Characterization of a Wheat-Dasypyrum breviaristatum Chromosome Addition and Its Derived Progenies Carrying Novel Dasypyrum-Specific Gliadin Genes
and Guangrong Li 1,*
Round 1
Reviewer 1 Report
The authors used ND-FISH and Oligo-FISH painting by oligo-pools of Synt1 to Synt7 to identify the linkage group of D. breviaristatum chromosomes in wheat-Dasypyrum breviaristatum lines and identified 6VbS.2VbL translocation chromosomes containing agronomically important genes. The transmission of 6VbS and 2VbL in D2138 and wheat hybrids were tested using three wheat cultivars viz., MY11, JM22 and CM62 where D2138 was used as a female parent. The F2 plants generated in the three crosses were screened using oligo and Synt probes. Four types of the homozygous 6VbS.2VbL deletions were recovered in the F3 progenies. Where all four types of 6VbS.2VbL deletion lines were confirmed by the sequential FISH with the Dasypyrum-specific probe Oligo-pDb12H. The four deletion lines were progressed to F4 generation by selfing to identify the agronomically important genes by molecular analysis. It was found that the sequences of α-gliadin genes were located on 6VbS approximately 20Mb to 170Mb region of 6VbS. Along with other important agronomic traits identified in 6VbS line this line can be used in the wheat breeding for quality improvement.
The authors did extensive work but the display of the work is very poor. In many instances the sentences convey wrong meaning or confusing. Clear projection of the results and discussion was missing. Serious English checking is needed. The authors should avoid unwanted discussion on the topic which is not related to the title of the manuscript. The correctness of the gene name, symbols and other details in the text and the figures need to be checked. Over all, exhaustive details with poor display of the information.
Author Response
The authors used ND-FISH and Oligo-FISH painting by oligo-pools of Synt1 to Synt7 to identify the linkage group of D. breviaristatum chromosomes in wheat-Dasypyrum breviaristatum lines and identified 6VbS.2VbL translocation chromosomes containing agronomically important genes. The transmission of 6VbS and 2VbL in D2138 and wheat hybrids were tested using three wheat cultivars viz., MY11, JM22 and CM62 where D2138 was used as a female parent. The F2 plants generated in the three crosses were screened using oligo and Synt probes. Four types of the homozygous 6VbS.2VbL deletions were recovered in the F3 progenies. Where all four types of 6VbS.2VbL deletion lines were confirmed by the sequential FISH with the Dasypyrum-specific probe Oligo-pDb12H. The four deletion lines were progressed to F4 generation by selfing to identify the agronomically important genes by molecular analysis. It was found that the sequences of α-gliadin genes were located on 6VbS approximately 20Mb to 170Mb region of 6VbS. Along with other important agronomic traits identified in 6VbS line this line can be used in the wheat breeding for quality improvement.
The authors did extensive work but the display of the work is very poor. In many instances the sentences convey wrong meaning or confusing. Clear projection of the results and discussion was missing. Serious English checking is needed. The authors should avoid unwanted discussion on the topic which is not related to the title of the manuscript. The correctness of the gene name, symbols and other details in the text and the figures need to be checked. Over all, exhaustive details with poor display of the information.
Response: Thank you for your suggestion. We agree to edit the description of sentences to avoid the grammatical and spelling errors. The discussion part was also adjusted in the version of revised manuscript. The description of the FISH characterization of the Dasypyrum breviaristatum species, and the possible effect of chromosome modification in D2138 derivatives were reduced. The discussion on gliadin traits improvement were added. The detailed gene names and symbols were also corrected. Indeed, more advanced population from further generations will be performed for the genetic resources to assess the breeding output.
Reviewer 2 Report
Impression:
The authors have extended their previous work on the amphiploid TDH_2 and disomic translation line D2138 (*), obtaining various wheat-D2138 hybrids in which part of the Dasypyrum breviaristatum chromosome 6 has been introgressed. The authors use various techniques to this end, including non-denatured fluorscent in-situ hybridization and chromosome painting with a number of probes previously proven to be fit for purpose. This introgression of chromosome 6 short arm (6VbS) in turn has had a favorable impact on the agronomic traits of these hybrids (obtained from field trials) as well as the gliadin protein profile. As also indicated by the authors in the conclusions, the resulting lines will be useful for further breeding.
*) https://doi.org/10.1139/gen-2020-0147
1) Main comments
Whilst the manuscript comprehensively reviews the experimental data on chromosome identification and analysis, and more concisely the outcomes of the agronomic and gliadin analysis, the manuscript would merit from including more context. This could include providing more details on, e.g.,
1a) the probes used for FISH e.g. pSc119 and pTa535 targeting specifically certain repetitive regions within cereal chromosomes,
1b) improvements in certain agronomic traits are highlighted, such as the increased protein contents, yet the increased number of tillers, which is often linked to higher yield (not included in the agronomic traits?) is not, which is a pity and should be included as well;
1c) the implications of the presence of the 3 alpha-gliadin genes found on introgressed chromosome fragment 6Vb for celiac patients and quality? One could check, for example, for the presence of a 33-mer highly immunogenic peptide fragment in the translated sequence (e.g., https://dx.doi.org/10.1111/tpj.15147)
1d) the potential benefit fo having a gametocidal gene in the introgressed 6VbS for wheat breeding
Minor comments:
Lines 277-279: sentence appears to be missing something
Line 287, Figure 6b: replace "glidin" with "gliadin
Author Response
The authors have extended their previous work on the amphiploid TDH_2 and disomic translation line D2138 (*), obtaining various wheat-D2138 hybrids in which part of the Dasypyrum breviaristatum chromosome 6 has been introgressed. The authors use various techniques to this end, including non-denatured fluorscent in-situ hybridization and chromosome painting with a number of probes previously proven to be fit for purpose. This introgression of chromosome 6 short arm (6VbS) in turn has had a favorable impact on the agronomic traits of these hybrids (obtained from field trials) as well as the gliadin protein profile. As also indicated by the authors in the conclusions, the resulting lines will be useful for further breeding.
1) Main comments
Whilst the manuscript comprehensively reviews the experimental data on chromosome identification and analysis, and more concisely the outcomes of the agronomic and gliadin analysis, the manuscript would merit from including more context. This could include providing more details on, e.g.,
1a) the probes used for FISH e.g. pSc119 and pTa535 targeting specifically certain repetitive regions within cereal chromosomes,
Response: We agree to add the reference for the probes pSc119.2 (a 120-bp family of tandem repeat), and pTa535 (a wheat specific tandem repeat).
1b) improvements in certain agronomic traits are highlighted, such as the increased protein contents, yet the increased number of tillers, which is often linked to higher yield (not included in the agronomic traits?) is not, which is a pity and should be included as well;
Response: We agree to add the statement for the significance of high yield improvement.
1c) the implications of the presence of the 3 alpha-gliadin genes found on introgressed chromosome fragment 6Vb for celiac patients and quality? One could check, for example, for the presence of a 33-mer highly immunogenic peptide fragment in the translated sequence (e.g., https://dx.doi.org/10.1111/tpj.15147)
Response: We agree to add the search of reported T cell stimulatory epitopes, the innate peptide p31-43 and the 33-mer peptide in the three sequences as suggested. The distribution and variation of the above epitops were not found, which provided probabilities to select less toxic germplasm for celiac disease patients.
1d) the potential benefit for having a gametocidal gene in the introgressed 6VbS for wheat breeding
Response: It is likely that the present 6VbS contained the gametocidal gene, which may cause more chromosomal variations of offspring for potentially useful of wheat-alien transfer. In fact, the plants with homozygous 6VbS in wheat displayed stable.
Minor comments:
Lines 277-279: sentence appears to be missing something
Line 287, Figure 6b: replace "glidin" with "gliadin
Response: We agree to make the corresponding corrections as suggested.
Reviewer 3 Report
In the manuscript entitled “Characterization of a wheat-Dasypyrum breviaristatum chromosome addition and its derived progenies carrying novel Dasypyrum-specific gliadin genes”, the authors have done a interesting study and identified novel genome segment from wild Dasypyrum and showed their functional role in improving the agronomic traits in cultivated wheat. They also showed potential to improve the breeding quality in cultivated wheat. The manuscript seems fine to me, however, I have some minor queries/ concerns regarding the study which are mentioned below:
1. Wheat is self-pollinated and Dasypyrum is cross pollinated species. D2138, is cross between TDH-2 and MY11. It will be interesting to know the nature of BC1F4 generation of the cross, which plant taken as male or female for backcross, and if there are any significant changes in the phenotypes of the progeny compared to the parents.
2. (Line 175-178): The deletions of telomeric region of wheat chromosomes were seen more in B-genome compared to A and D. Authors should shed light on this aspect?
3. (Table:2): After through screening of different lines and hybrids, the authors stated the involvement of 6VbS region in improving agronomic traits in wheat, it is clear through data shown in hybrids of D2138/MY11, D2138/JM22 and D2138/CM62. However, it will be interesting to know if there are differences in traits when plants compared for heterozygous (+6VbS/-6VbS) and homozygous (+6VbS/+6VbS) plants.
4. How many plants were screened for total plant height, spikelet lengths and Tillers per plants? And if all the individual plants were screened for the presence of 6VbS?
5. Authors should discuss the functional significance of α-gliadin gene in the manuscript, also its positive implication in seed protein quality in cultivating wheat.
6. The manuscript should be checked for grammatical and linguistic errors, such as (Line 325)-Determine spelling, (Line 289)-Figure 6a legends “gliain”, etc.
Author Response
In the manuscript entitled “Characterization of a wheat-Dasypyrum breviaristatum chromosome addition and its derived progenies carrying novel Dasypyrum-specific gliadin genes”, the authors have done a interesting study and identified novel genome segment from wild Dasypyrum and showed their functional role in improving the agronomic traits in cultivated wheat. They also showed potential to improve the breeding quality in cultivated wheat. The manuscript seems fine to me, however, I have some minor queries/ concerns regarding the study which are mentioned below:
- Wheat is self-pollinated and Dasypyrumis cross pollinated species. D2138, is cross between TDH-2 and MY11. It will be interesting to know the nature of BC1F4 generation of the cross, which plant taken as male or female for backcross, and if there are any significant changes in the phenotypes of the progeny compared to the parents.
Response: Thank you for your suggestion. D2138 is developed from the cross between TDH-2 and MY11. The TDH-2 is a wheat-D. breviaristatum partial amphiploid with genome AABBVbVb. It is self-pollination and chromosome stable. Therefore, BC1F4 generation from wheat (AABBDD) cross to AABBVbVb using wheat as backcrossing parents. The F4 generation gave rise to the stable line D2138. Each generation, the cytological identification was performed. The phenotype of D2138 were clearly different from the wheat parent MY11 by a later flowering time and loose plant-type.
- (Line 175-178): The deletions of telomeric region of wheat chromosomes were seen more in B-genome compared to A and D. Authors should shed light on this aspect?
Response: Thank you for your suggestion. The B- genome has high percentage of heterochromatin and rich of repetitive sequences, such as Oligo-pSc119.2 located regions, which cause the B genome chromosomes easy broken at the regions. We added the discussion on this aspect.
- (Table:2): After through screening of different lines and hybrids, the authors stated the involvement of 6VbS region in improving agronomic traits in wheat, it is clear through data shown in hybrids of D2138/MY11, D2138/JM22 and D2138/CM62. However, it will be interesting to know if there are differences in traits when plants compared for heterozygous (+6VbS/-6VbS) and homozygous (+6VbS/+6VbS) plants.
Response: Thank you for your suggestion. The plants with homozygous 6VbS and heterozygous 6VbS do not appear clear differences for observed agronomic traits.
- How many plants were screened for total plant height, spikelet lengths and Tillers per plants? And if all the individual plants were screened for the presence of 6VbS?
Response: About 15-30 plants were used to test the agronomic traits. All the individual plants were screened for the presence or absence of 6VbS by FISH of root tip mitotic cells.
- Authors should discuss the functional significance of α-gliadin gene in the manuscript, also its positive implication in seed protein quality in cultivating wheat.
Response: We agree to add the point in discussion. The present 6Vb derivative lines may introduce additional multi-copies of Dasypyrum gliadin genes. The gliadins present in the monomer or polymer fraction may have positive effects on the physical properties of wheat flour dough.
Round 2
Reviewer 1 Report
Still the manuscript requires English revisions with the help of native English speaker.
In labeling the individual chromosome of D. breviaristatum, 5Vb was missing in Figure 1d
Line 166 should be changed to “FISH revealed the presence of aneuploidy and chromosome rearrangements in introgression lines.
Lines 361-362: sentence formation not good. Suggest revising
Line 366: suggest deleting “however”
Line 415: why specifically CD patients? Overall it is gluten sensitive persons.
Line 418: suggest deleting “firstly”
Author Response
Still the manuscript requires English revisions with the help of native English speaker.
Response: The English was improved by Dr. Ian Dundas from Australia.
In labeling the individual chromosome of D. breviaristatum, 5Vb was missing in Figure 1d
Response: We add the mark of 5Vb in the figure.
Line 166 should be changed to “FISH revealed the presence of aneuploidy and chromosome rearrangements in introgression lines.
Response: We agree to make the changes.
Lines 361-362: sentence formation not good. Suggest revising
Response: We agree to edit the description.
Line 366: suggest deleting “however”
Response: We agree to the deletion.
Line 415: why specifically CD patients? Overall it is gluten sensitive persons.
Response: We agree to the description of “gluten sensitive persons” in all manuscript.
Line 418: suggest deleting “firstly”
Response: We agree to the deletion.
