Development of Simple Sequence REPEAT Markers for Genetic Diversity Analysis Based on the cDNA Sequences of Chinese Yam (Dioscorea spp.)
Round 1
Reviewer 1 Report
This article is very good for publish in the journal after revise in some points in text.
Comments for author File: 
Comments.pdf
Author Response
Reply to dear editor Sealey Xie and reviewers
Journal: Horticulturae
Manuscript ID: horticulturae-2026635
Title: Development of simple sequence repeat markers based on the cDNA sequences of Chinese yam (Dioscorea spp.) for genetic diversity analysis
Authors: Hong Wang, Yue Wang, Lingzhi Xiong, Yingde Chen, Jiali Sun, Changdong Ouyang, Baihua Li, Hanyi Zeng, Xiaorong Chen* and Mingbao Luan*
Dear editor Sealey Xie and Reviewers,
We are very grateful to the critical comments and thoughtful suggestions from you and the reviewers on our manuscript of ‘horticulturae-2026635’, the comments and suggestions are quite valuable for us. We have read and considered all the comments carefully, and tried our best to revise the original manuscript according to the comments of editors and reviewers.
According to your requirements, we have made point-to-point modifications to the questions and comments which were raised by the reviewers in the manuscript, and marked the modification of reviewers' comments in Red in the body of the revised manuscript.
We sincerely hope the Editor and Reviewers will satisfy with our revisions for the original manuscript. If you still have any questions about this paper, please don’t hesitate to contact me at the address below.
Yours sincerely,
Mingbao Luan
Yue Wang
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Xianjiahu West Road, Changsha 410205, Hunan Province, China
E-mail:[email protected]
E-mail: [email protected]
Responses to reviewer #1
Comment 1: Line 59, “Chinese yam”. （1）What is the local name of this species? Is it distribution only in China? Please explain? Add reference. （2）What is family of this plant? How many species of the genus Dioscorea? （3）Why do you study of this species, How many genetic variation problem?
Response: Dear reviewer, Thank you very much for your suggestion.
(1) Yam is distributed in Africa, Central and South America, parts of Asia, the Caribbean and the Pacific Islands. Chinese yam is the collective name for yams distributed in China, also called Huai yam, Huai-shan, Tu-shu, white yam, Yuyan and Shan-shu.
(2) Chinese yam belongs to the family Dioscoreaceae of the order Dioscoreales, and consists of about 600 species of domesticated and wild yams. However, only about 10 edible species are widely grown for food in tropical and subtropical regions of the world .
(3) Chinese yam, an important medicinal and food crop in China, is popular and has important economic, social and cultural values, because the appearance of many species of yam are similar in appearance, but their nutritional and medicinal components have large differences. It is difficult to identify quickly as the same species or the same taxa, which seriously hinders the selection and promotion of yam varieties, so there is necessary to analyze the genetic diversity of yam using molecular markers . The large amount of genetic variation distributed in yam populations, which controls traits such as flowering (FL), leaf shape (LS), leaf color (LC), stem color (SC), twining direction (TD), tuber shape (TS), root hair density (RHD), tuber skin color (TSC), flesh color (FC), and tuber flesh weight (TFW)
Regarding the answer to this question, I made a change in line 39-44 , line 46-49， line 64-70 and line 296-300 of the new manuscript.
Comment 2 : Line 53-56 “The differences existing…. in yam breeding.”Add reference.
Response: Dear reviewer, we add a reference in line 67-70 in the new manuscript.
Comment 3 : Line 85, “32 yam germplasm resources”. How is difference?
Response: The differences between the 32 yam germplasm resources we have described detaily in Table 1.
Comment 4 : Line 90 “a typical yam” Where are voucher specimens deposit?
Response: In the new manuscript, “a typical yam” is modified as GN_ZY_huaishan. These specimens were taken from Yichun, Jiangxi, and stored in the -80°C refrigerator at the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences in Changsha, Hunan.
Regarding the answer to this question, I made a change in line 90 and line 95-97 of the new manuscript.
Comment 5 : Table 1, please add dominant character of each variation of specimens.
Response: Thanks to your suggestion, we have described the characteristics of the 32 yam germplasm more detaily in Table 1 of the new manuscript.
Comment 6 : Table 1, add collector number of each voucher specimens.
Response: Dear reviewer, many of the 32 yams in our collection are cultivars, and the name of the yams was their collector number.
Comment 7 : Line 112-119, “Roots, stems and leaves of Yichun yam …,” Add References.
Response: Dear reviewer, We add a reference in line 112-119 in the new manuscript.
Comment 8 : Line 122-132, “Transcriptome sequencing ….” Add References
Response: Dear reviewer, We add a reference in line 122-132 in the new manuscript.
Comment 9 : Line 135-149, “SSRs were identified using….” Add References
Response: Dear reviewer, We add a reference in line 135-149 in the new manuscript.
Comment 10 : Line 152-164 “To analyze the genetic diversity….” Add References
Response: Dear reviewer, We add a reference in line 152-164 in the new manuscript.
Comment 11 : Line 236, discussion related with dominant characters of each variations in table 1
Response: Dear reviewer, Thank you very much for your questions. we add the discussion related with dominant characters of each variations in line 260-272 in the new manuscript.
Comment 12 : Line 273, “4.1 Transcriptome sequencing, assembly, and functional annotation” Discussion related with dominant characters of each variations in table 1
Response: We add the characters of GN_ZY_huaishan , which used for transcriptome sequencing, in line 278-284 in the new manuscript.
Comment 13: Line 284, “In this study, transcriptome sequencing of roots, stems, and leaves of Chinese yam from Yichun, Jiangxi, resulted in a CleanData of 6.35, 6.68, and 6.29 G, with marginal differences among them.” please check data again
Response: Dear reviewer, this data is consistent with line 178-179 in the results.
Comment 14: Line 294, “4.2 SSR markers” Discussion related with dominant characters of each variations in table 1
Response: Dear reviewer, We add a discussion of yam traits in line 295-302.
Comment 15: Line 308 “4.3 Phylogenetic analysis of yam” Discussion related with dominant characters of each variations in table 1
Response: Dear reviewer, We remove this subheading and add a discussion related to the Dioscorea spp traits Table 1, in line 319-336.
Comment 16: Line 346 “5. Conclusions” rewrite after edit discussion done
Response: Dear reviewer, Thank you very much for your suggestion. We revised the conclusion rewrite after edit discussion done in line 347-358.
Author Response File: 
Author Response.docx
Reviewer 2 Report
1. ABSTRACT/INTRODUCTION. Twenty-four SSR markers are fine for diversity analyses, but seem too few to visualize their use in marker-assisted selection in Dioscorea (in fact in any species).
2. INTRODUCTION, Lines 59-60. SSRs are not dominantly inherited; in fact they are recognized and appreciated by their co-dominant inheritance.
3. TABLE 1. For those readers unfamiliar with Chinese geography, it would be very useful to consult the geographical coordinates of provenance of the yam accessions, in addition to the name of the place name.
4. MATERIALS AND METHODS. Assessing the banding pattern in SSR as present or absent dismisses a lot of power inherent to this type of markers, given that they are usually co-dominant. How then is it recorded when two bands appear, as is the case with heterozygotes? Certainly the absence of bands also occurs in low frequency, but only in cases of null alleles, where the template DNA has undergone some mutation in the banding zone. Please justitfy/clarify futher the system used for calling alleles.
Author Response
Reply to dear editor Sealey Xie and reviewers
Journal: Horticulturae
Manuscript ID: horticulturae-2026635
Title: Development of simple sequence repeat markers based on the cDNA sequences of Chinese yam (Dioscorea spp) for genetic diversity analysis
Authors: Hong Wang, Yue Wang, Lingzhi Xiong, Yingde Chen, Jiali Sun, Changdong Ouyang, Baihua Li, Hanyi Zeng, Xiaorong Chen* and Mingbao Luan*
Dear editor Sealey Xie and Reviewers,
We are very grateful to the critical comments and thoughtful suggestions from you and the reviewers on our manuscript of ‘horticulturae-2026635’, the comments and suggestions are quite valuable for us. We have read and considered all the comments carefully, and tried our best to revise the original manuscript according to the comments of editors and reviewers.
According to your requirements, we have made point-to-point modifications to the questions and comments which were raised by the reviewers in the manuscript, and marked the modification of reviewers' comments in Red in the body of the revised manuscript.
We sincerely hope the Editor and Reviewers will satisfy with our revisions for the original manuscript. If you still have any questions about this paper, please don’t hesitate to contact me at the address below.
Yours sincerely,
Mingbao Luan
Yue Wang
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Xianjiahu West Road, Changsha 410205, Hunan Province, China
E-mail:[email protected]
E-mail: [email protected]
Responses to reviewer #2
Comment 1: 1. ABSTRACT/INTRODUCTION. Twenty-four SSR markers are fine for diversity analyses, but seem too few to visualize their use in marker-assisted selection in Dioscorea (in fact in any species).
Response: Dear reviewers, your suggestions are very helpful for our experimental design. In general, most researchers choose more SSR markers for genetic diversity analysis. However, as researchers become more proficient in the use of SSR markers, the fewer SSR markers can be used to separate different genetic backgrounds. Cheng et al. (2022) used 25 EST-SSR markers that can effectively distinguish Pistacia chinensis males and females, providing a solid framework for sex determination in plant seedlings. Li et al. (2020) classified 53 registered olives into two major groups based on 21 SSR markers. Narineh et al. (2022) conducted a genetic diversity and fingerprinting study using 5 SSR markers on 80 trees of 13 sweet orange varieties in Mazandara province, dividing the studied genotypes into two major distinct groups. Tiwari et al. (2019) used 14 SSR markers to analyze allelic variation in wild potato and classified 82 attributed SSR allelic profiles into five major clusters (I-V).In summary, fewer SSR markers can also be fine for diversity analyses.
Comment 2. INTRODUCTION, Lines 78. SSRs are not dominantly inherited; in fact they are recognized and appreciated by their co-dominant inheritance.
Response: Dear Reviewer, thank you for your suggestions and we made a change in the manuscript in line 78.
Comment 3. TABLE 1. For those readers unfamiliar with Chinese geography, it would be very useful to consult the geographical coordinates of provenance of the yam accessions, in addition to the name of the place name.
Response: Dear Reviewer, thank you for your suggestions and we add the geographical coordinates of the yam accessions in tabe l 1 of the manuscript.
Comment4. MATERIALS AND METHODS. Assessing the banding pattern in SSR as present or absent dismisses a lot of power inherent to this type of markers, given that they are usually co-dominant. How then is it recorded when two bands appear, as is the case with heterozygotes? Certainly the absence of bands also occurs in low frequency, but only in cases of null alleles, where the template DNA has undergone some mutation in the banding zone. Please justitfy/clarify futher.
Response: Dear reviewer, Thank you very much for your questions. SSR is a class of repetitive sequences consisting of a few nucleotides (1-6) as repetitive units up to several tens of nucleotides, which cause a high variability in SSR length due to the different number of repetitive units. Pairs of primers can be designed in a specific order with microsatellite regions. The polymorphism of SSR loci among different individuals can be revealed by PCR technique and polyacrylamide gel electrophoresis. When we record SSR bands, each pair of primers in different samples can amplify multiple bands of different lengths, which may be more than one, sometimes up to 10 or more, and these different length bands are polymorphic bands. In our experiments, my s4-1 primer has 3 polymorphic bands among 32 samples, and each sample has several polymorphic bands, which can be marked as "1" or "0" under this locus. The heterozygotes may have two bands from their parents, record "1" under the polymorphic band site of each of the two parents; or the size of the band is between the sizes of two parents bands, record "1" under another polymorphic site (not the polymorphic bands of the two parents). The position of SSRs on the genome varies, but the sequences at both ends are mostly conserved single-copy sequences, and the SSR primers are designed based on this principle, and the template DNA needs to have some large variation in the band region to cause the absence of bands, which also indicates that the sample is genetically distant.
Regarding the answer to this question, I made a change in line 166-169 of the manuscript.
Author Response File: Author Response.docx
