Induced Gynogenesis in Grass Carp (Ctenopharyngodon Idellus) Using Irradiated Sperm of Allotetraploid Hybrids

Mar Biotechnol DOI 10.1007/s10126-011-9365-8

ORIGINAL ARTICLE

Induced Gynogenesis in Grass Carp (Ctenopharyngodon idellus) Using Irradiated Sperm of Allotetraploid Hybrids

Hong Zhang & ShaoJun Liu & Chun Zhang & Min Tao & LiangYue Peng & CuiPing You & Jun Xiao & Yi Zhou & GongJian Zhou & KaiKun Luo & Yun Liu

Received: 24 July 2010 /Accepted: 4 January 2011 # Springer Science+Business Media, LLC 2011

Abstract Grass carp (Ctenopharyngodon idellus)eggs Keywords Grass carp . Gynogenesis . Microsatellite . Sex were activated by UV-irradiated diploid sperm of allotetra- determination ploid hybrids derived from red crucian carp (♀)×common carp (♂) and then duplicated by cold shock in 4–6°C water for 10–12 min. Different cold shock initiation times Introduction resulted in two types of diploid gynogenetic grass carp: meiotic gynogenetic (meiG) and mitotic gynogenetic As a valuable adjunct to fish genetics and aquaculture, (mitG). Over a 5-year period, a total of 17,170 meiG and gynogenesis has been successfully induced in many fish 1,080 mitG fry were produced and 6,862 meiG and 372 species, including rainbow trout (Oncorhynchus mykiss; mitG grass carp survived. The gynogenetic fish were Chourrout and Quillet 1982), common carp (Cyprinus confirmed by morphological characteristics, chromosome carpio; Komen et al. 1991), African catfish (Clarias examination, and microsatellite DNA analysis. The mor- gariepinus; Váradi et al. 1999), Japanese crucian carp phological traits of the gynogenetic grass carp were similar (Carassius cuvieri; Sun et al. 2006), red crucian carp to those of wild diploid grass carp. Normal gynogenetic (Carassius auratus, red variety; Sun et al. 2007), turbot fish were identified as diploid with 48 chromosomes by (Sxophthalmus maximus;Xuetal.2008), half-smooth chromosomal metaphases examination, while nonviable tongue sole (Cynoglossus semilaevis; Chen et al. 2009), abnormal embryos were detected as haploid with 24 and channel catfish (Ictalurus punctatus; Waldbieser et al. chromosomes. Microsatellite DNA analysis indicated that 2010). Gynogenesis can be achieved either by retention of after one generation of gynogenesis, the genetic purity of the second polar body (meiotic gynogenesis, meiG) or by meiG and mitG grass carp was significantly increased over suppression of the first cleavage (mitotic gynogenesis, that of wild grass carp. In addition, both meiG and mitG mitG) of eggs inseminated with irradiated sperm (Pandian grass carp groups were 100% female, and 88% of these and Koteeswaran 1998). The former can produce portions showed normal ovary development. Thus, the sex determi- of heterozygosity because of gene–centromere recombina- nation mechanism in female grass carp was homogamety. tion and more than three successive generations are needed The ability to establish pure all-female groups of meiG and to establish clonal inbred lines, while the latter results in mitG grass carp should be a valuable contribution to both completely homozygous diploid fish and pure lines can be fish genetics and grass carp breeding. established within two generations. Grass carp (Ctenopharyngodon idellus)isalarge herbivorous freshwater fish species of the family Cypri- : * : : : : : nidae. It is one of the most important species cultured in H. Zhang: S. Liu: ( ) C. Zhang: M.: Tao L. Peng C. You J. Xiao Y. Zhou G. Zhou K. Luo Y. Liu China and has been introduced to more than 40 other Key Laboratory of Protein Chemistry and Fish Developmental countries for aquatic weed control. Its fast growth rate, Biology of the Education Ministry of China, large size, lack of fine inter-muscular bones, and, most College of Life Sciences, Hunan Normal University, Changsha 410081, People’s Republic of China importantly, its feeding habits make this fish an ideal e-mail: [email protected] species for culture. Grass carp not only grow quickly but Mar Biotechnol also have a low requirement for dietary protein, and they Materials and Methods can be produced at low cost by feeding with aquatic weeds, terrestrial grasses, and by-products from grain processing Animal and Gamete Collection and vegetable oil extraction. However, farmed grass carp are readily susceptible to various kinds of diseases that Allotetraploid hybrids were obtained from the Protection cause high percentages of morbidity and mortality, leading Station of Polyploidy Fish in Hunan Normal University. to greatly reduced yields. Although antibiotics and drugs Grass carp were obtained from Hunan Xiang Yun Biology are typically effective treatments, the abuse of antibiotics Science and Technology Limited Company. and drugs may affect the food quality of the fish, which During the reproductive seasons (from May to June) each impacts negatively on consumers and pollutes the environ- year since 2006, sexually mature female grass carp were ment at the same time. induced to spawn by a single intraperitoneal injection of For these reasons, disease-resistant grass carp are highly human chorionic gonadotropin at a dose of 3–5 μg/g body desirable as they can greatly increase fish yields. However, weight for the first injection and 6–8 h later at a double dosage traditional methods for selecting disease resistance in pure for the second administration. Mature eggs could be harvested grass carp lines require several generations of sister brother in 10–16 h after the first injection at 23±2°C. matings, and since the grass carp takes more than 4 years to achieve sexual maturity, it would take at least several UV Irradiation for Sperm Inactivation decades to obtain a pure line. Moreover, the inbreeding line may vanish because of the close inbreeding retrogradation. The milt of male allotetraploid hybrids were stripped, One alternative method, gynogenesis, is considered to be an diluted with Hank’s solution (1:4), and then poured into efficient way to rapidly establish pure line. The genetic cold culture dishes to allow formation of a thin layer (0.1– purity of one generation of meiotic gynogenesis is 0.2 mm thick). UVC irradiation was performed using two estimated as approximately equivalent to that of eight to quartz UV lamps (ZSZ20D) emitting a wavelength of ten generations of sib matting. Two successive generations 253.7 nm. The total UV dosage administered to the sperm of meiotic gynogenetic fish can be used as breeders to was in the range 3,000–3,600 mJ/cm2, which corresponded provide seeds for mass production. In contrast, mitotic to an irradiation duration of 20–25 min. The milt gynogenesis can achieve homozygotes in one generation. suspension was continuously shaking at 30 rpm and 4°C Each of the mitotic gynogenetic fish can be a clonal during the exposure period. The irradiation was monitored founder capable of giving rise to an isoallelic clone, if by observing the vitality of the sperm under a microscope. reproduced gynogenetically. Therefore, mitotic gynogene- After UV irradiation, sperm were kept in glass tubes and sis, in combination with selective breeding, has been stored at 4°C. All processes were conducted in the dark. suggested as a shortcut to produce high-quality clonal fish lines with desired characteristics, such as higher growth Cold Shock Experiments rates and disease resistance. In the present study, using UV-irradiated sperm of Each batch of eggs, including three control groups, consisted allotetraploid hybrids derived from crucian carp (♀)and of a diploid control group (C1) of grass carp eggs and sperm; a common carp (♂), both meiotic and mitotic gynogenesis hybrid control group (C2) of grass carp eggs and allotetraploid were successfully induced in grass carp for the first time. hybrid sperm; a haploid control group (C3) of grass carp eggs Gynogenesis provided a means for analysis and further and UV-irradiated allotetraploid hybrid sperm without cold understanding of chromosome recombination and ex- shock; and an experimental group of grass carp eggs and UV- change, gene mapping, and the genetic mechanisms of irradiated allotetraploid hybrid sperm with cold shock. sex determination. In accordance with most other fish In the experimental group, eggs were activated by UV- species, gynogenesis in grass carp may also produce all- irradiated allotetraploid hybrid sperm and then subjected to a female offspring, which would be advantageous in elimi- cold shock treatment. In order to obtain the optimal protocol nating potential danger of excess reproduction, allowing for cold shock, three sets of experiments were designed. First, these fish to be used as biological tools to control nuisance cold shocks were given at a variety of temperatures, including aquatic plant growth. Outstanding individuals of gynoge- exposure to 0–4°C, 4–6°C, and 6–8°C water for 10- to 12-min netic grass carp can be used as breeders for artificial durations. Second, cold shocks were given at 4–6°C water for propagation. High-quality seeds would bring huge eco- durations of 8–10, 10–12, and 13–15 min. Third, cold shock nomic benefits in large-scale culture. Therefore, establish- at 4–6°C for 10–12 min was initiated at 1–30 min post- ment of pure all-female groups of meiG and mitG grass fertilization at 1- or 2-min intervals. All experiments were carp is of great significance in fish genetics and grass carp repeated more than three times. For each replicate, a different breeding. female was used. Mar Biotechnol

The percentages of embryos surviving to the 16–32 cell cleavage stage, incubation stage, and feeding stage, relative to the initial number of eggs in each group, were determined as the fertilization rate, the hatching rate, and the survival rate, respectively (Liu 1993).

Large-Scale Production of meiG and mitG Grass Carp

A large number of meiG and mitG grass carp were produced each year according to the methods developed in the present study (meiG: fertilized eggs with UV- irradiated allotetraploid hybrid sperm were cold shocked at 4–6°C for 10–12 min at 2–3 min after fertilization; mitG: cold shocked at 4–6°C for 10–12 min at 20–22 min after fertilization). Several batches of eggs have been used to Fig. 1 Effect of different temperatures of cold shock on gynogenesis induce gynogenetic fry each year since 2006. For each in grass carp. All eggs of experimental group were triggered at 2 min batch of eggs, one female was used. post-fertilization for 10–12 min. C1 diploid control, C2 hybrid control, C3 haploid control MeiG and mitG fry were calculated and then reared in a protected concrete pool. After 6 months of feeding, surviving fry were counted and transferred to earthen paper were described in a previous study (Liu et al. 2010). ponds. Amplification products were separated by electrophoresis on 8% polyacrylamide gels and sized against a pBR322 Morphological Traits DNA/Mspl ladder. Silver staining was used to visualize microsatellite DNA. At the age of 6 months, 20 meiG, 20 mitG, and 20 grass carp were morphologically examined. Both measurable and Gonad Observation and Sex Identification of meiG countable data were calculated. and mitG

Ploidy Examination by Chromosomal Preparation To observe the gonad development process and to identify the sex, meiG and mitG grass carp were sampled randomly To determine ploidy, 100 abnormal embryos with malfor- on a monthly basis between July 2007 and June 2008. Each mations similar to the C3 (haploids) and 20 normal month, four to eight fish were sampled for a total of 72 embryos were sampled at the tail bud stage. Chromosome individuals (including 47 meiG and 25 mitG grass carp) counts were also done on kidney tissue for each of ten 4- to throughout the year. The gonads were immediately fixed in 6-month-old meiG and mitG grass carp. Metaphase chromosome preparation of embryos and adults were performed essentially as previously described (Liu et al. 2010). For each type of fish, 200 metaphase chromosome spreads were analyzed.

Microsatellite Analysis

The total genomic DNA was isolated from whole blood collected from the caudal veins of 20 meiG grass carp, 20 mitG grass carp, 10 wild grass carp, and 10 allotetraploid hybrids according to the standard phenol–chloroform procedure (Sambrook et al. 1989). Eight pairs of microsatellite primers (MFW1, MFW5, MFW8, MFW15, hljy1516, hljy1920, hljy2728, hljy3940) were used with sequence repeat (CA)n according to published sequences (Crooijmans et al. Fig. 2 Effect of different duration times of cold shock on gynogenesis 1997;Gengetal.2006). Polymerase chain reaction in grass carp. All eggs were exposed to 4–6°C water 2 min post- (PCR) reaction conditions and components used in this fertilization. C1 diploid control, C2 hybrid control, C3 haploid control Mar Biotechnol

Fig. 3 Effect of different activation times of cold shocks on gynogenesis in grass carp. Cold shocks were applied at 4–6°C for 10–12 min. C1 diploid control, C2 hybrid control, C3 haploid control

Bouin’s solution for 24 h and then preserved in 70% shock at 4–6°C for 10–12 min, initiated at 2–3or20–22 min ethanol, paraffin-embedded, sectioned at 6 μm, and stained after fertilization, respectively. with hematoxylin and eosin. Compared to the control groups, both hybrid (C2) and haploid embryos (C3) were morphologically abnormal, showing a bent backbone, a short or almost no tail, and Results none survived to the first feeding stage. In the experimental group, two types of embryos were observed. Normal fry Optimal Cold Shock Conditions for meiG and mitG Grass (Fig. 4a) that had morphological characteristics similar to Carp normal diploid grass carp group (C1) were viable, whereas abnormal embryos (Fig. 4b) similar to C3 were nonviable. In the cold shock experiments, eggs fertilized with diluted but non-irradiated grass carp semen (C1), which served as Large-Scale Production of meiG and mitG Grass Carp an egg quality control, had an average survival to hatching stage above 80%. Eggs in all cold shock groups, as well as The yields of meiG and mitG grass carp for each production the haploid control (C3, the group that was activated by year are shown in Table 1. A total of 18,170 meiG and 1,080 UV-irradiated sperm without cold shock), had similar mitG fry were obtained during these 5 years. Of these, 6,862 fertilization rates, but the hatching rates and survival rates meiG and 372 mitG larvae survived. The meiG grass carp were extremely low compared to C1. Eggs inseminated produced in 2006 had reached sexual maturity and success- with UV-irradiated sperm had the highest survival rates fully reproduced in June 2010. when treated in 4–6°C water for 10–12 min (Fig. 1). The temperature of cold shock below 4°C (Fig. 1) or cold shock Morphological Traits duration longer than 12 min had severe negative impacts on egg viability and increased mortality (Fig. 2). Temperature The appearance traits of meiG and mitG grass carp are above 6°C or duration time less than 10 min did not allow shown in Fig. 5. Measurable traits and countable traits of effective retention of the second polar body or suppression of the first cleavage, and embryos with morphological malformation similar to the haploid control group (C3) were inviable. Two peaks were observed at 2–3 min and 20– 22 min post-fertilization (Fig. 3), with survival rates of 41% and 12.3%, respectively. Apparently, the initiation time of cold shock was very important for the diploidization of eggs, via retention of the second polar body (the first peak) or suppression of the first cleavage (the second peak). MitG grass carp had much lower survival rates compared to meiG Fig. 4 The appearance of normal and abnormal fry. a Normal fry of grass carp. Based on the experimental data, the optimum meiG grass carp. b Abnormal fry with enlarged pericardium, short, protocols for producing meiG and mitG grass carp were cold and bend tails Mar Biotechnol

Table 1 Number of gynogenetic fry produced and grass carp Year Fish type No. of fry obtained No. of grass carp survived surviving each year 2006 MeiG 600 200 2007 MeiG 5,000 1,682 2007 MitG 500 142 2008 MeiG 10,000 3,800 2008 MitG 100 50 2009 MeiG 800 300 2009 MitG 380 120 2010 MeiG 1,770 880 2010 MitG 100 60 meiG grass carp, mitG grass carp, and control wild grass 48). Figure 6 shows the chromosomes of meiG and mitG carp are presented in Table 2. MeiG and mitG grass carp grass carp. were completely identical in appearance and had almost the same measurable traits and countable traits as wild grass Microsatellite Analysis carp controls. Therefore, they were difficult to distinguish by morphological traits. Eight microsatellite loci were amplified in 20 meiG grass carp, 20 mitG grass carp, 10 common grass carp, and 10 Chromosomes allotetraploid hybrids. No identical allele of allotetraploid hybrids was examined in meiG and mitG grass carp groups Table 3 indicates the distribution of the chromosome in any of the eight loci examined, which indicated that number of 100 normal embryos (Fig. 4a) and 200 abnormal meiG and mitG grass carp showed exclusively maternal embryos (Fig. 4b) with haploid symptoms. The normal inheritance at these loci. In the three grass carp groups, the embryos bore chromosomes ranging from 45 to 49, in number of alleles per locus ranged from 1 to 7. At the which 90% of the metaphase analyses showed 48 chromo- hljy2728 locus, no individual polymorphism was observed somes, suggesting that they were diploids. The abnormal in the three grass carp groups. Heterozygous alleles were embryos with remarkable malformations had chromosome detected in the wild grass carp group in seven microsatellite numbers arraying from 20 to 28, and 78.5% of the primers (4/10 heterozygotes at MFW1 locus, 3/10 hetero- metaphases had 24 chromosomes, indicating that they were zygotes at MFW5 locus, 6/10 heterozygotes at MFW8 haploids. locus, 5/10 heterozygotes at MFW15 locus, 1/10 hetero- In the adult meiG and mitG grass carp, the chromosome zygotes at hljy1516 locus, 8/10 heterozygotes at hljy1920 numbers of somatic cells in metaphase of the mitosis were locus, and 7/10 heterozygotes at hljy3940 locus), whereas counted (Table 4). The chromosome numbers of meiG all meiG and mitG grass carp detected were homozygous at grass carp were mainly distributed from 45 to 48, with the eight loci (Table 5), except for one meiG grass carp metaphase spreads with 48 chromosomes accounting for (Fig. 7, lane 17) which was heterozygous at hljy3940. 96%, indicating that their chromosome number was 2n=48. These results indicated that after one generation of The chromosome number of mitG grass carp mainly ranged gynogenesis, genetic purity of meiG and mitG grass carp from 45 to 48, and 90.8% of these contained 48 was highly improved compared to the wild grass carp chromosomes, proving that they were also diploids (2n= group.

Fig. 5 The appearance of meiG and mitG grass carp. a Six- month-old meiG grass carp. b Six-month-old mitG grass carp Mar Biotechnol

Table 2 Phenotypes including measurable traits (the average ratios of length (HW/HL), tail width to tail length (TW/TL), head width to body length to whole length (BL/WL), body width to body length body width (HW/BW), and the countable traits (number of lateral (BW/BL), head length to body length (HL/BL), head width to head scales, number of dorsal fins in meiG, mitG, and wild grass carp)

Types of fish BL/WL BW/BL HL/BL HW/HL TW/TL HW/BW No. of lateral scales No. of dorsal fins

MeiG 0.85±0.03 0.27±0.02 0.25±0.04 0.77±0.03 0.88±0.03 0.74±0.03 38–44 III7 MitG 0.84±0.03 0.26±0.03 0.25±0.03 0.77±0.04 0.85±0.04 0.73±0.04 38–44 III7 Grass carp 0.84±0.04 0.26±0.04 0.25±0.04 0.77±0.03 0.86±0.03 0.73±0.03 36–44 III7

Gonad Observation and Sex Identification of diploid sperm for inducing gynogenesis has seldom been reported. In the present experiment, diploid sperm of A total of 72 individuals (including 47 meiG and 25 mitG allotetraploid hybrids were first used to activate the grass grass carp) were randomly examined. Primordial germ cells carp eggs and did so with high efficiency, with average (PGCs) transferred into the germinal ridge in 1 month fertilization rates above 90% and the highest survival rates (Fig. 8a, b), and the number of oogonia largely increased by for meiG and mitG grass carp at 41% and 12.3%, continuous mitosis over 2–3 months (Fig. 8c, d). Oocytes in respectively. The special characteristics of the diploid the early perinucleolar stage and in the perinucleolar stage sperm may contribute to the high efficiency. Diploid sperm could be observed at 4–12 months (Fig. 8e, f). Histological of allotetraploid hybrids showed normal shapes with head, sections suggested that the gonads of the examined mid-piece, and tail, but had a significantly larger head than gynogenetic grass carp were ovaries; 88% of gynogenetic normal haploid sperm (Liu et al. 2001). The concentration fish showed normal development of ovaries, while 12% of and motility of the diploid sperm was lower compared to individuals exhibited abnormal development. those of normal haploid sperm. With lower concentration and motility, much less irradiation time was needed for the diploid sperm to make the genome absolutely inactive but Discussion still be able to activate egg development. Less irradiation may result in the high efficiency of sperm in gynogenesis. In this study, protocols to induce meiotic and mitotic On the other hand, the reduction of irradiation time was gynogenesis were optimized and included UV irradiation of conducive to mass production. Moreover, the identification the diploid sperm of allotetraploid hybrids and the of gynogenetic fish was very simple since the hybrids of application of cold shock to the activated eggs. grass carp (♀) with allotetraploid hybrids (♂)were It is known that UV irradiation can cause direct DNA nonviable and malformed. The hybrid lethality is probably damage and a rapid loss of sperm fertilizing potential. due to the great imbalance in chromosomes between the Appropriate amount of UVC irradiation can entirely destroy two parental fish as grass carp has only 48 chromosomes the genomic components of sperm, but retain the ability to while allotetraploid hybrids possess 200 chromosomes. activate egg. In most gynogenesis studies, homogeneous or The initiation time, the shock temperature, and the heterogeneous sperm were used to activate egg develop- duration time are critical to the effectiveness of cold shock ment. Since it was hard to exclude the potential contami- in gynogenesis induction. In general, a cold shock treatment nant of sperm, gynogenetic fish were difficult to be for inducing meiotic gynogenesis should start between the identified when homogeneous sperm were used (Xu et al. time after gamete activation and before extrusion of the 2007; Lin and Dabrowski 1996). Suitable heterogeneous second polar body, whereas for inducing mitotic gynogen- sperm can solve this problem as the hybrids either showed a esis, the shock should start between the time after extrusion morphological marker (Sun et al. 2007) or are absolutely of the second polar body and before the first cleavage of the lethal (Rothbard et al. 1997; Stanley et al. 1975). Among all egg. Preliminary study of paraffin slices of grass carp of the heterogeneous sperm used for the induction of embryos showed that the second polar body had been gynogenesis, most were haploid sperm. However, the use excluded at 10–15 min after gamete activation, and the first

Table 3 Examination of chromosome number of embryos in Embryo type No. of embryos No. of metaphase Distribution of chromosome number the experimental group 20–24 24 24–28 45–47 48 49

Normal 100 200 0 0 0 12 180 8 Abnormal 200 400 25 313 62 0 0 0 Mar Biotechnol

Table 4 Examination of chromosome number of the meiG Fish type No. of fish No. of metaphase Distribution of chromosome number and mitG grass carp 45–47 48 49

meiG 10 500 18 480 2 mitG 10 250 22 227 1

cleavage furrow occurred at 40–45 min after fertilization. frequencies of some deleterious homologous recessive This was confirmed by the experimental results (e.g., genes and a lack of paternal DNA in the gynogenetic fish, Fig. 3) which showed that cold shock initiated at 2–3 and which might be essential for embryos to get through the 20–22 min post-activation resulted in the highest survival critical stages of early development. Furthermore, the rates for meiotic and mitotic gynogenesis, respectively. negative effects of cold shock associated with gynogenesis From the cold shock experiment, 4–6°C was considered to induction might also lead to the poor survival rate of be the best cold shock temperature for grass carp diploid gynogenetic fish. As a rule, the survival rate of gynogenesis; below 4°C was damaging to the eggs and mitotic gynogenesis was much lower than that of meiotic resulted in high mortality, whereas above 6°C did not allow gynogenesis. This was also confirmed by our experiments effective genome diploidization. Compared to the best cold with survival rate where only 12.3% of mitG grass carp shock temperature established for common carp (0–4°C) survived compared to 41% of meiG grass carp. The main (Linhart et al. 1986), the higher optimal temperature for reason for this phenomenon was that mitotic gynogenesis grass carp was possibly due to their semi-pelagic eggs was 100% homozygous and unmasked all recessive lethal which were less tolerant of cold. genes in one generation, while meiotic gynogenesis In the present study, shocks longer than 12 min did preserved portions of heterozygosis. not increase the number of gynogenetic fry under a Optimizing the cold shock protocol can largely given shock temperature and initiation time, while decrease damage to eggs and result in a relatively shocks shorter than 10 min resulted in poor diploidiza- higher survival rate. In the present study, optimal tion. These results were different from those reported protocols for both meiotic and mitotic gynogenesis were for other Cyprinidae species, such as loach, common determined. A total of 18,170 meiG grass carp and carp, and tench (Tinca tinca L.), among which the 1,080 mitG grass carp fry were produced over the duration time typically ranged from 20 to 60 min (Suzuki course of 5 years, and 6,862 meiG and 372 mitG grass et al. 1985;Linhartetal.1986, 1995;Wangetal.2006). carp have survived so far. The mitG grass carp showed However, the optimal duration in the present study was significantly different characteristics among individuals, similar to that reported for sea bass (Dicentrarchus labrax which means that outstanding individuals can be chosen L.) where 10-min shocks were sufficient to inhibit as founders of superior pure clonal lines. extrusion of the second polar body (Felip et al. 1997). Microsatellite markers have been viewed as powerful Low survival rate was a typical characteristic of tools for detecting genetic diversity and genetic linkage gynogenesis. This was probably because of the increased mapconstruction(LiuandCordes2004; Wright and

Fig. 6 Chromosome spreads at metaphase in meiG and mitG grass carp. a The 48 chromosomes of meiG grass carp. b The 48 chromosomes of mitG grass carp Mar Biotechnol

Table 5 Number of alleles, estimated size of alleles, the number of homozygotes and heterozygotes for the eight microsatellite loci in wild grass carp, meiG and mitG grass carp groups

Locus No. of alleles Estimated size No. of homozygotes/total no. of fish No. of heterozygotes/total no. of fish

Grass carp meiG mitG Grass carp meiG mitG

MFW1 2 170, 180 6/10 20/20 20/20 4/10 0/20 0/20 MFW5 2 138, 140 7/10 20/20 20/20 3/10 0/20 0/20 MFW8 2 160, 168 4/10 20/20 20/20 6/10 0/20 0/20 MFW15 2 150, 156 5/10 20/20 20/20 5/10 0/20 0/20 hljy1516 2 217, 242 9/10 20/20 20/20 1/10 0/20 0/20 hljy1920 7 244, 260, 262, 275, 288, 290, 310 2/10 20/20 20/20 8/10 0/20 0/20 hljy2728 1 170 10/10 20/20 20/20 0/10 0/20 0/20 hljy3940 4 238, 240, 260, 262 3/10 19/20 20/20 7/10 1/20 0/20

Bentzen 1994). Microsatellites have been widely used for Gynogenesis was an important approach for elaborat- the molecular identification of gynogenetic fish because of ing the sex determination mechanism by measurement their PCR-based, co-dominant Mendel inheritance pattern and of the sex ratio of gynogenetic diploids. The XY sex highly polymorphic characteristics (Li et al. 2008;Mcconnell determination system has been determined in most fish et al. 1995; Yan et al. 2005). Specific DNA fragments arising species (Chourrout and Quillet 1982;Gomelskyetal. from the paternal genome of blunt snout bream were 1994; Pongthana et al. 1995; Rougeot et al. 2005;Stanley detected in a gynogenetic red crucian carp by microsatellite et al. 1975) in which 100% females were obtained by markers (Liu et al. 2010). Similarly, exclusive maternal gynogenesis. However, a high proportion of males were inheritance and high heterozygosity were observed in a detected in diploid gynogenetic hybrid sturgeon (Omoto et meiotic gynogenetic large-scale loach (You et al. 2008). The al. 2005), large-scale loach (You et al. 2008), and half- microsatellite makers in the present study indicated that the smooth tongue sole (C. semilaevis;Chenetal.2009), gynogenetic grass carp were also derived exclusively by suggesting that a ZW sex determination system may maternal inheritance, with no contribution of the paternal operate in those limited species. In the present study, no genome of the allotetraploid hybrids detected in the males were found in any of the examined gynogenetic gynogenetic fish. The putative meiG grass carp group was grass carp, which indicated that female grass carp showed homozygous at all loci detected, except for one individual homogamety. This finding was in accordance with most that showed heterozygosity at hljy3940 locus. This sug- other fish species reported, such as common carp gested that these fish were true meiG grass carp which had (Gomelsky et al. 1994), loach (Suzuki et al. 1985), tench arisen by retention of the second polar body, and their (Wang et al. 2006), and rainbow trout (Krisfalusi et al. heterozygosity was probably due to a gene–centromere 2000). recombination. Putative mitG grass carp were 100% homo- Early-stage gonads were determined as putative zygous at all evaluated loci, thus suggesting that they were ovaries by the criteria of female precocious meiosis true mitG grass carp which were obtained by suppression of and the difference in number and arrays of germ cells the first cleavage. between ovaries and testes. Generally speaking, germ

Fig. 7 Electrophotogram of microsatellite DNA patterns produced by represented 20 mitG grass carp. Lanes 51–60 represented ten the primer hljy3940 in wild grass carp, meiG grass carp, mitG grass allotetraploid hybrids. Arrow showed heterozygous allele of meiG carp, and allotetraploid hybrids. Lanes 1–10 represented ten wild grass grass carp. K represented the negative control. M represented pBR322 carp. Lanes 11–30 represented 20 meiG grass carp. Lanes 31–50 DNA/Mspl marker Mar Biotechnol

Fig. 8 Histology of gonads of diploid meiG and mitG grass carp. a Histological section of 1- month-old meiG grass carp gonads showing PGCs translo- cated in the germinal ridge. b High magnification of (a)showing PGCs with one or two nucleolus. c Histological section of 3-month-old meiG grass carp gonads; the number of oogoniums were largely increased by mitosis. d High magnification of (c), mitosis of oogoniums. e Histological section of 6-month- old mitG grass carp gonads showing oocytes at stage I or early II. f Histological section of 12-month-old meiG grass carp gonads showing the developing oocytes in the perinucleolar stage

cells in putative ovaries outnumber those in putative In summary, UV-irradiated sperm of allotetraploid testes for most teleosts (Strüssmann and Nakamura hybrids were used to induce gynogenesis in grass carp. 2002; Nakamura et al. 1998). Abnormal ovarian develop- Optimal protocols for meiotic and mitotic gynogenesis in ment has been reported in many gynogenetic fish. For grass carp were determined and applied to large-scale example, in diploid gynogenetic rainbow trout, 30% of the production. Both meiG and mitG grass carp were obtained aberrant ovaries were aberrant and resembled triploid and showed good growth, allowing two mass groups of ovaries (Krisfalusi et al. 2000). Similarly, abnormal meiG and mitG grass carp to be established. The establish- ovaries, but at a lower percentage (12%), were detected ment of all-female pure groups of meiG and mitG grass carp in gynogenetic grass carp in the present study, which has important significance for fish genetics and grass carp reflects an increased homozygosity that unmasked dam- breeding. Because of their herbivorous habits, unisexual aging recessive alleles (Purdom 1983) or an incomplete gynogenetic grass carp also can be introduced as a form of inactivation of paternal chromatin (Krisfalusi et al. 2000; aquatic weed control without potential danger of excess Purdom 1983). reproduction. Mar Biotechnol

Acknowledgments This research was supported by grants from the application to the population genetics of Canadian east coast National Science Fund for Distinguished Young Scholars (no. stocks of Atlantic salmon. Aquaculture 137:19–30 30725028), the National Natural Science Foundation of China (no. Nakamura M, Kobayashi T, Chang XT, Nagahama Y (1998) Gonadal 30930071), and the State Key Basic Research Project of China sex differentiation in teleost fish. J Exp Zool 281:362–372 (abbreviate: 973 project, grant no. 2007CB109206). Omoto N, Maebayashi M, Adachi S, Arai K, Yamauchi K (2005) Sex ratios of triploids and gynogenetic diploids induced in the hybrid sturgeon, the bester (Huso huso female×Acipenser ruthenus References male). Aquaculture 245:39–47 Pandian TJ, Koteeswaran R (1998) Ploidy induction and sex control in fish. 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