Genetic Diversity of Paeonia rockii (Flare Tree Peony) Germplasm Accessions Revealed by Phenotypic Traits, EST-SSR Markers and Chloroplast DNA Sequences

Round 1

Reviewer 1 Report

The authors answered most of my comments and some critical points have been clarified. I find still however the focus of the paper difficult to follow. Some polishing regarding the questions raised and what the present results imply for the germplasm conservation is still needed.

 

I am wondering about the analysis of phenotypic data (especially the ANOVA), because three branches were taken from the same plant. This is not clearly stated in the methods section whether the mean values for each plants were considered in the analysis or whether all single measurements were considered. There is no guarantee at all plants from an accession exactly behave in the same way for phenotypic variation and only average values per plant should be used in the statistical analysis.

 

The interpretation of cpDNA analysis is difficult to understand. The authors are talking about high diversity within accessions whereas only 4 mutation sites and 3 haplotypes were identified. This is very low. The interpretation of the frequency of the three haplotypes in unclear: variation in cpDNA can only suggest high diversity within original wild populations or seed exchange. There is also no information about the origin of the accessions and the haplotypes found, while presence of spatial genetic structure was suggested.

 

Specific comments:

L 31-32 “The 282 accessions were classified into three…”

L32-34 Please rephrase

Author Response

Point 1: I am wondering about the analysis of phenotypic data (especially the ANOVA), because three branches were taken from the same plant. This is not clearly stated in the methods section whether the mean values for each plants were considered in the analysis or whether all single measurements were considered. There is no guarantee at all plants from an accession exactly behave in the same way for phenotypic variation and only average values per plant should be used in the statistical analysis. 


 

Response 1:

Thanks for the reviewer’s question.

In the original manuscript, all phenotypic data were analysed using the average values per plant, except the maximum and minimum values in Table 4 which used all measurements. After recalculating the maximum and minimum values according to the average values per plant, the revised table is shown：

 

Table 4. Descriptive statistics for quantitative traits measured in 180 accessions.

Trait	Mean	Standard deviation	Minimum	Maximum	F-value	P-value	Variable coefficient
1	7.54	0.93	4.80	10.07	15.96	0.00	12.33
2	7.41	1.19	2.73	9.80	18.11	0.00	16.06
3	2.71	0.63	1.57	4.37	16.09	0.00	23.25
4	1.97	0.58	0.67	3.77	18.43	0.00	29.44
5	15.41	1.90	9.67	19.63	6.97	0.00	12.33
6	32.55	35.39	9.00	238.33	22.72	0.00	108.73
7	5.28	1.05	0.00	10.33	8.08	0.00	19.89
8	125.92	22.56	71.33	206.67	7.42	0.00	17.92
9	149.02	32.42	78.67	276.00	4.15	0.00	21.76
10	163.84	34.41	75.33	277.00	5.37	0.00	21.00
11	28.24	18.88	0.00	97.33	2.99	0.00	66.86
12	32.04	18.40	4.33	86.00	1.56	0.08	57.43
13	21.02	19.03	1.00	169.33	7.71	0.00	90.53
14	36.98	6.24	20.67	55.84	9.46	0.00	16.87
15	23.13	5.57	11.67	45.07	7.26	0.00	24.08
16	12.00	2.52	6.97	21.57	9.96	0.00	21.00
17	15.15	3.41	9.00	31.00	14.87	0.00	22.51
18	6.95	1.46	3.53	11.13	10.99	0.00	21.01
19	7.06	2.07	3.13	15.30	5.21	0.00	29.32
20	478.26	408.66	25.33	2661.64	4.95	0.00	85.45
21	62.26	31.57	16.89	237.01	3.75	0.00	50.71
22	33.66	31.74	0.00	151.33	4.15	0.00	94.30
23	15.01	15.00	0.00	79.67	3.32	0.00	99.93
24	5.49	1.20	2.33	12.33	19.08	0.00	21.86
25	40.75	7.27	23.74	75.64	11.29	0.00	17.84
26	15.43	3.27	7.60	25.24	10.12	0.00	21.19
27	14.79	2.91	6.97	29.07	10.73	0.00	19.68
28	2.51	0.64	0.79	4.52	12.65	0.00	25.50

 

     The original manuscript was not clearly explained in the method, we have re-described these in the Materials and Methods section on Page 8, under the subtitle- 2.5.1, showing as: 

175-176 lines: “The average values per plant were used in the statistical analysis and the data were analyzed using the statistics software SPSS version 18.0.”

The coefficients of variation in Table 4 were recalculated based on the standard deviation and mean of the two decimal places (the values in the original manuscript are based on the three decimal places), and redescribed in Results section on Page 9, under the subtitle- 3.1.1, in Discussion section on Page 18, under the subtitle- 4.1showing as:

217-226 lines: “Descriptive statistical analysis was conducted at the whole population level, and it was found that the coefficient of variation of flower traits, stem and leaf traits, fruit traits ranged from 12.33% to 108.73%, with a very high degree of variation. Among the flower traits, the coefficient of variation ranged from 12.33%-108.73%. The variation of petal number (108.73%) was very obvious, but the other flower traits varied weakly in various accessions, among which the variation coefficients of flower diameter (12.33%) and petal length (12.33%) were the smallest. As to the branch and leaf traits, the variation coefficients of tillers’ number (90.53%), fruits (66.86%) and flowers (57.43%) per plant were relatively higher. Variation coefficient of the fruit traits ranged from 17.84% to 99.93% (avg. 48.50%). Among them, the variation coefficient of seed weight per fruit was the largest (99.93%), followed by seeds number per fruit (94.30%).”

556-557 lines: “By the genetic diversity of 28 quantitative traits of 180 accessions, this study revealed a genetic variation coefficient of 12.33%-108.73%, which was similar to Wu et al.”

564-566 lines: “In FTP used in this study, that the number of petals varied from 9 petals to 238.33 petals (Table 4) indicate that these accessions have diverse flower types from single though semi-double to double”.

569-572 lines: “Among these accessions used in this study, some of them with single flower, in most cases, are fertile for the formation of fruits (seeds) and can be screened as the high yield oil germplasms, in which the maximum fruit weight per plant was up to 2661.64 g (Table 4)”.

 

Point 2: The interpretation of cpDNA analysis is difficult to understand. The authors are talking about high diversity within accessions whereas only 4 mutation sites and 3 haplotypes were identified. This is very low. The interpretation of the frequency of the three haplotypes in unclear: variation in cpDNA can only suggest high diversity within original wild populations or seed exchange. There is also no information about the origin of the accessions and the haplotypes found, while presence of spatial genetic structure was suggested.

 

Response 2:

     Thanks for the reviewer’s question.

     The level of chloroplast diversity of accessions obtained in our study was compared with previous studies on the wild populations of P. rockii. The mean nucleotide haplotype diversity (Hd) and the mean nucleotide diversity (Pi) of FTP accessions in our study were 0.164 and 0.28×10^-3. Xu et al. measured the diversity of 214 individuals in 24 populations of P. rockii and obtained the results as Hd and Pi at the species level were 0.874 and 0.294×10^-2, which were significantly higher than Hd (0.1097) and Pi (0.383×10^-4) at the population level. By comparison with Xu et al.'s conclusions (the three chloroplast markers used in the two studies were identical), the chloroplast diversity of accessions in our study was higher than the population level and lower than the species level. This was discussed in the original manuscript:

574-585 lines: “The primary task of evaluating all kinds of samples in germplasm resources is to determine their genetic constitution in the repository. We can also compare newly collected cultivated or wild plants from various locations with existing germplasm accessions to facilitate identification. With the advent of molecular technologies and methods, it has become more efficient and scientific to use genetic information at the genome level to analyze accession differences in genetic resources and compare these samples with germplasm collections elsewhere ⁶².”

619-630 lines: “According to Yuan et al. ²⁸, the mean nucleotide haplotype diversity (Hd) and the mean nucleotide diversity (Pi) of the chloroplast (cpDNA) in 335 P. rockii wild individuals at the population level was 0.0686 and 0.765×10^-4, respectively, while Hd and Pi at the species level was 0.887 and 0.185×10^-2, which was significantly higher than population level. Based on the analysis of three cpDNA sequences. Xu et al. ³⁷ measured the diversity of 214 individuals in 24 populations of P. rockii and obtained the same results as Hd and Pi at the species level were 0.874 and 0.294×10^-2, which were significantly higher than Hd (0.1097) and Pi (0.383×10^-4) at the population level. This shows that wild P. rockii has a low genetic diversity at the population level and a higher genetic diversity at the species level. This situation was consistent with other tree peony species, P. delavayi ³³, P. ludlowii ³³, P. qiui ³⁷ and P. jishanensis ³⁷. However, Hd and Pi in FTP accessions in this study were 0.164 and 0.28×10^-3, which were higher than the population level and lower than the species level in populations of wild P. rockii.”

For haplotypes, in wild P. rockii studied by Xu et al., there were 10 haplotypes in the whole population, while each population contained 1 to 3 haplotypes, and many populations had only one haplotype. The number of haplotypes in our study was 3, which was also between the population level and the species level.

In the original manuscript, it was one-sided to state only the higher genetic diversity at the population level without emphasizing comparisons at the species level. We have re-described these in the Abstract section on Page 1, in the Discussion section on Page 19, under the subtitle- 4.3, in the Conclusions section on Page 21, showing as: 

32-35 lines: “the cpDNA data indicated that these accessions had a higher genetic diversity than the population level and a lower genetic diversity than the species level of wild P. rockii, and their existing spatial genetic structure of these accessions can be divided into two branches. ”

628-630 lines: “However, Hd and Pi in FTP accessions in this study were 0.164 and 0.28×10^-3, which were higher than the population level and lower than the species level in wild populations of P. rockii.”

689-691 lines: “The cpDNA data indicated that the genetic diversity of chloroplast genome in the FTP accessions had a higher genetic diversity than the population level and a lower genetic diversity than the species level of wild P. rockii, and they can be divided into two branches based on it”

Point 3: L 31-32 “The 282 accessions were classified into three…”，L32-34 Please rephrase.

Response 3:

Thanks for the reviewer’s question. The revised manuscript is as follows:

31-32 lines: “The 282 accessions were divided into three distinct groups;”

32-35 lines: “the cpDNA data indicated that these accessions had a higher genetic diversity than the population level and a lower genetic diversity than the species level of wild P. rockii, and their existing spatial genetic structure of these accessions can be divided into two branches.”

 

 

Reviewer 2 Report

Dear authors!
Your revised manuscript indicates that you were serious about my comments in the first review round. However, I had difficulty checking your corrections. It would be better if all the corrections were described in a cover letter with your comments for me.
In general, the manuscript has become much better. The table in Suppl. materials has greatly improved the perception of your results. I think, the manuscript may be recommended for publication after small corrections (although I emphasize that it is hardly interesting to a wide circle of readers). Tables 2, 4, 5, 6 should be in Suppls. The word “materials” should be replaced by the word “plants” or “samples” in part 4.2. In other parts too.

Author Response

Point 1: Tables 2, 4, 5, 6 should be in Suppls.

 

Response 1:

Thanks for the reviewer’s question.

The main content of this paper was to comprehensively evaluate the genetic diversity of these germplasm resources using phenotypic traits, EST-SSR markers and chloroplast DNA sequences. Table 2 showed the results of SSR markers, including some important indicators to evaluate genetic diversity, such as No. of Different Alleles (Na), No. of Effective Alleles (Ne). Table 4 showed the phenotypic variation indicators of these germplasm resources, including maximum, minimum and coefficient of variation. They were also direct indicators to evaluate the phenotypic diversity of germplasm resources.

Table 5 showed the principal component analysis of 28 quantitative traits. Its results  explained the variation distribution of phenotypic traits in the whole population. The combination of phenotypic traits and principal component analysis is also an important method for genetic diversity evaluation and germplasm screening. The main component analysis showed that the flower traits which had great influence on the phenotypic traits of P. rockii were petal length, petal width and flower diameter and so on. The leaf traits with greater influence were compound leaf length, petiole length, compound leaf width and so on. These phenotypic traits have certain reference value in the classification of P. rockii, and can be used as the main content of the research on phenotypic traits of P. rockii, thus saving the research time and improving the research efficiency.

Table 6 showed the correlation analysis of phenotypic traits. On the one hand, it can reflect the diversity of phenotypic variation and the rules between traits. On the other hand, understanding the interrelation between phenotypic traits is of great significance for further breeding. When highly correlated traits are selected, the modification of one trait can affect the others at the same time. Different improvement directions and selection criteria were provided for the selection of traits.

So I think these tables should be in the manuscript.

 

Point 2: The word “materials” should be replaced by the word “plants” or “samples” in part 4.2. In other parts too.

 

Response 2:

Thanks for the reviewer’s question. The word "materials" used in three places in the original manuscript has been corrected.

574-585 lines:“The primary task of evaluating all kinds of samples in germplasm resources is to determine their genetic constitution in the repository. We can also compare newly collected cultivated or wild plants from various locations with existing germplasm accessions to facilitate identification. With the advent of molecular technologies and methods, it has become more efficient and scientific to use genetic information at the genome level to analyze accession differences in genetic resources and compare these samples with germplasm collections elsewhere ⁶² .”

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript “Genetic Diversity and Population Structure of 2 Paeonia rockii (flare tree peony) Germplasm Accessions Revealed by Phenotypic Traits, EST-SSR 4 Markers and Chloroplast DNA Sequences” describes the genetic background of Paeonia rockii accessions in China. 282 accessions were collected in the main cultivation center and transplanted near Beijing. Genetic diversity was estimated at EST-SSR and chloroplast markers, phenotypic measurements were recorded over two consecutive summers.

This study represents a lot of work, but it is extremely difficult to judge the quality of the presented results. The English is poor and some methods are not described properly. Reading the manuscript gives the impression that there are caveats in the statistical analysis, whereas this could be a language problem. I would not trust the phenotypic measurements, as the plants has been transplanted from other populations and environmental effects can still affect traits after several years. Also, the authors mention measurements over two summers and do not take this into account in the analysis. If all measurements were not taken during the same season, then the phenotypic part of the study is flawed. To look at phenotypic differences among accessions, it is better to grow all plants from seeds with the same conditions to avoid environmental effects on phenotypic plasticity. I have further comments and questions below:

• What is the difference between your study and the one from Wu et al. 2019? Do you have a different focus?
• Cultivation center: are these localities the natural areas of the plants or provenance trails? The plants were transplanted to Beijing, but how many replicates per accession were used? Did you use a randomized design to avoid environmental effects? You also cannot exclude that the former environment influenced some parameters.
• If you selected 80 accessions for chloroplast sequencing according to floral shape, it means that there is a strong heritability of the flower traits. Is it correct? Otherwise I think this analysis is flawed.
• Statistical analysis: did you check for normality of data before ANOVA. You also have a hierarchical design (3 flowers per plant), how do you deal with this? And you used data from 2 years, how do take this into account, is there any annual variation?
• Correlation tests: you should use Bonferonni corrections.
• How do you explain the high and significant excess of heterozygosity? Did the plants originate from crossings? Or is it an effect of the mating system?
• It is strange to conduct STRUCTURE analysis on accessions if the plants are not derived from natural populations or when no information is available on their origin. The high heterozygosity could a sign that the accessions originated from former cultivation and crossings. In this case, STRUCTURE is not the right method, as you will detect family structure. Do the three groups correspond to different populations of origin? Further, there is no information on the parameters (number of replicates, iterations, model used…). Further, the high and significant heterozygosity violates the assumptions of HWE that is required for Bayesian clustering analysis.

 

 

Reviewer 2 Report

Dear authors!

After reading your manuscript, I came to the conclusion that I can recommend it for publication only after significant changes have been made. On the whole, the presented study have a low level of the originality and scientific novelty. And I have to share my opinion to the Editor who will decide on the acceptance/rejection of the manuscript. I see that the work had local, applied goals and is unlikely to be of interest to a wide circle of readers. My main comments (without which the manuscript cannot be accepted for publication) are follows. 1. The results are presented by clusters that are not clearly related. It is necessary to integrate the results of the assessment of phenotypic traits, microsatellite and chloroplast markers. Perhaps, it is better presented in the form of a table in the supplemental materials. The reader should clearly see what results were obtained for each collected tree. 2. All of the described groups, branches and haplotypes should be characterized. What phenotypes do they include?  The relationship between markers and phenotypic traits can significantly improve the scientific component of the work. Also, there are many other comments, which require reference to specific places of the manuscript. 1. Please check the linking throughout the manuscript. 2. Line 83. You wrote about 40 Wu's markers. If you used the same markers, then this must be reported (and the reasons why you used 34 out of 40 should be described too). If you used other markers, then I have a question, why Wu's markers were not used. 3. Figures 2-7 have a low quality. Letters and numbers must be clear. Figures must be improved. 4. Bulky tables need to be moved to supplemental materials. 5. Line 129. The word “materials” should be replaced by the word “plants” or “samples”. 6. Lines 146-147. You wrote that the 80 accessions can represent the typical flower shapes and colors from 282 accessions. This is not obvious. This must be proved. It is advisable to make an analysis with 282 samples. 7. Lines 229-240. Why was correlation analysis carried out? What is its value for work? It may be better to exclude these results. 8. Line 242. The word "bands" should be replaced by the word "fragments". 9. Lines 242-243. The text must be formulated in a different way. Primers do not amplify. An amplification is conducted with primers. 10. Line 256.The information about the progamme application must be in the methods (part 2). 11. Lines 262-266. The description of Figure 4 must be in the legend, not in the text. 12. Figures 4 and 5 should be united. 13. Lines 299-302. Remove to part 2.