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Download full report (630kb - HINT: right click and save the file to your hard disk before opening)
by Peter Oxley, Ben Oldroyd and Gladys Ho
May 2008
RIRDC Publication No 08/092 RIRDC Project No US-123A
Executive Summary
What the report is about
A genetic marker is any
genetic variant that is easily measured. These days most genetic markers
involve the detection of changes in a DNA sequence. These tiny differences
may have no bearing on phenotype, but can show strong genetic linkage to
genes that actually do affect phenotype. Thus we can potentially use a
genetic marker to help choose elite animals for breeding purposes. This
is known as Marker Assisted Selection or MAS. MAS has been successfully
developed in several animal species of agricultural importance, especially
dairy cattle and pigs where markers for milk and carcass quality parameters
respectively are known. The advantages of MAS over traditional breeding
methods is that animals can be selected for breeding at a relatively young
age without the need for extensive and expensive field trials. More often,
however, MAS is used as a useful adjunct to traditional field trials.
This report describes an attempt to develop the first genetic markers for MAS in the honeybee.
Although this goal was not achieved during the life of this project, tremendous progress has been made towards it. One marker was identified which shows a good correlation with hygienic behaviour, but we were unable to convert this marker to a simple diagnostic test. More importantly, a new framework for developing genetic markers for hygienic behaviour has been developed, and the research team is now pursuing this goal.
Who is the report targeted
at?
This report is primarily
aimed at policy makers within the honeybee industry. We hope that it will
help guide future policy and funding decisions regarding bee breeding efforts
in Australia. The report will also be of interest to queen breeders.
Background
The honeybee is a particularly
good candidate species for the introduction of MAS for the following reasons:
• It is difficult to assess the breeding value of colonies in a commercial
context, and this reduces the accuracy of selection.
Hygienic honeybee colonies
are those in which dead and diseased brood is rapidly removed from the
colony, thereby reducing the amount of inoculum present. Hygienic behaviour
is a trait present in about 20% of Australian honeybee colonies. Some researchers
claim that highly hygienic colonies are strongly resistant to the major
diseases of honeybees including American and European foul brood, Chalk
brood and Sac brood. Hygienic bees are also claimed to be resistant to
the parasitic mite Varroa. Hygienic behaviour is usually measured by using
liquid nitrogen to freeze-kill a small patch of brood. Hygienic colonies
uncap and remove the dead brood within 24 hours whereas this process takes
several days with non-hygienic colonies.
The first studies of hygienic behaviour were conducted in the 1960s. Walter Rothenbuhler crossed a strongly hygienic line with a strongly non-hygienic line. The resulting F1 colonies were not hygienic.
Rothenbuhler then raised daughters off an F1 queen backcrossed these to drones of the hygienic parent. He then evaluated these colonies for hygienic behaviour. The pattern of expression of hygienic behaviour among these backcross colonies suggested that the trait was controlled by two separate genes, one that controlled uncapping behaviour, the other which controlled removal behaviour.
In the 1990s there was renewed interest in hygienic behaviour as a means of control of the parasitic mite Varroa destructor. Professor Marla Spivak of the University of Minnesota was a lead player in this effort. Spivak selected a highly hygienic line, which she showed to be highly resistant to brood disease and to Varroa.
During the late 1990s with support from RIRDC, we imported Spivak’s hygienic line and a selected non-hygienic line in an effort to identify the actual genes that control hygienic behaviour, and to develop genetic makers for these genes. We created a backcross between these lines to produce ca. 68 colonies that showed varying (and repeatable) degrees of hygiene. Based on the fathers of the colonies we created a high-density linkage map using RAPDS – an early form of genetic marker. We then used Quantitative Trait Loci (QTL) interval mapping to find markers potentially associated with the hygienic trait. Seven such markers were identified.
In this current project we attempted to convert these suggestive loci into reliable genetic markers that could be used by Industry for MAS for hygienic behaviour.
Aims/Objectives
Methods used
Confirmation of the RAPD
markers
Field tests of hygienic
behaviour were carried out on random colonies from 2002-2004. The colonies
tested were both commercial and research colonies, unrelated to the original
hygienic backcross colony used to identify the suggestive QTLs (Lapidge,
Oldroyd et al. 2002). A total of 68 colonies were tested: 22 colonies were
evaluated in Minneapolis in 2002, and 46 colonies were evaluated in Richmond
Australia.
DNA was extracted from a sample of 24 workers from the colonies identified from field tests as being strongly hygienic or non-hygienic. These samples were used to determine if the strongest QTL identified in the genomic scan showed a correlation with hygienic behaviour in random colonies, as they would need to be useful in MAS. The RAPD primers studied were UBC123, UBC336 and UBC395.
Cloning and Sequencing
of the RAPD Band
To be usable in a simple,
reliable genetic test, it is necessary to identify the genomic sequence
that the RAPD primer pair amplifies. To do this we obtained the variable
RAPD bands from UBC123 and UBC336, cloned the DNA into a bacterium to amplify
it, and then sequenced the DNA of the clones.
Development of Primers
and Restriction Assay
The sequences obtained from
the cloned RAPD bands were aligned to the honeybee genome available on
the Internet. From these searches we identified the sequences that the
RAPD primers were amplifying, and developed new PCR primers that reliably
amplify the regions that were associated with hygienic behaviour. We then
identified the specific DNA base that differed between the fathers of the
hygienic colonies and the fathers of the non-hygienic colonies in our original
backcross.
The restriction endonuclease Msl I, was found to discriminate between the polymorphism that identified hygienic and non-hygienic fathers. From this we developed a CAPS assay for one gene related to hygienic behaviour.
In order to verify that the CAPS assay worked on colonies other than the backcross colonies, we performed the CAPS test on 19 hygienic and 17 non-hygienic colonies (24 workers from each colony) that we had identified in field trials.
Fine mapping hygienic
behaviour genes
We used the Honeybee Genome
sequence to help us map genes related to hygienic behaviour from the genomic
region identified by the RAPD bands.
Testing a new method of
controlled mating
We evaluated a single controlled
mating event set up by Mr. Horner. We genotyped 200 drones from the population
released by Mr. Horner, 20 feral workers collected from the area, and 260
offspring workers from 13 colonies. We showed that 99% of workers had genotypes
that were compatible with having been fathered by the released drones,
with only 1% incompatible. This shows that the method is highly effective.
Horner’s method has considerable advantages over artificial insemination,
and provides a viable alternative to island mating where no suitable island
is available.
Results/Key findings
The RAPD polymorphism for
UBC123 and UBC336 were strongly correlated with hygienic behaviour in the
36 random colonies (Figure 1) but UBC395 was not correlated, and so UBC395
was not considered further.
Figure 1: Frequencies of
workers from hygienic colonies (24 workers per colony, n=19, light grey)
and non-hygienic colonies (24 workers per colony, n=17, dark grey) carrying
the polymorphic bands associated with hygienic behaviour and amplified
by the markers UBC335 and UBC395. Workers from hygienic colonies carried
the UBC336 band at significantly higher frequency than those from non-hygienic
colonies (?2 = 40.1, P<0.001); whereas they carried the UBC395 band
at a lower frequency than those from non-hygienic colonies (?2 = 59.6,
P<0.001).
Having verified that RAPD markers UBC335 and UBC395 identified in the previous project are indeed associated with hygienic behaviour we proceeded to develop them further into a diagnostic test.
Both markers were successfully cloned and sequenced, and their location in the honeybee genome x determined. From this we developed a diagnostic test which successfully identified the hygienic and non hygienic fathers from our backcross population. Unfortunately, however, this test did not perform satisfactorily on colonies unrelated to the backcross. We suspected that this is due to additional alleles not present in the backcross population. We identified one additional allele, and a diagnostic test for it, but even then we concluded that we could not use our markers to identify hygienic individuals in random samples.
Implications for relevant
stakeholders:
At present there is only
a single genetic marker available that can be used for marker assisted
selection. It is now possible to use a relatively simple molecular test
to determine the sex allele(s) carried by potential mating partners, and
this information could potentially be used to reduce the effects of inbreeding.
This procedure is available in our laboratory. Such a test could be particularly
useful when importing stock through quarantine, when serious genetic bottlenecks
occur.
Beyond this application, there are currently no satisfactory tests that can be used for MAS in the honeybee. We believe, however, that MAS will be possible in the next few years, with markers for stinging behaviour and hygienic behaviour the most likely. Current research underway in Germany may reveal genes related to disease resistance, which are not related to hygienic behaviour.
Our research has shown that the Horner method of controlling mating by delaying the flight time of selected queens and drones provides excellent control of mating. The Australian Queen Bee Breeding Group, a consortium of Australian queen and honey producers is currently using artificial insemination to produce queens for field evaluation and for sale. Because of the potential cost savings and other benefits, we recommend that consideration be given to using Horner methods rather than artificial insemination.
General conclusion
The project confirmed that
the markers UBC336 and 395 were correlated with hygienic behaviour in our
backcross colonies. We successfully converted these markers to a simple
diagnostic test, which worked in the mapping population but did not work
in random colonies. Furthermore we were unable to locate the actual genes
that influence hygienic behaviour, probably because the mapping population
we had was too small to be able to pick up any but the strongest genes.
With Australian Honeybee Industry Council and Australian Research Council support we have now embarked on a new project in an attempt to locate the elusive hygienic behaviour genes. In this project we have performed the reciprocal cross so that variation in hygienic behaviour does not occur across colonies according to their fathering male, but among workers within a single colony. This new backcross was performed in Minnesota, and 400 hours of video recordings of the behaviour of 500 backcross workers are now being evaluated. The workers are currently being genotyped at 350 microsatellite loci across the honeybee genome. Whether this project will reveal genes that are correlated with hygienic behaviour or genetic markers that are useable by industry remains to be seen, but we are quietly optimistic.
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