The Genome sequences were acquired from the An. gambiae PEST strain (https://www.vectorbase.org/organisms/anopheles-gambiae/pest ) and An. sinensis (https://www.vectorbase.org/organisms/anopheles-sinensis/sinensis ). The PCR primers were designed using the Primer3 Program [21 (link)], based on the above genome sequences from An. gambiae and An. sinensis. Genomic DNA of An. lesteri was extracted from live fourth-instar larvae, using a DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany), and utilized as a template for PCR.
After the standard PCR procedures, amplified PCR products were loaded on agarose gel and cut for purification using a QIAquick Gel Extraction Kit (Qiagen). The DNA fragments were labeled with either Cy3.5-AP3-dUTP or Cy5.5-AP3-dUTP (GE Healthcare UK Ltd, Chalfont StGiles, UK), using a Random Primed DNA Labelling Kit (Roche Applied Science, Penzberg, Germany). In situ hybridization was performed using a previously described method [20 (link)]. Fluorescent signals were detected and recorded with a Zeiss LSM 710 laser scanning microscope (Carl Zeiss Microimaging GmbH, Oberkochen, Germany), and placed on the polytene chromosome map of An. lesteri.
After the standard PCR procedures, amplified PCR products were loaded on agarose gel and cut for purification using a QIAquick Gel Extraction Kit (Qiagen). The DNA fragments were labeled with either Cy3.5-AP3-dUTP or Cy5.5-AP3-dUTP (GE Healthcare UK Ltd, Chalfont StGiles, UK), using a Random Primed DNA Labelling Kit (Roche Applied Science, Penzberg, Germany). In situ hybridization was performed using a previously described method [20 (link)]. Fluorescent signals were detected and recorded with a Zeiss LSM 710 laser scanning microscope (Carl Zeiss Microimaging GmbH, Oberkochen, Germany), and placed on the polytene chromosome map of An. lesteri.