Telomere-to-Telomere Genome Assembly of Tibetan Medicinal Mushroom Ganoderma leucocontextum and the First Copia Centromeric Retrotransposon in Macro-Fungi Genome

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript described the sequencing, telomere-to-telomere assembly, and annotation of the Tibetan medicinal mushroom Ganoderma leucocontextum. Overall, it's very well executed study and the manuscript was clearly written and easy to follow. However, I do have a few comments and suggestions. 

1. Given that three genomes of this species have already been published, all with high coverage and reasonable assembly, it would be interesting to provide an overall assessment of the genomic differences among these four genomes. Such a comparison could elevate the present study above the previous ones and provide potential markers for future breeding purposes.

2. What's the mating type loci look like? Identifying them could provide information about its mating system.

3. The assembled genomes described only the nuclear chromosomes. What's its mitochondrial genome size, structure, and gene content?

Below are few minor comments:

line 29, change "fungi" to "fungus"

line 30, delete "be"

line 121 to 122: why include Arabidopsis here? And, give this is a monokaryon, why is heterozygosity predicted?

line 310, not clear, does this mean that 1,758 genes represent 99.7% of the complete genes. What is a "complete gene"?

line 352, one of the "submetacentric" should be changed to "subtelocentric".

line 412, the first sentence is incomplete.

 

Comments on the Quality of English Language

Overall, the quality of English is excellent.

Author Response

Response to reviewer 2

 

Comments and Suggestions for Authors

 

His manuscript described the sequencing, telomere-to-telomere assembly, and annotation of the Tibetan medicinal mushroom Ganoderma leucocontextum. Overall, it's very well executed study and the manuscript was clearly written and easy to follow. However, I do have a few comments and suggestions. 

R: Thanks very much for the positive comments and valuable suggestions on the Ms. We have revised the Ms. carefully following the suggestions one by one.

 

1.Given that three genomes of this species have already been published, all with high coverage and reasonable assembly, it would be interesting to provide an overall assessment of the genomic differences among these four genomes. Such a comparison could elevate the present study above the previous ones and provide potential markers for future breeding purposes.

R: We previously presented the assessment of these four genomes in supplemental Table S4, including information on genome quality, gene prediction, and so on. Following the suggestion, the comparison has now been moved to Table 1 and Table S4 has been deleted.

 

2. What's the mating type loci look like? Identifying them could provide information about its mating system.

R: Ganoderma leucocontextum is tetrapolar heterothallism. The mating type loci of strain GL72 have been analyzed and published in Journal of Fungal Research (https://doi.org/10.13341/j.jfr.2022.1516) by this team. We have identified HD1 and HD2 at MAT-A, and the flanking sequences of mip and β-fg genes. There were 8 putative pheromone receptor-encoding genes, 8 putative pheromone precursor-encoding genes at MAT-B.

 

3.The assembled genomes described only the nuclear chromosomes. What's its mitochondrial genome size, structure, and gene content?

R: In addition to the nuclear genome, we assembled a circular mitochondrial genome with a size of 89,684 bp, a GC content of 27.05%, comprising fifteen protein-coding genes, 2 ribosomal RNA genes, and 25 tRNA genes. The relevant results have been added in lines 261-264.

 

Below are few minor comments:

line 29, change "fungi" to "fungus"

R: Thanks and revised.

 

line 30, delete "be"

R: Agreed and revised.

 

line 121 to 122: why include Arabidopsis here? And, give this is a monokaryon, why is heterozygosity predicted?

R: Sorry for our mistake. Initially, we incorrectly thought that the findGSE software utilized Arabidopsis as a control. Now it has been deleted. Heterozygosity prediction was aimed to further confirm the monokaryon of strain GL72 and ensure the determination of assembly strategy.

 

line 310, not clear, does this mean that 1,758 genes represent 99.7% of the complete genes. What is a "complete gene"?

R: Here, complete gene is synonymous with complete BUSCOs, indicating genes within the genome that can be entirely matched with the BUSCO database, as opposed to being fragmented or missing. It was changed to ‘complete BUSCOs’ in line 312.

 

 

line 352, one of the "submetacentric" should be changed to "subtelocentric".

R: Agreed and revised.

 

line 412, the first sentence is incomplete.

R: Thanks. The complete sentence should be that “we tried to blast the high-quality genomes of G. leucocontextum strain CCMJ4170 using sequences of GlCEN427 and GlCEN706”.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The problem with the present manuscript is that there are at least three recently published papers dealing with the G. leucocontextum genome structure: ref 7 G3: Genes, Genomes, Genet. 2021, 11, jkab337, ref 8 J. Fungi 2022, 8, 311 and ref. 9. Mol. Ecol. Resour. 2022, 10.1111/1755-0998.13718.

The new ideas in the present manuscript are (1) to create a gapless genome, (2) the location of telomeres and (3) centromeres on the 13 chromosomes known from previous research. The previous publications   must have been useful in creating the present work. It would be nice for every sequenced fungal genome to have the genome arranged in chromosomes instead of a number of scaffolds.

 In the present manuscript the telomeres are located at both ends and the centromeres in each of the 13 chromosomes. It is good to publish the manuscript after some polishing, so the fungal community may certify the presented interpretations about the structure of telomeres and centromeres, although the other information about the genomic structure, gene number and especially the genes encoding secondary metabolites are already well reported in the previous publications.

The next sites need improvement

I wonder why the monokaryotic strain GL72 by protoplast monokaryogenesis technique. Why haploid spores from the fruiting body are not used? Is there some problem with haploid spore germination.

 Section 2.2. Library construction and sequencing is not well written. It is very difficult to follow or understand which sequencing method was used to obtain a specific result. The reason might be that the all the sequencing was done by a company. Reference 16 for Hi-C method is not right, in the Nature Genetics article the Hi-C method is from the article Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009). This awakes the question how the monokaryon GL72 was prepared for Hi- C method. In the manuscript there is no information, but in the manuscript the Hi-C method seems to have had an important role for all the structures presented in the first chapter of Results and Fig. 3.

Table 1. Global statistics for the genome of Ganoderma leucocontextum strain GL72

The results presented in Table 1 should be compared with those reported in references 7, 8 and 9. The comparison of genome features between GL72 and previously published strains should be moved from supplementary material Table S4 into the actual text to notify the previous research of G. leucocontextum. Especially since the authors state (lines 334-336) that “Overall, our newly sequenced G. leucocontextum genome surpasses the previously published genomes in terms of contiguity and assembly quality.

The previous work dealing with centromeric region in fungi is well referred to. It is also interesting to see whether the centromeric specific sequences CIGEN 407 and 706 will be present in other basidiomycetes than in species belonging to Polyporales. Instead references about the telomere research in fungi are absent, except reference 83.  It would be interesting to know whether the telomere associated sequences in the publication Fungal Genetics and Biology vol. 45: Pages S54-S62 are present also in Ganoderma genus.

 

 

Author Response

Response to reviewer 2

 

Comments and Suggestions for Authors

 

The problem with the present manuscript is that there are at least three recently published papers dealing with the G. leucocontextum genome structure: ref 7 G3: Genes, Genomes, Genet. 2021, 11, jkab337, ref 8 J. Fungi 2022, 8, 311 and ref. 9. Mol. Ecol. Resour. 2022, 10.1111/1755-0998.13718.

The new ideas in the present manuscript are (1) to create a gapless genome, (2) the location of telomeres and (3) centromeres on the 13 chromosomes known from previous research. The previous publications must have been useful in creating the present work. It would be nice for every sequenced fungal genome to have the genome arranged in chromosomes instead of a number of scaffolds.

In the present manuscript the telomeres are located at both ends and the centromeres in each of the 13 chromosomes. It is good to publish the manuscript after some polishing, so the fungal community may certify the presented interpretations about the structure of telomeres and centromeres, although the other information about the genomic structure, gene number and especially the genes encoding secondary metabolites are already well reported in the previous publications.

R: Thanks for your positive comments and valuable suggestions on the Ms. We have revised the Ms according to the comments and suggestions point by point.

 

The next sites need improvement

I wonder why the monokaryotic strain GL72 by protoplast monokaryogenesis technique. Why haploid spores from the fruiting body are not used? Is there some problem with haploid spore germination.

R: Yes, germination of the basidiospores of some Ganoderma species, including G. leucocontextum, is difficult. Few basidiospores of this species can germinate even under the optimized conditions.

 

Section 2.2. Library construction and sequencing is not well written. It is very difficult to follow or understand which sequencing method was used to obtain a specific result. The reason might be that the all the sequencing was done by a company. Reference 16 for Hi-C method is not right, in the Nature Genetics article the Hi-C method is from the article Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009). This awakes the question how the monokaryon GL72 was prepared for Hi- C method. In the manuscript there is no information, but in the manuscript the Hi-C method seems to have had an important role for all the structures presented in the first chapter of Results and Fig. 3.

R: This section has been modified, and some details on how the monokaryon GL72 was prepared for Hi- C method have been added in lines 94-105. Sorry for the wrongly cited reference 16. It has been corrected.

Table 1. Global statistics for the genome of Ganoderma leucocontextum strain GL72

The results presented in Table 1 should be compared with those reported in references 7, 8 and 9. The comparison of genome features between GL72 and previously published strains should be moved from supplementary material Table S4 into the actual text to notify the previous research of G. leucocontextum. Especially since the authors state (lines 334-336) that “Overall, our newly sequenced G. leucocontextum genome surpasses the previously published genomes in terms of contiguity and assembly quality.

R: Agreed. We have incorporated the key features related to the genome into Table 1 and Table S4 has been deleted.

 

The previous work dealing with centromeric region in fungi is well referred to. It is also interesting to see whether the centromeric specific sequences GlGEN407 and 706 will be present in other basidiomycetes than in species belonging to Polyporales. Instead references about the telomere research in fungi are absent, except reference 83.  It would be interesting to know whether the telomere associated sequences in the publication Fungal Genetics and Biology vol. 45: Pages S54-S62 are present also in Ganoderma genus.

R: Thanks. This is a very insightful question. GlCEN407 was only present in Polyporales as mentioned in lines 419-423 in the Ms. As for CIGEN706, upon conducting a blastn search in the NCBI genome database with a cutoff of e-5, we noted its occurrence not only in Polyporaceae but also various species of Basidiomycota, such as Schizophyllum commune, Mycena indigotica, Agaricus bisporus, and so forth. Notably, it was absent in Ascomycetes, and the relevant analysis has been incorporated into lines 424-427.

Additionally, in the publication "Fungal Genetics and Biology" vol. 45: Pages S54-S62, it was mentioned that adjacent to the telomere repeats is the occurrence of sequences encoding RecQ helicases, which may be unique to fungal genomes. We identified genes in our genome that potentially encode RecQ helicases based on the hidden markov model profile (hmm) for RecQ helicase (PTHR13710) using hmmsearch 3.3.2 with a cutoff of e-5. It was found that there were genes encoding RecQ helicases near some telomeres, including Chr04, 06, 08, 12. The relevant analyses have been incorporated into lines 599-605 and Table S11.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed my concerns. I recommend the acceptance of the manuscript for publication.