Biotechnology-Aquaculture Interface:

The Site of Maximum Impact Workshop

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Appendix
-Participants
-Steering Committee
-Program Committee

Workshop Report
-Preface
-Final Report

Markers for Genetic Selection of Desirable

Genetic Markers for Aquatic Domestication and Selective Breeding Programs

Leland Lai

Aquatic Stock Improvement Company (ASICo)

P.O. Box 5, Hawthorne, California 90251

Lelandlai@aquafauna.com

Abstract

Genetic markers have many applications in aquaculture breeding programs, including fingerprinting individual animals, relatedness tracking, determining genetic diversity, and assisting in selection of traits of economic importance. The hereditary information that defines living things is encoded in DNA, consisting of sub-units arranged in unique sequences. Within the genome of all organisms are segments where DNA sequences are repeated. These repeats are called "microsatellites" and are found in the same location along the genome of different animals of the same species, or even of closely related species. Microsatellites are found in all organisms and in bacteria. Most genes exist in a variety of forms, and each variant is referred to as an allele. The variability of many traits is due to variability in the alleles that control the trait. In essence, each animal can be fingerprinted when superior or desired performance under culture is determined. Molecular genetics have been used extensively in terrestrial agriculture, especially to improve crops and animals of high economic value. In contrast, the application of modern genetic technology to aquaculture is in its infancy. The US incurs billions of dollars in deficits from aquaculture product imports while trying to encourage high cost-based domestic production in competing lines. USDA can effect maximum impact in aquaculture production efficiency and cost structure improvements by shifting priority to the use of genetic biotechnology, moving the domestic industry towards better production stocks and/or to supply such technology to primary producers outside the US in the form of superior broodstock.

Keywords: microsatellites, genetic markers, polymorphic loci, genotypic breeding programs, Marker Assisted Selection (MAS), molecular tagging, fingerprinting, diversity tracking.

Introduction

Most agriculture sectors have not had sufficient funding to develop full genomic maps and for associating traits to the genes of economic importance. Genetic tools such as DNA microsatellites provide a relatively fast and cost effective way for generating breeding guidance by "marking" the trait loci that are important to the farmer. Microsatellite markers are already established for domestication and selective breeding programs throughout other sectors of terrestrial agriculture. In breeding, the use of markers has become very similar to using computer applications software. That is, while the general principles and equipment used in marker development are well known, the expertise of high throughput data extraction and guidance for breeding is a more closely held technology. Much of this expertise is currently in use in the private sector for competitive positioning, to effect changes for generating greater revenues, improving operating efficiencies, stabilizing the production and gaining a higher degree of process control over production.

Description of the Tool

DNA microsatellites are repetitive sequences of nucleotides that occur randomly throughout the genome of all organisms, including bacteria. In the last ten years, microsatellites have become the tool of choice for breeding applications and genetic fingerprinting due to their high information content. The highly polymorphic nature of microsatellites means they confer more information per unit assay and can be assayed more rapidly than any other marker system, thus reducing costs of the tool’s application. Microsatellites that are identified and associated with a trait become "markers" since they mark the presence of a gene responsible for the display of a trait and are co-inherited along with it.

What are Genetic Markers Used For?

Microsatellite markers are tools used for supporting breeding and domestication programs by: 1) Tracking diversity or establishing relatedness among and between individuals, families and populations; 2) Molecular or genetic tagging for strain identification; and, 3) Marker Assisted Selection for identifying and establishing associative links between markers and traits that may be economically important to the farmer (such as fast growth or survivability).

Why are Genetic Markers Important to Aquaculture?

The polymorphic information content in the form of allele frequency within a specific microsatellite location or locus allows us to distinguish organisms down to an individual level. Hence, when an aquatic organism displays superior trait performance, microsatellites identify whether these traits are phenotypic (probability of observation) or genotypic (molecular) in nature. The ability to genetically distinguish better performing animals allow identification of potential broodstock whose progeny may also display superior performance. There may also be gene combinations that account for phenotype that are different from the genetic combinations present in either of the parents. Finally, without molecular tools, breeders cannot accurately distinguish between environmental and genetic variances upon phenotypic expression. Genetically superior animals will out-perform the rest of the population even under poor environmental conditions. Microsatellite markers allow us to fingerprint animals for traits reflecting genotypic differences irrespective of the phenotype displayed and provide a molecular means of tracking the heritable elements. This guidance is important in the selection of broodstock since it improves the accuracy and quality of the breeding prediction for future generations. The better the prediction, the greater the magnitude and pace of genetic gain.

Impact on Aquaculture Industry

In the past 10 years, genetic markers have been used aggressively in agriculture for breeding guidance leading to genetic gains in almost every sector from chickens to the grains and malts used in beer. Nearly all terrestrial agriculture is conducted with plants and animal strains that are genetically selected for increased commercial performance and which show little resemblance to their wild ancestors (Knibb et.al., 1998). Aquaculture has so far remained almost untouched by the advances in applied breeding technology…aquaculture research in general and genetic improvement in particular have been hampered by short-term, scattered, and disjointed funding (Eknath et al., 1991). Application of such genetic tools in aquaculture is only a recent occurrence since there has been little perceived need or understanding of such technology. Consequently, aquaculture genetics today basically reflects the profiles of wild stocks in spite of estimated benefit to cost ratios from such genetic programs ranging from 5:1 to 50:1 (Gjerde, 1986). Along with genetic improvement should come the same gains in industrial efficiency and productivity, product quality, consistancy, availability and price reduction for consumers as has been obtained in all other agriculture sectors which have received these improvements through domestication. The long-term net benefits to aquaculture are growth and sustainability for the industry.

Some Practical Findings

ASICo has established genetic marker links related to "survivability" in L. vannamei when separately challenged with Taura Syndrome Virus (TSV) and with White Spot Syndrome Virus (WSSV), two viruses accounting for over US$1 billion/year in losses within the world shrimp industry.

  • Genetic diversity loss can be tracked in captive stocks. In less than twenty generations, Venezuelan captive stocks of L. vannamei lost more than half the diversity when compared to founding stocks from Panama, measured by the number of missing alleles in captive stocks.
  • TSV: Survivors from challenged animals off a single spawn were over 120 times more genetically diverse and heterozygous (contributory genetic components in loci are different from each parent), compared to the non-survivors which were predominantly homozygous (genetic components contributed by each parent are same and hence less diverse). Parent stock of these progeny were captive stocks from Venezuela known to have lost genetic diversity but not bred for any particular trait.
  • WSSV: There are no determinable differences in diversity to account for survivability performance. Ability to survive is most likely related to the combination of allele differences as markers have been identified in both survivors and non-survivors where frequencies of allele occurrences are statistically related to trait performance. All non-survivors tested PCR positive and all survivors tested PCR negative for WSSV (10,000 wild broodstock produced 200 million PL’s which were twice challenged in both pond and tank, producing 2,500 survivors or 1 survivor in every 80,000 animals tested).

Benefits

Genetic tools can benefit the U.S. industry and consumers in a number of ways even though domestic production may be relatively small. For example, US shrimp farmers produce a mere 1,500 mt per year worth some $10 million from 400+ hectares. While this accounts for less than 0.2% of the world’s aquaculture production tonnage, US demand for imported shrimp still exceeds $3 billion annually. Domestically, the farmer’s survival is highly dependent on niche marketing of higher margin fresh or live product, world production, U.S. market prices and annual cost of production. Competition on frozen product is marginal since domestic production must compete with 1.3 million hectares situated outside the U.S. with lower capital and operational cost per kilogram produced. Shrimp product value for human consumption is in the $5-10.00 per kilogram range. The equivalent value of broodstock easily exceeds $500.00 per kilogram. Phenotypically selected or disease-free stocks maintain even higher premiums and represent an industry in and of itself (broodstock for L. vannamei currently sell for $30-$50.00 each at 30-35 gram size, 25-30 count per kilogram). It is estimated that L. vannamei farms require 700,000 broodstock (males and females) to meet current post-larvae production for all of Latin America. Selected value of these stocks as a separate industry ranges from $42 million (at $15 per animal) to $140 million (at $50 per animal). In Thailand, the broodstock industry for P. monodon exceeds US$65 million with some 560,000 animals required to support its post-larvae production. Shouldn’t the U.S. be producing genetically improved broodstock as a technology exporter rather than competing with lower cost systems producing consumption oriented products?

Secondly, genetic gains from improved stocks reduce the cost of the product as the industry grows due to increased supplies. In other agricultural sectors, nominal increases in growth or survival of ten percent per year is realistic and readily achievable. The genetic gain in salmon has exceeded this rate for the last ten years. A ten percent decrease in import value resulting from lower prices for shrimp far exceeds the cost of funding genetic improvement programs targeting efficiency of production, even if the primary production is not performed in the United States.

Finally, genetic improvement provides the capability of culturing a better quality animal in less time, with greater survival, and at less cost than animals removed from the wild. The immediate benefit to the farmer is improved profits, realized as animals grow faster, more efficiently, having better survival and qualities the consumer is willing to pay more for.

Conclusions

The value to the U.S. aquaculture industry of using genetic markers is in application of the tool for improving aquatic stocks and not necessarily in the development of the tools themselves. For the most part, the tools and resulting breeding guidance from such technology already exist and only require competent deployment. This is already being done in most all sectors of general agriculture. Faster growth and better survivability will be the most important targets for genetic improvement in the next decade. Funding to achieve genetic improvements are directly related to reductions in import value for aquaculture products and for stimulating evolution of a domestic industry focused on use and export of genetically improved broodstock through selective breeding.

Genetic improvements in aquaculture increase the production of animal based protein from a sector that is sustainable. For many aquatic species Feed Conversion Ratios under 2:1 have already been attained. Currently, the price of aquacultured product is still higher than most other proteins produced from terrestrial sectors of agriculture. Further increases of worldwide production from genetic biotechnology will reduce consumer prices for these aquatic protein sources. As seen in salmon, increased efficiencies in production and supplies brought about by selective breeding technologies have created both a worldwide industry and lower import consumer prices for better quality year-around fresh product.

Future aquaculture production, however, will have to be achieved through intensification of management and technology wherein output is a consequence of production efficiency per unit area rather than increases in the area of production. It is estimated that total aquaculture production by 2025 will exceed 60 million metric tons, up from 15 million tons in 1990 (Hempel, 1993). There are no chemicals, physical methods, or nutrient additives throughout all agriculture sectors which can double crop production from the highest levels of output efficiency experienced in the 20th Century. This industry is also under heavy scrutiny for its impact upon the environment. Environmental conditions and selection pressures for survival, reproduction, and growth tend to differ between natural and captive culture conditions. There are no improvements for production efficiencies that can be expected from the continued use of wild broodstock. Looking for a few good genes?

Recommendations

Short-term (1-2 years):

  1. USDA should analyze the production factors of domestic aquaculture for cost effectiveness in long-term supply of finished product to the US consumer market. This should include the determination of growth in consumer demand for aquaculture products and whether the domestic industry can realistically have significance on fulfilling that demand. If not, can USDA influence profitable leadership using genetic biotechnology in aquaculture either as an industry in and of itself, or to reduce the ever-increasing deficit spending for such imported products. This will determine long-term viability for the industry and whether shear growth or intensification can provide solid ground for agency support of the domestic production sector.
  2. USDA should determine the economic benefits to be expected from use of genetic biotechnology in the form of tools commonly used in other agriculture sectors and the relative rate of genetic gains achieved. This will provide a basis of how funding is invested in such technologies and what returns can be expected. USDA should further determine whether such genetic biotechnology already exist in public or private sectors that can be immediately applied to domestic aquaculture or to re-define the economic targets for the industry.
  3. Agencies and industry groups should establish guidelines for breeding protocols that preserve long-term diversity for cultured aquatic species and to establish baseline genetic profiles for future references. This prevents bottlenecks as a result of captive breeding and provides reference points for managing and tracking captive stocks from those of natural fisheries. They should support the immediate development of a database profiling the relative diversity of the top ten species being commercially cultured to monitor long-term effects from traditional phenotypic breeding programs.

Mid-term (3-6 years):

6. USDA should provide support for guidance in breeding and domestication programs for the private sector through existing public institutions and extensions.

  1. USDA should support molecular programs (public or private) which complement traditional breeding programs in accelerated growth and better survival for the top 10 consumer oriented aquatic species being cultured in the U.S.

Long-term (7-10 years):

8) USDA should establish on-going databases to track cultured species as a result of selective breeding from those involving transgenic developments. This allows long-term isolation of captive stocks from gene contamination due to other forms of genetic biotechnology.

References

Eknath, A.E., Bentson, H.B., Gjerde, B., Tayamen, M.M., Abella, T.A., Gjedrem, T. et al. 1991.

Approaches to national fish breeding programs: Pointers from a tilapia pilot study. NAGA (the ICLARM Quarterly), 14: 10-12.

Gjerde, B., 1986 Growth and reproduction in fish and shellfish. Aquaculture, 57: 51-57.

Knibb, W.G., Gorshkova and S. Gorshkov. 1998. Genetic Improvement of Cultured Marine Fish: Case Studies. In: Tropical Mariculture, eds. S.S. De Silva and S.S. De Silva, pp. 111-137, Academic Press