# Patent

## Title
Method of parentage testing in North American bison, Bison bison and domestic cattle using microsatellite loci

## Abstract

A method for determining parentage of North American bison or domestic cattle offspring. In the method an offspring is tested to determine the alleles present in that offspring for selected loci. A pool of potential parents is also tested to determine which alleles of these loci are present in each parent. The likelihood that any potential parent is the parent of the offspring may then be determined by looking for the presence or absence of an allele in the offspring that is present in the parent or by some comparable method. The method may also be used to exclude all potential parents in a pool. Sixteen loci which may be used in such an analysis are BM1225, BM1706, BM17132, BM1905, BM2113, BM4440, BM720, BMS1862, BMS410, BMS510, BMS527, RM372, BMS1172, BMS2639, BM3628 and BMS2325. The method used to select these loci is also disclosed.

## Background
This application claims priority from a provisional application filed on Jan. 11, 2002, having Ser. No. 60/347,792, which is incorporated herein by reference.


This invention was made with Government support under ‘Validation of 15 Microsatellites for Parentage Testing is North America’, NSF Grant No. DEB-9622126, ‘Identification of Domestic Cattle Hybrids in Wild Cattle’, NSF Grant No. DEB-9622126, and ‘Nuclear Gene Marker for Cattle’, NSF Grant No. DEB-9622126 awarded by the National Science Foundation. The Government has certain rights in this invention.


BACKGROUND OF THE INVENTION
The present invention relates to a method of determining parentage in bison and domestic cattle using distinct microsatellite loci.
Bison once numbered in the millions in North America but due to the population bottleneck experienced in the late 1800's, bison numbers were reduced to no more than 300 individuals by 1880 (Coder 1975; Dary 1989). Almost all of the bison alive today can be traced back to five populations that were used to repopulate most of the extant public and private herds (Coder 1975). Current semi-wild bison populations are fragmented among public parks and sanctuaries throughout the United States and Canada. However, the vast majority of bison today reside on private ranches where they are raised for meat production. Recently, Mommens et al. (1998) demonstrated that bovine microsatellites are better suited for parentage testing in bison than conventional blood typing due to a greater degree of variation. However, their sample was limited to a single herd located in Belgium, which probably does not represent the actual genetic variation found in bison in North America.
Currently, parentage testing in domestic animals is based on exclusionary techniques using genetic markers. An offspring is tested assuming one known parent and one or a limited number of candidate parents. If only one candidate parent is left non-excluded, that parent is assigned parentage to the offspring. Although one non-excluded parent may be the true parent, there exists the possibility that other non-excluded candidate parents exist in the population but were not considered. A likelihood based testing procedure is more appropriate for situations in which there are many candidate parents and obtaining a known parent is difficult. Using likelihood based procedures, all potential parents are considered as candidates and there is no need to identify a known parent prior to testing.
SUMMARY OF THE INVENTION
The purpose of this study was to characterize, standardize and provide validation for a set of highly polymorphic microsatellites for use in routine parentage testing in North American Bison and domestic cattle.
Fifteen bovine microsatellites were evaluated for use in parentage testing in 725 bison from 14 public populations, 178 bison from two private ranches and 107 domestic cattle from 5 different breeds. The number of alleles per locus ranged from 5 to 16 in bison and from 5 to 13 in cattle. On average, expected heterozygosity, polymorphism information content (PIC) and probability of exclusion values were slightly lower in bison than in cattle. A core set of 12 loci was further refined to produce a set of multiplexed markers suitable for routine parentage testing. Assuming one known parent, the core set of markers provides exclusion probabilities in bison of 0.9955 and in cattle of 0.9995 averaged across all populations or breeds tested. Tests of Hardy-Weinberg and linkage equilibrium showed only minor deviations. This core set of 12 loci represent a powerful and efficient method for determining parentage in North American bison and domestic cattle.
Further tests have also indicated that two additional loci of the 15 originally identified, BMS1172 and BMS2639, may also be suitable for parentage testing. Two other loci, BM3628 and BMS2325, have subsequently been identified as suitable loci through testing in the two private bison herds.
The above summary provides a general outline of the invention. For a better understanding of the invention and its advantages, reference may be made to the following description of exemplary embodiments.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes a method of determining parentage in bison or domestic cattle using microsatellite loci. These loci were selected as set forth below and may be used in any type of parentage testing in which allelic variation is used to determine the likelihood of parentage. Preferred methods of detecting the specific alleles in the offspring and potential parents are described below, but alternative methods may also be employed. When used with the methods described below, the indicated levels of certainty are achieved. Alternative methods may result in differences of certainty if the methods change the probability that allele assignments are correct. One skilled in the art will be able to select alternative methods of detecting specific alleles which result in a desired level of overall parentage certainly.
In a preferred embodiment, 12 loci, BM1225, BM1706, BM17132, BM1905, BM2113, BM4440, BM720, BMS1862, BMS410, BMS510, BMS527, RM372, are used. However, one skilled in the art will understand that paternity may be determined using only a subset of these loci. The full set or any subset may also be used with any other loci useful in determining parentage. The four additional loci later identified as acceptable for parentage testing, BMS1172, BMS2639, BM3628 and BMS2325, may also be used in testing. Based upon known or readily determinable allele types and frequencies in a given population for each loci, a set of loci comprising the above loci and/or any other known loci may be selected for parentage testing. In general, the more loci selected, the greater the certainty of any parentage assignment will be. However, the invention by no means requires the use of all of the 12 loci originally identified, not does it require that a total of 12 loci be used in every embodiment. In a preferred embodiment, at least two loci are selected from the 16 identified above.
The goal of the study leading to the present invention was to characterize, standardize and provide validation for a set of polymorphic microsatellites for use in routine parentage testing in North American bison and domestic cattle. Table 3 demonstrates that the exclusion probabilities found in bison and cattle for these loci are comparable to other loci previously described (Glowatzki-Mullis et al. 1995; Peelman et al. 1998; Heyen et al. 1997; Mommens et al. 1998). Additionally, in cattle the core set of markers produces similar exclusion probabilities to the commercially available StockMarksTM kit (PE Biosystems). However, in bison the core set of markers offer higher exclusion probabilities than either the StockMarksTM kit or the ISAG approved set of markers (Mommens et al. 1998).
In order to validate the use of these markers for parentage testing in bison, the guidelines set forth for selecting loci for human parentage testing were followed (Parentage Testing Committee, American Association of Blood Banks, 1997). A total of 121 offspring were tested from two separate private populations. In every case the loci exhibited normal co-dominant Mendelian inheritance with no evidence of null alleles or mutaions. Similar validation studies were conducted using the BM3628 and BMS2325 loci similar results.
The development of an allelic ladder, which is used for each genotyping run, fulfills the requirement of a known DNA control and makes it possible to directly compare samples that are run at different points in time or even on different machines. In the case of parentage testing of domestic animals, this is a desirable feature since offspring will be tested years apart and re-running parents each year would be inefficient and costly. An allelic ladder also increases consistency between laboratories since each genotype is assigned relative to a known standard. Locus BMS510 exhibited single base pair differences in bison. Normally this would preclude this locus from being used as a marker for parentage testing due to the difficulty in allele assignment reproducibility. However, this problem was overcome by sizing alleles relative to the allelic ladder. The minimum and maximum standard deviation of allele sizes for this locus was 0.06 and 0.25 bp respectively with an average over all the alleles of 0.09 bp. These values represent between gel deviations. The within gel standard deviation, averaged across alleles, is reduced to only 0.06 bp. Smith (1995) demonstrated that values in this range were highly unlikely to produce incorrect allele assignment when an allelic ladder is used.
In order to use allele frequencies to calculate genotype frequencies and exclusion probabilities, allele frequencies from the populations tested must be consistent with HW expectations. 903 bison from 14 public populations and two private populations represent an adequate sample with which to estimate allele frequencies. Tests of HWE, although showing minor deviations for some locus population combinations, did not yield consistent deviations for the testing methods employed. The number of HW deviations observed in this study is similar to other studies in humans. Hammond et al. (1994) found 11.5% (18/156), Edwards et al. (1992) found 11.6% (7/60) and Thomson et al. (1999) found 5.5% (2/36) of the possible locus-population-test combinations showed deviations, which is comparable to the 7.5% (71/942) found in the current study. The lack of consistency in the observed deviations leads us to conclude that these loci are in HWE for the populations tested.
Tests of genotypic disequillibrium showed no consistent deviations in the populations which were not expected a priori to show deviations. In a production setting such as with the HHP and ABR populations, genotypic disequillibrium is expected because a limited number of bulls are used for breeding. However, in these cases typically the entire population will be tested and departures from genotypic equilibrium will have little effect on the final parentage analysis. Therefore, we conclude that the observed deviations in HWE and genotypic equilibrium are small enough that they will not significantly affect the calculations of genotypic frequencies or multilocus probabilities from allele frequencies.
The advantages of likelihood based parentage assignment over exclusionary methods have been demonstrated by Slate et al. (2000) for natural populations and extended to captive production populations in the current study. Indeed, even with highly developed sets of markers such as those presented here, genotyping errors occur. A likelihood-based system to assign parentage allows the lab to identify potential errors or mutations and make corrections before parentage is rejected.
PCR based methods in conjunction with highly variable microsatellite loci and fluorescent based genotyping provide the technologies needed to establish a new standard for parentage testing. The core set of 12 microsatellites presented here along with 4 additional microsatellites offers a powerful battery of markers for both parentage testing and individual identification. These loci are preferably used as the core set of 12 or in combination with one or more of the 4 additional loci, but they may be combined in whole or in part with other loci without substantial loss of identification power. These markers when used in likelihood based parentage testing will help to refine breeding programs and allow for improved genetic management by accurate determination of pedigrees in both bison and cattle.
The invention may be better understood through reference to the specific examples which follow. Although only preferred embodiments of the invention are specifically described above and in the examples, it will be appreciated that modifications and variations of the invention are possible without departing from the spirit and intended scope of the invention.


EXAMPLES
Example 1
DNA Source
Fourteen public bison herds, two private bison herds and five cattle breeds were sampled. Sample sizes and population locations are listed in Table 1. These herds represent most of the major public herds that have played a role in populating private bison herds around the world. Therefore, the majority of the genetic variation present in extant bison herds should be contained within these public herds.







TABLE 1







Bison populations and domestic cattle breeds sampled.













Sample


Public Herds
Abbreviation
Location
Size













Antelope Island State Park
AI
Utah
67


Custer State Park
CSP
South Dakota
37


Elk Island National Park
EIW
Alberta
25


(woods)


Elk Island National Park
EIP
Alberta
24


(plains)


Fort Niobrara National Wildlife
FN
Nebraska
24


Refuge


Finney Game Refuge
GC
Kansas
50


Henry Mountains
HM
Utah
21


Caprock Canyon State Park
CCSP
Texas
33


Mackenzei Bison Sanctuary
MBS
Canada
40


(woods)


Maxwell Game Refuge
MX
Kansas
35


National Bison Range
NBR
Montana
38


Wind Cave National Park
WC
South Dakota
152


Wood Buffalo National Park
WBNP
Canada
21


(woods)


Yellowstone National Park
YNP
Wyoming
158




Total
725


Private Herds


Arrowhead Buffalo Ranch, Ltd.
ABR
Ohio
135


Hidden Hollow Preserve
HHP
Kentucky
43




Total
178


Cattle Breeds


Angus
AN

54


Hereford
HE

16


Holstein
HO

12


Shorthorn
SH

12


Texas Longhorn
TLH

13



Total

107









Example 2
DNA Extraction
Genomic DNA was isolated from white blood cells by proteinase K treatment followed by phenol chloroform extraction (Sambrook et al. 1989) or by using the SUPER QUICK-GENE DNA Isolation kit (Analytical Genetic Testing Center, Inc. Denver, Colo.). DNA was also extracted from hair follicles using the following procedure. Approximately 8–12 hair follicles were cut from the switch of the tail using a razor blade and digested for four hours at 55° C. in 200 μl lysis buffer (500 mM KCl, 100 mM Tris-HCl pH 8.0, 0.1 μg/ml gelatin, 0.45% Triton X-100, 0.45% Tween-20, 0.5 mg/ml proteinase K). After digestion, samples were centrifuged at 5,000 g for two minutes. The clear aqueous layer was then transferred to a new tube and 0.5 μl of 10 mg/ml RNase A was added. The sample was then extracted once with phenol/chloroform/isoamyl alcohol (25:24:1) followed by a chloroform extraction. DNA was ethanol precipitated then resuspended in 50 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0).
Example 3
Loci
Bovine microsatellites were chosen from the USDA cattle mapping database (sol.marc.usda.gov) that fulfilled the following set of criteria in cattle:


    
    
        1. High PIC values, high heterozygosity and a large number of alleles
        2. Lack of known null alleles
        3. Loci non-syntenic or separated by more than 40 cM
        4. Allele size range
        5. Suitability for multiplex PCR.

Primer sequences flanking 15 microsatellites that fulfilled these criteria were synthesized with a fluorescent label attached to the 5′ end of each forward primer (Table 2).

    
    









TABLE 2







Chromosomal location and fluorescent dye used for each of the 15 loci


selected from the USDA database.












Locus
Chr.a
Pos.a
Dyeb
















BM1225
20
8.0
TET



BM1706
16
80.6
6FAM



BM17132
19
58.6
6FAM



BM1905
23
64.3
TET



BM2113
2
106.2
6FAM



BM4440
2
55.0
TET



BM720
13
38.6
TET



BMS1117
21
9.9
HEX



BMS1172
4
27.3
6FAM



BMS1862
24
32.8
HEX



BMS2639
18
57.0
6FAM



BMS410
12
0.0
TET



BMS510
28
22.1
HEX



BMS527
1
55.9
6FAM



RM372
8
19.1
HEX







aBovine chromosome and relative position (cM).



bABI fluorescent label used with forward primer.






Example 4
Multiplex PCR
Based on the results of genotyping approximately 500 bison and 50 cattle for these 15 loci, a core set of 12 loci were selected which could be amplified in two PCR reactions and co-loaded in a single lane of an ABI Prism377 sequencer or a single injection on an ABI Prism 310 capillary-based genetic analyzer. Core multiplex A consists of BMS510, BMS410, BM17132, RM372 and BMS527. Core multiplex B consists of BM4440, BM2113, BMS1862, BM1905, BM720, BM1706 and BM1225. PCR conditions for core multiplexes A and B are as follows: 25–100 ng template DNA, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1% Triton®-X, 3.0 mM MgCl2, 500 μm dNTPs, 0.05–0.3 μm each primer, 1X MasterAmp PCR enhancer (Epicentre Technologies), and 0.5U Taq DNA polymerase (Promega) in a 5 μl reaction. Thermal cycle parameters for core multiplex A and B were 2 minutes 96° C. followed by 35 cycles of (15 seconds 96° C., 15 seconds 54° C., 5 seconds 72° C.) with a final extension step of 20 minutes at 72° C. using a GeneAmp® PCR 9700 thermocycler (PE Biosystems). In later experiments a total of 16 loci, including the BMS1172, BMS2639, BM3628 and BMS2325 were amplified.
Example 5
Genotyping
PCR products were separated on an ABI Prism 377 DNA Sequencer (ABI377) or an ABI Prism 310 Genetic Analyzer (ABI310)(PE Biosystems) and sized relative to an internal size standard (GS500, PE Biosystems or MAPMARKER LOW, Bioventures). Fluorescent signals from the dye labeled microsatellites were detected using GENESCAN 3.1 software (PE Biosystems). Genotypes were assigned using Genotyper 2.0 software (PE Biosystems) by assigning both an integer value and the actual decimal value (called size) to each peak. After the allelic ladder was developed, the ladder was included on each gel (ABI377) or with each group of samples (ABI310) and genotypes were assigned relative to the actual sequence sizes of the allelic ladder. Previous samples that were genotyped without the allelic ladder were re-assigned genotypes based on the true sequence size of each allele.
Example 6
Cloning and Sequencing
Approximately one half of the bison alleles at each locus were cloned and sequenced. Samples were amplified individually and cloned using either the Original TA Cloning Kit or the Topo TA Cloning kit (Invitrogen) as per the manufacturer's protocol. Approximately 10–20 positive clones from each ligation were picked and grown overnight in 3 ml Terrific Broth containing 50 μg/ml ampicilin. A standard alkali-lysis mini-prep procedure was used to recover plasmid DNA (Sambrook et al. 1989). Plasmid DNA was diluted 1:50 with TE buffer and used as a source of template DNA for PCR. Each positive clone was amplified via PCR and genotyped using the ABI310. Clones that sized identical to one of the original alleles of the animal were used to make glycerol stocks. Cloned alleles were sequenced using the Big-dyeTM terminator cycle sequencing kit (PE Biosystems) and an ABI377 automated sequencer. Sequenced alleles were submitted to Genbank and have accession numbers AF213181 to AF213246. An allelic ladder was constructed by mixing equimolar amounts of DNA from the sequenced plasmids into a DNA mastermix. The combined plasmids were used as template DNA for the allelic ladder in each PCR multiplex.
Example 7
Data Analysis
Expected heterozygosity (Nei, 1987), exclusion probabilities and polymorphism information content (PIC) (Botstein et al, 1980) were calculated for each marker within each population. Two exclusion probabilities were calculated which correspond to different scenarios. Exclusion probability one (PE1) assumes genotypes are known for the offspring and a putative parent, but genotypes are not available for a known parent (one parent missing). Exclusion probability two (PE2) assumes genotypes are known for the offspring, one confirmed parent, and one putative parent (both parents genotyped). PE1 and PE2, as well as combined exclusion probabilities were calculated according to Jamieson & Taylor (1997). Tests of Hardy-Weinberg equilibrium (HWE) were performed using the program GENEPOP version 3.1d (Raymond and Rousset, 1995). Exact p-values were calculated for loci that had four alleles or less in a population. For loci that had more than four alleles present in a population, an unbiased estimate of the exact HW probability was calculated using the Markov chain method of Guo and Thompson (1992). Unbiased estimates of genotypic disequilibrium were calculated with GENEPOP using the Markov chain method. Parameters used for all Markov chain procedures were dememorization of 10,000 steps, 125 batches and 40,000 iterations per batch for a total Markov chain length of 5 million steps.
Example 8
Parentage Inference
Parentage testing was performed on the two pedigreed private bison herds to evaluate the actual effectiveness of the loci for determining parentage, verify Mendelian inheritance and check for the presence of null alleles. The accuracy of these pedigrees have previously been verified by genotyping over 200 microsatellites in these herds (unpublished data). The ABR sample contained 92 offspring and 44 potential parents. The HHP sample contained 29 offspring and 22 potential parents. Likelihood based parentage testing was performed using the program CERVUS 1.0 (Marshall et al., 1998) following the procedures outlined in the program. Analysis parameters used for simulations were as follows: 10,000 cycles, 45 candidate parents for the ABR herd and 22 candidate parents for the HHP herd, 95% of the candidate parents sampled, 100% of the loci typed, 1% typing error, 80% relaxed confidence and 95% strict confidence.
Unbiased expected heterozygosity, PIC, exclusion probabilities, allele frequencies, repeat length and the standard deviation in allele size calling are located in Table 4 and can be obtained via the internet at www.cvm.tamu.edu/derrlab/index.html. A total of 138 and 151 alleles were found in bison and domestic cattle respectively. The number of alleles per locus ranged from 5 to 16 in bison and from 5 to 13 in cattle. Of the 15 loci tested, 5 had a greater number of alleles in bison than in domestic cattle. For four of these loci, BM2113, BM1706, BMS1172 and BMS2639, this result is probably due to the limited number of cattle tested. According to published results, all four of these loci have an equal number of alleles or more in cattle compared to what was observed in bison (Stone et al. 1995; Bishop et al. 1994). The exception is BM1225 in which 16 alleles were observed in bison but cattle are reported to only have 11 alleles. Excluding BM4440 for the CCSP population, which was monomorphic, expected heterozygosities in bison ranged from 84.2% to 6.0% and from 85.4% to 39.8% for cattle. The overall mean heterozygosity across all populations and all markers was 62.17% for bison and 70.16% for cattle (Table 3). The only populations that failed to reach the 99% threshold for PE2 were Antelope Island and the CCSP population. This is most likely due to the fact that both of these herds were founded by a small number of individuals and have remained genetically isolated for much of their history (Coder 1975; Popov and Low 1950).







TABLE 3







Mean expected heterozygosity across all 15 loci and combined average


exclusion probabilities for all 15 loci and the core set of 12 loci.













Mean Expected
All Loci

Core Set













Population
Heterozygosity
PE1
PE2
PE1
PE2





AI
0.4496
0.8622
0.9858
0.8612
0.9820


CSP
0.6818
0.9953
0.9999
0.9901
0.9997


EIP
0.6666
0.9938
0.9999
0.9894
0.9997


EIW
0.5541
0.9663
0.9989
0.9506
0.9974


FN
0.6602
0.9943
0.9999
0.9860
0.9996


GC
0.6521
0.9923
0.9999
0.9828
0.9994


HM
0.5757
0.9777
0.9991
0.9599
0.9973


CCSP
0.4160
0.8457
0.9796
0.7768
0.9570


MBS
0.6334
0.9893
0.9998
0.9838
0.9996


MX
0.6729
0.9956
0.9999
0.9916
0.9998


NBR
0.6542
0.9902
0.9998
0.9714
0.9988


WBNP
0.6759
0.9950
0.9999
0.9886
0.9997


WC
0.6630
0.9943
0.9999
0.9859
0.9995


YNP
0.6340
0.9898
0.9998
0.9813
0.9993


ABR
0.6981
0.9972
1.0000
0.9920
0.9998


HHP
0.6591
0.9956
0.9999
0.9920
0.9995


AN
0.6907
0.9965
1.0000
0.9934
0.9999


HE
0.6359
0.9839
0.9995
0.9662
0.9981


HO
0.7279
0.9978
1.0000
0.9930
0.9999


SH
0.7029
0.9971
1.0000
0.9930
0.9999


TLH
0.7507
0.9993
1.0000
0.9984
1.0000


Mean Bison
0.6217
0.9734
0.9976
0.9610
0.9955


Mean Cattle
0.7016
0.9949
0.9999
0.9888
0.9995









Example 9
Hardy-Weinberg & Genotypic Disequilibrium Tests
Calculation of genotype frequencies and exclusion probabilities from allele frequencies depend on the underlying assumptions of HWE. However, the errors associated with using allele frequencies to calculate genotype frequencies and exclusion probabilities should be minimal as long as there is approximate agreement with HW expectations.
In order to test Hardy-Weinberg assumptions, three distinct tests of HWE were performed with the difference being the alternate hypothesis to equilibrium. Each locus within each population was checked for HWE for a total of 314 comparisons (CCSP was monomorphic at BM4440). Eight percent (25/314) of the locus/population combinations showed significant departure from HWE at p<0.05 for the probability test. In order to more precisely identify these deviations, score tests (U-tests) (Rousset and Raymond 1995) were performed with the alternative hypothesis of either heterozygote excess or deficiency. When the alternative hypothesis was heterozygote excess, 4.1% (13/314) of the locus/population combinations showed significant deviations from HWE at p<0.05. When the alternative hypothesis was heterozygote deficiency 10.5% (33/314) of the locus/population combinations were significant at p<0.05. There was no consistency between the three tests to indicate any specific locus/population was in disequilibrium.
Non-random association of gametes to form genotypes could also affect using allele frequencies to calculate genotype frequencies. In natural populations this is most likely due to population sub-structuring. Tests of genotypic disequilibrium within populations resulted in 1876 comparisons. Three populations (Al, ABR and HHP) were not tested for genotypic disequilibrium since all three of these populations use a limited number of breeding bulls. When these populations were eliminated from consideration because of known breeding structure, 6.4% (120/1876) of the combinations were significant at p<0.05. Tests of genotypic disequilibrium across populations resulted in 105 comparisons. No locus pairs showed significant disequilibrium across populations at p<0.05 (Bonferoni corrected).
Example 10
Parentage Inference
Both the ABR and HHP pedigrees were used to check the inheritance of the markers and to evaluate the effectiveness of both the markers and the likelihood testing procedure in a production setting. A total of 121 offspring were used to evaluate the test's effectiveness. For each offspring, every reproductively capable animal in the population was considered as a potential parent, allowing for the possibility of missing parents. The first cycle of parentage analysis resulted in eliminating all potential parents that showed incompatibilities at more than one locus. Parents that showed mismatches at one locus were considered as potential parents to allow for the possibility of either a mutation or a genotyping error. The potential parents that were left were then considered as known parents and this additional information was used to re-test the offspring against the original set of potential parents. After the second round of parentage analysis in the ABR herd, 87% (80/92) of the offspring were unambiguously assigned parentage to the correct sire and dam. A total of 12 offspring were not assigned parentage after the second cycle because these 12 cows were purchased as bred heifers; therefore the sires were unavailable. After inspecting the results from the first cycle of parentage analysis, the correct dam was assigned with >95% confidence in each case where the sire was unavailable. Parentage analysis results for the HHP population were much the same as for ABR. Every offspring was unambiguously assigned parentage except two offspring whose sire was not sampled, in which case, the correct dam was assigned with >95% confidence.
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    Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular cloning: a laboratory manual. (2nd ed). Plainview: Cold Spring Harbor Laboratory Press.
    Slate J., Marshall T. and Pemberton J. (2000) A Retrospective assessment of the accuracy of the paternity inference program CERVUS. Molecular Ecology 9, 801–808.
    Smith R. N. (1995) Accurate size comparison of short tandem repeat alleles amplified by PCR. Biotechniques 18, 122–128.
    Stone R. T., Pulido J. C., Duyk G. M., Kappes S. M., Keele J. W., & Beattie C. W. (1995) A small-insert bovine genomic library highly enriched for microsatellite repeat sequences. Mammalian Genome 6, 714–724.
    Thomson J. A., Pilotti V., Stevens P., Ayres K. L., & Debenham P. G. (1999) Validation of short tandem repeat analysis for the investigation of cases of disputed paternity. Forensic Science International 100, 1–16.









TABLE 4





Appendix A







BM1225






























Size1
Repeat2
Std. Dev.3
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





219

0.02



















0.125
0.077


215

0.10
















0.120


0.042


117

na




















0.018


119

0.20

0.149


0.196



0.025

0.024

0.161

0.049
0.105
0.120
0.633
0.375
0.208
0.169


241
16
0.24
0.136
0.191
0.354
0.063
0.478
0.527
0.119
0.465
0.138
0.529
0.431
0.167
0.122
0.457
0.459
0.663
0.759
0.567
0.331
0.112
0.116


243

0.15


0.125








0.028
0.003







0.115


145
18
0.16


0.063











0.008


147

0.16


0.063
0.011




0.050

0.014
0.083
0.003
0.003
0.015


249
20
0.28
0.015
0.068
0.063
0.083

0.031



0.043
0.111
0.028
0.046
0.007
0.060
0.023


251

0.01


0.042















0.292
0.083
0.038


253
22
0.27
0.127
0.243
0.125
0.183
0.043
0.061
0.313
0.145
0.650
0.114
0.056
0.472
0.201
0.199
0.146
0.047




0.038


255

0.05


0.061



0.143





0.001


259

0.38



0.042




0.013


261

na




















0.073


165
28
0.21
0.052
0.081


0.043
0.092
0.024


0.071

0.028
0.105

0.119


267

na



0.021


269
30
0.23
0.022
0.021

0.167
0.217
0.490
0.143
0.382
0.142
0.129
0.236
0.167
0.059
0.019
0.116
0.128


271

0.24

0.135
0.083



0.143

0.013



0.076
0.245
0.030
0.023


273
11
0.10
0.001



0.022

0.043


0.014
0.125




0.012


275

0.14


0.021
0.021


0.043




0.028



































Het4
0.580
0.806
0.827
0.615
0.689
0.614
0.818
0.615
0.546
0.656
0.732
0.721
0.803
0.708
0.736
0.516
0.398
0.472
0.677
0.633
0.793




PIC5
0.354
0.773
0.801
0.589
0.636
0.560
0.786
0.529
0.512
0.610
0.690
0.680
0.775
0.652
0.708
0.503
0.361
0.357
0.589
0.697
0.249




PE16
0.075
0.433
0.491
0.230
0.270
0.227
0.456
0.186
0.167
0.147
0.329
0.319
0.439
0.283
0.354
0.160
0.078
0.103
0.220
0.234
0.406




PE27
0.210
0.611
0.664
0.412
0.444
0.383
0.633
0.319
0.318
0.422
0.509
0.503
0.616
0.457
0.540
0.331
0.201
0.176
0.368
0.409
0.584







BM1106






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





232
11
0.14
0.246
0.054
0.104
0.560
0.146
0.150
0.100
0.136
0.112
0.200
0.316
0.267
0.174
0.076
0.060
0.302


234

0.11








0.013










0.042
0.035


236

na


















0.083


238
14
0.12
0.614
0.527
0.771
0.540
0.667
0.700
0.900
0.803
0.663
0.786
0.658
0.595
0.510
0.747
0.112
0.372
0.119

0.375
0.451


240

na








0.013


242

na












0.003




0.031


0.071


244

0.01














0.004


0.063
0.042
0.041
0.115


246
16
0.06

0.095








0.016

0.016


249

na











0.048








0.018


250
20
0.15
0.082

0.104


0.010

0.061
0.013



0.016
0.036
0.015
0.023
0.491
0.406
0.167
0.042


252

0.10
0.037
0.176
0.011
0.080
0.188
0.140


0.150
0.016

0.071
0.016
0.146
0.090
0.035
0.167
0.500
0.113
0.292
0.346


254

0.18

0.149

0.020




0.018


0.024
0.261
0.025
0.060
0.167
0.083



0.346


256

0.16
















0.120


0.125
0.035



































Het
0.661
0.662
0.388
0.578
0.504
0.470
0.182
0.335
0.527
0.345
0.470
0.576
0.642
0.332
0.189
0.705
0.683
0.589
0.727
0.699
0.751




PIC
0.519
0.619
0.357
0.491
0.448
0.425
0.164
0.109
0.491
0.293
0.380
0.514
0.583
0.308
0.370
0.634
0.614
0.494
0.662
0.636
0.696




PE1
0.222
0.253
0.076
0.167
0.125
0.111
0.016
0.055
0.152
0.059
0.109
0.175
0.223
0.055
0.081
0.265
0.267
0.173
0.295
0.274
0.339




PE2
0.371
0.431
0.209
0.293
0.265
0.255
0.082
0.169
0.316
0.152
0.202
0.326
0.383
0.175
0.224
0.430
0.469
0.295
0.466
0.445
0.511







BM17132






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





 75

na













0.010


 19

0.05



0.042




0.087


0.071






0.125


 81
13
0.06
















0.039


0.050


 81

0.10
0.881
0.230
0.085
0.104
0.152
0.561

0.015
0.275
0.371
0.092
0.238

0.062


0.031
0.251
0.167
0.050
0.211


 85
15
0.09
0.022
0.446
0.417
0.146
0.152
0.102
0.476
0.061
0.097
0.143
0.474
0.024
0.431
0.373
0.435
0.395
0.226
0.467
0.375
0.100
0.077


 87

0.09
0.082
0.311
0.250
0.616
0.543
0.245
0.214

0.125
0.314
0.434
0.452
0.336
0.142
0.069
0.301

0.063
0.542
0.200
0.346


 89
17
0.09
0.007
0.014
0.250
0.063
0.152
0.092

0.924
0.200
0.071

0.214
0.234
0.214
0.155
0.253
0.208


0.010
0.011


 91

na













0.029
0.141
0.070


 93

0.05






0.310


 95
20
na
0.007







0.025




0.130


0.009


 97

na
















0.009



0.018


 99

0.11
















0.066

0.042


101

0.04
















0.311
0.155
0.250
0.550
0.231


103

0.05
















0.123
0.031



































Het
0.218
0.656
0.701
0.551
0.642
0.609
0.639
0.143
0.768
0.704
0.582
0.675
0.649
0.776
0.663
0.702
0.395
0.671
0.765
0.650
0.315




PIC
0.206
0.582
0.619
0.513
0.691
0.554
0.558
0.135
0.725
0.641
0.437
0.636
0.573
0.739
0.597
0.615
0.757
0.605
0.712
0.604
0.125




PE1
0.024
0.217
0.267
0.165
0.221
0.199
0.199
0.010
0.566
0.269
0.167
0.268
0.209
0.384
0.236
0.264
0.410
0.245
0.551
0.243
0.564




PE2
0.111
0.368
0.434
0.316
0.197
0.360
0.346
0.070
0.544
0.434
0.284
0.438
0.355
0.564
0.392
0.428
0.598
0.410
0.530
0.421
0.542







BM1905






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





110

0.47



0.063




0.013


0.024








0.038


173
12
0.07
0.022
0.236
0.260
0.896
0.870
0.170
0.403
0.109
0.769
0.314
0.171
0.619
0.172
0.064
0.496
0.116


124

na




















0.058


176
14
0.45
0.246
0.709
0.604

0.130
0.720
0.592
0.469
0.141
0.557
0.579
0.310
0.311
0.795
0.158
0.779
0.019
0.063


178

0.15
















0.102
0.219
0.331
0.125
0.423


180
16
0.11
0.007















0.148
0.039

0.292
0.215


182

0.09
0.030

0.146
0.021




0.038

0.026


0.141
0.010


184
18
0.08
0.694
0.028

0.021

0.990

0.422
0.038
0.071
0.224
0.018


0.116
0.105


0.125
0.042


186

0.27
















0.019
0.123
0.208
0.083
0.038


188

0.24

0.028














0.602
0.563
0.333
0.333
0.269


192

na



















0.042


194

na
















0.028


198
25
0.10









0.057


0.017



0.083


0.083
0.017



































Het
0.458
0.444
0.557
0.195
0.229
0.443
0.489
0.595
0.168
0.587
0.589
0.524
0.316
0.318
0.613
0.173
0.603
0.625
0.114
0.783
0.739




PIC
0.396
0.383
0.487
0.185
0.201
0.397
0.366
0.503
0.358
0.514
0.528
0.442
0.275
0.790
0.637
0.339
0.367
0.570
0.663
0.338
0.683




PE1
0.106
0.099
0.152
0.019
0.026
0.093
0.116
0.174
0.073
0.178
0.178
0.137
0.050
0.050
0.194
0.068
0.209
0.212
0.291
0.189
0.323




PE2
0.226
0.216
0.292
0.100
0.101
0.230
0.183
0.297
0.211
0.319
0.331
0.256
0.143
0.160
0.314
0.192
0.388
0.380
0.464
0.565
0.506







BM2113






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





125

0.03









0.071


127

0.09

0.041
0.047

0.063



0.125


0.048
0.076

0.049
0.093
0.120
0.031
0.203
0.208
0.077


129
15
0.09
0.761
0.122
0.331
0.720
0.146
0.360
0.548

0.463
0.143
0.539
0.571
0.257
0.247
0.340
0.229


0.125

0.231


131

na












0.097







0.038


133
17
0.07

0.324




0.024

0.013
0.014
0.013

0.220
0.101
0.136
0.058


135

0.13

0.011



0.300


0.013
0.057


0.112



0.185
0.031
0.083
0.292
0.154


131

0.07
















0.370
0.459
0.500
0.042
0.023


139

0.07
















0.019
0.031
0.083
0.133
0.385


141

0.13



0.120




0.087


0.143
0.003



0.185
0.406

0.083
0.018


143
22
0.11
0.231
0.324
0.354
0.050
0.375
0.190
0.429
0.939
0.300
0.457
0.263
0.890
0.164
0.595
0.239
0.430
0.120
0.031

0.042


145

0.12

0.081


0.146
0.150

0.061

0.071

0.029
0.131

0.149
0.116


147

0.05

0.054
0.271
0.100






0.171
0.024
0.010
0.057
0.011
0.023


153
27
0.06
0.007
0.014


0.271




0.186
0.013



0.075



































Het
0.363
0.761
0.696
0.458
0.747
0.725
0.522
0.115
0.677
0.728
0.614
0.621
0.820
0.569
0.781
0.719
0.772
0.621
0.692
0.766
0.775




PIC
0.306
0.728
0.625
0.424
0.698
0.671
0.405
0.107
0.619
0.691
0.548
0.572
0.792
0.510
0.747
0.668
0.731
0.516
0.638
0.709
0.728




PE1
0.061
0.377
0.253
0.103
0.332
0.297
0.135
0.027
0.256
0.331
0.194
0.214
0.461
0.169
0.196
0.305
0.373
0.102
0.271
0.147
0.376




PE2
0.156
0.555
0.412
0.261
0.510
0.470
0.215
0.054
0.423
0.515
0.347
0.393
0.616
0.320
0.376
0.481
0.554
0.139
0.450
0.524
0.536







BM4440






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





121

na











0.025


123

0.19
0.007
0.027
0.063
0.146
0.167
0.120


0.038
0.113
0.066
0.125

0.032
0.038
0.035
0.185
0.344
0.458
0.457
0.269


125
15
0.22
0.015
0.230
0.104
0.271
0.104
0.030
0.405
1.030
0.188
0.147
0.197
0.250
0.342
0.566
0.313
0.244
296
0.053


0.154


127

0.31
0.716
0.027
0.375
0.188
0.229
0.590
0.071

0.150
0.118
0.429
0.150
0.049
0.266
0.146
0.198


129
11
0.25
0.246
0.581
0.208
0.333
0.333
0.190
0.405

0.439
0.279
0.039
0.400
0.560
0.120
0.299
0.477


131

0.21
0.001
0.103
0.219
0.063
0.146
0.070
0.119

0.175
0.162
0.276
0.050
0.026
0.016
0.131
0.023
0.111


0.083
0.016


133
19
0.27
0.003
0.023
0.021

0.021



0.013







0.324
0.469
0.157
0.167
0.115


137

na
















0.019


139

0.21
















0.019


0.167


141

na



















0.053


143
24
0.01









0.176


0.016

0.030
0.023
0.037

0.208
0.55
0.038


145

0.12

















0.031


0.231


141

0.81
















0.009
0.063
0.125

0.154


149

na

















0.031
0.042



































Het
0.427
0.600
0.756
0.763
0.384
0.599
0.661
0.000
0.721
0.821
0.706
0.746
0.561
0.630
0.770
0.680
0.765
0.663
0.716
0.766
0.827




PIC
0.163
0.549
0.709
0.713
0.752
0.555
0.587
0.000
0.619
0.790
0.651
0.697
0.482
0.566
0.731
0.621
0.720
0.594
0.639
0.719
0.784




PE1
0.092
0.198
0.342
0.350
0.387
0.199
0.230
0.000
0.312
0.456
0.283
0.334
0.163
0.210
0.374
0.355
0.361
0.241
0.291
0.361
0.449




PE2
0.199
0.362
0.526
0.328
0.567
0.369
0.183
0.000
0.491
0.612
0.454
0.512
0.289
0.365
0.553
0.423
0.539
0.400
0.469
0.546
0.626







BM720






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





203
 9
0.04

0.054


0.174




0.015




0.004
0.116


209

na




















0.115


213

0.08

0.095



0.052
0.167


0.029
0.091

0.224
0.166
0.097
0.023
0.009
0.031


215

0.02


















0.042
0.083
0.115


217

0.05
















0.500

0.083
0.333
0.135


219

0.05

















0.125


0.035


221

0.06


















0.167


223
19
0.11
0.022
0.189
0.083
0.152

0.521
0.024
0.128
0.105
0.124
0.131
0.211
0.089
0.142


225

0.11

0.143

0.152
0.022



0.092
0.015

0.132
0.193
0.003
0.239
0.093


227
21
0.02
0.061
0.054
0.438
0.543
0.239
0.052
0.524

0.474
0.294
0.081
0.474
0.102
0.267
0.067
0.213
0.009
0.063
0.083


229
22
0.10
0.455
0.297
0.375
0.087
0.391
0.229
0.286
0.094
0.250
0.324
0.622
0.184
0.257
0.307
0.164
0.070


0.167
0.031


231
23
0.09
0.453
0.054
0.104
0.065
0.174
0.146

0.578
0.039

0.081

0.132
0.047
0.429
0.384
0.157
0.156
0.167
0.041


233

0.03

0.014











0.068

0.070
0.157
0.313
0.167
0.081
0.038


235

0.05















0.012
0.281
0.031


0.154


237

0.06
















0.019
0.281
0.125
0.083
0.423


239

0.05
















0.037


0.292



































Het
0.511
0.104
0.657
0.654
0.717
0.653
0.623
0.554
0.692
0.708
0.580
0.689
0.812
0.793
0.721
0.775
0.693
0.790
0.876
0.791
0.769




PIC
0.491
0.771
0.534
0.610
0.634
0.636
0.551
0.470
0.644
0.644
0.545
0.632
0.782
0.737
0.676
0.736
0.652
0.744
0.841
0.743
0.727




PE1
0.172
0.433
0.229
0.245
0.313
0.240
0.197
0.151
0.279
0.273
0.190
0.260
0.446
0.409
0.510
0.388
0.287
0.394
0.549
0.197
0.375




PE2
0.291
0.610
0.382
0.424
0.489
0.411
0.147
0.274
0.455
0.436
0.367
0.433
0.621
0.588
0.538
0.568
0.469
0.572
0.711
0.575
0.563







BM51117






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





 87

0.03




0.022







0.007

0.011

0.039

0.250
0.083


 89
13
0.10
0.903
0.432
0.342
0.680
0.500
0.370
0.214
0.106
0.500
0.486
0.176
0.550
0.470
0.253
0.379
0.163


 91

0.07
0.073
0.351
0.487
0.240
0.213
0.160
0.214
0.894
0.397
0.229
0.095
0.225
0.421
0.228
0.383
0.538


 93

0.17
0.015
0.203
0.042
0.080
0.261
0.430
0.571

0.103
0.243
0.551
0.175
0.103
0.519
0.197
0.263
0.120
0.156
0.042
0.048
0.269


 95

0.12
0.007









0.027





0.528
0.375
0.292
0.208
0.385


 97

0.06










0.122





0.106
0.469
0.115
0.667
0.037


 99

0.01

0.014



0.040



0.043

0.050


0.030
0.038




0.038


101

0.06
















0.037

0.042

0.192


103

na




















0.038



































Het
0.179
0.653
0.537
0.478
0.641
0.654
0.389
0.191
0.585
0.656
0.618
0.672
0.593
0.617
0.673
0.622
0.618
0.625
0.723
0.514
0.745




PIC
0.169
0.577
0.428
0.414
0.370
0.387
0.517
0.172
0.494
0.591
0.568
0.560
0.505
0.545
0.605
0.555
0.548
0.536
0.634
0.456
0.691




PE1
0.016
0.215
0.141
0.112
0.209
0.224
0.169
0.018
0.169
0.226
0.208
0.201
0.177
0.188
0.240
0.199
0.199
0.189
0.284
0.133
0.127




PE2
0.089
0.363
0.235
0.257
0.362
0.375
0.385
0.056
0.290
0.385
0.382
0.362
0.101
0.334
0.356
0.154
0.349
0.329
0.452
0.278
0.508







BM51172






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





 86

0.10









0.029
0.176



0.030





0.015


 88
13
0.11
0.157
0.108
0.292
0.040
0.083
0.290
0.095
0.015
0.038
0.214
0.392
0.095
0.392
0.015
0.207
0.122


 90
14
0.13
0.067
0.473
0.333
0.760
0.646
0.490
0.313
0.015
0.738
0.686
0.162
0.595
0.294
0.627
0.481
0.451
0.500
0.500
0.500
0.308
0.131


 92
15
0.09




0.208
0.020



0.014


0.003

0.004

0.333
0.406
0.161
0.173
0.038


 94

0.09













0.006


0.028

0.250
0.125
0.077


 96

0.10
















0.139
0.094
0.083
0.042
0.115


 98

na











0.024


100

0.10

0.297
0.208

0.063
0.050

0.015
0.016
0.057
0.149
0.095
0.162
0.022
0.139
0.085


102
20
0.12
0.067

0.167
0.200

0.060

0.333
0.138

0.122
0.190
0.003
0.203
0.083
0.183


104

0.11
0.709
0.122



0.090
0.071
0.591




0.145
0.117
0.053
0.159



































Het
0.466
0.666
0.740
0.385
0.534
0.665
0.295
0.541
0.421
0.483
0.758
0.398
0.717
0.546
0.695
0.724
0.625
0.585
0.667
0.415
0.453




PIC
0.430
0.605
0.683
0.333
0.481
0.611
0.272
0.457
0.377
0.432
0.717
0.550
0.665
0.495
0.655
0.678
0.569
0.486
0.599
0.412
0.420




PE1
0.114
0.241
0.309
0.073
0.148
0.252
0.041
0.148
0.090
0.120
0.354
0.195
0.295
0.158
0.291
0.311
0.199
0.166
0.234
0.114
0.107




PE2
0.263
0.404
0.483
0.189
0.101
0.420
0.150
0.208
0.218
0.259
0.336
0.365
0.467
0.312
0.429
0.495
0.344
0.231
0.400
0.264
0.261







BM51862






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





142
 9
0.00
0.343
0.311
0.208
0.180
0.375
0.200
0.119

0.385
0.300
0.081
0.262
0.361
0.352
0.222
0.238


144

0.06



0.160

0.060



0.143

0.024


0.019


148

na




















0.035


152

na


154

0.10











0.048




0.278


0.300


156

0.06
0.127

0.042


0.250


0.013
0.051
0.284
0.024

0.017
0.049
0.107




0.131


158
17
0.05
0.007
0.392
0.208
0.100
0.025
0.030
0.071

0.218
0.200

0.041
0.248
0.145
0.203
0.107


160

0.07

0.135


0.125
0.720

0.641

0.129


0.238

0.038
0.119
0.389
0.188
0.083
0.087
0.154


162
19
0.07
0.015
0.034
0.042

0.050
0.060
0.071

0.017

0.054
0.119
0.030
0.062
0.079
0.036
0.009


0.042


164
10
0.05
0.485
0.027
0.104

0.250
0.050
0.357
0.141
0.051
0.014
0.189
0.048
0.010
0.079
0.162
0.036



0.041
0.033


166

0.09

0.027
0.042
0.440

0.120
0.048

0.141
0.043
0.054
0.119
0.036
0.173
0.053
0.250
0.157
0.063
0.208
0.203
0.077


168

0.09
0.022
0.014
0.271
0.120
0.175
0.010
0.167

0.315
0.114
0.338
0.262
0.060
0.110
0.177
0.107

0.061


0.192


170
23
0.09


0.083



0.167
0.219



0.048
0.017
0.041


0.009

0.560
0.033
0.192


172

0.11
















0.009
0.695
0.161
0.042


174

na
















0.009



0.038


190

na


















0.043


193

0.05
















0.139



0.018



































Het
0.632
0.703
0.825
0.731
0.757
0.828
0.800
0.526
0.768
0.821
0.762
0.834
0.741
0.793
0.842
0.840
0.734
0.492
0.634
0.702
0.354




PIC
0.563
0.649
0.793
0.631
0.709
0.801
0.764
0.465
0.731
0.791
0.719
0.803
0.704
0.764
0.319
0.808
0.683
0.443
0.627
0.654
0.317




PE1
0.212
0.283
0.466
0.321
0.346
0.480
0.424
0.136
0.378
0.463
0.260
0.481
0.341
0.425
0.310
0.492
0.316
0.123
0.260
0.294
0.507




PE2
0.361
0.457
0.610
0.503
0.525
0.653
0.604
0.276
0.559
0.618
0.538
0.660
0.519
0.605
0.679
0.664
0.491
0.271
0.435
0.476
0.676







BM52639






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





156

na




















0.018


164
14
0.12

















0.021
0.175
0.083


165

0.17
0.015


0.380




0.063
0.015

0.013




0.358
0.094
0.417
0.250
0.162


168

0.03
0.239
0.135
0.370

0.313
0.400
0.143
0.424
0.050
0.265
0.081
0.167
0.253
0.194
0.197
0.125


0.083

0.192


170
17
0.13
0.664
0.324
0.304
0.340
0.146
0.040
0.381

0.482
0.309
0.203
0.286
0.291
0.389
0.261
0.150


172

0.12
0.022
0.338
0.217
0.340
0.125
0.230
0.476
0.061
0.362
0.221
0.149
0.262
0.345
0.391
0.130
0.425
0.216
0.405
0.167
0.125
0.017


174
19
0.09
0.015

0.022
0.060
0.183
0.160


0.175
0.118
0.459
0.024


0.023
0.088
0.028
0.313

0.083
0.192


176

0.11
0.045
0.027

0.060
0.138
0.140

0.132
0.138
0.044
0.081
0.071
0.007

0.129
0.035


178
21
0.22

0.031

0.020
0.042
0.030





0.119
0.103

0.061
0.175
0.066
0.156
0.125
0.011


180

0.10
















0.301

0.031
0.175
0.038


184

na











0.024


186

0.08

0.095
0.037




0.364

0.029



0.025


190

na










0.027





0.039



































Het
0.501
0.751
0.723
0.720
0.802
0.743
0.615
0.666
0.752
0.775
0.717
0.809
0.727
0.652
0.766
0.751
0.726
0.719
0.770
0.777
0.728




PIC
0.446
0.106
0.664
0.663
0.363
0.701
0.527
0.597
0.715
0.732
0.676
0.773
0.674
0.375
0.726
0.709
0.669
0.652
0.724
0.727
0.655




PE1
0.131
0.346
0.294
0.302
0.416
0.338
0.195
0.236
0.356
0.375
0.312
0.433
0.102
0.214
0.365
0.349
0.300
0.285
0.369
0.111
0.330




PE2
0.269
0.523
0.464
0.474
0.595
0.517
0.316
0.391
0.578
0.553
0.491
0.610
0.474
0.361
0.544
0.552
0.470
0.456
0.531
0.553
0.479







BM5410






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





 81

0.10








0.013


0.071


0.003


 83
12
0.06
0.036
0.635
0.333
0.020
0.600
0.406
0.667
0.106
0.183
0.400
0.486
0.095
0.566
0.471
0.447
0.547
0.083
0.094
0.273
0.413
0.077


 85

0.11
0.262
0.081
0.042
0.080

0.156


0.138
0.243
0.041
0.048
0.158

0.056
0.015

0.031

0.042
0.269


 87

0.25

0.014


0.045

0.095
0.030
0.013


0.024

0.013





0.042


 89
15
0.09
0.638
0.257
0.604
0.840
0.455
0.229
0.024
0.470
0.538
0.329
0.284
0.667
0.276
0.252
0.346
0.384
0.019


 91

0.29








0.013

0.081





0.296

0.111
0.125
0.500


 93
17
0.19

0.014



0.083




0.014
0.024


0.094
0.012
0.028

0.056

0.038


 95

0.09
0.015

0.021
0.050

0.125

0.194
0.087
0.029
0.091
0.071

0.003
0.041
0.023
0.083

0.333
0.042


 97
19
0.24






0.214

0.013




0.258
0.008


 99

0.09
















0.065
0.003
0.056
0.161


103

0.03
















0.287
0.531
0.111
0.361
0.077


105

0.03
















0.056
0.186
0.056

0.038


107

na

















0.091


109

0.09
















0.083



































Het
0.519
0.327
0.527
0.287
0.543
0.739
0.506
0.617
0.653
0.617
0.670
0.519
0.581
0.670
0.669
0.559
0.803
0.671
0.800
0.766
0.676




PIC
0.454
0.464
0.459
0.269
0.436
0.695
0.430
0.534
0.611
0.608
0.616
0.512
0.510
0.601
0.610
0.453
0.775
0.626
0.747
0.719
0.617




PE1
0.136
0.142
0.138
0.041
0.147
0.131
0.129
0.194
0.247
0.237
0.256
0.162
0.167
0.293
0.253
0.156
0.445
0.262
0.401
0.364
0.257




PE2
0.269
0.279
0.251
0.152
0.241
0.511
0.271
0.311
0.425
0.392
0.424
0.395
0.388
0.383
0.415
0.269
0.621
0.444
0.580
0.347
0.428







BM5510






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





 82

na



















0.040


 84

na


















0.045


 88

0.14

















0.094


0.077


 90

0.25








0.015


 91
12
0.08
0.969
0.471
0.130
0.625
0.229
0.310
0.214
0.303
0.637
0.386
0.399
0.452
0.682
0.365
0.395
0.114


 92
   12A
0.14
0.008
0.382
0.500
0.195
0.188
0.370
0.143
0.697
0.097
0.214
0.292
0.239
0.197
0.331
0.301
0.267
0.370
0.544
0.409
0.333
0.423


 94
   13A
0.15

0.074
0.043
0.104
0.229
0.100
0.310

0.150
0.229
0.069
0.119
0.105
0.036
0.105
0.256
0.065


0.042
0.154


 95
14
0.06
0.023
0.074
0.326
0.083
0.354
0.220
0.333

0.050
0.171
0.250
0.190
0.095
0.197
0.199
0.163


 96

0.05
















0.009

0.091
0.333


 98

0.04
















0.037
0.039
0.045
0.042
0.192


100

na




















0.038


102

0.06

















0.031
0.045

0.071


104

0.06
















0.472
0.903

0.125


106

0.01
















0.046

0.273
0.063
0.035


108

na


















0.091



































Het
0.060
0.626
0.632
0.562
0.742
0.712
0.736
0.426
0.559
0.729
0.701
0.697
0.580
0.671
0.706
0.747
0.638
0.631
0.753
0.766
0.760




PIC
0.059
0.549
0.538
0.514
0.686
0.614
0.676
0.133
0.323
0.675
0.638
0.618
0.510
0.606
0.649
0.619
0.365
0.554
0.699
0.713
0.715




PE1
0.002
0.205
0.208
0.167
0.312
0.281
0.308
0.089
0.174
0.301
0.266
0.266
0.167
0.241
0.231
0.314
0.219
0.208
0.345
0.339
0.160




PE2
0.030
0.347
0.356
0.329
0.486
0.450
0.475
0.167
0.346
0.475
0.431
0.437
0.308
0.401
0.446
0.488
0.363
0.356
0.522
0.315
0.542







BM5523






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





159
11
0.05
0.119

0.021






0.014
0.029


163
13
0.06
0.033
0.135
0.146

0.053
0.110
0.024


0.186
0.103
0.095
0.010
0.047
0.038


165
14
0.13


0.208
0.040

0.110


0.141
0.041
0.092
0.071

0.019
0.011


167
15
0.08
0.037
0.054
0.063
0.240
0.184
0.120

0.331
0.167
0.129
0.013
0.333
0.010
0.023
0.143
0.021
0.170

0.125
0.042


169

na


















0.042

0.018


171

na




















0.077


173

0.09
0.419
0.294
0.229
0.140
0.342
0.400
0.543
0.045
0.106
0.280
0.551
0.262
0.247
0.259
0.485
0.500
0.056
0.063

0.083
0.108


175
19
0.10
0.188
0.150
0.271
0.060
0.289
0.160
0.256
0.676
0.167
0.214
0.158
0.119
0.160
0.463
0.139
0.384
0.009
0.031
0.208


177

0.11

0.297
0.063
0.320
0.132
0.100
0.143
0.045
0.218
0.129

0.119
0.573
0.180
0.184
0.081
0.259
0.031

0.500
0.115


179

0.06
















0.028
0.438

0.167
0.077


181

0.07















0.012
0.407
0.418
0.500
0.206
0.269


183

0.09
















0.102

0.083

0.115


185

na


















0.042


187

na
















0.009


189

na
















0.009



































Het
0.660
0.762
0.610
0.727
0.135
0.770
0.605
0.558
0.787
0.809
0.648
0.787
0.587
0.674
0.892
0.602
0.745
0.621
0.695
0.684
0.308




PIC
0.594
0.716
0.772
0.668
0.702
0.738
0.553
0.476
0.743
0.715
0.611
0.746
0.525
0.620
0.650
0.516
0.701
0.514
0.643
0.621
0.763




PE1
0.218
0.350
0.431
0.298
0.316
0.386
0.195
0.138
0.791
0.433
0.247
0.196
0.177
0.256
0.283
0.185
0.114
0.199
0.279
0.160
0.424




PE2
0.393
0.527
0.608
0.466
0.513
0.570
0.132
0.284
0.571
0.611
0.429
0.575
0.326
0.424
0.456
0.316
0.523
0.774
0.439
0.435
0.602







RM372






























Size
Repeat
Std. Dev.
AI
CSP
EIP
EIW
FN
GC
HM
JA
MBS
MX
NBR
WBNP
WC
YNP
ABR
HHP
AN
HE
HO
SH
TLH





114
 9
0.17

0.041

0.087
0.021



0.103


0.071
0.030
0.020
0.015
0.023


116

0.04

















0.063


0.192


118
11
0.01

0.365
0.152

0.229

0.167

0.038
0.071
0.043
0.048
0.293
0.534
0.135
0.244


122

na




















0.038


126

0.34
















0.151
0.061

0.167
0.077


128

0.22

0.108

0.217



0.091
0.051
0.086
0.029
0.143
0.140

0.113
0.116
0.150
0.375
0.136
0.411
0.108


130
17
0.10
0.412
0.122
0.457
0.587
0.146
0.638
0.222
0.121
0.531
0.586
0.329
0.524
0.260
0.106
0.338
0.031
0.208
0.250
0.545
0.161
0.018


132

0.09
0.008


0.087
0.031
0.018


0.115


0.071
0.020
0.137
0.004
0.015
0.094
0.074
0.091
0.041
0.154


134
19
0.14
0.164
0.149
0.391
0.022
0.271
0.260
0.611
0.788
0.126
0.729
0.443
0.143
0.127
0.373
0.256
0.302
0.170
0.156
0.227
0.205
0.192


136

0.09
0.414
0.135


0.250
0.100


0.013
0.029


0.080
0.022
0.132
0.151
0.047


138

0.54

0.081








0.157

0.050

0.008
0.047



































Het
0.633
0.797
0.622
0.599
0.791
0.528
0.557
0.359
0.656
0.596
0.673
0.680
0.803
0.757
0.774
0.811
0.793
0.763
0.639
0.741
0.315




PIC
0.555
0.767
0.335
0.548
0.743
0.462
0.489
0.328
0.623
0.545
0.610
0.641
0.773
0.712
0.737
0.776
0.754
0.721
0.574
0.684
0.778




PE1
0.201
0.429
0.189
0.195
0.391
0.140
0.151
0.064
0.260
0.194
0.247
0.277
0.437
0.149
0.383
0.440
0.404
0.363
0.213
0.915
0.430




PE2
0.341
0.609
0.324
0.360
0.572
0.175
0.294
0.185
0.446
0.357
0.405
0.462
0.614
0.526
0.562
0.613
0.584
0.543
0.379
0.494
0.601





1Allele size (does not include non-template adenine added to 3′ terminus by Taq polymerase)


2Number of repeats present in the sequenced bison allele.


3Standard deviation of called size assigned by Genescan software


4Expected heterozygosity


5Polymorphic information content


6Probability of excluding a putative parent when a confirmed parent's genotypes are unavailable.


7Probability of excluding a putative parent when a confirmed parent's genotypes are known.

## Claims

1. A method of determining parentage of a North American bison or domestic cattle offspring comprising:
i) determining the alleles present in the offspring for selected microsatellite loci;
ii) determining the alleles present in sampled potential parents of the offspring for the same selected microsatellite loci;
iii) determining the likelihood that any potential parent is the actual parent of the offspring based upon the presence or absence of an allele for each of the selected loci in the offspring that is present in the potential parent;
wherein the selected loci comprise BM1225, BM1706, BM17132, BM1905, BM2113, BM4440, BM720, BMS1862, BMS410, BMS510, BMS527, RM372, BMS1172, BMS2639, BM3628 and BMS2325.

2. The method of claim 1, wherein the selected loci consist of BM1225, BM1706, BM17132, BM1905, BM2113, BM4440, BM720, BMS 1862, BMS410, BMS510, BMS527, RM372, BMS1172, BMS2639, BM3628 and BMS2325.