Combining Ability and Gene Action Controlling Grain Yield and Its Related Traits in Bread Wheat under Heat Stress and Normal Conditions

Round 1

Reviewer 1 Report

The manuscript aimed at evaluating parental genotypes and their 27 crosses under normal and heat stress, exploring their diversity based on dehydration-responsive element-binding 2 gene.

The manuscript is well written and the experimental part is detailed and well described.

The experimental plan is well conceived and the two locations enough diverse, although a test in an environment different form Egypt might be important to further evaluate the tested genotypes.

Results are well presented and complete.

Maybe a table including the means of the lines presented in figure 3-4 can be useful as supplementary material.

In the discussion section as in the results , the authors highlight the presence of 57 SNPs among genotypes and conclude that P2 and P4 were the highest homologies for dehydrin gene  sequence with heat and drought-tolerant accessions Triticum aestivum, Triticum dicoccoides  and Aegilops tauschii). Interestingly, P2 and P4 exhibited consistent positive GCA effects as well as their hybrid combination P2×P4 showed positive and significant SCA effects for 587 grain yield under heat stress environment (E4).

However is there any relationship between the position of the SNP in the sequence and the increased resilience to heat stress? Can the authors include a little ( if known) of this in the discussion part?

Small minor revision

Table 2 and Table 3 replace

*and ** reveal p-value < 0.05 and 0.01, in the same order  

With

*and ** reveal p-value < 0.05 and 0.01, respectively

Line 509 rephrase “angles more than 90° suggest no to negative association among traits.” I believe authors want to say the angle suggests a negative association, please clarify this point.

Author Response

The manuscript aimed at evaluating parental genotypes and their 27 crosses under normal and heat stress, exploring their diversity based on dehydration-responsive element-binding 2 gene. The manuscript is well written and the experimental part is detailed and well described. The experimental plan is well-conceived and the two locations enough diverse, although a test in an environment different from Egypt might be important to further evaluate the tested genotypes. Results are well presented and complete.

Re: We would like to thank the Reviewer for his time dedicated to our manuscript and presenting positive aspects in our manuscript.

- Maybe a table including the means of the lines presented in Figures 3-4 can be useful as supplementary material.

Re: We thank the reviewer for this comment; we added Table 4 to the supplementary materials as suggested.

- In the discussion section as in the results, the authors highlight the presence of 57 SNPs among genotypes and conclude that P2 and P4 were the highest homologies for dehydrin gene sequence with heat and drought-tolerant accessions Triticum aestivum, Triticum dicoccoides  and Aegilops tauschii). Interestingly, P2 and P4 exhibited consistent positive GCA effects as well as their hybrid combination P2×P4 showed positive and significant SCA effects for grain yield under heat stress environment (E4). However is there any relationship between the position of the SNP in the sequence and the increased resilience to heat stress? Can the authors include a little ( if known) of this in the discussion part?

Re: More details have been added (please see lines 601-607)

Small minor revision

Table 2 and Table 3 replace *and ** reveal p-value < 0.05 and 0.01, in the same order  With *and ** reveal p-value < 0.05 and 0.01, respectively

Re: Replaced as suggested

Line 509 rephrase “angles more than 90° suggest no to negative association among traits.” I believe authors want to say the angle suggests a negative association, please clarify this point.

Re: The sentence has been modified (please see line 517)

Reviewer 2 Report

In their manuscript named ‘Combining Ability and Gene Action Controlling Yield Characteristics in Bread Wheat under Heat Stress and Normal Conditions’ Kamara et al. reported on the usage of a F1-hybrid population from a half-diallel crossing scheme of 6 parental lines to improve breeding for heat- and drought-tolerant varieties. Therefore, they tested the population and their parents at 2 different locations and two sowing dates at each location and calculated general and specific combining ability and heterosis with results of yield components and plant physiological traits to . Furthermore, they sequenced a dehydration-responsive element-binding 2 gene within the parental genomes to conclude in their heat- and drought-tolerance. The topic of this manuscript sounds interesting and is of great importance concerning the ongoing climate change. However, the title is not really reflecting the content of the manuscript and there are several major issues that have to be addressed. Furthermore, the study provides only weak results, which do not necessarily need to be published

 

Major issues:

 

1. Page 3, line 109: The genotype set consisting of 15 hybrids and their parental lines is very small for calculating GCA and SCA and especially for the calculation of combining ability x environmental interactions. In particular, by using a half diallel crossing scheme, the authors received only between one and five hybrids per cross, which is no base to receive resilient results for such complex traits that are furthermore under the influence of such strong environmental conditions. Therefore, the reviewer recommends using a bigger population for the calculation of combining ability and its interaction with the environment.
2. Page 3, line 134-136: The authors described the experimental plots as single rows with a length of 3m and spaces of 30cm between rows and 20cm between plants within the row. This could be compared with a single seed planting, which is not comparable with the normal way wheat is sown. The reviewer is not sure about the usual way wheat is sown in Egypt, but from an international point of view, the conclusions especially on yield, which were drawn therefrom on the real performance of the single hybrids, should be not very reliable.
3. Page 14, line 380: Why did the authors focus on exactly this DREB gene? As they explained in the introduction, there are many genes influencing heat and drought tolerance. Why is this single gene then so important that it has to be part of this manuscript.

 

Minor issues:

 

1. Due to some mistakes in grammar and spelling, the reviewer recommends proofreading and linguistic revisions by an English native speaker.
2. Figure 1: The authors should give a hint on the figure within the text.
3. Figure 1: Why did the authors use boxplots to describe the maximum and minimum temperatures? The reviewer guesses, that the boxplots should represent the daily values, but this does not get clear from the caption. Anyways, the boxplots should be changed to line charts, like it was made for the rainfall. Then the authors could mark the days, which showed the highest or lowest temperatures and could additionally mark important days of plant growth stages within this chart.
4. Page 7, line 228ff: Please give units for the mean values mentioned in the text.
5. Figure 2H: Wrong title for y-axis
6. Chapter 3.6-3.8: The part describing the results of GCA, SCA and Heterosis should contain the specific values. The reviewer recommends naming the most important values instead of only describing GCA or SCA being positive or negative. To make it even clearer, the authors could use relative values instead of just GCA or heterosis values.
7. Chapter 3.9: There is no further explanation for additive and dominance components and how they are used in calculation of heritability. The authors should make it a bit clearer by giving the formula and by explaining the method a little bit instead of just giving the references in chapter 2.4. That could make it a bit clearer to the reader. It is also not clear, where the authors get their knowledge from about the ratio of recessive and dominant genes in the parents
8. Page 24, line 584: The authors stated that parents P2 and P4 have the highest homologies for the dehydrin gene sequence with “heat and drought-tolerant accessions T. aestivum, T. dicoccoides and Ae. tauschii”. If the reviewer didn’t understand the content of the sentence totally wrong, these names stand for three species and not for just single accessions. So, how can the authors conclude, that the sequenced reference genomes of these species stand for drought or heat tolerant genotypes?

Author Response

In the manuscript named ‘Combining Ability and Gene Action Controlling Yield Characteristics in Bread Wheat under Heat Stress and Normal Conditions’ Kamara et al. reported on the usage of a F1-hybrid population from a half-diallel crossing scheme of 6 parental lines to improve breeding for heat- and drought-tolerant varieties. Therefore, they tested the population and their parents at 2 different locations and two sowing dates at each location and calculated general and specific combining ability and heterosis with results of yield components and plant physiological traits. Furthermore, they sequenced a dehydration-responsive element-binding 2 gene within the parental genomes to conclude in their heat- and drought-tolerance. The topic of this manuscript sounds interesting and is of great importance concerning the ongoing climate change. However, the title is not really reflecting the content of the manuscript and there are several major issues that have to be addressed. Furthermore, the study provides only weak results, which do not necessarily need to be published

Re: We would like to thank the Reviewer for his time dedicated to our manuscript and presenting positive aspects in our manuscript. We think the obtained results reflected the title of our manuscript. Since the parental genotypes and their crosses were evaluated at two locations (are different in soil and climate) under timely and late sowing date which provided high temperature during flowering and grain filling periods. Tolerant parental genotypes and cross combinations were identified based on grain yield, its attributes and heat tolerance indices. Furthermore, the obtained results were corroborated by DNA sequence analysis of dehydration-responsive element-binding 2 gene (DREB, stress tolerance gene in response to abiotic stress). Besides, the type of gene action controlling studied agronomic traits were determined. The designated tolerant genotypes and cross combinations could be exploited as a source of beneficial alleles for earliness and high yielding in breeding for heat tolerance. Since breeding for developing heat-tolerant and high-yielding wheat genotypes is essential current under abrupt climate change. Thereupon, we think all aforementioned aspects make our work deserves to be published.

 

 Major issues:

1- Page 3, line 109: The genotype set consisting of 15 hybrids and their parental lines is very small for calculating GCA and SCA and especially for the calculation of combining ability x environmental interactions. In particular, by using a half diallel crossing scheme, the authors received only between one and five hybrids per cross, which is no base to receive resilient results for such complex traits that are furthermore under the influence of such strong environmental conditions. Therefore, the reviewer recommends using a bigger population for the calculation of combining ability and its interaction with the environment

Re: Six diverse bread wheat genotypes )based on origin and level of tolerance to high temperature from previous preliminary screening trials( were crossed and generated 15 F₁ hybrids. In total 21 genotypes were evaluated which is an acceptable number in self-pollinated crops especially for starting a breeding program for stress tolerance. A close number of parents and cross combinations were generated using half diallel scheme and evaluated in several previous works as Yıldırım et al. (2013) Field Crops Res. 140, 9-17; Mia et al. (2017) Crop Pasture Sci. 68(6), 534-543; Bassuony and Zsembeli (2020) Cereal Res. Commun. 1-8 and Parkes et al. (2020) Agron. 10(12), 850. The combinig ability was utilized to identify tolerant and high-yielding cross combinations with expected transgressive segregants to follow the advanced generations.

 

2- Page 3, line 134-136: The authors described the experimental plots as single rows with a length of 3-m and spaces of 30-cm between rows and 20-cm between plants within the row. This could be compared with a single seed planting, which is not comparable with the normal way wheat is sown. The reviewer is not sure about the usual way wheat is sown in Egypt, but from an international point of view, the conclusions especially on yield, which were drawn therefrom on the real performance of the single hybrids, should be not very reliable

Re: Wheat is a self-pollinated crop requires emasculation manually which is labor-intensive and tedious work and simultaneously produces a small amount of seed for each hybrid. Moreover, we evaluated the genotypes at two sowing dates in two different locations in three replications. Accordingly, it was obligated to evaluate individual rows (3-m long) for each cross combination like all F_1s at all breeding programs worldwide. On the contrary, the plot size becomes larger in the following advanced generations with intensive planting.

3- Page 14, line 380: Why did the authors focus on exactly this DREB gene? As they explained in the introduction, there are many genes influencing heat and drought tolerance. Why is this single gene then so important that it has to be part of this manuscript.

Re: Dehydration Responsive Element Binding (DREB) proteins are important transcription factors specifically responsive to drought and heat stress. Accordingly thy could be used to improve plant stress tolerance due their ability to regulate the expression of downstream genes associated with stress tolerance (Wang et al., 2008. Plant Mol. Biol. 67, 589-602; Kidokoro et al., 2015. Plant J. 81, 505-518). They are broadly grouped as DREB1 and DREB2, involved in two separate signal transduction pathways under high temperature and dehydration. Consequently, for their importance in drought and heat tolerance, were applied to study the diversity in the investigated genotypes. Similarly, the association between DREB gene and stress tolerance was studied in several crops as Agarwal et al. (2006) Plant Cell Reports, 25, 1263-1274.; Lata, et al. (2011) J. Exp. Bot. 62, 3387-3401; Shavrukov et al. (2016) Front. Plant Sci. 7, 1736; Agarwal et al. (2017) J. Exp. Bot. 68, 2135-2148; Zotova, et al. (2018) Front. Plant Sci. 9, 1441.

 

Minor issues:

1-  Due to some mistakes in grammar and spelling, the reviewer recommends proofreading and linguistic revisions by an English native speaker.

Re: The manuscript has been carefully revised

2- Figure 1: The authors should give a hint on the figure within the text.

Re: It is mentioned in the text (please see lines 136-139)

3- Figure 1: Why did the authors use boxplots to describe the maximum and minimum temperatures? The reviewer guesses, that the boxplots should represent the daily values, but this does not get clear from the caption. Anyways, the boxplots should be changed to line charts, like it was made for the rainfall. Then the authors could mark the days, which showed the highest or lowest temperatures and could additionally mark important days of plant growth stages within this chart

Re: The figure has been modified  as the reviewer suggested

4- Page 7, line 228: Please give units for the mean values mentioned in the text.

Re: The units for each trait have been added as suggested

• Figure 2H: Wrong title for y-axis

Re: Thanks, y-axis has been corrected in Figure 2H

6- Chapter 3.6-3.8: The part describing the results of GCA, SCA and Heterosis should contain the specific values. The reviewer recommends naming the most important values instead of only describing GCA or SCA being positive or negative. To make it even clearer, the authors could use relative values instead of just GCA or heterosis values.

Re: Thanks for the suggestion, but the values are already presented in Tables 2-4, and repeating them in the text is redundant. 

7- Chapter 3.9: There is no further explanation for additive and dominance components and how they are used in calculation of heritability. The authors should make it a bit clearer by giving the formula and by explaining the method a little bit instead of just giving the references in chapter 2.4. That could make it a bit clearer to the reader. It is also not clear, where the authors get their knowledge from about the ratio of recessive and dominant genes in the parents

Re: Commonly genetic components and heritability are calculated as described by Hayman (1954) and Mather and Jinks (1971). Accordingly, presenting their formulas in the text is not common.

8- Page 24, line 584: The authors stated that parents P2 and P4 have the highest homologies for the dehydrin gene sequence with “heat and drought-tolerant accessions T. aestivum, T. dicoccoides and Ae. tauschii”. If the reviewer didn’t understand the content of the sentence totally wrong, these names stand for three species and not for just single accessions. So, how can the authors conclude, that the sequenced reference genomes of these species stand for drought or heat tolerant genotypes?

Re: The identified tolerant parents P₂ and P₄ exhibited high degree of similarity in heat shock transcription factors of the species Triticum dicoccoides and Aegilops tauschii. The species belongs to the Triticeae genus; Triticum and Aegilops are known as the main wild relatives of wheat containing a feasible source of gene pool for stress tolerance that could be utilized for conferring tolerance to abiotic stresses.

Reviewer 3 Report

The manuscript "Combining Ability and Gene Action Controlling Yield Characteris-2 tics in Bread Wheat under Heat Stress and Normal Conditions" described the research of using six parents and their 15 half-diallel F1s to  investigate yield-related traits of wheat under two location with two sowing dates. Experimental design and data analysis were both appropriate and the manuscript presented the detailed results. Some of my suggestions for improving this manuscript are:

1) The first paragraph of introduction should focus on wheat yield affected by climate change rather than the commonly knowledge of world production and human population.

2) The introduction of GCA and SCA in wheat heat tolerance was very limited and should include more literature and significant progress made in this research area.

3) Six wheat lines were used, but no performance description for those lines were mentioned. It is mentioned they were preliminary examined but should include a brief result for their heat tolerance from the unpublished data.

4) The plot used in research is a single 3-m row. It’s a very tiny plot for yield test. How many plants were grown in each plot? Or the seeding rate?

5) The results part provided much detailed analysis from different aspects. I would wish all those results are in some more logic order and can contribute to each other. Normally we would like to know how lines performed under different condition; GCA and SCA among genotypes for heat tolerance; environmental effects; groups according to heat index vs the groups through SNP marker of the Dehydrin gene; can Dehydrin gene be main factor for heat tolerance; and conclusion of which lines or crosses could be good.

6) Discussion in the manuscript mostly followed the logic order, but I would prefer the discussion emphasized more on conclusions of this research rather than on literature review.

Author Response

The manuscript "Combining Ability and Gene Action Controlling Yield Characteristics in Bread Wheat under Heat Stress and Normal Conditions" described the research of using six parents and their 15 half-diallel F1s to investigate yield-related traits of wheat under two location with two sowing dates. Experimental design and data analysis were both appropriate and the manuscript presented the detailed results.

Re: We would like to thank the reviewer for his time dedicated to our manuscript

1) The first paragraph of the introduction should focus on wheat yield affected by climate change rather than the common knowledge of world production and the human population.

Re: The first paragraph of the introduction has been modified as suggested (please see 61-70)

2) The introduction of GCA and SCA in wheat heat tolerance was very limited and should include more literature and significant progress made in this research area.

Re: This part has been modified (please see lines 81-87)

3) Six wheat lines were used, but no performance description for those lines were mentioned. It is mentioned they were preliminary examined but should include a brief result for their heat tolerance from the unpublished data.

Re: The heat tolerance reaction of six parental genotypes is presented in Table S1

4) The plot used in research is a single 3-m row. It’s a very tiny plot for yield test. How many plants were grown in each plot? Or the seeding rate?

Re: Re: Wheat is a self-pollinated crop requires emasculation manually which is labor-intensive and tedious work and simultaneously produces a small amount of seed for each hybrid. Moreover, we evaluated the genotypes at two sowing dates in two different locations in three replications. Accordingly, it was obligated to evaluate individual rows (3 m-long) for each cross combination like all F_1s at all breeding programs worldwide. On the contrary, the plot size becomes larger in the following advanced generations with intensive planting.

5) The results part provided much detailed analysis from different aspects. I would wish all those results are in some more logic order and can contribute to each other. Normally we would like to know how lines performed under different condition; GCA and SCA among genotypes for heat tolerance; environmental effects; groups according to heat index vs the groups through SNP marker of the Dehydrin gene; can Dehydrin gene be main factor for heat tolerance; and conclusion of which lines or crosses could be good.

Re: We think the results are presented in logic order as we started with the analysis of variance and performance of the cross combinations under different environments (lines 212-290, Table 1, Figures 2-5). Thereafter, their classification according to heat tolerance indices were presented (lines 292-302, Figure 6) and stability across different environments (lines 347-365, Figure 7). Then, the protein sequence of dehydrin gene (lines 370-400, Figures 7,8) and posteriorly, GCA and SCA among genotypes (lines 405-450 Tables 2-4).

The results of GCA and SCA, heat tolerance indices, cluster analysis, dehydrin gene were associated in the discussion section (593-599) as well as the importance of dehydrin gene for heat tolerance (lines 600-606). Finally, we conclude the best parents and cross combinations and presented in the conclusion (lines 683-693)

 

6) Discussion in the manuscript mostly followed the logic order, but I would prefer the discussion emphasized more on conclusions of this research rather than on literature review.

Re: The discussion has been revised and modified