400hd rox

Genomic DNA was extracted from the whole-body tissue of offspring and from the caudal fin of adults using a Chelex protocol20 (link)38 (link). The tissue samples obtained from 72 mothers, the putative fathers (n=60) and all of the offspring (n=532) were collected and stored in a freezer at −80 °C until used for analysis. We used five microsatellite loci to assign paternity to males within each of the 10 populations (see Table 5). PCR amplifications were performed using the GeneAmp PCR System 9700 Thermocycler (Applied Biosystems, CA, USA). The PCRs were performed with 7.64 μl BDH, 1 μl MgCl₂, 3 μl Taq buffer, 0.53 μl dNTPs, 0.38 μl primers (forward+reverse), 0.08 μl Taq DNA polymerase (Promega) and 2 μl DNA template. The cycling protocol included an initial denaturation step at 95 °C for 1 min, 30 cycles of 10 s denaturation at 95 °C, 30 s annealing (T listed in Table 5), 30 s extension at 72 °C and a final extension for 5 min at 72 °C. Amplified fragments were separated by electrophoresis on an ABI PRIMS DNA Analyzer 3100/3700 sequencer (ABI PRISM, Applied Biosystems), using 400 HD ROX (Perkin-Elmer, Applied Biosystems) as a size standard. PCR products were visualized using the Peak Scanner software ( www.appliedbiosystems.com) and paternity was assigned to offspring using CERVUS 3.0 (refs 39 (link), 40 (link)).

Thirty-five H. macrophylla accessions were used for the assessment of DNA markers for the double flower phenotype. Genotyping for J01 was performed as described above. Genotyping for S01 was performed by fragment analysis as follows. PCR amplification was performed in a 10-μL reaction mixture containing 5 μL of GoTaq Master Mix (Promega), 5 pmol FAM-labeled universal primer (5′- FAM-gctacggactgacctcggac -3′), 2.5 pmol forward primer with universal adapter sequence (5′- gctacggactgacctcggacCATCATTAATAGTGGTGACAG -3′), 5 pmol reverse primer, and 5 ng of template DNA. DNA was amplified in 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min; and a final extension of 5 min at 72°C. The amplified PCR products were separated and detected with a PRISM 3130xl Genetic Analyzer (Applied Biosystems, USA). The sizes of the amplified bands were scored against internal-standard DNA (400HD-ROX, Applied Biosystems, USA) with GeneMapper software (Applied Biosystems, USA).