Project description:Animals and samplings: Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described. Total RNA extraction and Reverse Transcription: Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer. Primers design: Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification. Real-time RT-PCR Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard. Keywords: ordered

Project description:The role of chromosomal inversions in adaptation and speciation is controversial. Historically, inversions were thought to contribute to these processes either by directly causing hybrid sterility or by facilitating the maintenance of co-adapted gene complexes. Because inversions suppress recombination when heterozygous, a recently proposed local adaptation mechanism predicts that they will spread if they capture alleles at multiple loci involved in divergent adaptation to contrasting environments. Many empirical studies have found inversion polymorphisms linked to putatively adaptive phenotypes or distributed along environmental clines. However, direct involvement of an inversion in local adaptation and consequent ecological reproductive isolation has not to our knowledge been demonstrated in nature. In this study, we discovered that a chromosomal inversion polymorphism is geographically widespread, and we test the extent to which it contributes to adaptation and reproductive isolation under natural field conditions. Replicated crosses between the prezygotically reproductively isolated annual and perennial ecotypes of the yellow monkeyflower, Mimulus guttatus, revealed that alternative chromosomal inversion arrangements are associated with life-history divergence over thousands of kilometers across North America. The inversion polymorphism affected adaptive flowering time divergence and other morphological traits in all replicated crosses between four pairs of annual and perennial populations. To determine if the inversion contributes to adaptation and reproductive isolation in natural populations, we conducted a novel reciprocal transplant experiment involving outbred lines, where alternative arrangements of the inversion were reciprocally introgressed into the genetic backgrounds of each ecotype. Our results demonstrate for the first time in nature the contribution of an inversion to adaptation, an annual/perennial life-history shift, and multiple reproductive isolating barriers. These results are consistent with the local adaptation mechanism being responsible for the distribution of the two inversion arrangements across the geographic range of M. guttatus and that locally adaptive inversion effects contribute directly to reproductive isolation. Such a mechanism may be partially responsible for the observation that closely related species often differ by multiple chromosomal rearrangements.

Project description:Animals and samplings: Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described. Total RNA extraction and Reverse Transcription: Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer. Primers design: Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification. Real-time RT-PCR Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.

Dataset Information

A large-scale chromosomal inversion is not associated with life history development in rainbow trout from Southeast Alaska.

Publications

A large-scale chromosomal inversion is not associated with life history development in rainbow trout from Southeast Alaska.

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