Development of SSR Markers Based on Transcriptome Sequencing and Verification of Their Conservation across Species of Ornamental Pennisetum Rich. (Poaceae)
and Ke Teng 2,*Round 1
Reviewer 1 Report
Pennisetum is widely used as forage, food crop, energy grass, ornamental plant around the world. SSR markers were developed in Pennisetum setaceum cv. ‘Rubrum’ through transcriptome sequencing. A total of 38 Pennisetum materials were further used to verify the transferability of these SSR markers. These data and results presented in this study are helpful to conserve and utilize the ornamental Pennisetum germplasm resources. Some points of this study should be carefully corrected and clarified before it can be accepted.
1）Are there any phenotype similarities among the Pennisetum materials? It is better to map these characters on the phylogenetic tree (Figure 2) to show the potential morphological and genetic correlation or consistency.
2）Are the transcriptome data of P. setaceum cv. ‘Rubrum newly produced for this study? If not, the transcriptome analysis and result should be simplified to avoid duplication with your previous studies [24].
3）The parts of genetic diversity in 38 Pennisetum materials should be strengthened.
4）The language throughout the manuscript needs further smooth.
5）I think the title of this manuscript is not clear.
Author Response
Response to Reviewer 1 Comments
Comments and Suggestions for Authors
Pennisetum is widely used as forage, food crop, energy grass, ornamental plant around the world. SSR markers were developed in Pennisetum setaceum cv. ‘Rubrum’ through transcriptome sequencing. A total of 38 Pennisetum materials were further used to verify the transferability of these SSR markers. These data and results presented in this study are helpful to conserve and utilize the ornamental Pennisetum germplasm resources. Some points of this study should be carefully corrected and clarified before it can be accepted.
Point 1: Are there any phenotype similarities among the Pennisetum materials? It is better to map these characters on the phylogenetic tree (Figure 2) to show the potential morphological and genetic correlation or consistency.
Response 1: Thank you for your suggestion.
Firstly, we agree with your suggestion that the match of phenotype and phylogenetic tree could better describe genetic correlation. However, at present, we did not obtain enough phenotypic data.
It is worth mentioning that there are partly similarities between the phenotypes of the Pennisetum material. Based on our group’s previous studies, Zhang et al. (2016) performed Nei’s genetic diversity analysis based on the phenotypes of nine Pennisetum cultivars, among which four materials were included in our research. We conducted UPGMA clustering for these 4 materials separately and compared to previous study (Figure 1). The results showed that phenotypic clustering results of P.alopecuroides, P.villosum, P.steaceum cv. ‘Rubrum’, P.setaceum from previous studies could be mapped to our molecular cluster at the molecular level. We will also carry out relevant studies in the future to further collect phenotypic data and provide a theoretical basis for the phylogenetic relationship and evolution of Pennisetum.
Figure 1．(A) Depictions of the Q-mode clustering analysis of Pennisetum taxa based on Nei’s genetic distances of the seven morphological traits Zhang et al. (2016).(B) UPGMA clustering analysis of P.alopecuroides, P.villosum, P.steaceum cv. ‘Rubrum’, P.setaceum .
Reference:
Zhang Y, Yuan X, Teng W, et al. Karyotype diversity analysis and nuclear genome size estimation for Pennisetum Rich. (Poaceae) ornamental grasses reveal genetic relationship and chromosomal evolution. Scientia Horticulturae, 2015, 193:22-31. Please provide your response for Point 1.
Point 2: Are the transcriptome data of P. setaceum cv. ‘Rubrum’ newly produced for this study? If not, the transcriptome analysis and result should be simplified to avoid duplication with your previous studies [24].
Response 2: Thanks for your suggestion. In our previous research, we constructed the full-length transcriptome of P. setaceum cv. ‘Rubrum. The second generation transcriptome sequencing data of P. setaceum cv. ‘Rubrum’ was newly assembled and utilized to develop molecular markers for P. setaceum species. We provided abundant transcripts for Pennisetum, and analyzed the genetic relationship of Pennisetum by SSR molecular markers.
Point 3: The parts of genetic diversity in 38 Pennisetum materials should be strengthened.
Response 3: Thanks for your suggestion. We re-analyzed and expanded the genetic diversity in 38 Pennisetum materials.
Lines 204-213: “A total of 312 polymorphic bands were amplified using 38 pairs of SSR primers, with an average of 8.21 bands per primers pairs. The PIC ranged from 0.16 (PaSSR-2) to 0.44 (PaSSR-47), with an average of 0.35(Table S2). 37 locus was medium level polymorphism (0.25 0.5) locus. SSRs displayed wide genetic variation among accessions. The average Na was 2.00. The Ne ranged from 1.19 (PaSSR-2) to 1.81 (PaSSR-47), with an average of 1.60. The H ranged from 0.16(PaSSR-2)-0.44(PaSSR-47), with an average of 0.35. The I were between 0.16(PaSSR-2) and 0.63 (PaSSR-47), with an average of 0.52(Table S2). The genetic similarity coefficient between accessions (Table S3) ranged from 0.39 to 1.00 with an average of 0.58(Table S3).”
Lines 315-329: “The genetic diversity in the collected Pennisetum samples was found to be moderate, which may be due to the materials having been collected in a relatively concentrated place where the genetic background difference may not be particularly large. In addition, based on the results of UPGMA clustering and PCoA analysis, we were able to determine the genetic background of different Pennisetum plant materials. And the results showed that the genetic relationship of Pennisetum samples was not closely related to the geographical distribution location. The phylogeny of P. clandestinum was classified into a separate category in UPGMA clustering and PCoA analysis. P. clandestinum is native to Africa and, thus, it perhaps not expected to have genetic similarity to other materials [47]. In addition, two kinds of P. alopecuroides cv. ‘Little Bunny’ materials from Beijing and Yunnan were clustered as one category, indicating that they are closely related. However, P. alopecuroides cv. ‘Ziguang’ from Beijing and P. alopecuroides cv. ‘Ziguang’ from Kunming, Yunnan, were classified into two categories. We speculated that this may be because they can cross with other local varieties when applied in different places.”
Point 4: The language throughout the manuscript needs further smooth.
Response 4: Thanks for your advice. We polished the language of the manuscript with the language service of the Editage Company (www.editage.com). All the revised content could be found in the manuscript in track-changes mode which I uploaded in the online system.
Point 5: I think the title of this manuscript is not clear.
Response 5: We revised the title to “Development of SSR Markers Based on Transcriptome Sequencing and Verification of Their Conservation Across Species of Ornamental Pennisetum Rich. (Poaceae)”
Thank you for your consideration and responsible review. We’d like to take this opportunity to thank you for your comments and constructive suggestion. Please feel free to inform us if there is still any problem.
Best regards,
Sincerely,
Ke Teng,
Corresponding Author
Beijing Academy of Agriculture and Forestry Sciences, Beijing, P.R. China
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Manuscript (agronomy-1776148) Guo et. al. is an interesting study on creating SSR markers using Pennisetum setaceum cv. ‘Rubrum’ transcriptome sequencing data. The relevance of this topic in the context of the analysis of Pennisetum genetic resources is obvious. These plants are used for fodder, biofuels and for ornamental purposes. In my opinion, the manuscript is rather good written and it is easy to read.
However, some concerns as followings should be noticed
Abstract
Lines 26-27: „ We selected 38 pairs of high polymorphism primers...“. Formally speaking, this phrase is not entirely correct. The authors study the polymorphism of the ssr loci (DNA sequence that is located between two primers). Oligonucleotide primers are always polymorphic. Primers are used to reveal polymorphic loci (markers).
Lines 34-37. Two sentences are very similar. “In addition, the fingerprint map was constructed and it improved the identification efficiency of Pennisetum resources.“ and “The construction of fingerprint helps to identify different resources of Pennisetum. Please, delete unnecessary. „
Line 73: “ Zhou et al. 2018 „ please, correct into „Zhou et al. [22]..“ Please, see MDPI references and citation style guide.
Line 75: „Wang et al. developed... “please, correct into „Wang et al. [23] developed...“
Line 305: Li et al. [42].
Line 76: “…28 pairs of polymorphic primers [23].“ please, correct into „...28 polymorphic loci [23].“ Or „...28 polymorphic markers [23].“
Line 125: “…S. viridis.were selected...“ please, correct into “...S. viridis were selected... “
Please, indicate in M&M how primers were designed.
Line 130: Please, indicate primer concentrations in PCR description.
Lines 139-141. Please justify in the text, why did you analyze SSR markers as binary dominant.
Results
The table that includes primer pairs developed in this study and used for analysis, number of alleles at each locus and alleles/bands size intervals, in my opinion, have to be included in the main text. This is valuable information for future research of Pennisetum germplasm by other researchers and according the title of the manuscript it is one of the main results. Supplementary files are not always available as in this case. For example, I did not find supplementary files in my reviewer panel.
The authors do not see any differences between genetic distance and genetic similarity, as they consider the scale shown in the figure as genetic distances in one place (lines 222, 223, 226) and genetic similarity in the other (line 219). In my opinion, the scale shown in Figure 2 shows genetic similarity. It would be logical to assume that Pennisetum variants were divided into one category at the genetic similarity of 0.89 (or genetic distance 0.11).
I think Pennisetum fingerprints can only be valuable if the plants are vegetatively propagated. If they can interbreed, as can be judged from some explanations of the authors in discussion, then the polyploid nature of these species (or at least part of them) does not allow one to accurately distinguish homozygotes from heterozygotes (except inbred lines). Therefore, segregation of SSR alleles and changes in fingerprint profiles in subsequent generations are possible if propagated by seeds.
The reference list does not meet the requirements of the journal. Please, correct.
Author Response
Response to Reviewer 2 Comments
Comments and Suggestions for Authors
Manuscript (agronomy-1776148) Guo et. al. is an interesting study on creating SSR markers using Pennisetum setaceum cv. ‘Rubrum’ transcriptome sequencing data. The relevance of this topic in the context of the analysis of Pennisetum genetic resources is obvious. These plants are used for fodder, biofuels and for ornamental purposes. In my opinion, the manuscript is rather good written and it is easy to read.
However, some concerns as followings should be noticed
Abstract
Point 1: Lines 26-27: “We selected 38 pairs of high polymorphism primers...”. Formally speaking, this phrase is not entirely correct. The authors study the polymorphism of the ssr loci (DNA sequence that is located between two primers). Oligonucleotide primers are always polymorphic. Primers are used to reveal polymorphic loci (markers).
Response 1: Thanks for pointing out the error. We agree with your suggestion that primers are used to reveal polymorphic loci (markers), instead of studying the polymorphism of primers. We have revised the “primers” to “markers”.
Lines 24-25: “We selected 38 pairs of highly polymorphic SSR markers from 50 randomly selected SSR markers.”
Point 2: Lines 34-37. Two sentences are very similar. “In addition, the fingerprint map was constructed and it improved the identification efficiency of Pennisetum resources.” and “The construction of fingerprint helps to identify different resources of Pennisetum”. Please, delete unnecessary.
Response 2: Thanks for your suggestion. We have deleted the duplicate statements. The following are retained:
Lines 28-29: “In addition, fingerprint maps were constructed to improve Pennisetum identification.”
Point 3: Line 73: “Zhou et al. 2018” please, correct into “Zhou et al. [22].” Please, see MDPI references and citation style guide.
Response 3: We have revised the literature citation format in accordance with MDPI references and citation style guide.
Line 66: “Zhou et al. [22] conducted genetic diversity analysis…”
Point 4: Line 75: “Wang et al. developed...” please, correct into “Wang et al. [23] developed...”
Response 4: We have revised it.
Line 68: “Wang et al. [23] developed 83,706 SSR markers…”
Point 5: Line 305: Li et al. [42].
Response 5: We have revised it.
Line 304: “Li et al. [42] developed”
Point 6: Line 76: “…28 pairs of polymorphic primers [23].” please, correct into “...28 polymorphic loci [23].” Or “...28 polymorphic markers [23].”
Response 6: Thanks for pointing out the error, we have revised the “primers” to “markers”.
Lines 69: “identified 28 pairs of polymorphic markers.”
Point 7: Line 125: “…S. viridis.were selected...” please, correct into “...S. viridis were selected... ”
Response 7: Thanks for your suggestion we have revised the “…S. viridis. were selected...” into “...S. viridis were selected...”.
Line 120: “…S. viridis were selected for use in screening...”
Point 8: Please, indicate in M&M how primers were designed.
Response 8: We have supplemented how the primers were designed in the Materials and methods. And we also added criteria for screening the ratio of repeat types of different SSR markers.
Line113-116: “SSR loci with dinucleotide to hexanucleotide repeat types are preferentially selected. The number of SSR markers selected for different repeat types is determined by their proportions. Using Primer3 (http://primer3.sourceforge.net/ releases.php) for each SSR primer design.”
Point 9: Line 130: Please, indicate primer concentrations in PCR description.
Response 9: Thanks for your suggestion. We have supplemented primer concentrations in PCR description.
Line 122: “The concentration of primers is 10µmol/L.”
Point 10: Lines 139-141. Please justify in the text, why did you analyze SSR markers as binary dominant.
Response 10: Thanks for your advice. Pennisetum is a plant with complex genetic background, inconsistent ploidy, and uncertain genetic relationship. Using binary dominant statistics is a better solution. We have added justification in the manuscript.
Lines 135-137: “Due to the complex genetic background of Pennisetum, aneuploidy and polyploidy exist widely in Pennisetum, and it is difficult to detect the peak value of each locus, so we analyzed SSR markers as binary dominant.”
Reference:
[1] Zhang Y, Zhu M L, and Dai S L. Analysis of karyotype diversity of 40 Chinese chrysanthemum cultivars. Journal of Systematics & Evolution, 2013, 51 (3):335-352.
[2] Zhang Y, Wang C, Ma H Z, et al. Assessing the Genetic Diversity of Chrysanthemum Cultivars with Microsatellites. Journal of the American Society for Horticultural Science, 2013, 138(6): 1-8
Results
Point 11: The table that includes primer pairs developed in this study and used for analysis, number of alleles at each locus and alleles/bands size intervals, in my opinion, have to be included in the main text. This is valuable information for future research of Pennisetum germplasm by other researchers and according the title of the manuscript it is one of the main results. Supplementary files are not always available as in this case. For example, I did not find supplementary files in my reviewer panel.
Response 11: Thanks for your advice. We have moved “The information of 50 pairs of SSR maker” (Table 4.) into the manuscript. It includes sequence information for SSR primers and whether they are polymorphic. In addition, “Band Statistics Raw Data” (Table S1), which contains information on the number of alleles and the allele size interval, is included in the attachment due to the large amount of data.
Point 12: The authors do not see any differences between genetic distance and genetic similarity, as they consider the scale shown in the figure as genetic distances in one place (lines 222, 223, 226) and genetic similarity in the other (line 219). In my opinion, the scale shown in Figure 2 shows genetic similarity. It would be logical to assume that Pennisetum variants were divided into one category at the genetic similarity of 0.89 (or genetic distance 0.11).
Response 12: Thanks for the correction. We have corrected the expression errors and distinguished differences in “genetic distance” and “genetic similarity”.
Lines :220-233. “When the genetic distance was 0.50, the 38 Pennisetum samples were divided into five categories. The Pennisetum variants were divided into one category at a genetic similarity of 0.89. When the genetic similarity was 0.81 (Figure 2), the P. purpureum Schum. variants were classified into one category. P. alopecuroides ‘Baimeiren’ from Kunming, Yunnan, and P. setaceum from Beijing are classified into one category, indicating that the two are closely related and could be distinguished when the genetic similarity is 0.47. The phenotypes of these variant materials have different degrees of variation, and it is impossible to accurately classify them only by phenotypic characteristics. The results confirmed their genetic relationship and further verified the reliability of the clustering results. P. clandestinum was branched separately at a genetic similarity of 0.48, indicating that it is distantly related to other Pennisetum samples. P. alopecuroides cv. ‘Little Bunny’ and P. alopecuroides cv. ‘Hameln’ have similar phenotypes at a certain growth stage, thus it is difficult to distinguish the two based on their phenotype. UPGMA clustering revealed that they branched at a genetic similarity of 0.48.”
Point 13: I think Pennisetum fingerprints can only be valuable if the plants are vegetatively propagated. If they can interbreed, as can be judged from some explanations of the authors in discussion, then the polyploid nature of these species (or at least part of them) does not allow one to accurately distinguish homozygotes from heterozygotes (except inbred lines). Therefore, segregation of SSR alleles and changes in fingerprint profiles in subsequent generations are possible if propagated by seeds.
Response 13: Thank you for your suggestion, we agree with your suggestion that the fingerprint of vegetatively propagated plants is more valuable. In our experiments, the five Pennisetum materials for fingerprinting were vegetatively propagated.
Point 14: The reference list does not meet the requirements of the journal. Please, correct.
Response 14: Thanks for your suggestion. We have changed the reference list to the correct format as requested using EndNote X9.2 software.
We’d like to take this opportunity to thank you for the review and helpful suggestions on our manuscript. The quality of revised manuscript is improved after we finished the recommended modifications. Thanks for your consideration. Best wishes!
Best regards,
Sincerely,
Ke Teng,
Corresponding Author
Beijing Academy of Agriculture and Forestry Sciences, Beijing, P.R. China
Author Response File: Author Response.pdf
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.
