Transcriptomic Analysis of Sex-Associated DEGs in Female and Male Flowers of Kiwifruit (Actinidia deliciosa [A. Chev] C. F. Liang & A. R. Ferguson)

Round 1

Reviewer 1 Report

Dear Authors,

This manuscript (horticulturae-1479148) entitled “Transcriptomic analysis of sex-associated DEGs in female and male flowers of kiwifruit (Actinidia deliciosa [A. Chev] C. F. Liang & A. R. Ferguson)” have proposed the identification of sex related genes in inflorescence of two different cultivars of A. deliciosa. This study is of great significance in kiwifruit research due to hermaphrodite nature of its flowers. I appreciate the original idea of the work; however, would like to put forward some suggestions/comments before editor’s final decision.

Comment 1: Kindly provide the background for selecting male plant material in this study.

Comment 2: Authors have collected samples on one developmental stage of flowers. In my opinion, they would have obtained more interesting results if there were more time points.

Comment 3: It would be more compelling if authors provide pictorial view of flowers at the time of sampling.

Comment 4: I would like to know about how did authors selected key regulatory pathway from KEGG analysis and how did they get set of genes shown in PPI network analysis?

Comment 5: How many biological replicates were used for RNA extraction?

Comment 6: In the discussion section, MYB is discussed under hormone-related DEGs. MYBs are transcription factors and in my opinion, they should be discussed with other transcription factors.

Comment 7: I will suggest to add heatmap for the genes used in PPI analysis. It will help to see the changes in their expression in different plant samples.

Comment 8: I cannot find expression profile for genes highlighted in abstract section (Achn131111 and Achn112341 or Achn206301). This is in contrast with PPI analysis as well. Kindly explain.

Comment 9: I failed to figure out why author is adding A. thaliana homologues gene IDs in front of some of transcript IDs. Kindly explain it.

Comment 10: Kindly use the full name for abbreviation when it is first used in MS.

Comment 11: Kindly revise the conclusion section. It looks more like an abstract.

Comment 11: I found it quite troublesome to see the gene IDs without their functional annotations and vice versa. Kindly keep it consistent throughout the MS.

Comment 12: Kindly revise your MS for grammatical mistakes and use Scientific language for standard expressions (i.e. KEGG pathways instead of KEGG route and Higher expression levels instead of overexpression), and rephrase some sentences if needed.

Author Response

Reviewer 1

Dear Authors,

This manuscript (horticulturae-1479148) entitled “Transcriptomic analysis of sex-associated DEGs in female and male flowers of kiwifruit (Actinidia deliciosa [A. Chev] C. F. Liang & A. R. Ferguson)” have proposed the identification of sex related genes in inflorescence of two different cultivars of A. deliciosa. This study is of great significance in kiwifruit research due to hermaphrodite nature of its flowers. I appreciate the original idea of the work; however, would like to put forward some suggestions/comments before editor’s final decision.

Thank you very much for all your suggestions and comments. I really appreciate them.

Comment 1: Kindly provide the background for selecting male plant material in this study.

Thank you for your comment. We have provided background about male plant material in (lines: 117-120). We have chosen a male hybrid from ‘Green Light’ x ‘Tomuri’ because inside of the kiwi breeding program of the University of Chile this genotype was a potential pollinator of the Hayward variety. This seedling was generated in 2010 in Curicó (VII region of Chile).

Comment 2: Authors have collected samples on one developmental stage of flowers. In my opinion, they would have obtained more interesting results if there were more time points.

I agree with your comment and of course, more phenological states would have been interesting to use. For further analysis, we will take into account more states and even use RNAseq from other publications.

Comment 3: It would be more compelling if authors provide pictorial view of flowers at the time of sampling.

A new figure has been added (Fig. 1) indicating the orchard where the crossing was made and the phenological states of female and male flowers.

Comment 4: I would like to know about how did authors selected key regulatory pathway from KEGG analysis and how did they get set of genes shown in PPI network analysis?

First, as we indicate (lines: 161-164), genes with a fold-change of ≥ 2 and a false discovery rate (FDR) < 0.05 were considered significant DEGs, so we used these genes as the main genes involved in the sex differentiation. On the other hand, as we explain (lines: 180-188), the PPI network was built taking into account the metabolic pathways of putative sex-associated genes in male and female flowers of other Actinidia species.

Comment 5: How many biological replicates were used for RNA extraction?

A sample pool of flowers from three random trees was used for male and female flowers considering one and three biological replicates from each pool respectively. As for qPCR validation, we considered three replicates for female and male flowers considering two technical replicates (lines: 128-131).

 

Comment 6: In the discussion section, MYB is discussed under hormone-related DEGs. MYBs are transcription factors and in my opinion, they should be discussed with other transcription factors.

I agree with your comment, MYBs are transcription factors but we decided to include its discussion in the section of Hormone-related genes because as we explain (lines: 467-481), the MYB family of genes participates in several signaling pathways such as the synthesis, signaling, and degradation of gibberellins which they are plant hormones involved the flowering.

Comment 7: I will suggest to add heatmap for the genes used in PPI analysis. It will help to see the changes in their expression in different plant samples.

Thank you for your suggestion. Heatmap figures (figures S1 and S2) have been added as supplementary material in order to visualize the gene expression.

Comment 8: I cannot find expression profile for genes highlighted in abstract section (Achn131111 and Achn112341 or Achn206301). This is in contrast with PPI analysis as well. Kindly explain.

I agree with your comment, and I understand that it would be necessary for an explanation. The expression profile of all genes including those mentioned above can be found in the supplemental material (Table S1). The fact that they do not appear in the PPI is because when the protein network was created there was a great lack of proteins connecting these genes with the rest of proteins because we depend of the protein database available, which needs to be completed. For this reason, is important to highlight some important genes as these, which are described by other authors which are involved with the kiwi sex differentiation in spite of they are not being included in the PPI. Anyway, we have decided to eliminate the comment in the abstract section, because really these genes showed a very low expression.

Comment 9: I failed to figure out why author is adding A. thaliana homologues gene IDs in front of some of transcript IDs. Kindly explain it.

Thank for your comment. As we known A. thaliana is often use as a plant model, so there to many genes and proteins described in this specie, especially as regards flowering. For this reason, some gene IDs are referenced to Arabidopsis, but in the current version of the manuscript we have tried to use only those referring to the Actinidia.

Comment 10: Kindly use the full name for abbreviation when it is first used in MS.

Thank you for your assessment. The manuscript has been revised again and the names of all abbreviation were indicated.

Comment 11: Kindly revise the conclusion section. It looks more like an abstract.

Thank you for your suggestion, the conclusions have been reorganized. Even we include a new finding regarding DEGs encoding methyltransferases.

Comment 11: I found it quite troublesome to see the gene IDs without their functional annotations and vice versa. Kindly keep it consistent throughout the MS

I agree, the gene IDs were revised in order to keep consistency and homogeneity of the names. The ID from the Arabidopsis were replaced to kiwi IDs in order to avoid misunderstanding.

Comment 12: Kindly revise your MS for grammatical mistakes and use Scientific language for standard expressions (i.e. KEGG pathways instead of KEGG route and Higher expression levels instead of overexpression), and rephrase some sentences if needed.

Thank you very much for your valuable comments. The English grammar was revised again and the mistakes were corrected.

As a final statement, we would like to point out that all modifications and new discussions were indicated in green color in the final version of the manuscript.

Reviewer 2 Report

 Brief Summary:

 

The manuscript “Transcriptome analysis of sex-associated DEGs in female and male flowers of kiwifruit (Actinidia deliciosa [A. Chev] C. F. 3 Liang & A. R. Ferguson) is a transcriptomics study of gene regulatory networks underlying sexual differentiation of flower organs in kiwifruit. They used pools of female and male flowers, prepared RNA-Seq libraries of them followed by deep sequencing of the libraries and NGS data analysis. The authors mainly made a gene regulatory network controlling the development of flower organs in female and male flowers. They found a set of key TFs as regulators of sex related flower development as well as several hormone signalling components previously shown to have function in controlling the flowering process in plants.   

 

Broad comments:

 

My main issues are with experimental design and presentation of the results. In general, the issue with experimental design is lack of proper number of biological replicates (both for sequencing and qPCR) and therefore poses skepticism on downstream analysis and conclusions drawn. The second issue is the way the results are presented mainly related to clarifications of DEGs that which ones are up- or down-regulated. The authors refer to DEGs and discuss them but do not clearly show in the figures how and in which genotype the DEGs are differentially expressed. I think it could be helpful for the reader to be able to see them in the figures as well. The discussion section also needs improvements; in some parts the discussion is poor and in some other parts there are too many speculations particularly in the hormone signaling section of discussion (examples are listed in the section below).

 

Specific comments:

 

Line 56: “In depth” instead of “in deep”

Line 113: As I understood there are no real biological replicates, also not clear that from how many trees per genotype the pools where prepared? This can of course affect all the downstream analyses. 

Line 195: “of which” instead of “which”

Table 1: The table is not self-explanatory and there is no text supporting some of the information in the table. For example, “Reads mapped Le. 197” or “Reads mapped Ri. 198”. Would be better to explain what they stand for wither in the legend or at least in the text.

Figure 1: I think this figure is also not self-explanatory and the way results are presented is somehow misleading. The legend should be more informative about colors (pink, purple and green) also the color coding for borders (p-value) is not provided in the figure. As I understood the pink, purple and green sections of the pie charts represent the genotypes and the common DEGs, but it is not clear which ones are up- and which ones are down-regulated.

I also don’t get what are the common DEGs (the green slice of the charts) in the Venn diagram, in other words since there is only one comparison (H vs GxT) then what are the common DEGs? What do they mean? Common DEGs are just the case when one has more than one comparison but in this case, there is only one comparison.

Figures 2: It is not clear which genes are up- or down-regulated in which genotype.

Line 302: “overexpressed” should be changed to upregulated.

Lines 316 -317: “promotes” and “encodes” better to change to “promote” and “encode”.

Line 327: Better be “WRKY family of proteins are important…”

Line 335: Weak discussion of the differences in the expression of AGL family between the male and female flowers.

Line 380: The sentence starting from “The determination of sex …….” needs rephrasing.

Line 388 to 403: I find the explanation about SyGl absence in the transcriptome data too much of speculation. I suggest the authors to rephrase this section.

Author Response

Reviewer 2

Brief Summary:

The manuscript “Transcriptome analysis of sex-associated DEGs in female and male flowers of kiwifruit (Actinidia deliciosa [A. Chev] C. F. 3 Liang & A. R. Ferguson) is a transcriptomics study of gene regulatory networks underlying sexual differentiation of flower organs in kiwifruit. They used pools of female and male flowers, prepared RNA-Seq libraries of them followed by deep sequencing of the libraries and NGS data analysis. The authors mainly made a gene regulatory network controlling the development of flower organs in female and male flowers. They found a set of key TFs as regulators of sex related flower development as well as several hormone signalling components previously shown to have function in controlling the flowering process in plants.  

Broad comments:

My main issues are with experimental design and presentation of the results. In general, the issue with experimental design is lack of proper number of biological replicates (both for sequencing and qPCR) and therefore poses skepticism on downstream analysis and conclusions drawn. The second issue is the way the results are presented mainly related to clarifications of DEGs that which ones are up- or down-regulated. The authors refer to DEGs and discuss them but do not clearly show in the figures how and in which genotype the DEGs are differentially expressed. I think it could be helpful for the reader to be able to see them in the figures as well. The discussion section also needs improvements; in some parts the discussion is poor and in some other parts there are too many speculations particularly in the hormone signaling section of discussion (examples are listed in the section below).

Thank you very much for your valuable comments and suggestions. Some figures show an overall view; however, we can check the expression of each gene in the supplementary file S1. Anyway, we have added a heatmap (Figure S1 and S2) as supplementary material in order to visualize better the gene expression of the main genes.

Specific comments:

Line 56: “In depth” instead of “in deep”

Ok, this expression was modified.

Line 113: As I understood there are no real biological replicates, also not clear that from how many trees per genotype the pools where prepared? This can of course affect all the downstream analyses.

Thank you for your assessment. The number of trees used to generate a pool of samples was three for each genotype (male and female), using for RNA extraction one replicate from the pool of samples for male (GxT) flowers and three biological replicates for female (H) flowers (lines: 128-131). The raw sequences of the biological replicates are available in the NCBI. It has been clarified in lines 146-147.

Line 195: “of which” instead of “which”

Ok, this expression was replaced.

Table 1: The table is not self-explanatory and there is no text supporting some of the information in the table. For example, “Reads mapped Le. 197” or “Reads mapped Ri. 198”. Would be better to explain what they stand for wither in the legend or at least in the text.

Thank you very much for your suggestion. “Reads mapped Le and Reads mapped Ri” were replaced by “Reads mapped Left and Reads mapped Right”. The reads were reflected in both directions.

Figure 1: I think this figure is also not self-explanatory and the way results are presented is somehow misleading. The legend should be more informative about colors (pink, purple and green) also the color coding for borders (p-value) is not provided in the figure. As I understood the pink, purple and green sections of the pie charts represent the genotypes and the common DEGs, but it is not clear which ones are up- and which ones are down-regulated.

I also don’t get what are the common DEGs (the green slice of the charts) in the Venn diagram, in other words since there is only one comparison (H vs GxT) then what are the common DEGs? What do they mean? Common DEGs are just the case when one has more than one comparison but in this case, there is only one comparison.

I agree with your comment. This figure has changed to Fig. 2 and the p-value has been added to the picture. In this figure we can not show which genes are up-regulated, here we only show a general view. In the Venn diagram, we can see common genes differentially expressed between female and male flowers (green color), while in pink color are referring to the genes expressed only in female (H), and in blue those genes expressed only in male (GxT). New supplementary material was added in order to clarify which genes are up-regulated or not (Heatmaps: Fig. S1 and S2). Anyway, the expression of all genes can be consulted in table S1.

Figures 2: It is not clear which genes are up- or down-regulated in which genotype.

Figure 2 has been renamed as Fig. 3. This figure provides an overall view of what pathways were more significant after KEGG analysis. In order to show up and down-regulated genes a new figure was added (Supplementary figures S1 and S2).

Line 302: “overexpressed” should be changed to upregulated.

Thank you for your comment. This expression was modified as you required.

Lines 316 -317: “promotes” and “encodes” better to change to “promote” and “encode”.

Ok, the manuscript was revised and all grammar mistakes were corrected.

Line 327: Better be “WRKY family of proteins are important…”

Thank you for your suggestion. The expression has been changed (line 370).

Line 335: Weak discussion of the differences in the expression of AGL family between the male and female flowers.

In order to clarify this section of the discussion, we refer to ABCDE model for flower development, and we have indicated what part is defined by each gene class (B, C and E) (lines: 378-384).

Line 380: The sentence starting from “The determination of sex …….” needs rephrasing.

This sentence was replaced by a new one: “The determination of sex in plants is a complex process, where the interaction of TFs, play a determinant role in the processes that regulate reproduction and growth” (lines: 478-480)

Line 388 to 403: I find the explanation about SyGl absence in the transcriptome data too much of speculation. I suggest the authors to rephrase this section.

I agree, in fact, if we consider that the genes identified in this work linked to the cytokinin pathway were very low expressed (could be due to different floral tissue) really does not make sense to highlight, but we mention them because of their importance in other works. On the other hand, we note other genes linked to methyltransferase and we have added new information in the discussion section. Some sentences of this paragraph were rephrased. 

As a final statement, we would like to point out that all modifications and new discussions were indicated in green color in the final version of the manuscript.

Reviewer 3 Report

Dear authors,

I have read the manuscript under title "Transcriptomic analysis of sex-associated DEGs in female and male flowers of kiwifruit (Actinidia deliciosa [A. Chev] C. F. Liang & A. R. Ferguson), written by Zapata et al.

I found the manuscript quite interesting and well written. Important genes such as Achn131111 and Achn112341 (AT1G50575) or Achn206301 (AT4G16340) were found to be over expressed in male flowers and proving that SyGI gene is sex-regulating genes involved in the cytokinins pathway.

Personally I am not so “fascinated” with just sequencing, transcription factors, genes and I believe that plant organism and metabolism (especially in hormonal signals) is more complicated and depends on many factors. In my opinion would be much better if the authors could connect this study with chemical analysis of hormones in female and male flowers during development, and then propose new models of flower development and sex determination in the Actinidia genus. So, next time, I encourage them to do so.

I wonder, what would happen if the authors analyzed sterile and/or hermaphrodite individuals? What would happen with plants of different ploidy? Please, write something about it.

No matter that I am not from English spoken country, I think that a native speaker should read the manuscript and correct some small mistakes.

 

Author Response

Reviewer 3

Dear authors,

I have read the manuscript under title "Transcriptomic analysis of sex-associated DEGs in female and male flowers of kiwifruit (Actinidia deliciosa [A. Chev] C. F. Liang & A. R. Ferguson), written by Zapata et al.

I found the manuscript quite interesting and well written. Important genes such as Achn131111 and Achn112341 (AT1G50575) or Achn206301 (AT4G16340) were found to be over expressed in male flowers and proving that SyGI gene is sex-regulating genes involved in the cytokinins pathway.

Personally I am not so “fascinated” with just sequencing, transcription factors, genes and I believe that plant organism and metabolism (especially in hormonal signals) is more complicated and depends on many factors. In my opinion would be much better if the authors could connect this study with chemical analysis of hormones in female and male flowers during development, and then propose new models of flower development and sex determination in the Actinidia genus. So, next time, I encourage them to do so.

I wonder, what would happen if the authors analyzed sterile and/or hermaphrodite individuals? What would happen with plants of different ploidy? Please, write something about it.

No matter that I am not from English spoken country, I think that a native speaker should read the manuscript and correct some small mistakes.

Thank you very much for your valuable comments and suggestions, and of course connecting chemical analysis of hormones with female and male flowers during fruit development would be an interesting approach. As for your comment about what would happen when we have plants of different ploidies, we have added a comment in the introduction section (lines: 75-86) where we explain the difficulty of retaining the polymorphism linked to the kiwi sex determination due in part to kiwi genome complexity.  

As a final statement, we would like to point out that all modifications and new discussions were indicated in green color in the final version of the manuscript.

Reviewer 4 Report

The manuscript “Transcriptomic analysis of sex-associated DEGs in female and 2 male flowers of kiwifruit (Actinidia deliciosa [A. Chev] C. F. 3 Liang & A. R. Ferguson)” reported sex associated differential expressed genes in female and male flowers of kiwifruits and proposed sex associated transcriptional regulation networks using RNA-seq analysis. Overall, the manuscript is well written and technically sound. However, major concerns come from quality of the transcriptome and analysis.

 

1. Authors didn’t mention about quality and statistics of RNA-seq and assembly such as error %, GC content, Q30/Q20 and unigenes statistics etc., Further, authors should compare their RNA-Seq quality with the previous published transcriptome studies in Kiwi fruits (Pang et al, 2017 and Lie et al., 2015a).

 

2. Authors must mention number of biological replicates for each sample used for sequencing.

 

3. Authors should provide a comparative table depicting number of transcripts, especially TFs sequentially involved in different steps of the pathways regulating floral initiation and sex differentiation, using previous published kiwi transcriptomes, Arabidopsis and different plants and then discuss the common and unique patterns in term of sex differentiation.

 

4. Sex associated genes are polymorphic and segregate between genders in many dioecious plants. Identification of SNPs and corresponding loci will help in better understanding of sex determination in kiwi fruits.

 

5. Authors should provide heatmaps related to differentially expressed transcription factors between male vs female flowers. Further, include a table of over expressed transcription factors associated with sex differentiation in male and female flowers.

 

6. DNA methylation play a major role in flowering initiation. In the current study, gene such as methyltransferase1 and DNA methyl transferases are differentially expressed between male and female flowers (Suppl file 1). Authors should analyze the data and comment about effect of methylation on sex dertmination.

 

7. Quality of figures should be improved for publication. 

Author Response

Reviewer 4

The manuscript “Transcriptomic analysis of sex-associated DEGs in female and 2 male flowers of kiwifruit (Actinidia deliciosa [A. Chev] C. F. 3 Liang & A. R. Ferguson)” reported sex associated differential expressed genes in female and male flowers of kiwifruits and proposed sex associated transcriptional regulation networks using RNA-seq analysis. Overall, the manuscript is well written and technically sound. However, major concerns come from quality of the transcriptome and analysis.

1. Authors didn’t mention about quality and statistics of RNA-seq and assembly such as error %, GC content, Q30/Q20 and unigenes statistics etc., Further, authors should compare their RNA-Seq quality with the previous published transcriptome studies in Kiwi fruits (Pang et al, 2017 and Lie et al., 2015a).

Statistics of RNA-seq and assembly have been added and were compared with other studies as you mentioned (Pang et al, 2017 and Lie et al., 2015a).

Thank you for your valuable comment. I agree, therefore we have added new information in Table 1, including sequencing quality scores such as Q20 and Q30 as well as the average GC content (Table 1). In addition, the sequencing results were compared with a similar transcriptomic comparison (Tang et al., 2017) (lines: 212-217).

 

2. Authors must mention number of biological replicates for each sample used for sequencing.

Thank you for your comment, I understand that this statement must be clarified. In the RNAseq, we used for female and male a pool of flowers from three random trees. From each pool, we used three and one biological replicates for Hayward (female) and GxT (male) respectively. In the qPCR validation, we did three biological replicates and two technical for both genotypes. The explanation was added in the Materials and Methods section (lines: 128-131).

3. Authors should provide a comparative table depicting number of transcripts, especially TFs sequentially involved in different steps of the pathways regulating floral initiation and sex differentiation, using previous published kiwi transcriptomes, Arabidopsis and different plants and then discuss the common and unique patterns in term of sex differentiation.

Thank you for your comment, I really appreciate it. Of course, I would like to be able to synthesize all of the transcripts involved on different steps linked to sex differentiation, in fact in the discussion section we try to do it. However, the complexity of this matter makes it difficult to offer more clarity in the discussion, where we need to take information from other species such as Arabidopsis. Regarding this concern, new information was added in the discussion section. All new information and changes have been added in green color.

 

4. Sex associated genes are polymorphic and segregate between genders in many dioecious plants. Identification of SNPs and corresponding loci will help in better understanding of sex determination in kiwi fruits.

I agree with the comment, the SNP identification could help us to identify loci linked to sex determination but not only SNPs, we could add many modifications of sequences such as INDELs or deletions. In this work, we have focused mainly on gene expression, pathways, and protein-protein interaction networks, but of course, searching SNPs would be an interesting approach in order to generate molecular markers useful to apply in different progenies. We really appreciate this comment and will try to apply it for further analysis.

5. Authors should provide heatmaps related to differentially expressed transcription factors between male vs female flowers. Further, include a table of over expressed transcription factors associated with sex differentiation in male and female flowers.

Thank you very much for your contribution. A heatmap considering the most important genes was added comparing male and female flowers. The table of overexpressed genes was previously included in the supplementary material (Table S1).

6. DNA methylation play a major role in flowering initiation. In the current study, gene such as methyltransferase1 and DNA methyl transferases are differentially expressed between male and female flowers (Suppl file 1). Authors should analyze the data and comment about effect of methylation on sex determination.

Thank you very much for your appreciation, because this result is evidencing that genes such as Achn260751 linked to DNA methyltransferases are overexpressed in male flowers which could be due to epigenetic mechanisms linked to sex differentiation. Therefore a new discussion was added.

As a final statement, we would like to point out that all modifications and new discussions were indicated in green color in the final version of the manuscript.

 

Round 2

Reviewer 4 Report

Authors have addressed most of my concerns and revised the article accordingly.

Minor comment

Authors used one biological replicate for male flower and three biological replicates for female flowers. Authors should take same number of replicates for comparative studies. Authors should justify how they normalized the replicates data between male and female flowers.

 

Author Response

Minor comment

Authors used one biological replicate for male flower and three biological replicates for female flowers. Authors should take same number of replicates for comparative studies. Authors should justify how they normalized the replicates data between male and female flowers.

Dear Reviewer

Thank you very much for your appreciation. I agree it had been convenient to use three biological replicates of female and male flowers. However, we had a lack of resources and we finally decided to use only one biological replicate for male flowers, but at least we considered this biological replicate from a pool of flowers in order to obtain a "biological average" of the phenological state. On the other side,  being flowers (female and male) completely different from the sexual point of view, the biological differences are assured and are consistent. 

As for the sample normalization, DEseq2 package v2.11.40.2 was used in order to normalize to FPKM (Fragments Per Kilobase of transcript per Millon mapped reads) (Lines 157-164). There are RNAseq experiments without replicates that have been analyzed with a non-parametric method based on bootstrapping, also Fisher's exact test, GFOLD, edgeR, and Cuffdiff in which show high accuracy and can be compared to other methodologies. 

In our case, we used Cuffdiff and his tool Cuffnorm to normalize the data. Cuffldiff consists of a setting tool for the gene expression analysis of the RNAseq.