Research Article |
Corresponding author: Xiaoting Huang ( xthuang@ouc.edu.cn ) Academic editor: Virmantas Stunžėnas
© 2018 Liping Hu, Liming Jiang, Ke Bi, Huan Liao, Zujing Yang, Xiaoting Huang, Zhenming Bao.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hu L, Jiang L, Bi K, Liao H, Yang Z, Huang X, Bao Z (2018) Genomic in situ hybridization in interspecific hybrids of scallops (Bivalvia, Pectinidae) and localization of the satellite DNA Cf303, and the vertebrate telomeric sequences (TTAGGG)n on chromosomes of scallop Chlamys farreri (Jones & Preston, 1904). Comparative Cytogenetics 12(1): 83-95. https://doi.org/10.3897/CompCytogen.v12i1.14995
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Mitotic chromosome preparations of the interspecific hybrids Chlamys farreri (Jones & Preston, 1904) × Patinopecten yessoensis (Jay, 1857), C. farreri × Argopecten irradians (Lamarck, 1819) and C. farreri × Mimachlamys nobilis (Reeve, 1852) were used to compare two different scallop genomes in a single slide. Although genomic in situ hybridization (GISH) using genomic DNA from each scallop species as probe painted mitotic chromosomes of the interspecific hybrids, the painting results were not uniform; instead it showed species-specific distribution patterns of fluorescent signals among the chromosomes. The most prominent GISH-bands were mainly located at centromeric or telomeric regions of scallop chromosomes. In order to illustrate the sequence constitution of the GISH-bands, the satellite Cf303 sequences of C. farreri and the vertebrate telomeric (TTAGGG)n sequences were used to map mitotic chromosomes of C. farreri by fluorescence in situ hybridization (FISH). The results indicated that the GISH-banding pattern presented by the chromosomes of C. farreri is mainly due to the distribution of the satellite Cf303 DNA, therefore suggesting that the GISH-banding patterns found in the other three scallops could also be the result of the chromosomal distribution of other species-specific satellite DNAs.
GISH banding, FISH, Cf303 , telomere, scallop
Chromosomal bandings were used to identify chromosomes and provided insight into the substructure and organization of whole chromosomes (
Genomic in situ hybridization (GISH) has been successfully used for discrimination of genome constitutions in hybrids and allopolyploids (
Repetitive DNA refers to DNA sequences that occur in multiple copies and makes up the major proportion of the nuclear DNA in most eukaryotic genomes. Changes in repetitive DNA likely contribute to the karyotypical features and variations, as well as genome sizes (
In the present study, GISH was carried out on chromosomal slides of interspecific hybrids C. farreri × P. yessoensis, C. farreri × Argopecten irradians (Lamarck, 1819) and C. farreri × Mimachlamys nobilis (Reeve, 1852). Chromosomal distributions of the randomly repeated DNA sequences were revealed by GISH-banding in the four scallop species (C. farreri, P. yessoensis, A. irradians and M. nobilis). In order to verify the sequences constitution of GISH-banding, FISH with the satellite DNA Cf303 and vertebrate telomere sequence (TTAGGG)n probes were performed to compare the GISH-banding of C. farreri. Our results provided the first application of GISH-banding in Pectinidae, and first physical mapping of the satellite DNA Cf303 and vertebrate telomere sequence (TTAGGG)n in C. farreri, aiding to understanding chromosome distribution and composition of the repetitive DNA sequences in the studied scallops.
The sexually mature scallops, C. farreri, P. yessoensis, A. irradians and M. nobilis, were obtained from hatcheries in Shandong Province, China. The interspecific hybrids C. farreri × P. yessoensis, C. farreri × A. irradians and C. farreri × M. nobilis were carried out in the laboratory. Eggs and sperm were collected from sexually mature scallops. Eggs were fertilized by adding sperm suspension. After fertilization, excessive sperm was removed by rinsing with seawater on a 20 µm screen (
Telomeric (TTAGGG)7 probes were synthesized and 5’-end labelled with biotin-16-dUTP (Invitrogen). Plasmids were isolated from a fosmid clone containing the satellite DNA Cf303 by standard laboratory methods (
GISH and FISH were performed according to the methods of
Using labeled total genomic DNA from C. farreri as probe, the hybridization signal (yellow-green) covered only chromosomes of C. farreri, but not P. yessoensis, A. irradians and M. nobilis, on the mitotic chromosomes of C. farreri hybrids with these scallop species (Fig.
Metaphase chromosomes and karyotypes of scallop hybrids. A1, A2, D1, D2 C. farreri × P. yessoensis B1, B2, E1, E2 C. farreri × A. irradians C1, C2, F1, F2 C. farreri × M. nobilis A1, A2, B1, B2, C1, C2 the chromosomes originating from C. farreri were painted in yellow-green using the labeled genomic DNA probes from C. farreri D1, D2 - the chromosomes originating from P. yessoensis were painted in yellow-green using the labeled genomic DNA probes from P. yessoensis. Nucleolus organizer regions (NORs) in P. yessoensis were marked with arrows in (D1). E1, E2 the chromosomes originating from A. irradians were painted in yellow-green using the labeled genomic DNA probes from A. irradians. F1, F2 the chromosomes originating from M. nobilis were painted in yellow-green using the labeled genomic DNA probes from M. nobilis. Scale bars: 5µm.
Considering that the GISH-bandings were mainly discovered in the telomeric and/or peri-telomeric regions of some chromosomes in C. farreri, the vertebrate telomeric sequence (TTAGGG)n, as well as satellite DNA Cf303 were used as probes to compare the signal distribution. Telomeric repeats were hybridized to the ends of all C. farrerichromosomes, the signal intensity was weak and varied among different chromosomes, and no interstitial hybridization signal was observed (Fig.
Constitutive heterochromatin has been defined as a structurally distinct kind of chromatin comprising noncoding, largely repetitive DNA, which is permanently not transcribed (
Using GISH,
Highly repeated DNA exists within each eukaryotic genome. Satellite DNAs, as the tandem arrays of repeated units, are chiefly localized at heterochromatic regions of chromosomes (
Vertebrate-type telomeric sequences (TTAGGG)n located at terminal regions of each chromosome of C. farreri in our study. The signal intensities were weak and varied among different chromosomes; no interstitial hybridization signal was observed. This is the first report about the chromosomal distribution of telomeric sequences in C. farreri. The locations of these sequences were coincident with the results reported in P. yessoensis and A. irradians (
Satellite DNAs are highly repetitive DNA sequences that can be located in pericentromeric (
In contrast to the location of telomere sequence in scallops, the satellite DNA Cf303 existed only on the chromosomes of C. farreri, but not on the chromosomes of closely related species P. yessoensis, A. irradians and M. nobilis, which suggested that the satellite DNA Cf303 was species-specific. After comparing the signal distribution and intensity of GISH-banding, vertebrate telomeric sequences and satellite DNA Cf303, we found the GISH-banding pattern in C. farreri was not consistent with the result of telomeric repeats. Interestingly, the GISH-bands overlapped the FISH signals obtained with satellite DNA Cf303. Generally, satellite DNAs are chiefly localized at heterochromatic regions of chromosomes (
This research was funded by the National Natural Science Foundation of China (41676132), the Fundamental Research Funds for the Central Universities (201762016, 201562018), the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-49), the key Research and Development Program of Shandong Province (2016ZDJQ0208), and the Seed Improvement Project of Shandong Province.