Results
and discussion
The
overall objectives of PiGMaP were to develop a 20 centiMorgan
genetic (linkage) map covering 90% of the genome; to produce a
physical map with at least one distal and one proximal landmark
locus mapped on each chromosome arm and to plan experiments to
map the so-called quantitative trait loci (or QTLs) [refs: 5,
6, 8, 31,
32, 65, 66,
67, 68, 70,
73, 108, 110,
111, 115, 118,
121, 123, 157].
Genetic
(linkage) mapping
The
genetic (linkage) map was developed by a group of seventeen laboratories
within the collaboration. Reference pedigrees were established
in five centres (Scotland (01),
France (06), Germany (05),
the Netherlands (08) and Sweden
(12)) [refs: 19,
69, 71, 116,
152]. These pedigrees take the form of three-generation
families in which grandparents from genetically divergent breeds
were crossed to produce the parental (F1) generation which were
subsequently intercrossed. In the Scottish, French and Dutch pedigrees
the founder grandparental breeds were the Chinese Meishan and
the European Large White (Yorkshire). The Swedish and German pedigrees
had European Wild Boar and European improved breeds as their grandparents.
The karyotypes of some of the founder animals were checked for
abnormalities [refs: 7, 74,
76]. DNA samples from 118 F2 pigs plus their
respective parents and grandparents were distributed to fourteen
different laboratories (including one in Australia and one in
the United States) for genotyping.
Several classes of molecular genetic markers were developed and
used in the genetic mapping studies. First, restriction fragment
length polymorphisms (RFLPs) were established by Southern blot
analysis. Most of the probes used to reveal RFLPs were expressed
sequences in the form of cDNA clones. Both homologous (mainly
cDNA) and heterologous (human, rodent, and other mammalian) probes
were used to develop these RFLP markers. The diverse origins of
the founding breeding stock meant that many loci screened in this
manner did indeed prove to be polymorphic. Over fifty such RFLPs
have been characterised by the Bologna, Edinburgh, Foulum, Iowa,
Oslo, Sydney and Uppsala groups [refs - 14,
15, 52, 72,
75, 86, 92,
93, 94, 96,
97, 98, 103,
105, 124, 126,
127, 134, 135,
136, 137, 138,
153, 158, 159,
160, 162, 181,
182, 183]. Second, single
strand conformational polymorphisms (SSCPs) have been developed
for the 3'-untranslated regions of some genes [ref: 64].
These expressed sequences provide the basis for aligning the pig
gene map with the maps of other species, in particular with those
of the "map rich" species - man and mouse [refs: 56,
64, 174].
Hypervariable markers based upon both minisatellite and microsatellite
loci have also developed. The Merelbeke, Hohenheim and Leicester
groups have isolated and characterised sixteen new locus specific
minisatellite (VNTR) loci [refs - 50, 81,
83, 90, 166,
167, 168, 169].
Simple tandem repeat (STR) or microsatellite loci are the markers
of choice for QTL-mapping studies as they are abundant, evenly
distributed and readily genotyped using the polymerase chain reaction
(PCR). The structure, number of repeat blocks and chromosomal
distribution of porcine microsatellitte loci (dG-dT)n - (dC-dA)n
have been found to exhibit the same desirable features as their
human counterparts [refs - 36, 49,
79, 87, 88,
89, 91, 95,
125, 163, 171,
172, 173, 175,
176]. Participants 01, 02,
03, 04, 05,
06, 08, 09,
and 12 isolated and sequenced several hundred
microsatellite loci. Primers for polymerase chain reaction amplification
and genotyping have been designed and the highly polymorphic nature
of the loci confirmed. Over 150 of these microsatellite loci have
been genotyped in the shared mapping pedigrees. [refs - 3,
4, 18, 19,
20, 21, 22,
23, 39, 40,
51, 58, 102].
Markers with the combined benefits for comparative mapping of
expressed sequences and high polymorphic information content (PIC)
as provided by microsatellite repeats have been developed by scanning
the EMBL and GenBank databases for sequences that have both these
attributes. Amongst the loci for which such markers have been
developed are CGA, DAGK, IGF1, and SPP1.
Markers based on polymorphic poly(A) tracts adjacent to known
genes have also been established [ref: 101].
Finally, protein polymorphisms, erythrocyte antigen variants and
SLA genotypes have also been determined in some of the reference
pedigrees.
Genotypic data on animals in the shared panel of reference families
were sent to the Edinburgh laboratory by the typing laboratories
and entered into a central relational database built with INGRES
software. This database (ResPig) is accessible across the Internet
to all the participants who have provided genotyping data. The
data have been subjected to two-point and multipoint linkage analysis
using the Crimap suite of programs.
A linkage map of the porcine genome has been developed by segregation
analysis of 245 genetic markers [ref: 4]. Eighty-two
of these markers correspond to known genes. Linkage groups have
been assigned to all eighteen autosomes plus the X chromosome.
As sixty-six of the markers on the linkage map have also been
mapped physically, there is significant integration of linkage
and physical map data. Six informative markers failed to show
linkage to these maps. As in other species, the genetic map of
the heterogametic sex (male) was significantly shorter (~16.5
Morgans) than the genetic map of the homogametic sex (female)
(~21.5 Morgans). The sex averaged genetic map of the pig was estimated
to be ~18 Morgans in length. Mapping information for 59 Type I
loci (genes) enhances the contribution of the pig gene map to
comparative gene mapping. As the linkage map incorporates both
highly polymorphic Type II loci, predominantly microsatellites,
and Type I loci it will be useful both for large experiments
to map quantitative trait loci and for the subsequent isolation
of trait genes following a comparative and candidate gene approach.
Physical
(cytogenetic) mapping
Participants
in Copenhagen, Toulouse, Uppsala and Utrecht have assigned genes
to chromosomes by in situ hybridisation techniques [refs:
10, 11, 12,
13, 28, 29,
30, 34, 43,
44, 52, 59,
60, 61, 77,
82, 84, 85,
86, 112, 113,
124, 131, 140,
141, 142, 154,
155, 161, 175,
177, 178, 184].
Although radioactive methods of in situ hybridization have
been used for some assignments, especially where a short heterologous
cDNA probes was used, most experiments now employ fluorescent
in situ hybridization (FISH). Regional assignments have
been made for one hundred and thirty loci, including sitxy-nine
anonymous DNA segments[ref: 62]. The physical
mapping of functional genes to chromosomes is essential to the
alignment of the porcine gene map with the maps of other species,
in particular with those of humans and mice. The anonymous DNA
sequences that have been mapped include cosmid and P1 clones from
which microsatellite markers have been developed for linkage studies
[refs: 41, 42, 60,
61, 80, 104].
The Toulouse laboratory have developed a method termed SLIM-PCR
for the isolation of microsatellites from cosmids without subcloning.
Synteny mapping has been effected by analysis of somatic hybrid
cell lines. The cell lines available at the outset did not constitute
a mapping panel and therefore further hybrid cell lines were developed
by the Copenhagen, Cambridge, Toulouse, Ulm and Utrecht groups
[ref: 54, 63]. However,
analysis of the new pig-rodent somatic hybrid cell lines created
for the project, indicate that fragmentation and rearrangement
of the porcine chromosomes in the hybrid lines is a problem. The
use of alternative fusion partners is being explored. A fluorescent
in situ hybridization procedure with a porcine SINE (short
interspersed elements) probe has been developed which enables
the identification of pig chromosomes in pig x rodent hybrid cells
[ref - 24, 63]. Synteny
mapping studies have confirmed the localization of twelve loci.
DNA from a hybrid cell line presumed to have retained the p-arm
of chromosome 12 as the only pig component has been used to establish
a library enriched for sequences from this region of the pig genome.
A physical (cytogenetic) map of the pig genome has been developed
by the PiGMaP collaborators [ref: 62]. Physical
mapping information is available for a total of 142 loci, including
69 anonymous DNA segments. Of these 142 loci, 130 have been regionally
mapped on chromosomes by in situ hybridization and 12 have
been assigned to chromosomes using somatic cell hybrids. Landmark
loci have been identified on each chromosome arm of all 18 autosomes,
the Y chromosome and the pseudoautosomal region of the X and Y
chromosomes.
Flow
cytometry
The
polydisperse nature of the porcine karyotype allows the chromosomes
to be sorted effectively by the use of a dual laser FACS machine.
The DNA content of the haploid porcine genome (~2770 Mb) has been
estimated from the flow karyotype [ref: 164].
The DNA content of individual chromosomes ranging from 295 Mb
for chromosome 1 to 68 Mb for chromosome 18 (the Y chromosome
is only 47 Mb) has also been estimated.
The groups (02 and 06), that
established flow cytometry of pig chromosomes have determined
the chromosomal identity of the flow sorted peaks [refs: 35,
38, 48, 57,
78, 99, 106,
107, 132, 133,
143, 145, 150,
165, 179, 180].
Material corresponding to a single peak isolated by preparative
flow cytometry was amplified by the polymerase chain reaction
(PCR) using degenerate primers and fluorescent label incorporated
into the PCR product [ref: 144]. This fluorescently
labelled DNA was then used to probe or paint metaphase chromosomes
and thus identify the chromosomal origin of the flow sorted peak.
The identity of all twenty peaks, corresponding to the eighteen
autosomes plus the X and Y sex chromosomes has been determined
by both groups [refs: 38, 180].
The resulting fully characterised and confirmed flow karyotype
will be useful for future studies directed at particular chromosomes.
Already the flow sorted material has been used to develop chromosome
specific libraries for chromosomes 1, 6, 7, 11, 13, 16 and 18
[refs: 16, 36, 37,
46, 149]. Markers isolated
from the chromosome 1, 6 and 13 libraries have been mapped by
linkage analysis and in situ hybridization. One subchromosomal
region specific library for 6p1.1-q1.2 has been established by
coincident cloning using DNA isolated from flow sorted chromosome
6 and from a somatic hybrid cell line containing, amongst other
porcine chromosome fragments, 6p1.1-q1.2 [refs: 25,
27].
Repetitive
sequences
Repetitive
sequences and the physical components of chromosomes - telomeres
and centromeres have also been studied [refs: 26,
45, 47, 55,
148, 170]. The characterisation
of porcine SINE sequences has proved to be useful in the amplification
of the limited amounts of DNA produced by flow cytometry or chromosome
scraping and in the characterisation of the chromosome content
of somatic hybrid cell lines [refs: 24, 26,
63]. A novel genotyping method named 5' and
3' SINE-PCR was developed to genotype the variable dinucleotide
repeats found at the 3' end of about 12% of pig SINE sequences
[refs: 146, 147]. As the
method uses one arbitary primer and a SINE primer no sequencing
or cloning is required to develop the markers. Conventional SINE
and LINE-PCR products are also polymorphic in the mapping pedigrees.
New repeat families have been characterised and mapped to chromosome
3 and 10 [ref: 53].
Quantitative
trait loci (QTL) mapping
QTL mapping studies have been initiated at each of the five centres
that have established mapping populations [refs: 1,
152]. The design of effective QTL mapping
experiments have been considered [refs: 109,
117, 151, 156].
New statistical methods for locating quantitative trait loci have
been developed [refs: 33, 119,
120, 122, 128,
129, 130]. Already regions
of chromosome 4 that influence growth rate, fatness and intestinal
length have been identified in a Wild Boar / Large White cross
[ref: 1]. Fatness, whether measured as the percentage
of fat in the abdominal cavity or backfat thickness, appears to
be under the control of QTLs that map to the proximal end of chromosome
4. The QTLs for intestinal length and growth rate (from birth
to 70 kg) are located distal to the "fatness" QTLs.
Major
scientific breakthroughs and / or industrial applications:
A
genetic (linkage) map of the pig genome covering all 18 autosomes
with a resolution of at least 20 centiMorgans has been established
[ref: 4]. This linkage map which combines both
highly polymorphic anonymous DNA markers and polymorphisms within
or close to known genes will be an invaluable resource for future
studies to map genes influencing economically important traits.
A physical (cytogenetic) map with landmark loci identified on
each chromosome arm provides a valuable framework for the subsequent
isolation of trait genes on the basis of their map positions [ref:
62].
A collaborative study involving the PiGMaP and Nordic pig gene
mapping groups established that genes located on pig chromosome
4 have significant effects on growth, fatness and intestinal length
[ref: 1]. The resolution of pig chromosomes
into 20 discrete fractions, corresponding to the 18 autosomes,
plus the X and Y sex chromosomes was not only elegant, but also
provides the means to focus mapping future mapping studies on
particular chromosomes [refs: 38, 180].
Already chromosome specific libraries have been developed for
such studies.
