Rainbow trout (Oncorhynchus mykiss
) are the most-widely cultivated cold freshwater fish in the world and are considered by many to be the "aquatic lab-rat". Interests in the utilization of rainbow trout as a model species for genome-related research activities focusing on carcinogenesis, toxicology, comparative immunology, disease ecology, physiology, transgenics, evolutionary genetics, and nutrition have been well documented [1
]. Coupling great interest in this species as a research model with the need for genetic improvement for aquaculture justifies the continued development of genome resources facilitating selective breeding.
Genome size estimates derived from molecular weight of DNA per cell for rainbow trout and other salmonids vary from 2.4 to 3.0 × 109
]. As with most salmonids, rainbow trout experienced a recent genome duplication event resulting in a semi-tetraploid state [4
]. Our physical mapping experience with BACs from the Swanson library has demonstrated that duplicated loci can be detected by DNA fingerprinting [5
]. Additionally, BACs that represent one of two duplicated loci were shown by fluorescent In-situ
hybridization (FISH) to distinctly hybridize to a specific chromosome pair [6
]. Therefore, it is likely that the vast majority of the duplicated loci contain enough sequence variation to allow correct assembly of a physical map using BAC DNA fingerprinting.
Current genomic resources available for rainbow trout research include multiple bacterial artificial chromosome (BAC) libraries [5
]; doubled haploid (DH) clonal lines [8
]; genetic maps [3
]; a large EST database [16
]; and DNA microarrays [18
Seven rainbow trout BAC libraries were constructed to date. Two libraries constructed in Japan [7
] contain average insert sizes of 58 kb and 110 kb, and provide haploid genome coverages of 6.7 fold and 5.3 fold, respectively. However, they have not been arrayed in plates and library screening tools are not available. One BAC library from the Swanson male homozygous line and one from the OSU female homozygous line were commercially constructed by Amplicon Express Inc. in 2002. Both libraries were prepared from partial digestions with HindIII. The OSU BAC library has 96,768 clones with an average insert size of 110 kb (4.5× coverage). The Swanson BAC library has 184,704 clones with an average insert size of 130 kb (10× coverage). HindIII BAC DNA fingerprinting for local physical mapping of 27 Type-I markers in the Swanson library demonstrated the library's utility for identifying duplicated loci and confirmed its 10× coverage [5
]. Both libraries have been used for genomic sequencing and genetic mapping of loci of interest [20
]. An additional 5× genome coverage Swanson DH YY male library (92,160 clones) was constructed at the Children's Hospital Oakland Research Institute (CHORI-220; http://bacpac.chori.org/library.php?id=405
) in 2005 using EcoRI partial digestion of genomic DNA with an estimated average insert size of 159 kb. An additional two new libraries were prepared by Amplicon Express in 2008. The two new 5× genome coverage Swanson DH YY male libraries (110,592 clones each) were prepared using BamHI and EcoRI partial genomic digestion to complement the 10X HindIII Swanson library and have estimated insert sizes of 140 kb. The four Swanson DH YY male libraries described above were prepared using genomic DNA from the same Swanson doubled-haploid clonal line that is propagated and maintained at the lab of Gary Thorgaard in Washington State University.
Two genetic maps with improved marker densities were recently developed for rainbow trout by INRA [12
] and the NCCCWA [15
]. The INRA map is based on a panel of two DH gynogenetic lines. It has more than 900 microsatellites over 31 linkage groups and a total length of 2,750 cM (average resolution of 3 cM). The NCCCWA map is based on a panel of five families that represent the starting genetic material of the NCCCWA selective breeding program. It has 1,124 microsatellite loci over 29 linkage groups and a total length of 2,927 cM (average resolution of 2.6 cM).
The rainbow trout haploid karyotype is composed of 52 chromosome arms, but chromosome numbers can vary among rainbow trout populations in concordance with their native geographic distribution [28
]. Therefore, anchoring the genetic linkage groups to the physical chromosome arms was a crucial task accomplished by Phillips et al. [6
] using BACs as FISH probes. The range of the haploid chromosome number (N) is between 29 and 32 [28
]. The karyotype of the Swanson DH line is composed of 2N = 58 [29
]. The offspring of "hybrids" between strains with different chromosome number are viable and they can be used for genetic mapping as two uni-armed (acrocentric) chromosomes from the parent with 2N = 60 will align with a di-armed (metacentric) chromosome from the parent with 2N = 58. A comparative cytogenetic map of the rainbow trout and Atlantic salmon using FISH with BACs that harbor Type-I markers and microsatellites is being developed in a coordinated effort [30
]. This cytogenetic map and the comparative genetic map of Danzmann et al. [31
] provide a frame-work for future high resolution trout-salmon comparative genome maps.
Qualitative/quantitative trait loci (QTL) mapping experiments in rainbow trout have been very successful because of their high fecundity, external fertilization, and ease of gamete handling and manipulation. Many QTL have been identified for production and life-history traits including resistance to the parasite C. shasta
], resistance to IHNV [33
] and to IPNV [35
], Killer cell-like activity [36
], upper thermal tolerance [37
], embryonic developmental rate [8
], spawning time [41
], confinement stress response [43
] and smoltification [44
]. The availability of a BAC physical map integrated with the genetic map will facilitate fine mapping of QTL, the selection of positional candidate genes and the incorporation of marker-assisted selection (MAS) into rainbow trout breeding programs. A major shortcoming of QTL studies is that they are limited to the variation present in a limited number of families and typically do not detect loci with small effect. This can be overcome by whole genome association studies and other approaches that capture effects of most QTL that contribute to the population-wide variation in a trait such as genomic selection. Recently we demonstrated the feasibility of low resolution LD association studies in rainbow trout [46
]. In the absence of whole genome sequence assembly, the robust integrated physical and genetic map that we aim to construct will provide better resolution than the current genetic maps for ordering of genetic markers and estimating physical distances between markers, thus facilitating whole genome association studies rainbow trout.
Several BAC-based physical maps were constructed in recent years for economically important aquaculture species including tilapia [47
], Atlantic salmon [2
] and catfish [48
]. Here we report the construction of the first physical map of the rainbow trout genome using a 10× genome coverage BAC library derived from the Swanson DH clonal line.