Phenotypic Diversity Analysis of the Progeny Variation of a ‘Mosaic Leaf’ Loropetalum chinense var. rubrum Based on Flower Organ Characteristics

Round 1

Reviewer 1 Report

Diversity review for the manuscript titled “Phenotypic diversity analysis of the progeny variation of a 'mosaic leaf' Loropetalum chinense var. rubrum based on flower or- 3 gan characteristics”.

 

Introduction

Line 70: see marked text. Is that a correct information?

 

Materials and methods:

The applied statistical analysis should be presented in this section, such as: CV (line 157), genetic diversity index (line 156), PCA analysis (line 174), Correlation analysis (Line 195), flower color classification cluster analysis (Line 227). We strongly suggest the authors to create subheads in material and methods sections according to the subheadings created in results section.

Principal component analysis by accessions dispersions should be used only with the first two Principal component (PC) accumulate 70% of the variability. In this manuscript the authors described six PC accumulating 79.55% (Line 177) and the figure 1 presented the dispersion based in the first three component (line 194), in this case, there is a bigger error associate with the analysis. Please see,

Arunachalam, V. 1981. Genetic distance in plant breeding. Indian Journal Genetics & Plant Breeding. 41:226-236.

Do Rego, E. R., Do Rêgo, M. M., Cruz, C. D., Cecon, P. R., Amaral, D. S. S. L., & Finger, F. L. (2003). Genetic diversity analysis of peppers: a comparison of discarding variable methods. Crop Breeding and Applied Biotechnology, 3(1), 19-26.

 

Results

Materials and methods, results and discussion sections should be present the same subheads.

 

This Discussion section is very poor.

 

Best regards,

 

The reviewer.

 

Comments for author File: Comments.pdf

Author Response

Point 1:  Line 70: see marked text. Is that a correct information?

Response 1: Thank you for your guidance. The sentence is that we didn't express it clearly. We have modified the sentences of line70. For line 70, The wild and cultivated types of L. chinense and L. chinense var. rubrum also showed phenotypic diversity in the number genetic structure of flower [27-28], and the law of totality variation is present cultivated variety > early cultivated variety [29].

 

Point 2: The applied statistical analysis should be presented in this section, such as: CV (line 157), genetic diversity index (line 156), PCA analysis (line 174), Correlation analysis (Line 195), flower color classification cluster analysis (Line 227). We strongly suggest the authors to create subheads in material and methods sections according to the subheadings created in results section.

Response 2: Thank you for your suggestion. We have reintroduced applied statistical analysis in materials and methods. We rewrite the passage of 2.6. Statistical Analysis: Using Excel 2019 for data statistics. SPSS 22.0 was used to calculate the mean, maximum, minimum, standard deviation(SD), coefficient of variation(CV) and genetic diversity index (H') of the phenotypic traits in the flower organs of L. chinense var. rubrum. CV is a normalized measure of the discrete degree of probability distribution, the formula is: CV = (SD/Mean) × 100%. H' is based on Shannon-wiener Information Index, the formula is: H' =-∑ Pi∙InPi, Pi is the probability of the first character of a character. Origin 2021 was used for principal component analysis and correlation analysis of flower color phenotypic traits. The flower color classification used European distance method, and was analyzed by Origin2021. The distribution of anthocyanins in petals,calyx and nectaries were plotted by Adobe Photoshop CC2019. Given that the methodology and statistical analysis need to be explained in detail in order to better illustrate the methodology and statistical analysis, it is not consistent with the results and the subheadings discussed.

Point 3: Principal component analysis by accessions dispersions should be used only with the first two Principal component (PC) accumulate 70% of the variability. In this manuscript the authors described six PC accumulating 79.55% (Line 177) and the figure 1 presented the dispersion based in the first three component (line 194), in this case, there is a bigger error associate with the analysis. Please see,

Arunachalam, V. 1981. Genetic distance in plant breeding. Indian Journal Genetics & Plant Breeding. 41:226-236.

Do Rego, E. R., Do Rêgo, M. M., Cruz, C. D., Cecon, P. R., Amaral, D. S. S. L., & Finger, F. L. (2003). Genetic diversity analysis of peppers: a comparison of discarding variable methods. Crop Breeding and Applied Biotechnology, 3(1), 19-26.

 

Response 3: Thank you for your suggestion. We have carefully read the 2 articles you recommended and thank you very much for your guidance. Two papers focus on genetic distance and clustering methods of principal components, but we focus on the influence factors of principal components. It has been reported that in principal component analysis, the number of principal components can be determined according to the principle that the eigenvector value is greater than 1.0. Therefore, we extracted principal components with eigenvector values greater than 1.0 from 15 phenotypic traits and analyzed their main influencing factors. We referred to several literature reports:

LV Wei,HAN Junmei,WEN Fei, et al. Phenotypic Diversity Analysis of Sesame Germplasm Resources, Journal of Plant Genetic Resources,2020,21(1):234-242,251.

JING Lingxia, BU Chaoyang, LI Chunniu, et al. Genetic Diversity of Phenotypic Traits in 25  Jasminum  Germplasm Resources,Chinese Journal of Tropical Crops ,2020, 41(9): 1762-1769.

Wang Lifei, Xu Jiajie, Huang Xiaoxia, et al. Phenotypic Traits and Comprehensive Evaluation of 57 Modern Chinese Rose Germplasm Resources,Journal of Southwest Forestry University(Natural Science),2022, 42(1): 83-90. 

 

Point 4: Materials and methods, results and discussion sections should be present the same subheads.

 

Response 4: Thank you for your suggestion.We have changed the subheadings of the results and discussion sections to match. However, we considered that the methodology and statistical analysis needed to be elaborated, and in order to better illustrate the methodology and statistical analysis, there was no agreement with the results and the subheadings discussed.

 

Point 5: This Discussion section is very poor.

Response 5: Thank you for your guidance. The discussion section and the conclusion were revised and supplemented. We supplemented Line361~ 368; 397 ~ 402; 429 ~ 443;457 ~ 462.

For Line361~ 368, Compared with other horticultural crops, ornamental plants have been studied more in phenotypic determination and pigment analysis based on flower organs. As a major ornamental part and sexual reproduction organ, improving the variation of flower organs in offspring can help enrich flower color breeding. In this study, the offspring of a self-selected chimera (L. chinense var. rubrum 'Huayejimu1') had obvious variation in flower color, and the offspring had the mosaic flower color trait of the genetic mother parent, but there were also variations that separated different flower colors, showing the phenotypic diversity of flower organs.

397 ~ 402, It can be seen from the above that among the varieties of L. chinense var. rubrum produced by L. chinense var. rubrum 'Huayejimu1' with flower color mosaicism, the most important one is that there are many flower color variations, and the differences are obvious. This unique flower color mosaicism can produce new flower color varieties efficiently with a certain degree of character separation when it is passed on to offspring.

429 ~ 443, In the past, studies on the color of ornamental plants, including color rendering mode, pigment chemistry, gene function and breeding strategies, mainly focused on some traditional famous flowers in China. In peony, orchid, lily and other plants, flower color classification, anthocyanin distribution, and flower color content have been analyzed, and the related research on anthocyanin metabolism pathway is also more in-depth [35, 52-54]. These achievements are the basis for the research of new flower color breeding. It can also be found from this study that the flower color of L. chinense var. rubrum is not only red or purplish red. By using this special variety L. chinense var. rubrum, we can obtain white flowers similar to the flower of L. chinense, and obtain the purplish pink and purplish red flowers similar to the flower of L. chinense var. rubrum. In addition, we can obtain polychrome flowers mixed with the flower color of L. chinense and L. chinense var. rubrum. With the increase in the breeding work in L. chinense var. rubrum over the last decade, the variation in flower color has gradually diversified. In the future, we can consider to select and breed more varieties with different colors from this kind of special materials to obtain new germplasm more quickly.

457 ~ 462, It must be mentioned that this unique variety of L. chinense var. rubrum ‘Huayejimu1’ with dual-colored ‘red + green’ leaves and dual-colored ‘white + red’ petals has generated a large number of variations in the genetic process of its offspring, which are mainly reflected in the color characteristics of flower organs, thus greatly saving the time for flower color breeding to cultivate more varieties of flower colors.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript is a descriptive work on the phenotypic plasticity of Loropetalum chinense var. rubrum. The data are solid and shown in informative tables and figures.

The study would be much more interesting for a broader readership if important terms like L*, a*, b*, C*, H, V, and C are better explained in the methods and if the legends of figures and tables explain all the terms and part figures that are shown. For example, what means "saturation along the red-green axis" should be explained and illustrated.

One major issue needs thorough revision. In Table 2 the “petal number” is given in cm, which makes no sense; this has to be clarified. In the Methods section it has been determined that petal number is the number of petal per flower; the maximum petal number thus must be a whole number.

More major and minor comments are listed in the attached PDF.

Comments for author File: Comments.pdf

Author Response

Point 1: The study would be much more interesting for a broader readership if important terms like L*, a*, b*, C*, H, V, and C are better explained in the methods and if the legends of figures and tables explain all the terms and part figures that are shown. For example, what means "saturation along the redgreen axis" should be explained and illustrated.

 

Response 1: Thank you for your guidance. We have made the corresponding changes to explain the terms L*, a* , b*, C* , H, V, and C in methods, as well as the terms and partial numbers shown in the legend of the figures and tables. We rewrite the sentences of line124 ~ 131,and 134 ~ 135. For line 124 ~ 131, CIELab color space consists of lightness (L*) , redness (a*) and yellowness (b*). L* denotes the change in brightness from black to bright; a* represents the color change from green to red; b* represents the color change from blue to yellow. L*, a* and b* were measured directly by grating spectrometer (YS3010, 3nh, China). The center position of petals was measured with a D65 light source and a 10°angle of view,and each flower was measured seven times to get an average. Calculated according to the formula: chroma C* = (a* ² + b* ²) 1 / 2. C* value represents the vertical distance to the L*axis, and the greater the distance, the greater the chroma.

Line134 ~ 135,munsellinterpol model to convert them into H (Munsell Hue), V (Munsell Value) and C (Munsell Chroma) values..

 

Point 2: One major issue needs thorough revision. In Table 2 the “petal number” is given in cm, which makes no sense; this has to be clarified. In the Methods section it has been determined that petal number is the number of petal per flower; the maximum petal number thus must be a whole number.

 

Response 2: Thank you for your guidance. We have also found our error and have changed the centimeter to the millimeter. We have made changes in table 2 . The millimeters are the units of measurement for the anthocaulus length, corolla diameter, corolla longitudinal diameter, petal length and petal width data. The maximum and minimum of the number of flower number, petal number, sepal number, stamen number, nectary number and pistil number are all integers. We have also added note in table 2.

 

 

Point 3: More major and minor comments are listed in the attached PDF.

 

Response 3: Thank you for your guidance. We have modified each comment in the attached PDF accordingly. Modify as follows:

Point(1): the punctuation behind round

Response(1): Line 47, We change the punctuation after round from“, ”to“;”.

Point(2): What is the difference between "with colorful leaves" and "brightly colored"?

Response(2): "with colorful leaves" refers to the color-leafed plants," brightly colored" refers to the bright color of the flowers. Line 48 ~ 49，We have changed“is brightly colored”with“has bright flower colors”.

 

Point(3): citation needed for ""few studies... especially in floral organs"

Response(3): The latter sentence is not citation, it is not a coordinate conjunction, is a adversative conjunction. Line 55, We have changed“and” with “However”.

 

Point(4): What is pattern color? Does it belong to green leaves or flowers?

Response(4): "pattern color" refers to the color phenotypes. It belongs to the flower. We rewrite the sentences of line 76：but they cannot provide a precise definition or realize the standardization classification of the flower color phenotypes.

 

Point(5): Table 1. Anthocaulus : the first letter needs to be small letter；insert blank before and after =

Response(5): Table 1.We have changed“Anthocaulus” with “anthocaulus”. We inserted blanks before and after” = ”.

 

Point(6): insert blank before (

Response(6): Line 149，We have made changes accordingly.

 

Point(7): Table 2. Flower number cannot have a Maximum in cm or an Minimum in cm. Same is valid for petal number, sepal number, stamen number, nectary number

Response(7): We have modified table 2 and added explanations in the note.

 

Point(8): Table 5. Why are only 6 samples out of 60 considered here?

Response(8): In table 5, sample number has 60,the total number of samples adds up to 60.

 

Point(9): 3.2.2 In the first paragraph：L. chinense var. rubrum needs to be in italics

Response(9): We have made corresponding changes. P3：Line117,122;  P8: Line 253~255;  P9:Line267,278,291; P10:Line294 ~ 295,302; P11:Line337,340 ~ 341; P12:Line360,365,369,373 ~ 374; P13:Line378,389,391 ~ 392,395,398,409,415 ~ 417,419.Others that require italics have also been modified.

 

Point(10): 3.2.2The first sentence of the second paragraph: It is not a phylogenetic tree, but a cluster analysis

Response(10): Line 256,We have changed “phylogenetic tree” with “cluster analysis”；Line 256, We have changed“phylogenetic tree” with “cluster diagram”.

 

Point(11): Figure 3. Explain the colors in the cluster

Response(11): Line 258~259，clustered into five groups: I polychromatic, II purplish pink, III purplish red, IV green-white and V yellowish white.

 

Point(12): 3.2.3 This explanation should be given in the Methods section

Response(12): We have modified the L*, a*, and b* values in the methods section.Line 124 ~ 131.

 

Point(13): Figure 4. a* in the second panel of A should be turned for 90°；Better split Fig. 4A into three panels；Explain what is shown in Fig 4A, 4B, 4C；too much empty space

Response(13): We have modified figure 4 and changed the description in the note. We also modified the spacing of the headings.

 

Point(14): 3.2.4 In the first paragraph：This term is not defined in the manuscript

Response(14): We have modified in the methods section. Line143-145, We added a explanation about Color depth. Color depth indicates the brightness level of the color. The higher the content of anthocyanin, the more pigment distribution, the darker the color.

 

Point(15): 3.2.4 In the second paragraph: anthocyanin content thresholds,explain in the method section

Response(15): We have modified in the methods section. Line151-152，We added a explanation about anthocyanin content thresholds. Anthocyanin content thresholds refer to the numerical range of anthocyanin content.

 

Point(16): Figure 5. Explain what is shown in a, b, c, d,e, and o (first row)

Response(16): In figure 5. Note: a: polychromatic group; b: green white group; c: yellowish white group; d: purplish red group; e: purplish pink group.

 

Point(17): Table 8. 1 decimal place is sufficient

Response(17): We considering that all the other tables are 2 decimal places,So this is also the last two decimal places. We have also adjusted the table format.

 

Point(18): Fig.6 Insert blank after .

Response(18): Line346,We have changed“ Fig.6” with “Fig. 6”. 

 

Point(19): Figure 6. explain waht is shown in the five columns

Response(19): In figure 6. we added note,Note: Lc1 represents the polychromatic group; Lc5 represents the yellowish white group; Lc51 represents the green-white group; Lc26 represents the purplish pink group; Lc29 rrepresents the purplish red group.

 

Point(20): 4.1 What does this mean?

Response(20): The performance of coefficient variation and diversity index was not consistent on the same trait, indicating that the variation range was large.We rewrite the sentences of  Line383-385,Therefore, the performance of coefficient variation and diversity index was not consistent on the same trait, indicating that the variation range was large. This is similar to the phenotypic results of Sesame, Rosa hybrid, Lotus and Chestnut [45-48].

Please see the attachment，thank you!

Author Response File: Author Response.docx

Reviewer 3 Report

The presented manuscript is concise and very well written. My suggestion would be to try and edit the discussion section and conclusions since there is some repetitiveness there. 

Please pay attention to the following issues:

1. Taxon's official name is not written in italics in many places in the text: e.g. L118, 239-241, 264, 279, 291, 347, 352, 357, 367, 370, 373 and many places on page 13. 

2. Name of the taxon in tables and figures should also be given in full, not in short version

3. There are some formating issues in Figure 4, Table 8, Table A1.

4. P8L242-243 This is not a phylogenetic tree, it's a phenogram/cladogram/cluster tree since it uses phenetic data. A phylogenetic tree is based on synapomorphies or DNA data.

Author Response

Point 1:The presented manuscript is concise and very well written. My suggestion would be to try and edit the discussion section and conclusions since there is some repetitiveness there.

Response 1: Thank you for your guidance. The discussion section and the conclusion were revised and supplemented. We supplemented Line361~ 368; 397 ~ 402; 429 ~ 443;457 ~ 462.

For Line361~ 368, Compared with other horticultural crops, ornamental plants have been studied more in phenotypic determination and pigment analysis based on flower organs. As a major ornamental part and sexual reproduction organ, improving the variation of flower organs in offspring can help enrich flower color breeding. In this study, the offspring of a self-selected chimera (L. chinense var. rubrum 'Huayejimu1') had obvious variation in flower color, and the offspring had the mosaic flower color trait of the genetic mother parent, but there were also variations that separated different flower colors, showing the phenotypic diversity of flower organs.

397 ~ 402, It can be seen from the above that among the varieties of L. chinense var. rubrum produced by L. chinense var. rubrum 'Huayejimu1' with flower color mosaicism, the most important one is that there are many flower color variations, and the differences are obvious. This unique flower color mosaicism can produce new flower color varieties efficiently with a certain degree of character separation when it is passed on to offspring.

429 ~ 443, In the past, studies on the color of ornamental plants, including color rendering mode, pigment chemistry, gene function and breeding strategies, mainly focused on some traditional famous flowers in China. In peony, orchid, lily and other plants, flower color classification, anthocyanin distribution, and flower color content have been analyzed, and the related research on anthocyanin metabolism pathway is also more in-depth [35, 52-54]. These achievements are the basis for the research of new flower color breeding. It can also be found from this study that the flower color of L. chinense var. rubrum is not only red or purplish red. By using this special variety L. chinense var. rubrum, we can obtain white flowers similar to the flower of L. chinense, and obtain the purplish pink and purplish red flowers similar to the flower of L. chinense var. rubrum. In addition, we can obtain polychrome flowers mixed with the flower color of L. chinense and L. chinense var. rubrum. With the increase in the breeding work in L. chinense var. rubrum over the last decade, the variation in flower color has gradually diversified. In the future, we can consider to select and breed more varieties with different colors from this kind of special materials to obtain new germplasm more quickly.

457 ~ 462, It must be mentioned that this unique variety of L. chinense var. rubrum ‘Huayejimu1’ with dual-colored ‘red + green’ leaves and dual-colored ‘white + red’ petals has generated a large number of variations in the genetic process of its offspring, which are mainly reflected in the color characteristics of flower organs, thus greatly saving the time for flower color breeding to cultivate more varieties of flower colors.

Point 2:  Taxon's official name is not written in italics in many places in the text: e.g. L118, 239-241, 264, 279, 291, 347, 352, 357, 367, 370, 373 and many places on page 13. 

Response 2: Thank you for your guidance. Because of our carelessness, there are indeed many details that need to be improved. In future writing, we will be more careful. These need to be written in italics, we have made corresponding changes. Since the changes are based on the word, there is a difference in the number of lines compared to the PDF, Please refer to the modified word.P3：Line117,122;  P8: Line 253 ~ 255;  P9:Line267,278,291;  P10:Line294 ~ 295,302; P11:Line337,340 ~ 341; P12:Line360,365,369,373 ~ 374; P13:Line378,389,391 ~ 392,395,398,409,415 ~ 417,419.

 

Point 3: Name of the taxon in tables and figures should also be given in full, not in short version

Response 3: Thank you for your suggestion. We have completed the names of taxa in the tables and figures.

Point 4: There are some formating issues in Figure 4, Table 8, Table A1.

Response 4: Thank you for your suggestion. We have modified figure 4 and changed the description in the note. We also modified the spacing of the headings. We have adjusted the table 8 and table A1 formats.

 

Point 5: P8L242-243 This is not a phylogenetic tree, it's a phenogram/cladogram/cluster tree since it uses phenetic data. A phylogenetic tree is based on synapomorphies or DNA data.

Response 5: Thank you for your guidance. We also noticed the problem and made some changes accordingly. Line 256,We have changed “phylogenetic tree” with “cluster analysis”；Line 256, We have changed“phylogenetic tree” with “cluster diagram”.

Please see the attachment, thank you!

Author Response File: Author Response.docx