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1 Introduction 83
2 The social and genetic architecture of a honeybee colony 84
2.1 Biological level 1: the gene 85
2.2 Biological level 2: the individual 87
2.3 Biological level 3: the patriline 89
2.4 Biological level 4: the colony 91
2.5 Implications of the four biological levels for honeybee breeding 92
2.6 The architecture of disease resistance 92
3 Biological properties of honeybees that facilitate breeding 94
4 Biological properties of honeybees that hinder breeding 97
4.1 Mating behaviour 97
4.2 Inbreeding and the sex locus 98
5 Methods to address the difficulties in honeybee breeding 100
5.1 Marker-assisted selection 102
5.2 Increasing control of mating 104
6 Minimizing inbreeding and loss of brood viability 105
7 Success stories 107
8 Concluding remarks 108
References 108
1 Introduction
The western honeybee (Apis mellifera) has been utilized by humans for honey
and wax production for millennia, yet unlike most other domesticated animals,
the biology, physiology and behaviour of domestic honeybees have changed
little during this time. For most of this period, bees were not managed so much
as kept: people provided rudimentary containers (which were often destroyed
during honey harvesting), and hoped that wild bee colonies would take up
residence (Crane, 1983). Active management and manipulation of honeybee
colonies has only become possible more recently due to a few transformational
developments in husbandry. These were the discovery of the bee space and
ADVANCES IN INSECT PHYSIOLOGY VOL. 39 # 2010 Elsevier Ltd. All rights reserved.
ISBN 978-0-12-381387-9
DOI: 10.1016/S0065-2806(10)39003-5
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invention of the moveable frame hive in 1851 (Langstroth, 1853), the honey
extractor in 1867 (Langstroth, 1867) and the queen excluder in 1849 (Dadant,
1975).
The first significant advance in bee breeding was the development of proce-
dures that allowed the production of large numbers of queens from a single
queen mother (Doolittle, 1889). For the first time, it was possible to produce
large numbers of queens from superior colonies. The discovery that queens and
drones mate in flight outside the hive (Huber, 1814) had important implications
for honeybee breeding, for it showed that paternity was random. Isolation of
desired breeding colonies occurred at least as early as 1928 (Weatherhead,
1986). Complete control of mating was made possible by the development of
instrumental insemination (II) by Watson in 1927, though it was not until
additional developments by Laidlaw in 1944 that instrumental insemination
became routinely reliable (Laidlaw, 1944; Cale and Rothenbuhler, 1975).
Application of the techniques of queen propagation and artificial insemina-
tion has permitted the establishment of bee breeding programmes. Most bee
breeding programmes have focussed on honey production, temperament and
disease resistance (for descriptions and examples, see Rothenbuhler, 1958;
Ruttner, 1988; Szabo, 1988; Manning, 1996; Rinderer et al., 1999; van
Engelsdorp and Otis, 2000; Rinderer et al., 2001; Spivak and Reuter, 2001b;
Harbo and Harris, 2005; Harris, 2008). Sadly, few bee breeding programmes
have been successful in the long term, constrained by limited progress in trait
improvement, the detrimental effects of inbreeding and poor returns on invest-
ment. However, recent advances in honeybee genetics (Weinstock et al., 2006;
Bienefeld et al., 2007; Oldroyd and Thompson, 2007) have allowed greater
understanding of the genetic architecture of the honeybee colony, and have
provided new opportunities to utilize novel genetic techniques for the enhance-
ment of honeybee breeding. These advances may usher in a new era for bee
breeding in which cheaper and more refined molecular methods are used to
identify and propagate superior individuals.
A comprehensive review of honeybee genetics and its implications for breed-
ing was last undertaken by Rinderer (1986). In light of the new technologies that
have arisen since then, a new review is timely. Here, we consider the social and
genetic architecture of honeybee colonies, and how this relates to issues of
honeybee genetic improvement. We also review recent developments in honey-
bee genomics and the promise that these hold for future efforts in bee breeding.
Honeybee breeding requires identifying colonies that show superior traits and
ensuring that the alleles that contribute to these traits are passed on to the next
generation. However, the relationships between genes and desired commercial
traits are more complex in honeybees than in other livestock.
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Colony
Queen Drone fathers
First generation
0.5 1.0
Second generation
0.5 0.25 0.75 0.75
Honeybee colonies are not a single individual, but a family of many related
individuals (Fig. 1). The performance of a colony is therefore dependent upon
the work of its many members and the interactions among them. Furthermore,
a honeybee colony can be considered to consist of four levels of biological
organization (sensu Reeve and Keller, 1999) and genetic architecture: the gene,
the individual, the patriline and the colony (Fig. 2). Each level of biological
organization can be considered to express a set of observable characteristics that
are analogous to the phenotype of an individual organism. This phenotype
arises from the influence of lower biological levels (forward effects, Fig. 2),
environmental effects (including feedback from higher and lower biological
levels) and interactions within each level.
Forward effects of
phenotype:
Resource availability
Climate
Backward effects
Pests/diseases
(environmental):
FIG. 2 The biological organization of a honeybee colony. Direct effects of each biological level are indicated with vertical green arrows.
Influences of lower levels on higher levels is indicated by horizontal green arrows. Environmental effects and influences of higher levels on lower
levels are indicated by horizontal blue arrows, while interactions within a biological level are indicated by vertical blue arrows. (For interpretation
of the references to colour in this figure legend, the reader is referred to the Web version of this chapter.)
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Lobo et al., 2003; Arechavaleta-Velasco and Hunt, 2004), body size (Hunt
et al., 1998), age of first foraging and foraging preference (Hunt et al., 1995;
Page et al., 1998; Page et al., 2000; Rueppell et al., 2004; Ruppell et al., 2004;
Rueppell, 2009), hygienic behaviour (Oxley et al., 2010a), worker sterility
(Oxley et al., 2008) and sex determination (Beye et al., 2003).
One of the most extensively studied behavioursforaging behaviourhas
revealed a complex genetic architecture, which is influenced by multiple loci of
moderate to small effect (Page et al., 2000; Rueppell et al., 2004; Ruppell et al.,
2004), and which exhibit significant dominance and epistatic effects. Further-
more, these QTL have been shown to affect multiple traits (Hunt et al., 2007), a
fact further reinforced by studies showing significant genetic correlations
between a variety of traits related to foraging and food storage (Collins et al.,
1984; Rueppell et al., 2004).
A negative genetic correlation between two traits (e.g. between hoarding and
time to sting; see Collins et al., 1984) means that a beneficial change in one trait
will lead to a detrimental change in the other. A trade-off between two traits will
reduce the rate or even limit the extent to which the traits can be improved by a
breeding scheme. Negative correlations between traits may exist as a result of
either linkage or pleiotropy. Linkage arising from non-random mating (such as
the introduction of unrelated individuals into a population) decreases with each
successive generation after mixing (Hazel, 1943). Pleiotropy, in contrast, will
permanently hinder the breeders ability to improve one trait without altering
others affected by the same genes. As any pleiotropic genes that cause a positive
genetic correlation quickly reach fixation in a breeding population under selec-
tion (Ruane and Colleau, 1995), pleiotropic genes that cause a negative genetic
correlation increase in their relative impact on the ability of the breeder to
improve performance over time (Lerner, 1950).
Factors affecting gene expression operate at different biological levels. Lar-
vae exposed to pathogens upregulate a number of antimicrobial peptides
(Evans, 2004). Genes regulating worker maturation and behaviour are influ-
enced by the social environment of the colony (Grozinger et al., 2003). Most
significantly, the diet fed to young female larvae influences the expression of
many genes (Evans and Wheeler, 1999; Evans and Wheeler, 2000; Hepperle
and Hartfelder, 2001), which in turn determines the developmental fate of the
offspring. Recent work suggests DNA methylation may also have a significant
role in determining gene expression in relation to caste determination
(Kucharski et al., 2008; Elango et al., 2009).
Differences in the tasks performed in the colony by each caste are reflected in
their differences in size (Winston, 1987), morphology and longevity (Page and
Peng, 2001).
Drones do not contribute to colony-level traits of economic importance,
except, perhaps, in the negative sense of resource consumption. However,
they do influence a colonys reproductive fitness as determined by the number
of drones that successfully mate and contribute genes to the next generation.
The size of the population of drones within a colony is highly variable, with
numbers varying between zero and several thousand, depending on the time of
year and condition of the colony (Allen, 1958, 1963; Free and Williams, 1975).
The number of drones produced by colonies selected for breeding is particularly
important when queens are allowed to mate in areas containing unselected
colonies.
A honeybee colonys queen, with few exceptions (Anderson, 1963; Barron
et al., 2001), is the sole reproductive female in a colony. The rate at which the
queen is able to produce eggs therefore influences the growth of a colony.
Workers are facultatively sterile, activating their ovaries only when their colony
becomes queenless and broodless. This division of reproductive tasks has
allowed the evolution of strongly divergent caste traits, with queens and workers
expressing physiological adaptations suited to their respective roles.
The worker population can be subdivided according to the tasks currently
being performed by individual workers. These include nursing, comb building,
cell cleaning, nest defence and foraging (Seeley and Kolmes, 1991). Workers
progress through these tasks in an age-dependent manner (mediated particularly
through titres of juvenile hormone and octopamine circulating in the blood;
Robinson, 1987; Huang and Robinson, 1992; Sullivan et al., 2000; Schulz et al.,
2002). The rate at which workers progress through these tasks is influenced by
the individuals genotype (Page and Robinson, 1991; Ben-Shahar et al., 2002;
Jones et al., 2004) and by the number of workers that are available to perform
the task (Robinson, 1987; Leoncini et al., 2004; Chapman et al., 2007a).
Honeybee colonies have two generations present for most of their exis-
tencethe reproductive queen and her worker offspring. Through the pro-
duction of pheromones, the queen influences worker behaviour such as the
age at which workers commence foraging (Pankiw et al., 1998), foraging
activity (Jaycox, 1969), the rearing of replacement queens (Pettis et al., 1995;
Melathopoulos et al., 1996) and the propensity to swarm (Phamdelegue et al.,
1993; Pankiw et al., 1998; Beggs et al., 2007). The interactions that arise
between castes contribute significantly to colony-level phenotype (Oldroyd
et al., 1990). Because phenotypic correlations between worker and queen
effects on some traits are negative (Bienefeld and Pirchner, 1991), gains made
from selecting on colony-level phenotype alone are less than gains made with
selection accounting for effects of both castes (Bienefeld and Pirchner, 1991).
Drones that mate with a queen do not contribute any genetic material to the
drones produced by a colony, and his worker offspring are normally sterile.
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Thus, a drones genetic legacy is mostly via the fathering of new queens. Each
queen produced will only inherit the genetic contribution of one of the drones
that mated with her queen mother. As a result, only 1/k (where k is the effective
number of matings; Boomsma and Ratnieks, 1996) of the paternal genetic
contribution to a colonys performance is inherited by any one offspring
queen, and only half of this is subsequently represented in her worker offspring.
Without identification of a virgin queens paternity, her predicted breeding
value is inaccurate. After mating, the predicted performance of a queens
worker offspring is affected by genotypes of the drones she mates with. New
queens may even be the offspring of unselected drones, potentially undoing
previous benefits gained from selection. If the contribution of the drones to
colony performance is ignored, and evaluation is based solely on the merit of the
mother, the selection response achieved in a programme will be significantly
decreased (Henderson, 1984).
FIG. 3 Coefficients of genetic relationship between a worker and the possible drone
offspring of a colony. Numbers indicate the average relatedness between the individual
and the reference worker (top left). Arrows indicate pathways of inheritance of genetic
material.
0.4
0.35
Average relatedness
0.3 Relationship
0.25 of worker
to: queens
0.2
0.15 Worker
0.1 offspring
0.05
0
0 5 10 15 20 25
Number of drone fathers
(Jones et al., 2004), are more disease resistant (Palmer and Oldroyd, 2003;
Seeley and Tarpy, 2007) and show improved foraging efficiency (Mattila et al.,
2008), food storage and colony growth (Oldroyd et al., 1992b; Mattila and
Seeley, 2007). Increased performance is believed to be a result of genetically
influenced differences in the propensity of workers to perform particular tasks
(reviewed in Beshers and Fewell, 2001; Myerscough and Oldroyd, 2004;
Oldroyd and Fewell, 2007). Variation in worker propensities to perform a task
means that for a certain level of stimulus to perform a task, only a particular
subset of workers will engage in that task. This modulation of the number of
workers performing any particular behaviour allows more efficient allocation of
workers to tasks (Myerscough and Oldroyd, 2004; Graham et al., 2006). Work-
ers with a sufficiently low threshold for a task may even become specialists, to
the point of delaying their maturation and progression to other colony tasks
(Arathi et al., 2000; Beshers and Fewell, 2001).
Some tasks, such as hygienic behaviour and colony defence, are frequently
performed by workers from a small subset of the total number of patrilines
(Arathi et al., 2000; Hunt et al., 2003). Thus, when queens are chosen based
only on colony-level phenotype, they may not be from the patrilines contribut-
ing to the desired trait. Smaller breeding populations therefore risk losing rarer
desirable alleles before they can become fixed in the population. As continued
selection in a closed population increases the genetic homogeneity, colony
performance arising from patrilineal genetic diversity will decrease, reducing
the gains made by selection.
Besides perhaps stinging behaviour, the performance of any one worker is not
considered important to the beekeeper. The commercially important traits of
honeybeeshoney and wax production and pollination efficiencyare measured
at the level of the colony. Other traits such as disease resistance, overwintering
ability and swarming tendency are of concern predominantly for the way in which
they influence the primary traits, or prevent the loss of the colony. While the queen
can be considered the only individual of any lasting importance in a colony, she
does not play a direct role in any of the primary traits, but rather influences them
through her production of eggs, the contribution of her genes to the worker
population and the influence of her pheromones on worker behaviour. Breeders
therefore tend to treat a colony as a single entity. Of all the biological levels,
colony-level traits are assuredly the most important commercially.
Colony performance is an emergent property of its social architecture: the
multiple levels of biological organization that arise as a result of its multiple
generations and varied degrees of relatedness among individuals, both within
and between castes, and, ultimately, the genes that these individuals carry
(Fig. 2).
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The accuracy of any estimate of breeding merit is limited by the accuracy with
which the phenotype of interest can be measured. Many commercially impor-
tant traits of honeybees are behavioural (e.g. aggression, tendency to swarm,
foraging efficiency) and are influenced by the interactions that occur within and
between each of the four biological levels. Honeybee breeders need to identify
those aspects of a colonys performance that are genetically variable and
determine how the genomes of different individuals interact in the next genera-
tion to influence colony-level performance. Selection of colonies with superior
genotypic merit for breeding is therefore rarely straightforward.
By way of example, we describe in the following section the interactions that
have been identified at the different biological levels for disease resistance.
Disease resistance is an economically important trait for bee breeders
(Sammataro et al., 2000; van Engelsdorp and Otis, 2000), and mechanisms
behind various aspects of disease resistance have been intensively studied
(reviewed in Spivak and Gilliam, 1998; Wilson-Rich et al., 2009). Examining
the genetic architecture of disease resistance therefore illustrates the scope of
the interactions that occur between the four biological levels, and some of the
issues that need to be addressed in breeding for disease resistance.
TABLE 1
Heritability estimates for commercially important traits of honeybees
Trait Heritability
w
Refers to estimated worker effect; q Refers to estimated queen effect
a
Bienefeld and Pirchner (1990)
b
Collins et al. (1984)
c
Decanini et al. (2007)
d
Harbo (1992)
e
Moritz (1985)
f
Moritz et al. (1987)
g
Oldroyd et al. (1987)
h
Kraus et al. (2005)
i
Soller and Bar-Cohen (1967)
j
Vesely and Siler (1963)
k
Bar-Cohen et al. (1978)
l
Harbo and Harris (1999a)
m
Lapidge et al. (2002)
n
Boecking et al. (2000)
More positively, a high recombination rate means that genetic mapping studies
are able to locate genetic markers that are closely linked to a gene of interest,
aiding the development of gene-assisted selection (which is not affected by
linkage disequilibrium or recombination rates). The honeybee therefore stands
to gain much from the application of genomic technology.
Honeybee queens mate in flight (Koeniger and Koeniger, 1991) with drones
originating from colonies up to 15 km away (Jensen et al., 2005). Drones gather
at specific geographical locations known as drone congregation areas (DCAs)
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(Loper et al., 1987; Pechhacker, 1994), which may contain as many as 15,000
drones (Koeniger et al., 2005). Queens fly to these DCAs, where they mate with
between 10 and 50 drones (Palmer and Oldroyd, 2000) over the course of one to
three mating flights (Neumann et al., 1999).
Honeybee mating biology means that queens mate with drones sourced
from a large geographic range. Jensen et al. (2005) noted that only 18% of
matings occur between drones and queens from the same apiary. This makes
it extremely difficult to reproductively isolate selected lines from unselected
and wild colonies. Nonetheless, because the queens direct phenotype and
contribution to the genotype of workers contribute greatly to a colonys pheno-
type (Oldroyd et al., 1990; Bienefeld et al., 2007), the use of selected queens
and unselected drones can still provide high-performing colonies for commer-
cial use. However, the contribution of unselected alleles to new generations of
queens impairs the potential rate of improvement in a breeding population.
Identification of the paternal genetic contribution to the phenotype of a
queens colony is further hindered by multiple mating. Since only a fraction
of the colonys paternal genome is passed on to offspring queens, neither the
remaining drone contributions nor the emergent properties arising from the
interactions between the patrilines are inherited. Single drone inseminations
can be used as a tool to eliminate this problem (Harbo, 1999), but the resulting
queens have reduced useful life, and do not show the full emergent properties of
more diverse colonies (Jones et al., 2004; Mattila and Seeley, 2007; Seeley and
Tarpy, 2007; Mattila et al., 2008). The possibility of selecting queens from a
patriline that did not contribute to colony phenotype further hinders selection
efforts. Thus, the genetic contribution from drones evades the breeders control
at both the queen-mating and the queen-rearing level.
Drone
Queen
father
A B B
A B A B B B
FIG. 5 Sex determination in honeybees. Letters represent two possible alleles of the
complementary sex determination (csd) locus. Viable drones arise from unfertilized eggs
(Beye et al., 2003). Diploid drones, though capable of developing, are killed by workers
(Woyke, 1963), rendering them effectively non-viable. In this example, half of all
offspring sired by the drone father will therefore be non-viable.
Because diploid males are killed by the workers, these offspring are effec-
tively non-viable. Therefore, half of all the offspring sired by a drone carrying
an allele at the sex locus that is identical with one of the queens alleles will be
non-viable. This leads to spotted brood as workers remove all the homozygous
larvae, which reduces the population growth rate achievable by the colony
(Tarpy and Page, 2001).
The average brood viability (V) in a population of honeybee colonies is a
function of the number of csd alleles in the population, given by
V 1 1=a; 1
where a is the number of alleles (Page and Marks, 1982). The number of alleles
maintained at equilibrium in a population is dependent on the effective popula-
tion size and the mutation rate (Yokoyama and Nei, 1979). Since the average
brood viability is greater than 85% when there are seven or more alleles, it
would seem advantageous to maintain at least this many alleles in a breeding
population. However, maintaining seven alleles at equilibrium in a randomly
mating closed population requires an effective population size of approximately
200 (Yokoyama and Nei, 1979), which is equivalent to approximately 93 colo-
nies (assuming 10 matings per queen, using the formula of Wright (1933) and
Moran (1984)). Any population smaller than this, or any breeding design that
does not maximize maintenance of csd allelic diversity, will therefore
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A C
ps
ps
B D
ps ps ps
FIG. 6 Four primary methods of accounting for multiple matings in BLUP model of a
honeybee breeding programme. (A) All paternal contributions are ignored. (B) Males
considered a genetic group and contribute a common effect to each individual in a
generation. (C) Drones from related sister queens are combined to form a pseudo-father
(indicated by subscript ps), and then added to pedigree. (D) When multiple pseudo-
fathers are used, they each contribute equally to the breeding value of their offspring
(using average relatedness).
drones and the average relatedness between them. Such a model was success-
fully implemented by Bienefeld et al. (2007) for a closed breeding programme
in Germany. The pseudo-fathers represent a group of sister queens that are all
the offspring of a single queen. This simplification means that the genetic
relatedness of the drones can be calculated (Bienefeld et al., 1989), as well as
the likelihood of desirable genes being passed to the next generation (i.e. the
relatedness path coefficient). Pseudo-fathers can be incorporated into the BLUP
model with their own estimated breeding value and pedigree, having a single
mother (the mother of the sister drone producing colonies) and a single father
(another pseudo-father).
Another method of incorporating unknown fathers into the breeding pedigree
is to calculate the average breeding value inherited from all possible fathers
(through creation of the average numerator relationship matrix (NRM); Kerr
et al., 1994). This provides the biomatrician with the ability to account for
drones that are not descended from full sisters, which is the case in most
breeding schemes. This method is most likely to be of benefit when combined
with the creation of pseudo-fathers for all sister drone producing colonies, as
this reduces the total number of fathers that need to be accounted for, improving
the accuracy of the average NRM model (Kerr et al., 1994), as well as being
logistically simpler.
disequilibrium (due to being located very close to the QTL), or are causative
mutations, have the greatest utility, but it is substantially more difficult
to identify such markers, and requires screening of a much larger number
of potential markers.
Because the honeybee has an extremely high recombination rate (Beye et al.,
2006), a QTL with a confidence interval of a given size will result in a smaller
number of gene candidates than in species with a lower rate of recombination.
Nonetheless, the use of standard linkage mapping will rarely provide a resolving
power greater than 1020 cM, except in cases where thousands of individuals
are mapped (Darvasi and Soller, 1997; Weller and Soller, 2004). This interval
can still generate up to 40,000 genes/4000 cM 20 cM 200 gene candidates,
depending on the density of genes within the confidence interval of the QTL. It
is therefore necessary to rank primary candidates from those located in the QTL
region.
Ron and Weller (2007) proposed four criteria for identifying primary gene
candidates following a QTL study:
Ron and Weller (2007) concede that most identified genes would fail to
meet all four of their criteria successfully. Nevertheless, their sifting schema
provides a useful method for determining the most likely gene candidates from a
list, and indicates several directions that can be taken to verify the role of the
candidates in affecting phenotype.
Most markers are based on genetic variation in a population, as described
above. However, it is also possible to use the expression profiles of known
candidates. Abaecin expression is correlated with colony-level P. larvae resis-
tance (Evans and Pettis, 2005) and is the only known expression candidate for a
commercial honeybee trait. Although abaecin expression has not been used as a
selection criterion for disease resistance in any breeding programme, assess-
ment of its protein expression levels may in theory be used as a complement to
selection based on sequence level variation at other loci.
Markers can be incorporated into a selection strategy in two primary ways.
Individuals can be initially screened for a molecular marker (or set of markers),
and from this selected subset of the population individuals are then chosen
based on phenotype or estimated breeding value. Alternatively, both phenotypic
and molecular information can be considered together, using a selection index
to give appropriate weighting to the two components.
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For single traits, simulation studies have shown that MAS gives greatest
improvement when a trait has a low heritability (Lande and Thompson, 1990;
Ruane and Colleau, 1995). Selectionboth with and without MASleads to
the superior alleles becoming fixed in the selected population. Directly selecting
these alleles, therefore, leads to a faster rate of genetic improvement. It also
reduces the possibility that the superior alleles are lost due to genetic drift before
they have a chance to become fixed in the breeding population. However, after
three or four generations of MAS, most individuals in a population will carry the
preferred allele (Ruane and Colleau, 1995; Pong-Wong and Woolliams, 1998).
From this point, the incorporation of marker information in the selection scheme
becomes detrimental to the selection process, and standard BLUP actually
performs better (Ruane and Colleau, 1995; Dekkers and Van Arendonk, 1998).
For selection of multiple traits, MAS will enhance selection for a longer
period due to the lower selection intensity on each trait. When more loci are
being selected, there is also a greater probability that superior alleles will be lost
if MAS is not used. MAS provides the greatest benefit over other selection
methods when the traits being selected have a high economic weighting, have
negative genetic correlation (but not between their QTL) or their heritability is
large (Togashi and Lin, 2009).
Hygienic behaviour and aggression exhibit many of these properties. They
are both given substantial economic weighting by beekeepers (van Engelsdorp
and Otis, 2000), have negative genetic correlation (Guzman-Novoa et al.,
2002a), and show moderate to high heritability (Moritz et al., 1987; Harbo
and Harris, 1999a). Furthermore, both these traits are close to having identified
molecular markers available (Lobo et al., 2003; Oxley et al., 2010a). Therefore,
honeybee breeders appear to stand to benefit greatly from the use of MAS.
Gaining control over the mating of queens and drones can be achieved through
three primary mechanisms: the use of instrumental insemination, geographical
isolation of breeding colonies using islands or mountains and control of mating-
flight time.
Instrumental insemination (II), although the most technically difficult of the
three methods, provides the greatest degree of control over mating. Indeed, II
even allows the breeder to achieve mating outcomes that are biologically
impossible in vivo. Semen from a single drone can be used to inseminate
multiple queens, or the semen from hundreds of drones can be homogenized
and used to inseminate many hundreds of queens, providing complete unifor-
mity in the paternal contribution to each colony in the population (Kaftanoglu
and Peng, 1980; Kuhnert et al., 1989). Instrumental insemination also allows for
the opportunity to genotype the drones prior to semen collection (Oldroyd and
Thompson, 2007).
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Having established a closed breeding population, there are several broad meth-
ods available to minimize inbreeding, particularly at the sex locus. These
methods involve some or all of the following components: maintaining a large
population size, using queen replacement or line breeding, identifying and
monitoring the csd alleles in the population, selecting for brood viability and
importation of unrelated stock. Each of these methods can be used indepen-
dently of the others, or combination (for a detailed analysis of many of these
methods, see Page and Laidlaw, 1982b).
The most straightforward method is to maintain a sufficiently large closed
population that can maintain enough csd alleles to ensure high brood viability.
Page and Laidlaw (1982a) have shown that an effective population size of
107 has a greater than 90% chance of maintaining brood viability greater than
85% over 40 generations. The effective population size can be increased
through artificial insemination of homogenized semen from a large number of
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drones (Moritz, 1984). This has the added advantage of eliminating the variation
between colonies that arises from differences in paternity that occur when
semen is not homogenized. Because selection tends to favour more closely
related individuals, selection within the population will require a substantially
larger population size to offset the effects of selection on inbreeding (Omholt
and Adnoy, 1994). This means that large population size alone is unlikely to be
sufficient to maintain high brood viability in a selection programme.
It is possible to increase the effective population size, and reduce loss of csd
alleles, through various forms of population subdivision (Page and Laidlaw,
1982b). The least constrained system consists of maintaining two reproduc-
tively isolated populations, which are then crossed to increase the heterozygos-
ity across all alleles. The most constrained involves the selection of at least one
daughter queen from the existing population to constitute the next generation. In
each of these schemes, the level of inbreeding generally decreases with increas-
ing subdivision, but the level of trait improvement is simultaneously decreased.
Simulations by Omholt and Adnoy (1994) predict that the loss of viability from
selection with queen replacement will be similar to that in a population with
random mating of individuals and no selection.
With the identification of the csd locus (Beye et al., 2003), it is now possible
to genotype an individuals sex locus (Hasselmann et al., 2008). This allows the
number of alleles in a population to be monitored directly, and can ensure that
every allele present initially in the population is passed on in each subsequent
generation. However, even with limited genotyping within a population, this
method may not provide sufficient benefit in most schemes to merit the eco-
nomic cost of genotyping. Furthermore, tracking sex alleles will not reduce the
level of inbreeding at other loci, which may still lead to undesirable losses in
productivity.
It is also possible to select for csd allelic diversity indirectly, by selection for
high brood viability. This procedure is effective because queens carrying rare
csd alleles are unlikely to mate with drones carrying identical alleles, and will
therefore have a higher brood viability. Selection for high brood viability
therefore selects for the rarer alleles in the population, reducing the probability
that they are lost due to genetic drift. When combined with mass selection (no
population subdivision, queen replacement, etc.), this method gives greater trait
improvement and higher brood viability than using selection with queen
replacement (Omholt and Adnoy, 1994).
The final method of reducing inbreeding is the introduction of new alleles to
the population from external stock. This procedure has the advantage that new
traits not present in the current population can be introduced directly, if found in
suitable external stock. Because this method does not merely reduce the rate of
loss of alleles, it can be used to maintain a breeding population indefinitely.
Further, it will not only prevent loss of alleles at the sex locus, but also across
the genome, effectively reducing the effects of inbreeding depression.
Author's personal copy
7 Success stories
While there have been many successful honeybee breeding programmes over
the years, we highlight two that have been successfully operating for many
years. These examples have incorporated several of the techniques we have
described in this review.
A breeding scheme established on Rottnest Island, west of Perth in Western
Australia, has successfully maintained a closed population honeybee breeding
scheme since 1979. To prevent inbreeding, replacement queens are reared from
each of 20 separate lines each generation. Furthermore, drones from four
evaluated colonies that are external to the scheme are introduced each year.
Between 1983 and 1991, semen from drones from the external colonies, and
from each of the 20 lines, was homogenized and used to inseminate the
replacement queens (Allan and Carrick, 1988). Colony evaluation in 1991
(Manning, 1996) showed an average 34.27% greater productivity than unse-
lected colonies from outside the programmea 3.1% increase per year. Since
1991, instrumental insemination was abandoned in favour of isolated mating on
Rottnest Island. After more than 25 years as a semi-closed population, there is
no evidence that the breeding lines have greater homozygosity at random
microsatellite loci relative to feral colonies (Chapman et al., 2008).
A programme run by the German Beekeepers Association (DIB) has been
using a number of islands in the North Sea for isolated mating of colonies since
1972. Drones used in each mating are the offspring of 410 full sisters, each the
Author's personal copy
8 Concluding remarks
Honeybee breeders now have at their disposal the most advanced genome
technologies, similar to those available for any livestock, as well as appropriate
statistical models for breeding value estimation. As our understanding of the
genetics of honeybee behaviour continues to improve, we will begin to see that
genetic markers for commercially important traits become an economically
viable option for evaluation of colonies. Our ability to control mating with
instrumental insemination and geographical or temporal isolation also continues
to improve. Combined with MAS and breeding value estimation, honeybee
breeding is beginning to realize significant increases in trait improvement.
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