Combining Ability and Testcross Performance for Carotenoid Content of S₂ Super Sweet Corn Lines Derived from Temperate Germplasm

Round 1

Reviewer 1 Report

The manuscript deals with sweet corn biofortification which is of great significance. Authors have analysed the genetic effect and combining ability of S2 super sweet corn lines on carotenoid contents including provitamin A. They reported non-additive gene action is the key for inheritance of carotenoid contents and identified promising inbreds and hybrids. The study is interesting and precisely written. Congratulations on good work.

 

It is to suggest following few revisions to include before publication.

1. Authors have justified the use of single cross hybrid as tester (Female) in the manuscript. Among the 40 test cross hybrids (three way cross hybrids), did they noticed the higher grain yield performance compared to commercial checks? Because, they have mentioned in Line 26: Testcross hybrids with good performance, desirable SCA estimates, and ……defined? If yes, highlight the points in discussion section.
2. Briefly explain the extraction protocol of Schaub et al. [31] method, and also mention the slight modifications authors did in the protocol?
3. Similarly, briefly explain the protocol described by Gupta et al. [32] and also mention the slight modifications authors did in the protocol?
4. There are other published reports on sweet corn biofortification using molecular breeding of crtRB1. If possible, authors are advised to include and improve the manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The study is about to estimate the genetic effect  and combining ability of S2 super sweet corn lines on carotenoid contents. This study is useful for the researchers for formulating breeding strategies for improvement of carotenoids in sweet corn. The manuscript is well written with clear cut hypothesis, materials and methods and results and discussion.

However, the English language of the manuscript is poor and not upto the mark in a scientific community. Improve the language throughout the manuscript. The sentences are confusing for readers. Rewrite the sentenses, keep eyes on comma, full stop (.) and sentense breakdown to make the manuscript clear for the readers as many of the readers are not from the same subject.

My specific comments are given below:

Introduction:

1. Line 27-29: In table S1, highest concentration of beta carotene is shown by hybrid T2 x L2 and not by Hybrid T2 x L9. Please confirm. Add the content of each carotenoid fraction in bracket for clear picture.

2. Add one line on the type of gene present in super sweet corn, its function and concentration of total soluble sugars compared to other types of sweet corns.

3. Line 52: Like lutein and zeaxanthin, add the function of provitamin A carotenoids (β-carotene and β-cryptoxanthin).

4. Line 80-82: The genes PSY1, CYP97C and HYD3 are involved in carotenoids biosynthesis, while ZmCCD1 is involved in carotenoids degradation. Here the authors have written that PSY1, CYP97C and HYD3 expressed during late grain filling stage and ZmCCD1 at early stage. But, before synthesis of carotenoids, how carotenoids degradation gene expressed. I think ZmCCD1 gene express at late grain filling stage. Please confirm from some gene expression related studies at different stages of kernel development in sweet corn and field corn.

Materials and methods:

5. In table 1, the authors have written that the Total carotenoid contents were analyzed at maturity stage (35 days after anthesis). But, sweet corn is harvested at about 20 days after anthesis, and after that sweet corn quality reduces drastically. So analyzing carotenoids at this stage (35 days) may not represent the true nutritional value of sweet corn. Please confirm the date of analyzing the carotenoids, if it has been done on 20 days after anthesis as in Field experiment subheading, it has written that ears were harvested at 20 days after pollination.

6. Line 162: Write the estimation procedure of different fractions of carotenoids also or the specific wavelengths used for detection.

7. Line 165: Rewrite the formula as V is missing. Insert proper spaces.

8. Line 190-191: Delete the sentence ‘General combining ability (GCA) and specific combining ability (SCA) were estimated [35]’ as it has written well in next paragraph.

Results and discussion:

9. Line 217-218: Write the name of some promising experimental hybrids from this study specific for dry and wet season separately for higher provitamin A carotenoids and higher non-provitamin A carotenoids, separately. Also name some hybrids performed better in both the environments.

10. Line 232-233: In table 2, it can be seen that the effect of environment is very high (more than the hybrid effects). So the gene effect estimates derived in this study may not be reliable enough for choosing the breeding strategy for improving carotenoids content in sweet corn. Please clarify.

11. Line 267-268: Rewrite the sentence. It can be as 'On the other hand, previous studies reported medium to high broad-sense heritability estimates for these traits.

12. Line 301-303: Instead of selecting top ten hybrids, mention the name of few (2 or 3) hybrid combination possessing either higher non-provitamin A carotenoids (lutein+zeaxanthin) or provitamin A carotenoids (B-CT+half of the B-CX). You can also mention the name of 2-3 hybrids with higher total carotenoids.

13. Line 328-329: The sentence ‘A genotype with high GCA such in L20 presented a large number of favorable alleles of carotenoids existed’ is not clear, rewrite it.

14. Line 357-358: Sentence is not clear, rewrite it. Also it cannot be validated in Table 2. It might be Table 3. There are only top 4 genotypes involving T2 tester. Fifth one involves T1 tester. Please verify.

15. Line 359-360: The sentence ‘Moreover, T2 tester identified two lines forming testcrosses showing significant and positive GCA estimate for all studied, whereas T1 tester did not identify any line (Table 3)’ cannot be validated from Table 3. Enlist the cross combinations here for clear picture.

16. Line 372-373: You have mentioned five hybrids, but enlisted only two each for lutein and zeaxanthin. Add the others also. If not, write two instead of five.

17. Line 376: There are two tables with same number as Table 3. Please re-number the tables and also change the table numbers in text accordingly.

18. Line 381-382: The tester used in this study is a hybrid, so how you would expect complementation of favourable alleles? Explain.

Besides, some comments are also attached in manuscript.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

 

I am not an expert in crop breeding or agronomy so my comments and critique are restricted on the molecular and biochemical aspects (or the lack thereof) of the study. Any study aiming to increase nutrients for biofortification purposes, in any organism (including plants) needs to start with a thorough understanding of the underling biosynthetic pathway leading to that product or group of products. This is because without this understanding it is near impossible to draw meaningful conclusions from any study that does not explicitly establish a basis for the major limiting factors which determine levels of important nutrients. More importantly, it is critical to state the recommended daily intake for each and every target nutrient as without this information the relevance of the study, no matter how successful experimentally, will be near useless in the end in terms of the final objective, i.e. generating useful and commercially relevant bio fortified crops. Finally, it is also important to discuss other studies which have used alternative technologies, including advanced engineering and molecular biology and their outcomes, to compare levels of nutrient increases achieved using different techniques, again for the sole purpose of establishing relevance of the study in question. The current manuscript falls short on all the above points.

Major flaws in the study:

I based my critique in the light of the stated objective of this study (lines 89-92), which purports “to determine the genetic effect...of superior sweet corn lines on carotenoid contents”.

Corn, including sweet corn, contains only traces of pro-vitamin A (beta carotene) so unless one follows the approach of Harjes (Harjes CE, et al.,2008 Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification. Science 319:330–333) and their subsequent development of the orange corn within the Harvest Plus program, any endeavor will fall short of the final outcome. The two major carotenoids in corn, including sweet corn are the non-pro vitamin A carotenoids lutein and zeaxanthin. It is correct that sweet corn contains high levels of these two carotenoids and this is useful in the context of dealing with macular degeneration, as the authors of the manuscript acknowledge.

For comparison purposes, Golden Rice 2 and High carotenoid corn, produce ca: 35 ug/g and 60 ug/g dry weight beta carotene, respectively. The highest level of this nutrient in the current study is around 3 ug/g. Placing these levels with the context of the RDI for beta carotene make the study completely irrelevant in practical terms.

The other major issue is stability of carotenoids upon storage. Has this aspect been looked at?

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf