**7. Conclusions**

*Emerging Technologies, Environment and Research for Sustainable Aquaculture*

Advantages and utility of this kit are summarized below:

• Useful for screening of hatcheries for genetic contamination

• Potential for seed certification hatchery accreditation

• Highly sensitive and specific for rohu × catla hybrids and reciprocal crosses

• An essential tool for government/private agencies to ensure purity of seed

A total of 685 samples of different life stages (spawn and fry) from different hatcheries of four Indian states, Odisha, Gujarat, Bihar, and West Bengal (**Table 1**), were collected and tested using the species-specific designed primers with the same

**State Details of hatchery Samples (n) Hybrid (n) Hybrid (%)** Odisha State Fisheries Farm, Kausalyaganga 150 6 4 Odisha State Fisheries Farm, Bhadrak 30 0 0 Odisha State Fisheries Farm, Balasore 30 2 7 Odisha Balakati Private Hatchery 25 0 0 Odisha Balakati Private Hatchery 25 3 12 Gujarat Private Hatchery 45 23 51

Bihar Koshi Fish Hatchery, Madhubani 30 5 17 Bihar Ganga Fish Hatchery, Madhubani 30 4 13 Bihar Yamuna Fish Hatchery, Madhubani 30 6 20

*Presence of hybrid seeds (spawn/hatchlings and fry) in hatchery populations in four Indian states (Odisha,* 

Naihati Market 200 137 68.5

30 0 0

30 3 10

30 2 7

**6.3 Validation of primers by screening unknown samples from hatcheries**

• PCR and agarose gel-based detection kit

• Takes only 4–6 h to get the results

• Positive control DNA 100 ng

• Nuclease-free water

• No sequencing required

thermal cycler condition [60].

Bihar Shri Tripura Chaudhary, Matsya Farm,

Bihar Asha Fish Breeding Centre, Baheri

Bihar Kamla Fish Hatchery, Jhajarpur,

*Gujarat, Bihar and West Bengal), n = 685.*

Vaishali

Block, Darbhanga

Madhubani

• 100 bp ladder

**32**

West Bengal

**Table 1.**

There have been numerous studies on hybridization of fishes, and certainly not all of the hybrids reported are contributing to commercial aquaculture production. 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.

Intergeneric hybrids between the species rohu (*Labeo rohita*) and catla (*Catla catla*) are being produced in Indian carp hatcheries without any monitoring. The parental species belong to the more representative genus of the family Cyprinidae, which is an important freshwater fish group that is widespread throughout India, and are important fishery resources to specific communities. Some of the hatcheries are practicing multispecies breeding (mainly rohu and catla) in the same breeding pool and at the same time leading to interspecific hybridization. There is every chance of these hybrids escaping to natural waters which would lead to pollution of the genetic material in the wild, leading to non-availability of the pure strains of the carps in the future. There seems to be a misunderstanding regarding the culture of mixed species for composite fish culture with mixed breeding of carps by the hatchery managers and fish farmers. When the farmers are demanding mixed seeds (mixture of pure rohu and catla), the hatchery managers are mixing the spawners to produce hybrids.

Hybrid identification based on morphology, ecology, and behavior can be difficult and, most of the time, confusing and uncertain. Multiplex PCR strategy has proven to be an efficient methodology that could be quickly and inexpensively executed, which would allow diagnoses through simple PCR based upon singlenucleotide polymorphisms.

Morphological differences of hybrids are only minor and need close examination by experienced workers, and it is difficult to verify the genuine hybrids from interspecific hybridization. Allozymes were used to detect the genetic difference between the hybrids and their parental species; the use of allozyme loci failed to provide a sufficient genetic basis of hybrids, probably due to the less informative nature of allozyme loci (i.e., limited number of polymorphic loci available and low level of polymorphisms). Mutation at the DNA level that causes a replacement of a similarly charged amino acid may not be detected by allozyme electrophoresis, although allozymes represent actual gene products. Molecular techniques have been applied in the worldwide aquaculture, allowing for an adequate management of several cultivated species and providing a huge number of molecular markers that have been applied successfully for hybrid identification and detecting genetic introgression in fish. Nuclear molecular markers have supplied valuable information in the detection of hybridization events as well as the identification of reciprocal hybrids. Since mitochondrial DNA in animals has the characteristic of maternal inheritance [61], they are not suitable to detect hybrids. On the other hand, nuclear DNA serves as an efficient tool for hybrid identification.

The use of multiplex PCR marker revealed a distinct electrophoretic pattern between *Labeo rohita*, *Catla catla*, and their hybrid. The advantage of multiplex PCR is that it does not require the additional step of restriction enzyme digestion, which can eliminate any post-PCR analyses as well as additional time and costs. On the other hand, there can be limitations to the primer designs that should be taken into consideration, where these mainly reflect the ability of the primers to have good specificity and reliability in the application [62].

Finally based on the personal experience and inferences from other related studies, the following policy guidelines are recommended:

