Quality Green Tea (Camellia sinensis L.) Clones Marked through Novel Traits

Round 1

Reviewer 1 Report

The manuscript covers the aim and scope of the Journal; however it needs correction before acceptance for publication. The Authors should read their manuscript carefully because there a lot of mistakes (e.g. line 61 “corbon”, line 140 “phorosyntheis”), editorial errors (lack or unnecessarily spaces, unnecessarily capital letters) and misleading statements. Some examples are below.

Line 92-93: “Polyphenolic compounds were extracted from finely ground sample with 10ml 70% methanol  using a modified Folin–Ciocalteu’s method (…)” - Folin–Ciocalteu’s method is not a method for extraction. This is a method to assess the antioxidant activity based on  total polyphenolic content.

Line 95: “Theanine was estimated in HPLC by extracting (…) – it  should be: “samples were prepared by extracting…)

Line 98: “ (…) C18 column with 1.0 ml flow rate using water as eluent…” - flow rate is not column parameter. It should be: using water as eluent at flow rate of ..

Line 99:  “Theanine was detected by comparing with Theanine standard through a previously  prepared calibration curve” – it is completely unclear. What parameter was compared? Was calibration curve prepared for quantity analysis (lack of detail, e.g. standard concentration)?

Moreover:

Lack of experimental data, e.g.: data of chromatographic column (length, particle size, producer), data of HPLC apparatus (e.g. type of detector, producer), temperature of thermostat; how many samples were weighed? how many water was taken for extraction?; data was statistically analysed using …

Ref. 10 is cited in wrong place. Lines 98-99 describe chromatographic condition and cited work is on colorimetric method. Lack of references for chromatographic condition.

It would be interesting to show the results for TPC assay and theanine determination separately (not only ratio of both)

English correction of text is needed

Author Response

Response to Reviewer 1 Comments

Point 1: The manuscript covers the aim and scope of the Journal; however it needs correction before acceptance for publication. The Authors should read their manuscript carefully because there a lot of mistakes (e.g. line 61 “corbon”, line 140 “phorosyntheis”), editorial errors (lack or unnecessarily spaces, unnecessarily capital letters) and misleading statements.

 Response 1:

Line 61 “carbon” Line 140 “photosyntheis” Corrected editorial errors

Point 2: Line 92-93: “Polyphenolic compounds were extracted from finely ground sample with 10ml 70% methanol using a modified Folin–Ciocalteu’s method (…)” - Folin–Ciocalteu’s method is not a method for extraction. This is a method to assess the antioxidant activity based on total polyphenolic content.

Response 2:

Polyphenolic compounds are extracted from finely ground sample with 10ml 70% methanol. The extraction is carried out in a water bath set at 70°C over 10 minutes time. Total polyphenol content of leaf tea was then determined by a colorimetric assay using Folin-Ciocalteu phenol reagent by following the method of ISO 14502-1 (ISO, 2005). Total polyphenols content of the samples are expressed as gallic acid equivalent.

Point3. Line 95: “Theanine was estimated in HPLC by extracting (…) – it should be: “samples were prepared by extracting…)

Response 3:

Extraction of theanine was carried by brewing the finely powdered sample with hot water for 5 min using a magnetic stirrer. The brew was filtered through filter paper and allowed to cool. Cooled extract is filtered with 0.45µm membrane filter and 20 µl is injected into a Phenomenex C18 5µ 250mm x 4.6mm reverse phase column.

Point 4. Line 98: “(…) C18 column with 1.0 ml flow rate using water as eluent…” - flow rate is not column parameter. It should be: using water as eluent at flow rate of ..

Response 4:

Theanine estimation was carried out in   UHPLC DIONEX Ultimate 3000 UHPLC system. The flow rate was 1.0 ml per min using water as eluent, UV detection at 210 nm and column oven temperature was maintained at 25⁰

Point 5. Line 99:  “Theanine was detected by comparing with Theanine standard through a previously prepared calibration curve” – it is completely unclear. What parameter was compared? Was calibration curve prepared for quantity analysis (lack of detail, e.g. standard concentration)?

 Response 5:

A calibration curve was prepared using theanine standard (Sigma, Aldrich) at concentrations 10, 20, 50, 100 and 150ppm. Theanine was detected and estimated by comparing the retention time and peak area with theanine calibration curve (Draft International Standard, ISO/DIS 11287)

Point 6 Moreover:

Lack of experimental data, e.g.: data of chromatographic column (length, particle size, producer), data of HPLC apparatus (e.g. type of detector, producer), temperature of thermostat; how many samples were weighed? How many water was taken for extraction?; data was statistically analysed using …

Response 6:

Phenomenex C18 5µ 250mm x 4.6mm reverse phase column. UHPLC DIONEX Ultimate 3000 UHPLC system. UV detection at 210 nm and column oven temperature was maintained at 25⁰ Theanine was detected and estimated by comparing the retention time and peak area with theanine calibration curve (Draft International Standard, ISO/DIS 11287).

Point 7 Ref. 10 is cited in wrong place. Lines 98-99 describe chromatographic condition and cited work is on colorimetric method. Lack of references for chromatographic condition.

Response 7:

Lines 98-99 describe chromatographic condition. The reference for chromatographic condition is

ISO. DIS 19563: Determination of theanine in tea and instant tea in solid form using high-performance liquid chromatography 2016.

Yes we agreed, finally we arranged the reference at proper place with new numbering.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript describes the tea clone qualification based on germplasm. The manuscript is well written,  but some points should be revised. please make the comments against the following comments,

1) in abstract, please add the space in seasons and Two in line 22.

2) in line 61, and 134, what is 'corbon'? Is this carbon?

3) in lines 62-64, you said the importance of treatment of seasonal changes and some protocol. Can you say the discussion so far discussed. Pleas ecite some papers regarding the analysis of other papers.

4) in lines 118-120, Please mention more how to treat some data.

5) In Figure 1, do not abbreviate the y axis.

6) in Figure 3, firstly, please explain coordination of 1 and 2, and if coord. 1 increased, what does it mean?

Author Response

Response to Reviewer 2 Comments

Point 1: In abstract, please add the space in seasons and Two in line 22.

Response 1:

Space added between seasons. Two

Point 2: In line 61, and 134, what is 'corbon'? Is this carbon?

Response 2:

carbon

Point3. In lines 62-64, you said the importance of treatment of seasonal changes and some protocol. Can you say the discussion so far discussed. Please ecite some papers regarding the analysis of other papers.

Response 3:

For green tea, seasonal variation (i.e. flushes) along with manufacturing protocol and planting material (clones or germplsm) greately influence the tea quality thats why we did the study accordingly and setup the best clone out of six slection and manufacturing protocol.

Point 4. In lines 118-120, please mention more how to treat some data

Response 4:

Regarding genotyping of the tea germplasm, SNPs from EST database of tea was designed as described by [12]. SNP sequences were submitted to the Assay Design Group at Fluidigm Corporation (South San Francisco, CA, USA) for design and manufacture of primers for a SNPtypeTM genotyping panel. The assays were based on competitive allele-specific polymerase chain reaction (PCR) and enable bi-allelic scoring of SNPs at specific loci (KBioscience Ltd, Hoddesdon, UK). The Fluidigm SNPtypeTM Genotyping Reagent Kit was used according to the manufacturer’s instructions. DNA was extracted from dried tea leaves of the 8 tea clones/germplasm with the DNeasyH Plant Mini kit (Qiagen Inc., Valencia, CA, USA) and isolated DNAs were subjected to Specific Target Amplification (STA) using these primers in order to enrich the SNP sequences of interest. Genotyping was performed on a nanofluidic 96.96 Dynamic ArrayTM IFC (Integrated Fluidic Circuit; Fluidigm Corp.). The End-point fluorescent images of the 96.96 IFC were acquired on an EP1TM imager (Fluidigm Corp.) and data was analyzed with Fluidigm Genotyping Analysis Software [12, 13]. Principal Coordinates Analysis (PCoA), based on the pairwise distance matrix. Distance-based multivariate analysis was used to assess the relationship among the individual farmer cultivars, as well as their relationship with reference clones from international genebanks. Pairwise genetic distances were computed using the DISTANCE procedure implemented in GenAlEx 6.5 [14, 15]. The same program was then used to perform Principal Coordinates Analysis (PCoA), based on the pairwise distance matrix. Both distance and covariance were standardized.

Point 5. In Figure 1, do not abbreviate the y axis.

Response 5:

Sir, abbreviated both the axis

For x- axis: Selected clone

For y- axis- parameters like

light (PAR), net photosynthesis (Pn), stomatal conductance (gs), evaporation (E), leaf temperature (Lt) and water use efficiency (WUE).

Point 6. In Figure 3, firstly, please explain coordination of 1 and 2, and if coord. 1 increased, what does it mean?

Response 6:

The principal component analysis PcoA, Coordinate 1 and 2 actually indicate the two of three dimensional plane of observation. You will get a separate distribution of the germplasm (points) if the coordinate is different say Coordinate 2&3 or Coordinate 1&3.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript has been corrected; thus I recommend it for publication in current form

Reviewer 2 Report

The manuscript is well revised against the reviewer's comments.