Fine Mapping of qWCR4, a Rice Chalkiness QTL Affecting Yield and Quality
, Yun Zhu, Peng Yun, Guangming Lou, Lu Wang, Yipei Wang, Guanjun Gao *, Qinglu Zhang, Xianghua Li and Yuqing He *
Xuehui Li
Round 1
Reviewer 1 Report
In this manuscript, the authors finely mapped a rice chalkiness QTL, qWCR4, to a 35 kb region on chromosome 4. The authors also identified two candidate genes in the region through gene expression analysis. The Materials and Methods section need to be rephrased. The mapping population development, field experimental design, and data analysis are not clearly described. Some comments and edits in detail see below.
Page 2 line 75-82: More information about the NIL population is needed here. how the NIL population was developed? How many lines were evaluated in the field each generation each year? What was field experimental design? Suggest to describe the populations used for validation of the qWCR4 and the populations used for fine mapping separately.
Page 3 line 113-114: What is IGV software? A citation is needed.
Page 3 line 115-123: What population or lines were used for the gene expression analysis?
Page 3 line 132-135: How many lines from BC5F2 population? What traits? What statistical model used for phenotypic data analysis? Phenotypic data analysis, linkage analysis, and QTL fine mapping analysis is not clearly described here.
Page 3 line 142-152: How the homozygous and heterozygous NILs were determined?
Author Response
Response to reviewer #1
(1) Page 2 line 75-82: More information about the NIL population is needed here. How the NIL population was developed? How many lines were evaluated in the field each generation each year? What was field experimental design? Suggest to describe the populations used for validation of the qWCR4 and the populations used for fine mapping separately.
Response: We are very sorry for our negligence. We have added the basic information of populations in Line 78-96.
(2) Page 3 line 113-114: What is IGV software? A citation is needed.
Response: Thank you for your suggestion. The citation has been added in Line 133-134.
(3) Page 3 line 115-123: What population or lines were used for the gene expression analysis?
Response: We are very sorry for our negligence. Information of NILs were added in Line 138-140.
(4) Page 3 line 132-135: How many lines from BC5F2 population? What traits? What statistical model used for phenotypic data analysis? Phenotypic data analysis, linkage analysis, and QTL fine mapping analysis is not clearly described here.
Response: We are very sorry for our negligence. We have added the relevant information to Line 156-165. The phenotypic traits analyzed in this study were listed in Section 2.2.
(5) Page 3 line 142-152: How the homozygous and heterozygous NILs were determined?
Response: Sorry for this confusion. A BC5F2 population derived from a self-pollinated BC5F1 plant was defined as NIL-F2 population. We have added more information in determining NILs in Line 181-188.
Reviewer 2 Report
The authors performed fine-mapping of qWCR4 using BIL and validated using NILs.
The method is very reasonable and the writing is also good.
But, there is one major thing to verify.
In fig. 2A, how to author decided the candidate region?
The region (M23 ~ M24) showed the same pattern as the region M24~M25.
Also, it looks there are no significant differences between the WCR of ZY02, ZY09, ZY32, ZY21.
Please check fig2 again.
See the following article:
Figure 1 in Wu et al. 2022, Natural variation in WHITE-CORE RATE 1 regulates redo[ homeostasis in rice endosperm to affect grain quality. Plant Cell.
One more thing.
In fig 3. It is better to check the sequence variation of NILs instead of parents.
Author Response
Response to reviewer #2
(1) In fig. 2A, how to author decided the candidate region? The region (M23 ~ M24) showed the same pattern as the region M24~M25. Also, it looks there are no significant differences between the WCR of ZY02, ZY09, ZY32, ZY21. Please check fig2 again.
Response: Sorry for this confusion. We have carefully checked Fig 2A.
The candidate region was obtained through the progeny test method. T test was performed to compare the WCR differences between plants with two different homozygous genotypes in each recombinant progeny line. if the p-value was less than 0.05, the candidate gene was considered to be located in the heterozygous fragment, otherwise it was considered to be located in the homozygous fragment. This method has been added in the Materials and methods part (Line 159-165) and the legend of Figure 2 has been modified.
The genetic background of ZY04 and its progeny test result determinate that qWCR4 should locate in the M24~M25 region, but not in the M23~M24 region.
In Fig 2a, significant WCR difference was detected in progeny line of ZY02, the progeny result should be H, and as same method, the progeny test result of ZY09, ZY32 and ZY21 should be homozygous.
(2) In fig 3. It is better to check the sequence variation of NILs instead of parents.
Response: Thank you for your suggestion. 4 sequencing markers (Seq1, Seq2, Seq3 and Seq4) were developed to sequence parents and NILs in LOC_Os04g50060 and LOC_Os04g50070 regions. Relevant primers were listed in Supplementary Table 1, and a sequence consistency of parents and NILs could be seen in Supplementary Fig 3, Please check it.
Reviewer 3 Report
Figure 2: define “H, A and B” of the progeny test.
Lines 181-183: please be specific which were the three genes you focused here. Since there were six annotated genes. Two had no expression in endosperm, three were focused. What about the sixth one? And which was the sixth, and why the sixth one was not studied?
In Figure2b: mark the relative positions of the 6 genes within the span of 35.26kb.
Lines 276-279: be specific which one coding a GRAS… and which one coding a C2H2 zinc figure…… Also, the way it was written, it appears that it is the expression in endosperm led to the suggestion of one coding a GRAS… Shouldn’t it be the DNA sequences of the genes show the encoding of transcription factors? What were the % sequence homology between your target genes and GRAS family transcription factor and C2H2 zinc finger protein?
Line 293-296: here instead of suggesting these two genes were transcription factors, the authors suggested the variations in the promotor of these two genes might be the biding sites for transcription factors….. This statement contradicted what was written above.
A few grammatic mistakes.
Author Response
Response to reviewer #3
(1) Figure 2: define “H, A and B” of the progeny test.
Response: Thank you for your suggestion. The method of how to define H, A and B of the progeny test has been added in the materials and methods part (Line 156-165), and the legend of Figure 2 has been modified.
(2) Lines 181-183: please be specific which were the three genes you focused here. Since there were six annotated genes. Two had no expression in endosperm, three were focused. What about the sixth one? And which was the sixth, and why the sixth one was not studied?
Response: Sorry for this confusion. According to the annotation, the sixth gene in this region encoded a putative retrotransposon, which not considered a function gene in general, and this reply has been added to Line 224-225.
(3) In Figure2b: mark the relative positions of the 6 genes within the span of 35.26kb.
Response: Thank you for your suggestion. We have added these genes to Figure 2c.
(4) Lines 276-279: be specific which one coding a GRAS… and which one coding a C2H2 zinc figure…… Also, the way it was written, it appears that it is the expression in endosperm led to the suggestion of one coding a GRAS… Shouldn’t it be the DNA sequences of the genes show the encoding of transcription factors? What were the % sequence homology between your target genes and GRAS family transcription factor and C2H2 zinc finger protein?
Response: Thank you for your suggestion. The mistake in written has been corrected in Line 324-332. the % sequence homology between target genes and GRAS family transcription factor and C2H2 zinc finger protein were shown in added Supplementary figure 4 and Line 238-240, please check it.
(5) Line 293-296: here instead of suggesting these two genes were transcription factors, the authors suggested the variations in the promotor of these two genes might be the biding sites for transcription factors…. This statement contradicted what was written above.
Response: Sorry for this confusion. We have corrected our sentence in Line 347-348, please check it.
