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Copyright 0 1990 by the Society of America

Assessment of Inbreeding by DNA Fingerprinting: Development of a Calibration Curve Using Defined Strains of Chickens

U. Kuhnlein,* D. Zadworny," Y. Dawe,* R. W. Fairfull+ andJ. S. Gavora'

*Department of Science, Macdonald College of McGill University, Ste. Anne de Bellevue, Quebec H9X ICO, Canada and j-Animal Research Centre, Agriculture Canada, Ottawa, Ontario KIA OC6,Canada Manuscript received September 1 1, 1989 Accepted for publication February 2, 1990

ABSTRACT By analyzing DNA fingerprints of chickens from seven well-defined genetic groups, a calibration curve was established relating the degree of inbreeding with the average band frequency, allelic frequency and band sharing. The probe used was bacteriophage M13 DNA and digestion of the genomic DNAwas carried out with the MspI restriction enzyme. The analysisalso provided an estimate of the average allelic frequency at a hypervariable locus and the average frequency per locus and generation. The values of 0.24 and 1.7 X respectively, are similar to the estimates for humans using other probes and hybridization protocols. It is suggested that the calibration curve established can be used for determining inbreeding not only in chickens, but also in other .

YPERVARIABLE minisatellite regions or vari- groups of chickens as a model, we havepreviously H ablenumber of tandemrepeat (VNTR) loci shown thatan index of , based on give rise to multiple at a high frequency (JEF- DNAfingerprinting pattern correctly reflected the FREYS 1987; NAKAMURAet al. 1987). Such loci consist history and relationships of the different of tandem repeats of short segments of DNA and (KUHNLEINet al. 1989). alleles arise from variations in the number of repeats A furtherapplication of DNA fingerprinting in (JEFFREYS,WILSON and THEIN1985a). Different al- genetics is the assessment of inbreeding. leles canbe distinguished by cuttingDNA with a In this communication we report on the analysis of restriction enzyme which recognizes sites flanking the strains and lines of chickens, with known degrees of region of repetitive DNA and thus yields DNA frag- inbreeding, by DNA fingerprinting and theestablish- ment of a calibration curve relating the inbreeding ments whose lengths vary with the number of repeats. coefficient to band variability and band sharing. It is Minisatellites from different loci fall into families suggested that this calibration curve can be used to whose sequences are related (NAKAMURAet al. 1987). assess inbreeding in experimentaland commercial Southern blots hybridized with a minisatellite probe populations of chickens, as well as in rare or localized therefore yield a complexbanding pattern called breeding populations of any . DNA fingerprint. A singlehybridization usually re- solves about 30 bands.Since in heterozygotes both MATERIALS AND METHODS alleles of a locus are generally visible, this represents Genetic groups: All genetic groups studied were White at least 15 loci out of the 1000-1500 hypervariable Leghorns. Their origin and characteristics are described in loci estimated to be present per (WYMAN and Table 1,and their inbreeding coefficients are listed in Table WHITE 1980; WONG et al. 1986;NAKAMURA et al. 3. All these strains and lines are currently maintained at the 1987; KNOWLTONet al. 1986). Animal Research Centre of Agriculture Canada in Ottawa, Canada, except lines 6, and 7, which are kept at the Re- DNAfingerprints are highlyindividual specific gional Poultry Research Laboratory of the U.S. Department ( JEFFREYS,WILSON and THEIN198513) and have there- of Agriculture, East Lansing, Michigan. The inbreeding forefound wideapplications in forensicmedicine coefficients of strains 7, 8, 9, S and WG were computed on individual pedigree basis up to 1980 and subsequently esti- ( JEFFREYS, BROOKFIELDand SEMEONOFF1985; GILL, mated from the size of populations and type of JEFFREYS and WERRETT 1985), as well as ecological mating (FALCONER1960). The contribution of the initial studies aimed at establishing mating behavior (WET- inbreeding of the founder population to the current in- TON et al. 1987; BURKEet al. 1989). However, DNA breeding coefficient has been estimated to be <0.00 1. The fingerprinting can be used not only to establish rela- highly inbred lines 6:3 and 7, were derived by brother-sister (STONE1975). The current inbreeding coefficient tionships among individuals, but is alsoa powerful is >0.98 (L. B. CRITTENDEN,personal communication). tool for researchin . Using genetic DNA fingerprinting: DNA was extracted from 100 hI of

(;t.twtic.\ 125: 161-16.5 (\fa\, 1990) 162 U. Kuhnlein et al.

TABLE 1 TABLE 2 Description of genetic groups Evaluation of band frequencies and allelic frequencies in S S White 1.eghorn strainselected for susceptibility to hlarek's disease at Cornell University until 1971. Chicken Maint;~inedsince at Ottawawithout selection (Hum Band fre- Allelic #I #2 #3 #4 #.i #6 quency" frcquencyh :nld COLE1957; GAVORA,EMSLEV and COLE1979) 7 Fortned fromfour commercial WhiteLeghorn strains 1 +'+" +' - - 0.500 0.360 in 195X mld maintained since without selection 2 +' +' +' +' +' +' 1.000 1 .000 (GOWF. ;rnd FAIRFULL 1980) :1 +' +' +' +'+' +' 1.000 1.000 X Drrived from strain 7 in 1969 and selectedsince for 4+'"" +' 0.J33 0.?26 high eggnumber and related traits (GOWE and 5 +'+"--- 0. ?I 3 ?I 0.226 FAIRFULL1980) 6 +' +' +' +' +' +' 1.000 1 .000 9 Derivcdfronl str;Iin 7 in 1969 and selected for high 7 +' +'+' +' +' +' 1.000 1.000 egg production rate ;rnd 1-elated traits ((;OWE and 8 +' - +' +' - +' 0.667 0.51 4 FAIRFULL1980) 9-+' - +r - +' 0.500 0.360 UT(; Inbred line derived from strain 9 (GAVORA,KUHNLEIN 10 - f' - +' +' - 0.3600.500 ;\lid SPENCER 1989) 11 - - +r- - - 0.167 0.108 (il Highly inbred White Leghorn line derivedatthe I2 - + +' + +' + 0.667 0..514 Regional PoultryResearch Station of the USDA, 13 + - +' + +' + 0.839 0.707 F~stLansing, Michigan, USA (STONE 1975). Resist- 14"" +' +' 0.333 0.226 ant to Marek's disease I5 - + - - +' + 0.500 0.360 7y Smnr as line 6.1,except Marek's disease susceptible Average band frequency 0.62 -+ 0.07 (STONF.1975) Average allelic frequency 0.53 -+- 0.08 " Average and standard error.Analyzing five chickens instead of heparinizedblood accordingto JEFFREYS and MORTON six gave average band frequencies between 0.61 and 0.69, with the (1987) anddissolved in 5 mM Tris-HCI, 0.1 mM EDTA (pH average being 0.63 f 0.01. Scoring the seven nmst intense bands 7.5). A 2O-pl mixture containing 5 118 of DNA and 18 units only gave an average band frequency of 0.62 f 0.07. of MspI restriction enzyme and MspI buffer was incubated iAllelic frequency calculated from the band frequency(see text). at 37" for 2-4 hr, electrophoresed in a 1% agarose gel (1 ' Eight bands which had the highest intensity in the particular V/cm for 16.5 hr) and transferred to nitrocellulose mem- l~llle. branes by capillary blotting. Prehybridization, hybridization and washing were carried out according to the method of will then be VASSARTet al. (1987) using "'P-labeled M13mp9 single b = (NU+ u/A)/(No + U) strand DNA as a probe (Pharmacia). Labeling was carried out with an oligo-prime labeling kit (Pharmacia) andyielded The two equations can be solved for u, yielding the expres- specific activities between 5 X 10' and lo9 dpm per gg of sion DNA. Fifty nanograms of probe were used per hybridization ZJ = (1 - b)/(PAb - 2) reaction. The filters were exposed to Kodak XAR-5 film at -70" for 24-62 hr with one or two Cronex intensifying In our determination six were scored and the aver- screens. age band frequency extrapolated to 100% inbreeding was Evaluation of DNA fingerprints: DNA fingerprints of 0.983. This yields a mutation rate of 1.7 X IO-'' per locus six randomlyselected chickens per genetic group were and generation. scanned with a computer-linked densitometer and the eight Regression analyses and the determinationof confidence most intensive bandswere marked on eachDNA finger- intervals were carried out according to standard procedures print. The scans were then compared for the mutual pres- (ZAR 1974). ence of these bands. Bands were scored as identical (ie., representing the same allele) if they had the same apparent rnolecular weight and relative intensities differing by less RESULTSAND DISCUSSION tll;un a f'actor of two (homozygote versus heterozygote). An example of this evaluation is given in Table 2. The molec- To study the dependence in the variability of the ular weights of the bands scored were between 25 and 2.5 DNA fingerprinting pattern on the degreeof inbreed- kb. ing, we examined 7 different breeding populations of Calculation of the mutation rate to new alleles: Extrap- olation of the dependence of the average band frequency chickens whose degree of inbreedingranged from on inbreeding to 100% inbreeding provides an estimate of 2.5% to >98% (Tables 1 and 3). The DNA finger- the mutation rate (v) to a new allele per generation and printing patterns of chickens with inbreeding coeffi- locus. Forv << 1 in a 100% inbredline, the expected number cients of >98% and 39%, respectively, are shown in of new bands (u) per generation is Figure 1. The banding patterns of chickens from the U = 27JNIJd4 highly inbred line were identical, whereas considera- ble variation was observed among chickens of the less N,, is the number of bands which would have been scored in the absence of mutation (each representing two copies of . ;I minisatellite locus dueto homozygosity) and A is the As an index of the uniformity (U) of the DNA nunlber of aninlals tested. The average band frequency (b) fingerprintingpattern within astrain, the average Inbreeding and DNA Fingerprints 163 A B v " An alternative measure for band variability within a strain is band sharing (WETTONet al. 1987). Band sharing (S) for two individuals is computed as S = ~NAB/(NA+ NR) where NAB is the numberof bands shared andNA and NB are thetotal number of bands scored in individual A and B, respectively. This measure can be extended to several individuals by averaging over all possible pairwise combinations. As expected,average band sharing increases with the inbreeding (Table 3). The relationship between band sharing and the inbreeding coefficient is nonlinear and has to be fittedby a higher FIGURE1 ."DNA fingerprints of chickens from two strains with order approximation. different inbreeding coefficients. kmel A, strain S with an inbreed- If the inbreedingcoefficient (f)of a strainis known, ing coefficient of 39%. Panel B, highly inbred line 72 withan the allelic frequencies (pi)can be estimated from the intweetling coefficient >W%. band frequencies (vi) based on the equation (FAL- band frequency was computed according to the equa- CONER 1960): tion vi=(pi)*+fpi(l -pi)+2pi(l -pi)-2fpi(l -pi) A' homozygotes heterozygotes U = (1/N) vi i= I This equation can be solved for pi. The average value of pi for the different strainsare listed in Table 3. Its where N is the number of different bands scored and dependence on inbreedingis close to linear (Figure 2; vi is the frequency of band in the breeding popula- i correlation coefficient of 0.999). tion. Assuming that each band represents an allele of The intercept atf= 0 (no inbreeding) provides an a hypervariable minisatellite region, this index is equal estimate for the average frequency of alleles which to the average frequency of genotypes which have a particular allele in common. With an increasing de- can be distinguished at hypervariable loci. A value of gree of inbreeding one would expect that more and 0.243 was obtained (95% confidence intervalf0.026). more alleles become fixed, resulting in the extreme A second estimate of the same parameter can be case in an average genotypic frequency approaching obtainedfrom thenumber of differentbands ob- one. In an outbred population on the other hand, the served and the number of bands scored per chicken average genotypic frequency would be determined by (Table 3). Assuming that different alleles at a locus the genetic variability of the founder population and will always result in hybridization signals of compa- the average number of alleles per locus which are rable intensity-an assumption which appears to be distinguishable in the DNA fingerprinting pattern. substantiated by WYMAN andWHITE (1980),WONC et It is obviously desirable to score as many bands and al. (1 986), NAKAMURAet al. (1987) and JEFFREYS et individuals as possible. However, the number of lanes al. (1988)"we expect that in the less inbred strains on a gel are limited, distant lanes are hard to compare which contain few homozygotes each fingerprint will and scoring of faint bands is unreliable. We therefore reveal two alleles per locus. Hence, the average of restricted ourselves to analyzing six chickens per ge- 10.6 bands scored per chicken (strains 7, 8 and 9, netic group and limited the number of bands scored Table 3) represent 5.3 loci. Since an average of 22.5 in each chicken (see MATERIALS AND METHODS and different alleles were scored per strain the average Table 2 for details). Evaluation of a duplicate DNA allelic frequency was 0.236. fingerprint in one of the genetic groups (Table 3) In comparison,NAKAMURA et al. (1 987) determined indicated that this scoring method was reproducible the number of different alleles at 76 differenthyper- and nearly identical values where obtained when five variable minisatellite lociin 60 to a 120 unrelated chickens or fewer bands were analyzed (footnote to human subjects. Approximating the allelic frequency Table 2). at each loci by the reciprocal value of the number of Table 3 lists the average band frequencies of seven different alleles observed and averaging over all loci, strains or lines of chickens. As expected, it increases an overall average allelic frequency of 0.255 is ob- with thedegree of inbreeding.Linear regression tained (95% confidence interval k0.028). A second analysis yields a correlation coefficient of 0.996 (P < estimate by JEFFREYS, WILSONand THEIN(1985a) 0.01), indicating thatthe dependence of the band using fewer probes and subjects yielded an average of frequencyon inbreeding iswell represented by a 0.322 (95% confidence interval k0. 144). linear approximation (Figure 2). Extrapolation of the band frequency tof= 1 (1 00% 164 U. Kuhnlein et al.

TABLE 3 Average band frequencies, allelic frequencies and band sharing in seven genetic groups of chickens with different degrees of inbreeding

Inbreed- Genetic" ing No. of different Average No. bands Band Allelic group coefficient scoredbands scored per chicken frequencyb frequency' Band sharing 7 25 0.026 10.70.43 f 0.7 f 0.05 0.27 f 0.03 0.44 k 0.05 8 10.2 21 0.103 -t O.tid 0.48 f 0.05 0.330.51 -t 0.05 f 0.03 9 210. 126 10.5 f 0.5 0.50 k 0.05 0.33 f 0.04 0.52 k 0.04 9' G. 126 22 11.3 f 0.5 0.52 f 0.05 0.34 f 0.04 0.53 f 0.04 S 0.39 15 9.3 t 0.3 0.62 f 0.07 0.53 k 0.08 0.69 f 0.02 WG 0.762 13 10.5 f 0.4 0.81 f 0.05 0.78 f0.81 0.05 f 0.02 7, >0.98 8 8.0 f 0.0 .001 .OO 1k 0.00 t 0.00 .00 1 f 0.00 6, >0.98 8 8.0 f 0.0 1 .00 f 0.00 1 .OO 1f 0.00 .OO -e 0.00

a Genetic groups are describedin Table 1. The average band frequency is equal to the average frequencyof genotypes which carry an allele corresponding to a band. ' Calculated from the band frequencies as indicated in the text. The reciprocal value of the average allelic frequency is equal to the average number of alleles per locus. '' Average and standard error. ' Duplicate DNA fingerprint of strain nine chickens.

surprising that our values obtained by extrapolation are in such goodagreement with those obtained through direct experimental analysis. It indicates that the average band frequency adequately reflects the degree of inbreeding and that the curve of the rela- tionship between band variability and coefficients of inbreeding can be used to determine the degree of inbreeding in unknown populations by DNA finger- printing. This may be of practical usein assessing inbreeding in commercial flocks of chickens or popu- 0 lations of livestockspecies, thus providing information zl a ~L--.-~~l_i about genetic variability and potential response to * 0 0.2 0.4 0.6 0.8 1 selection. Further, it can be used to determine the INBREEDING degree of inbreeding in geographically isolated - FIGURE 2,"Dependence of band variability (0)and apparent ing populations or among . allelic variability (0)on inbreeding.Linear regression analysis through the data points from seven strains (Table 3) yielded cor- Whether the same calibration curve can be used for relation coefficients of 0.996 and 0.999, respectively. The inter- other species or families of minisatellites remains to cepts ;It 0 inbreeding were 0.417 (k0.035) and 0.243 (+0.026), and bedetermined. However, the good agreement be- the slopes were 0.566 (+0.059) and 0.744 (k0.044). The values in tween mutation rate and allelic frequencies observed parenthesis define the 95% confidence interval. in this study with the M 13 probe and in humans with other probes indicates that such extrapolation may be inbreeding) provides an estimate of the frequency of meaningful. new alleles arising per generation and locus (see MA- TERIALS AND METHODS). A value of 1.7 X IO-' was Thisresearch was supported by grantsfrom the Conseil des obtained. This mutation rate is close to the theoretical Recherchesen Peche et Agro-alimentdire duQuebec and the Natural Sciences andEngineering ResearchCouncil ofCanadd. value of JEFFREYS, WILSONand THEIN(1985b) who The expert technical assistance of L. VOLKOVand the supply of estimated that, based on population simulations, new bloodsamples by L. B. CRITrENDEN andthe staff of the ARC alleles should arise at a frequency of 0.5-1.5 X isolation facility are gratefully acknowledged. per gamete and generation for a minisatellite 10 kb long. It is also close to the experimentally determined LITERATURECITED rate reported by JEFFREYS et al. (1988). In the latter BURKE,T., N. B. DAVIES,M. W. BRUFORD andB. J. HATCHWELL, study an extensive analysis of five different-tandem 1989 Parental careand mating behaviour of polyandrous repetitive hypervariable loci in 40 families comprising dunnocksPrunella modularis related to paternity by DNA a total number of 475 subjects yielded mutation rates fingerprinting. Nature 338: 249-25 1. FALCONER,D. S., 1960 Introduction to Quuntitutive Genetics. Oliver between 0 and 0.052 with an average of 1.2 X & Boyd, Edinburgh. per locus and generation. GAVORA,J. S., A. EMSLEYand R. K. COLE,1979 Inbreeding in Considering the differences in the sizes of the pop- 35 generations of development of Cornell S strain of Leghorns. ulations and the classesof loci analyzed, it is quite Poult. Sci. 58: 1 133-1 136. Inbreeding and Inbreeding DNA Fingerprints 165

GAVORA,J. S., U. KUHNLEIN and J.L. SPENCER,1989 Absence of KELLER,1986 Useof highly polymorphic DNA probes for endogenous viral genes in an inbred line of Leghorns selected genotypicanalysis following bone marrow transplantation. for high eggproduction and Marek’s disease resistance. J. Blood 68: 378-385. Anim. Breed. Genet. 106: 2 17-224. KUHNLEIN,U., Y. DAWE,D. ZADWORNY andJ. S. GAVORA, GILL, P., A. J. JEFFREYS and D. J. WERRETT, 1985 Forensic 1989 DNAfingerprinting: a tool for determining genetic application of DNA fingerprints. Nature 318: 577-579. distances between strains of poultry. Theor. Appl. Genet. 77: GOWE,R. S., and R.W. FAIRFULL, 1980 Performanceof six 669-672. longterm multi-trait selected Leghorn strains and three control NAKAMURA,Y., M. LEPPERT, P. O’CONNELL,R. WOLFF,T. HOLM, strains, and a strain cross evaluation of the selected strains in, M. CULVER,C. MARTIN, E. FUJIMOTO,M. HOFF, E. KUMLIN Proceedings of the 1980 South Pacajk Poultry Science Convention, and R.WHITE, 1987 Variablenumber of tandem repeat Auckland, N.Z., pp. 141-162. (VNTR)markers for humangene mapping. Science 235: HUTT, F. B.,and R. K. COLE, 1957 Controlof leukosis in the 1616-1622. fowl. J. Am. Vet. Med. Assoc. 131: 491-495. STONE,H. A,, 1975 Useof highly inbred chickens in research. JEFFREYS,A. J., 1987 Highlyvariable minisatellites andDNA Tech. Bull. No. 1514, U.S. Department of Agriculture. fingerprinting. Biochem. SOC. Trans.15: 309-3 17. WETTON,J. H., R. E. CARTER,D. T. PARKIN andD. WALTERS, JEFFREYS, A. J., J. F. Y. BROOKFIELDand R.SEMEONOFF, 1987 Demographic study of a wild house sparrow population 1985 Positive identification of an immigration test-case using by DNA fingerprinting. Nature 327: 147-149. human DNA fingerprints. Nature 317: 818-819. WONG, Z., V. WILSON,A. J. JEFFREYS and S. L. THEIN, JEFFREYS, A. J., and D. B. MORTON,1987 DNAfingerprints of 1986 Cloning a selected fragment from a human DNA ‘fin- dogs and cats. Anim. Genet. 18: 1-15. gerprint’: isolation of an extremely polymorphic minisatellite. JEFFREYS, A. J., V. WILSONand S. L. THEIN,1985a Hypervariable Nucleic Acids Res. 14: 4605-4616. minisatellite regions in human DNA. Nature 314: 67-73. WYMAN, A., andR. WHITE, 1980 A highly polymorphic locus in JEFFREYS, A. J., V.WILSON and S. L. THEIN,1985b Individual- human DNA. Proc. Natl. Acad. Sci. USA 77: 6754-6758. specific fingerprints of human DNA. Nature 316 76-79. VASSART,G., M. GEORGES,R. MONSIEUR, H. BROCAS,A. S. LE- QUARRE and D. CHRISTOPHE, A sequence in M phage JEFFREYS, A. J., N. J. ROYLE,V. WILSON and Z. WONG, 1987 13 1988 Spontaneousmutation rates to new length alleles at detects hypervariable minisatellitesin human and animal DNA. tandem-repetitive hypervariable loci in human DNA. Nature Science 235: 683-684. 332: 278-280. ZAR, J. H., 1974 BiostatisticalAnalysis. Prentice-Hall,Englewood Cliffs, N.J. hNOWLTON, R. G., V. A. BROWN,J. C. BRAMAN,D. BARKER,^. W. SCHUMM, C. MURRAY, T. TAKVORIAN, J. RITZDONIS- and H. Communicating editor: W.-H. LI