MAIN
ACHIEVEMENTS
- regions
of the genome influencing economically important traits, including
fatness and growth rate have been identified
- the
RN gene which affects meat quality has been mapped to chromosome
15
- the
resolution and genome coverage of the PiGMaP linkage maps have
been improved
- a
five-fold genome coverage large fragment genomic library has
been established in Yeast Artificial Chromosome (YAC) vectors
- large
fragment genomic libraries have been established in alternative
bacteriophage P1 and bacterial artificial chromosome (BAC) vectors
- DNA
sequence signatures (or expressed sequence tags, ESTs) have
been established for >2,000 genes
- over
200 genes or ESTs have been added to the pig genome map
- minisatellite
loci have been shown to predominantly telomeric in the pig
- genetic
diversity in 12 (common and rare) pig breeds has been examined
in a pilot study
Mapping trait loci -
Animal breeding genetic models often assume that complex traits
such as growth and carcass composition are controlled by a large
number of unlinked genes (quantitative trait loci, or QTL) each
with only a small effect on the trait. The alternative to this
"infinitesimal" model or hypothesis is that there are individual
genes with significant effects on the trait of interest. The development
of genome maps based on highly polymorphic molceular genetic markers
allows these alternative hypotheses to be tested. QTL-mapping
involves searching for associations between the segregation of
marker alleles and performance. Thus, QTL-mapping requires populations
of animals that exhibit genetic variation in the traits of interest
and genotyping of these populations for a panel of polymorphic
genetic markers covering the genome. The necessary mapped polymorphic
markers were developed in the original PiGMaP collaboration (Archibald
et al., 1995). The specific objectives and targets for
mapping trait loci were -
- to
produce resource populations of pigs in three-generation intercross
pedigrees and to isolate and store DNA from them
- to
record phenotypic information on the traits of interest for
1800 F2 pigs
- to
genotype the resource populations for 100 selected polymorphic
genetic markers spaced at approximately 20 cM intervals
- to
analyse the data for associations between traits and markers
- to
produce new genetic markers and improve the genome coverage
of the PiGMaP linkage maps
To
produce resource populations of pigs in three-generation intercross
pedigrees and to isolate and store DNA from them
Three-generation QTL-mapping populations were established in which
purebred pigs were crossed to create F1 animals. These F1 animals
were then mated inter se to generate large numbers of F2 pigs
which were recorded for the traits of interest (Table 1). The
populations developed by the Roslin Institute, INRA and Wageningen
Agricultural University were based on crosses between genetically
divergent (Chinese) Meishan and (European) Large White breeds.
Further crosses between a) European Wild Boar and Pietrain or
Meishan and b) Meishan and Pietrain were established by the Hohenheim
laboratory. Finally, crosses between Wild Boar and Swedish Yorkshire
(Large White) pigs had been established earlier by the Swedish
University of Agricultural Sciences, Uppsala. Genetically divergent
founder breeds were used in order to maximise the chance of identify
regions of the genome (QTL) that influence the genetic control
of these traits. For example, the Meishan differs greatly from
European breeds (e.g. 40% greater litter size, puberty at half
the age, resistance to stress and K88 E. coli, docile temperament,
half the growth rate and twice the subcutaneous fat thickness).
To
produce resource populations of pigs in three-generation intercross
pedigrees and to isolate and store DNA from them
Trait recording and preparation of DNA samples was completed for
>2500 F2 pigs. Although these pigs have been performance tested
for a wide range of traits, the analyses were limited to the following
traits - birth and weaning weights, growth rate to slaughter,
subcutaneous fat depths and some measures of meat quality. Eighteen
hundred animals were selected from these populations for the QTL-mapping
analysis.
To
genotype the resource populations for 100 selected polymorphic
genetic markers spaced at approximately 20 cM intervals
In order to map quantitative trait loci in these pig populations
it was necessary to genotype the pigs for genetic markers that
provide good coverage of the porcine genome. The pigs in the QTL-populations
were genotyped for 100 markers at 20 centiMorgan (cM) intervals
(see Table 1). Microsatellite markers are ideal for QTL mapping
studies as they are highly polymorphic and can be relatively rapidly
using the polymerase chain reaction (PCR) and automated fluorescent
DNA fragment analysers (sequencers). Panels of markers were selected
from the >1500 micorsatellites mapped by the PiGMaP Linkage Consortium
and the parallel mapping activities at the USDA Meat Animal Research
Center. Markers were selected on the basis of their map locations
and ease of use. As the markers were also selected for their degree
of informativeness or heterozygosity in the target populations
different markers were used in the various QTL-mapping populations.
Table1: Summary of QTL mapping
To
analyse the data for associations between traits and markers
A total genome scan for QTL detection was carried out for growth,
fatness, meat quality, body proportion and immunological traits
using phenotypic records from the F2 generation of the Uppsala
Wild Boar - Large White intercrosses (see Andersson et al.,
1994). Further characterization of the major QTLs on pig chromosome
4 affecting growth and fatness traits is being effected by backcrossing
to Large White pigs. The presence of one or more QTLs on chromosome
4 was confirmed by data on 85 BC2 (second backcross) progeny in
two half-sib families.
A QTL analysis of chromosome 4 in the Roslin Large White - Meishan
pedigrees provides preliminary evidence for at least one QTL for
growth. However, this QTL appears to be located more proximally
than the growth QTL detected in the Uppsala pedigrees.
QTLs with effects on traits including carcass composition, meat
quality, fattening and stress reaction have been mapped to chromosome
6 in the Hohenheim Wild Boar - Pietrain and Meishan - Pietrain
crosses. Most of these QTLs are in the region of the 'halothane'
locus which encodes a skeletal muscle sarcoplasmic reticulum calcium
release channel (CRC). There are also indications of QTL in the
P3-EAO interval towards the telomeric end of the long arm of chromosome
6. Further chromosome 6 markers are being genotyped in these pedigrees
to increase the resolution of the QTL mapping.
QTLs influencing 12 performance traits have been mapped to chromosome
7 in the Hohenheim Meishan - Pietrain population. Evidence that
these QTLs are of varying importance in different populations
is provided by the failure to detect QTLs influencing these twelve
traits in the Wild Boar - Pietrain crosses. The QTLs for carcass
composition were mapped to a 40 cM interval between S0064 and
S0066. The Meishan alleles at these QTLs were associated with
better meat quality, smaller meat area and surprisingly with a
leaner carcass than the Pietrain alleles. Using a single marker
analysis of variance and multipoint maximum likelihood methods,
some significant chromosome 7 effects have been found in the INRA
Large White - Meishan crosses for - growth rate (SLA), fat androsterone
level (SLA) and plasma cortisol levels (S0101).
Effects of the region around S0058 (chromosome 14) on average
backfat thickness were also revealed by single marker analysis
in the INRA Large White - Meishan pedigrees.
A pilot joint QTL analysis was undertaken with respect to chromosome
4, which has been shown in earlier studies to harbour genes influencing
growth and fat levels. Genotypes and trait (growth) data for 1800
animals each genotyped for up to 17 polymorphic microsatellites
on chromosome 4 were pooled and subjected to a joint analysis.
This pilot study allowed the evaluation of methods for the analysis
of pooled data.
In addition to the search for quantitative trait loci, some major
genes were also studied. For example, the RN gene, which has a
major effect on meat quality, was mapped to chromosome 15 (Milan
et al., 1995, 1996; Le Roy et al., 1996). The effect
of the estrogen receptor gene (ESR) on prolificacy was examined
in two Large White lines of pigs (Legault et al., 1996).
Associations between variation in the growth hormone locus (GH1)
and performance traits were examined in the Hohenheim pedigrees.
In the Meishan - Pietrain crosses eight traits were associated
with GH1 genotypes, but no effects were detected in the Wild Boar
- Pietrain crosses. The differences between the extremes in the
Meishan - Pietrain F2 pigs were ~10 mm for back fat depth, ~1
kg for back fat weight, 6% for lean and fat cut percentages and
0.3% for lean to fat ratio. Although associations between PIT1
genotypes and some performance traits were found in the Wild Boar
- Pietrain and the Meishan - Pietrain pedigrees, these findings
were not significant if a genome-wide significance threshold is
used.
To
produce new genetic markers and improve the genome coverage of
the PiGMaP linkage maps
In order to make the best use of the available microsatellite
markers from both the PiGMaP and USDA maps for QTL and trait gene
mapping it was necessary to align these maps. Alignments of PiGMaP
and MARC linkage maps have been published for chromosomes 1, 2,
3, 5, 9, 10, 11, 13, 14 and 15 (Zhang et al., 1995; McQueen
et al., 1995; Riquet et al., 1995; Davies et
al., 1995; Kapke et al., 1996; and Milan et al.,
1996). The PiGMaP, USDA and Nordic collaboration linkage maps
of chromosome 6 and 7 have been fully integrated by pooling and
analysing all the available genotyping data (Paszek et al.,
1995; Rohrer et al., in press).
Targetted improvements of poorly mapped chromosomes was effected
by establishing chromosome-specific libraries from which to isolate
new markers. For example, a flow-sorted chromosome 11 specific
libary was developed in collbaoration between the CEA group at
Fontenay-aux-Roses and INRA Toulouse (Riquet et al., 1995).
This library was critical to the improvement of the linkage map
of chromosome 11.
A second release of the PiGMaP Linkage Consortium map of the porcine
genome has been submitted for publication (Archibald et al.,
in preparation). The expanded data set includes more than 700
polymorphic genetic markers. The additional markers include ~100
selected from the USDA-MARC maps. The resolution of the linkage
maps has been enhanced by merging the PiGMaP and Nordic collaboration
genotyping data. Thus, there are up to 600 informative meioses
for some markers.
Minisatellites are a class of often highly polymorphic tandem
repeats clustered towards chromosome ends in humans. Thus, they
have contributed to the definition of the ends of human genetic
(linkage) maps. Pig minisatellite sequences were cloned, characterised
and mapped in order to determine whether this class of loci might
be similarly useful for defining maps ends in the pig. Forty-four
minisatellite containing cosmids were isolated from gridded arrays
of 15,000 pig cosmid clones. Polymorphic markers developed from
these clones have been mapped in the PiGMaP reference pedigrees.
In collaboration with the INRA Toulouse group physical (cytogenetic)
map locations were established for 30 minisatellite containing
cosmids. These data demonstrate that, as in humans, minisatellites
are clustered towards the telomeres. Interestingly, a cluster
was also found on chromosome 6 at an ancestral chromosome fusion
site.
Tools
for trait gene identification and cloning -
QTL mapping studies identify chromosomal regions that contain
genes controlling traits of economic and biological significance.
There are two strategies for the subsequent identification and
isolation of such trait genes - 'positional cloning' or the 'positional
candidate gene approach'. We have developed key resources necessary
for implementing these strategies - large fragment genomic libraries
and catalogues of (mapped) expressed sequences (or genes). The
specific objectives and targets were -
- to
produce a 3-5 fold genome coverage YAC library
- to
produce a 3 fold genome coverage P1 library
- to
develop 1000 expressed sequence tags (ESTs) from cDNA clones
- to
map 200 expressed sequences (genes and/or ESTs)
- to
develop comparative pig-human and pig-mouse comparative maps
with a resolution of 10 cM
To
produce a 3-5 fold genome coverage YAC library
Large fragment genomic clones are an essential resource for positional
cloning as they allow the isolation and characterisation of regions
of genomic DNA =100 kb containing, for example, a marker known
to flank the trait gene of interest. Three large fragment library
resources were developed - a a library constructed in a yeast
artificial chromosome vector and two libraries constructed in
bacterial cloning vectors.
A porcine YAC library of about 34,000 clones was established by
the Laboratoire de Radiobiologie Appliquée. The average
size of the cloned inserts is ~300 kb. Thus, as the pig genome
is estimated at 3 x 109 bp, the 34,000 clones provide
about 3-fold coverage of the genome. Fluorescent in situ hybridization
analysis of 30 clones indicates that up to half of the clones
in the library may be chimeric. The clones have been individually
picked into 96-well microtitre plates and stored at -70oC.
The library has been organised into 43 superpools, with each superpool
containing 768 clones (i.e. 8 plates of 96 wells). Low density
filters (22 membranes with each clone represented once) have been
prepared for screening by hybridization. Clones have been exchanged
with Professor Brennig (Göttingen) who has ~10,000 pig YAC
clones and Dr. Lehrach (Berlin) who has 8,000 pig YAC clones.
These pooled YAC libraries contain ~50,000 clones are are equivalent
to 5-fold coverage of the porcine genome.
The complexity of the library was tested by PCR screening for
35 distinct genes, including 28 on behalf of 10 other laboratories,
including Uppsala, Ghent, Toulouse, Limoges and Iowa. Twenty-six
of the PCR primer pairs (75%) led to the isolation of at least
one YAC clone. A total of forty-four YAC clones were isolated.
YACs containing sequences from the porcine major histocompatibility
complex (MHC) have been isolated and a contig constructed for
this region encompassing 7 clones covering 1 Mb. This contig contains
at least five MHC class I sequences and seven non-MHC genes. The
physcial map of the SLA complex, including the class II region,
already comprises 44 characterized genes, and represents the best
defined chromosomal segment in the pig.
To
produce a 3 fold genome coverage P1 library
A porcine genomic library of about 50,000 clones with an average
insert size of ~80 kb was established in the bacteriophage P1
vector by the Roslin group (McQueen et al., 1995). Individual
clones were picked into 384-well microtitre plates and stored
at -70oC. This library represents ~1.3 fold coverage
of the porcine genome. However, as handling of the clones is difficult
and the preparation of DNA from the clones is not trivial further
development of this library was abandoned. A replacement bacterial
based large fragment genomic library was constructed in the bacterial
artificial chromosome (BAC) vector pBeloBAC11. Clones established
in this BAC vector are easier to handle than P1 clones and the
average insert size is >100 kb. Half the clones will carry BamHI
(partial) fragments and the other half of the library will contain
HindIII (partial) fragments. Clones were picked in duplicate
into 384-well plates and are being stored at -70oC.
One hundred thousand clones will be picked in order to give about
3-fold genome coverage. The development and exploitation of the
BAC library will continue in the context of an EC INCO Copernicus
project.
To
develop 1000 expressed sequence tags (ESTs) from cDNA clones
In order to increase the gene content of the porcine genome maps,
expressed sequences as represented by cDNA clones are being characterised
and mapped. A total of 1230 clones from a small intestine cDNA
library have been sequenced from both the 3' end (poly(A) tail)
and 5' end and a further 570 sequenced from the 3' end only (Copenhagen,
Oslo) (Winterø et al., 1996). Searches against the
DNA sequence databases have yielded tentative identities for 585
of these transcripts and a further 449 clones have been scored
as anonymous. One hundred and eighty-nine clones selected from
a granulosa cell library (Toulouse) have been partially sequenced
from both their 5' and 3' ends to generate further ESTs (expressed
sequence tags). Further muscle cDNA clones have also been characterised
by sequence analysis (Bologna). The target of 1000 cDNA clones
sequenced has been significantly exceeded. Sequence data from
these clones have been lodged in the EMBL sequence database.
Table 2: Summary of cDNA sequence (Expressed Sequence Tags, ESTs)
To
map 200 expressed sequences (genes and/or ESTs)
The pig genome probably contains about 50-100,000 genes. Thus,
only a small proportion of porcine genes have been even partially
characterised. The ESTs developed within this project more than
doubles the number of partially characterised pig genes. If these
partially charcaterised genes are to be useful in the identification
of trait genes, then it is necessary to map the genes / ESTs.
The target of 200 mapped expressed sequences (genes and/or ESTs)
has been surpassed. Over 200 genes or expressed sequences have
been mapped by linkage analysis, synteny mapping in somatic cell
hybrids, in situ hybridization or pulse field gel electrophoresis
(see Table 3). Polymorphic markers (restriction fragment length
polymorphisms (RFLPs), single or double strand conformational
polymorphisms (SSCP, DSCPs)) have been developed for 96 genes
(or ESTs) and mapped in the PiGMaP and Nordic reference pedigrees
(Bologna, Copenhagen, Foulum, Iowa, Roslin, Uppsala).
DNA from further informative somatic cell hybrid panels has been
distributed by the Toulouse and Utrecht laboratories, respectively.
The Toulouse panel has been characterised in sufficient detail
to allow the assignment of genes and markers to sub-chromosomal
regions. A World Wide Web facility has been developed at INRA
Toulouse to aid mapping in this resource ( http://www.toulouse.inra.fr/lgc/pig/hybrid.htm).
Over seventy genes or ESTs have been mapped in this resource.
Forty-five genes have been localised on chromosomes by in situ
hybridization by the Copenhagen, Toulouse, Ulm, Uppsala Utrecht
groups including cDNA clones. Fine scale physical mapping studies
using pulse field gel electrophoresis and the development of YAC
contigs have focussed on the major histocompatibility complex
(Jouy-en-Josas, Merelbeke).
Table 3: Summary of genes mapped
To
develop comparative pig-human and pig-mouse comparative maps with
a resolution of 10 cM
These additions to the porcine gene map facilitate the comparison
of the porcine genome with better mapped species including humans
and mice. The conservation of synteny between pigs and humans
has been examined by heterologous chromosome painting (Zoo-FISH).
The initial results from the Ulm group, who identified 47 chromosomal
segments that are conserved between humans and pigs (Rettenberger
et al., 1995), have been confirmed by the Uppsala laboratory
and by the Toulouse group in collaboration with the Fontenay-aux-Roses
laboratory. These latter groups have also painted human chromosomes
with porcine chromosome-specific probes (Goureau et al.
1996). Chromosome homology between the domestic pig and the babirusa
(also from the Suidae family) has been evaluated by heterologous
chromosome painting with pig chromosome-specific probes (Bosma
et al., 1996).
Heterologous chromosome painting experiments have also been conducted
to establish a swine-bovine comparative map. For example, pig
chromosomes 5, 11, 12 and 13 appear to be well conserved in cattle
as each corresponds to only one or two bovine chromosomes - SSC13
and BTA1 + BTA22; SSC5 and BTA5; SSC11 and BTA12, SSC12 and BTA19.
Some bovine chromosomes are completely painted by probes from
only one pig chromosome (BTA6 by SSC8; BTA9 by SSC1; BTA18 by
SSC6; BTA20 by SSC16; BTA21 by SSC7; BTA26 and BTA27 by SSC14;
BTA29 by SSC3). This extension of the comparative maps to multiple
dimensions - human:pig, pig:human, human-cow; pig:-cow, human-mouse
- will maximise the opportunities for transferring mapping information
from one species to another. Thus, the candidate genes to explain
a trait mapped in species A may be provide by gene mapping information
on homologous regions in species B, C and D.
The additions to the gene maps of the pig as descibed above have
allowed the development of a pig-human and pig-mouse comparative
map with an average resolution of 10 cM. Further refinement of
these comparative maps is required in order to fully exploit the
gene-rich maps of humans and mice as sources of candidate genes
for trait genes in pigs.
Pilot
study on genetic diversity The breeds for the genetic
diversity pilot study have been identified and DNA is being prepared.
The protocols for sampling and marker analysis have been agreed.
A panel of 27 microsatellite markers has been selected for genetic
diversity studies. After extensive testing by the Toulouse and
Wageningen laboratories the markers were chosen on the basis of
their high degree of polymorphism, genome distribution, absence
of non-amplified alleles and ease of use on fluorescent DNA fragment
analysers. Stocks of the necessary fluorescent and unlabelled
PCR primers have been prepared and distributed by the US Pig genome
coordinator (Dr. M. F. Rothschild). This panel of markers has
been adopted by FAO for pig genetic diversity studies.
DNA fingerprinting offers an alternative to microsatellite markers
for evaluating breed genetic diversity and genetic variation.
For example, the minisatellite marker S0322 reveals higly informative
multi-locus DNA profiles. This probe was used to examine diversity
between three groups of unrelated pigs (group 1 = Large Whites;
group 2 = Chinese Meishan; group 3 = Pietrain, Landrace, Duroc,
British Saddleback and a commercial synthetic) A total of 110
polymorphic fragments were scored (on average 14.4+-2.8 fragments
per individual). Although the sample size for each group was quite
small there was evidence of breed specific fragments. This DNA
fingerprinting approach was also used to compare outbred and inbred
lines of Large White pigs. The inbred lines showed a significant
loss of both variation and large DNA fragments.
