**Abstract**

Hybridization is the mating of genetically differentiated individuals or groups and may involve crosses within a species or between separate species. Hybridization can be natural or human-mediated. Reproductive barriers prevent excessive introgression in the former, whereas more often introgression and genetic pollution happen in the latter. Hybridization is more widespread among members of Cyprinidae than any other groups of freshwater fishes. In many carp hatcheries in India, breeders of catla (*Catla catla*) and rohu (*Labeo rohita*) are kept in the same tank for breeding, resulting in production of hybrid seeds. Fish hybrids can pose a serious threat to the aquatic environment biodiversity. Consequently, genetic monitoring of organisms is entailed to unambiguously identify parental species and their hybrids. Adopting a multiplex PCR using β-actin gene primers, a kit has been developed to distinguish between the hybrids from their parental species. Agarose electrophoresis revealed one band of about 100 bp in size specific for rohu, another at 300 bp specific for catla, and both bands in hybrid. The kit was tested successfully with the samples collected from many hatcheries located in four Indian states. The rohu-catla early hybrid identification PCR kit could serve as a stepping stone for carp seed certification and hatchery accreditation.

**Keywords:** hybridization, introgression, carps, hatchery, PCR

## **1. Introduction**

Hybridization is defined as the mating of genetically differentiated individuals or groups and may involve crosses within a species (also known as line crossing or strain crossing) or crosses between separate species [1]. Issues related to hybridization are complex, making the job of conservation biologists tougher. Interspecific and intergeneric hybridization do happen naturally, and it is considered to play an important role in evolution process [2]. Indian major carps (IMCs) comprising of *Labeo rohita*, *Catla catla*, and *Cirrhinus mrigala*, owing to their fast growth and taste, enjoy a prime position in the Indian aquaculture scenario. These carps attain a marketable size of 800–1000 g in less than a year and are generally propagated on an extensive and/or intensive scale in a polyculture system [3]. Among the three IMCs, catla and rohu are generally chosen for freshwater aquaculture due to their faster growth. IMCs, though originally inhabitants of the Ganga River network in North India and the rivers of Pakistan, Bangladesh, Nepal, and Burma, are also transplanted into other rivers in central as well as peninsular India and also in

aquaculture systems of Nepal and Sri Lanka. In 2005 global carp production reached 28.8 million tons, accounting for 37.5% in quantity and 25.6% in value of total aquaculture production [4]. China with 21.05 and India with 3.9 million tons were the top carp producers in the world. Indian major carps catla (2.76 million tons), rohu (2.91 million tons), and mrigal (0.47 million tons) were among 29 species with production over 100 t in 2005 [4].

It was noted that the growth of IMCs affected the grow-out culture phase and the profitability in carp farming is decreasing [5, 6]. The growth of carps is affected largely due to poor quality and mixed seed of carps produced by breeding carps of different species at the same time in the spawning pools of carp hatcheries that make easy hybrids. To make more profit, hatchery managers practice this in breeding programs in hatcheries when there is paucity of brood fish of desired species that is either males or females. This practice has been rampant in many parts of the Indian subcontinent. Unlike other agricultural crops and domestic land animals, the hybrids did not grow better than their native natural parents. Good quality seed of catla and rohu are in great demand in the Indian subcontinent. Seed is one of the indispensable resources needed for aquaculture. Taking the advantage of seed demand of these two species, many hatchery owners and seed producers supply hybrid seed (catla × rohu or rohu × catla) of these two species in the name of pure rohu or catla during the young stages. These hybrids cannot be easily differentiated from each other morphologically at early stages of development, e.g., hatchling and early fry stages. Among the most pressing issues concerning seed in global aquaculture development include inadequate and unreliable supply of quality seed, genetic quality, and inadequate hatchery technology.

For production of quality seed in aquaculture, many methods were tried in the past, including intergeneric and interspecific hybridization. Hybridization technique was used by aquaculturists in the hope of producing aquatic organisms with specific desirable traits or general improvement in performance. Intergeneric and interspecific hybridization programs have been applied in fish farms with the purpose of producing animals that perform better than the parental species (hybrid vigor) [1].

Nevertheless, many species are jeopardized by hybridization and genetic introgression, and these are particularly prevalent threats to the diversity of freshwater fish [7–10]. If fertile, hybrids can genetically contaminate natural and farmed stocks through genetic homogenization. They may also compete in several ways with the native parental lineages [2, 11, 12].

Currently, several genetic markers have been developed for different species that are used in aquaculture programs [13, 14]. PCR-RFLP and multiplex PCR are considered fast, simple, and inexpensive, but they have rarely been used in the characterization of hybrids or in aquaculture in general. These techniques are important tools in species identification, especially in studies related to the biological conservation and forensic identification [15, 16]. Natural occurrence of both interspecific and intergeneric hybrids of Indian major carps has been reported mostly from reservoirs and other natural ecosystems. From natural ecosystems such as reservoir and dry bundhs, several hybrids have been recorded [17]. Many of these hybrids were found to be intermediate in characters of the parent species. Only a few hybrids, both artificially produced and naturally occurring, have been studied in detail for their cultural qualities and adaptability to various environments. Several interspecific and intergeneric hybrids of Indian major carps are *Catla catla, Labeo rohita, Cirrhinus mrigala*, and *Labeo calbasu* [18, 19], and those of Indian major carps with exotic carps, viz., common carp [20] and silver carp [21], have been artificially produced through hypophysation. These hybrids were not popular due to poor survival and many undesired traits in aquaculture.

**25**

**Figure 1.**

*Hybridization in Carps and Early Detection of Carp Hybrids Using PCR-Based Kit*

Quality seed is a fundamental prerequisite for sustainable and successful aquaculture, be it small-scale or commercial farming. Inadequate supply of quality seed is often suggested as a major constraint for aquaculture in many parts of the world [22]. The issue of quality comes to the attention of producers only after a certain period of time when performance indicators (e.g., growth, production, survival, and disease) consistently point a finger towards seed quality. Factors which contribute to production of poor quality seed would have become established as a normal

Field level morphological identification of seeds of commercially important carp species, viz., catla and rohu, at early stage is a difficult task as experience has shown. Many times fish farmers get cheated by unethical practices by hatchery owners as they sell these hybrids in the name of pure rohu or catla seed. At that point of time, it is almost impossible for the farmers to recognize the differences. So after nursery rearing, poor survival and growth of these seeds become a bane for them. Even though they approach the fishery officials for law enforcement, they also feel helpless in the absence of a genuine identification method at this

An attempt has been made to review hybridization in carps (Cyprinidae), focusing on the advantages and disadvantages of human-mediated hybridization in cultured species of carps and detection methods of carp hybrids. Further, the development of a novel multiplex PCR-based approach for the identification of seeds of rohu and catla and their hybrids (parents and hybrids), using some molecular markers, and its field testing with hatcheries in four Indian states are described.

The major purpose behind human-mediated hybridization is genetic improvement in cultured species. This is expectedly achieved through combining desirable traits of parental species, resulting in heterosis or hybrid vigor in the progeny. In fishes human-mediated hybridization is around 50% [23]. In natural hybridization reproductive barriers prevent introgression to happen. That is, chances of F1 hybrid mating with parental species are remote. On the other hand, in human-mediated hybridization in which the rate is higher and the number of escapees is more, the

**2. Hybridization: natural and human-mediated**

*Schematic representation of natural and human-mediated hybridization.*

*DOI: http://dx.doi.org/10.5772/intechopen.91946*

practice in the system.

early stage.

#### *Hybridization in Carps and Early Detection of Carp Hybrids Using PCR-Based Kit DOI: http://dx.doi.org/10.5772/intechopen.91946*

Quality seed is a fundamental prerequisite for sustainable and successful aquaculture, be it small-scale or commercial farming. Inadequate supply of quality seed is often suggested as a major constraint for aquaculture in many parts of the world [22]. The issue of quality comes to the attention of producers only after a certain period of time when performance indicators (e.g., growth, production, survival, and disease) consistently point a finger towards seed quality. Factors which contribute to production of poor quality seed would have become established as a normal practice in the system.

Field level morphological identification of seeds of commercially important carp species, viz., catla and rohu, at early stage is a difficult task as experience has shown. Many times fish farmers get cheated by unethical practices by hatchery owners as they sell these hybrids in the name of pure rohu or catla seed. At that point of time, it is almost impossible for the farmers to recognize the differences. So after nursery rearing, poor survival and growth of these seeds become a bane for them. Even though they approach the fishery officials for law enforcement, they also feel helpless in the absence of a genuine identification method at this early stage.

An attempt has been made to review hybridization in carps (Cyprinidae), focusing on the advantages and disadvantages of human-mediated hybridization in cultured species of carps and detection methods of carp hybrids. Further, the development of a novel multiplex PCR-based approach for the identification of seeds of rohu and catla and their hybrids (parents and hybrids), using some molecular markers, and its field testing with hatcheries in four Indian states are described.

## **2. Hybridization: natural and human-mediated**

The major purpose behind human-mediated hybridization is genetic improvement in cultured species. This is expectedly achieved through combining desirable traits of parental species, resulting in heterosis or hybrid vigor in the progeny. In fishes human-mediated hybridization is around 50% [23]. In natural hybridization reproductive barriers prevent introgression to happen. That is, chances of F1 hybrid mating with parental species are remote. On the other hand, in human-mediated hybridization in which the rate is higher and the number of escapees is more, the

*Emerging Technologies, Environment and Research for Sustainable Aquaculture*

production over 100 t in 2005 [4].

quality, and inadequate hatchery technology.

with the native parental lineages [2, 11, 12].

due to poor survival and many undesired traits in aquaculture.

aquaculture systems of Nepal and Sri Lanka. In 2005 global carp production reached 28.8 million tons, accounting for 37.5% in quantity and 25.6% in value of total aquaculture production [4]. China with 21.05 and India with 3.9 million tons were the top carp producers in the world. Indian major carps catla (2.76 million tons), rohu (2.91 million tons), and mrigal (0.47 million tons) were among 29 species with

It was noted that the growth of IMCs affected the grow-out culture phase and the profitability in carp farming is decreasing [5, 6]. The growth of carps is affected largely due to poor quality and mixed seed of carps produced by breeding carps of different species at the same time in the spawning pools of carp hatcheries that make easy hybrids. To make more profit, hatchery managers practice this in breeding programs in hatcheries when there is paucity of brood fish of desired species that is either males or females. This practice has been rampant in many parts of the Indian subcontinent. Unlike other agricultural crops and domestic land animals, the hybrids did not grow better than their native natural parents. Good quality seed of catla and rohu are in great demand in the Indian subcontinent. Seed is one of the indispensable resources needed for aquaculture. Taking the advantage of seed demand of these two species, many hatchery owners and seed producers supply hybrid seed (catla × rohu or rohu × catla) of these two species in the name of pure rohu or catla during the young stages. These hybrids cannot be easily differentiated from each other morphologically at early stages of development, e.g., hatchling and early fry stages. Among the most pressing issues concerning seed in global aquaculture development include inadequate and unreliable supply of quality seed, genetic

For production of quality seed in aquaculture, many methods were tried in the past, including intergeneric and interspecific hybridization. Hybridization technique was used by aquaculturists in the hope of producing aquatic organisms with specific desirable traits or general improvement in performance. Intergeneric and interspecific hybridization programs have been applied in fish farms with the purpose of producing animals that perform better than the parental species (hybrid

Nevertheless, many species are jeopardized by hybridization and genetic introgression, and these are particularly prevalent threats to the diversity of freshwater fish [7–10]. If fertile, hybrids can genetically contaminate natural and farmed stocks through genetic homogenization. They may also compete in several ways

Currently, several genetic markers have been developed for different species that are used in aquaculture programs [13, 14]. PCR-RFLP and multiplex PCR are considered fast, simple, and inexpensive, but they have rarely been used in the characterization of hybrids or in aquaculture in general. These techniques are important tools in species identification, especially in studies related to the biological conservation and forensic identification [15, 16]. Natural occurrence of both interspecific and intergeneric hybrids of Indian major carps has been reported mostly from reservoirs and other natural ecosystems. From natural ecosystems such as reservoir and dry bundhs, several hybrids have been recorded [17]. Many of these hybrids were found to be intermediate in characters of the parent species. Only a few hybrids, both artificially produced and naturally occurring, have been studied in detail for their cultural qualities and adaptability to various environments. Several interspecific and intergeneric hybrids of Indian major carps are *Catla catla, Labeo rohita, Cirrhinus mrigala*, and *Labeo calbasu* [18, 19], and those of Indian major carps with exotic carps, viz., common carp [20] and silver carp [21], have been artificially produced through hypophysation. These hybrids were not popular

**24**

vigor) [1].

reproductive barriers are broken down, leading to introgression which may eventually lead to genetic extinction (**Figure 1**).
