Chapter 4 Progress in Carp Genetics Research David J. Penman1 4.1 Introduction been carried out on many of the cyprinid species of interest to aquaculture in Asia. Much of this Ten years ago, very little was known about the information, and equivalent information on genetics of most of the cyprinid species of interest other cyprinids (as well as other groups of fish), is to aquaculture in Asia, for example the Chinese summarized by Klinkhardt et al. (1995). It is carps, silver carp (Hypophthalmichthys molitrix), evident from a frequency distribution of diploid bighead carp (Aristichthys nobilis) and grass carp chromosome numbers for cyprinid species taken (Ctenopharyngodon idella); Indian major carps, from this database that 2n = 50 is by far the most catla (Catla catla), rohu (Labeo rohita) and mrigal common diploid chromosome number in (Cirrhinus cirrhosus); and the silver barb cyprinids, and also that polyploidisation (Barbonymus gonionotus). The exceptions to this (tetraploidy and hexaploidy) has occurred in would have been the common carp (Cyprinus several cyprinids (Fig. 4.1). Ancestrally tetraploid carpio) and the goldfish Carassius( auratus). Still species (approximately 100 chromosomes) are relatively little is known about the genetics of found in the genera Carassius, Cyprinus, Spinibarbus most of these species, but significant progress has and Tor, while Schizothorax taliensis and been made in the last decade. Valuable S. yunnanensis are ancestrally hexaploid (with 148 information about the genetic structure of wild chromosomes). In addition to being ancestrally and captive populations and traits of interest to tetraploid, C. auratus and C. gibelio also have aquaculture are now becoming available and our forms that are triploid (relative to the species’ knowledge of these areas should increase greatly ancestrally tetraploid chromosome complement, over the next decade, as should the applications i.e. they have approximately 150 chromosomes). of genetics to the aquaculture of cyprinids. Reddy (1999) described karyological studies on Indian cyprinid species in some detail. Griffith et This chapter reviews progress in genetics research al. (2003) demonstrated an increase in the that is relevant to aquaculture of cyprinid species lifespan of fish with larger genomes, from an in Asia. It begins with cytogenetic (4.2) and analysis of a wide range of species. Within molecular (4.3) techniques that can be used to individual orders such as the Cypriniformes the provide basic genetic descriptions of cyprinid relationship was positive but not significant, species and populations, as well as having possibly because of the limited number of applications in aquaculture genetics. Hybridiza- samples analyzed. tion studies, of which many have been carried out, are dealt with next (4.4). Quantitative Perhaps curiously, the number of fundamental genetics, the main focus of this publication (4.5), chromosome arms (NF) described by various is divided into four subsections (inbreeding and authors in the database of Klinkhardt et al. (1995) negative selection; current status; stock/strain showed much more intraspecific variation than comparisons; and selective breeding). Chromo- the diploid chromosome counts. It is not clear if some set manipulations (4.6), sex determination this reflects genuine polymorphism or differences and its manipulation (4.7), gene transfer (4.8) in interpretation by different authors. and finally applications of cryopreservation (4.9) and tissue culture (4.10) to aquaculture genetics Nucleolus organizer regions (NORs) have been research complete the review. studied in several species of Asian cyprinids (e.g. Carman et al. 1993; John et al. 1993; George et al. 4.2 Cytogenetics 1994; Anjum and Jankun 1998). George et al. (1994) showed that the NORs varied between Basic cytogenetics studies, describing Indian major carp species. Lakra and Krishna chromosome numbers and morphology, have (1996) reviewed chromosome banding 1 University of Stirling, U.K. 82 WorldFish Center | Carp Genetic Resources for Aquaculture in Asia CHAPTER 4 | Progress In Carp Genetics Research 83 Fig. 4.1. Frequency distribution of the diploid number of chromosomes in cyprinid species (worldwide). Source: Klinkhardt et al. (1995). Where more than one value is given per species in the database, the most common value is shown (in the case of a tie, the lower value is shown). The only cyprinid species in the database that is not shown is Ptychobarbus dipogon (2n = 446) techniques for fish and described G-banding for semi-automated erythrocyte cell volume the first time in Indian major carps. Luo (1998) quantification (Harrell et al. 1998). Such methods used trypsin digestion and Giemsa staining to are generally used in C. carpio due to the large produce chromosome-specific banding in the number of chromosomes in this species (Cherfas grass carp. This technique may go some way to et al.1993; Klinkhardt et al. 1995). overcoming some of the general difficulties in obtaining high resolution banding patterns in Chromosome counts are a good way of verifying fish chromosomes (Goodier and Davidson haploid controls in gynogenesis or androgenesis 1993). experiments, and are generally used in combination with morphological examination Chromosomes can be used as genetic markers in (haploid embryos show a typical “haploid studies on hybridization, induced polyploidy, syndrome”) and scoring survival rates (nearly all gynogenesis and androgenesis. While the haploid embryos die before diploid embryos techniques involved to prepare metaphase reach the first feeding stage). Restoration of spreads from large numbers of fish or embryos diploidy via temperature or pressure shocks given are fairly laborious, they are also well to the fertilized eggs following UV treatment of standardized. sperm or eggs can be verified by comparing the karyotypes of shocked batches (should be diploid) Distinguishing triploid and diploid fish using compared to the UV controls (should be haploid). metaphase spreads prepared from hatched However, to verify uniparental inheritance in embryos is fairly routine and allows analysis at an diploid gynogenetic or androgenetic fish and early stage (Kligerman and Bloom 1977). biparental inheritance in diploid controls, However, assessment of polyploidy may be more molecular genetic techniques such as DNA easily carried out using other methods such as fingerprinting are more informative. 82 WorldFish Center | Carp Genetic Resources for Aquaculture in Asia CHAPTER 4 | Progress In Carp Genetics Research 83 Interspecific hybridization may not produce the where the product of a gene rather than the DNA expected diploid animals with one haploid set of sequence itself is examined (polymorphism is chromosomes from each parental species detected in the form of bands in different (Chevassus 1983; see 4.4). Altered ploidy levels positions in an electrophoresis gel after staining in such hybridizations should be easily detected, for enzyme activity in the case of allozymes). but for parental species with similar karyotypes, With the development of a variety of techniques analyzing offspring metaphase spreads may not for working directly with DNA sequences, it is detect some of the more unusual outcomes of now possible to detect DNA polymorphisms interspecific fertilizations, such as gynogenetic or directly, both in the nuclear genome and the androgenetic progeny. In many situations, mitochondrial genome. allozyme or DNA markers, possibly in combination with karyotype analysis, may be Standard techniques can be used for isozymes, more powerful. multilocus DNA fingerprinting, RAPDs and mitochondrial DNA in different species with Chromosome polymorphisms have been found relatively little adaptation or prior knowledge of in population genetics studies in some species of the species, while other markers such as fish (e.g. variation in B chromosome frequency in microsatellite DNA loci may require considerable the characid Astyanax scabripinnis: Néo et al. 2000) preliminary research to isolate relevant DNA but are of limited general application. sequences and develop appropriate PCR primers (some microsatellite loci will, however, cross- Cyprinids are gonochoristic and several amplify in related species). aquaculture species have been shown genetically to have apparently fairly simple sex determination A variety of polymorphic genetic markers, systems (generally but not always XX/XY – see primarily allozymes and different types of DNA 4.7). However, in common with most other markers, can be used for such purposes as species of fish, heteromorphic sex chromosomes phylogenetics, species and hybrid discrimination, appear to be absent or very rare in cyprinids (e.g. population genetic analysis, comparison of levels Manna and Khuda-Bukhsh 1977; Buth et al. of genetic variation in hatchery and wild stocks, 1991). pedigree analysis in hatchery and experimental breeding, and gene mapping. Although studies of cyprinid karyotypes yielded some interesting information and applications – Knowledge of the genetic structure of wild for example insights into the role of populations can be important in conserving polyploidization in the evolution of this group, genetic resources and managing capture fisheries and chromosome counts can be used as markers and identifying potential source populations for in chromosome set manipulation experiments – the development of aquaculture brood stock. otherwise limited progress has generally been Allozyme analysis has been used to study made