Genome-Wide Association Study (GWAS) Identifies Key Candidate Genes Associated with Leaf Size in Alfalfa (Medicago sativa L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this article, the authors delve into the genetic basis of alfalfa leaf morphology, which is an important characteristic of this non-model plant species and valuable crop. As the process of alfalfa leaf development is not well-known, the authors employ GWAS for leaf morphology to identify candidate genes associated with this trait.

I have some remarks:

Line 51. In the sentence “In Arabidopsis, the REVOLUTA transcription factor involved in leaf development is regulated by miR165”. It has been described that both mir165 and mir166 target Class III HD-ZIP, including REVOLUTA (doi.org/10.1186/s12870-017-0983-9)

Line 59-60 The meaning of QTL has been mentioned before (Line 52). Let's not use this term again.

Line 109. Can you clarify how you calculated the genetic variance and residual variance?

Line 126. Please explain why you used the general linear model (GLM) approach instead of a mixed linear model.

Line 99-100. Indicate that the abbreviations: “LL” and “LW” are used in combination with the years 2018, 2019 and 2020 (LL18, LL19, LL20 and so on) in order to clarify the results shown in line 143

Line 152. In Figure A, it would be helpful to include an image of the leaf alfalfa indicating the middle leaflet of the third or fourth fully expanded trifoliate leaf, counted from the shoot tip.

Line 146 Pearson correlation analysis is not indicated in Materials and Methods

Line 172 There is a missing space in "follows:3".

Line 175. Include an explanation of how the phenotypic variance explained (PVE) was calculated in the material and methods section.

Line 181. Table 2. Explain in the material and methods section what the Superior Allele is, its importance and how it was obtained. You mention it again until line 195

Lines 183 and 184. Eliminate “on chromosome 4” and “on chromosome 5”. This is redundant because you mention it in the coordinate of each SNP (chr4__10421186 and chr5__24099977)

Line 184. There is no Figure 3c and 3d; only there is a description of the whole of Figure 3.

Line 186. Change this sentence: “This suggests that these markers have a significant effect on alfalfa leaf size” to “This suggests that these markers could have a significant effect on alfalfa leaf size”. An experimental validation is necessary to confirm the statement.  

Line 193 In the Materials and Methods section, please provide a detailed description of the methods used for the haplotype analyses.

Line 210. Please mention the names of the 13 Chinese cultivars or provide a reference citation for them.

Line 217. "What is the meaning of the abbreviation 'PI'?"

Line 263. The year-to-year variation of LL and LW could assist in predicting alfalfa leaf size under future climate conditions?

Line 275-276 Please mention the genomic region of the haplotypes (T/T, A/G and A/A)

 

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Author Response

Response to the comments of Reviewer 2

(1). It is surprising to note that only 6 candidate genes are influencing the leaf length and width

in Medicago sativa despite variations in leaf size and development.

Response: It is not necessary that only six genes influence leaf length and width, but these six candidate genes have been shown to have roles in leaf development in other species. Due to the different genetic mechanisms, the role of these genes in alfalfa needs to be further verified. The six candidate genes identified in this study can be used as references to explore the mechanism of alfalfa leaf size.

 

(2). Lines 260-261: Among the population and environment specific associations with leaf

development, which one predominate the other?

Response: The heritability of the leaf length and leaf width calculated in this study were 65.09% and 56.62%, respectively. A heritability of 1 indicates that the phenotypic variation is entirely caused by genetic factors, while a heritability of 0 indicates that the phenotypic variation is entirely caused by environmental influences. Therefore, in the context of our study, genetic factors have a greater influence on leaf length and leaf width.

 

(3). T/T haplotype appears as the frequent superior allele over others. Is there any specific reason

for this?

Response: There is no particular reason for this. Only in the context of our study do T/T haplotypes appear most frequently.

Reviewer 3 Report

Comments and Suggestions for Authors

The article reports the results of highly comprehensive and systematic results of GWAS analysis of leaf length and leaf width of 222 accessions of alfalfa phenotyped over three years using SNPs. 26 SNPs mapped on seven of the eight chromosomes associated with LL and LW have been identified. Six candidate genes some of which are associated with chloroplast and chlorophyll development have also been identified none of which has been previously identified to be associated with leaf size in alfalfa in other studies using other accessions, populations, and molecular markers for QTL analysis. The nature and source of the accessions used, plot size used and whether sown from the same seed source over years or maintained over three years in the same plots, and the number of plants selected in each accession for recording phenotypic data and DNA extraction and sequencing should be given as there could be a lot of heterozygosity and heterogeneity being the cross-pollinated crop. How could one of the chloroplast candidate genes be identified on chromosome 4 using SNPs based on the reference sequence data of the nuclear genome?

Author Response

Response to the comments of Reviewer 3

The article reports the results of highly comprehensive and systematic results of GWAS analysis of leaf length and leaf width of 222 accessions of alfalfa phenotyped over three years using SNPs. 26 SNPs mapped on seven of the eight chromosomes associated with LL and LW have been identified. Six candidate genes some of which are associated with chloroplast and chlorophyll development have also been identified none of which has been previously identified to be associated with leaf size in alfalfa in other studies using other accessions, populations, and molecular markers for QTL analysis. The nature and source of the accessions used, plot size used and whether sown from the same seed source over years or maintained over three years in the same plots, and the number of plants selected in each accession for recording phenotypic data and DNA extraction and sequencing should be given as there could be a lot of heterozygosity and heterogeneity being the cross-pollinated crop. How could one of the chloroplast candidate genes be identified on chromosome 4 using SNPs based on the reference sequence data of the nuclear genome?

Response: Thank you very much for your questions, which are very important. The phenotypes from different individuals of the same germplasm resources used by us were more consistent, and there were great differences among the germplasm resources. Specific information on the 220 materials is explained in the citation inserted. Because alfalfa is a perennial crop, it can survive in the field for many years. We chose to sequence a representative strain from each material and measured the phenotype of all individual strains from each material. We have made additions to the Materials and Methods section.

For SNPS found by GWAS, all genes were identified within 20 kb of each of the upper and lower reaches of significant SNPS using the Zhongmu-4 reference haploid genome. Then, the gene was searched using NCBI's BLASTP function. Then, the functional annotation of the genes found was made by referring to the literature.