Research Article |
Corresponding author: Jianguo Xiang ( xiangjianguo055x@hunau.edu.cn ) Academic editor: Rafael Noleto
© 2024 Liaoruilin Zhang, Jianguo Xiang, Juan Li, Jie Zhou, Jinliang Hou, Yanfei Huang, Hong Li.
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:
Zhang L, Xiang J, Li J, Zhou J, Hou J, Huang Y, Li H (2024) Karyotype analysis of Quasipaa spinosa David, 1875 (Anura, Dicroglossidae) with conventional cytogenetic techniques. Comparative Cytogenetics 18: 97-103. https://doi.org/10.3897/compcytogen.18.116806
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The current study analyzed the chromosomal karyotype of Quasipaa spinosa David, 1875 from Hunan Province, China. The karyotype, C-banding, BrdU-banding pattern were characterized using direct preparation of bone-marrow cells and hemocyte cultures. The findings indicated that Q. spinosa was a diploid species (2n = 26) that lacked heteromorphic chromosomes and secondary constrictions. C-banding analysis revealed an abundance of positive signals in the centromere regions, while the BrdU-banding pattern showed three phases in both male and female, occurring consistently and in chronological sequence during S-phase. Notably, there was no asynchronous replication in the late phase. This study enhanced our understanding of the karyotypic structure of Q. spinosa by conventional cytogenetic techniques, thus providing essential scientific insights into the cytogenetics of Q. spinosa.
BrdU-banding, C-banding, karyotype, Quasipaa spinosa
Quasipaa spinosa David, 1875 (Anura, Dicroglossidae) is an amphibian native to and widely distributed in southern and southeastern China, where it thrives in forests and hilly areas at altitudes of 500–1500 meters (
Chromosome karyotype studies are valuable in elucidating the phylogeny of the species studied as well as assisting in its classification (
This study presents an analysis of the karyotype of Q. spinosa using conventional cytogenetic techniques, aiming to contribute novel and valuable karyotypic information while enhancing the existing cytogenetic database for Q. spinosa.
The study utilized 15 healthy adult male and 15 female Q. spinosa, each weighing between 90–100 g, sourced from farm located in Taiping Town (29°58'42"N, 111°05'25"E), Shimen County of Hunan Province, China. Animal treatment and the study protocol strictly adhered to ethical guidelines formulated by the Animal Protection Committee (APC) of Hunan Agricultural University (201903297) (ethics license: No. LSK 202-3-D106). Specimens were transported to the laboratory via a specialized vehicle designed to maintain breeding environment temperatures thereby reducing mortality due to temperature-induced stress.
Bone marrow-cell suspensions were prepared as previously described by
The animals were anesthetized and blood was collected from the heart. The blood was injected into human peripheral blood lymphoid medium (0.2 ml of blood for every 5 ml of culture medium) which was then incubated for 90 hours in a thermostatic incubator at 28 °C in the dark. The cells were treated with 100 μg/ml bromodeoxyuridine (BrdU) 9 h before the end of the culture period, followed by 40 μg/mL fluorodeoxyuridine (FdU) for 6 h and the addition of colchicine to a final concentration of 0.03 μg/ml for 2 h. Acridine orange was added at a final concentration of 10 μg/mL 1 h before harvesting (
Q. spinosa exhibited a diploid number of 2n = 26 lacking heteromorphic chromosomes and secondary constrictions (Fig.
Chromosome number (CN), relative length (RL), arm ratio (AR), and chromosomal classification (CC) of mitotic chromosome. M = metacentric chromosome; SM = submetacentric chromosome. LHV= lower and higher values for each chromosome.
CN | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
RL | 15.38± 0.84 | 12.46± 0.81 | 11.65± 0.82 | 10.75± 0.62 | 10.04± 0.56 | 7.45± 0.58 | 6.98± 0.55 | 5.93± 0.55 | 4.97± 0.61 | 4.17± 0.13 | 3.88± 0.21 | 3.48± 0.11 | 2.96± 0.85 |
LHV | 13.88~ 16.51 | 11.11~ 13.95 | 10.31~ 12.69 | 9.83~ 11.68 | 9.16~ 10.65 | 6.64~ 8.41 | 6.15~ 7.92 | 5.06~ 6.93 | 4.15~ 5.95 | 3.97~ 4.37 | 3.54~ 4.17 | 3.32~ 3.67 | 2.81~ 3.07 |
AR | 1.25± 0.05 | 1.85± 0.12 | 1.50± 0.05 | 2.39± 0.10 | 1.25± 0.06 | 1.34± 0.05 | 1.34± 0.05 | 1.88± 0.08 | 1.15± 0.02 | 2.23± 0.07 | 1.28± 0.05 | 1.06± 0.03 | 1.41± 0.04 |
LHV | 1.20~ 1.34 | 1.70~ 2.03 | 1.40~ 1.58 | 2.18~ 2.54 | 1.15~ 1.33 | 1.24~ 1.42 | 1.26~ 1.43 | 1.76~ 2.04 | 1.11~ 1.19 | 2.12~ 2.34 | 1.19~ 1.36 | 1.02~ 1.12 | 1.33~ 1.5 |
CC | M | SM | M | SM | M | M | M | SM | M | SM | M | M | M |
After BrdU infiltration, abundant chromosomal division phases and replicative banding patterns were observed as the cell culture progressed. During the S-phase, chromosomes that had completed DNA synthesis were stained dark purplish-red or dark, while segments that were still undergoing synthesis post-BrdU infiltration appeared lavender-colored or light purplish-blue. Consequently, the replication bandings were classified into three periods based on the proportion of dark and light staining during the intermediate stage. First, in the early-replication stage at the time of BrdU treatment, the bands showed a roughly 1:1 ratio between early-replicated dark-stained bands and late-replicated light-stained bands. Second, in the mid-replication stage, dark-stained areas predominated while the chromosomes nevertheless remained distinguishable, and third, in the late replication phase, the chromosomes were almost entirely dark-stained following the completion of replication.
The replication timing of each of the 13 Q. spinosa chromosomes ranged from the very early to the late stages (Fig.
Q. spinosa in Hunan exhibited a karyotype comprising nine pairs of metacentric and four pairs of submetacentric chromosomes (4sm+9m), consistent with the finding reported by
The C-banding positive region was predominantly localized in the centromere region of Q. spinosa, with chromosomes 1–5 exhibiting the most pronounced positivity. Moreover, C-banding positivity was also observed within the respective centromere regions of Limnonectes taylori Matsui, Panha, Khonsue et Kuraishi, 2010 (
In conclusion, Q. spinosa was a diploid species (2n=26) with the absence of heteromorphic chromosomes and secondary constrictions. Notably, heterochromatin in the centromere region and patterns of change in the BrdU-banding were observed. In this study, the karyotypic structure of Q. spinosa was analyzed, providing further genetic information on Q. spinosa.
This research was supported by the National Natural Science Foundation of China grant/award number (31772832). Genetics Laboratory, College of Fisheries, Hunan Agricultural University, China. The author thanks Dr. Wu for the linguistic corrections of the text.