Comparative Transcriptome Analysis to Identify Candidate Genes Related to Chlorogenic Acid and Flavonoids Biosynthesis in Iridaceae

Round 1

Reviewer 1 Report

This manuscript presents a Comparative transcriptome analysis to identify candidate genes related to chlorogenic acid and flavonoids biosynthesis in iridaceae. The authors identify 761 metabolites, including 202 flavonoids, 106 phenolic acids based on metabolome profiling. Most of flavonoids were highly accumulated in I. pallida. Besides, author identified many key genes involved in chlorogenic acid and flavonoids biosynthesis. Generally speaking, the data presented by authors is important and informative and lays the foundation for future study of abiotic chlorogenic acid and flavonoids biosynthesis in iris rhizome. However, some points should be addressed to improve the manuscript:
1. Please provide scale bar in Figure 1
2. The format of reagent information provided in the paper method is not unified. Some provide manufacturer information, some do not; some provide manufacturer's city information, and some do not. Please provide the manufacturer information of RNase-free DNase I in line 127.
3. What’s the y-coordinate in Figure 5b?
4. The writing format of the Dof gene in the text is not uniform, sometimes uppercase (DOF, line 284), sometimes lowercase (Dof, line 327).
5. The format of ‘I. germanica’ shoult italic in line 177.
6. The typeface of manuscript should be unified. In current version, some characters use the new Roman font, and some us Palatino Linotype.

Author Response

This manuscript presents a Comparative transcriptome analysis to identify candidate genes related to chlorogenic acid and flavonoids biosynthesis in iridaceae. The authors identify 761 metabolites, including 202 flavonoids, 106 phenolic acids based on metabolome profiling. Most of flavonoids were highly accumulated in I. pallida. Besides, author identified many key genes involved in chlorogenic acid and flavonoids biosynthesis. Generally speaking, the data presented by authors is important and informative and lays the foundation for future study of abiotic chlorogenic acid and flavonoids biosynthesis in iris rhizome. However, some points should be addressed to improve the manuscript:
1. Please provide scale bar in Figure 1

Answer: We added the scale bar in revised Figure 1.
2. The format of reagent information provided in the paper method is not unified. Some provide manufacturer information, some do not; some provide manufacturer's city information, and some do not. Please provide the manufacturer information of RNase-free DNase I in line 127.

Answer: Thank you for your suggestion, we provide the manufacturer information of RNase-free DNase I, and unified the format of reagent information according to your suggestion.
3. What’s the y-coordinate in Figure 5b?

Answer: The y-coordinate in Figure 5b represented for relative expression level. We redrawn the Figure 5b and 6b, and provided the y-coordinate title in figure 5b and 6b according to your suggestion.
4. The writing format of the Dof gene in the text is not uniform, sometimes uppercase (DOF, line 284), sometimes lowercase (Dof, line 327).

Answer: We modified the wrong format (DOF) in new version.
5. The format of ‘I. germanica’ should italic in line 177.

Answer: we revised this mistake.
6. The typeface of manuscript should be unified. In current version, some characters use the new Roman font, and some use Palatino Linotype.

Answer: We have unified the font of all characters in the text into Palatino Linotype.

Reviewer 2 Report

 Some candidate genes related to CGA and flavonoids biosynthesis in rhizome were found in this study. It is helpful to explore the potential molecular regulatory mechanism for CGA and flavonoids biosynthesis.

1、The sampling stage of rhizome should be clearly described. Is it at the flowering stage？

2、Four ANR genes were got from rhizome in this study. But many plants have two ANR genes involved in the flavonoid pathway. Please show your opinion in the discussion.

3、Are you sure Figure 1B is I. pallida? The beard color of I. pallidais described in the Flora should be yellow. 

Author Response

Some candidate genes related to CGA and flavonoids biosynthesis in rhizome were found in this study. It is helpful to explore the potential molecular regulatory mechanism for CGA and flavonoids biosynthesis.

1. The sampling stage of rhizome should be clearly described. Is it at the flowering stage？

Anwer: We sampled the iris rhizome in mid August. In this stage, the iris has finished flowering. We provided the sampling time in revised method.

2. Four ANR genes were got from rhizome in this study. But many plants have two ANR genes involved in the flavonoid pathway. Please show your opinion in the discussion.

Response: Thank you for your suggestion. In my opinion, more ANR genes identified in iris genome may be caused by much larger genome size. In our study, a total of 94,461 transcripts were identified using single molecular long-read sequencing, including four ANR transcripts. Excluding the influence of multiple transcripts (isoforms) produced by one gene which caused by the effect of selective promoters and alternative splicing, all four ANR transcripts were produced by four different gene sets. It is reported that genome size of iris  is 4494.1 Mb, much larger than those of apple (742.3 Mb) and Glycine max (941 to 1,374 Mb), which harbored two ANRs, respectively. It was also larger than those of  Arabidopsis  (135 MB) and rice (389  MB),  which harbored only one ANR, respetively. Thus, larger genome size harboring more gene sets resulted in more ANR genes.

Reference:

Zhu, Min.; Wang, Y. P.，Xiong, W. B.; Luo Y. G.; Duan. L. X. et al. Genome size estimation for three iridaceae plants by using flow cytometry. Journal of Chinese Medicinal Materials 2018, 41, 2306-2310.

Han Y.P.; Sornkanok V.; Ruth E.S.; Schuyler S.K. Introduction of apple ANR gene into tobacco inhibits expression of both CHI and DFR gene in flowers, leading to loss of anthocyanin, J. Exp. Bot. 2012, 63, 2437-2447.

Nicolas, D.;Jean-Marc, C.; Gareth, L.; Claude, B.; Nathalie C, et al. High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat. Genet. 2017 49, 1099-1106.

Kovinich, N.; Saleem, A.; Arnason, J. T.; Miki, B. Identification of two Anthocyanidin Reductase genes and three red-brown soybean accessions with reduced Anthocyanidin Reductase 1 mRNA, activity, and seed coat proanthocyanidin amounts. J. Agr. Food Chem. 2012, 60, 574-584.

Liu, Y. C.; Du, H. L.; Li, P. C.; Shen, Y. T.; Peng, H. et al.  Pan-Genome of Wild and Cultivated Soybeans. Cell 2020, 182, 162-176.

Bok, K. Y.; Aye, T. A.; Yeji, K.; Li, X.; Woong, C. J., et al. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and measurement of catechin and epicatechin in Tartary Buckwheat. The scientific world journal 2014, 2014:726567.

Xie, D. Y.; Sharma，S. B.; Paiva，N. L.; Ferreira, D.; Dixon, R. A. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 2003, 299, 396-9.

3. Are you sure Figure 1B is I. pallida? The beard color of I. pallidais described in the Flora should be yellow.

 Anwer: Thank you for your reminding. We checked our figures, and confirmed that Figure 1B is I. pallida. The beard color of I. pallidais in Figure 1B is canary yellow, especially at bear tip.