New Flowering and Architecture Traits Mediated by Multiplex CRISPR-Cas9 Gene Editing in Hexaploid Camelina sativa

Round 1

Reviewer 1 Report

This research produced some laudable results of mutations in Camelina in flowering time and plant architecture that may increase the diversity of traits in this crop for breeding desired traits for sustainable production. The genes of interest, including SVP, FLC, LHP1, TFL1 and ELF3, have been shown to be involved in regulating flowering time and sometimes plant growth habits such as shoot branching in Arabidopsis. As Camelina is closely related to Arabidopsis, similar effects were expected in mutants generated by CRISPR mutagenesis, however not certain. The T-DNA used in the study included all five genes, transgenic plants contain different alleles or homeologs that have been mutated. The authors therefore might be able to isolate a suite of mutants containing different genes and determine their effects in Camelina. Characterization of their individual functions may provide more effective methods to generate targeted traits and avoid undesired ones, as were observed in the transgenics generated in the current study.  

Other comments:

1. Presentation of mutant genotypes and phenotypes can be more clearer. The current formats are somehow different to track. 

2. Results (e.g., section 3.2) have redundant descriptions of Methods 

3. Line 99: FT protein, not mRNA, is transported from leaves to shoot apical meristems

 

Author Response

Reviewer 1

Please find below, answers and comments (A) to reviewer’s remarks (Q) that have been numbered for clarity.

Q1 : Presentation of mutant genotypes and phenotypes can be more clearer. The current formats are somehow different to track. 

A1 :We agree with the reviewer and we simplified the line and plant names with a new Table listing the names and the actual pedigree. We also explained in material and method section the naming process allowing the tracking of parental origin of every plant.

Q2 : Results (e.g., section 3.2) have redundant descriptions of Methods 

A2 :The reviewer refers to the paragraph describing preliminary experiments testing the possibility of outcrossing between neighboring plants in the same tray during the screen. We think that it is relevant to the result section since it describes original finding and not only a method. We therefore would prefer leaving this section as it is. However, we have modified the paragraph to better describe the objective of the experiment and also included “data not shown” to stress that it was original data.

Q3 : Line 99: FT protein, not mRNA, is transported from leaves to shoot apical meristems

A3 : We have modified the text by mentioning “signal molecules” since there is still some debate about the signaling role of FT protein and its mRNA

Reviewer 2 Report

This manuscript describes directed research on Camelina sativa to shorten its vegetative phase through Targeted Induced Genetic Variation with CRISPR technology. Five genes are identified as targets based on orthology to the closely related Arabidopsis thaliana and previous knowledge that includes a wiring diagram of flowering in Arabidopsis.  The genes chosen act as repressors of FT in Arabidopsis or have been shown to modify growth habit to favor synchronous flowering. 

The relevance of this approach to sustainability goals is clearly articulated and relates to the ability of Camelina to fit into a double cropping system when planted in the late spring/early summer.

The manuscript presents a state of the art approach to molecular breeding that is particularly applicable to polyploids such as Camelina where some sub-functionalization or expression biases among homeologs may be present that increases potential for combinatorial effects among alleles and for complex phenotypes.  These could be caused by disfunctional alleles or combinations of alleles that effectively create a spectrum of possible variation including hypomorphic variation. Classic mutation breeding in polyploids is not always productive and lack of standing variation make traditional backcross strategies less attractive.

The downside of this approach is that with polyploids, genome-specific PCR primers are not always possible to develop such as here with the SVP gene SG-2 copy. This makes interpretation of the genotype more difficult.

As seen in this paper, it can also be extremely difficult to follow up on all variants. These include those without phenotypes or with possible off target mutations or large deletions that may not be amplified. Bias has been incurred through selection in this experimental approach that cannot be easily avoided.  More follow-up, demonstrating co-segregation or association of selected genotypes with early flowering, late flowering, dwarfism, branching or determinant growth would have been desirable and provide more convincing evidence to link genotype and phenotype. This could be accomplished via several different methods involving bulked sampling or individual genotyping.

The T-DNA used has multiple repetitive sequences.  Please describe in the methods how the 10 sgRNA transcriptional units were verified in both E. coli and Agrobacterium to ensure that the constructs were not rearranged.

The figures are all informative in both the main body and supplemental file.  I believe that the authors have provided a sound basis for their further analysis under field conditions of the families that were selected.

Author Response

 Reviewer 2

Please find below, answers and comments (A) to reviewer’s remarks (Q) that have been numbered for clarity.

Q4 : As seen in this paper, it can also be extremely difficult to follow up on all variants. These include those without phenotypes or with possible off target mutations or large deletions that may not be amplified. Bias has been incurred through selection in this experimental approach that cannot be easily avoided. 

A4 : We agree that such gene editing method could induce allelic biais that could modify the expected phenotype. However, the selection process was carried out independently of the genotypes and only based on the phenotype. Different phenotypes would have probably achieved with different mutagenesis strategy.

Q5 : More follow-up, demonstrating co-segregation or association of selected genotypes with early flowering, late flowering, dwarfism, branching or determinant growth would have been desirable and provide more convincing evidence to link genotype and phenotype. This could be accomplished via several different methods involving bulked sampling or individual genotyping.

A5 : Indeed, segregation analysis of the mutations would have allowed to identify the causal alleles of the different phenotypes but that was not the scope of this work. We aimed in demonstrating the possibility of selecting in polyploid species like camelina complex traits by using multiplex gene editing. The identification of causal alleles will be the subject of future work.

Q6 : Please describe in the methods how the 10 sgRNA transcriptional units were verified in both E. coli and Agrobacterium to ensure that the constructs were not rearranged.

A6 : It has been done detailed in the material and method section

Q7 : Please provide detail explanation of all the statistical procedures used.

A7 : It has been detailed in the material and method section

Reviewer 3 Report

Dear Bellec et al., 

This is an interesting and engaging study, which provides useful information and represents a valuable contribution about an important subject.  The paper is generally well written and structured. The authors have provided adequate background information and encompassed all relevant references in the introduction part.   They used appropriate research design and described methodology section very well. The result part was nicely presented. The authors compared and contrasted their findings from related previous studies, using data from other crops too.  However, the overall quality of this manuscript can be improved by addressing some of the issues raised by the reviewers, especially for the data analysis part. Please provide detail explanation of all the statistical procedures used.

Comments and suggestions for improving the overall quality of the manuscripts are:

Please provide citation line 36, ......such as rapeseed and sunflower.

More about agronomic properties, its uses and adaptability in changing climate, you can refer to newly review article on Camelina (https://doi.org/10.3390/plants11060772)

The authors mentioned R software was used for the statistical tests (line 174). Provide more explanation of how statistical analysis was performed.

All tables and figures should “stand alone” meaning that they can be interpreted in isolation, without having to refer to information in the text. Figure 3 to me is not very clear. For example, legends for Line 6-2-7-28-12 and 6-2-109-20-2 seem very similar and have same issue with others. The authors said along with other phenotypic features......line 280. What do you mean by along with other .......? Please mention them here as well.

Figure 4 can be improved. * Not clear on Figures. Figure 5 and figure 6 are not the figures, they are tables. Tables must be improved, especially Table 2 (so called figure 6 here).

 

 

 

Author Response

Reviewer 3

Please find below, answers and comments (A) to reviewer’s remarks (Q) that have been numbered for clarity.

Q8 :  Please provide citation line 36, ......such as rapeseed and sunflower.

More about agronomic properties, its uses and adaptability in changing climate, you can refer to newly review article on Camelina (https://doi.org/10.3390/plants11060772)

A8 : The reference was included in introduction section

Q9 : The authors mentioned R software was used for the statistical tests (line 174). Provide more explanation of how statistical analysis was performed.

A9: It has been detailed in the material and method section. We have used non-parametric Wilcoxon test for non-paired data.

Q10 : All tables and figures should “stand alone” meaning that they can be interpreted in isolation, without having to refer to information in the text. Figure 3 to me is not very clear. For example, legends for Line 6-2-7-28-12 and 6-2-109-20-2 seem very similar and have same issue with others.

A10 : We have clarified the figure by using different drawing art and have detailed the basis of line naming in material and method section.

Q11 : The authors said along with other phenotypic features......line 280. What do you mean by along with other .......? Please mention them here as well.

It refered to basal branching, determinate flowering and shorter stature as described in Table1. A11 : We however agree that it is confusing to refer to the other phenotypes in Fig.3 so the sentence was deleted from Fig.3 legend.

Q12 :  Figure 4 can be improved. * Not clear on Figures. Figure 5 and figure 6 are not the figures, they are tables. Tables must be improved, especially Table 2 (so called figure 6 here).

A12 : The Figure 5 and 6 have been modified in Table 1 and 2.

Round 2

Reviewer 1 Report

My comments have been addressed