**4. Threats and concerns of human-mediated hybridization**

The harmful effects of hybridization, with or without introgression, have led to the extinction of many populations and species in many plant and animal taxa. Hybridization is especially problematic for rare species that come into contact with other species that are more abundant [2]. Hybridization can decrease diversity through the breakdown of reproductive barriers, the merger of previously distinctive evolutionary lineages, and the extinction of populations or species. There are two main mechanisms by which hybridization can lead to extinction. If hybrid fitness is strongly reduced relative to that of parental individuals (i.e., outbreeding depression), and hybridization is common, population growth rates of one or both parental lineages may decline below replacement rates due to wasted reproductive effort, leading to extinction [41, 45]. Hybridization involving captive-bred individuals can have harmful consequences beyond the loss of genetic integrity [46]. In many cases, the stocked individuals differ genetically from the target population, which can result in outbreeding depression following hybridization [45].

Inadvertent hybridization and backcrossing can lead to unexpected and undesirable results in hybrid progeny, such as failure to produce sterile fish, loss of color pattern, and reduced viability. Uncontrolled and unintentional hybridization could undermine the performance of cultured stocks and restrict future use of the contaminated stocks as broodstock. The level of unintentional or accidental hybridization has important considerations for the conservation of aquatic biodiversity and will influence risk assessment on the use of hybrid fishes in aquaculture and fisheries [1]. Continued hybridization may eventually lead to a breakdown of species barriers, thereby compromising the genetic integrity of the species in the wild and leading to production losses in aquaculture [30]. Hybrid introgression in major carp species is very likely to have negative consequences, as a result of loss of distinct feeding strategies of the pure species, which are the basis of successful polyculture systems [46].

In Indian major carps, inadvertent production of hybrids out of mixed spawning of species has been well documented. Actually the farmers are on the lookout for "mixed seeds," meaning a certain proportion of catla, rohu, and mrigal along with other exotic carps for polyculture [47]. For the sake of time and economy, the hatchery producers keep broodfish of different species, particularly rohu and catla,

*Emerging Technologies, Environment and Research for Sustainable Aquaculture*

carcass composition, meat yield, etc. were carried out [28].

**3.1 Purposes and merits of human-mediated hybridization**

ally lead to genetic extinction (**Figure 1**).

**3. Hybridization in Cyprinidae**

were carried out [23, 39].

tion of new hybrid lineages [40, 41].

reproductive barriers are broken down, leading to introgression which may eventu-

Hybridization is more widespread among members of Cyprinidae than any other groups of freshwater fishes [23]. Several permutation and combinations of interspecific and intergeneric crosses were carried out among Indian major carps (*Catla catla*, *Labeo rohita* and *Cirrhinus mrigala*) and exotic carps (*Hypophthalmichthys molitrix*, *Ctenopharyngodon idella*, *Aristichthys nobilis* and *Cyprinus carpio*) with the major objective to achieve hybrid vigor in economic traits [24–31]. The Indian major carp species are known to be able to hybridize, and hybrids are fertile and can be backcrossed to the parental species [32–34]. Hybridization has been shown to have a significant impact on production-related traits (notably growth), with some studies reporting a growth rate of F1 hybrids intermediate between that of the parent species [35, 36]. Hybrids are reported to have lower rates of growth than either of the parental species of Indian carps, other studies reporting growth rates lower than for either of the parental species [37]. Intergeneric hybrids between catla (*Catla catla*) and fimbriatus (*Labeo fimbriatus*) were produced by employing the technique of hypophysation and dry stripping. Detailed investigations on their embryonic and larval development, taxonomic characters, and aquaculture potential in terms of growth, feed utilization, body

A catla-rohu hybrid produced by hypophysation was found to be intermediate in general appearance to the parent species. Gut content analysis revealed that the hybrid was mainly phytophagous in its diet. Growth rate was observed to be faster than in rohu. It matured within 3 years and was equally responsive to hypophysation [38]. In other cyprinid species, also such efforts of hybridization

It has long been recognized that hybridization can have a variety of evolutionary

Hybridization in carps was being carried out to increase growth rate, transfer desirable traits between species, combine desirable traits of two species into a single group of fishes, reduce unwanted reproduction through production of sterile fish or mono-sex offspring, take advantage of sexual dimorphism, increase harvestability, increase environmental tolerances, and increase overall hardiness in culture conditions. Hybrids constitute a significant proportion of some countries' production for certain taxa, for example, hybrid striped bass in the USA, hybrid clarid catfish in Thailand, hybrid characids in Venezuela, and hybrid tilapia in Israel. Hybridization has been used in tandem with polyploidization to improve developmental stability in hybrid progeny [1]. Intergeneric hybrids between catla (*Catla catla*) and fimbriatus (*Labeo fimbriatus*) combined desirable qualities such as the small head of the fimbriatus and the deep body of the catla and exhibited heterosis in terms of meat yield with higher flesh content than either of the parents. Hence the hybrids appear to be of considerable importance to aquaculture [28]. It is also believed that hybrids of parental genotypes might be able to explore ecological niche unavailable

outcomes, including outcomes that maintain or increase diversity such as stable hybrid zones, the evolutionary rescue of small inbred populations, the origin and transfer of adaptations, the reinforcement of reproductive isolation, and the forma-

**26**

in the same breeding pool, resulting in unintentional production of the hybrid seeds [34, 48]. The intergeneric hybrids are fertile, and they can breed (backcross) with parental species to produce introgressed F2 hybrids. The thoughtless and injudicious ways of fish breeding are likely to affect the "gene pools" of these prized food fishes badly [27].

Silver carp and bighead carp sometimes are hybridized inadvertently because of their similar appearance and because of shortage of "the correct" species at spawning time due to differences in maturation times between male and female carp. This hybridization often results in a fish that does not feed efficiently as its gill rakers are intermediate in shape between those of the silver carp that eats phytoplankton and those of the bighead carp that consumes zooplankton [1]. The rohu-catla reciprocal hybrids are reported to have limited economic value [27]. These hybrids are also reported to be more susceptible to parasitic infection than the parental species [49]. Hybridization between silver carp (*Hypophthalmichthys molitrix*) and bighead carp (*Aristichthys nobilis*) suggests further generations of hybridization or introgression between the species in hatcheries, with potentially damaging consequences for the integrity of these stocks and their performance in aquaculture [50]. Pecos pupfish (*Cyprinodon pecosensis*) is threatened with replacement by its hybrids with sheepshead minnow (*C. variegatus*) [12]. Continued hybridization between invasive bigheaded carps (*Hypophthalmichthys nobilis*) and silver carp (*Hypophthalmichthys molitrix*) has indicated reduced nutritional performance of their progeny [29].

### **5. Detection of hybrids**

Accurate identification of hybrids is important not only for sustainable aquaculture development, guiding aquaculture domestication efforts, assessing aquaculture production, and identifying useful crosses, but also to allow for a better understanding of biodiversity issues. It would be unfortunate to experience a widespread loss of pure species in aquaculture as happened with tilapia as a result of widespread introduction and subsequent hybridization; it would be also a significant cause for concern if hybrid Thai catfish or the hybrid Venezuelan characids pose more of a threat to local species than the pure species [51].

Before 1966 only morphology-based methods were used to identify hybrids. Subsequently followed by morphology (45%), allozymes (35%), mtDNA (12%), nDNA (4%), and karyology (2%) were used till the late 1990s. Genetic markers and population genetic theory have provided powerful tools facilitating the description of hybridization events and serve as sources of evidence for factors underlying occurrence, direction, and extent of introgression between fish taxa [23]. VNTR minisatellite and microsatellite loci, SINE's, RAPD, AFLP, and ISSR assume dominance, whereby individuals are characterized by the presence or absence of amplification products of specific size. The number of alleles producing a product (one for heterozygotes and two for a homozygote) cannot be directly determined. Thus, the per-locus information context of dominant markers is less than for codominant loci. Mitochondrial DNA cannot be used alone to detect hybrids because of the marker's haploid and matrilineal mode of inheritance. However, mtDNA can be a powerful tool to establish directionality when used in conjunction with nuclear genetic markers [23]. Genetic markers, such as allozymes, mtDNA, and nuclear DNA, were used to confirm hybrid status and to determine directionality of the hybridization event [12]. Multiple markers were employed to determine if an Icelandic population of eels (*Anguilla anguilla*) included hybrid individuals from matings of parents originating from populations in North America and Europe [43].

**29**

**Figure 2.**

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

Historically, meristic and morphometric measurements were the primary means of identifying naturally occurring hybrids. The introduction of allozyme electrophoresis provided a methodology whereby individuals of most species could be assayed for biochemical markers with a demonstrable heritable basis [52]. The use of mtDNA was first cited in the surveyed literature in analyses of fish hybridization in the mid-1980s [4]. In the late 1980s, nuclear DNA was started to be used for

Documentation of hybridization often has been based on meristic or morphological criteria that can be misleading when used as the sole source of inference, particularly for hybrid individuals beyond the F1 generation [55]. Morphology, allozymes, and mtDNA were used in the analysis of *Notropis chrysocephalus* and *N. cornutus* hybrid zones in Michigan drainages [56]. Genomic RFLP was used to detect hybrids of Indian major carps, and the results indicated that intergeneric hybridization did occur during "mixed spawning" of these carps and the hybridization frequency was appreciable, at about 10% [34]. Utilizing an integrated approach, which incorporates geometric morphometrics, life history, and molecular genetic analyses, the levels and processes of hybridization in two species of cyprinids were determined [39]. The extent of intergeneric hybridization in Indian major carps was studied using allozymes [30]. DNA fingerprinting using RAPD-ISSR assay was used

**6. PCR-based kit for detection of early hybrids of rohu-catla reciprocal** 

For the parental lineages, 50 individuals of *Labeo rohita* (rohu) and 50 individuals of *Catla catla* (catla) were genetically analyzed. Crosses performed by mating females of catla and males of rohu and vice versa resulted in the intergeneric hybrid (**Figure 2**). Twenty-four hybrid individuals were included in the genetic analysis. Spawns of reciprocal hybrids were collected for further genetic analysis. DNA was extracted from the fin clips of adults of parental species using standard phenol-

*Catla (PCc), rohu (PLr) parents, F1 hybrid (CcLrF1Hy), and F2 backcross hybrids (bcF2Hy).*

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

identifying hybridization process [53, 54].

to detect hybridization in Indian major carps [57].

**crosses**

**6.1 Development**

chloroform method [58].

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

Historically, meristic and morphometric measurements were the primary means of identifying naturally occurring hybrids. The introduction of allozyme electrophoresis provided a methodology whereby individuals of most species could be assayed for biochemical markers with a demonstrable heritable basis [52]. The use of mtDNA was first cited in the surveyed literature in analyses of fish hybridization in the mid-1980s [4]. In the late 1980s, nuclear DNA was started to be used for identifying hybridization process [53, 54].

Documentation of hybridization often has been based on meristic or morphological criteria that can be misleading when used as the sole source of inference, particularly for hybrid individuals beyond the F1 generation [55]. Morphology, allozymes, and mtDNA were used in the analysis of *Notropis chrysocephalus* and *N. cornutus* hybrid zones in Michigan drainages [56]. Genomic RFLP was used to detect hybrids of Indian major carps, and the results indicated that intergeneric hybridization did occur during "mixed spawning" of these carps and the hybridization frequency was appreciable, at about 10% [34]. Utilizing an integrated approach, which incorporates geometric morphometrics, life history, and molecular genetic analyses, the levels and processes of hybridization in two species of cyprinids were determined [39]. The extent of intergeneric hybridization in Indian major carps was studied using allozymes [30]. DNA fingerprinting using RAPD-ISSR assay was used to detect hybridization in Indian major carps [57].
