Application of Genomics to Genetic Improvement of Channel Catfish

Geoffrey C. Waldbieser and William R. Wolters

USDA-ARS Catfish Genetics Research Unit

Thad Cochran National Warmwater Aquaculture Center

P.O. Box 38

Stoneville, Mississippi 38776

gwaldbieser@ars.usda.gov; bwolters@ars.usda.gov

Abstract

Genetic improvement of channel catfish is essential for long-term viability of the U.S. catfish industry. High levels of phenotypic variation due to lack of sustained selection and large family sizes permit a high selection intensity in this species. Breeders must improve assays to quantitate performance and estimate genetic contribution to important traits such as growth rate and feed conversion efficiency, disease resistance, carcass yield and quality, and reproductive performance. Utilization of genomics tools will increase the efficiency of selective breeding programs. Microsatellite markers are abundant in the catfish genome and demonstrate high levels of polymorphism in catfish populations. These markers have been used for family and strain identification, and to produce a first generation genetic linkage map. Effective use of genomics tools in catfish selective breeding programs will require a marker-dense genetic linkage map and a physical map useful for identification of genes controlling important traits. Bioinformatics capacity must be enhanced to assist marker development and mapping, identification of quantitative trait loci, identification of superior germplasm and development of breeding strategies using genomics technologies.

Keywords Catfish, Genetics, Genomics, Breeding, Marker assisted selection

Introduction

Production of channel catfish for human consumption is now the largest sector (46%) of commercial finfish production in the United States (FAO 1997), utilizing 78,000 ha of production ponds (USDA 2000). The U.S. catfish industry now processes 600 million pounds annually, an increase of 36% since 1994 and 75% since 1989 (USDA 2000). Channel catfish account for almost all of commercial production while a closely related species, blue catfish, is also cultured. Low feed costs, consistent prices paid to producers by processors, successful marketing, and effective industry infrastructure have resulted in steady growth and sustained profitability of the catfish industry. Research on reproduction, nutrition, and pond management has also supported industry growth, but genetic improvement programs leading to improved catfish lines are only beginning to be applied.

Catfish require unique rearing systems compared with traditionally species like cattle, swine, and poultry, and are cultured either in ponds or tanks. Interspecific hybridization is possible between channel and blue catfish via manual spawning, but hybrids are not yet efficiently produced for large scale commercial application. Genome manipulations such as gynogenesis, triploidy, sex reversal, and transgenesis have been accomplished in channel catfish. However, these techniques are performed at low efficiency and used only as specific breeding aids. The greatest gains will probably be made using traditional selective breeding strategies incorporating molecular genetic technologies.

The lack of sustained genetic selection in channel catfish has effectively maintained a high level of phenotypic variation in commercial and research populations. Catfish breeders also have access to feral broodstock to increase the genetic variation in populations, if necessary. Large numbers of offspring (10,000-20,000 full sibs) permit high selection intensity. Selection of high-level performers, along with moderate heritability estimates for some traits (Tave 1986), will permit breeders to realize greater genetic gains in early generations of select populations. However, catfish in ponds are more difficult to observe and measure than traditional animal species, and methods must be improved for the efficient identification of families and strains because of the similar physical characteristics of catfish populations.

Catfish breeders can take advantage of some technologies used in mammalian genetic improvement programs, although information on the physiological processes involved in important production traits in fish, such as growth rate, disease resistance, spawning success, and carcass composition is currently limited. Furthermore, breeders must develop better assays to quantify performance for these traits (accurately measure phenotypes) and characterize sources of genetic variation and trait correlations.

Traditional selective breeding programs will serve as the foundation for utilizing catfish quantitative genetic variation for agricultural production (Bondari 1984; Dunham and Smitherman 1987; Wolters and Johnson 1995). The selective breeding program at Auburn University has produced selected Kansas and Auburn lines, and channel catfish x blue catfish hybrids, and the MSU2 line was released by Mississippi State University. The USDA-ARS Catfish Genetics Research Unit was established by Congress to provide long-term genetic improvement of catfish germplasm for use in commercial production, and has developed and released the NWAC103 line. Genomics research will provide tools to increase the efficiency of the existing selective breeding programs.

Catfish Genomics

The goal of an agricultural genomics program is to provide useful molecular tools for the identification of genes that control economically important traits. Molecular markers that identify beneficial alleles within these genes can be used to increase the efficiency of broodstock selection. Traditional selection by performance is difficult for many traits. A trait may be sex-limited, such as identifying males that will pass on beneficial traits for their daughters’ spawning success. Exposure of broodstock to test disease resistance depends on the ability to perform controlled experiments with pathogens, often difficult for catfish diseases. In some cases performance testing is lethal, such as measurement of carcass quality. A rapid molecular test, for example a DNA test or protein assay, can be used to identify broodstock that have inherited useful genetic variants without the need for performance testing. The tools needed to perform these tests in catfish include molecular DNA markers, genetic linkage and physical maps, reference and resource families, and bioinformatics capacity.

Many types of molecular markers have been developed in catfish. Low levels of polymorphism and lack of correlation with phenotypes negates the use of isozyme markers in catfish selective breeding (Dunham and Smitherman, 1984; Hallerman et al., 1986; Carmichael et al., 1992). Anonymous DNA markers such as amplified fragment length polymorphisms and random amplified polymorphic DNA markers were developed in catfish (Liu et al. 1998a, 1998b). These are dominant markers that reveal genomic variation between blue and channel catfish, but low levels of variation within channel catfish limits their use in applied genomics. The most useful molecular markers for catfish genome analysis are microsatellites, which demonstrate high levels of allelic polymorphism within and between strains and families. To date, researchers at the USDA-ARS Catfish Genetics Research Unit and the Auburn University Fish Molecular Genetics and Biotechnology Laboratory have published 390 catfish microsatellite markers (Waldbieser and Bosworth, 1997; Liu et al., 1999a,b; Tan et al., 1999; Waldbieser et al., 2001). Both groups are sequencing cDNA clones from several tissues in order to develop markers for catfish genes, and the Auburn researchers have placed more than 3500 catfish sequences in the GenBank EST database.

Both groups have developed reference families for genetic linkage mapping; ARS researchers have focused on channel catfish families and Auburn researchers have focused on blue x channel hybrid catfish families. An initial genetic linkage map has been produced from two families of NWAC 103 x Norris strain crosses to maximize the number of informative meioses in the offspring. This map consists of 263 microsatellite markers arrayed in 32 linkage groups (Waldbieser et al., 2001), providing an average intermarker distance of 9 centimorgans (9% recombination between markers). As more markers are developed and placed on the map there will be 29 linkage groups, one for each chromosome. Only 8% of the markers came from sequences encoding genes, the rest were developed from non-coding genomic DNA unique to catfish. Many catfish cDNA sequences contain short tandem repeats in the 5’ or 3’ untranslated region, therefore some cDNA libraries have now been enriched for clones that contain short tandem repeats in order to develop markers for genes (Liu et al., 1999; Nonneman et al. 2001). Increasing the marker density on the linkage map with genes that are conserved between species will allow researchers to compare catfish with "map rich" species, especially zebrafish (Gates et al., 1999; Shimoda et al., 1999; Woods et al., 2000).

Efforts are now under way to produce a physical map of the catfish genome. A cooperative effort between ARS and the University of Mississippi Medical Center has led to the development of a large insert BAC library for channel catfish. This library has been arrayed into microtiter plates with enough clones for 7.5 fold coverage of the catfish genome. Cooperative research between the ARS and Auburn laboratories will identify BAC clones that contain genes placed on the linkage map. One way of linking the genetic map and the physical map is to localize BAC clones to chromosomes via in situ hybridization. Using DNA fingerprinting techniques and bioinformatics, the BAC clones are linked to form a physical map of the genome. This powerful tool will assist breeders in the identification of DNA sequence variation influencing performance for economically important traits.

Genomics has already been applied to genetic improvement in the catfish industry. Microsatellite markers have been used to determine parentage of spawns collected from communal ponds (Waldbieser and Wolters, 1999). This marker technology allows breeders to use large half- and full-sib families from natural spawning conditions to estimate genetic components of phenotypic variation. The markers have also been used to provide a genetic fingerprint of the NWAC 103 catfish line. Coincident with the release of this catfish line is the development of a strain certification program by the catfish industry. Genetic markers will help producers maintain the genetic integrity of the NWAC 103 line, and other lines of catfish released in the future. (MAFES 1996; MSIA, 2000).

Conclusions and Recommendations

Channel catfish have many characteristics that are amenable to genomics research, such as small genome size (about 1/3 the size of mammalian genomes) and an abundance of microsatellite loci in gene-encoding regions. Large family sizes in channel catfish will increase the power to order closely linked markers and increase the power of statistical methods used to detect quantitative trait loci (QTL) controlling economically important traits. Catfish genomics research should be directed within the goals of selective breeding programs. Molecular markers and a genetic linkage map are currently available. The map will need ~1000 evenly distributed markers to be efficiently used for localization of genomic regions controlling important traits. The physical map must also be developed. Comparative mapping with zebrafish, and other species with well-developed maps, will be useful for identification of candidate genes to the extent that gene location and order are conserved between species. Bioinformatics capacity will need to be developed to efficiently handle the large data sets involved in marker development, linkage analysis, and QTL analysis.

Short term goals include increasing the number of markers on the genetic linkage map to improve map density and genomic coverage, development of a comparative genetic map with zebrafish, development of QTL resource populations, and improvement of bioinformatics capacity. Microsatellite alleles within known (candidate) genes can be tested for association with performance. Molecular markers should be used for family and individual identification to assist traditional breeding programs and strain identification. Also, assays for measurement of quantitative traits such as reproductive performance, disease resistance and carcass quality need to be refined.

Mid-term goals include QTL scans to identify regions of the genome involved in important traits. Regions must then be "fine mapped" with more markers to identify candidate genes, and development of a genomic BAC clone map will assist this effort. Informatics methods should be developed for efficient identification of superior broodstock and efficient use of marker-assisted selection. Marker-assisted identification schemes should be improved to increase the efficiency of detecting catfish lines. Transgenesis in model species will assist breeders in evaluating catfish gene constructs and the potential use of this technology.

Long-term goals include the determination of the complete DNA sequence of the catfish genome. Molecular markers should be used from QTL studies to select superior broodstock and assist breeders in introgression of beneficial alleles from other Ictalurid species.

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