Sixteen embryos were randomly selected from each capsule for DNA extraction which was performed according to the following protocol: each embryo was incubated for 3 h at 56°C with 20 µl of lysis buffer (10 mM Tris-HCl PH8.3, 50 mM KCl, 0.5% Tween-20, 500 µg/ml proteinase K), followed by 15 min at 95°C [35] (link). Samples were then centrifuged at 3000 rpm for 2 min to pellet cellular debris.
Genotyping of microsatellites was conducted using fluorescently-labeled primers and an automated ABI3730 XL DNA sequencer. Five microsatellite loci (TB19, QR43, R12, R13, RV11), were amplified following the PCR protocol described in [36] (link). PCR products were electrophoresed on an ABI3730 XL DNA sequencer with the LIZ-500 size standard (Applied Biosystems). Data were analyzed using GeneMarker v2.2 (SoftGenetics, State College, PA, USA). Probably because of low DNA quality, two of the loci (R12 and RV11) were not genotyped successfully in some families (Locus R12 for Family 2, 6, 9 and 10, Locus Rv11 for Family 1, 3, 4 and 15). These loci were excluded for the subsequent analyses of parentage for these families.
Genotyping of microsatellites was conducted using fluorescently-labeled primers and an automated ABI3730 XL DNA sequencer. Five microsatellite loci (TB19, QR43, R12, R13, RV11), were amplified following the PCR protocol described in [36] (link). PCR products were electrophoresed on an ABI3730 XL DNA sequencer with the LIZ-500 size standard (Applied Biosystems). Data were analyzed using GeneMarker v2.2 (SoftGenetics, State College, PA, USA). Probably because of low DNA quality, two of the loci (R12 and RV11) were not genotyped successfully in some families (Locus R12 for Family 2, 6, 9 and 10, Locus Rv11 for Family 1, 3, 4 and 15). These loci were excluded for the subsequent analyses of parentage for these families.
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