This document discusses the application of biotechnologies in ex situ conservation, characterization, and utilization of fish genetic resources. It covers several key points: 1) Biotechnologies like cryopreservation of gametes and cells, and molecular characterization can help conserve genetic diversity of fish outside their natural habitats and aid breeding programs. 2) Characterizing genetic diversity within and among fish species is important for conservation efforts, understanding evolution, and improving aquaculture. Molecular tools are crucial for tasks like species discovery, genetic stock identification, and selection programs. 3) Emerging techniques like genome sequencing and stem cell technologies provide new opportunities for conservation and utilization of fish genetic resources, but also pose challenges like standardization and preventing

1. Applications of Biotechnologies in Ex Situ
Conservation, Characterization and Utilization
of Fish Genetic Resources
J.K. Jena & K.K. Lal*
Indian Council of Agricultural Research, New Delhi, India
*ICAR-National Bureau of Fish Genetic Resources, India

2. Sustainable intensification targets:
• Increase productivity
o Improvement in species
o Efficient husbandry systems
o Minimum disease risk
• Sustained genetic diversity during domestication
• Safeguard Biological diversity
• Compliance to International Policy Frameworks & Conventions
Aquaculture: Dependable Way for Future Food Production
• Rise in aquaculture production meeting >50% fish demand - out of
151.2 mmt fish consumed, 80 mmt comes from aquaculture
• 7-9% consistent rise in last 2 decades
• Hope for future - Global fish production to reach 201 mmt by 2030,
aquaculture 109 mmt (≈60%)
• Sustainable intensification - Focus & Concern

3. Capitalizing fully on the enormous potential of aquatic genetic
resources requires recognizing & overcoming:
• Current lack of information on genetic characteristics,
performance, location & accessibility of fish genetic resources,
and regarding threats to their survival
• Inadequate national programs & information systems for aquatic
genetic resources
• Lack of a global policy & management approach to aquatic
genetic resources.
Commission of Genetic Resources for Food & Agriculture (CGRFA)
Major Impediments
A Blue Revolution in the Twenty-First Century

4. Ex situ conservation
• Cryo-conservation of gametes
• Development of cell lines (Somatic & Stem cells)
Molecular characterization
• Interspecific diversity
• Intra-specific diversity & Genetic stocks
• Safeguarding farmed stocks
• Genetic improvement
• Genomics & Gene mining
Germplasm repositories
Role of Biotechnology

5. Breeding Programs Management
• Sperm available to help
asynchronous maturity
• Genetic selection program
• Exchange between farms for
outbreeding
• Assisted breeding with brood
stock sourced from wild
• Marketing of evaluated, disease
free & quality sperm
• Sperm mediated transgenic
Genetic Resource Banking (GRB)
• Preserving selected stocks from
outbreaks, disasters & genetic
drift.
• Storage of stocks or species in
danger of extinction
• Preserve germplam created for
research or commercial production
(polyploids, transgenics etc)
• Rehabilitation of extinct species,
with availability of cryopreserved
sperm
Cryo-Conservation of FGR

6. Sperm • Successful protocol for over 200 fish species
• Preservation only haploid paternal Germplasm
 Cross breeding (only paternal contribution)
 Diploidy through Androgenesis (very low success)
Oocyte • Difficult and no success achieved with fish
• Allows the preservation of haploid female Germplasm
 Cross breeding with cryopreserved sperm
Embryos • Difficult in fish & some success in shellfish like
Molluscs/Balanus etc.
• Preservation of diploid genome
 Post-thaw normal development
Cells for Cryoconservation in Breeding Plans & GRB

7. Blastomeres • Good survival rates
• Require complementary reproductive technology
(chimera)
• Preservation of diploid genome
 Grafting in embryos or Nuclear transfer
Stem Cells
• Primordial germ
cells
• Spermatogonia
• Relatively easy to freeze
• Potential application through surrogate Broodstock
• Preservation of diploid genome
 Grafting in Surrogate: success in few species
Somatic adult
cells
• Somatic cells can be cultured and frozen.
• Preservation of diploid genome
• Require complementary reproductive technology
(chimera)
 Nuclear transfer
 Induced Pluripotency combined with surrogate
broodstock
Cells for Cryoconservation in Breeding Plans & GRB

8. JHS Blaxter 1953; Herring
• Only method available at present in fish
• Need species-specific protocol to keep sperm
in quiescent stage
• Research expansion horizontal - Over 200 sp
• Experimental upscale: Blue channel catfish,
Rainbow trout, European seabass, Sea bream &
Turbot
• Upscaling program (NBFGR-NFDB) initiated in
India for exchange between carp hatcheries
• Protocol developed for 18 species in India
• 23 years old frozen sperm evaluated to yield 52%
hatching
Cryopreservation of Fish Sperm

9. 7. Post-Thaw
Activation & Hatching
1. Sperm
Collection
3. Dilution in
Specific
Extender
2. Quality
Evaluation
4. Sperm
Loading 4°C
Straws Cryovials
NaCl
KCl
Egg yolk
DMSO---
5. Vapor
Phase -80°C
6. LN2 Storage
Sperm
Maintained in
Quiescent State
• Enhancing predictability in
quality & quantity of gamete
collection
• Improved & standardized of
freezing/ thawing protocols
under scale-up conditions in
commercial use
Challenges (Biological and Procedural)
For upscaling
Cryopreservation Process & Challenges

10. Stem Cells: Surrogate Broodstock Bevelopment
Fish Cell Lines (Embryonic &
Somatic)
Repository with 59 Cell lines
Donor stem cell colonization
Surrogate sperm Surrogate eggs
Stem cells were observed to undergo
proliferation, vertical differentiation inside the
common carp gonads and produced surrogate
gametes (18 weeks after the cell transplantation)
• Isolated spermatogonial stem cells
(from goldfish (Carassius auratus)
• Transplanted in Cyprinus carpio (host)
Ex Situ Conservation - Native Germplasm

11. Diversity Characterization
Inter-specific Divergence
Molecular
Taxonomy
Conventional
Phylogeny
• Intra-specific Divergence
• Population Genetics - Genomics
• Phylogeography
Species Strains/Stocks Genome
Greater Understanding on
Sequencing of a uniform
mtDNA fragment (COI)
Mitochondrial & Nuclear
markers

12. Between 1997-2018, average near 380 new species added per year
By William N. Eschmeyer and Jon David Fong
http://researcharchive.calacademy.org/research/ichthyology/catalog/speciesbyfamily.asp
FISH BOL
Species Bar-coded : 11227
Unnamed barcode clusters found : 2432
• Taxonomy capacity a challenge
• Molecular taxonomy complementing morphology is way forward
• Many species may undergo unrecognized extinction unless pace of
precise systematics is increased
Discovering & Describing New Species

13. N. hexagonolepis
N. chillynoides
T. mosal
T. mussallah
T. khudree
T. tor
T. mosal
mahanadicus
T. putitora
South Asia Mahseer
Phylogeny
Needs Understanding
of Correct Taxonomy
Organisms Diversity Evolution (2014)
Same
lineage
Different
climates
Effective
Conservation &
Management of
Biodiversity
Taxonomic Instability: Challenge to Conservation

14. Genetic Stock identification and knowledge on performance considered
critical before they are lost (CGRFA & GTI, CBD)
• Indicate genetic diversity in the species
• Adaptive variability
• Track gene flow and exchange
• Evolutionary pressures
Concern
In 72% of cultured finfishes, 80% molluscs and 80% crustaceans, wild
genetic diversity is studied, but markers are not consistent (Benzie 2010)
• Genetic stocks in wild relatives need to be brought for
on-farm performance evaluation
• Knowledge is critical to evolve strategies for selection
program & enhance utilization
Characterizing Genetic Stocks

15. Catfish: n=410 3 distinct Clades
Molecular markers
• Allozyme (not common now)
• Microsatellite DNA
• Mitochondrial genes such as cytochrome b;
ATPase 6/8
• With genome sequencing generated Single
Nucleotide markers will find use
Need for standardized marker panel with gene indices to characterize &
monitor genetic alterations happening in farm & domestication
• Important Species characterized in Asia: Asian seabass, Hilsa, Indian
major carps, Tiger shrimp, White Shrimp, Pangasianodon hypopthalamus
• India implemented dedicated Network Program on Genetic Stock
identification
• 32 species wild populations are characterized
Characterizing Genetic Stocks

16. • Threat to Sustainability of Aquaculture
• Impact quality input – Seeds
• Genetic management of broodstock
• Genetic fitness
• Production performance
Vulnerable Groups
• Genetically improved
• Introduced with Low Founder Base
A concern as domestication rises
Genetic Erosion & Inbreeding

17. • Characterization of farmed populations using molecular &
biological tools & performance evaluation
• Quantitative assessment of hatcheries with respect to inbreeding
levels, loss of genetic variation, genetic contamination
• Sperm banking to assist planned germplasm exchange
• Elite germplasm in some farms, a possibility, will help in further
improvement of farmed population & deserve conservation
• Seed of authenticated origin & standard quality for aquaculture
• Development of Brood banks
Safeguarding Farmed Stocks from Genetic Erosion

18. Opportunity
• Continued genetic gain
• Gains made are permanent
• Gain transmitted to next generations
• Gains in a nucleus: multiplied in
millions of fish
• Key to improve production efficiency
• Overcome the limits of resources in
aquaculture intensification
• 8.2% aquaculture production is based on
systematic breeding program
• Characterization help breeding plans
Major Programs
Common carp 8
Rohu carp 1
Catla carp 1
Silver Barb 1
Nile Tilapia 20
Tilapia O. shiranus 1
Stripped Catfish 1
Catfish 1
Blunt Snout Bream 1
Freshwater Prawn 2
Selective Breeding

19. • Population Genomics: Transcriptome associated markers to study
population variation
• Combines genomic concepts & technologies with the population
genetics to understand evolution
• Technical ability to Improve stocks through quantitative trait
association linkage (QTL) mapping for marker assisted selection
(MAS) in genetic improvement programs
• De novo whole genome sequencing
• Genome wide selection for multiple traits
• Search targets: Mining of adaptive alleles
3rd Generation Sequencer
read >33000 bp; 99%
SMRT : Real Molecule Sequencing
ICAR-NBFGR
New Trends - Genome

20. Project Country Organism
1 10 K genome Singapore, China
& multi-country
2 1k Transcriptome China
3 Allele
Bioprospecting
India Catfish, Rohu, Tiger Shrimp
4 Genomic Seq
platform
India Rohu, Magur, Hilsa, Catla,
White shrimp
5 Genome Seq Japan Fugu, Medaka, Anguilla sp
6 Genome Seq South Korea Notothenia coriiceps
7 Transcriptomics Singapore Lates calcarifer
8 Genome Seq Bamgladesh Hilsa
Some Projects in the Region

21. Voucher
Specimen
Live Gene
Bank
Retrieve Diploid
Genome for
Species Recovery ;
Genomic Research
Embryonic &
Somatic Cells, Cell
Lines, Sperm Stored
at -196ºC;
as per Specific
Protocol
For Prioritized
Species With Known
Breeding Protocols
(Sperms/Embryo)
•Resource for future
research
AqGR Soverign Rights of the Nations
Reference for
Source of
DNA/Cell
Accessions.
To resolve
taxonomic
conflicts.
Useful for
Propagation
Assisted
Rehabilitation
Facilitate
Aquaculture
Diversification
Tissue Samples
Frozen at
-80ºC,
Ethanol preserved
at 4ºC
Fast mode of
Developing
Germplasm
Repository
Total DNA Isolates
Genomic Libraries
cDNA Libraries &
EST Clones.
Gene Constructs
Probes for Specific
Genes
Other Genomic
Materials
Collection of
Preserved
Specimens
Formalin/
Iso-propyl Alcohol
or as per protocol
Live Captive Brood
Stock
of Prioritized
Species in Farm
Represent Natural
Genetic Variation
Cryopreserved
Cells
Tissues DNA
Bank
•Source of DNA to
Retrieve Genetic
Information
Germplasm & Genomic Repository in the context of FGR

22. Thanks