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. 2019 Mar 26;14(3):e0214028.
doi: 10.1371/journal.pone.0214028. eCollection 2019.

Molecular Cytogenetic Characterization of Repetitive Sequences Comprising Centromeric Heterochromatin in Three Anseriformes Species

Free PMC article

Molecular Cytogenetic Characterization of Repetitive Sequences Comprising Centromeric Heterochromatin in Three Anseriformes Species

Yoshinobu Uno et al. PLoS One. .
Free PMC article


The highly repetitive DNA sequence of centromeric heterochromatin is an effective molecular cytogenetic marker for investigating genomic compartmentalization between macrochromosomes and microchromosomes in birds. We isolated four repetitive sequence families of centromeric heterochromatin from three Anseriformes species, viz., domestic duck (Anas platyrhynchos, APL), bean goose (Anser fabalis, AFA), and whooper swan (Cygnus cygnus, CCY), and characterized the sequences by molecular cytogenetic approach. The 190-bp APL-HaeIII and 101-bp AFA-HinfI-S sequences were localized in almost all chromosomes of A. platyrhynchos and A. fabalis, respectively. However, the 192-bp AFA-HinfI-L and 290-bp CCY-ApaI sequences were distributed in almost all microchromosomes of A. fabalis and in approximately 10 microchromosomes of C. cygnus, respectively. APL-HaeIII, AFA-HinfI-L, and CCY-ApaI showed partial sequence homology with the chicken nuclear-membrane-associated (CNM) repeat families, which were localized primarily to the centromeric regions of microchromosomes in Galliformes, suggesting that ancestral sequences of the CNM repeat families are observed in the common ancestors of Anseriformes and Galliformes. These results collectively provide the possibility that homogenization of centromeric heterochromatin occurred between microchromosomes in Anseriformes and Galliformes; however, homogenization between macrochromosomes and microchromosomes also occurred in some centromeric repetitive sequences.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1
Giemsa-stained karyotypes of female bean goose (A. fabalis) (A) and whooper swan (C. cygnus) (B). Arrowheads indicate the positions of the centromeres in chromosome 4 and the Z and W sex chromosomes.
Fig 2
Fig 2
C-banded metaphase spreads of A. fabalis (A) and C. cygnus (B) females. Arrows indicate the Z and W sex chromosomes.
Fig 3
Fig 3
Chromosome painting with chicken (G. gallus, GGA) chromosome-specific DNA probes to Hoechst-stained chromosome spreads of A. fabalis (A‒C) and C. cygnus females (D‒E). DIG-labeled GGA1 (red) and biotin-labeled GGA9 (green) hybridized to chromosomes 1 and 9, respectively (A, D). DIG-labeled GGA4 (red) hybridized to chromosome 4 and a pair of microchromosomes, and biotin-labeled GGA6 (green) hybridized to chromosome 6 (B, E). DIG-labeled GGA3 (red) hybridized to chromosome 3, and biotin-labeled GGA7 (green) hybridized to chromosome 7 (C). DIG-labeled painting probe of 20 microchromosome pairs hybridized to approximately half of the microchromosomes (F). Scale bars represent 10 μm.
Fig 4
Fig 4. Comparison of the nucleotide sequences of APL-HaeIII, AFA-HinfI-S, AFA-HinfI-L, and CCY-ApaI sequences with their homologous sequences.
Dot matrix analysis between the consensus sequences of 190-bp APL-HaeIII and 192-bp AFA-HinfI-L (A). The gray region on AFA-HinfI-L shows the 12–17-bp T-rich and A-rich motif that is conserved in the CNM repeat sequence family of Galliformes [14,16,18], and squares on the sequences indicate the A3–5 or T3–5 internal repeats in this motif. Dot matrix analysis of the 101-bp AFA-HinfI-S consensus sequence (B). Dot matrix analysis was performed in the condition of the scoring matrix, 200PAM/K = 2 and threshold score = 22 (E = 0.00805). Alignment of the APL-HaeIII consensus sequence and partial sequence at nucleotide position 60–192 of the AFA-HinfI-L consensus sequence with the RBMII sequences of A. platyrhynchos (APL) (X61424) and Aix sponsa (ASP) (X61410) (C). Alignment of the partial sequences at nucleotide position 76–95 of the AFA-HinfI-L consensus sequence and at position 24–43 of the APL-HaeIII consensus sequence (D) and the partial sequence at positions 179–223 of the CCY-ApaI consensus sequence (E) with the four CNM sequence homologs in Galliformes, viz., CNM repeat in chicken [14], TM repeat in turkey (M. gallopavo) [16], CCH-S in Blue-breasted Quail (C. chinensis) [17], and ACH-Sau3AI in chukar partridge (A. chukar) [18]. Squares indicate the A3–5 or T3–5 internal repeats in the 12–17-bp T-rich and A-rich motifs conserved in the CNM repeat sequence family of Galliformes [14,16,18].
Fig 5
Fig 5. Chromosomal distribution of four families of repetitive sequences on metaphase spreads.
Chromosomal distribution of the biotin-labeled APL-HaeIII-04 fragment on the PI-stained metaphase chromosome spread of A. platyrhynchos female (A). Hybridization pattern of the biotin-labeled CCY-ApaI-05 fragment to the Hoechst-stained metaphase spread of C. cygnus female (B). Hybridization patterns of the biotin-labeled AFA-HinfI-S03 fragment (green) (C) and DIG-labeled AFA-HinfI-L04 fragment (red) (D) to the Hoechst-stained metaphase spread of A. fabalis female and their merged image (E). Scale bars represent 10 μm.
Fig 6
Fig 6. Southern blot hybridization patterns of four repetitive sequence families.
Southern blot hybridization of A. platyrhynchos genomic DNA probed with the APL-HaeIII-04 fragment (A). Southern blot hybridization of A. fabalis genomic DNA probed with the AFA-HinfI-S03 (B) and AFA-HinfI-L04 (C) fragments. Southern blot hybridization of C. cygnus genomic DNA probed with the CCY-ApaI-05 fragment (D). A mixture of λ DNA–HindIII and ϕX174 DNA–HaeIII was used as a molecular size marker for (A, B, D), and a mixture of λ DNA–HindIII digest and 100-bp ladder digest was used for (C).
Fig 7
Fig 7. Slot blot hybridization of four repetitive sequences to genomic DNA of 17 species.
The fragments used for this experiment are as follows: APL-HaeIII-04 (A), AFA-HinfI-S03 (B), AFA-HinfI-L04 (C), and CCY-ApaI-05 (D). Genomic DNA used for this experiment was obtained from the following avian species of 10 orders: (1) Struthioniformes, SCA (S. camelus) and DNO (D. novaehollandiae); (2) Tinamiformes, EEL (E. elegans); (3) Galliformes, NME (N. meleagris), CJA (C. japonica), and GGA (G. gallus); (4) Anseriformes, AFA (A. fabalis), CCY (C. cygnus), and APL (A. platyrhynchos); (5) Gruiformes, GLE (G. leucogeranus); (6) Pelecaniformes, PMI (P. minor); (7) Strigiformes, BBL (B. blakistoni); (8) Accipitriformes, PHA (P. haliaetus) and NNI (N. nipalensis orientalis); (9) Psittaciformes, PAT (P. aterrimus) and AAU (A. autumnalis); and (10) Passeriformes, HRU (H. rustica). The locations of genomic DNA on membranes used for slot blot hybridization are represented at the bottom of the figures.

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Grant support

This work was supported by a Grant-in-Aid for Scientific Research (B) (no. 22370081) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.