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Research work in the fisheries genetics and breeding in India

IntroductionInland aquaculture in India is highly dependent on carps, i.e., Indian major carps, catla (Catla catla), rohu (Labeo rohita), mrigal (Cirrhinus mrigala), Chinese carps and common carp (Cyprinus carpio). Until the mid-1950s, fish culturists depended on wild collections of Indian carps seed from rivers. However, the success of hypophysation technique made spawning these fish under captivity possible. This has helped farmers in procuring seed from hatcheries. However, in an attempt to produce more seed to meet the increasing demand, the seed producer did not pay attention to the quality. Consequently, the carp seed produced from many hatcheries was reported to show signs of inbreeding depression, such as poor survival and growth, susceptibility to disease, deformities, etc. Hence it became essential to carry out research on genetic improvement of these carps through various approaches. In the 1960s and 1970s, fish genetic research was mainly restricted to cytogenetic studies and hybridization. Later, chromosome manipulation, developing breeding programs for important carp species and application of molecular techniques were emphasized. Several national and central institutions and state agricultural and other universities undertake fish genetic research. Some of these are the Central Institute of Freshwater Aquaculture (CIFA), National Bureau of Fish Genetic Resources (NBFGR), Central Institute of Fisheries Education (CIFE), University of Agricultural Sciences (UAS), Bangalore, College of Fisheries, Mangalore, Centre for Cellular and Molecular Biology (CCMB), Madurai Kamraj University (MKU). Development in fisheries and aquaculture genetics can be studied under different field of achievement as following -

HybridizationSoon after the success of induced breeding of the Indian major carps, work on genetic improvement of these carps was initiated in the late fifties starting with simple interspecific and intergeneric hybridization to produce and evaluate their useful traits for aquaculture, since hybridization was regarded to be one of the methods for combining desirable traits of selected species. Accordingly, hybridization work was carried out between different species of a genus (interspecific) and species of different genera (intergeneric) among the Indian major carps and Indian and Chinese major carps, including the common carp (Cyprinus carpio variety communis, Linnaeus,) to study whether the hybrids acquired any useful traits from their respective parent species, such as smaller head, wider body and more flesh content as in rohu-catla hybrid, pond breeding habit as in Cyprinus carpio, or advantageous feeding habits. (Chaudhuri, 1959 and 1973; Alikunhi and Chaudhuri, 1959; Alikunhi et al., 1963a and 1972; Naseem Humsa, 1971 and 1972. Naseem Hamsa and Alikunhi, 1971; Varghese and Sukumaran, 1971; Ibrahim, 1977; Konda Reddy, 1977; Varghese and Shantharam, 1979; Basavaraju and Varghese, 1980a, 1980b; 1981, 1983; Basavaraju et al., 1989; 1990; 1994 and 1995; Ibrahim et al., 1980; Bhowmick et al., 1981; John and Reddy, 1986; Kesavanath et al., 1980; Konda Reddy and Varghese, 1980, 1980a, 1980b and 1983; Khan et al., 1989 and 1990; Jana, 1993). Altogether, six interspecific and 13 intergeneric hybrids were produced among the four species of Indian major carps belonging to three genera, i.e., Catla, Labeo and Cirrhinus (Reddy 1999).

List of interspecific hybrids between Indian major carps


FEMALE MALE HYBRID (rohu-kalbasu) (kalbasu-rohu) (rohu-bata) (kalbasu-bata) (gonius-kalbasu) (fimbriatus-rohu) COMMENTS Hybrid grows faster than Kalbasu and fertile. Hybrid grows faster than Kalbasu and fertile. Hybrids exhibited poor hatching and slow growth rate. Hybrids exhibited poor hatching and slow growth rates. Hybrids exhibited poor hatching and slow growth rates. Hybrids grow faster than fimbriatus and have better feeding efficiency.

1 L.rohita L.calbasu

(Chaudhuri, 1971).
2. L.calbasu L. Rohita

(Chaudhuri, 1971).
3. L.rohita L. bata 4. L.calbasu L. bata 5. L. gonius L.calbasu 6. L. fimbriatus L.rohita

(Basavaraju et al.1990)

Of the 13 intergeneric hybrids produced only four hybrids, L. rohita-C. catla, Cirrhinus mrigala-C.catla, L.rohita-Cirrhinus mrigala, L. fimbriatus-C. catla and the reciprocal hybrids have been found to possess useful traits in terms of growth (Ibrahim, 1977; Basavaraju and Varghese, 1980 b).
List of intergeneric hybrids between Indian major carps
FEMALE 1. C. catla L.rohita MALE HYBRID (catla-rohu) COMMENTS Growth rate of hybridsvariable, grow better than both the parents (Verghese and Sukumaran, 1971), grow slower than both the parents (Konda Reddy & Verghese, 1980), fertile hybrids. Hybrids grow faster than rohu. The meat yield in hybrids is higher than in parents. The hybrids are fertile. (Bhowmick et al., 1981 and Jana.

2. L.rohita C. catla

(rohu-catla)

1993).
3. C. catla C. mrigala 4. C. mrigala C. catla 5. L.rohita C. mrigala (catla-mrigal) Hybrids grow slower than the parents and are fertile. ( Ibrahim 1977) (mrigal-catla) Hybrids grow better than either parents and are fertile. (Ibrahim,

1977).
(rohu-mrigal) Growth rate of hybridsvariable, grow better than the parent species (Ibrahim, 1977), grow slower than both the parents (Basavaraju and Verghese, 1980b). The hybrids are fertile. Hybrids grow slower than both the parents and are fertile. Hybrids grow faster than kalbasu and they have broader feeding spectrum & adaptability. Hybrids exhibited low viability, need further study with regard to survival & performance. Fertilization in the hybrid cross was normal (8090%) with 60%

6. C. mrigala L.rohita 7. C. catla L.calbasu 8. L. calbasu C. catla 9. L.calbasu C. mrigala

(mrigal-rohu) (catlakalbasu) (kalbasucatla) (kalbasu-

mrigal) 10. L.rohita C. reba 11. L.calbasu C. reba 12. C. catla L. fimbriatus 13. L. fimbriatus C. catla (rohu-reba) (kalbasureba) (catlafimbriatus) (fimbriatuscatla)

hatching rate. The hybrids are fertile. Hybrids exihibit low viability. Hybrids exihibit low viability. Hybrids grow much faster than L. fimbriatus. The meat yield is higher than the parents. Hybrids grow much faster than L. fimbriatus. The meat yield is higher than the parents. (Basavaraju et al. 1994)

Intergeneric hybrids between Indian major carps and Common carp


FEMALE MALE HYBRID (rohu-common carp) (mrigal-common carp) (common carprohu) (common carpcatla) (common carpmrigal) COMMENTS Hybrids exhibited poor survival and are sterile. Poor survival. The hybrid tends to resemble C. carpio and are sterile. Higher survival rate than the reciprocal-cross and dominated by mostly C. carpio traits. The hybrids are sterile (aneuploid), grow better under monoculture system. Higher survival rate than the reciprocal-cross and dominated by mostly C. carpio traits. The hybrids are sterile (aneuploid), grow better under monoculture system. Hybrids are inferior to both the parents in growth and are sterile.

1. L.rohita C. carpio 2. C. mrigala C. carpio 3. C. carpio L.rohita

(Khan et al., 1990)


4. C. carpio C. catla

5. C. carpio C. mrigala

Chromosome manipulationChromosome manipulation as a tool to improve genetic status of fish was initiated at CIFA and in other institutes. Meiotic gynogenesis was successfully induced in Indian major carps for the first time in L. rohita, C. catla (John et al. 1984) and C. mrigala (John et al. 1988). The first report on successful induction of mitotic gynogenesis in rohu was that of Reddy et al., 1993, followed by Hussai, et al., 1994. Reddy et al., 1993 used irradiated milt of common carp, Catla catla and also Cirrhinus mrigala, to activate eggs of rohu (Labeo rohita) to produce mitotic gynogen rohu. Irradiated milt of Chinese grass carp (Ctenopharyngodon idella) was also used by Reddy et al., 1997 (unpublished) to induce mitotic gynogenesis in rohu, catla, mrigal and kalbasu. Use of milt from heterologous species,

preferably having low compatibility to hybridize may be more desirable for the easy elimination of paternal genetic input with irradiation. Therefore, the milt from Chinese carps was used, as they are incompatible to produce viable hybrids with Indian carps. Induction of androgenesis in Indian major carps was not very successful as egg consists of huge mass of cytoplasm and yolk that prevent the proper exposure of the chromosomes to UV rays. Some preliminary attempts have been made to induce artificial induction of triploidy and tetraploidy in Indian major carps with varying degrees of success (Reddy et al. 1987; 1990). The genetics division of CIFA succeeded in inducing triploidy in L. rohita and tetraploidy in L. rohita and C. catla. Common carp triploid showed 60-100% more growth than its diploid siblings (Reddy et al. 1998). Triploid C. idella can be utilized in open water reservoirs to control aquatic weeds without propagating itself as it is supposed to be sterile. Triploidy was successfully induced in C. carpio through heat shock treatment to produce sterile individuals as an approach to control unwanted reproduction in growout ponds. Heat shock at 40C, 1-3 min after fertilization for 1.5 min resulted in 100% triploidy. The triploid C. carpio was found to grow as fast as diploid and yield 15-18% extra edible meat because of low GSI in case of triploids (Basavaraju et al. 1998). An attempt to induce sterility in freshwater catfish Heteropneustis fossillis through heat shock was partially successful. Heat shock for 3 min at 40C, 4 min after fertilization yielded 46% triploids. Current research in chromosomal engineering in Indian major carps aims to produce allotriploids of carp hybrids (C. catla x L. rohita and L. calbasu x L. rohita) and to evaluate them for resistance against parasitic infection. In C. carpio, the objective of chromosome manipulation is to produce sterile fish to optimize production in aquaculture.
Details of induced gynogenesis of carps at CIFA
Species Gynogenesis Shock treatment Results

C.S. = cold shock H.S. = heat shock Nature C.catla C.catla L.rohita L.rohita C.mrigala C.mrigala L.calbasu L.calbasu Meiotic Meiotic Meiotic Meiotic Meiotic Meiotic Meiotic Meiotic Mitotic Mitotic Mitotic Mitotic Mitotic Mitotic Mitotic Mitotic C.S. H.S. C.S. H.S. C.S. H.S. H.S. C.S. Intensity 12C 39C 12C 39C 12C 39C 40C 12C Duration 10 min 1 min 10 min 1 min 10 min 1 min 1 min 10 min Diploid gynogenesis Diploid gynogenesis Diploid gynogenesis Diploid gynogenesis Diploid gynogenesis Diploid gynogenesis Diploid gynogenesis Diploid gynogenesis

Some preliminary attempts have been made to induce artificial induction of triploidy and tetraploidy in Indian major carps with varied degrees of success (Reddy et al., 1987; Reddy et al., 1990). Reddy et al. (1987), made the first attempt of inducing polyploidy in one of the Indian major carps, Labeo rohita by using colchicine. They could induce only tetraploid and diploid mosaics. However, Reddy et al. (1990) successfully induced triploidy in rohu, L.rohita, and tetraploidy in L.rohita and Catla catla by using thermal shocks. Similarly successful induction of tetraploidy was also reported in the case of Cirrhinus mrigala and L.rohita (Zhang, 1990).
Details of induced triploidy/tetraploidy in major carps at CIFA
Species Ploidy Shock treatment Nature C.catla L.rohita L.rohita C.mrigala Triploid Tretraploid Tretraploid Tretraploid Heat shock Heat shock Heat shock Heat shock Intensity Duration 40C 42C 30C 39 40C 2 min 12 min 2 min 2 min Percentage of tetraploids & triploids obtained 3055% 12% 70% 1040%

Sex Manipulation

Research on manipulation of sex (production of sterile and monosex population) as a tool for increasing production has been in progress in many institutes in India. Administration of 17- methyltestosterone (MT) through diet to eight-day old fry for a period of 45 days induced complete sterility in C. carpio. Compared with normal fish, sterile fish grew faster (21%) and had better resistance against bacteria (Mohire et al. 1999). In progress at UAS, Bangalore, are studies on the production of monosex population of C. carpio through hormone sex reversal and progeny testing as an alternative approach to overcome sexual maturation and unwanted reproduction. UAS, in collaboration with the International Centre for Living Aquatic Resources Management (ICLARM), is carrying out research to evaluate performance of crossbreeds of Indian major carp and to assess inbreeding of C. Catla from different hatcheries of Karnataka state and the usefulness of sterile triploid and monosex C. carpio in aquaculture. Selective breeding of Indian tiger shrimp was done at cife Mumbai.

Selective BreedingIn aquaculture ever since the success of induced breeding of the Indian major carps through hypophysation, the technique has been gradually popularized throughout the Indian subcontinent as well as neighbouring countries such as Pakistan, Bangladesh, Burma, Nepal etc. Consequently, many hatcheries have developed in almost all these countries. However, in over four decades, attention has been paid only to produce fish seed to meet a pre-targeted quantity with very little regard to quality (Eknath 1991). A survey of carp hatcheries in the state of Karnataka in South India was made (Eknath and Doyle 1990) because of the growing concern that

hatchery stocks may be inbred, as suggested by rapid deterioration of their performance. Eknath and Doyle (1990) found that the rate of the accumulation of inbreeding (F) was particularly high for the three most desirable carp species in the country, catla, rohu and mrigal. The most important reasons for this appeared to be that all the hatcheries function as isolated genetically closed units, raising their own stocks of brood fish. Moreover, the individuals for replenishment of brood stock in the hatchery are those which were retained after the hatchery had reached its seed production target for a given year (Eknath and Doyle 1990). It was also reported that small hatcheries appear to rapidly inbreed their stocks. The tendency to breed slow growing and later maturing individuals was also observed in some of the hatcheries (Eknath and Doyle, 1985), which is undersirable. The

survey Basavaraju et al. (1997) conducted particularly for catla in the three major seed producing hatcheries in the state of Karnataka, namely Tunga Bhadra Dam (TBD), Bhadra Reservoir Project (BRP) and Kabiri Reservoir Project (KRP), also indicated that the practices followed by these hatcheries are likely to result in both inbreeding and negative selection. Maheshwari and Biradar (1998) reported decline in fertility of hatchery bred Labeo rohita due to inbreeding. A study was conducted at Powarkheda, Freshwater Fish Farm of the Central Institute of Fisheries Education (ICAR), India where induced breeding of L. rohita was carried out between 1982 and 1992 to assess fertility. It has been reported that breeding of related individuals up to the sixth generation led to 71% decline in fertility level as compared to base population. It was also observed that the range, standard deviation and coefficient of variation were highest for fecundity in the first generation. This indicates, according to the authors, the scope for improvement through selection.

CIFA, in collaboration with the Institute of Aquaculture Research of Norway (AKVAFORSK), undertook genetic improvement of L. rohita through selective breeding. The project started in 1992, and the first phase was completed in 1997 with the release of Indias first genetically improved fish Jayanti rohu.. The second phase is in progress. Currently two projects are in operation on carp selection. One on Labeo rohita as already mentioned, the other on Catla catla at the University of Agricultural Sciences, Bangalore, Karnataka State

Genetic CharacterisationIdentification and genetic characterization of wild and hatchery populations are important since broodstock with good genetic background is necessary for a successful breeding program. The knowledge from such investigations can be used in optimizing and sustaining yield, stock management and conservation of genetic diversity. The work being undertaken in the biotechnology division of CIFA involves molecular genetic characterisation of Indian major carp using different types of DNA markers.

Genetic Markers-

Screening of L. rohita founder stocks used for genetic improvement undertaken by CIFA in collaboration with NBFGR indicated the presence of adequate genetic variability and absence of genetic

contamination. Screening of two populations of L. rohita, from Ganga and Gomati Rivers with isozymes and isoelectric focusing of eye lens proteins revealed polymorphism in XDH, PGM-1, PGM-2, EST-1G6PDH and ACP out of 20 isoenzymes studied at NBFGR. With the use of a cost-effective package of genetic markers to quantify genetic contamination of hatchery stocks of Indian major carps, significantly higher levels of introgressions than

with phenotypic markers were revealed. In Indian major carps, two mt-DNA genes were successfully amplified with polymerase chain reaction and amplified reaction fragment pattern for 2.5 kb was obtained for five restriction enzymes. Polymorphism was indicated in the pattern obtained with Eco R1 (NBFGR).

Gene BankingA compendium entitled Fish biodiversity of India containing information on systematics, habitat and distribution of 2118 finfishes has been made by NBFGR. The bureau has also achieved some success in using cryopreserved sperm to transfer germplasm for

crossbreeding program between distant and discrete populations. The farmed C. carpio at Bilaspur (Himachal Pradesh) was crossed with frozen sperms of wild stocks of Ooty (Tamil Nadu) and Rewalsar. Rewalsar stock was found to be superior, and Himachal Pradesh State Fisheries was advised to replace existing farmed stocks of C. carpio, which was found to be contaminated with goldfish genome. A similar program is in progress for rainbow trout at Ooty, Tamil Nadu. The NBFGR genebank has sperms of eleven species: C. catla, L. rohita, C. mrigal, C. carpio, Salmo gairdneri, Salmo trutta, T. putitora, T. khudree, Tenualosa ilisha, Labeo dussumieri and Horabagus brachysoma.. The improvement of the technique over time resulted in hatching with a range of 65%-100% with majority between 80%90%. Hatchlings have been produced from five-year old frozen sperms of L. rohita. Crossbreeding of Nilgiris rainbow trout with faster growing stock from Himachal Pradesh to achieve better growth was successfully undertaken. The fingerlings produced from crossbreeding of Tamil Nadu rainbow trout with Himachal Pradesh stocks using cryopreserved milt have exhibited higher growth. These results showed that cryopreserved milt could be utilized to transfer genome. Sperm cryopreservation protocols were developed for the

first time for the above species by NBFGR. Viable larvae were produced from cryopreserved milt raising the possibility of stock enhancement in depleted areas. Other institutes like CCMB and the National Institute of Immunology and universities such as MKU and Jawaharlal Nehru University (JNU) are developing transgenic fish technology, including construction of genomic and C-DNA libraries of Indian carps C. catla, L. rohita and Indian catfish H. fossilis and Clarias batrachus, and isolation of growth hormone gene from these libraries. At CCMB, construction of c-DNA libraries from pituitary glands of C. catla and L. rohita and sequencing of c-DNA clones of C. catla were carried out. At JNU, construction of genomic DNA libraries of C. catla and L. rohita, cloning and characterization of genomic clones and encoding interferon gamma gene(s) in these species are being undertaken. MKU is also undertaking c-DNA cloning of growth hormone (GH) gene of H. fossilis and L. rohita; screening and isolation of fulllength c-DNA of growth hormone of H. fossilis and L. rohita; and mapping and sequencing of full length c- DNA of GH gene of these two species

Transgenic fishThe research on fish transgenics in Indian is relatively recent (Ayyappan et al., 2001). The first Indian transgenic zebrafish was generated in 1991, by Pandiyan et al followed by first triploid transgenic Brachydanio rerio in 1995, using borrowed constructs from foreign sources (Sheela et al., 1998; Pandian et al., 1991; Pandian & Marian, 1994). To construct transformation vectors for the indigenous fishes, growth hormone genes of rohu (Labeo rohita) (Venugopal et al., 2002 a, b) and catfish, Heteropneustes fossilis (Anathy et al., 2001) were isolated, cloned, sequenced and confirmed in prokaryotic and eukaryotic systems. A vector was made with grass carp _-actin promoter driving the expression of r-GH. The sperm electroporation technique was

standardized (Venugopal et al., 1998) to ensure 25% hatchling survival and 37% presumptive transgenics. Southern analysis confirmed genomic integration in 15% of the tested individuals. Transgenic rohu and singhi grew faster than the respective controls and converted the food at a significantly higher efficiency (Pandian, 2003). At Centre for Cellular and Molecular Biology (CCMB), Hyderabad, autotransgenic Catla catla and Labeo rohita were generated using the growth hormone gene constructs (both cDNA and genomic DNA) fully developed from these species, thus giving a Swadeshi touch to the entire experiment (Majumdar, 2002, pers. comm.; CCMB 2002). Efforts are on way to isolate and characterize salt-resistant genes from marine environment in a collaborative project between Central Institute of Fisheries Education (CIFE) Mumbai and CCMB, Hyderabad. Similarly, attempts have been initiated at M. S. Swaminathan Research Foundation, Chennai, Central Marine Fisheries Research Institute (CMFRI), Kochi, National Bureau of Fish Genetic Resources (NBFGR) and Central Institute of Fisheries Technology (CIFT), Kochi to isolate and characterize salt tolerance genes from mangrove plants like Avicenia sp., sea grasses and marine microbes. India has developed experimental transgenics of rohu fish, zebra fish,cat fish and singhi fish.Genes,promoters and vectors of indigenous origin are now available for only two species namely rohu and singhi for engineering growth. Transgenic rohu recently produced from indigenous construct at Madurai Kamraj University has proved to be eight times larger than the control siblings. In India , research in transgenic fish was initiated in Madurai Kamraj University,centre for Cellular and Molecular Biology,Hyderabad and National Matha College Kollam with borrowed construct from foreign scientist(Sharma , 2004)

CYTOGENETIC STUDIES The study of fish chromosome (karyotype) was initiated in India from 1960s by using basically the methodologies available for mammals. Gradually modified fish chromosome methodologies have been developed for obtaining quality metaphase chromosomes (Reddy and John 1986; Banerjee, 1987; Reddy and Tantia, 1992 and Nagpure and Barat, 1997). Of about 2,000 species of inland and marine fish analysed for karyological information (Das and Barat, 1995), over 200 species belong to India, which include both freshwater as well as marine species. Most of these studies are related to the analysis of diploid karyotypes. Karyological studies on Indian major carps have been carried out by many workers (Khuda-Bukhsh and Manna, 1974; Majumdar and Ray Chaudhuri, 1976; Zhang and Reddy, 1991; Jana, 1993). However, regarding Indian major carps, the investigations, besides karyotype studies, also pertain to a comparative account of parental species and their hybrids which were attempted to interpret hybrid viability, fertility and sterility (Reddy et al, 1990a, Zhang and Reddy, 1991 and Jana, 1993).
Comparative karyological studies on Indian major carps as reported by various authors
Species Type of chromosomes Number of fundamental arms Authors

2n Metacentric Submetacentric Subtelocentric L.ronu 50 50 50 18 6 10 10 8 26 16 18 18 C. catla 50 4 24 24 18 24 22 22 22 A/t 78 88 Manna & Prasad (1971) Majumdar and Ray Chaudhuri (1976) Gui et al., (1986) Zhang & Reddy (1991) Jana, 1993 Khud-Bukhsh & Manna (1976)

50 50 50 50 C.mrigala 50 50 50 L.calbasu 50 50

8 6 12 12 6 6 12 6 6

16 32 16 16 8 26 18 8 32

26 12 22 22 36 10st = 22A/t 18 (A/t) 20 St/t 36 12 80 78 88

Manna (1977) Majumdar & RayChaudhuri (1976) Zhang & Reddy (1991) Jana (1993) Manna & Prasad (1971) Majumdar & RayChaudhuri (1976) Zhang & Reddy (1991) Manna & KhudaBukhsh 1977 Majumdar & RayChaudhuri, 1976

Comparative karyological studies on some hybrids of Indian carps as reported by various authors
Hybrid type L.rohita C.catla C.catla L.rohita L.calbasu C.catla L.calbasu C.catla C.carpio L.rohita C.carpio C.catla C.carpio C.mrigala Type of chromosomes 2n Metacentric Submetacentric Subtelocentric 50 4 50 12 50 12 50 10 50 76 74 75 24 10 10 14 22 28(18st+10t) 28 (16st+12t) 26 90 Khuda-Bukhsh & Manna 1976 88 Jana, 1993 Khuda-Bukhsh & Rege, 1975 Krishnaja & Rege, 1975 Reddy et al., 1990 Reddy et al., 1990 Reddy et al., 1990 NF Authors

ConclusionInvolvement of genetics and biotechnology is now becoming only option to overcome several problems of aquaculture such as inbreeding depression, poor disease resistance and growth rate stock mixing etc . Even though India is not pioneer in development of fisheries and aquaculture genetics but significant work has been done on Indian major carp and other indigenous species which has benefitted this sector allot.

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