Inheritance and Genetic Mapping of Late-Bolting to Early-Bolting Gene, BrEb-1, in Chinese Cabbage (Brassica rapa L.)
, Yanyan Zhao 1, Shuangjuan Yang 1, Henan Su 1, Zhiyong Wang 1, Saleh Ahmed Shahriar 3, Jundang Li 1, Zhiyuan Yang 4, Yuxiang Yuan 1,* and Xiaowei Zhang 1,*Round 1
Reviewer 1 Report
In this study, authors studied the inheritance and genetic mapping of Late-bolting to Early- 1 bolting gene, BrEb-1, in Chinese Cabbage (Brassica rapa L.). In my opinion, the study is planned nicely, and the findings are interesting. The manuscript is also written well. However, I did not find the supplementary data associated with this manuscript. If authors provide the supplementary data, then I would be happy to review it again. My other comments are below:
- There are many typographical, and errors throughout the manuscript. Authors need to check them carefully.
- The quality of figures is bad. Please increase the font size of the text used in the figures.
- Authors used both names Chinese cabbage and B. rapa throughout the manuscript. I would suggest authors to maintain uniformity in using either Chinese cabbage or B. rapa. Authors may use both names in the beginning.
- Please create a separate section for conclusion and include future prospects of the study.
- How was the expression level in qRT-PCR results calculated? Which sample was used as reference sample for calculating relative quantification if 2-ΔΔCT method was used? Please mention this in the manuscript.
Author Response
Reviewer 1:
In this study, authors studied the inheritance and genetic mapping of Late-bolting to Early- 1 bolting gene, BrEb-1, in Chinese Cabbage (Brassica rapa L.). In my opinion, the study is planned nicely, and the findings are interesting. The manuscript is also written well. However, I did not find the supplementary data associated with this manuscript. If authors provide the supplementary data, then I would be happy to review it again. My other comments are below:
- There are many typographical, and errors throughout the manuscript. Authors need to check them carefully.
Author’s response: We are grateful to reviewer 1 and all the typos and errors have been corrected.
- The quality of figures is bad. Please increase the font size of the text used in the figures.
Author’s response: Figure quality has been improved.
- Authors used both names Chinese cabbage and B. rapa throughout the manuscript. I would suggest authors to maintain uniformity in using either Chinese cabbage or B. rapa. Authors may use both names in the beginning.
Author’s response: The uniformity for naming has been updated where possible. This is because there are various subspecies of B. rapa. Therefore we presented in such a way.
- Please create a separate section for conclusion and include future prospects of the study.
Author’s response: Thank you for your kind suggestion. We have presented the conclusion in a separate paragraph.
- How was the expression level in qRT-PCR results calculated? Which sample was used as reference sample for calculating relative quantification if 2-ΔΔCTmethod was used? Please mention this in the manuscript.
Author’s response: At first we normalized by housekeeping gene ‘BrGAPDH’ and then SY2004 was used for reference sample for 2-ΔΔCT method.
Reviewer 2 Report
Please see my comments in the attached file
Comments for author File: 
Comments.pdf
Author Response
Reviewer 2:
This is an interesting piece of work and whilst the science is of high standard the manuscript is difficult to read at times and would really benefit from being thoroughly edited by a more proficient writer of English. In particular, there are very many instances where the space between words is omitted - even in the title ("...GeneticMapping...", line 1 page 1), but littered throughout the whole manuscript.
Author’s response: There is an error caused during email communication with manuscript among authors. However, we have corrected the error throughout the manuscript.
Abstract
P1 Line 20 omit "to the seed market" as this is confusing.
Author’s response: ‘to the seed market’ has been omitted.
P1 Line 21 omit "a four-generation family" as this is confusing. You only test parents, F1 and F2 - these are three generations.
Author’s response: Abstract has been updated by omitting "a four-generation family".
P1 line 32 more correct to say "...BraA07g030360 which code..."
Author’s response: The sentence has been corrected by adding ‘which’.
Introduction
p1 line 48-49 replace "morphotypes types" with "morphotypes" Section on Arabidopsis flowering time pathway
Author’s response: "morphotypes types" has been replaced with "morphotypes"
p2 lines 53-64 is difficult to read and needs rewriting. I suggest: "The regulation of flowering time is well studied in Arabidopsis, a close relative of Brassica species, with over 180 genes identified through functional analysis [12,15]. At least six major pathways are involved, such as photoperiod, vernalization, ambient temperature, age, autonomous, and gibberellin [9,12,15]. It has been reported that flowering time in Arabidopsis is regulated by the transcriptional activation of FLOWERING LOCUS T (FT). This is repressed by FLOWERING LOCUS C (FLC), through interaction with genes associated with vernalization pathway, but activated by the zinc finger transcription factor CONSTANS (CO), through interactions with the genes related to the circadian clock and the photoperiod pathway [16,17]. Schiesslet al. identified 35 flowering related genes in Brassica species, five of which were orthologous to known Arabidopsis genes in the flowering regulating pathway [16]."
Author’s response: Thank you very much for your kind correction. We have updated this part as per reviewer’s suggestion.
Six genes were identified as candidates for late bolting in Chinese cabbage (p2 lines 73-78). The annotations for 1-4 and 6 are given, but not for the fifth (BraA08g012630). Please just state "unknown" for this.
Author’s response: We used ‘unknown’ for the fifth gene.
P2 lines 79-93 also needs simplifying. Perhaps start with an introductory sentence for example "Previous studies have detected WTLs for flowering time across most chromosomes in both B rapa and the closely related vegetable B oleracea. For example, in B rapa......"
P2 lines 94-98 contextualises why flowering time is important in these vegetables and should be moved to earlier in the introduction, perhaps before P2 line 51. The sentence at the start of P2 line 99 is not needed.
P2 line 103 make it clear that BSA is on the F2 generation.
Author’s response: We conducted BSA in F2 which has been updated in the manuscript.
Materials and methods
P3 lines 108-115. Not clear if P1 is also vernalization-insensitive; also please make it clear which parent was used as female/male (presumably P1=female?). For completeness also state dates during which plants were grown in field.
Author’s response: P1 (CX14-1) was male and SY2004 was female.
P3 lines 117-121. I understand what you mean, but you state "height of .....plants was measured to analyse the inheritance pattern of early-bolting trait... (Table 1)" but you don't give any height data in Table 1, just your classification into early/mid/late bolting. Also, as in the abstract, you refer to "four generation family" (line 122) but only have 3 generations (parents, F1, F2).
Author’s response: We did a typing mistake, it will be three generation family (Parent, F1 and F2). The height data has been placed as supplementary data.
Line 124 incorrect spelling of segregant
Author’s response: The spelling mistake has been corrected.
Line 130 - presumably the 30 early bolting and 30 late-bolting lines were selected at random from each class (90 early and 126 late)?
Author’s response: Yes, we polled genomic DNA of 30 early bolting and 30 late-bolting lines for BSA.
Page 4 line 157. Please state at what approximate developmental stage the plants were at when leaves were sampled for DNA extraction. If the early-bolting P1 was sampled on the same date as the late bolting P2 then the apparent differential expression might simply be due to differences in their developmental stage. I would also have liked the F1 to have been included in this experiment.
Author’s response: We sampled the young leaves when we calculated the stem height, that means it was at the same time when P1, P2 and F2 were sampled.
Results
P5 line 193 express as a % so “113,067 (7.15%) were non-synonymous..”
Author’s response: We have updated the manuscript as % as suggested by reviewer.
Line 194 same thing, “8,722 (5.2%) were non-synonymous”
Author’s response: We have updated the manuscript as % as suggested by reviewer.
Figure 2 please consider increasing the font size of the chromosome names as I had to zoom in to read these and they are too small in a printed version
Author’s response: The font size for Figure 2 has been updated.
Lines 205-218 would be simplified by another figure which shows the physical maps of the candidate regions and how the intersections from different analyses (parents: SNPs, ED and InDels; bulks: SNPs, ED and InDels
Author’s response: Thank you very much for your kind comments. Figure 3-6 already cover these information.
Lines 223, 230, 235 please state the value for the significance threshold in each case
Author’s response: Thank you very much for your kind comments. We have added the threshold according to reviewer’s comments.
P6 line 241 – please correct to “The threshold line (red) was 0.99;”
Author’s response: “The threshold line (red) was 0.99;” has been corrected.
Figure 7 (p9) should be included in the results instead of the discussion. These three graphs look suspiciously similar to each other, unfortunately I cannot see the accompanying supplementary data.
Author’s response: Figure 7 has been placed in the result section.
Discussion
P8 lines 258 -263 can be simplified, for example: “Incomplete dominance inheritance patterns have been seen for traits in diverse species including B. napus [33,34], rice [35] and tomato [36].
Author’s response: We have simplified the sentences in lines 258-263 as suggested by reviewer.
Lines 264-268 do not make sense. “Bulked segregant analysis (BSA) was first used to map disease resistance genes in lettuce [38]. For BSA, bulked DNA samples are prepared from individuals within a segregating population which contrast for a particular trait, for example bulks of resistant and susceptible individuals. Comparing the genotype of these bulks across the genome can identify markers linked to the trait under investigation [37-39].
Author’s response: Lines 264-268 have been removed and updated considering reviewers suggestion.
Lines 273-276. You have added together the SNPs detected between parents (2321) to the SNPs detected between bulks (1526) to make a total of 3847. You have also done the same for InDels (229+131=360). I believe that this is an incorrect simplification, surely the bulks (1526 SNPs plus 131 InDels) are a subset of those detected between the parents, and not in addition to them?
Author’s response: Lines 273-276, the sentences have been updated via deleting the incorrect simplification.
Lines 289 to 301 are a clumsy and long-winded introduction to your candidate genes. You also need to introduce some abbreviations sooner than you do. How about: “AGAMOUS-Like MADS-box protein AGL12 is involved in late-flowering phenotypes and root development in Arabidopsis [42]. The Arabidopsis gene LONG VEGETATIVE PHASE 1 (LOV), which codes for a NAC transcription factor, regulates flowering time and cold response [43], and other Arabidopsis NAC genes have also been reported to regulate flowering time [44]. Pentatricopeptide repeat-containing proteins (PPR) play important developmental roles in plants [45-48]. During our searches of gene annotations in the BrEb-1 candidate region we identified three promising candidate genes. BraA07g029500, BraA07g029530 and BraA07g030360 code for AGL-12, PPR and NAC transcription factor 29, respectively.”
Author’s response: Thank you very much for your kind comments. We fully agree with your suggestion and accordingly updated in the manuscript.
Lines 306-309 should be in the Results section not the discussion. Perhaps for the discussion just include the line “The transcript levels of the three candidate genes were all up-regulated in late-bolting compared to early-bolting genotypes (Figure 7.)
Author’s response: We agree with the reviewer’s suggestion and accordingly updated in the manuscript.
Round 2
Reviewer 1 Report
Authors have addressed my comments satisfactorily. The manuscript may be accepted for publication in its current form.
