Protocol detail

Metaphase Chromosome Preparation and FISH

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The preparation of metaphase chromosomes was performed as described (Zhu and Gui 2007 ; Li et al. 2016b ). Six DNA probes were used to perform FISH analysis according to standard procedures as described previously. The BAC-DNAs marking chromosome 5A (Chr5A), Chr5B, Chr17A, and Chr17B (Li et al. 2014 (link); Mou et al. 2021 (link)) were labeled by DIG-Nick Translation Mix and Biotin-Nick Translation Mix (Roche), respectively. To investigate the sex determination system of the novel amphitriploids and its offspring, the fragments specific to sex chromosomes (Chr22B) were amplified by PCR and labeled by DIG-Nick Translation Mix (Roche, Switzerland) (Lu et al. 2021 (link)), and the genomic DNA of C. auratus labeled by Biotin-Nick Labeling System were used to distinguish X and Y chromosomes. The chromosomes were stained with DAPI (Sigma, USA). The sections were observed and photographed by Zeiss Axio Imager2 (Analytical & Testing Center, IHB, CAS).

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Lu M., Li Z., Zhu Z.Y., Peng F., Wang Y., Li X.Y., Wang Z.W., Zhang X.J., Zhou L, & Gui J.F. (2022). Changes in Ploidy Drive Reproduction Transition and Genomic Diversity in a Polyploid Fish Complex. Molecular Biology and Evolution, 39(9), msac188.

Publication 2022

DIG-Nick Translation Mix Biotin-Nick Translation Mix DAPI

Biotin Chromosomes Dapi Dna probes Fish procedures Genomic Metaphase Sex chromosomes Sex determination Y chromosomes

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Variable analysis

independent variables

Preparation of metaphase chromosomes
Six DNA probes used to perform FISH analysis
Labeling of BAC-DNAs marking chromosome 5A, Chr5B, Chr17A, and Chr17B using DIG-Nick Translation Mix and Biotin-Nick Translation Mix
Amplification and labeling of fragments specific to sex chromosomes (Chr22B) using DIG-Nick Translation Mix
Labeling of genomic DNA of C. auratus using Biotin-Nick Labeling System

dependent variables

Observation and photography of chromosomes stained with DAPI

control variables

Standard procedures for FISH analysis as described previously

positive controls

BAC-DNAs marking chromosome 5A, Chr5B, Chr17A, and Chr17B

negative controls

Not explicitly mentioned

Annotations

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