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Abstract In order to obtain cytogenetic data of Odontobutis potamophila， head-kidney cells were collected as experimental materials to prepare chromosome specimen. The karyotypes of O. potamophila and the distribution and amount of transcriptionally active nucleolar organizer regions on chromosomes were analyzed by Giemsa staining and Ag-NORs. The results showed that the number of diploid chromosomes of O. potamophila was 44； the genome of O. potamophila was composed of 44 telocentric chromosomes； the karyotype formula was 2n=44t， NF=44； Ag-NORs were found in the paracentromeric region of chromosome. The results may lay the foundation for revealing the genetic pattern and chromosomal evolution of O. potamophila and contribute to further genetic breeding of O. potamophila.
Key words Odontobutis potamophila； Karyotype； Ag-NORs
Odontobutis potamophila， belonging to the family Odontobutidae in order Perciformes， is a type of small freshwater benthic carnivorous fishes that are distributed in Poyang Lake， Nanchang， Jiujiang， Yichun， Yingtan and other regions in Jiangxi Province[1-2]， which is delicious， nutritious and well received by consumers with great market demand and high economic value[3]. A large number of studies have been conducted about the biological characteristics， isoenzymes， embryonic development， development of prelarva fish， postlarva fish and juvenile fish， and organ differentiation of O. potamophila[4-7]， while the karyotypes of O. potamophila were rarely reported[8]. However， no studies have been reported yet on silver staining of nucleolar organizer regions （Ag-NORs） of O. potamophila. Chromosomes are the carriers of genes， which contain not only DNA as the physical basis of heredity， but also a large amount of proteins and a small amount of RNA that form a complex chromosome structure with DNA. It is precisely because the chromosome determines the genetic characteristics of the species， there is certain specificity in the number and morphology of chromosomes， behavior status in meiosis process and characteristics of chromosome banding of different species. Therefore， carrying out studies of fish chromosomes can not only reveal the genetic composition， genetic variation patterns and developmental mechanisms， but also contribute to interspecific hybridization and identification of polyploid breeding results[9]. In this study， karyotype characteristics of O. potamophila were investigated by Ag-NORs， aiming at providing the theoretical basis for studying cytogenetics and germplasm resource protection of O. potamophila， revealing the chromosomal evolution of Odontobutis species and further genetic breeding of O. potamophila. Materials and Methods
Materials
O. potamophila， weighing 50 g， was obtained from Wuyuan County in Jiangxi Province in May 2016， which was raised in the breeding laboratory of Jiangxi Fisheries Research Institute. Randomly 10 individuals were selected for karyotype analysis （6 males and 4 females）.
Methods
According to the method proposed by Cai et al.[10] with slight modifications， blood samples were collected from the tail vein at 10 μl/g body weight and then phytohemagglutinin （PHA） was injected into the base of the pectoral fin at 10 μg/g body weight. After 4 h， colchicine was injected into the base of the pectoral fin at 1 μg/g body weight. Bloodletting and gill removing were performed 15 min later， and head-kidney was placed in a 10 ml centrifuge tube to prepare the cell suspension. Cells were collected by centrifugation at 500 r/min， added with 0.075 mol/L KCl solution and placed in a 37 ℃ water bath for 30 min for hypotonic incubation. After centrifugation， the mixture was added with newly prepared Carnoy’s fluid and fixed at room temperature for 20 min. The fixing step was repeated twice. After centrifugation， the precipitate was collected and added with an appropriate amount of newly prepared Carnoy’s fluid to resuspend cells. The cell suspension was collected with a pipette and dropped on a glass slide， passed through the flame of an alcohol lamp three times， and naturally dried before use.
The chromosome specimen for karyotype analysis was stained with Giemsa stain and placed under a microscope for observation and photographing. Subsequently， 100 well-distributed metaphases from different individuals were selected and counted under a microscope. After statistical analysis， the diploid chromosome number of O. potamophila was determined. Multiple well-distributed metaphases with clear morphology， moderate length， well-defined centromeres and two moderately separated monomers were selected for microphotography and measurement. The relative length and arm ratio were calculated for classification， pairing and karyotype analysis according to the criteria proposed by Levan[11].
Ag-NORs banding was performed according to the method proposed by Cai et al.[10] with slight modifications. The filter paper was unfolded on the bottom of the petri dish， moistened with distilled water， pressed with two toothpicks， and heated in a 60 ℃ water bath. The prepared fresh specimen was placed faceup on a petri dish， added with 20 μl of gelatin developer on the slide， added with 40 μl of 50% silver nitrate solution， covered with the coverslip， and heated in a water bath until the specimen turned golden brown （about 13 min）. The specimen was rinsed rapidly with distilled water. After removal of the coverslip， the specimen was observed and photographed under a microscope. Results and Analysis
Karyotypes of O. potamophila
One hundred well-distributed metaphases from different individuals of O. potamophila were selected and counted under a microscope. The results indicated that 80% metaphases of O. potamophila harbored 44 diploid chromosomes， suggesting that the number of diploid chromosomes of O. potamophila was 44 （Table 1）.
Idiogram and karyotype
Metaphases with clear morphology and well-defined centromeres were selected and observed to obtain the idiogram （Fig. 1） and karyogram （Fig. 2） of O. potamophila. The results indicated that O. potamophila genome was completely composed of telocentric chromosomes； the karyotype formula was 2n=44t； chromosome arm number was 44； there was no secondary constriction or satellite.
Ag-NORs patterns
The silver-stained loci of O. potamophila were clearly visible （Fig. 3）. Statistical analysis was performed for 40 metaphases and 80 interphase nucleus. The number of Ag-NORs of O. potamophila was 1-3. There were significant differences among the occurrence frequency of Ag-NORs with polymorphism and different amounts in different cells. In interphase nucleus， chromosomes with two silver-stained loci accounted for the highest percentage （accounting for 70%）， followed by those with three silver-stained loci （accounting for 20%）； chromosomes with one silver-stained locus accounted for only 10%. In mitotic phase， chromosomes with one silver-stained locus accounted for 90% and those harboring two silver-stained loci accounted for 10%. Silver-stained loci were close to centromeres.
Conclusion and Discussion
According to records， there are at least four Odontobutis species in China， including O. potamophila， O. sinensis， O. haifengensis and O. yaluensis. At present， chromosome arm number of three Odontobutis species， of which the basic karyotypes have been investigated， ranges from 44 to 48. Specifically， O. potamophila and O. yaluensis maintain the original karyotype （2n=44t） and arm number （NF=44）； the karyotype of O. haifengensis is 2n=44t and the arm number is 48[8， 12-14]. Studies have shown that the unchanged chromosome number and increased chromosome arm number are caused by the reversed position of chromosome centromeres[15]. Li[16] reported that among specific taxonomic groups， groups with more chromosome arms are more specialized than those with fewer chromosome arms. Accordingly， it is generally considered that among Odontobutis species， groups with NF=44 are more primitive， while those with NF>44 are more specialized. Therefore， O. potamophila is a relatively primitive group among Odontobutis species according to the chromosome arm number of basic karyotype. Studies have shown that nucleolar organizer regions and heterochromatin may play an important role in chromosome evolution， in addition to the basic karyotype of chromosomes. Ag-NORs （NOR， nucleolar organizer region） are distribution regions of 18S rRNA and 28S rRNA genes in cells， which can reveal the number and distribution site of transcriptionally active rRNA genes in cells. The transcriptional activity of rRNA genes is mainly affected by the number and transcription rate of transcriptionally active genes. The size and intensity of Ag-NORs are related to the activity of NOR. Therefore， the polymorphism of Ag-NORs actually reflects the polymorphism and chromosome evolution rate of transcriptional activity of rRNA genes[17-18]. Previous reports suggest that the position and number of Ag-NORs vary among different species. The occurrence frequency of Ag-NORs also exhibits polymorphism among different varieties （groups） of the same species. Thus， the polymorphism of Ag-NORs can be used as a genetic marker in animal genetic breeding research[19-20]. So far， very few studies have been reported about cytogenetics of Odontobutis species， indicating a great potential for development of Odontobutis resources. In this study， the silver staining of nucleolar organizer regions （Ag-NORs） of O. potamophila is reported for the first time for Odontobutis species， which not only lays the theoretical foundation for analyzing the karyotypes and phylogenetic evolution of other Odontobutis species， but also provides reference for artificial breeding， genetic breeding and germplasm resource protection and development of Odontobutis species.
References
[1] WU HL， ZHONG JS. Fauna Sinica： Osteichthyes， Perciformes （V）， Gobioidei[M]. Beijing： Science Press， 2008.（in Chinese）
[2] WU HL， WU XQ， XIE YH. A revision of the genus Odontobutis from China with description of a new species[J]. Journal of Shanghai Fisheries University， 1993， （1）：52-61.（in Chinese）
[3] ZHANG YP， WANG HH， WU B， et al. Research progress on protection and germplasm resource of Odontobutis[J]. Jiangsu Agricultural Sciences， 2015， 43（2）：230-232.（in Chinese）
[4] QIAO DL， HONG J. Preliminary Study on morphological character and taxonomic status of Odontobutis obscurus in Huaihe[J]. Freshwater Fisheries， 2007， 2：20-23.（in Chinese）
[5] HU XC， LUO Y， ZHAO YL， et al. Variation of the composition and contents of the soluble proteins during embryonic development of Odontobutis potamophila[J]. Freshwater Fisheries， 2008， 1：20-22.（in Chinese） [6] HU XC， ZHAO YL. Visual organ development and its relationship with feeding of Odontobutis potamophila[J]. Chinese Journal of Zoology， 2007， 5：41-48.（in Chinese）
[7] XU XD， LIAO HS， WANG HH， et al. Six kinds of isozymes in different tissues of Odontobutis potamophila[J]. Chinese Agricultural Science Bulletin， 2017， 33（24）：1-5.（in Chinese）
[8] ZHANG J， TANG J， SHEN SD， et al. Karyotypic study on Odontobutis potamophila Günther[J]. Jiangsu Agricultural Sciences， 2010， （2）：253-256.（in Chinese）
[9] YU XJ， ZHOU T， LI YC， et al. Chromosomes of Freshwater Fishes in China[M]. Beijing： Science Press， 1989， 1-3.（in Chinese）
[10] CAI Y， ZHOU YC， XIE RM， et al. A study on the karyotype， Ag-NORs and C-banding in Epinephelus bleekeri[J]. Journal of Fisheries of China， 2012， 36（5）：647-651.（in Chinese）
[11] Levan A. Nomenclature for centrometic position on chromosomes[J]. Hereditias， 1964， 52（2）： 201-220.（in Chinese）
[12] GUI JF， LI YC， LI K， et al. A comparative study on karyotypes of two species of Gobioid fishes[J]. Zoological Research， 1984， （s2）：71-72， 105.（in Chinese）
[13] ZHANG KJ. Studies on the karyotypes of three freshwater fishes （Coreosiniperca roulei， Sarcocheilichthys nigripinnis nigripinnis and Odontobutis obscurus）[J]. Journal of Fisheries of China， 1989， 13（1）：52-58.（in Chinese）
[14] ZHANG LJ， ZHU F， YIN SW， et al. Karyotypes of Odontobutis potamophila （♀） × Odontobutis yaluensis （♂） and their hybrid progeny[J]. Marine Fisheries， 2013， 35（2）：183-188.（in Chinese）
[15] Perazzo G， Noleto RB， Vicari MR， et al. Chromosomal studies in Crenicichla lepidota， and Australoheros facetus， （Cichlidae， Perciformes） from extreme Southern Brazil[J]. Reviews in Fish Biology & Fisheries， 2011， 21（3）：509-515.
[16] LI SS. Cytotaxonomy of fish[J]. Biological Science Dynamics， 1981.（in Chinese）
[17] REN XH， CUI JX， YU JX. Studies on NORs of 6 species in Cyprinidae （Pisces）[J]. Hereditas （Beijing）， 1993， 15（4）：11-13.（in Chinese）
[18] Galetti Jr PM， Molina WF， Affonso PRAM， et al. Assessing genetic diversity of Brazilian reef fishes by chromosomal and DNA markers[J]. Genetica， 2006， 126（1-2）：161-177.
[19] WANG RDF， SHI LM， HE WS. A comparative study of the Ag-NORs of Carassius auratus from different geographic districts[J]. Zoological Research， 1988， 9（2）：165-169.（in Chinese）
[20] REN XH， YU QX， WEI P. Polymorphisms of silver-stained NORs in rice-field eels （Monopterus albus Zuiew）[J]. Acta Genetica Sinica， 1991（4）：304-311.（in Chinese）
Key words Odontobutis potamophila； Karyotype； Ag-NORs
Odontobutis potamophila， belonging to the family Odontobutidae in order Perciformes， is a type of small freshwater benthic carnivorous fishes that are distributed in Poyang Lake， Nanchang， Jiujiang， Yichun， Yingtan and other regions in Jiangxi Province[1-2]， which is delicious， nutritious and well received by consumers with great market demand and high economic value[3]. A large number of studies have been conducted about the biological characteristics， isoenzymes， embryonic development， development of prelarva fish， postlarva fish and juvenile fish， and organ differentiation of O. potamophila[4-7]， while the karyotypes of O. potamophila were rarely reported[8]. However， no studies have been reported yet on silver staining of nucleolar organizer regions （Ag-NORs） of O. potamophila. Chromosomes are the carriers of genes， which contain not only DNA as the physical basis of heredity， but also a large amount of proteins and a small amount of RNA that form a complex chromosome structure with DNA. It is precisely because the chromosome determines the genetic characteristics of the species， there is certain specificity in the number and morphology of chromosomes， behavior status in meiosis process and characteristics of chromosome banding of different species. Therefore， carrying out studies of fish chromosomes can not only reveal the genetic composition， genetic variation patterns and developmental mechanisms， but also contribute to interspecific hybridization and identification of polyploid breeding results[9]. In this study， karyotype characteristics of O. potamophila were investigated by Ag-NORs， aiming at providing the theoretical basis for studying cytogenetics and germplasm resource protection of O. potamophila， revealing the chromosomal evolution of Odontobutis species and further genetic breeding of O. potamophila. Materials and Methods
Materials
O. potamophila， weighing 50 g， was obtained from Wuyuan County in Jiangxi Province in May 2016， which was raised in the breeding laboratory of Jiangxi Fisheries Research Institute. Randomly 10 individuals were selected for karyotype analysis （6 males and 4 females）.
Methods
According to the method proposed by Cai et al.[10] with slight modifications， blood samples were collected from the tail vein at 10 μl/g body weight and then phytohemagglutinin （PHA） was injected into the base of the pectoral fin at 10 μg/g body weight. After 4 h， colchicine was injected into the base of the pectoral fin at 1 μg/g body weight. Bloodletting and gill removing were performed 15 min later， and head-kidney was placed in a 10 ml centrifuge tube to prepare the cell suspension. Cells were collected by centrifugation at 500 r/min， added with 0.075 mol/L KCl solution and placed in a 37 ℃ water bath for 30 min for hypotonic incubation. After centrifugation， the mixture was added with newly prepared Carnoy’s fluid and fixed at room temperature for 20 min. The fixing step was repeated twice. After centrifugation， the precipitate was collected and added with an appropriate amount of newly prepared Carnoy’s fluid to resuspend cells. The cell suspension was collected with a pipette and dropped on a glass slide， passed through the flame of an alcohol lamp three times， and naturally dried before use.
The chromosome specimen for karyotype analysis was stained with Giemsa stain and placed under a microscope for observation and photographing. Subsequently， 100 well-distributed metaphases from different individuals were selected and counted under a microscope. After statistical analysis， the diploid chromosome number of O. potamophila was determined. Multiple well-distributed metaphases with clear morphology， moderate length， well-defined centromeres and two moderately separated monomers were selected for microphotography and measurement. The relative length and arm ratio were calculated for classification， pairing and karyotype analysis according to the criteria proposed by Levan[11].
Ag-NORs banding was performed according to the method proposed by Cai et al.[10] with slight modifications. The filter paper was unfolded on the bottom of the petri dish， moistened with distilled water， pressed with two toothpicks， and heated in a 60 ℃ water bath. The prepared fresh specimen was placed faceup on a petri dish， added with 20 μl of gelatin developer on the slide， added with 40 μl of 50% silver nitrate solution， covered with the coverslip， and heated in a water bath until the specimen turned golden brown （about 13 min）. The specimen was rinsed rapidly with distilled water. After removal of the coverslip， the specimen was observed and photographed under a microscope. Results and Analysis
Karyotypes of O. potamophila
One hundred well-distributed metaphases from different individuals of O. potamophila were selected and counted under a microscope. The results indicated that 80% metaphases of O. potamophila harbored 44 diploid chromosomes， suggesting that the number of diploid chromosomes of O. potamophila was 44 （Table 1）.
Idiogram and karyotype
Metaphases with clear morphology and well-defined centromeres were selected and observed to obtain the idiogram （Fig. 1） and karyogram （Fig. 2） of O. potamophila. The results indicated that O. potamophila genome was completely composed of telocentric chromosomes； the karyotype formula was 2n=44t； chromosome arm number was 44； there was no secondary constriction or satellite.
Ag-NORs patterns
The silver-stained loci of O. potamophila were clearly visible （Fig. 3）. Statistical analysis was performed for 40 metaphases and 80 interphase nucleus. The number of Ag-NORs of O. potamophila was 1-3. There were significant differences among the occurrence frequency of Ag-NORs with polymorphism and different amounts in different cells. In interphase nucleus， chromosomes with two silver-stained loci accounted for the highest percentage （accounting for 70%）， followed by those with three silver-stained loci （accounting for 20%）； chromosomes with one silver-stained locus accounted for only 10%. In mitotic phase， chromosomes with one silver-stained locus accounted for 90% and those harboring two silver-stained loci accounted for 10%. Silver-stained loci were close to centromeres.
Conclusion and Discussion
According to records， there are at least four Odontobutis species in China， including O. potamophila， O. sinensis， O. haifengensis and O. yaluensis. At present， chromosome arm number of three Odontobutis species， of which the basic karyotypes have been investigated， ranges from 44 to 48. Specifically， O. potamophila and O. yaluensis maintain the original karyotype （2n=44t） and arm number （NF=44）； the karyotype of O. haifengensis is 2n=44t and the arm number is 48[8， 12-14]. Studies have shown that the unchanged chromosome number and increased chromosome arm number are caused by the reversed position of chromosome centromeres[15]. Li[16] reported that among specific taxonomic groups， groups with more chromosome arms are more specialized than those with fewer chromosome arms. Accordingly， it is generally considered that among Odontobutis species， groups with NF=44 are more primitive， while those with NF>44 are more specialized. Therefore， O. potamophila is a relatively primitive group among Odontobutis species according to the chromosome arm number of basic karyotype. Studies have shown that nucleolar organizer regions and heterochromatin may play an important role in chromosome evolution， in addition to the basic karyotype of chromosomes. Ag-NORs （NOR， nucleolar organizer region） are distribution regions of 18S rRNA and 28S rRNA genes in cells， which can reveal the number and distribution site of transcriptionally active rRNA genes in cells. The transcriptional activity of rRNA genes is mainly affected by the number and transcription rate of transcriptionally active genes. The size and intensity of Ag-NORs are related to the activity of NOR. Therefore， the polymorphism of Ag-NORs actually reflects the polymorphism and chromosome evolution rate of transcriptional activity of rRNA genes[17-18]. Previous reports suggest that the position and number of Ag-NORs vary among different species. The occurrence frequency of Ag-NORs also exhibits polymorphism among different varieties （groups） of the same species. Thus， the polymorphism of Ag-NORs can be used as a genetic marker in animal genetic breeding research[19-20]. So far， very few studies have been reported about cytogenetics of Odontobutis species， indicating a great potential for development of Odontobutis resources. In this study， the silver staining of nucleolar organizer regions （Ag-NORs） of O. potamophila is reported for the first time for Odontobutis species， which not only lays the theoretical foundation for analyzing the karyotypes and phylogenetic evolution of other Odontobutis species， but also provides reference for artificial breeding， genetic breeding and germplasm resource protection and development of Odontobutis species.
References
[1] WU HL， ZHONG JS. Fauna Sinica： Osteichthyes， Perciformes （V）， Gobioidei[M]. Beijing： Science Press， 2008.（in Chinese）
[2] WU HL， WU XQ， XIE YH. A revision of the genus Odontobutis from China with description of a new species[J]. Journal of Shanghai Fisheries University， 1993， （1）：52-61.（in Chinese）
[3] ZHANG YP， WANG HH， WU B， et al. Research progress on protection and germplasm resource of Odontobutis[J]. Jiangsu Agricultural Sciences， 2015， 43（2）：230-232.（in Chinese）
[4] QIAO DL， HONG J. Preliminary Study on morphological character and taxonomic status of Odontobutis obscurus in Huaihe[J]. Freshwater Fisheries， 2007， 2：20-23.（in Chinese）
[5] HU XC， LUO Y， ZHAO YL， et al. Variation of the composition and contents of the soluble proteins during embryonic development of Odontobutis potamophila[J]. Freshwater Fisheries， 2008， 1：20-22.（in Chinese） [6] HU XC， ZHAO YL. Visual organ development and its relationship with feeding of Odontobutis potamophila[J]. Chinese Journal of Zoology， 2007， 5：41-48.（in Chinese）
[7] XU XD， LIAO HS， WANG HH， et al. Six kinds of isozymes in different tissues of Odontobutis potamophila[J]. Chinese Agricultural Science Bulletin， 2017， 33（24）：1-5.（in Chinese）
[8] ZHANG J， TANG J， SHEN SD， et al. Karyotypic study on Odontobutis potamophila Günther[J]. Jiangsu Agricultural Sciences， 2010， （2）：253-256.（in Chinese）
[9] YU XJ， ZHOU T， LI YC， et al. Chromosomes of Freshwater Fishes in China[M]. Beijing： Science Press， 1989， 1-3.（in Chinese）
[10] CAI Y， ZHOU YC， XIE RM， et al. A study on the karyotype， Ag-NORs and C-banding in Epinephelus bleekeri[J]. Journal of Fisheries of China， 2012， 36（5）：647-651.（in Chinese）
[11] Levan A. Nomenclature for centrometic position on chromosomes[J]. Hereditias， 1964， 52（2）： 201-220.（in Chinese）
[12] GUI JF， LI YC， LI K， et al. A comparative study on karyotypes of two species of Gobioid fishes[J]. Zoological Research， 1984， （s2）：71-72， 105.（in Chinese）
[13] ZHANG KJ. Studies on the karyotypes of three freshwater fishes （Coreosiniperca roulei， Sarcocheilichthys nigripinnis nigripinnis and Odontobutis obscurus）[J]. Journal of Fisheries of China， 1989， 13（1）：52-58.（in Chinese）
[14] ZHANG LJ， ZHU F， YIN SW， et al. Karyotypes of Odontobutis potamophila （♀） × Odontobutis yaluensis （♂） and their hybrid progeny[J]. Marine Fisheries， 2013， 35（2）：183-188.（in Chinese）
[15] Perazzo G， Noleto RB， Vicari MR， et al. Chromosomal studies in Crenicichla lepidota， and Australoheros facetus， （Cichlidae， Perciformes） from extreme Southern Brazil[J]. Reviews in Fish Biology & Fisheries， 2011， 21（3）：509-515.
[16] LI SS. Cytotaxonomy of fish[J]. Biological Science Dynamics， 1981.（in Chinese）
[17] REN XH， CUI JX， YU JX. Studies on NORs of 6 species in Cyprinidae （Pisces）[J]. Hereditas （Beijing）， 1993， 15（4）：11-13.（in Chinese）
[18] Galetti Jr PM， Molina WF， Affonso PRAM， et al. Assessing genetic diversity of Brazilian reef fishes by chromosomal and DNA markers[J]. Genetica， 2006， 126（1-2）：161-177.
[19] WANG RDF， SHI LM， HE WS. A comparative study of the Ag-NORs of Carassius auratus from different geographic districts[J]. Zoological Research， 1988， 9（2）：165-169.（in Chinese）
[20] REN XH， YU QX， WEI P. Polymorphisms of silver-stained NORs in rice-field eels （Monopterus albus Zuiew）[J]. Acta Genetica Sinica， 1991（4）：304-311.（in Chinese）