Download The Chicken Genetic Map and Beyond Hans H. Cheng USDA

The Chicken
Genetic Map and Beyond
Hans H. Cheng
USDA, AgriculturalResearch Service
Avian Diseaseand OncologyLaboratory
3606 East MountHope Road
East Lansing,MI 48823
Introduction
Under the Chinese calendar, 1993 is the year of the chicken which
symbolizes a new beginningor rebirth. It is fittingthat the official start of the
National Animal Genome Research Program (NAGRP) and its commitment to
developing molecular genetic maps in several species including the chicken
should begin in 1993. With funding specifically designated for the chicken
genetic map, we will see the beginning of a new era that promises great
advances in science and technologywith the chicken. In this discussion,I will
describe the new types of markers being placed on the genetic map, compare
the relative advantagesand disadvantagesof each type of marker, and present
howthe genetic map can be utilizedto identifygenes involvedin complextraits.
The Chicken Genetic Map
A genetic map is simply an ordered array of genetic markers showing
genetic distances between each locus. The first classicalgenetic map of the
chicken was publishedin 1936 by Hutt and regular revisionsof the map have
appeared since then (for review, see Bitgoodand Somes, 1990). The majority
of the genetic markers placedon the classicalmap were morphologicalmarkers
suchas yellowskinor pea comb.
The reliance on morphologicalmarkers has restricted the utility of the
classical genetic map. Due to the limited number of morphological markers
available, few linkage groups have been established resulting in incomplete
coverage of the genome. In addition,chickensused in crosses are unlikelyto
carry more than a few of the morphologicalmarkerswhich restrictsthe number
of loci beingfollowedeven more. Finally,morphologicalmarkers are subjectto
environmental or developmental influences and are frequently limited by
epistasis and pleiotrophy.
Due to the limitationof the classicalmap, advances in genetic mapping
of whole genomes, and the initiationof the NAGRP, a program was established
in the United States at East Lansingto make a molecular genetic map of the
chicken. Concurrently,programsin other countrieshad begun with the same
goal. The enormoussize of the projecthas led to internationalcollaborationsto
developthe chickengeneticmap.
In constructingthe moleculargenetic map, it was apparent to the East
Lansinggroupthat utilityto boththe scientistand the breeder be a high priority.
To achieve maximum usefulness of the genetic map, the genetic markers
should satisfy several criteria and have certain characteristics. First, the
markers should be highly abundant and evenly distributed throughout the
genome which ensuresthat all regionsof the genome can be marked. Second,
the markers should be highly polymorphicwhichenables them to be useful in
almost any cross. Third, the markers must be easily scored to permit a large
39
number of loci or progeny to be typed. Finally, the markers should be in a form
that can be easily disseminated among laboratories.
The _ew molecular genetic map will rely on DNA-based markers which
will increase the usefulness of the map. DNA-based markers can satisfy all the
requirements of an ideal genetic marker. First, there is an almost limitless
number of potential markers available. Second, the markers can be sensitive to
single-base changes making them highly polymorphic between individuals.
Third, through the use of the polymerase chain reaction (PCR), the markers can
be easily typed. Fourth, distribution of the markers is easily achieved through
DNA sequence information or probes cloned into bacteria. Finally, DNA is
easily obtained from the blood of chickens and is generally not affected by the
environment or the developmental stage of the chick.
A variety of DNA-based markers known by their acronyms have been
and are being developed for molecular genetic maps. Each type of marker has
its own relative advantages and disadvantages. Since many types of markers
are being placed on the chicken genetic map, a short discussion of how these
markers are developed and their relative strengths and weaknesses is merited.
Restriction fragment length polymorphisms or RFLPs are the oldest type
of DNA-based marker. First proposed as a genetic marker in 1980 by Botstein
et al., RFLPs have fueled the drive towards developing saturated linkage maps
in many species. RFLPs are developed as depicted in Figure 1. DNA isolated
from each individual is cut using a restriction enzyme that typically recognizes a
unique 6 base-pair sequence. Differences in the DNA sequences between
individuals result in differences in DNA fragment lengths after enzyme
treatment. These differences are detected by separating the DNA fragments by
size on an agarose gel and visualization of the appropriate fragment by
Southern blot hybridization. RFLPs have the major advantage of detecting
related or conserved genes from other species, thus, enabling comparative
mapping between species (see Table 1). However, the technology is very labor
intensive, relatively slow, and requires the use of radioisotopes. Furthermore,
only single- or low-copy DNA can be assayed resulting in the inability to follow
a large portion of the genome that contains high-copy DNA. Although a useful
scientific tool, it is unlikely that RFLPs will be routinely used commercially.
Random amplified polymorphic DNA or RAPD markers are based on
PCR (Williams et al., 1990). In PCR (Saiki et al., 1988; see Figure 2), specific
regions of the genome are exponentially amplified in 30-40 repetitive cycles of
three steps per cycle. In the first step, the DNA strands are denatured by
heating to 94°C. In the next step, short stretches of synthetic DNA known as
primers pair to complementary sequences of the template DNA when the
reaction is cooled to the appropriate temperature. Finally, DNA polymerase
replicates DNA starting from these primers as the temperature is elevated to
72°C. Through repetitive cycles, PCR is able to selectively amplify the region
between the primers. The amplified product can be quickly visualized by gel
electrophoresis and staining.
RAPD markers are generated by using a single primer of 10 bases in
length in the PCR (see Figure 3). Since the primers are very short, they are
able to bind to numerous sites in the genome and many products are formed. If
the individuals being compared are identical at the amplified locus, then the
DNA of each individual will direct the production of a band of identical size.
40
Differences between individuals at several loci are easily detected by the
presence or absence of similar-sized bands in stained agarose gels.
RAPD markers offer many advantages (see Table 1). First, the procedure
is very quick with results obtained in one day and without the need for
radioisotopes. Second, inexpensive commercial sets of primers are available
(Operon Technologies, Alameda, CA) enabling the potential detection of many
hundreds of loci. Third, multiple loci are assayed simultaneously in one test
and these loci can be in both high- or low-copy DNA. Finally, unique to RAPD
markers is the ability to target specific loci or phenotypes which makes tagging
genes of simple traits relatively easy. The main disadvantages are that RAPD
markers are dominant markers, thus, they cannot distinguish between a
homozygous or heterozygous individual.possessing the allele. Also, RAPD
markers are very sensitive to PCR conditions resulting in problems with
reproducibility.
The third type of marker is the simple sequence repeats or SSRs, also
known as microsatellites (Litt and Luty, 1989; Weber and May, 1989). These
markers are based on the observation that there are numerous tandem repeats
of 1 to 6 base-pairs sequences throughout the genome, e.g. (CA)12. Using PCR
with primers that flank these tandem repeats, amplified products are generated
(see Figure 4). Polymorphisms in the number of repeats occur frequently and
can be detected by visualization on acrylamide or agarose gels. These types of
markers are becoming very popular since they are very easy to use, give high
reproducibility, and are highly polymorphic between individuals (see Table I).
The main disadvantages are that marker development is labor intensive and
radioisotopes are normally used in detection of polymorphisms.
Another marker that is specific for the chicken is based on the chicken
repeat element, CR1. It also relies on PCR to produce the DNA polymorphisms
which enhances its ease of use. More information on this type of marker will be
published soon by Levin, Crittenden, and Dodgson.
All types of DNA-based markers have been developed for the molecular
genetic map. Two backcross populations have been internationally recognized
as the reference populations for which all the markers will be scored upon.
Both reference populations have been previously described (Bumstead and
Palyga, 1992; Crittenden et al., 1992).
Currently on the East Lansing map, 188 markers have been scored. Of
these, 158 of the markers have been resolved into 26 linkage groups with an
average distance of 8 cM per marker (see Figure 5). The preliminary map
contains about 1253 cM within linkage groups; it is estimated that the entire
chicken genome contains 2000-3000 cM. The saturation of markers on the map
is fairly good as 85-90% of randomly selected markers fall into one of the
existing groups. The Compton map (United Kingdom) is very similar in the
number of markers scored, coverage, and saturation..Efforts are underway to
consolidate the two maps into one.
A major advantage of the molecular genetic map is that additional
markers can be placed on the map using the same reference population. Since
the DNA from each individual has been isolated and stored, there is no need to
produce another population to score more DNA polymorphisms. Instead, the
molecular genetic map will be continually expanded with more genetic markers
41
screened on the existing reference populations which will result in a more
complete and saturated map.
Strategy for Identifying Quantitative Trait Loci (QTLs)
The main purpose of the chicken genetic map is to identify and map
genes involved in traits of economic importance. The vast majority of these
traits are multigenicand complex in nature. The chicken genetic map will allow
for the systematic analysis of the entire genome to see where genes that
contribute to the trait of interest are located and how much these genes
contributeto the trait. Successful identificationof these QTLs will lead to the
developmentof linkedmarkers for possibleuse in marker-assistedselection.
Several procedures have been .developed to use genetic maps to
identifyQTLs. Lander and Botstein(1989) have developed one popularlyused
method called interval mapping. In this procedure,crosses are made between
individualsor groupsthat differgreatlywithrespectto the trait of interest. These
resource populations produce segregating populations for the trait. All the
individuals are scored for the phenotype of the trait. The more extreme
individualsare then screened with 50-150 evenly-spacedpolymorphicmarkers.
Each positionin the genome is hypothesizedto containa QTL for the trait. Next,
the most likely phenotypic effect of the putative QTL is determined for each
genotype at that locus and the odds ratio calculated (the chance that the data
would resultfrom a QTL dividedby the chance that it wouldresultfrom no QTL).
For convenience, the IOglO of the odds ratio is reported and called the LOD
score which is proportional to the likelihoodof the existence of a QTL at that
position. A threshold LOD score determines when an,actual QTL is present.
This methodologyhas been successfullyapplied in the identificationof genes
involved in agronomic traits of tomato (Paterson et al., 1988), hypertensionin
the rat (Jacob et al., 1991), heterosisof corn (Stuberet al., 1992) and plant and
plant pathogeninteractionsin the commonbean (Nodoriet al., 1993). A similar
methodology has identified quantitative traits involved in the growth and
reproductiverates of soybean (Keim et al., 1990), and low-phosphorusstress in
corn (Reiter et al., 1991).
We hope to apply this method towards identification of genes that confer
resistance to Marek's disease (MD). It is known that certain alleles of the major
histocompatibility complex (MHC) or B complex confer genetic resistance to MD
(Briles et al., 1977; Bacon, 1987; Bacon and Witter, 1992) which has resulted in
some use of haplotyping to indirectly select for favorable birds. This markerassisted selection overcomes many obstacles associated with dealing with
hazardous pathogens which results in substantial time and money savings to
the commercial breeder.
Besides the MHC, other genetic factors are known to exist that have a
major influence on resistance to MD. For example, line 6 and line 7 chickens
share the same B 2 haplotype yet differ greatly with respect to resistance to MD.
Recent results suggest that the contribution of resistance to MD by non-MHC
genes may play a larger role than the MHC. Using three commercial White
Leghorn lines, sires that were heterozygous for MHC haplotype from a
susceptible or resistant line were mated to dams that were homozygous for
MHC haplotype from a moderately resistant line (Groot and Albers, 1992). 1359
chicks were produced, scored for MHC haplotype, and infected with Marek's
42
disease virus (MDV) at one day of age. Measuring mortality, it was determined
that sire line had a larger effect than either maternal or paternal MHC haplotype
suggesting that non-MHC genes play a more prominent role in disease
resistance than the MHC. This data is consistent with what is known in the
mouse where breeding experiments have estimated that the MHC accounts for
only 12-26% of the interline genetic differences in antibody response (Biozzi et
al., 1980).
Our strategy to detect non-MHC genes that confer resistance to MD is to
make an F2 population using the resistant line 6 and the susceptible line 7 as
the parents. Each chick will be inoculated with MDV and the level of resistance
measured using a variety of traits. Evenly-spaced markers from the chicken
genetic map will be used to genotype each chick and interval mapping will
declare when a QTL is found and where is the location. Each putative QTL will
be further confirmed in other populations that segregate for resistance to MD.
Once markers have been confirmed to be linked to genes conferring
resistance, efforts will be made to test these markers in commercial populations.
If the markers are found to be tightly linked in commercial lines, then markerassisted selection can be applied. In the case of MD, introgression of genes
conferring resistance by marker-assisted selection would complement existing
vaccinal efforts for control (Gavora and Spencer, 1979).
Although of limited application in chicken breeding, marker-assisted
selection has had a long history of success in plant breeding. Prior to the
discovery of DNA markers, plant breeders were able to use linked isozyme
markers, which detect difference in enzymes, in marker-assisted selection. A
classic example is the use of the alkaline phosphatase marker to detect
nematode resistance in tomato. The use of this marker eliminated the need to
directly screen the plants with the pathogen. Tomato breeders quickly realized
that the marker was more reliable, quicker, less expensive, and easier to use
than the traditional test. In addition, the marker eliminated the need to work with
a pathogenic organism. The use of marker-assisted selection in plants has
been extended with the development of many DNA-based markers that are
linked to various genes of interest.
Summary
Molecular biologyhas recentlygiven us the tools to generate a limitless
number of genetic markers. With a large number of markers in hand, it is
possibleto develop highly-saturatedgenetic maps. A moleculargenetic map of
the chicken is rapidlybeing completed. As a consequence of this genetic map,
it will be possibleto identifyand map genes involvedin complex traits such as
production and disease resistance. Once identified, markers linked to the
genes of interest can be developed for commercial use in marker-assisted
selection whichwill result in substantialsavingsof time and money.
43
Figure 1. Restriction Fragment
Length Polymorphism
(RFLP).
Double-strandedDNA is illustratedby the two horizontallines with arrows;the
arrow indicates the 3' end. The vertical lines represents the pairing of
complementarybases between strands. DNA isolatedfrom individualsA and B
is cut using a restrictionenzyme. Recognitionsites for the restrictionenzyme
are indicated by the short arrows pointingdown. Agarose electrophoresis
separates the DNA fragments by size. The open box indicateswhere the DNA
was loaded. Smaller fragmentsmigratefaster than the larger fragmentsto the
bottom of the gel. The appropriate fragment illustrated by the filled box is
detected by a process knownas Southern blothybridization. In this process, a
radiolabeledpiece of DNA knownas the probe is used to detect complementary
DNA. As shown,individualA has an extra recognitionsite for the enzyme when
compared to individualB resultingin a smallerfragment beingdetected by the
probe.
Figure 2. Polymerase Chain Reaction (PCR). The symbols are the
same as in Figure 1. DNA is denatured by elevatingthe temperatureto 94°C.
The reaction is cooled and the primers represented by the short horizontal
arrows bind to complementary sites. Elevation of the temperature to 72°C
allowsthe DNA polymeraseto replicatethe DNA startingfrom the 3' end of the
primer. These three steps are repeated for 25-40 cycles. The region between
the primersis selectiveamplifiedover 100,000-fold.
Figure 3. Random Amplified Polymorphic DNA (RAPD). The symbols
are the same as in Figure 1. DNA from individualsA and B is used as template
in separate PCRs with a single primer of 10 bases in length. The relative
shortness of the primer allows it to bind to numeroussites in the genome; the
bindingof only four sites by a primer in each PCR is depicted. If the two sites
are within 5 kilobases of each other and oriented withthe 3' ends facing each
other, then an amplified DNA product is made. This can be visualized by
agarose gel electrophoresisand staining. Differences in the banding patterns
of the individualsindicates genetic differences; each band of a particular size
represents a different locus. As illustrated, individualA amplifies a band of
smaller size not observed in individual B. On the other hand, individual B
amplifies a band of larger size not observed in individualA. The other three
bands are common between the two individualsindicating that the two are
identicalat those loci.
Figure 4. Simple Sequence Repeats or Microsatellites. The symbols
are the same as in Figure 1. DNA from individualsA and B is used separately
as template in a PCR with primersthat flank a regioncontaining a CA-repeat.
Gel electrophoresis of the PCR product indicatesthat individualB directs the
synthesisof a larger DNA productwithone more CA-repeatthan individualA.
44
Figure 5. Autosomal Genetic Linkage Map of the Chicken. The
current autosomal genetic linkage map of the chickenis based on a panel of 52
backcross progeny from the East Lansing reference population. Loci are in
italics and the type of locus or probe is given in parentheses. The numbers on
the left are approximate map distances given in centiMorgans (cM).
(C)=classical locus, (F)=RFLP, (A)=RAPD, (R)=CR1 repeat, (M)=microsatellite,
(V)=single-locus minisatellite or VNTR. ADL stands for anonymous loci typed at
the Avian Disease and Oncology Laboratory, and MSU stands for loci typed at
Michigan State University, Department of Microbiology. Other loci are named
for the gene represented. Loci that are marked by the probe type only were
typed in other laboratories and the data is not yet published.
Table 1.
Properties of DNA-Based Markers.
45
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46
Figure 2: Polymerase Chain Reaction (PCR)
ANNEAL PRIMERS
DENATURE AND
re=..=
PRIMER EXTENSION
AND EXTEND
DENATURE, ANNEAL,
25-40 CYCLES
REPEAT FOR
DNA AMPLIFIED >100,000 FOLD
47
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Table 1:
Properties of DNA-Based Markers
RFLPs
RAPDs
_SSRs
Distribution
Ubiquitous
Ubiquitous
Ubiquitous
Part of genome surveyed
Low copy
All
All
Level of polymorphism
Moderate
to High
Moderate
to High
High
Type of genetic marker
Codominant
multiple alleles
Dominant
two alleles
Codominant
multiple alleles
# of loci detected
1-3
1-10
1
Detection of homologous loci
Yes.
No
?
Technical difficulty
High
Low
Low
Reliability
High
Low-High
High
Speed
Slow
Very fast
Fast
DNA quantity
1000+ ng
10-50 ng
10-50 ng
50+ kb
Pure
10+ kb
Relatively pure
5+ kb
Crude
Use of radioisotopes
Yes
No
Yes
Cost
High
Moderate
Moderate
Ease of dissemination
Difficult
Easy
Easy
- DNA quality
51
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complexon disease
resistance and productivity. PoultrySci. 66:802-811.
Bacon, L.D., and Witter, R. 1992. Influenceof turkeyherpesvirusvaccinationon
the B-haplotypeeffect on Marek's disease resistance in 15.B-congenic
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Bitgood, J.J., and Somes, R.G. 1990. Linkage relationships and gene
mapping. In: Poultry Breeding and Genetics, R.D. Crawford (ed.),
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Biozzi,G, Siquera, M., Stiffle,C., Ibanez, O.M., Mouton,D., and Ferreira, V.C.A.
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Botstein,D., White, R.L, Skolnick,M, and Davis, R.W. 1980. Constructionof a
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53
Questions to Dr. Hans H. Cheng
,
Question (Dr. Bob Gowe): I understood your slide to indicate that there would
be no epistasis associated with quantitative trait loci (QTLs) identified by DNA
markers?
Answer: I think that there is a slight misunderstanding here. Procedures such
as interval mapping used to identify QTLs with a molecular genetic map do not
place any judgments with regards to the gene interactions of identified loci.
Where I think the confusion arises is that the DNA markers themselves are not
subject to epistatic or environmental effects since they do not change unless
rare mutations occur. Thus, DNA markers are considered "neutral" and for this
reason can be used at any time. In contrast, morphological markers often show
epistasis or development effects, e.g., slow-feathering is observed only early in
feather development.
Question (Dr. Milton Boyle): Could you explain what you meant by targeting
your probe to a specific gene or trait using RAPD markers?
Answer: RAPD markers are the only DNA markers available that one can
quickly use to generate linked markers to simple traits, phenotypes, or genetic
locations. Using a procedure called bulked segregant analysis (BSA; see
Michelmore et al., PNAS 88:9828, 1991), a population that is segregating for
your trait, gene, or genetic locus is generated and scored. The characteristic
being scored then allows you to divide your population into 2 groups. Then you
simply screen various RAPD markers to see which ones are polymorphic. Any
polymorphic marker observed must be linked to your trait, gene, or genetic
locus.
For example, let's assume that you would like a marker linked to the dominant
white gene. Since the white feather color is dominant, an F2 population would
yield 3 white chicks for every non-white chick. The white phenotype would
separate the chicks into 2 groups, the white chicks and the non-white chicks.
RAPD markers would be used to screen for DNA polymorphisms. Since all the
other genetic loci that are not linked to the white gene are segregating
independently, these unlinked loci will be present in both groups. Thus any
DNA polymorphism observed will be linked to the white gene.
This procedure is extremely fast and simply, taking less than 2 weeks to identify
and confirm linkage of a genetic marker. I have used this procedure numerous
times to target markers to simple traits in plants.
54