Báo cáo Nghiên cứu khoa học Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp

Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp Christopher M Austin Pham Anh Tuan Thai Thanh Binh Le Quang Hung Nguyen Thi Tan School of Science and Primary Industries Faculty of Health, Education and Science Charles Darwin University, Casuarina Campus, Darwin, Northern Territory 0909, Australia Research Institute for Aquaculture No I (Ministry of Fisheries) Dinh Bang, Tu Son, Bac Ninh Vietnam June 2007 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 2 CONTENTS List of Figures..................................................................................................................4 List of Tables ...................................................................................................................6 Summary..........................................................................................................................8 1. Introduction...............................................................................................................11 1.1 Taxonomy and distribution................................................................................11 1.2 Biology of common carp .....................................................................................13 1.3 Common carp culture .........................................................................................14 2. Common carp culture in Vietnam...........................................................................17 2.1 Summary of socio-economic survey of common carp culture in Vietnam ....19 2.1.1 Carp farmer family profile .............................................................................21 2.1.2 Common carp culture.....................................................................................21 2.1.3 Suggestion of fish farmers .............................................................................27 3. Common carp breeding programs in Vietnam. .....................................................28 3.1 Introduction.........................................................................................................28 3.2 Crossbreeding of common carp .........................................................................29 3.3 Mass selection of common carp .........................................................................31 3.4 Family selection of common carp ......................................................................37 3.5 Combination of family and individual selection of common carp..................38 4. Overview molecular genetic studies on common carp in the world .....................42 5. Genetic diversity of common carp in Vietnam using direct sequencing and SSCP analysis of the DNA control region..................................................................45 5.1 Objective ..............................................................................................................47 5.2 Materials and Methods .......................................................................................47 5.2.1 Sample collection...........................................................................................47 5.2.2 DNA extraction and sequencing of control region ........................................49 5.2.3 Single Strand Conformation Polymorphism (SSCP) amplification...............50 5.2.4 Data analysis ..................................................................................................51 5.3 Results ..................................................................................................................52 5.3.1 Control region sequences and SSCP variation...............................................52 5.3.2 Genetic differentiation and relationships among populations........................54 5.4 Discussion.............................................................................................................59 5.4.1 Control region sequence variation and utility of the SSCP techniques .........59 5.4.2 Genetic diversity of common carp in Vietnam ..............................................60 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 3 5.4.3 Insights into the dissemination of cultured common carp in Vietnam ..........63 5.4.4 Conservation of wild carp stocks ...................................................................65 5.4.5 Conclusion .....................................................................................................68 6. Genetic diversity of common carp in Vietnam using four microsatellite loci .....69 6.1 Objectives.............................................................................................................72 6.2 Materials and Methods .......................................................................................72 6.2.1 Sample collection and DNA extraction..........................................................72 6.2.2 Data collection ...............................................................................................72 6.2.3 Genetic diversity analysis ..............................................................................73 6.2.4 Genetic differentiation between populations .................................................74 6.3 Results ..................................................................................................................75 6.3.1 Within population variation ...........................................................................75 6.3.2 Genetic differentiation and relationships among populations........................79 6.4 Discussion.............................................................................................................85 6.4.1 Genetic diversity within common carp populations.......................................85 6.4.2 Differentiation between populations ..............................................................88 6.4.3 Comparison of microsatellite and mitochondrial DNA data..........................90 7. Expanded genetic analysis of common carp stocks from private hatcheries, farmer ponds and market places .....................................................................93 7.1 Introduction.........................................................................................................93 7.2 Materials and Methods .......................................................................................94 7.2.1 Sample collection and data set .......................................................................94 7.2.2 DNA extraction and Single Strand Conformation Polymorphism (SSCP) amplification ...........................................................................................................95 7.2.3 Data analysis ..................................................................................................95 7.3 Results ..................................................................................................................96 7.3.1 SSCP variation ...............................................................................................96 7.4 Discussion...........................................................................................................104 7.4.1 Additional insights into diversity of common carp in Vietnam...................104 Appendix ......................................................................................................................108 Reference......................................................................................................................112 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 4 List of Figures Figure 1.1. Distribution of common carp in the world (FAO 2003)...............................12 Figure 1.2. World production of common carp (FAO 2003)..........................................15 Figure 2.1. Varieties of common cap in Vietnam (A: Bang Giang River, B&F:Dak Lak, C: Son River red, D: Bac Kan) ...........................................................................18 Figure 2.2. Types of common carp culture in Vietnam (A: Pond culture, B: Rice-fish culture, C: Integrated aquaculture)......................................................................19 Figure 2.3. The locations at which stakeholders were interviewed for the socio- economic survey..................................................................................................20 Figure 2.4. Type of common carp culture in Vietnam....................................................22 Figure 2.5. Percent fish species stocked into household ponds in Vietnam ...................23 Figure 2.6. Water source used for common carp culture ................................................23 Figure 2.7. Common carp sources in the farms in Vietnam............................................24 Figure 2.8. Farmer evaluation quality of seed.................................................................26 Figure 2.9. Demand for common carp in Vietnam .........................................................26 Figure 3.1. Mass selection of three blood common carp (Thien and Thang, 1992) .......33 Figure 5.1. Collection localities for Cyprinus carpio L. samples in Vietnam.................49 Figure 5.2. Silver stained polyacrylamide gel showing the eight SSCP variants detected in common carp populations in Vietnam (a). Neighbour-joining tree reconstruction derived from CR sequences, using HKY+I+G model of evolution. Bootstrap values are based on 1,000 replicates. Bootstrap value is given for nodes with at least 50% or more support (b).......................................................66 Figure 5.3. Relationships among common carp from wild and hatchery population in Vietnam using the unbiased genetic distance of Roger (1972) and UPGMA joining method. A, B, and C are SSCP haplotypes which predominate in each cluster. .................................................................................................................67 Figure 5.4. MDS plot of pairwise Fst value among hatchery and wild populations of common carp in Vietnam. Population codes are given in Table 5.1...................68 Figure 6.1.Allele number and distribution of allele frequencies for Hungarian, Indonesian and Vietnamese experiemtal common carp populations at each microsatellite locus. ............................................................................................80 Figure 6.2. UPGMA dendrogram of common carp populations in Vietnam based on matrixes of genetic distance (Nei et al., 1983)....................................................84 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 5 Figure 6.3. MDS plot of paiwise Fst value among experimental, hatchery and wild populations of common carp in Vietnam, (a) based on microsatellite data, (b) mitochondrial DNA data. Population codes are given in Table 5.1. ..................86 Figure 7.1. Collection localities for Cyprinus carpio L. samples in Vietnam. Population code are given in Table 7.1. ................................................................................98 Figure 7.2. Haplotype diversity of Common carp groups in Vietnam base on SSCP analyses. ............................................................................................................101 Figure 7.3. Relationships among common carp from experimental, provincial hatchery, private hatchery, private pond, market, wild populations in Vietnam using the unbiased genetic distance of Rogers (1972) and UPGMA joining methods. A, B, C are SSCP haplotypes which predominate in each cluster. Population codes are given in Table 7.1..............................................................................................102 Figure 7.4. Multidimension scaling (MDS) plot of pairwise Fst values among common carp from experimental, provincial hatchery, private hatchery, private pond, market, wild populations in Vietnam. Population codes are given in Table 7.1. A, B, C are main groups.........................................................................................103 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 6 List of Tables Table 2.1. Number of fish farms surveyed......................................................................21 Table 2.2. Frequency changed common carp broodstocks in hatcheries are changed....25 Table 3.1. Survival rate (%) for fry and fingerling of Vietnamese common carp (V), Hungarian common carp and their reciprocal hybrids (VH, HV) (Thien and Thang, 1992) .......................................................................................................30 Table 3.2 Morphology and morphometrics of common carp varieties and their hybrids (H: Hungarian; Y: Indonesian Yellow; V: Vietnamese White) (Nguyen et al., 2005) ...................................................................................................................31 Table 3.3. Adjusted average body weight of the common carp in an experiment on determination of realized heritability in 1988 (Thien and Thang, 1992)............32 Table 3.4. Summary data of the mass individual selection of the hybrid stocks of common carp (V: Vietnamese, H: Hungarian and Y: Indonesian yellow common carp) (Tran and Nguyen, 1992)...........................................................................34 Table 3.5. Heritability (h2) of the body weight of hybrid common carp Hungarian x (Vietnamese x Yellow) (Tran and Nguyen, 1992)..............................................35 Table 3.6. Growth rate of common carp of the F3 and F5 selected generations in a grow- out pond; Stocks [Hungarian x (Vietnamese x Yellow)] in Communal stocking in 1995 (Nguyen, 2005). .....................................................................................36 Table 3.7. Body weight of common carp in pure stocks and their hybrids reared in the same pond, 1995 (Thien and Thang, 1992). .......................................................37 Table 3.8. Current crossbreeding between carp lines to produce 86 families (Nguyen, 2005) ...................................................................................................................40 Table 5.1. Location, code and number of samples sequenced and analysed by the SSCP technique. ............................................................................................................48 Table 5.2. Number of haplotypes and haplotype diversity in each common carp population. Population code given in Table 5.1..................................................57 Table 5.3. Pair-wise estimate of variance of haplotype frequencies (Fst) among of samples. Population codes given in Table 5.1. ...................................................58 Table 5.4. AMOVA results for three groups (experimental, hatchery, wild) of 20 common carp populations base on SSCP data. (Intra = intrapopulation, Inter = interpopulation, values are %).............................................................................59 Table 6.1. Characteristics of Cyprinus carpio microsatellite loci tested.........................73 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 7 Table 6.2. Genetic variability of four microsatellite loci in 20 populations for common carp in Vietnam. Population codes given in Table 5.1. ......................................77 Table 6.3. Number of private alleles at four microsatellite loci in 20 common carp populations and group. The population codes are given in Table 5.1.................78 Table 6.4. Pairwise Fst values between 20 common carp populations in Vietnam based on four microsatellite loci. Population codes given Table 5.1............................81 Table 6.5.Results of assignment test (self-classification) of common carp individuals based on four microsatellite loci. Population code are given in Table 5.1. ........82 Table 6.6. Genetic diversity of experimental, hatchery and wild common carp groups in Vietnam based on variation at four microsatellite loci (Intra = intrapopulation, Inter = interpopulation, values are %).................................................................85 Table 7.1. Location, code and number of common carp sample fro Vietnam analysed by the SSCP technique.............................................................................................97 Table 7.2. Number of haplotype (N) and haplotype diversity (Hd) in each common carp population in Vietnam.........................................................................................99 Table 7.3. Number of population (N), haplotype (n) and haplotype diversity (Hd) of six common carp groups in Vietnam. .....................................................................100 Table 7.4. AMOVA results for six groups (experimental, provincial hatchery, private hatchery, private pond, market and wild) of common carp population in Vietnam base on SSCP data. (N = number of population was analysed, Intra = intrapopulation, Inter = interpopulation, value are %)......................................104 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 8 Summary This document brings together, for the first time, information from diverse sources relating to common carp breeding and genetic improvement strategies in Vietnam. These sources include the results of a survey of common carp farmers and hatchery operators, published and unpublished reports in Vietnamese on fish breeding and genetic improvement programs and experiments and the results of a comprehensive molecular genetic analysis of common carp stocks throughout Vietnam. The survey of common carp farmers and hatchery operators was conducted through interviews using standardised questionnaires and workshop. These activities confirmed common carp as the principal species for freshwater aquaculture in north Vietnam and that farmers were well informed about the significance of making use of genetically improved carp to improve productivity. The responses to the survey indicated that the majority of farmers believed they were utilising government produced genetically improved carp strains. Farmers emphasised difficulties in acquiring general knowledge and new skills associated with carp breeding and indicated they would welcome greater opportunities for training and participating in workshops. A key outcome of the survey was that while farmers were aware of the importance of using genetically improved strains they had difficulty in accessing such stocks in sufficient quantities to make an impact on the gene pool of their farmed stocks. The key information in this regard is that farmers were only accessing new genetically improved stocks on from a 1-4 year cycle and even when introducing new stocks these would only make a 10% contribution to farm's or hatchery's existing broodstock. This, coupled with the fact that farmers and hatchery operators spawn their fish in communal ponds where there is no control over matings means that new broodstock may make only minimal contributions to the production of fry and fingerlings. This problem could be circumvented if farmers had the skills and equipment to undertake controlled and induced spawning of carp broodstock using the techniques of gamete stripping and artificial fertilisation. A survey of the Vietnamese literature on the genetic improvement of carp for pond aquaculture production indicates that a large scale and ongoing selective breeding and broodstock development and management programme has been in place for over 30 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 9 years supported by the Vietnamese government. This program has included a survey of indigenous Vietnamese carp genetic resources, the importation of carp strains from Indonesia and Europe, selective breeding trials and genetic improvement programmes involving cross breeding and mass and family selection. Growth and heritability data collected as part of these programmes support a significant increase in the productivity of the genetically improved carp strains. However these trials have been undertaken exclusively in well managed research ponds. Thus an important requirement is the conduct of trials of the relative performance of different carp strains within farmer’s ponds as these present very different biological, physical and management environments to those in research ponds. Further, while the Vietnamese government genetic improvement program was implemented to improve the livelihoods of carp farmers there does not seem to have been a concerted program to widely disseminate genetically improved carp to farmers. The application of modern molecular genetics to an analysis of genetic diversity within and between Vietnamese carp stocks provides very important and novel insights into the management and dissemination of carp stocks through out Vietnamese. An important preliminary finding was that the three main experimental lines or strains of common carp could be readily distinguished from each other and that all three experimental lines had reduced variability. This means that the patterns of genetic variability within populations and the relationships between populations provide information on the mixing and origin of stocks. The overall, conclusions from the genetic analysis is that the dissemination of genetically improved carp strains has only been a partial success. While a number of common carp stocks from provincial government hatcheries had elevated diversity levels and represented mixtures of Indonesian and Vietnamese carp most hatcheries appear to be largely or completely derived from Vietnamese carp stocks and some of these had very low levels of diversity indicating inbreeding and poor broodstock management. Surprisingly, there was very little evidence that introduced Hungarian carp had contributed to gene pools in hatcheries or in the market place given the strongly held believe of that this strain has positive characteristics especially in relation to growth and is thought to has been widely dissemination. However, the genetic data are consistent with pond trials which indicated this strain has poorer survival compared to the other stains. In fact, the genetic profiles of all the carp sampled from the markets was consistent with their derivation from Vietnamese stocks and that Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 10 small scale farmers may not be benefiting from the significant investment in the development of genetically improved common carp. There are three important recommendations from a synthesis of the information from the farmer survey, the Vietnamese carp breeding program and the genetic data. Firstly, there is the need for the provision of a greater level of technical training and workshops for farmers specifically relating to carp breeding and broodstock management. Secondly, more effective strategies are needed to be developed for the dissemination of genetically improved broodstock to ensure the benefits of better performing stocks are made available, especially to small scale farmers. Thirdly, on-farm trials need to be conducted to evaluate the performance of different breeds and strains of common carp under different grow-out conditions and at a range of locations. This last recommendation would have the added benefit of encouraging the participation of small scale farmers in the research and the evaluation process of different strains. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 11 1. Introduction 1.1 Taxonomy and distribution Common carp (Cyprinus carpio L.) occur in water ways throughout Eurasia, from Western Europe through to China, Korea, Japan and South East Asia and from Siberia, south of latitude 600 N, to the Mediterranean and India (Kohlmann and Kersten, 1999). The actual origin and natural distribution of common carp is disputed by some authors due to its long history of domestication in both Europe and Asia which have led to many translocations over a significant period of time (Balon, 1995). Because of the long documented cultivation history of common carp in China, some scientists considered that the ancestor of European domestic carp were derived from Asian common carp stocks, during ancient Greek and Roman periods (Chiba et al., 1966; Vooren, 1972). Others consider that the common carp is indigenous to Europe and in fact postulate a European origin for wild carp and subsequent dispersal east to Siberia and China (Balon, 1995; Kottelat, 1997). Due to its popularity as an aquaculture and ornamental species, common carp has been widely translocated, outside its European and Asian distribution. As a result of these transfers and introductions, it is now perhaps the most widespread species of freshwater fish in the world, with naturally reproducing populations established in many countries in both northern and southern hemispheres. FAO (1998) lists introductions of common carp into 124 countries, of which 81 are recorded as having established feral populations or viable aquaculture stocks. Cyprinus carpio is taxonomically a most confusing species with over 30 synonyms listed on FishBase ( Based on just recent Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 12 taxonomic treatments, opinions vary widely: Balon (2004) and Kirpitchnikov (1999) recognised three subspecies: European common carp (Cyprinus carpio carpio Linnaeus, 1758), and Far Eastern common carp (C. carpio haematopterus Temminck & Schlegel, 1845), South East Asian common carp (C. carpio viridiviolaceus Lacepede, 1803), whereas Kottelat (2001) recognised European common carp as Cyprinus carpio Linnaeus, 1758 and Asian common carp Cyprinus rubrofuscus Lacepede, 1803. These taxa are distinguished mainly by morphology, such as number of gill rakers and scales, shape and color characteristics. However, there is often overlap in these traits which may be in part due to stock mixing and hybridization which may have blurred taxonomic boundaries. Moreover, the domestication of common carp has not only led to changes in body proportion, scalation and colour, but also in physiological characteristics (Balon, 1995; Baruš et al., 2002). Figure 1.1. Distribution of common carp in the world (FAO 2003) The taxonomic uncertainties, compounded by translocations, and the development of phenotypically distinctive domesticated lines, invites the use of molecular markers to elucidate the taxonomy of common carp and genetic relationships among stocks (Balon, Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 13 1995; Lever, 1996). There is also a clear need to examine the status of possible wild common carp gene pools to determine if there is a need to conserve genetic resources in this species. Lever (1996) considered that the wild ancestor of the common carp may be a single species, Cyprinus carpio, widely distributed from the Danube to Amur Rivers. While the existence of several self-sustaining wild populations of carp have been documented (Kohlmann and Kersten, 1999; Paaver and Tammert, 1993), Komen (1990) believes that truly self-sustaining wild populations of common carp are probably rare. 1.2 Biology of common carp Common carp is a largely benthic species that prefers shallow water habitats covered with aquatic weeds and grasses. It is an omnivorous fish that mostly feeds on the bottom but can exploit all levels in the water column. The natural diet of carp is dominated by chironomids, snails, young clams, shrimps and other benthic animals. This species also consumes aquatic plants, filamentous algae, seeds of plants and organic detritus. Under pond culture conditions, common carp will take soybean and peanut cakes, rice and wheat bran (Zhong, 1989). The common carp may be sexually mature as early as the end of its first year; however it typically requires three to four years to reach this stage (Cooper, 1987). According to Linhart et al. (1995) common carp have a high fecundity for a freshwater species producing 100,000 to 300,000 eggs per kg body weight with reports of as many as 360,000 to 599,000 eggs per female. The eggs are sticky in nature, and attach to aquatic weed and other material after spawning. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 14 Common carp reach 0.6 to 1.0 kg body weight within one season in the polyculture fish ponds of subtropical/tropical areas (FAO, 2004). Currently the world record for the largest carp stands at 34.3 kg. In their natural range carp can live up to 15 years, however they have been reported in some areas living to over 24 years of age with males often living longer than females (Balon, 1995). 1.3 Common carp culture Common carp is the most extensively cultivated freshwater fish species in the world (Chiba et al., 1966; Komen, 1990; Wohlfarth, 1984; Zhou et al., 2004b). This fish has several advantages that make it popular for commercial culture: (1) very fast growth rate, (2) high environment tolerance, (3) ease of handling, (4) ability to be raised in high density, (5) ability to utilise artificial diet with relatively low protein content, and (6) occurrence of highly productive strains and breeds produced from long-term domestication and selective breeding (Kirpitchnikov, 1999). Culturing and breeding of common carp has a long history dating back about 4,000 years in China and close to 2,000 years in Europe. Several special breeding centres have been developed in different regions of Europe, like the Czech Republic, Germany and Hungary, as well as Russia and Ukraine. China and Japan are the ancient culturing centers in Asia, but during the last decades India, Indonesia and Vietnam have started to culture common carp as a result of deliberate fish importation and acclimatization activities (Bakos and Gorda, 2001). Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 15 Annual production of common carp worldwide is over three million metric tons (FAO, 2003) (Fig 1.2). In Asian countries, common carp contributed 17% to total carp production from aquaculture in 2001 (Gupta et al., 2005). Figure 1.2. World production of common carp (FAO 2003). Li (2001) reported that production of common carp reached 2.05 million tons in 1999 and accounted for 20% of total freshwater fish output in China. In Indonesia, common carp production reached 178,362 tons in 1996, and accounts for 54.3% of total cultured freshwater fish in this country (Hardjamulia et al., 2001). In Europe, common carp is by far the most important freshwater fish with annual aquaculture yield of about 220,000 tons, which is higher than the European yield of rainbow trout (Linhart et al., 2002). Breeding and culture of common carp has been the backbone of fish farming in many European countries. For example, in 2001 common carp production reached 14,000, 10,500, 17,000 and 21,000 tons in Hungary, Germany, Czech, and Poland respectively. Common carp are cultured in a wide range of environments including ponds, cages, Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 16 tanks, reservoirs and rice-fields, as monoculture and polyculture with a variety of other species (FAO, 2001). In addition to production for food, common carp have been selectively bred for a variety of colors and color patterns for the ornamental fish market (Balon, 1995). Best known varieties are Koi carps which are called “swimming flowers” and are among the most expensive of ornamental fish species. Amano (1968) recorded an annual production of some 10 million fish amounting 1,000,000,000 yen in Japan. Although originally developed in Japan, Koi carps are now cultured in many parts of the world, including China, Europe and America (Balon, 1995). Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 17 2. Common carp culture in Vietnam Common carp (Cyprinus carpio L.) is thought to be indigenous to northern Vietnam, and translocated to southern Vietnam (Nguyen and Ngo, 2001). Eight local varieties have been recognised in Vietnam: white carp, Bac Kan carp, high body carp, Ho Tay carp, South Hai Van carp, red carp, violet carp, and reduced scale carp. These varieties differ in morphology, colour, distribution, and some other biological characteristics (Tran, 1983). The white carp is one of the most popular and important fish in aquaculture in Vietnam. In the study by Tran (1983) it was found that indigenous strains of common carp have poor growth rates and highly variable color and scale phenotypes. For example, the Bac Kan strain is unusually elongated in shape and is morphologically distinct from other strains. It commonly attains a weight of only 70-80 g in first year in polyculture and 160-200 g in the second year when grown in low input rice-field. Nevertheless, it is an important strain as it is adapted to the rice-field environment and farmers can maintain broodstock that do not depend on wild seed. The most important traits of the Bac Kan strain are that it can be cultured in shallow water and is tolerant of fluctuations of water temperature that characterises this environment. Another useful trait for culture in terraced rice-field, in which water flows from terraced field to the next below, is that the fish rarely leave the fields even during flooding when water spills across dikes (Edwards et al., 2000). The local “rice field” common carp strains are called “resident fish” or “fix- home fish” because of this useful characteristic (Tran, 1983) (Fig 2.1). Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 18 Figure 2.1. Varieties of common cap in Vietnam (A: Bang Giang River, B&F:Dak Lak, C: Son River red, D: Bac Kan) In general, Vietnamese common carp is considered a good aquaculture species because it exploits natural pond productivity, has good survival rate and the flesh has low fat content (Bakos and Gorda, 2001). There are however concerns about wild carp stocks which are thought to be in decline because of excessive harvesting and crossbreeding with introduced carp stocks (Nguyen and Ngo, 2001). Common carp have been cultivating in polyculture system in Vietnam including pond culture with a range other species, rice-fish, cage and integrated aquaculture with poultry and pig (Fig 2.2). Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 19 Figure 2.2. Types of common carp culture in Vietnam (A: Pond culture, B: Rice-fish culture, C: Integrated aquaculture). 2.1 Summary of socio-economic survey of common carp culture in Vietnam Socio-economic data were collected by interviews, using standardised questionnaires, with farmers, private hatchery owners and provincial hatchery managers from 21 provinces representing a total of 133 interviewees mostly in the highland (Appendix 1, Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 20 Figure 2.3). Data were analysed using SPSS 11. The break down of stakeholders surveyed is presented in Table 2.1. Figure 2.3. The locations at which stakeholders were interviewed for the socio- economic survey. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 21 Table 2.1. Number of fish farms surveyed Farm N Percentage (%) Farmers 91 68.4 Provincial hatchery managers 30 22.6 Private hatchery owners 12 9.0 Total 133 100.0 2.1.1 Carp farmer family profile The age of the farmers ranged from 38.2 to 50 years old in the 21 provinces surveyed. Over 55% of farmers are women, and 15% from ethnic backgrounds. Farmers learn to culture fish by many ways, media (television, newspaper), and farmer training. However, most farmers mainly learn fish culture techniques from each other, especially their families. The survey results indicated that over 76% of the farmers had not received any formal training in common carp culture. 2.1.2 Common carp culture • Type of culture Common carp is one of the main aquaculture species in Vietnam and they are cultured in ponds, rice fields, and cages. The results of the survey indicated that farmers have been predominately using ponds (79%) and with a much smaller proportion using ponds with rice-fields (12.8%) or just rice-fields (6.8%) for culturing common carp (Fig 2.4). Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 22 78.95% 6.77%12.78% 1.50% Rice field Pond Pond & rice field Cage Figure 2.4. Type of common carp culture in Vietnam • Common carp in polyculture pond Most farmers surveyed use polyculture techniques. Common carp are cultivated along with Chinese carp and Indian carp in both ponds and rice fields. More than 8 different fish species are stocked with common carp (Cyprinus carpio) including grass carp (Ctenopharyngodon idellus), silver carp (Hypophthalmichthys molitrix), bighead carp (Aristicchthys nobilis), rohu (Labeo rohita), mrigal (Cirrhinus cirrhosus), tilapia (Oreochromis niloticus), black carp (Mylopharyngodon piceus), pirapitinga (Piaractus brachypomus), other species. Of these species common carp (30.9%) is the dominant species in polycuture ponds (Fig 2.5). Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 23 9.03% 10.87% 11.24% 2.33% 3.38%0.87% 18.53% 30.88% 9.75% 3.12% Common carp Grass carp Silvel carp Bighead carp Rohu Mrigal Tilapia Black carp Pirapitinga Other Figure 2.5. Percent fish species stocked into household ponds in Vietnam • Pond management Since approximately 78.9 % of the farmers surveyed had access to irrigation canals, they were able to exchanged pond water at regular intervals. Only 3.8% of the farmers rely solely on rainwater for aquaculture and were seriously restricted in their capacity to exchange water (Fig 2.6). Common carp are generally fed directly with soybean, corn, cassava or indirectly though pond fertilization. 3.80% 78.90% 17.30% Irrigation water Rainwater River, stream water Figure 2.6. Water source used for common carp culture Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 24 • Seed sources Common carp cultured in the farms are derived from many different sources. Over 55.6% of seed stocks were obtained directly from private or provincial hatcheries and 20.4% from retailers who buy from a range of sources including from private and provincial farmers hatcheries, and possibility also from fishermen who obtain stocks from the wild. 4.93% 20.43% 4.23% 55.62% 14.79% River Hatchery Retailers Other Retailers & hatchey Figure 2.7. Common carp seed sources in the farms in Vietnam • Common carp strains Common carp have been cultured in Vietnam for at least a thousand years. The strains of common carp that are cultured include indigenous carp from rivers and lakes, and genetically improved carp. Based on farmer’s claims about 79.2% of cultivated carp in the farms are three blood carp strains that come from government research institutes and represent several genetically improved forms of carp. The remaining farmers grow carp from the wild or from unknown sources. Most farmers produce fingerlings in their ponds form commercial sale and also use these stocks their own farm ponds. A total of 96.2% of the farmers are selling seed to other smaller scale farms. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 25 • Hatchery establishment and broodstock management Most hatcheries have been established since 1984 with common carp being the major fish for spawning. Carp broodstooks are spawned about 1-3 times per year and are maintained on a diet of soybean, cassava, bud rice and artificial food. All hatcheries (96%) induce common carp breeding using tradition methods involving mostly reproduction in ponds or tanks or rice-fields using broodstock mostly composed of mixed strains and using fibrous material for egg attachment. Most hatcheries change broodstock every 1-2 year (Table 2), although hatcheries only change from10 to 20% of their broodstock at any one time. Table 2.2. Frequency changed common carp broodstocks in hatcheries are changed Change (Year) Percentageof hatcheries (%) 1 41.2 2 17.6 3-4 32.4 >4 8.8 Like common carp seed, broodstock in the farms and hatcheries are derived from different sources. Three blood genetically improved broodstock is most common, mainly obtained from Research Institute for Aquaculture No 1, Bac Ninh (RIA1) (73.9%) with 4.3% of broodstock representing the Hungarian strain. • Quality of seed Quality of fingerlings depends on quality of broodstocks and breeding techniques. Only 25.5% of farmers reported the quality of seed is good (Fig 9) though 62.4% of farmers did not have any comment. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 26 11.30% 25.60% 0.60% 62.50% Good Moderate Not good No comments Figure 2.8. Farmer evaluation of quality of seed • Demand for common carp Farmers generally consider the demand for common carp is increasing in Vietnam. About 36.8% of the farmer reported the demand of common carp is increasing (Fig 2.9) compared to 0.7% who thought the converse. It should be noted that 60.2% had no comment. 2.26% 60.15% 36.84% 0.75% Increase discrease No comment No change Figure 2.9. Demand for common carp in Vietnam Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 27 2.1.3 Suggestion from fish farmers The suggestions of farmer were collected from 133 interviewees and from 75 participants in two workshop for fish hatchery managers and farmers which were organised in RIA1 2005 and Thai Nguyen Aquaculture department in 2006. The suggestions of farmer include: • Quality of broodstock and seed of common carp should be improved • Providing good common carp stocks for farmers • More training fish breeding and cultured techniques for farmers • Research on reduce common carp seed cost Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 28 3. Common carp breeding programs in Vietnam. 3.1 Introduction Traditional knowledge supports the existence of there are 8 local varieties of common carp in Vietnam and the introduction of three exotic strains. The local varieties, identified on the basic of morphology and coloration are, white scaled, Bac Can, Ho Tay, South Hai Van, Red, Violet, High Body Depth and Scattered Scale varieties (Trong, 1983). The introduced common carps are Hungarian scaled, Hungarian mirror and Indonesian yellow strains. A government supported common carp selective breeding program has been in place in Vietnam for over 30 years with the aim of developing and disseminating strains with high growth and survival rates to fish farmers (Tran and Tran, 1995). In 1970 and 1975 the mirror and scale strains of Hungarian common carp were introduced to Vietnam. Indonesian yellow carp was introduced to the south of Vietnam before 1975 and then transferred to the north in 1978. In the 1970s experiments on hybridization of Vietnamese white carp with the Hungarian carps were carried out (Tuong and Thien, 1979; Thien and Tuong, 1983; Thien, 1993). Experiments found hybrid common carp (F1) to have fast growth and high survival and the best productivity in farm ponds was obtained from raising hybrid carp. However due to improper broodstock management, the base stocks of common carp in almost all hatcheries over the country were gradually losing their diversity, thus decreasing the effectiveness of breeding programs. Since 1981, research programs have focused on selection of common carp with the intention of creating a fish breed with stable genetic qualities. In the first phase (1981-1985) the program focused on the assessment of carp Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 29 stocks for selection. In the second phase (1986-1995) mass selection was carried out among the hybrid stocks over six generations. Continued family selection was carried out over two generations in the period of 1996 to 2000, making up the third phase. 3.2 Crossbreeding of common carp Initially eight varieties of local common carp were investigated, of which white carp, a variety with high viability is the most popular (Trong 1983). However, this carp and other varieties of Vietnamese common carp presented slow growth and early maturity. Attempts aimed at obtaining heterosis by crossing among these varieties were not successful. Under Vietnamese conditions, the Hungarian carps showed fast growth and late maturation but were easily infected with diseases and possessed low viability. The first hybrid generation (F1) crossing between Vietnamese white carp and Hungarian carp showed the best characteristics from their parents i.e. high survival rate, fast growth and attractive appearance. The survival rate of hybrid fry and fingerling was much higher than that of Hungarian carp (Table 3.1). At the same time survival rate of the hybrids and Vietnamese carp was similar. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 30 Table 3.1. Survival rate (%) for fry and fingerling of Vietnamese common carp (V), Hungarian common carp and their reciprocal hybrids (VH, HV) (Thien and Thang, 1992) . Survival rate (%) Stage Crossing 1974 1975 1976 Fry V VH HV H 51.6 61.6 60.4 22.3 - 70.0 44.3 40.0 71.2 80.0 78.0 37.6 Fingerling V VH HV H 85.9 ± 9.4 94.9 ± 1.9 81.4 ± 7.5 45.7 ± 5.2 - 76.2 ± 2.9 76.7 ± 2.3 38.6 ± 2.4 78.3 ± 0.2 90.0 ± 3.3 73.0 ± 11.3 46.3 ± 5.1 A more sophisticated cross breeding strategy was subsequently adopted to bring together a number of positive qualities from different varieties and to improve the genetic variability of the initial stock for mass selection. At first three hybrid strains were constructed by crossing Vietnamese carp with Hungarian carp (VH), Vietnamese carp with yellow carp (VY) and Hungarian carp with yellow carp (HY). Then the males of each single hybrid were crossed with females of the third strain. The double hybrids obtained in these crossings have been evaluated and used as stock for further selection (Fig. 3.1). Assessments of this selection program included comparisons using morphological, physiological and biochemical characteristics among the above mentioned pure common carp varieties and their hybrids (Table 3.2). Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 31 Table 3.2 Morphology and morphometrics of common carp varieties and their hybrids (H: Hungarian; Y: Indonesian Yellow; V: Vietnamese White) (Nguyen et al., 2005) Descriptions Variaties Hybrids H Y V HxV HxY VxY Body weight (g) 24.30 ± 1.41 22.20±1.27 16.30±0.61 21.20±0.91 22.8±1.21 12.90±0.89 Standrd length (cm) 8.20±0.16 3.3±0.18 7.9±0.09 8.1±0.11 8.2±0.15 6.9±0.15 As% of standard length: Maximal body height 38.1± 0.19 35.1±0.32 30.9±0.25 35.5±0.25 37.3±0.28 32.2±0.30 Minimal body height 14.30±0.13 13.0±0.13 11.9±0.10 14±0.16 13.9±0.17 14.4±0.17 length of head 43.10±0.24 31.90±0.13 31.6±0.16 34.4±0.38 33.1±0.30 32.1±0.40 Dorsal spine to tip most dorsal ray 36.30±0.24 36.6±0.25 33.5±0.16 36.6±0.38 34.4±0.14 37.1±0.30 Intestine length 174.00±1.60 185.7±1.8 145.0±1.3 175±1.4 186±2.20 As% of head length: Diameter of eye 26.60±0.32 24.0±0.37 29.6±0.49 25.9±0.45 25.4±0.30 27.9±0.50 length of barbell 17.80±0.29 18.6±0.25 18.0±0.21 19.2±0.21 18.2±0.26 17.4±0.30 No. of lateral line scales 37.70±0.20 32.9±0.25 32.0±0.14 32.6±0.12 33.8±0.16 33.4±0.15 No. of Dorsal rays 18.90±0.12 18.3±0.16 20.4±0.16 18.0±0.08 18.2±0.26 19.0±0.15 No. Anal rays 5 5 5 5 5 5 No. of branched stamens in first bow 24.9±0.20 19.7±0.17 7.7±0.18 20.2±0.20 22.7±0.23 20.2±0.18 No. of vertebrae 35.80±0.12 35.10±0.08 34.10±0.08 34.4±0.13 35.00±0.16 34.70±0.20 3.3 Mass selection of common carp In order to carry out mass individual selection about 5-10 families (one family included 1 female and 3-4 males) of each hybrid stock were bred on the same day. Their eggs were incubated under the same conditions. The rearing of fry and fingerlings and culturing them to marketable size were done under similar environmental conditions. On an average, about 20% of total number marketable fish in each stock were selected based on the body weight and appearance (big body and small head). The selection effectiveness was estimated according to Falconer (1960). R= Sh2 = iδh2 R: effectiveness of selection ; i: intensive of selection S: selection differential ; δ: average square variation h2: heritability of the trait (body weight) Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 32 To estimate the coefficient of realized heritability of fish body weight in each selected generation, the experiments were implemented according to the schema in Figure 3.1. Before mass selection of marketable fish was done, a randomly collected control population was kept. Then another group was collected by selecting for big body weight (experimental group). The differential in the average body weight between experimental group and control group give the selection difference (S). In the next year the off-spring of the two groups were obtained by the same method and the fingerlings of control and experimental groups were reared by communal stocking in the same pond to a marketable size. The difference in the average body weight between two offspring groups gives the effectiveness of selection for one generation (R). The heritability was calculated according to formula: The data collected during the process of mass selection showed that the number of experimental fish in each stock was limited due to a limited number of ponds. Even though the scale of selection is small (Table 3.3) the indices obtained proved to be acceptable. Table 3.3. Adjusted average body weight of the common carp in an experiment on determination of realized heritability in 1988 (Thien and Thang, 1992) Group of fish Stocking weight (g) Harvesting weight (g) (150 days) Weight after adjustment (g) Experiment 71±4 365±9 335 Control 51±3 286±9 316 h2 = R S Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 33 Thus in the first generation the number of fish obtained for mass selection in one of the stocks was 1720, in each of two other stocks was 400 where as in second generation the number of fish in each stock was only 250 but the selection differential in second generation was even higher than that in F1. In the F3 generation field trials, due to poaching of some stocked fishes the total number of fish obtained for selection was reduced. So around 33% of fishes in each stock was selected (Table 3.4). It led to declining of the selection intensity and the selection differential. In the next generations following the recommendation of some geneticist-selectionists (Kirpichnikov, 1987) about 20% of fishes was kept to establish the broodstocks. As a result the selection indices were stabilized at an acceptable level (Fig 3.1). Figure 3.1. Mass selection of three blood common carp (Thien and Thang, 1992) Vietnamese (white) common carp Hungarian (scale) common carp Indonesian (yellow) common carp V H Y HY VY VH V(HY) V(HY) V(HY) V(HY) V(HY) V(HY) ♀V x HY♂ ♀Y x VH♂♀H x YV♂ H(VY) H(VY) H(VY) H(VY) H(VY) H(VY) Y(VH) Y(VH) Y(VH) Y(VH) Y(VH) Y(VH) M A S S S E L E C T I O N M A S S S E L E C T I O N M A S S S E L E C T I O N M A S S S E L E C T I O N M A S S S E L E C T I O N F1 (Single) F1 (Double) 1986 F2 1988 F3 1989 F4 1991 F5 1993 F6 1995 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 34 Table 3.4. Summary data of the mass individual selection of the hybrid stocks of common carp (V: Vietnamese, H: Hungarian and Y: Indonesian yellow common carp) (Tran and Nguyen, 1992). Year Stocks Total Body Indices collected through selection generation and place No. of fish weight (g) Severity V (%) Intensity i (S/δ ) Differenti al S (g) 1986 F1 RIA.1 H x (YxV) V x (YxH) Y x (HxV) 400 400 1720 162±6 178±4 187±8 12.5 12.5 7.5 2.77 1.66 1.94 99 84 82 1988 F2 RIA.1 H x (YxV) V x (YxH) Y x (HxV) 248 258 253 152±7 104±5 148±9 10.1 9.7 9.9 1.76 2.03 1.60 117 177 164 1989 F3 RIA.1 H x (YxV) V x (YxH) Y x (HxV) 75 243 74 149±8 155±12 310±16 33.3 32.9 33.8 1.25 0.80 0.77 52 62 41 1991 F4 RIA.1 H x (YxV) V x (YxH) Y x (HxV) 200 209 189 260±6 197±5 299±6 20.0 19.1 25.9 1.26 1.75 1.24 74 124 47 1992 F5 RIA.1 H x (YxV) V x (YxH) Y x (HxV) 229 235 175 314±12 300±9 350±10 21.8 21.3 22.0 1.28 1.72 1.72 97 69 93 1993 F5 RIA.1 H x (YxV) V x (YxH) Y x (HxV) 257 263 243 226±10 300±14 318±11 19.5 20.9 22.6 1.39 1.06 1.44 85 101 114 1995 F6 all stocks Brooders Average for 5 generations 228 19.8 1.52 93 After five selected generations the common carps of all three stocks have been clearly improved in growth rate and appearance. However, in order to estimate the genetic gain it was necessary to know the coefficient of heritability. The data obtained in the experiments to determine the realized heritability of body weight showed that the Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 35 indices in the first two generations were suitable for individual selection, but they decreased from F1 to F4 generation. In F1 generation the realized heritability was 0.29. In F2 generation experiments, the results were analysed and adjusted according to the methodology of Wohlfath and Moav (1972), because of the difference in body weight between two groups of fingerling when stocked (Table 3.5). Table 3.5. Heritability (h2) of the body weight of hybrid common carp Hungarian x (Vietnamese x Yellow) (Tran and Nguyen, 1992). Generation Parent's body weight (g) Offspring's body weight (g) Heritability Control Stock Selected Stock Control Stock Selected Stock h2=R/S F1 F2 162±6 218±10 261±9 312±21 180±4 316 209±6 335 + 0.29 + 0.20 The realized heritability of body weight was 0.20 for the F2 generation. In the F4 generation this index declined to around 0. In fact, the effectiveness of individual selection of common carp in the last two generations was low. The experiment to compare growth rate of carps obtained from breeders in F3 and F5 selected generation (Table 3.6) showed that the difference in body weight was 7% only. It is expected that improvement of selection effectiveness in the next phase of the breeding program should be done by applying another method, for example, family selection. The average coefficient of realized heritability calculated for each generation was 0.16. So, based on average index of selection differential, the response to selection for increasing body weight in each generation could be estimated and it should be around Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 36 15g or 6.6%. So, the total genetic gain for body weight in the common carp breeding program after 5 selected generations is estimated to be 33%. Table 3.6. Growth rate of common carp of the F3 and F5 selected generations in a grow-out pond; Stocks [Hungarian x (Vietnamese x Yellow)] in Communal stocking in 1995 (Nguyen, 2005). Generation 6 July 1995 Stocking 18 Aug. 1995 average 5 Oct. 1995 average 16 Nov. 1995 average 14 Dec. 1995 average 9 Feb. 1996 weight (g) weight (g) weight (g) weight (g) weight (g) Average (g) % F5 15.7 60 170±4 264±7 317±6 409±10 107 F3 15.7 60 170±8 232±7 305±9 382±9 100 The progressive decrease in the genetic gain could be caused by inbreeding. To avoid inbreeding depression it was recommended to cross among the three stocks to producing hybrid seed for grow-out farmers. Preliminary data obtained in the experiments following this direction (Table 3.7) showed that the heterosis effect was present in both experiments but it was more significant in the experiment No.2. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 37 Table 3.7. Body weight of common carp in pure stocks and their hybrids reared in the same pond, 1995 (Thien and Thang, 1992). No. Experiment Stocks and their hybrids Stocking weight (g) Harvesting weight (g) Heterosis effect g % H x (VY) 20 365±10 + 21 5.8 1 Y x (HV) 20 377±10 + 9 2.4 Hybrid 19 386±12 H x (VY) 16 367±12 + 127 34.6 2 Y x (HV) 17 415±10 + 79 19.0 Hybrid 17 494±15 3.4 Family selection of common carp Family selection of common carp has been conducted at RIA1 since 1998, with the main objective to obtain a common carp line with fast growth and high survival to improve its production in aquaculture. Using the 5 th generation of common carp from mass selection as the initial materials, the programme has succeeded in producing two new generations through induced breeding and rearing of juvenile in hapas. The first generation, with about 375 brood fish, were selected from the 5 best families (out of 24). Following up the breeding workplan for the second generation, induced breeding was undertaken successfully with 40 families of the selected generation and 23 pairs of the base population. Rearing of juveniles has been carried out entirely in hapas. After harvest, 2000 fingerlings of the second generation were reared to marketable size for further selection. In the middle of 2001, fish in the grow-out pond were harvested for estimation of genetic parameters. Based on breeding value (A), 400 fish (200 females and 200 males) were selected as brood stock for the third generation of selection. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 38 Family selection of common carp has been conducted for three years (1998-2000). Following up the selection plan, the programme has succeeded in selection of the first generation and from then the second generation have been produced. Selection of individuals and families in the second generation was undertaken when the size of fish in growout ponds reached marketable size. In order to assess the result of selection after two selected generations, the realized heritability of body weight (h2) at the second selected generation needs to be determined. These calculations indicated a 5 % increase in growth rate in each selected generation. 3.5 Combination of family and individual selection of common carp On going selective breeding experiments are using a combination of family and individual selection. The most recent program utilised six common carp (Cyprinus carpio L.) lines in this breeding program consisting of: (1), Hungarian 6th generation of mass selection (2), Yellow 6th generation of mass selection (3), Vietnamese 6th generation of mass selection (4), Hungarian scale carp (5), Indonesian yellow carp (6). The brood-fish were 2-3 years old with mean body weight of 1.5-3.0 kg. All the spawners were healthy, free from disease and deformity. Broodstock were induced to spawn and gametes were stripped and mixed to create 101 families. These were produced in 3 batches of 12, 38, 51 families, over a 22 day period. All male and female broodstock were marked by PIT tags (AVID Microchip) to identify the individuals and family. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 39 After hatching, the larvae were gathered in fine mesh hapas for nursing. Swim-up fry of each family were stocked in 1 m3 hapas with the stocking density of 1,000fry/hapa. The fry were fed daily with the pelleted feed containing 40% crude protein at a rate of 10% body weight. Rearing period was for 2 months (April-May). Fingerlings of each family were reared in 5.0 m3 hapas, 500 fry stocked per a hapa. The fry were fed daily with pelleted feed (30% crude protein) at 7% body weight. Rearing period lasted from May to June. As it is known that common carp do not growth well in hapas environment because of its natural benthic feeding habit the experiment was designed to culture carp in ponds by tagging fish using Code Wide Tag (CWT). Application of CWT for fingerlings mean that 5 families could be stock in the same pond with marks at 5 different positions on tail, abdomen, back, left and right cheek. The growout stage for marked CWT fish was conducted in earthen ponds by dividing them into units of 120m2 area for each of 500 individuals from 5 families. Fish were grown in these ponds from June to August and reached 100-150g. Over this time, some families were lost because of low survival rate. Other families were removed as the morphology such as yellow colour and no scale phenotype are not preferred by consumer in Vietnam. Position of family tags are identified by CWT scanner. It is anticipated that results of these trials will be available very soon. In addition the RIA1 is still importing new strain from Hungary in an effort to provide better performing carp strains for small scale farmers (Table 3.8). Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 40 Table 3.8. Current crossbreeding between carp lines to produce 86 families (Nguyen, 2005) Maternal parent Paternal parent Family selection, 2nd gener. Hungarian 6th gener. H(VY) Hungaria n scale carp Yellow 6th gener. Y(VH) Indonesia n Yellow Vietnamese line 6th gener. mass selection Family selection, 2nd gener. 8 9 7 5 1 3 Hungarian 6th gener. H(VY) 9 7 4 1 2 Hungarian scale carp 6 4 4 - Yellow 6th gener. Y(VH) 5 3 3 Indonesian Yellow 3 3 Vietnamese line 6th gener. mass selection 2 Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 41 In conclusion, while there has been significant effort to produce genetically improve carp by Vietnamese government over many years a major concern is the distribution of seed stock to farmers which may only have been partly successful due to the mixing of stocks in hatcheries and on farms. Inbreeding depression and undesirable genetic effects may also be a problem as many farmers report carp growth is slow and fish mature when they are a small size. Furthermore, the viability of breeding experiment may have been compromised because pure stocks of experimental common carp strains kept in separate ponds at RIA1 may have been mixed (A. T. Pham pers.comm). Thus there are a number of important issues that need to be addressed concerning Vietnamese common carp stocks including their genetic status and history, the extent of dissemination of selectively bred stocks and the conservation status of wild stocks. These issues can be addressed by obtaining molecular genetic information which can provide insights into genetic diversity and therefore, inbreeding and the extent of stock mixing in both cultured and wild stocks. In addition, molecular genetic data can contribute to the resolution of issues relating to the taxonomy, evolution, and biogeography of common carp and help identify genetically divergent wild stocks of conservation significance. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 42 4. Overview molecular genetic studies on common carp in the world Because it is one of the most important farmed freshwater fish in the world, common carp have been subject of numerous molecular genetic studies. The foci of these studies have been questions relating to phylogenetics, population structure, taxonomy and aquaculture and the impact of domestication. In the past, traditional protein markers had been used to study the genetics of common carp populations on regional levels. For instance, allozyme information is available for domesticated and wild population culture and samples in Hungary (Csizmadia et al., 1995), Czech Republic (Desvignes et al., 2001; Slectova et al., 2002), Uzbekistan (Murakaeva et al., 2003), Italy (Cataudella et al., 1987), Japan (Macaranas et al., 1986), Indonesia (Sumantadinata and Taniguchi, 1990), Israel (Ben-Dom et al., 2000), Estonia (Paaver and Gross, 1991), Poland (Anjum, 1995), Germany (Kohlmann and Kersten, 1999) and Australia (Davis et al., 1999). More recently mitochondrial DNA (Davis et al., 1999; Froufe et al., 2002; Mabuchi et al., 2005; Zhou et al., 2003), microsatellite (Bartfai et al., 2003; David et al., 2001; Desvignes et al., 2001; Lehoczky et al., 2005; Tanck et al., 2000), RADP (Bartfai et al., 2003; Wang and Li, 2004), and AFLP data (David et al., 2001) have been used to examine genetic variation in common carp populations. To date, there are few studies of common carp populations across its full geographical range in Eurasia. Those studies that have been conducted have not sampled comprehensively, but nevertheless suggest that carp may be divided into European and Asian groups (Brody et al., 1979; Kohlmann et al., 2003; Kohlmann et al., 2005); Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 43 although this interpretation is not consistent with the actual data presented in some studies (Kohlmann et al., 2005). The complete mitochondrial DNA nucleotide sequence of common carp was described by Chang et al. (1994) (GenBank accession number: X61010). This helped provide primers for sequencing mitochondrial DNA genes and fragments which is more and more popularly used to investigate relationships of common carp strains or populations. For example, the molecular phylogeny of three subspecies of common carp in China was analysed by Zhou et al. (2004b) using sequence of Cytochrome b (Cyt b) and control (CR) gene regions. Mabuchi et al. (2005) discovered a genetically divergent form of common carps from Lake Biwa, using mitochondrial DNA CR sequence data. Phylogenetic relationships of ornamental (koi) carp, Oujiang color carp and Long-fin carp were analysed by Wang and Li (2004) using mitochondrial DNA cytochrome c oxidase subunit II (COII) gene sequences. Currently there are several published studies of common carp that give primers for microsatellite loci (Crooijmans et al., 1997; Sun and Liang, 2004; Yu and Guo, 2004). Thirty two microsatellite markers of poly (CA) type in common carp were described by Crooijmans et al. (1997). While these authors stated that these loci will be valuable as genetic marker for use in population genetic, breeding and evolution studies, they did present any population genetic analyses (Kohlmann et al., 2005; Lehoczky et al., 2005; Zhou et al., 2004a). Kohlmann et al. (2003; 2005) have presented the only population genetic studies of common carp using four microsatellite loci and found significantly greater variation than was apparent from allozyme studies. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 44 Studies of QTLs in common carp are limited. There is only one gene linkage map of common carp which mapped of loci associated with cold tolerance (Sun and Liang, 2004), using the segregation of 272 markers, including 105 gene markers, 110 microsatellites, and 57 RAPD markers. Despite the large number of genetic studies of common carp, none have addressed genetic diversity and genealogical relationships on a global basis. Views on the taxonomy and origin of carp are questionable and require detailed investigation. In addition the study of common carp from certain regions have been neglected, including Vietnam (south East Asia), which is home to indigenous common carp that have not been examined using modern molecular genetic methods to any significant extent. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 45 5. Genetic diversity of common carp in Vietnam using direct sequencing and SSCP analysis of the DNA control region Common carp is one of the main aquaculture species in Vietnam (Nguyen and Ngo, 2001) and is cultivated along with Chinese carp and Indian carp in polyculture ponds and rice-fields. Thesis species together makes up one of the most important food and income resources in rural communities in Vietnam (Edwards et al., 2000). The enhancement of common carp production in Vietnam has focused substantially upon the development of genetically improved strains (Tran and Tran, 1995). For this purpose, Hungarian and Indonesian carp strains were imported into Vietnam almost 30 years ago for crossbreeding and mass selection programs with local Vietnamese carp. The genetic improvement strategy adopted for common carp in Vietnam involved the development of hybrid common carp by crossbreeding among three genetic lines (Vietnamese white, Hungarian scale and Indonesian yellow common carp) coupled with mass selection. The ongoing development of farmed common carp stocks in Vietnam requires that several important issues are addressed. These included the possibility that: (1) stocks of experimental or genetically improved lines have become mixed with other stocks in ponds at Research Institute for Aquaculture No 1 (RIA1) or provincial hatcheries; (2) distribution of these lines as seed stock or broodstock to regional hatcheries or farms has been ineffective in increasing genetic diversity, and (3) reduction in effective population sizes (Ne) in genetically improved or hatchery maintained stocks has occurred, leading Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 46 to loss of genetic variation, gene frequency changes and possibly inbreeding depression. This last possibility is consistent with reports by many farmers of slow growth and early maturity in their cultured stocks of common carp (T. A. Pham, personal communication). In addition to the genetically improved lines of common carp produced at RIA1, eight local varieties have been recognised in Vietnam with reported useful features for culture and for marketing (Bakos and Gorda, 2001). There is now the possibility that distinct wild common carp stocks are in decline because of excessive harvesting and interbreeding with introduced common carp strains (Nguyen and Ngo, 2001). Therefore, it is important to identify and characterise genetically distinct local varieties of cultured fish for conservation purposes in general and specifically for common carp populations (Mabuchi et al., 2005; Murakaeva et al., 2003). A wide range of molecular marker systems have been used to study genetic diversity in aquaculture species (Chen et al., 2004; Diniz et al., 2005; Garoia et al., 2004; Sato et al., 2005; Sotka et al., 2005; Valles-Jimenez et al., 2004; Yu and Guo, 2004). As one of the most important aquaculture species in the world, genetic variation in carp has been examined in many parts of the world using microsatellites (Desvignes et al., 2001; Kohlmann et al., 2005), RAPDs (Bartfai et al., 2003), AFLPs (David et al., 2001), allozymes (Kohlmann and Kersten, 1999), RFLPs (Gross et al., 2002; Zhou et al., 2003) and direct sequencing of mtDNA fragments (Froufe et al., 2002; Zhou et al., 2004b). While these have included studies of both European and Asian common carp so far there have been no detailed studies of molecular genetic variation in common carp in Vietnam nor has the technique of Single Strand Conformation Polymorphism (SSCPs), Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 47 which is being increasingly used to assay for genetic variation in fish species (Aurelle and Berrebi, 2001; Liu and Cordes, 2004), been applied to common carp. 5.1 Objective In this study, we investigate genetic diversity of Vietnamese common carp strains and populations by using direct DNA sequencing and SSCP analysis of the mtDNA CR . This region of the mtDNA molecule was chosen because it has a very high nucleotide substitution rate, making it particularly useful for estimating the genetic population structure of closely related animal populations (Sivasundar et al., 2001; Vigilant et al., 1991). These data are then used to examine a range of questions relating to genetic diversity and management of wild and domesticated populations of common carp in Vietnam. 5.2 Materials and Methods 5.2.1 Sample collection Common carp were collected in 2003 and 2004 from broodstock populations maintained by eleven hatcheries, three experimental common carp lines maintained at RIA1, and by sampling six wild populations, using seine and lift nets. Samples of common carp strains from China, Indonesia, Japan, Hungary and India were obtained for comparative purposes. Tissue samples were taken as fin clips and preserved in 90% ethanol. Locality and sample size details are provided in Table 5.1 and Fig 5.1. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 48 Table 5.1. Location, code and number of samples sequenced and analysed by the SSCP technique. Population Code Location Type Sequencing SSCPs Hungarian scale-RIA11 HUS Tu Son, Bac Ninh, Vietnam E 4 50 Indonesian yellow-RIA1 IDY Tu Son, Bac Ninh, Vietnam E 6 50 Vietnamese white-RIA1 VNW Tu Son, Bac Ninh, Vietnam E 4 50 Vinh Phuc VIP Me Linh, Vinh Phuc, Vietnam H 4 50 Thai Nguyen THN Cu Van, Thai Nguyen, Vietnam H 3 50 Son La SOL Son La town, Son La, Vietnam H 4 50 Bac Kan BAK Bach Thong, Bac Kan, Vietnam H 6 50 Tuyen Quang TUQ Hoang Khai, Tuyen Quang, Vietnam H 4 50 Yen Bai YEB Van Chan, Yen Bai, Vietnam H 3 50 Hoa Binh HOB Hoa Binh town, Hoa Binh, Vietnam H 6 50 Ha Tinh HAT Duc Long, Ha Tinh, Vietnam H 4 50 Can Tho CAT Cai Rang, Can Tho, Vietnam H 4 36 Sai Gon SAG Binh Chanh, Sai Gon, Vietnam H 4 35 Thac Ba Reservoir TBR Yen Binh, Yen Bai, Vietnam H 3 50 Bang Giang River BGR Cao Bang town, Cao Bang, Vietnam W 6 50 Lo River LOR Yen Son, Tuyen Quang, Vietnam W 4 50 Red River RER Van Giang, Hai Hung, Vietnam W 4 50 Lam River LAR Nam Dan, Nghe An, Vietnam W 3 50 Son River SOR Bo Trach, Quang Binh, Vietnam W 4 47 Dak Lak DAL Ea Kao, Dak Lak, Vietnam W 4 50 Xingguonensis XIG Jaing xi China 3 5 Wananensis WAN Jaing xi China 3 5 Wuyuanensis WUY Jaing xi China 3 5 Color COL Jaing xi China 3 5 Red Koi REK Komaki Japan 3 21 Wild Amur WAR Karnataka, India 3 5 Majadanu MAJ Sukamandi, Indonesia 3 5 Rajadanu RAJ Sukamandi, Indonesia 3 5 Widan WID Sukamandi, Indonesia 3 5 GenBank GBK Taiwan2 1 Goldfish GOF Unknown 1 Population size (n) 1 Research Institute for Aquaculture No 1; 2 Origin of sample not provided; E: Experimental group; H: Hatchery group; W: Wild group Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 49 Figure 5.1. Collection localities for Cyprinus carpio L. samples in Vietnam 5.2.2 DNA extraction and sequencing of control region The total genomic DNA was extracted using the protocol from Crandall et al. (1999). Between three and six individuals from each populations or strain were first analysed by direct sequencing. The mitochondrial CR was amplified using primers Carp-Pro (5’ Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 50 AAC TCT CAC CCC TGG CTA CCA AAG 3’) and Carp-Phe (5’ CTA GGA CTC ATC TTA GCA TCT TCA GTG 3’) designed from the common carp whole mitochondrial genome sequence (GenBank, AC: X61010). PCR was carried out in 50 µl reactions volumes (1 X reaction buffer, 2 mM dNTP, 1.5 mM MgCl2, 0.5 µM of each primer, 0.5 units Taq DNA polymerase, and approximately 200 ng DNA template). Thermal cycling comprised 95 oC for 3 min, followed by 34 cycles of 95 oC for 30 s, annealing at 55 oC for 30 s, and an extension temperature of 72 oC for 1 min. This was then followed by a final extension of 72 oC for 3 min. PCR products were purified using the QIA quick PCR purification kit (Qiagen Hiden Germany), following ABI PRISM BigDye Terminator (Foster city, CA, USA) sequencing protocols. For each individual, sequencing reactions were performed using both forward and reverse primers, resulting in a consensus fragment of 745 bp in length. 5.2.3 Single Strand Conformation Polymorphism (SSCP) amplification A short highly variable fragment of the CR was selected from the common carp sequences obtained as described above. This fragment was approximately 230 bp and was amplified by the primers F1 (5’ GCA GGT ACA TAA TAT TAA 3’) and R1 (5’ CAG ATG CCA GTA ATA ATT 3’). PCR was carried out in 10 µl reaction volumes (1 X reaction buffer, 2mM dNTPs, 1.5mM MgCl2, 0.5µM of each primer, 0.1 units Taq DNA polymerase, and approximately 10ng DNA template). Thermal cycling comprised 94 oC for 3 min followed by 34 cycles 94 oC for 20 s, annealing at 55 oC for 20 s, and an extension temperature of 72 oC for 50 s, followed by a final extension of 72 oC for 3 min. PCR product size was estimated using the Promega DNA/Hae III marker. Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 51 To obtain SSCP phenotypes, PCR product (1µl) was added to 7 µl of loading buffer (99% formamide, 1mM NaOH, 0.2% w/v bromophenol blue and xylene cyanol), denatured for 2 min at 94 oC and placed directly on ice. Samples were loaded onto an 8% polyacrylamide (37.5: 1 acrylamide-bisacrylamide) gel (16 cm long, 1.5 mm thick) containing 5% glycerol and 0.5 X TBE, and run at 5W for 12h at 4 oC. SSCP products were visualised by silver staining using the method of Mirol et al. (2002). The resultant bands were scored by comparison with five standard carp haplotypes (representing the five most frequently encountered haplotypes), which were included on each gel for reference. Rare haplotypes were subsequently run side by side to ensure they were correctly scored. 5.2.4 Data analysis Nucleotide (π) and haplotype diversity (h) were calculated from the sequence data using DNASP 4.10 (Rozas et al., 2003). Sequences were aligned using the program Clustal X (Thompson et al., 1997). The most suitable model of evolution was obtained using Modeltest 3.06 (Posada and Crandall, 1998). This model was used to calculate pairwise sequence distances between haplotypes and for the construction of a Neighbour-Joining dendrogram (NJ) using PAUP*4.0b.10 (Swofford, 2000). Confidence levels in the resulting relationships were assessed using the bootstrap procedure with 1,000 pseudoreplicates (Swofford, 2000). The corresponding control region sequence for common carp from GenBank (Chang et al., 1994) was included in the data set for comparative purposes, and the Carassius auratus control sequence (GenBank accession NC_002079) was used as the outgroup. For the SSCP data set, haplotype diversity for each population was calculated using ARLEQUIN (Scheneider et al., 2000). Genetic divergence between samples was Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 52 estimated by pairwise Fst analysis using the same program. Patterns of overall genetic relationships among populations were summarised using UPGMA clustering of Roger’s (1972) genetic distance as implemented in TFPGA (Miller, 1997). Multidimension scaling (MDS) was performed on matrices of genetic distance to test for the presence of nonhierarchical patterns of relationships among populations using SPSS 10.0. The partitioning of genetic diversity within and between populations and populations grouped according to origin (experimental line, hatchery and wild), was undertaken by an analysis of molecular variance (AMOVA) for binary data, using the GenALEX add- in for Microsoft Excel (Peakall and Smouse, 2001). Multiple comparisons were subjected to sequential Bonferroni correction to control the Type I error rate (Rice, 1989). 5.3 Results 5.3.1 Control region sequences and SSCP variation Sequences for a 745 bp fragment of the mtDNA CR were obtained from 111 fish representing 41 populations or strains. A total of 19 haplotypes with 78 variable and 30 phylogenetically informative sites were identified. The nucleotide composition was A + T rich (A= 31%; T= 32%), and variation consisted predominantly of transitions (Ti : Tv = 2.56). All sequences have been deposited in GenBank (AC: AY597942-AY597976; DQ354144-DQ354149). Vietnamese carp populations have high haplotype diversity (mean = 0.92±0.02), but low nucleotide diversity (mean = 0.01±0.00). The most divergent Vietnamese haplotypes differed by only 9 base pairs. Diversity and relationships among haplotypes are depicted in Fig 5.2 together with the 14 corresponding SSCP phenotypes determined from the shorter (230 bp) fragment. From this figure it can be seen that the SSCP Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 53 technique was successful in resolving a significant proportion of the nucleotide variation detected in sequencing the longer control fragment. It is noteworthy that four of these haplotypes allow the discrimination of Vietnamese white (haplotype C), Hungarian (haplotype A) and Indonesian yellow (haplotypes B & D) carp strains from RIA 1. In addition, common carp samples from China (haplotypes I & R) and Indonesian (haplotypes B, D &J) and Koi carp (haplotype L) were all distinguishable from Vietnamese carp. The summary of relationships among control haplotypes (Fig 5.2) shows that, apart from two of the Chinese strains, which share the same haplotype, and are quite divergent from the other carp samples, there are a large number of haplotypes that are closely related to each other. Minor exceptions are the Hungarian carp haplotype (A) and a haplotype found in Bak Kan and Dak Lak (haplotype E), the Bang Giang River (haplotype F) and the Lo River (haplotype G) and Koi carp (haplotype L). A total of 968 individuals from both wild and hatchery populations were scored for SSCP variation. In addition to the non-Vietnamese strains that had seven distinguishable SSCP phenotypes, five SSCP haplotypes were distinguishable among Vietnamese common carp samples. Comparison with the nucleotide sequences revealed that these SSCP haplotype differ by 3-8 bp. Haplotype frequencies and diversity estimates are summarized in Table 5.2 for 20 common carp populations. Three haplotypes, Hungarian (A), Indonesian (B) and Vietnamese (C), predominated in common carp samples and five (D, E, F, G and H) were relatively rare or occurred only at low frequencies. Intra-population diversity varies widely among the populations ranging from populations with a single haplotype (h =0) to six haplotypes (h = 0.55). Fish breeding practices and stock improvement strategies in Vietnam in relation to common carp ___________________________________________________________________________ ___________________________________________________________________________ Christopher M Austin, Tuan Anh Pham, Binh Thanh Thai, Hung Quang Le, Tan Thi Nguyen 54 The experimental strains from RIA1 have the lowest diversity (h= 0-0.28), the hatchery stocks with the exception of Thai Nguyen, have high diversity (h = 0.49-0.64) and the wild stocks have an intermediate level of diversity (h = 0.26-0.41) (Table 5.2). The three experimental strains from RIA1 are also highly differentiated from each other. The Hungarian scale strain is fixed for haplotype A, the Indonesian yellow strain is dominated by haplotype B (84%), while the Vietnamese white strain in dominated by haplotype C (94%). All three of these haplotypes are found in almost all hatchery and wild carp populations, however in the Vietnamese white strain haplotype C dominate (55%) followed by the predominate Indonesian haplotype B at 25% and the Hungarian haplotype A at 12%. The six wild common carp populations (RER, LOR, LAR, SOR, DAL, and BGR) have generally similar haplotype profiles and like the Vietnamese experimental strain, haplotype C predominates. Six of the hatchery stocks have haplotype profiles largely similar to the wild populations (haplotype C = 0.52-0.94). The five other hatchery samples, in contrast, have haplotype profiles dominated by the Indonesian haplotype B (0.50-0.84), although it is noteworthy that they also all possess the Vietnamese haplotype C, albeit at a lower frequency (0.04-0.39). Interestingly, almost all hatchery and wild samples have the Hungarian haplotype (A