be considered for publication.

• The study was conducted over two growing seasons (2023–2024). Were the results analyzed separately for each year before pooling the data? If significant year-to-year differences were observed, please explain how environmental variation may have influenced the reported agronomic and nutritional traits.

• Could the authors provide more detailed information regarding climatic conditions during the two cultivation years? Temperature, rainfall, and other environmental factors may significantly affect sugar accumulation, protein content, and especially isoflavone biosynthesis.

• The manuscript frequently refers to “genetic diversity”. Since no molecular markers or genomic analyses were performed, would “phenotypic diversity” or “agronomic and nutritional diversity” be more appropriate terminology throughout the manuscript?

• The clustering analysis identified four groups. What specific characteristics biologically differentiate Group 3 and Group 4 from the remaining germplasm? Beyond the dendrogram, could the authors explain which traits contributed most strongly to the separation of these groups?

• The PCA explained 79.61% of the total variance. Could the authors provide a PCA score plot showing the distribution of all accessions and highlighting the superior genotypes discussed later in the manuscript?

• Plant height, node number, and effective pod number were strongly associated in PC1. Do the authors consider these relationships to result from genetic linkage, common developmental regulation, or simple morphological dependence? This aspect deserves further discussion.

• Several accessions produced more than 100 effective pods per plant. Could the authors explain whether these accessions also maintained superior seed size and nutritional quality, or whether yield advantages were accompanied by quality trade-offs?

• Table 3 indicates significant positive correlations between vegetative vigor and pod production. Could the authors discuss whether these correlations could be used as indirect selection criteria in breeding programs?

• The manuscript identifies ‘Heiyan Susudou’ as having the highest soluble sugar content and ‘Xiangshui Maodou’ as having the highest free amino acid and vitamin C content. Have these accessions previously been used in breeding programs, and do they possess any agronomic limitations that may restrict their practical use?

• The discussion repeatedly links soluble sugars and amino acids to consumer preference. Since no sensory evaluation was performed, could the authors moderate these conclusions or support them with additional references specifically correlating these biochemical parameters with sensory acceptance in vegetable soybean?

• The average protein content reported in the present study appears relatively high. Could the authors compare these values with those reported for modern commercial edamame cultivars cultivated in China, Japan, and other major producing countries?

• The biological significance of the observed vitamin C variation deserves additional discussion. For example, would the difference between the lowest and highest accession be nutritionally meaningful from a consumer health perspective?

• The isoflavone results are among the most interesting findings of the study. Could the authors discuss possible mechanisms responsible for the nearly seven-fold variation observed among accessions? Are these differences mainly genotype-dependent, or could environmental adaptation and geographic origin also contribute?

• Genistin and daidzin accounted for the majority of total isoflavones. Could the authors elaborate on the nutritional and technological implications of this composition profile compared with germplasm characterized by higher aglycone contents?

• The manuscript identifies ‘Bayuejiao Maodou’ and ‘Hei Maodou’ as elite high-isoflavone accessions. Could the authors provide additional information regarding their agronomic performance? High phytochemical content alone may not be sufficient for breeding if yield characteristics are poor.

• Have the authors investigated correlations between total isoflavones and other nutritional traits such as protein, sugars, amino acids, or saponins? Such analyses could provide valuable information for future breeding programs targeting multiple quality traits simultaneously.

• Since the stated objective includes identifying superior genotypes combining high yield and desirable nutritional traits, could the authors provide a final ranking or selection index highlighting the most promising accessions based on all evaluated parameters?

• The Conclusions section would benefit from more specific recommendations. Which 3–5 accessions do the authors consider the most valuable candidates for future breeding programs, and for which specific traits should each of these accessions be prioritized?

• Finally, several linguistic inconsistencies and grammatical issues remain throughout the manuscript. A careful English language revision is recommended before publication.

Author Response

We greatly appreciate the reviewer’s positive and constructive comments on our manuscript. We have carefully addressed all concerns raised and revised the manuscript accordingly. The point-by-point responses are provided below.

 

Comments 1: The study was conducted over two growing seasons (2023–2024). Were the results analyzed separately for each year before pooling the data? If significant year-to-year differences were observed, please explain how environmental variation may have influenced the reported agronomic and nutritional traits.

Response 1: Thank you for pointing this out. We conducted separate statistical analyses for the 2023 and 2024 growing seasons prior to data pooling. No significant year-to-year differences were found in agronomic and nutritional traits. The environmental conditions were consistent during the two seasons and had no obvious impact on the measured indicators. We have added this explanation in the revised manuscript.

 

Comments 2: Could the authors provide more detailed information regarding climatic conditions during the two cultivation years? Temperature, rainfall, and other environmental factors may significantly affect sugar accumulation, protein content, and especially isoflavone biosynthesis.

Response 2: Thank you for your valuable and constructive comments. We have added comprehensive meteorological records for Minhang District, Shanghai during the 2023 and 2024 growing seasons. The annual precipitation was 1226.1 mm in 2023 and 1475.0 mm in 2024, while temperatures remained comparable between the two years. Notably, there was more rainfall, frequent rainstorms, and inadequate sunshine in 2024. Despite these environmental variations, only slight fluctuations were observed in sugar, protein, and isoflavone accumulation, and no significant inter-annual differences were detected in agronomic or nutritional characteristics (P3, Line 95-102).

 

Comments 3: The manuscript frequently refers to “genetic diversity”. Since no molecular markers or genomic analyses were performed, would “phenotypic diversity” or “agronomic and nutritional diversity” be more appropriate terminology throughout the manuscript?

Response 3: Thank you for pointing this out. We have thoroughly revised the manuscript and replaced all inappropriate instances of “genetic diversity” with “phenotypic and nutritional diversity” across the text. The revised terminology conforms to our research objectives and results. All changes are marked in red.

 

Comments 4: The clustering analysis identified four groups. What specific characteristics biologically differentiate Group 3 and Group 4 from the remaining germplasm? Beyond the dendrogram, could the authors explain which traits contributed most strongly to the separation of these groups?

Response 4: Thanks for your comment. Group 3 (‘Heiyan Susudou’ and ‘Jinhui Maodou’) showed the highest main stem node number and first pod height, while Group 4 (‘Chongming Zhoupidou’) had the shortest growth period and the smallest stem diameter, standard pod length, and standard pod width. By cross-referencing Supplementary Table 2, which documents the agronomic and yield-related traits of the 30 soybean varieties, we identified the distinctive characteristics that differentiate these accessions from the remaining germplasm (P9, 282-303). All changes are marked in red.

 

Comments 5: The PCA explained 79.61% of the total variance. Could the authors provide a PCA score plot showing the distribution of all accessions and highlighting the superior genotypes discussed later in the manuscript?

Response 5: Thank you for your valuable and constructive comments. According to the comment, we have supplemented a PCA score plot (Figure 3, Figure S1, Table S3) that visually displays the distribution pattern of all tested accessions based on the four principal components, which together accounted for 79.61% of the total phenotypic variation. Meanwhile, the descriptions related to superior varieties have been supplemented in the revised manuscript and highlighted in red for reference.

 

Comments 6: Plant height, node number, and effective pod number were strongly associated in PC1. Do the authors consider these relationships to result from genetic linkage, common developmental regulation, or simple morphological dependence? This aspect deserves further discussion.

Response 6: Thank you for this insightful comment. The strong correlation among plant height, main stem node number, and effective pod number per plant reflected by PC1 represents a synergistic outcome of morphological dependency, genetic linkage, and co-developmental regulation operating at multiple levels, rather than being determined by any single mechanism in isolation.

First, at the morphological level, plant height is primarily determined by main stem node number combined with internode length, while soybean pods are predominantly borne at nodes. Consequently, main stem node number serves as the morphological foundation determining the potential effective pod number. Extensive statistical analyses, including those from the present study, have consistently confirmed highly significant positive correlations between plant height and main stem node number, as well as between main stem node number and effective pod number per plant, reflecting the inherent morphological dependency among these traits.

Second, at the genetic level, all three traits are typical quantitative traits controlled by multiple genes/QTLs. Previous studies have demonstrated that QTLs governing different traits frequently exhibit co-localization on chromosomes (i.e., residing in identical or adjacent regions), providing direct genetic evidence for linkage among these traits. Furthermore, pleiotropic effects occur where a single gene influences multiple traits simultaneously. For instance, the Dt1 gene located at the qHub_5 locus not only regulates plant height and node number but also concurrently affects internode length and pod-bearing range, clearly demonstrating the capacity of one gene to coordinately modulate plant height, node number, and pod distribution and, thus, serving as compelling evidence for co-developmental regulation. For details, please refer to the Discussion section of the revised manuscript, where the revised text is marked in red (Lines 437-460).

 

Comments 7: Several accessions produced more than 100 effective pods per plant. Could the authors explain whether these accessions also maintained superior seed size and nutritional quality, or whether yield advantages were accompanied by quality trade-offs?

Response 7: Thank you for this insightful comment. Regarding the accessions with over 100 effective pods per plant (‘Heidou’, ‘Changshengguo Maodou’, ‘Qingsu 7’, ‘Jinhui Maodou’, ‘Hei Maodou’, ‘Lvxiang Susudou’, and ‘Bianqing Maodou’), we conducted a specific analysis of yield components and quality traits.

For seed size, we simultaneously measured both fresh 100-seed weight and dry 100-seed weight. fresh weights ranged from 33.3 to 60.8 g, while dry weights ranged from 54.73 to 110.63 g. Compared with medium-yield accessions, these lines did not show a uniform decrease in seed size. The 100-seed fresh weights of ‘Qingsu 7’, ‘Heidou’, ‘Hei Maodou’, and ‘Jinhui Maodou’ were higher than the average, while the 100-seed dry weights of ‘Qingsu 7’, ‘Heidou’, and ‘Hei Maodou’ exceeded the average. Notably, ‘Bianqing Maodou’, which had the highest number of effective pods per plant, exhibited 100‑seed fresh weight and dry weight that were both significantly lower than the mean.

For nutritional quality trade-offs, our data indicated that high yield did not lead to a universal decline in all quality traits but, rather, showed trait-specific distribution patterns. For instance, ‘Qingsu 7’ showed a synergistic relationship between high yield and high fructose/saponin content; ‘Changshengguo Maodou’ maintained the highest total sugar and glycitin content; ‘Heidou’ possessed high protein content. Conversely, we observed an antagonistic case in ‘Jinhui Maodou’, which had the lowest amino acid content but the highest daidzin content among all accessions.

In conclusion, there is no single linear trade-off between pod number and quality. We acknowledge that the underlying physiological and genetic mechanisms governing the coordination between yield and nutritional quality in these landraces remain complex and warrant further systematic investigation. The current dataset provides a valuable foundation for identifying accessions that combine high yield potential with targeted nutritional attributes for breeding applications.

 

Comments 8: Table 3 indicates significant positive correlations between vegetative vigor and pod production. Could the authors discuss whether these correlations could be used as indirect selection criteria in breeding programs?

Response 8: Thank you for pointing this out, and we fully agree that the significant positive correlations between vegetative vigor and pod production hold potential value for breeding. In early-generation breeding, using pod number per plant (typically assessed at R6 stage) as a direct selection criterion faces two major practical constraints: (i) it requires waiting until the reproductive phase, prolonging the selection cycle; and (ii) the large number of individual plants in early generations makes precise yield-trait assessment extremely laborious and inefficient. In contrast, vegetative vigor can be measured rapidly, visually, and non-destructively during the early vegetative phase. Thus, employing it as an indirect selection criterion could significantly shorten the selection cycle and reduce measurement costs, making it particularly suitable for large-scale screening in early generations.

Nevertheless, we are fully aware that a strong phenotypic correlation does not guarantee reliable indirect selection. Before applying this approach in practical breeding programs, it is essential to validate the broad-sense heritability of vigor traits, their genetic correlation with pod yield, and to conduct cost–benefit analyses to quantify the expected genetic gain from indirect selection. Moreover, potential negative trade-offs, such as delayed maturity or reduced harvest index due to excessive vegetative growth, should also be considered.

 

Comments 9: The manuscript identifies ‘Heiyan Susudou’ as having the highest soluble sugar content and ‘Xiangshui Maodou’ as having the highest free amino acid and vitamin C content. Have these accessions previously been used in breeding programs, and do they possess any agronomic limitations that may restrict their practical use?

Response 9: Thank you for pointing this out. According to the publicly available literature and institutional breeding records, neither ‘Heiyan Susudou’ (No. 21) nor ‘Xiangshui Maodou’ (No. 18) has been systematically utilized in commercial vegetable soybean breeding programs. This indicates that their genetic potential, particularly for nutritional quality traits, remains largely untapped.

As illustrated in Figure 1, both accessions display normal phenotypic appearance with no obvious morphological defects, suggesting their suitability as crossing parents. However, as shown in Table S2, both accessions exhibited below-average fresh 100-seed weight and dry 100-seed weight compared to the overall mean of all tested materials. Specifically, ‘Heiyan Susudou’ and ‘Xiangshui Maodou’ ranked in the lower quartile for yield-related traits, which may limit their direct deployment as commercial cultivars (Table S2). Despite these yield limitations, we believe these two accessions hold considerable value as elite donor parents for nutritional quality improvement. By introgressing their superior nutritional quality alleles into elite high-yielding cultivars, it is feasible to develop novel fresh-edible soybean varieties that combine desirable agronomic traits with enhanced nutritional value.

 

Comments 10: The discussion repeatedly links soluble sugars and amino acids to consumer preference. Since no sensory evaluation was performed, could the authors moderate these conclusions or support them with additional references specifically correlating these biochemical parameters with sensory acceptance in vegetable soybean?

Response 10: Thank you for pointing this out, and we fully agree with and accept this revision suggestion. In the Introduction section of the original manuscript (Lines 52–60), we cited References 10–16 to summarize previous studies on the association of soluble sugars and amino acids with consumer preferences. The existing literature has demonstrated that sucrose is the main contributor to sweetness, while free amino acids can enhance both sweetness and umami taste; sweetness and umami are crucial determinants of consumer preference. Crude fat and crude protein significantly affect texture and mouthfeel, and isoflavones may introduce undesirable bitterness in some products.

In this revision, we have toned down the direct linkage between soluble sugars/free amino acids and consumer preference. We have clearly defined them as potential key flavor biochemical indicators affecting the sensory palatability of vegetable soybean, rather than compounds that directly determine consumer preference, thereby strictly limiting the scope of inference and improving the rigor of the discussion. All revisions in the manuscript have been highlighted in red (P15, Section 4.2).

 

Comments 11: The average protein content reported in the present study appears relatively high. Could the authors compare these values with those reported for modern commercial edamame cultivars cultivated in China, Japan, and other major producing countries?

Response 11: Thank you for pointing this out. Upon careful re-examination of the raw data, we identified a labeling error in the original manuscript: the crude protein content was inadvertently reported as g/100 g FW instead of the correct unit g/100 g DW. We sincerely apologize for this oversight, which arose during data compilation, and have corrected it in the revised manuscript (Table 4, Table S4).

To address the reviewer’s question regarding comparability with modern commercial cultivars: when converted to a fresh weight basis—assuming a typical moisture content of 60–65% in fresh vegetable soybean pods, as reported for edamame—the protein content in the present study translates to approximately 12.9–16.7 g/100 g FW. This range is consistent with the values reported for major commercial cultivars such as the U.S. varieties ‘Jack’ and ‘William 82’ (10.46–15.60 g/100 g FW) [Ref. 30].

We also note that China is the world’s largest producer of vegetable soybean, yet comprehensive nutritional profiling of domestic commercial cultivars remains limited. The relatively low coefficient of variation (CV) for protein content observed across the 30 Shanghai-native accessions in this study further supports the notion that protein accumulation is a relatively stable quality trait in Glycine max, with minor CV among diverse germplasm.

 

Comments 12: The biological significance of the observed vitamin C variation deserves additional discussion. For example, would the difference between the lowest and highest accession be nutritionally meaningful from a consumer health perspective?

Response 12: Thank you for pointing this out. It should be noted that vitamin C was not discussed as a focal point in our study, primarily for the following reasons: vitamin C is synthesized only in limited amounts in fresh edamame and germinated soybean seeds, and its content is markedly lower than that of common fruits and vegetables such as oranges, rendering it less competitive in comparative nutritional profiling.

Nevertheless, we acknowledge your perspective that the observed vitamin C variation holds noteworthy biological significance. In our study, the vitamin C content among the 30 evaluated landraces ranged from 10.51 to 19.33 mg/100 g FW, with a mean of 14.85 ± 2.12 mg/100 g FW and a coefficient of variation of 14.27%. This CV value indicates moderate genetic diversity among the evaluated accessions, which carries important implications for breeding programs aimed at enhancing crop nutritional quality. Specifically, the accession with the highest vitamin C content (19.33 mg/100 g FW) could serve as a valuable genetic resource for the development of high-vitamin C vegetable soybean cultivars.

From a consumer health perspective, according to the dietary recommendations established by the Chinese Nutrition Society, the recommended nutrient intake (RNI) of vitamin C for adults is 100 mg per day. Based on a typical serving size of 100 g fresh vegetable soybean, the lowest-content accession would provide approximately 10.5% of the daily RNI, whereas the highest-content accession would contribute approximately 19.3%. This difference is equivalent to an additional 8.8 mg of vitamin C per 100 g serving. Although the absolute value is modest, it may still hold nutritional significance for consumers seeking to maximize their nutrient intake through dietary choices, particularly for populations with insufficient fruit and vegetable consumption. We have incorporated this discussion into the revised manuscript (Discussion, Section 4.2, lines 508-515).

 

Comments 13: The isoflavone results are among the most interesting findings of the study. Could the authors discuss possible mechanisms responsible for the nearly seven-fold variation observed among accessions? Are these differences mainly genotype-dependent, or could environmental adaptation and geographic origin also contribute?

Response 13: Thank you for pointing this out. All accessions evaluated in this study were Shanghai landraces, grown in the same experimental field under uniform cultivation management practices. Therefore, we consider that the observed variation in isoflavone content is predominantly attributable to genotypic effects. Second, non-random human selection may have intensified population differentiation. Farmers in different townships of Shanghai have practiced seed saving over generations based on local consumption habits, sensory preferences, and traditional uses, and this directional selection pressure may have driven allele frequency shifts in different local populations, resulting in significant divergence of quality traits such as isoflavone content at the population level. Finally, historical habitat adaptation may have left metabolic imprints. Stable differences in soil type, water and fertilizer conditions, light microclimate, and cultivation traditions persist among different districts and counties, and landraces may have gradually developed metabolic profiles matched to their native habitats through long-term adaptive processes, leading to differentiation in isoflavone accumulation levels.

 

Comments 14: Genistin and daidzin accounted for the majority of total isoflavones. Could the authors elaborate on the nutritional and technological implications of this composition profile compared with germplasm characterized by higher aglycone contents?

Response 14: Thank you for pointing this out. Previous studies have demonstrated that glycosylated isoflavones are generally less bitter than their aglycone counterparts and exhibit higher stability during fresh consumption and mild processing [37].

Vegetable soybean is primarily consumed in fresh form, and the markedly lower bitterness of glycosylated isoflavones compared with aglycone forms allows maximal retention of isoflavone functional activity while minimizing negative impacts on the characteristic sweet and tender taste of vegetable soybean, thereby enhancing consumer sensory experience.

Prior to consumption, vegetable soybean requires typical thermal treatments such as blanching or short-term steaming. The superior thermal stability of glycosylated isoflavones ensures more effective preservation of isoflavone bioactivity during these conventional processing steps, whereas germplasm rich in aglycones may be more susceptible to thermal degradation losses under identical processing conditions. Compared with germplasm characterized by higher aglycone contents, the glycoside-dominant profile of the landraces evaluated in this study is particularly well-suited for fresh-market and minimally processed vegetable soybean products.

 

Comments 15: The manuscript identifies ‘Bayuejiao Maodou’ and ‘Hei Maodou’ as elite high-isoflavone accessions. Could the authors provide additional information regarding their agronomic performance? High phytochemical content alone may not be sufficient for breeding if yield characteristics are poor.

Response 15: Thank you for pointing this out. As shown in Table S2, the standard pod length and width, as well as the fresh and dry 100-seed weights, of ‘Bayuejiao Maodou’ and ‘Hei Maodou’ were below the population mean but did not represent the lowest values observed. This indicates that their yield-related traits, while not attaining superior levels within this germplasm collection, nevertheless fall within a moderate and potentially exploitable range. In vegetable soybean quality breeding programs, such accessions can serve as valuable specialty donor parents for enhancing the functional nutritional components of conventional cultivars, with the ultimate goal of combining high-isoflavone content with desirable agronomic traits through hybridization.

 

Comments 16: Have the authors investigated correlations between total isoflavones and other nutritional traits such as protein, sugars, amino acids, or saponins? Such analyses could provide valuable information for future breeding programs targeting multiple quality traits simultaneously.

Response 16: Thank you for pointing this out. We performed correlation analyses between total isoflavone content and major nutritional quality traits, including protein, soluble sugars, free amino acids, and total saponins. The results revealed varying degrees of correlation between total isoflavones and several of these traits (Table S7). The corresponding data have been added to the revised manuscript and are highlighted in red for easy identification (P14, Line 412-416).

 

Comments 17: Since the stated objective includes identifying superior genotypes combining high yield and desirable nutritional traits, could the authors provide a final ranking or selection index highlighting the most promising accessions based on all evaluated parameters? The Conclusions section would benefit from more specific recommendations. Which 3–5 accessions do the authors consider the most valuable candidates for future breeding programs, and for which specific traits should each of these accessions be prioritized?

Response 17: Thank you for pointing this out. We have made the revisions and highlighted them in red (P16, Conclusion).

Based on the comprehensive assessment of 11 agronomic/yield traits, 10 nutritional traits, and 6 isoflavone components across 30 vegetable soybean accessions, we have identified six priority accessions for future breeding programs.

1. ‘Xiangshui Maodou’—Priority trait: Flavor and nutritional quality

This landrace exhibited the highest free amino acid content (6.00 mg/g) and the highest vitamin C content (19.33 mg/100 g FW) among all 30 accessions. Free amino acids are key contributors to umami taste and flavor complexity in vegetable soybean, while vitamin C is an essential antioxidant with health-promoting properties.

2. ‘Heiyan Susudou’—Priority trait: Sweetness

This landrace displayed the highest total soluble sugar content (53.61 mg/g FW) and the highest sucrose level (32.63 mg/g), which is the primary determinant of sweetness in vegetable soybean.

3. ‘Hei Maodou’—Priority trait: Dual-purpose (yield+functional food quality)

This accession combined high yield potential (>100 effective pods per plant) with exceptionally high total isoflavone content (>1500 μg/g DW). Isoflavones possess antioxidant and anticarcinogenic properties, positioning this accession as a valuable dual-purpose germplasm for developing high-yielding functional food cultivars.

4. ‘Dashu Maodou’, ‘Baimang Liuyuebai’, and ‘Caojing Huadou’—Priority trait: Seed size

These three accessions shared the largest seed size, with fresh 100-seed weights exceeding 100 g and dry weights approaching 50 g. Large seed size is a commercially desirable trait for edamame marketability, and these accessions may serve as valuable donors for seed size enhancement in breeding programs.

 

Comments 18: Finally, several linguistic inconsistencies and grammatical issues remain throughout the manuscript. A careful English language revision is recommended before publication.

Response 18: We sincerely thank the reviewer for pointing out the language issues in our manuscript. We fully agree that careful English revision is essential for clarity and professional presentation. There, the entire manuscript has been carefully re-edited by a native English-speaking colleague with expertise in food science and technology, who paid special attention to grammatical correctness, terminology consistency, and overall readability. All revised sentences and paragraphs have been highlighted in red (or tracked changes) in the revised manuscript for easy identification.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I reviewed the manuscript titled “Comprehensive Evaluation of Agronomic Traits and Nutritional Composition in Summer-Sown Vegetable Soybean Varieties from Shanghai, China. The manuscript is well written, particularly results section. Methodology should be added with clear and detailed info.

Abstract

Sub-headings like background, methods, and results words should be removed

Keywords should be revised

Introduction

62-73: add citation

2.3. Analysis for protein, free amino acids, fatty acids, isoflavnoes: authors should add the detailed methodology of each method in this section

Detailed HPLC methodology is needed

Line 113: the citation format is wrong : by Hijrah et al. (2024) [1].

Detailed methodology on PCA and hca should be added in statistical analysis

Figure 1:  increase the font size of those numbers

Figure 2: increase the font size

PCA plots should be added

Tables 4 and 5: results are duplicated in text as well. Same info is presented in Table and Results. I advise authors to discuss the Table results in a more engaging way

5. Conclusions: should be revised and include the nutritional aspects.

References should be cross-check and should meet the journal format

Author Response

We greatly appreciate the reviewer’s positive and constructive comments on our manuscript. We have carefully addressed all concerns raised and revised the manuscript accordingly. The point-by-point responses are provided below.

 

Comments 1: Sub-headings like background, methods, and results words should be removed.

Response 1: Thank you for this suggestion. In response, we have removed all standalone subheadings (Background, Methods, and Results) from the revised manuscript.

 

Comments 2: Keywords should be revised.

Response 2: Thank you for pointing this out. We have revised the keywords.

 

Comments 3: 62-73: add citation

Response 3: Thank you for pointing this out. We have added citations in the original lines 62–73, which are now lines 67–78.

 

Comments 4: 2.3. Analysis for protein, free amino acids, fatty acids, isoflavnoes: authors should add the detailed methodology of each method in this section. Detailed HPLC methodology is needed.

Response 4: Thank you for pointing this out. Detailed methods have been added to Section 2.3 of the manuscript, and the new text is indicated in red for easy identification (Page 3-4).

 

Comments 5: Line 113: the citation format is wrong: by Hijrah et al. (2024) [1].

Response 5: Thank you for pointing this out. We have revised the reference format (Line 581).

 

Comments 6: Detailed methodology on PCA and hca should be added in statistical analysis

Response 6: Thank you for pointing this out. Methodology on PCA and hca have been added to Section 2.4 of the manuscript, and the new text is indicated in red for easy identification (Page 4).

 

Comments 7: Figure 1:  increase the font size of those numbers

Response 7: Thank you for pointing this out. We have increased the font size in Figure 1 (Page 5).

 

Comments 8: Figure 2: increase the font size

Response 8: Thank you for pointing this out. We have increased the font size in Figure 2 (Page 5).

 

Comments 9: PCA plots should be added

Response 9: Thank you for pointing this out. We have added the PCA plot in Figure S1.

 

Comments 10: Tables 4 and 5: results are duplicated in text as well. Same info is presented in Table and Results. I advise authors to discuss the Table results in a more engaging way.

Response 10: Thank you for pointing this out. The descriptions in the Results section for Tables 4 and 5 have been modified (P11-13).

 

Comments 11: 5. Conclusions: should be revised and include the nutritional aspects.

Response 11: Thank you for pointing this out. We have revised the Conclusion section (Line 540-543).

 

Comments 12: References should be cross-check and should meet the journal format.

Response 12: Thank you for pointing this out. We have cross-checked the references against the original sources.

Author Response File: Author Response.pdf