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Limits to genetic rescue by outcross in pedigree dogs

Article  in  Journal of Animal Breeding and Genetics · June 2018

DOI: 10.1111/jbg.12330

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Received: 23 February 2018
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DOI: 10.1111/jbg.12330

ORIGINAL ARTICLE

Limits to genetic rescue by outcross in pedigree dogs

J.J. Windig | H.P. Doekes

Livestock Research, Animal Breeding

and Genomics, Wageningen University &
Research, Wageningen, the Netherlands
Correspondence
J.J. Windig, Wageningen University &
Research, Livestock Research, Animal
Breeding and Genomics, P.O. Box 338,
6700 AH, Wageningen, the Netherlands.
Email: jack.windig@wur.nl
Summary
Outcrossing should reduce inbreeding levels and associated negative effects in
highly inbred populations. In this study, we investigated the effectiveness of dif-
ferent outcrossing schemes using computer simulations. The inbreeding rate esti-
mated for a 25-year period of 2.1% per generation in a highly inbred dog breed
reduced to 1.8% when a single litter was produced by an outcross without back-
crosses. To reduce the inbreeding rate below 1%, more than eight of the 14 litters
born yearly in the recipient breed had to be outcrossed. However, outcrossing in
pedigree dogs is usually followed by backcrossing and generally involves one or
a few litters. Backcrossing reduced the effect of outcrossing considerably. When
two litters were produced by an outcross followed by one generation of backcross,
the inbreeding rate was 2.0% per generation. Continuously outcrossing was more
effective than a single or a few outcrosses. When each newborn litter during
25 years had a 5% chance of being produced by an outcross, the inbreeding rate
reduced to
0.2%. To investigate the possibility that new alleles were introduced
from the donor population into the recipient population, the fate of different type
of alleles (varying from completely lethal to beneficial) before and after an out-
cross was investigated by first simulating 80 years of natural selection prior to the
outcross and then different types of outcross. Because natural selection reduced
the frequency of lethal alleles before outcrossing, the introduction of a lethal allele
that was segregating in the donor breed but not in the recipient breed occurred
rarely. Introduction of slightly detrimental alleles or neutral alleles occurred more
frequently. In conclusion, outcrossing only had a limited short-term effect unless
repeated continuously. Nevertheless, it may help to buy time in which the popula-
tion structure can be changed so that the effective population size increases.

KEYWORDS
conservation genetics, dogs, genetic defects

1 | INTRODUCTION outcrossing has been proposed to restore genetic diversity

and to overcome a high prevalence of genetic disorders, for
In highly inbred populations, genetic diversity may be so example, in the Norwegian Lundehund (Stronen et al.,
low, and genetic disorders so common, that the only possi- 2017) and the Griffon Bruxellois (Knowler, Berg, McFa-
bility for the breed to survive is outcrossing with individu- dyen, La Ragione, & Rusbridge, 2016). In wild popula-
als from other populations. In pedigree dog breeds, tions, several examples exist where a single individual
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© 2018 The Authors. Journal of Animal Breeding and Genetics published by Blackwell Verlag GmbH.

238 | wileyonlinelibrary.com/journal/jbg J Anim Breed Genet. 2018;135:238–248.

WINDIG AND DOEKES
restored genetic diversity, for example, in the wolf (John-
son et al., 2010), in the Florida panther (Vila et al., 2003)
and in the common hamster (La Haye, Koelewijn, Siepel,
Verwimp, & Windig, 2012). Such successful outcrossing is
called genetic rescue. However, genetic rescue in wild pop-
ulations does not always seem to work (Tallmon, Luikart,
& Waples, 2004). For example, in the grey wolf, after ini-
tial positive effects of a single individual entering a highly
inbred population, the population is now again on the verge
of extinction (Hedrick, Peterson, Vucetich, Adams, &
Vucetich, 2014). According to Whiteley, Fitzpatrick, Funk,
& Tallmon, (2015), “Genetic rescue may not save imper-
illed populations over the long term . . . but can buy time
by improving fitness and increasing population sizes in the
short term.” In dogs, research is lacking on when outcross
can be successful or not.
Outcrossing can work because individuals from different
breeds are less or even unrelated, compared to individuals
within the breed. Inbred offspring are produced by mating
related individuals, and the more related the parents are,
the higher the inbreeding coefficient of the offspring. Unre-
lated individuals always produce offspring with an inbreed-
ing coefficient (F) of 0, regardless of their own level of
inbreeding. Consequently, when individuals from different
breeds are mated, their offspring will be less or even not
inbred. Inbreeding depression is mainly caused by recessive
deleterious alleles (Lynch & Walsh, 1998; Roff, 1997).
These alleles may reach relatively high frequencies through
genetic drift in populations with a small effective popula-
tion size. However, due to the random nature of genetic
drift, generally different alleles drift to high frequencies in
different populations, unless in rare cases when there has
been (indirect) selection in both breeds for the same delete-
rious allele, for example, when there is a closely linked
allele under selection. Thus, with an outcross generally, the
frequency of a specific deleterious allele with a high fre-
quency is, at least theoretically, expected to be reduced.
The downside of outcrossing is that the recipient breed
may lose its distinctiveness. Half of the genome of offspring
of an outcross originates from the donor breed, and conse-
quently, they may deviate phenotypically considerably from
the breed standard. To avoid changing the breed characteris-
tics too much, a related breed is used as donor breed and the
resulting offspring is carefully examined and selected
according to the breed standard, and then backcrossed with
animals of the recipient breed. This may be repeated for sev-
eral generations, and with each generation of backcrossing,
the contribution of the donor breed is halved. However, with
each backcross, the benefits of the outcross are also halved.
It is thus important to find the right balance between
outcrossing, backcrossing and mating within the breed itself.
Various aspects may influence the success of an
outcrossing scheme, including the number of backcross
| 239
generations, the number of litters produced by outcross,
whether all outcross litters are produced in a single genera-
tion and/or if outcrosses are repeated over time, and the
number of pups selected from outcross litters. It is not pos-
sible to experimentally determine variation in the outcome
of an outcross in pedigreed dogs, as repetition is hard to
realize. Therefore, we have to rely on theoretical results. In
this study, we used computer simulations to determine the
effects of different outcrossing schemes. We were particu-
larly interested in the average effect of different outcrossing
schemes on the mean level of inbreeding and inbreeding
rate, and in the possible variation around this average.
More in detail, we examined the effect of:
• The number of backcrosses after the initial outcross
• Repeated outcrosses
• Size of outcross: number of nests and pups selected for
further breeding
We also investigated to what extent alleles that are pre-
sent in the donor breed but absent in the recipient breed
are introduced into the recipient breed during an outcross.
Next to that, we investigated the opposite situation: to what
extent alleles that are present in the recipient breed but
absent in the donor breed are eliminated from the recipient
breed due to an outcross. These last simulations were done
with different outcross schemes for alleles with effect vary-
ing from completely lethal to beneficial.
2 | MATERIAL AND METHODS
The effects of outcrossing on inbreeding and allelic varia-
tion were assessed with a simulation program that was pre-
viously developed for the analysis of genetic management
in pedigreed dogs (Windig & Oldenbroek, 2015). The
Saarloos Wolfhond breed was used as a template for the
breed to be outcrossed. This is a Dutch breed originating
from a cross between a Wolf and a German Shepherd
made around 1935. The breed was recognized in 1975 by
the Dutch Kennel Club. Numerically, the breed has
remained small with at most around 25 litters born per
year. Inbreeding rates were high up to 10% per generation
between 1996 and 2001 (AVLS, 2012). Since then, several
genetic disorders appeared and litter size decreased. In
2016, one of the breeding organizations started an outcross
project to counter the negative effects of inbreeding.
2.1 | Simulations
Reproductive parameters extracted from pedigree data of
the breed from 2000 to 2010 were used to determine the
input for the simulations. In this period, the population
240
| WINDIG
consisted of on average 28 breeding females and 11 breed-
ing males, producing 14 litters per year with a total of 68
pups. The average age of parents at the birth of their pups
was 4.1 years (=generation interval), with a maximum of
9 years for fathers and mothers and a minimum of 2 years
for mothers and 1 year for fathers. Litter size was on aver-
age 4.9 pups with a maximum of nine pups. These values
were used as input for the simulation program. The breed
was simulated for 100 years, and each simulation was
repeated 50 times to determine variation across repeats.
Fifty runs were chosen after a trial run with 1000 runs and
determining the variation in inbreeding rates between
groups of 50 runs, which was maximally 0.14%. In all
these simulations, the breed was simulated without outcross
in the first 80 years, while in the last 20 years, outcrosses
could be simulated. Various outcross schemes, plus no out-
cross as a control, were simulated in these 20 years.
The simulation program consists of four steps [details in
(Windig & Oldenbroek, 2015)]. In the first step, the popu-
lation is set up according to data provided by the user. The
population size and age structure are kept constant, as is
generally the case in captive populations when no changes
occur in breeding policy or demand for (young) animals. In
the second step, animals are mated. Each year the same
number of litters is born (as determined by the user). To
produce a litter, a female is chosen at random from the
available breeding females and mated at random with a
breeding male. Females can have only one litter per year,
whereas males can have multiple litters. The distribution of
litter size is determined by input from the user and remains
the same throughout the simulation. In the third step, the
inbreeding level of newborn pups is calculated, as well as
their relatedness with all other animals alive in the popula-
tion. In the last step, old animals are culled and replaced
by young animals. All remaining animals become 1 year
older. Culling of animals is determined by the age struc-
ture, and which animals are culled within an age class is
determined at random. By specifying more age classes,
overlap of generations is increased. Culled animals are
replaced by one of the pups born that year, which one is
determined at random. Average inbreeding level per year
and average age of parents are produced as output from
which inbreeding rates per generation can be calculated.
The simulation program was extended to simulate sev-
eral (sub)populations simultaneously, with exchange
between the subpopulations determined by input of the
user. To simulate the outcross, two populations were simu-
lated: the Saarloos Wolfhond as recipient population and a
donor population. The donor population had the same age
structure and litter size distribution as the recipient popula-
tion but consisted of 50 breeding males and 50 breeding
females, and its effective population size was thus consid-
erably larger. Both populations were first simulated
AND DOEKES
separately for 80 years so that kinships and inbreeding
levels built up to different values in the two populations. In
the 80th year, the outcross was started.
Results of the simulation, in particular the inbreeding
rate and the genetic contribution of donor ancestors to the
recipient population, are evaluated for year 75–100, which
is about the time period breed associations consider for an
outcross project. Inbreeding rate (DF) is calculated over
these 25 years following Falconer and Mackay (1996)
using

1 !
1
F100 25
DF ¼ L 1

1
F75
where L is the generation interval and F75 and F100 the
average inbreeding level in the population in, respectively,
year 75 and 100. The genetic contribution of donor ances-
tors to an animal is calculated as the average of the donor
contribution of both parents. A pup resulting from a cross
between a 100% (=pure) donor father and 100% recipient
mother thus has a donor population contribution of 50%.
2.2 | Outcrossing schemes
Several outcrossing schemes were simulated, which dif-
fered in the number of outcrosses and the combination with
backcrossing (Figure 1).
2.3 | Single outcross, no backcross
The simplest scheme was a single outcross whereby a sin-
gle litter was sired by a father from the donor population.
Pups produced by the outcross were treated as all other
pups. This implied that, in the year following the outcross,
culled animals were replaced with pups from either out-
cross or non-outcross matings (both with same probability).
Next, the number of litters sired by fathers from the donor
population was increased, up to all 14 litters in that year
being sired by the donor population.
2.4 | Single outcross, with backcross
The second simulated scheme was an outcross followed by
one or more backcross generations. In the first year of the
outcross event, either two or four litters were sired by a
father of the donor population. From each of the litters pro-
duced, two pups were selected for breeding in the next year
and mated with a sire or dam from the recipient population.
If only one pup was born (9% of the litters), just that pup
was selected. As the number of litters born from females of
the recipient breed remained constant, less litters were born
with both parents from the recipient breed in years where
females were used in the outcrossing scheme. The initial
WINDIG AND DOEKES
F I G U R E 1 Schematic representation of four types of outcross
schemes simulated. Donor population (on left) consisted of 50 males
and 50 females producing 25 litters annually, recipient population
consisted of 11 males and 14 females producing 14 litters. [Colour
figure can be viewed at wileyonlinelibrary.com]
outcross was followed by 1, 2, 3 or 4 years of backcrosses.
Pups produced by the final backcross were treated as all
other pups.
2.5 | Repeated outcross
In these schemes, the effect of repeating an outcross was
simulated. This was done for an outcross involving two lit-
ters, either not followed by a backcross, or followed by
two backcrosses. An outcross was repeated either once or
twice. The first outcross started in year 80, the first repeat
in year 85 and the second repeat in year 90.
2.6 | Continuous outcross
Continuously outcrossing was the last scheme simulated. In
this case, each year all litters in the recipient population
had a 1% chance of being sired by a father from the donor
population. Consequently, in 20 years’ time with 14 litters
per year, on average 2.8 litters were sired by a father from
the donor population, which is about the same number as
| 241
produced in a single outcross of a single litter repeated
three times. The main difference is that with the continuous
outcrossing scheme more or less than three outcrosses may
occur and that these outcrosses are randomly distributed
over the years. Continuously outcrossing was simulated to
mimic the situation in which occasionally crossings are
allowed, or done secretly, with dogs without an official
pedigree (so-called look-a-likes). Continuously outcrossing
was also simulated with a 5% and a 10% chance of being
sired by a father from the donor population.
2.7 | Fate of alleles
In addition to the effect of outcrossing on inbreeding, we
evaluated the fate of alleles. For this purpose, the simula-
tion software was expanded with biallelic loci without link-
age between loci. At the start of the simulation, each
animal received two alleles per locus, where the probability
that a specific variant occurred was based on its starting
allele frequency (provided by the user). In the following
years, each newborn pup received at random one allele
from each parent. In the first 80 years of the simulation
without outcross, allele frequencies changed because of nat-
ural selection and genetic drift so that frequencies built up
to realistic frequencies depending on their influence on fit-
ness.
Five different types of alleles were simulated in this
research: (i) neutral alleles, (ii) detrimental alleles which
reduced survival of newborn pups by 20% when
homozygous, (iii) beneficial alleles which were the com-
plement of detrimental alleles and thus increased pup
survival by 25% when hetero- or homozygous compared
to homozygous non-beneficial alleles, (iv) lethal alleles
for which animals died at 3 years of age when homozy-
gous, and (v) completely lethal alleles for which all
homozygous animals died at birth. Mating and inheri-
tance of alleles were random, but selection against alleles
occurred because pups being homozygous for lethal alle-
les died (either at birth or after 3 years or with a chance
of 20% for depending on the type). Simulations were
first performed with the allele only segregating in the
recipient population and absent in the donor population.
This was done to determine whether an outcross could
eliminate or decrease the frequency of the allele. Next,
simulations were performed with the allele segregating
only in the donor population and absent in the recipient
population. This was done to determine how often the
allele was introduced by outcross into the recipient popu-
lation and, if so, which frequency it could reach in the
next 20 years. To attain realistic frequencies of the dif-
ferent alleles, simulations were first run for 80 years
without outcross, so that selection and genetic drift chan-
ged the initial frequencies. All alleles started at a
242
| WINDIG AND DOEKES

frequency of 50%, and the expectation was that selection T A B L E 1 Inbreeding rate (DF in %; between year 75 and year
decreased the frequency of the detrimental and lethal 100) and genetic contribution of donor population in year 100 for a
alleles and increased the frequency of the beneficial alle- single outcross, not followed by backcrosses. Outcross took place in
les. Changes due to genetic drift were random in direc- year 80 where one or more males from a donor population were used
to produce one or more litters
tion and larger in the recipient population than in the
donor population, due to the larger effective population DF and Ne year 75 to 100 Donor contribution
size of the latter. For each allele type, five loci were (%) in year 100 (%)
Number
simulated and simulations were repeated 25 times, so of litters Mean (95% c.i.) Ne Mean (Min.–Max.)
that, in total, 125 alleles per type were simulated. Thus, 0 2.1 (1.0–3.0) 25 0.0 (0–0)
in a given simulation, five unlinked loci of the same
1 1.7 (0.7–2.8) 29 1.5 (0–6.7)
type were simulated.
2 1.7 (0.5–2.8) 29 2.7 (0–15.3)
Allele frequency changes were evaluated for two of the
simulated outcrossing schemes. The first was the outcross 4 1.4 (0.2–2.6) 37 4.3 (0–15.6)
involving two litters, followed by two backcrosses and 8 0.9 (
0.7 to 2.5) 54 7.7 (0–23.3)
repeated twice, as this is the type of outcross the breed 14 0.1 (
1.5 to 1.8) 398 13.2 (0.9–29.4)
society is considering. The second type was the continuous
outcross where each litter had a 10% chance to be sired by
a father from the donor population, as this was the most on average 13.2% when all 14 litters were outcrossed.
efficient type analysed. The variation in donor contribution was always large,
with for all number of litters, except all 14 litters out-
crossed, runs occurring where the donor contribution
3 | RESULTS was completely eliminated.
3.1 | No outcross
The inbreeding rate in the recipient breed (the simulated
3.3 | Single outcross, with backcrossing
Saarloos Wolfhond) without outcross was 2.1% per genera- A single outcross followed by one or more generations
tion, resulting in an average inbreeding level of 0.36 in of backcrossing decreased the effect of the outcross, or
year 75 and 0.45 in year 100 (Figure 2). As expected, the even resulted in higher inbreeding rates compared to no
inbreeding rate in the donor population was with 0.8% con- outcross (Table 2). In case of two litters being out-
siderably lower. Inbreeding rates varied considerably across crossed, there was hardly an effect of outcrossing when
simulations, for the recipient population between 1.5% and followed by one generation of backcrossing (average
3.2%, and for the donor population between 0.7% and inbreeding rate decreased from 2.1% to 2.0%), no effect
1.0%. The simulation thus confirmed that inbreeding rates with two or three backcrosses and a slight increase (to
for the recipient population will be high if no measures are 2.2%) with four backcrosses. For all these schemes, the
taken. variation in inbreeding rates across simulation runs was
large, with rates ranging from approximately 1%–3%. In
case four litters were outcrossed, the inbreeding rate still
3.2 | Single outcross, no backcrossing decreased when one or two backcrosses were used, but
A single outcross, with a single male producing a sin- increased with three or four backcrosses. Variation across
gle litter and no backcross, reduced the inbreeding rate runs was always large, especially in the scheme with
to on average 1.7% (Table 1). However, there was con- four backcrosses, where the maximum inbreeding rate
siderable variation between simulation runs, with a min- was 3.2%. With each backcross, the average donor con-
imum inbreeding rate of 0.3% and a maximum of 3.2%. tribution to the recipient population decreased, down to
Outcrossing with more males further reduced average 0.5% or less when three or more backcrosses were used.
inbreeding rates, down to 0.1% when all 14 litters in Complete elimination of donor contribution occurred in
the breed in that year were the result of an outcross. all backcross schemes, while the maximum observed, for
Variation in inbreeding rates was considerable. In a few an outcross involving four litters followed by one back-
cases, for eight and 14 litters outcrossed, the inbreeding cross, was just above 9%.
level even decreased. On the other hand, inbreeding
rates well above 2% occurred as well, for up to eight
litters produced by outcross. The genetic contribution of
3.4 | Repeated outcross
the donor population to the recipient population Repeated outcrossing reduced inbreeding rates further
increased with the number of litters outcrossed, up to (Table 3). When the outcross was carried out three times
WINDIG AND DOEKES | 243

T A B L E 2 Inbreeding rate (DF) and DF year 75 to 100 Donor contribution in

level (F) for a single outcross followed by Number of
(%) year 100
one or more generations of backcrossing. Number backcross
Outcross took place in year 80 where two of males generations Mean (95% c.i.) Ne Mean (Min.–Max.)
or four males from a donor population 0 no outcross 0 2.1 (1.0–3.0) 24 0.0 (0–0)
were used to produce four litters. From 2 0 1.7 (0.5–2.8) 29 2.7 (0–15.3)
each litter, two pups were selected and
1 2.0 (0.8–3.2) 25 1.1 (0–6.5)
backcrossed in the next year to the
recipient population 2 2.1 (1.1–3.1) 25 0.7 (0–3.2)
3 2.1 (1.3–3.1) 24 0.2 (0–1.4)
4 2.2 (1.2–3.1) 22 0.1 (0–0.5)
4 0 1.4 (0.2–2.6) 35 4.3 (0–15.6)
1 1.6 (0.4–2.8) 31 2.8 (0–9.4)
2 2.0 (0.9–3.1) 25 1.4 (0–4.0)
3 2.1 (1.2–3.0) 23 0.5 (0–1.9)
4 2.2 (1.2 - 3.1) 22 0.4 (0 –1.1)

T A B L E 3 Inbreeding rate (DF) and Number of DF year 75 to 100 Donor contribution in

level (F) for schemes without or with one backcrosses (%) year 100 (%)
or more outcrosses. In each scheme, two following each Number of
males produced two litters from which outcross outcrosses Mean (95% c.i.) Ne Mean (Min.–Max.)
two pups per litter were selected to be
0 no outcross 0 2.1 (1.0–3.0) 24 0 (0–0)
backcrossed or added to the pool of
breeding animals 0 1 1.7 (0.5–2.8) 29 2.7 (0–15.3)
2 1.5 (
0.1 to 3.0) 34 3.8 (0–12.3)
3 1.0 (
0.6 to 2.7) 48 6.1 (0–18.0)
2 1 2.1 (1.1–3.1) 24 0.7 (0–3.2)
2 1.9 (0.7–3.1) 26 1.3 (0–8.9)
3 1.8 (0.9–2.8) 28 1.8 (0–4.9)

with two litters being outcrossed, the inbreeding rate without
backcrossing reduced to 1.0% (Table 3, Figure 3). The
reduction was smaller when the outcross was followed by
two backcrosses. The average inbreeding level was then
1.8% for three repeats. In all cases, variation across
runs was considerable and inbreeding rates above 2.5%
occurred. The contribution of the donor population to the
recipient population in year 100 increased, compared to no
repetitions. In some runs, however, complete elimination of
the donor contribution occurred for each scenario evaluated.
3.5 | Continuous outcrossing
Continuously crossing some animals with the donor popu-
lation reduced inbreeding rates and in most cases also
inbreeding levels (i.e., inbreeding rate <0). With a 1%
chance for a litter being sired by a male of the donor popu-
lation, so on average once every 7 years an outcross litter,
the inbreeding was on average comparable to one or two
single outcrosses. With 5% or 10% exchange, inbreeding
rates were negative on average (Table 4). Variation in
inbreeding levels across runs was considerable with
continuous outcrossing. For a 10% outcrossing chance, the
inbreeding level immediately dropped to the level in the
donor population in some runs, whereas in others, the
inbreeding level in year 100 was still larger than that in the
donor population (Figure 3). The contribution of the donor
population was with a 10% outcrossing rate on average
25.2% in year 100 with a minimum of 12.5% and a maxi-
mum of 45.5%.
3.6 | Allelic diversity
3.6.1 | Alleles originally segregating in
recipient population only
In the first 75 years of the simulation, allele frequencies
in the recipient population changed because of genetic
drift and natural selection. The frequency of neutral alle-
les on average hardly changed (0.506 in year 75, com-
pared to the initial 0.5). There was, however,
considerable variation in allele frequency between differ-
ent loci, from 0% (eliminated) to 100% (fixed), with all
intermediate frequencies occurring at about the same rate
244
| WINDIG AND DOEKES

F I G U R E 2 Inbreeding level (F) for recipient population (top

lines) and donor population (bottom lines) across years without
outcrossing. Average (solid lines), minimum (dotted lines) and F I G U R E 3 Inbreeding levels in the recipient population without
maximum (dashed lines) of all 50 runs. [Colour figure can be viewed outcross (solid squares), with outcross in year 80 followed by two
at wileyonlinelibrary.com] backcrosses and repeated in year 85 and year 90 (open squares), and
with a continuous outcrossing scheme from year 80 onwards (open
T A B L E 4 Inbreeding rate (DF) and level (F) for a continuous circles). Average of 50 simulation runs is shown. In the repeated
outcrossing scheme. In each generation, a litter from the recipient outcross scheme, 2 out of 14 litters per outcross were sired by a
population has a chance of 1%, 5% or 10% to be sired by a male father from the donor population. In the continuous scheme, each
from the donor population litter had a 10% probability of being sired by a father from the donor
DF year 75 to 100 Donor contribution population. [Colour figure can be viewed at wileyonlinelibrary.com]
Percentage litters
(%) in year 100 (%)
sired by continued to occur and after the outcross, 24% of the
donor breed Mean (95% c.i.) Mean (Min.–Max.) beneficial alleles were fixed versus 28% without outcross.
0 no outcross 2.1 (1.0–3.0) 0 (0–0) The percentage of lethal allele that was eliminated was
1% 1.7 (
0.3 to 3.4) 2.7 (0–13.2) the same with and without outcross. Changes in frequen-
5%
0.2 (
2.6 to 2.1) 14.6 (0–35.5) cies were larger when the recipient population was con-
tinuously outcrossed to the donor population. For
10%
1.8 (
3.6 to 0.0) 25.2 (12.5–45.3)
example, none of the beneficial alleles remained fixed
after continuous outcrossing, and 91.2% of the lethal
(Figure 4). The completely lethal allele on the other hand alleles were eliminated (Figure 4).
disappeared for 70.8% of the simulated alleles and its
average frequency was 0.034 in year 75. When the allele
3.6.2 | Alleles originally segregating in donor
was lethal after 3 years, it disappeared for 63.5% of the
population only
simulated alleles The detrimental allele disappeared for
29.4% of the simulated alleles, and the beneficial allele In the simulations where the allele initially segregated in
was never lost. The beneficial allele was, however, fixed the donor population, the average frequency after 75 years
in 17.9% of the cases, while alleles of all other types was very similar to that in the recipient population for sim-
were never fixed before outcrossing. ulations in which the allele initially only segregated in the
When no outcross was performed, genetic drift and recipient population (Figure 5, first row). The variance,
natural selection continued to change allele frequencies. however, was lower, in line with the smaller effective pop-
More alleles were eliminated for all types except benefi- ulation size and higher genetic drift in the recipient popula-
cial alleles, for which more alleles became fixed. For tion.
example, the percentage of eliminated lethal alleles After a repeated outcross (39 outcross, each followed
increased from 70.8% after 75 years to 91.2% after by two backcrosses) to the recipient population where the
100 years. The percentage of beneficial alleles that were allele was absent, the allele remained absent in the recipient
fixed increased from 17.9% to 28% without an outcross. population in most cases. When outcrossing occurred, con-
Allele frequencies after a repeated outcross (39 outcross, tinuously alleles were more often introduced into the recipi-
each followed by two backcrosses) with a donor popula- ent population. Beneficial alleles were introduced in all
tion in which the allele was absent, were very similar to simulations. On the other hand, the lethal allele remained
the frequencies observed without outcross. For example, absent in 98.2% of the simulations after 39 outcross fol-
the beneficial allele disappeared in only one of the simu- lowed by two backcrosses, and in 80.4% after continuous
lations after the outcross, while fixation by selection outcrossing.
WINDIG AND DOEKES
4 | DISCUSSION
In this study, we evaluated the effect of different outcross-
ing schemes on the level and rate of inbreeding and on the
fate of neutral, beneficial, lethal and detrimental alleles. In
general, outcrossing had a limited effect on inbreeding
levels. Especially when an outcross was followed by one
or more backcrosses, the effect was small. When four back-
crosses were used, the average inbreeding level even
increased somewhat. This was due to the smaller number
of litters available for pure breeding. When outcrossing
was followed by backcrossing, it had to be repeated (in
case of two outcrossed litters at least twice) to have a nota-
ble effect. Inbreeding rates below 1% only occurred with
continuous outcrossing.
The effect of outcrossing is often limited despite the
fact that the F1 has an inbreeding level of 0%. There are
several reasons for this limited effect. The main reason is
that the effect of the outcross is halved with each genera-
tion of backcrossing. For example, if the F1 is crossed with
a purebred dog, three of the four grandparents are from the
recipient population, and the expected inbreeding level is
half the kinship before the outcrossing. The second reason
is that when the effective population size remains small,
the inbreeding rate returns to the level before outcrossing.
In cases where a large proportion of the animals is out-
crossed, the number of purebred dogs decreases, and when
only a few remain for the backcrosses the effective popula-
tion size may even decrease, as was the case when an out-
cross was followed by four generations of backcrossing.
The inbreeding rate varied widely after outcrossing. In
the simulations, mating and survival after an outcross were
random, and consequently, reproduction of descendants of
the outcross varied across runs. In some runs, descendants
were hardly used and this resulted in higher inbreeding
rates, and complete elimination of donor alleles. In other
runs, descendants were frequently used and inbreeding
rates were lower and donor contributions higher. It is thus
important to manage the population after an outcross in
order to increase the effective population size. There are
several options for genetic management, but one of the
most effective is the mean kinship method, that is, to
exclude animals from breeding that have a higher than
average kinship with all other candidates for breeding
(Lewis & Windig, 2017). This method may be particularly
effective after an outcross, because descendants from an
outcross will initially have a low mean kinship, but once
the genetic contribution of a dog used in an outcross has
reached a too high value, its descendants can be excluded
from breeding. We tested the mean kinship method for the
scenario of two outcrosses each followed by three back-
crosses, for which we simulated the mean kinship method
after the last backcross, thus from year 92 to 100. The
| 245
inbreeding rate of 1.8% without mean kinship (Table 3, last
line) reduced to on average 1% (with a 95% c.i. of 0.2%–
1.8%), while the contribution from the donor population
increased to 4.4% on average, ranging from 0.5% to 4.0%.
Thus, good genetic management after an outcross is needed
to profit from the introduced genetic diversity.
Inbreeding rates and effective population sizes vary
across breeds (Wijnrocx, Francois, Stinckens, Janssens &
Buys, 2016). The Saarloos Wolfhond simulated in this
study is at the lower end of the effective population sizes
and was chosen because typically outcrosses are initiated
for such breeds. For breeds with a higher effective popula-
tion size, an outcross will even have less effect. An unre-
lated breed was simulated as a donor breed. In practice,
related breeds will be used. Consequently, the effect of an
outcross may even be lower than simulated in this study.
However, for pedigreed dogs, even dogs of related breeds
have never been allowed as parent so common ancestors
occur many generations ago. For example, the Saarloos
Wolfhond simulated in this study, originated more than
80 years ago, which is on average 20 generations ago. So
consequently, the diminished effect of outcrossing with a
related breed instead of an unrelated breed on inbreeding
levels and rates after an outcross will be marginal.
When outcrossing is used with the aim to eliminate (or
introduce) one or more alleles in the population, genomics
may help to determine which animals to use in backcross-
ing. Procedures have been developed for such genomic-
assisted introgression (Hospital, Chevalet, & Mulsant,
1992; Wall, Visscher, Hospital, & Woolliams, 2005) and
may speed up the process. Using genomics to support
outcrossing with the general aim to increase the genetic
diversity and prevent further inbreeding depression will be
more challenging. Ideally, unwanted alleles should be
replaced without introducing other unwanted alleles. How-
ever, identifying genes of interest is a major challenge.
After identifying a breeding goal such as an index that
combines the breed standard with longevity and fertility,
combining outcrossing with genomic selection may be the
best way forward in this respect as introgression combined
with genomic selection proved in simulations to be suc-
cessful in a breeding program combining selection for
genetic merit with introgression of a single allele (Gaspa,
Veerkamp, Calus, & Windig, 2015).
The unwanted introgression of (unknown) deleterious
alleles from a donor breed is often used as an argument
against outcrossing. Since probably all animals carry reces-
sive deleterious alleles, this may indeed be the case. How-
ever, the opposite, introgression of beneficial alleles is
possible as well. The chance that different alleles are pre-
sent in the donor breed depends on how closely related the
breed is. The more closely related the breed, the larger the
chance that the same alleles are present, unwanted or not.
246
| WINDIG AND DOEKES

F I G U R E 4 Allele frequency in recipient and donor population before outcrossing and after 25 years in recipient population with no
outcrossing, outcrossing three times with two backcross generations or with continuous outcrossing for different types of alleles. Each panel is
based on 25 simulations with five alleles per simulation. Starting frequency distribution in the donor population is shown in the leftmost column.
Second column contains starting frequency in the recipient population. The middle column shows the frequency after 25 years in the recipient
population without outcrossing, the 4th column shows frequency after three repeats of an outcross, each followed by two backcrosses. The
rightmost column shows frequency after 25 years continuously outcrossing in 33% of matings. Within each graph, the leftmost bar (red) is the
frequency of alleles eliminated from the population, the rightmost bar (green) fixed in the population, and intermediate bars (blue) are
segregating alleles. [Colour figure can be viewed at wileyonlinelibrary.com]

Consequently, the introduction by outcross of alleles not
present in the breed may be even lower than simulated in
this study, and frequencies of introduced alleles derived
here can be seen as an upper limit.
Another important aspect to realize is that natural selec-
tion in the past will have influenced current frequencies so
that deleterious alleles generally occur in lower frequencies
than beneficial alleles. In this study, we show that because
of this, it is for alleles present in the donor breed and
absent in the recipient breed in fact more likely to
introduce beneficial alleles than deleterious alleles. Lethal
alleles can only remain at low frequencies in a population
with a high effective population size, because of selection
against them at higher frequencies.
5 | CONCLUSION
Unless outcrossing is repeated regularly, or continuously,
it has a limited effect on inbreeding and allele
WINDIG AND DOEKES | 247

F I G U R E 5 Allele frequency in donor and recipient population before outcrossing and after 25 years recipient population outcrossing three
times with two backcross generations or with continuous outcrossing for different types of alleles The allele frequency before the outcross was
0% in the recipient population. Starting frequency distribution in the donor population is shown in the leftmost column. Other details as in
Figure 4. [Colour figure can be viewed at wileyonlinelibrary.com]

frequencies. Although initially pups are born with
inbreeding coefficients of 0, inbreeding levels and rates
can quickly return to previous levels. It thus remains
important to increase the effective population size of the
recipient breed with other methods. An outcross may
help to reset inbreeding levels to lower levels as a start-
ing point to permanently reduce inbreeding rates, for
example, by changing the population structure. In this
respect, outcrossing in pedigree dogs can buy time by
improving fitness in the short term. This time then
should be used wisely by setting up genetic management
that increases the effective population size more perma-
nently. The introduction of unwanted alleles from a
donor population into the recipient population can occur;
however, alleles with negative effects of fitness will
generally have low frequencies in the donor population
due to natural selection.
ACKNOWLEDGEMENTS
We are grateful to the Dutch breed society of the Saarloos
Wolfdog (AVLS) for discussions, information on popula-
tion size and plans for outcrossing in the breed. Thanks are
also due to Manon Pippel who ran some of the initial sim-
ulations for her MSc project.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest
regarding the publication of this article.
248
| WINDIG
ORCID
J.J. Windig http://orcid.org/0000-0002-7485-7424
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How to cite this article: Windig JJ, Doekes HP.
Limits to genetic rescue by outcross in pedigree
dogs. J Anim Breed Genet. 2018;135:238–248.
https://doi.org/10.1111/jbg.12330