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• W1937514770 abstract "We are reporting, for the first time in livestock, the development of an allelic ladder for the standardization of allele calls by STR copy number2. Multiplex PCR composed of eleven microsatellite markers plus two gender markers (Table S1) was established by our group. Initially, we collected samples from individuals that were homozygous for the 119 alleles that appeared in the accumulated Korean cattle (Hanwoo) genotype. After each allele was independently amplified by PCR, the amplicon was cloned into the pCR®2.1-TOPO vector (Invitrogen, USA). The cloned amplicons were sequenced using the M13 forward and reverse primers. The clones that contained a correct insert, no PCR error, and the expected STR copy number, were selected. Plasmid DNA from 119 selected positive clones were extracted and subjected to PCR amplification using the STR primer set that corresponded to each insert. The PCR products were then mixed in a tube in which each allele should be present in a similar amount. As shown in Fig. S1, the 119 mixed alleles were clearly resolved by an injection, followed by capillary electrophoresis on an ABI3130xl Genetic Analyzer (PE Applied Biosystems, USA). The allelic ladder resolved 96 times. We collected DNA sizing data for each allele and used it for defining a fixed bin. The fixed bin was defined by the minimum and maximum sizing values from 96 runs3. In Fig. 1a, 2000 data points of ETH10 from 1000 Hanwoo individuals were plotted and the linearity was estimated by allele number. In Fig. 1b, data points within a fixed bin of allele 22 are shown. There was a good linearity (R2 = 0.9998) and perfect inclusion of the data points within the fixed allele bin (no error), which indicated that the DNA size-to-STR copy number calibration was accurate. In Fig. 1c, data points within the ±3 modified standard deviation (SD) are illustrated. A total of 1998 data points of 2000 (99.9%) were located within the ±3 modified SD, which indicated that the calibration was very precise. Similar levels of accuracy and precision were maintained in the calibrations of the other 11 STR loci (Table S2). Accuracy (a and b) and precision (c) of the ETH10 allelic ladder and genotyping data. In (a), 2000 data points from 1000 heads of Hanwoo were plotted by each allele defined by STR copy number (x-axis) and DNA size (y-axis). The upper and lower limit of each fixed allele bin was marked together with the corresponding data group. The correlation coefficient was 0.9998, indicating a very good linearity of the allele groups spaced by 2-bp intervals. In (b), allele 22 was shown in detail. In (c), the data points were distributed within the ±3 modified standard deviation and ±0.5 bp. Probability of identity (PI) and probability of identity of random half-sibs (PIhalf-sibs) estimated using the api-calc version 1.0 software1 were 6.00 × 10−14 and 1.13 × 10−10, respectively. As a result, there is little possibility that an identical individual will appear within the entire population of Hanwoo (numbering 1.67 × 107 individuals), assuming that the entire Hanwoo population is the enormous half-sib group formed by the absolute genetic contribution of a few nuclear bulls. A nationwide cattle traceability law using ear tags with individual ID numbers implemented by the Korean Ministry of Food, Agriculture, Forestry and Fisheries has been effective since 2009. If it becomes necessary to supplement and confirm the traceability system, traceability-related legislation could enact a provision for applying a DNA test using the STR makers and the calibration method developed in this study. The allelic ladder and calibration method developed in this study may contribute to the establishment of a better nationwide standard of DNA testing for tracing Hanwoo. Acknowledgements: This work was supported by grant no. 20070501034006 from the BioGreen 21 Program, Rural Development Administration, Korea. C. K. Yoo was supported by a scholarship supported by a grant from the BK 21 Program, the Ministry of Education, Science and Technology, Korea. Figure S1 Electrophoregram resolving the allelic ladder composed of 117 alleles from 11 bovine STR markers by an injection, followed by capillary electrophoresis. Table S1 Information on the 11 STR and 2 gender markers used in this study. Table S2 Precision data for the STR markers used in this study. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W1937514770 date "2010-12-01" @default.
• W1937514770 modified "2023-09-23" @default.
• W1937514770 title "An allelic ladder for eleven short tandem repeat loci for use in the cattle identity test" @default.
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• W1937514770 doi "https://doi.org/10.1111/j.1365-2052.2010.02142.x" @default.
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