XX/XY Heteromorphic Sex Chromosome Systems in Two Bullhead Catfish Species, Liobagrusmarginatus and L. Styani
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Original Article Cytogenet Genome Res 122:169–174 (2008) DOI: 10.1159/000163095 XX/XY heteromorphic sex chromosome systems in two bullhead catfish species, Liobagrus marginatus and L. styani (Amblycipitidae, Siluriformes) a b a a, c b a a J. Chen Y. Fu D. Xiang G. Zhao H. Long J. Liu Q. Yu a b College of Life Science, Wuhan University, Wuhan , Hubei; Yangtze River Fisheries Institute, Jinzhou , Hubei c Department of Biology, Hubei College of Traditional Chinese Medicine, Wuhan , Hubei (P. R. China) Accepted in revised form for publication by M. Schmid, 30 July 2008. Abstract. Karyotypes and chromosomal characteristics osis of L. marginatus suggested the presence of heteromor- of two species of bullhead catfish of the genus Liobagrus , phic XX/XY sex chromosome systems. The results of namely L. marginatus and L. styani were examined by (TTAGGG)n FISH and CMA3 -staining suggested that Rob- means of conventional (Giemsa and CMA3 staining) and ertsonian fusion might play an important role in chromo- molecular (FISH with telomeric, 5S and 18S rDNA probes, some differentiation among these species of Liobagrus . The respectively) cytogenetic techniques. The diploid numbers 5S rDNA situated on the sex chromosomes of L. marginatus of L. marginatus and L. styani were 2n = 24 and 30 respec- and 18S rDNA on the sex chromosomes of L. styani were tively. The karyotypes were: L. marginatus , 20m+2sm+2st located by FISH, and the origins of heteromorphic sex chro- in females and 19m+2sm+2st+1a in males; L. styani , 16m+ mosomes via chromosomal reversion or unequal crossing 10sm+4st in both sexes. The karyotypes of the two species over of heterochromatin were hypothesized. and the delayed-pairing of synaptonemal complexes in mei- Copyright © 2008 S. Karger AG, Basel The catfish family Amblycipitidae contains the three re- Cytogenetics of these catfishes remains poorly known cently recognized genera Amblyceps, Liobagrus and Xiuren- with karyotypes of five species ( Liobagrus andersonii, L. bagrus with about 26 species (Nelson, 2006). The represen- mediadiposalis, L. nigricauda , L . marginatoides and L. margi- tatives of bullhead catfish genus Liobagrus occur mainly in natus ) in Korea and China (Ojima, 1985; Yu et al., 1989). China’s Yangtze River, Zhujiang River and other rivers Although the karyotypes of the five species are different, along the southeast coast and in Taiwan. In addition, a few they all suggest the presence of heteromorphic XX/XY sex species are distributed in Korea and North Vietnam. Those chromosome systems. localized in China have been classified into nine species In the present study, karyotypes and chromosomal char- (Chu, 1999). acteristics of two species of bullhead catfish of the genus Liobagrus , namely L. marginatus and L. styani were exam- ined by means of conventional (Giemsa and CMA3 stain- This work was supported by the National Natural Science Foundation of China ing) and molecular (FISH with telomeric, 5S and 18S rDNA (Grant No. 30400044). probes, respectively) cytogenetic techniques. Moreover, we Request reprints from Jiangdong Liu examined the chromosome pairing in synaptonemal com- College of Life Science, Wuhan University plexes of L. marginatus , to confirm the presence of sex chro- Wuhan, Hubei 430072 (P. R. China) telephone: +86 27 87860370; fax: +86 27 68752560 mosome regions. e-mail: [email protected] or [email protected] Fax +41 61 306 12 34 © 2008 S. Karger AG, Basel Accessible online at: E-Mail [email protected] 1424–8581/08/1222–0169$24.50/0 www.karger.com/cgr www.karger.com a b Fig. 1. Karyotypes of female (left) and male (right) L. marginatus ( a ) and L. styani ( b ), arranged from Giemsa-stained chromosomes. Bars = 5 m. Materials and methods Fluorochrome staining CMA3 fluorescent staining was carried out according to Sola et Materials al. (1992). Photographs were observed by an Olympus BX50 micro- Mature individuals of L. marginatus and L. styani were collected scope with a G excitation filter, and recorded by an Olympus c7070 from rivers in Ya’an, Sichuan Province, China. All analyzed specimens camera. were deposited as vouchers in the Museum of Aquatic Organisms, In- stitute of Hydrobiology, Chinese Academy of Sciences, under codes of F I S H 13500046 for L. marginatus and 13500001 for L. styani . For telomeric (TTAGGG)n FISH, the probe was produced by PCR in the absence of a DNA template (Ijdo et al., 1991), and then Chromosome preparation and karyotype labeled with biotin-11-dUTP by nick translation (Roche, Cat. No. Mitotic chromosomes were prepared from kidneys using the stan- 11745824910). dard air-drying procedure according to Yu et al. (1989). More than 20 For FISH of ribosomal genes, a pair of primers, 5SA (5 -TAC GCC Giemsa-stained (4%, pH 6.8) metaphases per specimen were photo- CGA TCT CGT CCG ATC-3 ) and 5SB (5 -CAG GCT GGT ATG GCC graphed and karyotyped using the chromosome nomenclature system GTA AGC-3 ), designed from the 5S rDNA sequence of rainbow trout by Levan et al. (1964). (Komiya and Takemura, 1979), was used to amplify 5S rDNA repeats Meiotic chromosomes were prepared following the technique de- from the genomes of the species, containing putative coding regions scribed by Yi et al. (2002) with some modification. The testes were im- for the 5S rRNA and also the NTS. Two PCR products of approximate- mersed in 0.75% KCl for 2 h and fixed in methanol:acetic acid (3:1) for ly 250 bp and 450 bp were obtained for each species. The fragments 20 min. Then the tissue was snipped adequately and the suspension was were cloned into PMD18-T vector (TaKaRa, Cat.No.CK2601A) and se- dropped onto slides and air-dried. The bivalents were stained with quenced. Then the probe was labeled with digoxigenin-11-dUTP using Giemsa and observed under a microscope. a random primed labeling kit (Roche, Cat. No. 11004760001). To pre- pare the 18S rDNA probe, an approximately 1500 bp fragment, a por- tion of 18S rDNA repeats of zebrafish (Danio rerio ), was produced by PCR using the primer pair 18SA (5 -TCT GCC CTA TCA ACT TTC 170 Cytogenet Genome Res 122:169–174 (2008) Fig. 2. ( a ) Bivalents of a pachytene sper- matocyte of L. marginatus . The perfect pair- ing of homologous autosomes formed the lin- ear bivalents. However, in the bivalent of sex chromosomes, some parts formed a loop (ar- row). This phenomenon is explained as de- layed pairing of nonrecombining regions. Such regions may represent sex associated fragments. ( b–d ) Similar phenomena in other spermatocytes. Bar = 5 m. -GAT-3 ) and 18SB (5 -AGT TTG ATC GTC TTC TCG GC-3 ). The tial hybridization signals were detected on a large m chro fragment was cloned, sequenced, and then labeled with biotin-11- mosome pair in the karyotype of L. marginatus ( Fig. 4 ). dUTP by nick translation. The 5S rRNA genes of L. marginatus were located on the In situ hybridizations were carried out according to the proto- col of Pinkel et al. (1986) with some modification. For biotin-labeled X and Y chromosomes at the same distance to the centro- th probes, hybridization sites were detected using CY3-streptavidin meres while 18S rRNA genes were found on the 4 m chro- and biotinylated anti-streptavidin (Sigma, Cat. No. S6402 & S6390). mosome pair. The 5S rRNA genes of L. styani were located Anti-dig-fluorescein Fab fragments (Roche, Cat. No. 11207741910) on the 1 st sm chromosome pair, 18S rRNA genes on the sex and fluorescein anti-sheep IgG (Vector, Cat. No. FI-6000) were chromosomes ( Fig. 5 ). used for digoxigenin-labeled probes. The chromosomes were counter- stained with 20 g/ml DAPI. Images were captured by a SenSys 1401E cooled CCD camera, and combined using MetaMorph software. Discussion R e s u l t s The diploid numbers of species of Liobagrus reported up to now are 2n = 24, 28, 30 and 42 with FN = 46, 52, 56 and The diploid number of L. marginatus was 2n = 24 and of 80 respectively (Ojima, 1985; Yu et al., 1989). Such diversity L. styani was 2n = 30. The karyotypes of L. marginatus were of karyotypes of these species indicates the extensive dif- 20m+2sm+2st in females and 19m+2sm+2st+1a in males. ferentiation of chromosomes, as in some other fish groups The karyotypes of L. styani were 16m+10sm+4st in both such as Salmonidae (Phillips and Ráb, 2001). In the present sexes ( Fig. 1 ). The X chromosome of L. marginatus was work, our results showed that the characteristics of chromo- metacentric, and the Y was acrocentric of similar size. In somes of the two Liobagrus species were largely different, contrast, the X and Y chromosomes of L. styani were het- including those of their heteromorphic sex chromosomes. eromorphic in size, and had a similar p/q ratio. In a metaphase of L. marginatus hybridized with In the pachytene phase of the L. marginatus spermato- (TTAGGG)n probes ( Fig. 4 a), there is a pair of inter- cyte, some delayed-pairing segments of sex chromosomes stitial signals on a large m chromosome pair. Although showed a circular configuration ( Fig. 2 ). (TTAGGG)n repeats at non-telomeric sites may be a part of The sex chromosomes in the karyotypes of the two spe- satellite DNA (Pagnozzi et al., 2000), the interstitial sites of cies were stained with CMA3 ( Fig. 3 ). In addition, there were such sequences have been proven to provide a greater flex- dual CMA3 -positive sites on a large m chromosome pair of ibility for karyotype changes (Kilburn et al., 2001). Here we L. marginatus . consider it to be the result of chromosomal fusion, taking FISH with the (TTAGGG)n probe showed labeling in the account of the presence of a dual CMA3 -positive-sites ends of each chromosome in the karyotypes of the two spe- chromosome pair of L.