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\begin{document}
\begin{frontmatter}
  \title{{\Huge \bf PART I---Revision Note}}%\\Establishment of quantitative PCR methods for the quantification of geosmin-producing potential and \textit{Anabaena} sp. in freshwater systems}
\end{frontmatter}
  \iffalse
\author[lab1]{Ming Su}
\ead{ming.su@live.com}
\author[lab2,lab3]{Virginie Gaget}
\ead{Virginie.Gaget@sawater.com.au}
\author[lab2,lab4]{Steven Giglio}
\ead{Steven.Giglio@healthscope.com.au}
\author[lab2]{Michael Burch}
\ead{mike.burch@sawater.com.au}
\author[lab1]{Wei An}
\ead{anwei@rcees.ac.cn}
\author[lab1]{Min Yang\corref{cor1}}
\ead{yangmin@rcees.ac.cn}
\cortext[cor1]{Corresponding author. State Key Laboratory of Environmental Aquatic Chemistry, Research Center for
Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Rd, Haidian, Beijing 100085. Tel: +86 10 6284
9175; Fax: +86 10 62923541.}
\address[lab1]{State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences,
Chinese Academy of Sciences, 18 Shuangqing Rd, Haidian, Beijing 100085}
\address[lab2]{South Australia Water Corporation, Australian Water Quality Centre, 250 Victoria Square, Adelaide, SA,
5000, Australia}
\address[lab3]{Current address: Ecology, Evolution and Landscape Sciences, School of Earth and Environment Sciences,
Adelaide University, Adelaide, Australia}
\address[lab4]{Current address: To be added. Adelaide, Australia}
%wrtemp
\begin{abstract}

  The revise-required manuscript entitled ``\textit{\textbf{Establishment of quantitative PCR methods for
    quantification of geosmin-producing potential and \textit{Anabaena} sp.~in freshwater systems}}" with a previous
    manuscript number of \textbf{WR21535} has been carefully revised, according to the reviewers and editors' comments.
    Briefly, the following major changes have been made:
    \begin{enumerate}
      \item We have rechecked the specificity of \textit{ARG} primer by testing it on 15 \textit{Anabaena} strains and 2
        other strains using conventional PCR and gel electrophoresis;
      \item We have revised the data analysis and improved them;
      \item We have improved the Results and discussion section using statistic analysis; moreover, we have discussed
        the applicability of both qPCR assays;
      \item We have carefully checked the references, and made some changes.
    \end{enumerate}
    We believe that the quality and readability of this revised manuscript have been significantly improved.

\end{abstract}
\end{frontmatter}
\newpage
\fi
    \section*{\Large Introduction}

    The change list is shown in \S1 Change list; at the same time, the point to point responses to the comments are
    shown in \S2 Ponit to point responses, with the normal text indicates the original comments and the bold text
    indicates our responses.\\\\

    Title:

    \textbf{Establishment of quantitative PCR methods for the quantification of geosmin-producing potential and
      \textit{Anabaena} sp.~in freshwater systems}
      \\

      Authors:

      Ming Su:\qquad\quad\qquad\qquad\qquad\quad\ \ ming.su@live.com

      Virginie Gaget: \ \qquad \qquad\qquad\quad\ \ virginie.gaget@sawater.com.au

      Steven Giglio:\qquad\quad \qquad\qquad\quad\ \ steven.giglio@healthscope.com.au

      Michael Burch: \ \qquad \qquad\qquad\quad\ \ mike.burch@sawater.com.au

      Wei An: \qquad\qquad\quad \ \qquad\qquad\quad\ \ anwei@rcees.ac.cn

      Min Yang: \qquad\qquad \ \qquad\qquad\quad\ \ yangmin@rcees.ac.cn
      \\

      Content:

      Body text

      Figures \qquad\qquad\qquad\qquad\qquad\quad\ 5

      Tables \qquad\qquad\qquad\qquad\qquad\quad\ \ 4

      Supporting Information\qquad\qquad\quad1



      \newpage
      \section{\Large Change list}
      \label{chlist}

      \textbf{According to the comments and suggestions, the manuscript has been carefully revised.  The changes in the
      revised manuscript are listed here.\\}

      \subsection{Content changes}

      \begin{enumerate}[(1)]

        \item \zhypera{w1} A grammar error was corrected: ``are showed in Figure A.2'' has been changed to ``are shown
          in Figure A.2.''\\
          
        \item \zhypera{dis1} ``It should be noted that, the three frequently reported bloom-forming strains of
          AWQC-ANA318 \textit{A.~circinalis}, FADC-0001 \textit{A.~spiroides} and \textit{A.~flos-aquae} could be well
          amplified by the \textit{ARG} primers.'' has been changed to ``It should be noted that, the $rpoC_1$ gene
          of three frequently reported bloom-forming species of \textit{A.~circinalis} (strain AWQC-ANA318),
          \textit{A.~spiroides} (strain FADC-0001) and \textit{A.~flos-aquae} could be well amplified by the
          \textit{ARG} primers.''\\

        \item \zhypera{dis2} ``In addition, the melting curve analysis results exhibited distinct different in the
          melting temperatures (T$_\textrm{m}$) between the amplicon of \textit{Nostoc} sp.~(86 $^\circ$C) and those of
          the four AWQC \textit{Anabaena} strains (83 $^\circ$C), showing that the melting analysis may also be used for
          differentiating the \textit{Anabaena} sp.~from the other species.'' was replaced by ``In addition, the
          melting curve analysis results exhibited distinct different in the melting temperatures (T$_\textrm{m}$)
          between the amplicon of \textit{Nostoc} sp.~(86 $^\circ$C) and those of the four AWQC \textit{Anabaena}
          strains (83 $^\circ$C), showing that the melting analysis may also be used for differentiating
          the \textit{Anabaena} species between each other.''\\

        \item \zhypera{dis3} A new paragraph was added to describe more about the novelty of our study distinguished
          from other studies. The added text is ``In 2003 a sesquiterpene protein domain in \textit{Streptomyces
          coelicolor A3(2)} was linked to the presence of the taste and odor compound geosmin \citep{Cane2003,
          Gust2003}. Since then the gene responsible for the biosynthesis of geosmin (\textit{geo} gene) has been
          extensively characterized \citep{cane2006, Jiang2006, jiang2007}. In 2007 two \textit{geoA}-like genes were
          detected in a strain of \textit{Phormidium} (cyanobacteria, Oscillatoriales) \citep{ludwig2007identification}
          but their function in cyanobacteria were not elucidated until full characterization of the \textit{geoA} gene
          was performed in Nostoc punctiforme \citep{Steven2008}. Since then the expression of these genes has been
          studied in a single strain of \textit{Anabaena} \citep{Giglio2011}, which was demonstrated that the
          expression of the geosmin gene appears to be constitutive in nature. While in recent years some studies
          established quantitative PCR assays to detect and measure the production of geosmin by \textit{Streptomycetes}
          in the environment \citep{auffret2011establishment, lylloff2012detection}; to the best of our knowledge, no
          peer reviewed study has displayed a quantitative PCR assay to detect the production of geosmin specifically by
          cyanobacteria. The present study is the first qPCR assays for the detection and quantification of the
          cyanobacterial geosmin synthase in waters. Moreover, the ARG qPCR assay allows the detection of several
          \textit{Anabaena} species, major bloom-forming genus and confirmed geosmin producers. Combined, these two
          assays allow the monitoring of the population of \textit{Anabaena} at the same time as the production of
          geosmin. The comparison of the two results therefore assists in determining if \textit{Anabaena} is
          responsible for the release of geosmin in the environment.''\\

        \item \zhypera{con1} ``The present work provided two qPCR assays able to identify \textit{Anabaena} sp., a major
          taste and odor producing cyanobacteria as well as the \textit{geosmin synthase} gene in freshwater system.''
          was replaced by ``The present work provided two qPCR assays able to identify \textit{Anabaena} sp., a major
          taste and odor producing cyanobacteria as well as the \textit{geosmin synthase} gene in freshwater system for
          the first time in a peer reviewed study.''\\ 

        \item \zhypera{w2} A spelling error was corrected: ``grately'' has been replaced by ``greatly''.\\
        
        \item Three new references were added for comparison with our study. 
          
          \zhypera{ref1} \citep{ludwig2007identification}: ``In 2007 two \textit{geoA}-like genes were detected in a
          strain of \textit{Phormidium} (cyanobacteria, Oscillatoriales) \citep{ludwig2007identification} but their
          function in cyanobacteria were not elucidated until full characterization of the \textit{geoA} gene was
          performed in Nostoc punctiforme \citep{Steven2008}.''; 
          
          \zhypera{ref2} \citep{auffret2011establishment, lylloff2012detection}: `` While in recent years some
          studies established quantitative PCR assays to detect and measure the production of geosmin by
          \textit{Streptomycetes} in the environment \citep{auffret2011establishment, lylloff2012detection};''\\

      \end{enumerate}

      \newpage

      \section{\Large Point to point responses}
      \label{p2pre}

%      \textbf{Response to the editor and reviewers' comments} \\[4ex]
      \subsection*{\textbf{Reviewer \#3}}

      In this study titled 'Establishment of real-time PCR methods for the quantification of geosmin-producing potential
      and \textit{Anabaena} spp. in freshwater systems', the authors established reliable qPCR-based methods which can detect and
      quantify \textit{Anabaena} spp. and geosmin synthesis. 

      They designed PCR primer sets for $rpoC_1$ gene and geosmin synthase gene which are specific to \textit{Anabaena}
      spp., and proved effective in the study. Furthermore, reliabilities of the two qPCR-based methods were confirmed
      by comparing to direct measuring methods such as counting Anabaena spp. by microscopy and measuring geosmin by
      using gas chromatography-mass spectrometry. 

      The qPCR-based methods for quantifying geosmin-related microorganisms in environments are not novel ideas since
      some recent studies dealing with similar topics already have existed. However, this study is considered to have a
      novelty of quantifying both of geosmin and \textit{Anabaena} spp. as major producer by using only qPCR processes.
      In that sense, I strongly ask the authors describe more about novelty of their study distinguished from the others
      in the discussion or conclusion part.

      The manuscript is well written, and provides detailed information of the experimental procedures and results.
      Overall, I am recommending the publication of this manuscript in 'Water Research'.\\[4ex]
          \begin{bf}
            Response/Action: (\zhypera{dis3})\\
            Thank you for your valuable comments. According to your suggestion, we have made add a new paragraph in
            \S\ref{new-resdiss:applicability} \nameref{new-resdiss:applicability} for emphasizing the novelty by
            comparing with other studies. The detailed changes are shown as follows:\\[1ex]

            ```In 2003 a sesquiterpene protein domain in \textit{Streptomyces coelicolor A3(2)} was linked to the
            presence of the taste and odor compound geosmin \citep{Cane2003, Gust2003}. Since then the gene responsible
            for the biosynthesis of geosmin (\textit{geo} gene) has been extensively characterized \citep{cane2006,
            Jiang2006, jiang2007}. In 2007 two \textit{geoA}-like genes were detected in a strain of \textit{Phormidium}
            (cyanobacteria, Oscillatoriales) \citep{ludwig2007identification} but their function in cyanobacteria were
            not elucidated until full characterization of the \textit{geoA} gene was performed in Nostoc punctiforme
            \citep{Steven2008}. Since then the expression of these genes has been studied in a single strain of
            \textit{Anabaena} \citep{Giglio2011}, which was demonstrated that the expression of the geosmin gene
            appears to be constitutive in nature. While in recent years some studies established quantitative PCR assays
            to detect and measure the production of geosmin by \textit{Streptomycetes} in the environment
            \citep{auffret2011establishment, lylloff2012detection}; to the best of our knowledge, no peer reviewed study
            has displayed a quantitative PCR assay to detect the production of geosmin specifically by cyanobacteria.
            The present study is the first qPCR assays for the detection and quantification of the cyanobacterial
            geosmin synthase in waters. Moreover, the ARG qPCR assay allows the detection of several \textit{Anabaena}
            species, major bloom-forming genus and confirmed geosmin producers. Combined, these two assays allow the
            monitoring of the population of \textit{Anabaena} at the same time as the production of geosmin. The
            comparison of the two results therefore assists in determining if \textit{Anabaena} is responsible for the
            release of geosmin in the environment.''\\\\

          \end{bf} 
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\end{document}