Map** and Candidate Gene Analysis of the Low-Temperature-Sensitive Albino Gene OsLTSA8 in Rice Seedlings
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study titled "Map** and Candidate Gene Analysis of the Low Temperature-Sensitive Albino Gene OsLTSA8 in Rice Seedlings" aims to elucidate the genetic basis underlying the low temperature-induced albino phenotype in rice seedlings. The current study evaluates a comprehensive analysis using integrated molecular biology, phenotypic analysis, genome map**, RNA-Sequencing and qPCR validation. This multi-faceted approach enhances the robustness and reliability of the findings.
The authors have meticulously crafted each section of the manuscript, including the introduction, materials and methods, results, and discussion, ensuring clarity and consistency throughout. However, there are several critical points that need to be addressed before the current article can be considered for final acceptance in the journal Current Issues in Molecular Biology (CIMB).
Full form for “PPR proteins’ at first sight
Line 36-39: need references to support the statement.
Line 167-171: Should be in materials and methods.
Line 280: observed thus far “observed so for”
There is a need to conclude the study results and implication of the study at the end of the discussion section.
There is a need to add the growth conditions for the experiment in the materials and methods section.
The study lacks phenoty** analysis for the leaf color, SPAD reading or chlorophyll measurements???
Initial map** using SSR, and insertion deletion (InDel) was also missing.
RNA sequencing data to SRA to NCBI not deposited and mentioned in the M & M.
Most of the software hyperlinks used for analysis was missing in the materials methods.
Author Response
Dear Editors and Reviewers,
Thank you for giving me the opportunity to revise this manuscript. We have made careful revisions as required by the editor. The point-to-point responses to reviewer’s comments are listed below. Moreover, we have sought professional editing service to improve the English writing. We hope the revised manuscript can be accepted for publication and we are looking forward to hearing from you soon.
Best regards,
**nghai Yang
Reviewers' comments:
Reviewer #1: The study titled "Map** and Candidate Gene Analysis of the Low Temperature-Sensitive Albino Gene OsLTSA8 in Rice Seedlings" aims to elucidate the genetic basis underlying the low temperature-induced albino phenotype in rice seedlings. The current study evaluates a comprehensive analysis using integrated molecular biology, phenotypic analysis, genome map**, RNA-Sequencing and qPCR validation. This multi-faceted approach enhances the robustness and reliability of the findings.
The authors have meticulously crafted each section of the manuscript, including the introduction, materials and methods, results, and discussion, ensuring clarity and consistency throughout. However, there are several critical points that need to be addressed before the current article can be considered for final acceptance in the journal Current Issues in Molecular Biology (CIMB).
Q1: Full form for “PPR proteins’ at first sight.
Response: Thank you. We have added the full form for PPR protein.
Q2: Line 36-39: need references to support the statement.
Response: Thanks for your suggestion. We have cited relevant references.
- Zhang,Z.; Tan,J.; Shi,Z.; **e,Q.; **ng,Y.; Liu,C.; Chen,Q.; Zhu,H.; Wang,J.; Zhang,J.; Zhang,G. Albino Leaf1 that encodes the sole octotricopeptide repeat protein is responsible for chloroplast development. Plant Physiol. 2016, 172, 1182-1191.
- Wang,Y.; Wang,J.; Chen,L.; Meng,X.; Zhen,X.; Liang,Y.; Han,Y.; Li,H.; Zhang,B. Identification and function analysis of yellow-leaf mutant (YX-yl) of broomcorn millet. BMC Plant Biol. 2022, 22, 463.
Q3: Line 167-171: Should be in materials and methods.
Response: Thanks. We have revised this part (line 403-409 in tracked manuscript).
Q4: Line 167-171: Should be in materials and methods.
Response: Thanks. We have corrected this error.
Q5: There is a need to conclude the study results and implication of the study at the end of the discussion section.
Response: Thanks for your suggestion. We have added the conclusion (line 295-302 in tracked manuscript).
Q6: There is a need to add the growth conditions for the experiment in the materials and methods section.
Response: Thanks for your suggestion. we have added this part (line314-315 in tracked manuscript).
Q7: The study lacks phenoty** analysis for the leaf color, SPAD reading or chlorophyll measurements???
Response: Thanks for your suggestion. we have added the determination of the chlorophyll content (line 317-330 in tracked manuscript) and result (line 317-330 and Figure 1C in tracked manuscript).
Q8: Initial map** using SSR, and insertion deletion (InDel) was also missing.
Response: Thank you. Previous studies have shown that SNPs can occur at any position in the genome and serve as next-generation molecular markers, with advantages such as large quantity, uniform distribution, rich polymorphism, and high accuracy [3]. In the monocotyledonous model plant rice, it is 1 SNP/336 bp [4], and in the dicotyledonous model plant Arabidopsis, it is 1 SNP/333 bp [5]. Therefore, we directly selected SNPs to analyze rice albino leaf genes in this study.
- Ganal,W.; Altmann,T.; Röder,M.S. SNP identification in crop plants. Curr Opin Plant Biol. 2009, 12, 211-217.
- Schmid,K.; Sorensen,T.R.; Stracke,R.; Torjek,O.; Altmann,T.; Mitchell-olds,T.; Weisshaar,B. Large-scale identification and analysis of genome-wide single-nucleotide polymorphisms for map** in Arabidopsis thaliana. Genome Res. 2003, 13, 1250-1257.
- Yu,J.; Wang,J.; Lin,W.; Li,S.; Li,H.; Zhou,J.; Ni,P.; Dong,W.; Hu,S.; Zeng,C.; Zhang,J.; Zhang,Y.; Li,R.; Xu,Z.; Li,S.; Li,X.; Zheng,H.; Cong,L.; Lin,L.; Yin,J.; Geng,J.; Li,G. The Genomes of Oryza sativa: a history of duplications. PLoS Biol. 2005, 3, e38.
Q9: RNA sequencing data to SRA to NCBI not deposited and mentioned in the M & M.
Response: Thanks. The QTL-seq and RNA-seq data that support the findings of this study have been deposited to Na-tional Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) with the acces-sion code PRJNA1125040 (SRA no. from SRR29442753 to SRR29442758) and PRJNA1125039 (SRA no. from SRR29442741 to SRR29442752).
Q10: Most of the software hyperlinks used for analysis was missing in the materials methods.
Response: Thanks. We have added the software hyperlinks in this study. RSEM (http://deweylab.biostat.wisc.edu/rsem/); DESeq2 (http://bioconductor.org/packages/stats/bioc/DESeq2/); SAMtools (https://github.com/samtools/samtools.git); SnpEff ( https://pcingola.github.io/SnpEff/).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors1. You can give more information about SNPNUM method.
Author Response
Dear Editors and Reviewers,
Thank you for giving me the opportunity to revise this manuscript. We have made careful revisions as required by the editor. The point-to-point responses to reviewer’s comments are listed below. Moreover, we have sought professional editing service to improve the English writing. We hope the revised manuscript can be accepted for publication and we are looking forward to hearing from you soon.
Best regards,
**nghai Yang
Reviewers' comments:
Reviewer #2: Comments and Suggestions for Authors
Q1: You can give more information about SNPNUM method.
Response: Thanks for your suggestions. We have added this part (line 355-374 in tracked manuscript).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors employed reciprocal crosses to obtain albino mutants, followed by QTL-Seq methods to identify potential gene regions associated with albinism on Chromosome 8. Integrating RNA-Seq findings, they identified four candidate genes, subsequently validated by qRT-PCR. While the study provides insight into candidate genes related to albino seeding, the description of materials requires clarification and expansion.
Additional comments are provided below for consideration.
The introduction solely focuses on albino characteristics but lacks a discussion of relevant literature regarding the suitability of QTL-Seq for their research.
The material section lacks detail, particularly regarding the rice variety (Japonica or indica) and its origin.
Do C6 and C13 represent normal green-leaf plants? It's puzzling that reciprocal crosses involving them both produce albino mutants.
The timing of RNA extraction, critical for RNA-seq experiments, remains unspecified; clarification regarding the plant stage during RNA extraction is necessary.
The discussion section requires significant enhancement. Many sentences (e.g., L214-254) are more suited for the introduction, not the discussion. These points should be addressed to improve the clarity and comprehensiveness of the study.
Author Response
Dear Editors and Reviewers,
Thank you for giving me the opportunity to revise this manuscript. We have made careful revisions as required by the editor. The point-to-point responses to reviewer’s comments are listed below. Moreover, we have sought professional editing service to improve the English writing. We hope the revised manuscript can be accepted for publication and we are looking forward to hearing from you soon.
Best regards,
**nghai Yang
Reviewers' comments:
Reviewer #3: The authors employed reciprocal crosses to obtain albino mutants, followed by QTL-Seq methods to identify potential gene regions associated with albinism on Chromosome 8. Integrating RNA-Seq findings, they identified four candidate genes, subsequently validated by qRT-PCR. While the study provides insight into candidate genes related to albino seeding, the description of materials requires clarification and expansion.
Additional comments are provided below for consideration.
Q1: The introduction solely focuses on albino characteristics but lacks a discussion of relevant literature regarding the suitability of QTL-Seq for their research.
Response: Thanks for your suggestions. We have added this part (line 82-83 in tracked manuscript). The relevant references are as follows:
- Qin,D.; Dong,J.; Xu,F.; Guo,G.; Ge,S.; Xu,Q.; Xu,Y.; Li,M. Characterization and fine map** of a novel barley Stage Green-Revertible Albino Gene (HvSGRA) by Bulked Segregant Analysis based on SSR assay and Specific Length Amplified Fragment Sequencing. BMC Genomics. 2015, 16, 838.
- Ye,S.; Yang,J.; Huang,Y.; Liu,J.; Ma,X.; Zhao,L.; Ma,C.; Tu,J.; Shen,J.; Fu,T.; Wen,J. Bulk segregant analysis-sequencing and RNA-Seq analyses reveal candidate genes associated with albino phenotype in Brassica napus. Front Plant Sci. 2022, 13, 994616.
- Ke,X.; Shen,J.; Niu,Y.; Zhao,H.; Guo,Y.; Sun,P.; Yang,T.; Jiang,Y.; Zhao,B.; Wang,Z.; Wu,T.; Wang,H.; Li,Z. Cucumber NUCLEAR FACTOR-YC2/-YC9 target translocon component CsTIC21 in chloroplast photomorphogenesis. Plant Physiol. 2023, 192, 2822-2837.
Q2: The material section lacks detail, particularly regarding the rice variety (Japonica or indica) and its origin.
Response: Thank you. We have revised this part ((line 307-312 in tracked manuscript).
Q3: Do C6 and C13 represent normal green-leaf plants? It's puzzling that reciprocal crosses involving them both produce albino mutants.
Response: Thank you. Both F2 populations of ‘C6×C13’ and ‘C13×C6’ can produce albino plants. The construction of populations ‘C6×C13’ and ‘C13×C6’ is to demonstrate that albino seedlings are controlled by nuclear genes.
Q4: The timing of RNA extraction, critical for RNA-seq experiments, remains unspecified; clarification regarding the plant stage during RNA extraction is necessary.
Response: Thanks for your suggestions. We have added relevant contents (line 406-408, 422 in tracked manuscript).
Q5: The discussion section requires significant enhancement. Many sentences (e.g., L214-254) are more suited for the introduction, not the discussion. These points should be addressed to improve the clarity and comprehensiveness of the study.
Response: Thanks for your suggestions. We have modified this section and added a conclusion(line 223-319 in tracked manuscript).
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsAuthors responds well to each comment for the improvment of the article. I endorsed the article for publication in CIMB-MDPI.
