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

#### **6.1 Development**

*Emerging Technologies, Environment and Research for Sustainable Aquaculture*

fishes badly [27].

performance of their progeny [29].

threat to local species than the pure species [51].

originating from populations in North America and Europe [43].

**5. Detection of hybrids**

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

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

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

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

**28**

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 phenolchloroform method [58].

Total genomic DNA from spawn was isolated using DNeasy blood and tissue kit, Qiagen. DNA quantity was determined against a molecular marker standard (λ-DNA 25 ng, Fermentas) by electrophoresis in a 0.8% agarose gel. β-actin sequences of carps available in GenBank were downloaded (Accession numbers: AF415205, M24113, GU338376, AY531753, AF415206) and aligned using Clustal W program implemented in the software Bioedit version 7.0.5.3 [59], and conserved primers for the amplification of a fragment size of ~ 1000 bp in Indian major carps and minor carps were done manually. Genomic DNA (~20–100 ng) from both species of IMCs was amplified in a 25 μl PCR volume containing 10 picomoles of each conserved primer, 2.5 mM of each dNTP, and 0.25 U of *Taq* polymerase with a thermal regime of 94°C (5 min), 35 cycles at 94°C (0.5 min), 60°C (0.5 min) and 1 min at 72°C (1 min) and final extension of 72°C (5 min). PCR products were purified using Qiagen PCR purification kit followed by bidirectional cycle sequencing on ABI 3100 PE automated capillary sequencer.

A total of 20 sequences of both the species (10 *Labeo rohita* and 10 *Catla catla*) were aligned using Clustal W program in Bioedit software. Species-specific reverse primers for both species were designed, taking the species-specific mutation into account. A touchdown PCR was carried out with a 25 μl PCR volume containing 10 picomoles of each species-specific reverse primers (one rohu and one catla) and 20 picomoles of universal forward primer, 2.5 mM of each dNTP, and 0.25 U of *Taq* polymerase with the PCR condition of 94°C (5 min), 2 cycles at 94°C (0.5 min), 68°C (0.5 min) and 1 min at 72°C (1 min), 2 cycles at 94°C (0.5 min), 66°C (0.5 min) and 1 min at 72°C (1 min), 2 cycles of at 94°C (0.5 min), 64°C (0.5 min) and 1 min at 72°C (1 min), 25 cycles at 94°C (0.5 min), 62°C (0.5 min) and 1 min at 72°C (1 min) and final extension of 72°C (5 min). The PCR products were checked in a 2% agarose gel.

Partial sequences of the nuclear β-actin gene amplified using a set of primers BAF (5′GTAGGCACGACATTGAATGGG3′) and BAR (5′AGACAAAGGAAGTCCCTCTGC3′) generated a total of 820 bp which revealed some differences in the nucleotide composition between *Labeo rohita* and *Catla catla*. Single-nucleotide polymorphism was found between the species which were used to design species-specific internal primers.

Two primers were designed specific for each species considering the polymorphic sites in the sequences. Both primers designed were in the reverse direction: primer BALRR (5′-CTTGAAAACTGTACAATCACGTTC-3′) was specific for *Labeo rohita*, and BACCR (5′-GCTAGCTAATAGACGTAATCATTTAG-3′) was specific for *Catla catla*. Amplification of these primers (BAF, BALRR, and BACCR) established different electrophoretic banding patterns when run in a 2% agarose gel. The result revealed one band at about 100 bp specific for *L. rohita* and another band at 300 bp specific for *C. catla*. In the rohu × catla hybrid, a heterozygote pattern was observed with two diagnostic bands, with each one inherited from one parental strain. Using these species diagnostic primers, a PCR-based rohu-catla hybrid identification kit was developed which has received provisional Indian patent number "343/KOL/2013 of 26.3.2013." For the validation of the developed kit, a total number of 685 samples from different places were screened which revealed that 54 out of them were hybrids (**Figure 3**).

#### **6.2 Features of the kit**

The use of a multiplex PCR marker in the present study revealed a distinct electrophoretic pattern between rohu and catla and their hybrid. The advantage of multiplex PCR is that it does not require the additional step of restricted enzyme digestion and can thus eliminate any post-PCR analyses as well as additional time and costs. On the other hand, there are limitations to the primer designs that should be taken into consideration. The primers should be specific and reliable in

**31**

**Figure 3.**

*hybrid).*

**Figure 4.**

with the help of the kit (**Figure 4**).

*Rohu-catla early hybrid identification kit.*

• Species-specific primers

• dNTP mix (2.5 mM each)

• Taq DNA polymerase 3 U/μl

• 10X Taq DNA buffer

• Universal primer

Contents of the kit are mentioned below:

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

binding. This study can serve as a basis for further study on the introgression of these hybrids with their parental species. Genetic monitoring of mixed spawning and unintended hybridization of Indian major carps in hatcheries can be carried out

*PCR test of hatchery spawn samples (300 bp marker in catla, 100 bp marker in rohu, and both markers in* 

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

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

#### **Figure 3.**

*Emerging Technologies, Environment and Research for Sustainable Aquaculture*

on ABI 3100 PE automated capillary sequencer.

used to design species-specific internal primers.

Total genomic DNA from spawn was isolated using DNeasy blood and tissue kit, Qiagen. DNA quantity was determined against a molecular marker standard (λ-DNA 25 ng, Fermentas) by electrophoresis in a 0.8% agarose gel. β-actin sequences of carps available in GenBank were downloaded (Accession numbers: AF415205, M24113, GU338376, AY531753, AF415206) and aligned using Clustal W program implemented in the software Bioedit version 7.0.5.3 [59], and conserved primers for the amplification of a fragment size of ~ 1000 bp in Indian major carps and minor carps were done manually. Genomic DNA (~20–100 ng) from both species of IMCs was amplified in a 25 μl PCR volume containing 10 picomoles of each conserved primer, 2.5 mM of each dNTP, and 0.25 U of *Taq* polymerase with a thermal regime of 94°C (5 min), 35 cycles at 94°C (0.5 min), 60°C (0.5 min) and 1 min at 72°C (1 min) and final extension of 72°C (5 min). PCR products were purified using Qiagen PCR purification kit followed by bidirectional cycle sequencing

A total of 20 sequences of both the species (10 *Labeo rohita* and 10 *Catla catla*) were aligned using Clustal W program in Bioedit software. Species-specific reverse primers for both species were designed, taking the species-specific mutation into account. A touchdown PCR was carried out with a 25 μl PCR volume containing 10 picomoles of each species-specific reverse primers (one rohu and one catla) and 20 picomoles of universal forward primer, 2.5 mM of each dNTP, and 0.25 U of *Taq* polymerase with the PCR condition of 94°C (5 min), 2 cycles at 94°C (0.5 min), 68°C (0.5 min) and 1 min at 72°C (1 min), 2 cycles at 94°C (0.5 min), 66°C (0.5 min) and 1 min at 72°C (1 min), 2 cycles of at 94°C (0.5 min), 64°C (0.5 min) and 1 min at 72°C (1 min), 25 cycles at 94°C (0.5 min), 62°C (0.5 min) and 1 min at 72°C (1 min) and final exten-

sion of 72°C (5 min). The PCR products were checked in a 2% agarose gel. Partial sequences of the nuclear β-actin gene amplified using a set of primers BAF (5′GTAGGCACGACATTGAATGGG3′) and BAR

(5′AGACAAAGGAAGTCCCTCTGC3′) generated a total of 820 bp which revealed some differences in the nucleotide composition between *Labeo rohita* and *Catla catla*. Single-nucleotide polymorphism was found between the species which were

Two primers were designed specific for each species considering the polymorphic sites in the sequences. Both primers designed were in the reverse direction: primer BALRR (5′-CTTGAAAACTGTACAATCACGTTC-3′) was specific for *Labeo rohita*, and BACCR (5′-GCTAGCTAATAGACGTAATCATTTAG-3′) was specific for *Catla catla*. Amplification of these primers (BAF, BALRR, and BACCR) established different electrophoretic banding patterns when run in a 2% agarose gel. The result revealed one band at about 100 bp specific for *L. rohita* and another band at 300 bp specific for *C. catla*. In the rohu × catla hybrid, a heterozygote pattern was observed with two diagnostic bands, with each one inherited from one parental strain. Using these species diagnostic primers, a PCR-based rohu-catla hybrid identification kit was developed which has received provisional Indian patent number "343/KOL/2013 of 26.3.2013." For the validation of the developed kit, a total number of 685 samples from different places were screened which revealed that 54 out of them were hybrids (**Figure 3**).

The use of a multiplex PCR marker in the present study revealed a distinct electrophoretic pattern between rohu and catla and their hybrid. The advantage of multiplex PCR is that it does not require the additional step of restricted enzyme digestion and can thus eliminate any post-PCR analyses as well as additional time and costs. On the other hand, there are limitations to the primer designs that should be taken into consideration. The primers should be specific and reliable in

**30**

**6.2 Features of the kit**

*PCR test of hatchery spawn samples (300 bp marker in catla, 100 bp marker in rohu, and both markers in hybrid).*

**Figure 4.** *Rohu-catla early hybrid identification kit.*

binding. This study can serve as a basis for further study on the introgression of these hybrids with their parental species. Genetic monitoring of mixed spawning and unintended hybridization of Indian major carps in hatcheries can be carried out with the help of the kit (**Figure 4**).

Contents of the kit are mentioned below:


Advantages and utility of this kit are summarized below:

