BACKGROUND INFORMATION REGARDING RAINBOW TROUT GENETIC MAP DATASET

Segregation analysis was performed using 76 doubled haploid (DH) rainbow trout produced by androgenesis from a hybrid between the 'OSU' and 'Arlee' androgenetically-derived homozygous lines. Four hundred and seventy-seven markers segregated into 31 major linkage groups and 10 small groups (<4 markers/group). The DH rainbow trout lines and linkage map present a foundation for further genomic studies.

Production of doubled haploid rainbow trout: Homozygous parental lines and doubled haploid rainbow trout were produced by androgenesis. Successful androgenesis results in a diploid organism that contains two sets of paternal chromosomes. Androgenesis was accomplished by irradiating rainbow trout eggs with 40,000 rads of gamma radiation from a Cobalt-60 source, destroying the maternal nuclear genome. The eggs were then fertilized with normal sperm from a chosen male to initiate development of a haploid zygote. Diploidy was restored by applying a heat shock of 31.5ƒ C for 5 minutes at 210 minutes after fertilization. Male rainbow trout are the heterogametic sex, so both male (YY) and female (XX) homozygote individuals were produced in the first generation of androgenesis. The homozygous parental lines, which were clones of the original first generation homozygote, were produced by androgenesis or gynogenesis (with polar body retention) from the gametes of the first generation homozygous males or females, respectively. The parental lines were confirmed homozygous by DNA fingerprinting .

DH fish were produced by androgenesis using sperm from an F1 individual (OSU x ARL) from a cross of OSU (XX) and ARL (YY) parentals. Doubled haploid fish were grown in recirculating systems until they reached 4-6 inches, then tagged with passive integrated transponder (PIT) tags which allowed each fish to be individually identified.

Molecular Markers:
Segregation of molecular markers was analyzed in 76 DH individuals. AFLP and multilocus DNA fingerprinting were used to produce the majority of the 477 molecular markers analyzed. Other markers included single locus microsatellites and randomly amplified polymorphic DNA (RAPDs).

AFLP markers were named so the primer combination that produced the marker and the resulting band size could be identified from the locus name. The first three letters represented the +3 nucleotides for the EcoRI primer, the second three letters represented the +3 nucleotides of the MseI primer, the number represented the size in base pairs (bp) of the detected band and the last letter represented the parental line that the band was detected in (o or a) or if it was codominantly inherited (c).

The VNTR oligo probes included the simple repeats ATCC, ATC, ATAG, CTT, GCT and CGC. Minisatellite probes included Jeffreys 33.6 , M13 and Per . SINE loci were detected using oligonucleotide probes complementary to the 5' and 3' ends of the HpaI element and a probe complementary to the 5' end of the FokI element . Three restriction enzymes were used for the Southern blot analysis; HaeIII (h), RsaI (r) and DpnII (d). Fingerprint markers were named so that the probe, enzyme, band size and parental line that the band was detected in could be determined. For example, the name ATCCh8.6o designated a marker that was detected by the probe ATCC using the restriction enzyme HaeIII and produced a band that was 8.6 kilobases (kb) in size and detected in the OSU parent.

Five single-locus microsatellite primer pairs were analyzed; FGT-1, oneu 2 and oneu 6, and MS-73 and MS-35.

RAPD Primer sources were either from Operon, Inc. (CS) or University of British Columbia (UBC). The four RAPD primers that detected polymophisms and their sequences were CS32 (CCCACGGATC), CS40 (GACTGCTCGG), CS45 (CACGTCGGAG) and UBC516 (AGCGCCGACG), which detected two polymorphisms. The name used on the map contains the source of the primer (CS or UBC), the molecular weight of the polymorphic band and the parental origin of the band (a, o or c).

Rainbow trout linkage map: Linkage analysis using 477 markers (476 molecular markers and sex) produced a genetic map comprising 41 linkage groups and covering a distance of 1997.5 cM. The majority of the markers segregated into 31 large linkage groups (greater than 6 markers/group) with the additional markers segregating into 1 small group of 3 markers and 9 marker pairs. There were 9 markers that remained unlinked following the analysis.

Additional marker terminology:

The markers have been sorted by linkage group they have been assigned to. The alleles found in individuals are designated as A if the allele from the Arlee parent was present, and B is the allele from the OSU parent was present. Markers in the large groups are indicated by number and markers in the small groups have letters designating which markers they co-segregate with. Unlinked markers have a u and markers that were not analyzed for technical reasons have an x. Markers followed by symbols demonstrated ratios that significantly deviated from expected Mendelian ratios towards either the male allele (a-*) or the female allele (o-^).

Dataset for rainbow trout genetic map produced using doubled haploid analysis,
(Large file size ~2MB, slow download.)

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