Download 1995 Broad et al: CURRENT STATE OF THE NEW ZEALAND

Proc. Aust.Assoc. Anim. Breed. Genet. Vol. 11
CURRENT STATE OF THE NEW ZEALAND SHEEP GENOME PROGRAMME
TE BROAD, GW MONTGOMERY, AM CRAWFORD, AND DF HILL
AgResearch Molecular Biology Unit, Department of Biochemistry, University of Otago, PO Box 56,
Dunedin New Zealand.
SUMMARY
The main focus of the NZ Sheep Genome Programme has been the construction of a primary map of the
sheep genome to be used as a tool for the discovery of genes encoding economically important traits, by the
process of “reverse genetics” or positional cloning. The first fruits of this approach were realized last year
by fmdmg DNA markers linked to the Booroola fecundity gene on sheep chromosome 6. The region of the
X chromosome containing the Inverdale gene has also been defined by DNA markers.
Since then, work continues to complete the construction of the primary map which will integrate the
physical map (of about 150 markers for known genes on the human, mouse and cattle maps), and the
genetic map of the sheep (of about 220 microsatellite markers isolated from sheep and cattle DNA).
The mapping effort is increasingly being redirected to the search for markers for wool quality (fibre
diameter, fleece weight), resistance to disease (facial eczema and nematodes), and meat quality (fat depth,
muscle area) traits, and into the isolation of the genes for the Booroola and Inverdale fecundity traits.
The resources of the Programme, which include mapping data and reagents, flocks, a sheepyeast artificial
chromosome library, aud computing expertise, are available to collaborators.
INTRODUCTION
The long term prosperity of the New Zealand sheep industry depends on increasing the financial margins
between costs and prices in a sustainable manner. This will be achieved by the development and
application of new technologies to produce high value products which enjoy good market demaud without
market access restrictions related to perceptions of product quality.
The Sheep Genome programme is an integral part of New Zealand’s strategy to achieve this acceptability
and profitability. The programme is directed towards developing technologies for increasing the flexibility
of sheep production systems, improving the health of animals, and the quality and diversity of their
products. An understanding developed of the molecular basis of genetic variation will be exploited by
marker assisted selection using specially developed DNA markers to improve current selection procedures
which have been the basis of the steady genetic improvement in the productivity of the NZ sheep industry
over the last 25 years.
Foremost in the requirements to develop these tools has been the construction of a “primary map” of the
sheep genome. The strategy for constructing this map has been outlined in a recent review (Broad and Hill
1994). The aim has been to produce an integrated physical and genetic map which provides sufficient
coverage of the sheep genome such that a mapped marker will co-segregate with a trait of value to the
sheep industry. Such a map will serve to localize the genes responsible for the trait to a defmed
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chromosomal segment, and this will eventually lead to the isolation and characterization of the genes
involved.
The value of the “whole genome screening” approach was demonstrated by the fmding of markers linked to
the Booroola high fecundity (Fe&) gene (Montgomery et al. 1993). (Regress towards developing markers
for identifying carriers of this trait, and the identification of the FecB gene is summarimd below).
The increased fecundity of sheep with the Booroola gene is attributable to a mutation at a single locus
which segregates in the classical Mendelian fashion. However, it is known that many of the production and
performance traits segregate in a more complex, quantitative manner. The “whole genome screening”
approach appears to have succeeded in localizing quantitative trait loci for growth and famess in pigs
(Andersson et al. 1994) to particular chromosomal segments, but more testing of the applicability of this
approach for identifying quantitative trait loci is still required. Exploration of such loci affecting wool
quality, disease resistance and meat quality traits of sheep is now underway using the low-density “primary
map”, so it is expected that the general applicability of the approach will soon become apparent
As the nascent maps of livestock species have grown, interest in the evolution of genome function and
organization has also increased. Comparative genome analysis has emerged as a powerful tool for
piupointing genetic lesions discovered first in the human genome. Homologues of different loci in the
human genome producing mabgnant hyperthermia in pigs, leucocyte adhesion deficiency in Holstein cattle,
and hyperkalemic paralysis in Quarter horses have been identified (Womack 1992). The burgeoning
genomic and cDNA sequencing information of the human genome is also expected by the animal gene
mapping community to be of immense value as they come to grips with positional cloning studies in their
species. However, human medicine has been quick to exploit the benefits of animal models for
understanding and treating diseases of humans. In sheep, studies are underway of neuronal ceroid
lipofuscinosis as a model for human Batten’s disease, of cystic fibrosis, and of inherited deafness but these
are outside the scope of this review.
The Sheep Genomics programme depends on the GENES programme at AgResearch Invermay
Agricultural Centre, Mosgiel, for the maintenance of animals, their phenotypic evaluation, quantitative
genetic analysis, and statistics. The dependence of the Programme on research collaborations with other
research institutions will be apparent in the summary of research that now follows.
SUMMARY OF RESEARCH AREAS
1. Current status of the sheen man
The cytogenetic map consists of Giemsa-banded chromosomes of the sheep on which kuown genes and
cloned segments have been mapped. Errors and inconsistencies in the existing numbering of the sheep
chromosomes were formally recognised at a meeting of the Sheep Karyotype Standardization Committee at
Guelph in July 1994, and a table outlining these has been published (see Ansari et al. 1994).
As at January 1995, about 140 loci had been mapped to the sex chromosomes and 24 of the 26 pairs of
sheep autosomes by cell hybrid analysis and/or by in situ hybridization. A further thirteen had been
assigned using uncharacterized cell hybrids to 10 syntenic groups (whose chromosome number is
unknown).
Most of these loci (designated as Tyne I by O’Brien et al. 1993) are known coding genes for which cDNA
probes are available. Their sequence and function is conserved across a wide range of mammalian species,
and many have now been mapped in the human, mouse, and cow genomes. They now serve as cross459
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reference points between the genomes of sheep and these species, enabling conserved segments in their
chromosomes to be defined and knowledge of their genetic constitution and function to be extrapolated
between them. They have been named according to the conventions established for the human genome.
Crawford et al. (1995) have recently published a genetic map covering 2070 CMof the estimated 2770 CM
of the autosomes of the sheep genome (Chapman and Bruere 1977). This was generated from the linkage
analysis of 246 highly polymorphic markers in nine, three generation, full-sib pedigrees which comprise
the AgResearch International Mapping Flock.
The markers (designated by O’Brien et al. (1993) as Type II loci) were mainly DNA microsatellites from
bovids. Some 86 anonymous microsatellites were derived from the sheep genome, 126 were from cattle, 1
was from deer, and 33 markers (consisting mainly of microsatellites and a few RFLPs) were associated with
kuown genes. By linkage analysis, these markers were assigned to 26 liige
groups, each of which
represents a different sheep autosome. As the chromosomal location of most of the cattle markers was
known, and as the location on sheep chromosomes of the 33 markers associated with known genes was also
kuown, the chromosomal location of all these linkage groups could be deduced.
Work to “fill-in” the unmapped 700 CM on the sheep autosomes is now well advanced. Microsatellite
markers from the linkage groups are being screened over the cell hybrids to confirm their deduced
chromosomal location in the sheep. Also using these markers, the sheep YAC library is being screened for
chromosome-specific clones whose location is being physically determined by FISII. All of this work will
complete the construction of an integrated “primary map” of the sheep genome.
Meanwhile, fine mapping of the sheep X chromosome and chromosome 6 continues apace to locate the
Fecund@ Znverdale (FecX*Z)and Booroola (FecB) genes, respectively.
2. Single eene exoloration studies
The Booroola gene was located on one of the 26 autosomes after an extensive screening of 144 markers
(Montgomery et al. 1993). Once linkage was established, extensive use was made of comparative mapping
data and physical mapping resources in the Programme, to refine the position of the gene to a region of 19
CMon sheep chromosome 6 (Montgomery et al. 1994). The gene is flanked by a microsatellite marker and
an RPLP marker
The search for markers linked to the Inverdale gene was more focussed as family segregation studies had
established that it was located on the sheep X chromosome (Davis et al. 1991). A large number of
mammalian X chromosome markers have been screened in sheep and a provisional liige map of the X
chromosome completed (Dr SM Galloway, personal. communication).
3. Multi-factorial trait exnloration
Wool quality traits have been selected as a model system to assess the potential of the genome screening
approach for defining major contributing genes in quantitative trait loci. Super-fine Merinos have been
crossed with high fleece weight Romney sheep to enable the genes for these traits to be identified. These
breeds differ by 7-12 phenotypic standard deviations in fibre diameter, follicle density, secondary to
primary follicle ratio, and crimp frequency. In 1991,4 family lines were established. Four Fl rams (one
from each sire line) were backcrossed to Merino ewes in 1992 and 1993 to generate large half-sib families
with 100 progeny per sire. The sires have been mated to Romney ewes in 1994 to produce a further 100
progeny by the reciprocal backcross. DNA samples from the flock are being screened with the mapped
microsatellites from the sheep linkage map. Markers from sheep chromosomes 1 and 11 which carry the
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genes for high glycine-tymsine
screened first.
keratins and type I intermediate filament keratins, respectively, will be
Searches for disease resistance genes are underway in flocks selected for resistance and susceptibility to
facial eczema, and nematodes. Facial eczema is a mycotoxicosis caused the hepatotoxin sporedesmin. Two
lines of sheep have been selected at AgResearch Ruakura Research Centre, Hamilton, since 1975 (Morris et
al. 1989) for either resistance or susceptibility to sporedesmin challenge (Towers and Stratton 1978). The
two selection lines are now so different in their challenge response (which is determined by measuring the
circulating level of gamma glutamyl-transferase 3 weeks after sporedesmin treatment) that they have to be
challenged at two different doses:(0.15 mg/kg body weight for the resistant line versus 0.04 mg/kg for the
susceptible line). The Fl progeny produced by crossing the lines show responses to challenge that are
intermediate between the parental lines. Backcross F2 progeny generated by reciprocal crosses with sires
from either parental lines are being screened with 130 mapped microsateLlites spread at approxiamately 20
CM intervals across the sheep genome. Screening is also underway with four candidate genes known to be
associated with resistance in humans and mice, namely, cellular (Cu-Zn) and mitochondriaJ (Mn)
superoxide dismutases, catalase, and the membrane glycoprotein (P-gp), which belongs to a small gene
family associated with multiple drug resistance in cultured cells.
For nematode resistance, two lines have been selected since 1984 at AgResearch Wallaceville Research
centre, Upper Hutt, by Dr S Bisset and AgResearch Invermay by Dr JC McEwan, on the basis of faecal egg
counts under field challenge. These animals now exhibit a six-fold difference in their mean faecal egg
counts when run together under the same field challenge. Because actual parasite burdens are difficult to
estimate, phenotyping of the half-sib Fl progeny will now include 2 field challenges with 3 faecal egg
counts per challenge followed by slaughter and adult worm counts, identified by genus, in both abomasum
and small intestine.
The search for loci contributing to meat quality is beginning this year using a candidate gene approach to
determine whether there is any evidence for allelic associations in selected candidates with fatness aud
leanness in sheep. The flocks on which this work is focussed are fat, lean and control Coopworth lines of
120 ewes per line that have been developed since 1980 by Dr JC McEwan. AgReearch lnvermay.
A
difference of 4 standard deviations exists between the fat and lean lines in subcutaneous fat depth. DNA
polymorphisms in the candidate genes, such as the obese gene recently cloned in mouse (Zhang et al.
1994), will be examined in the half-sib Fl progeny.
4. Develonment of diaanostic eenotvning tests
Considerable technical development has been implemented to increase the rate of genotyping so that the
large scale genetic analyses required to define loci for multigenic traits is now possible. The process of
setting up PCR reactions for microsatellite analysis has now been automated aud the redesign of
electrophoretic systems has resulted in a rate of genotyping that covers up to 4000 individuals per month.
Efforts are underway to reduce the costs of individual genotyping assays, and to launch a commercial
parentage analysis service called GenomNZ.
RESOURCES
1. SheeD with Robertsonian translocations
Sheep derived from the New Zealand Romney breed which carry either single or multiple centric fusions
have been maintained as a nucleus flock. Its foundations were purchased in the mid-1980s from a larger
flock established in 1969 by Professor Neil Bruere, Massey University, Pahnerston North, New Zealand.
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This flock, which began with the first reported centric fusion (MI) in sheep (Bruere 1969), was
subsequently enlarged with the discovery of two other centric fusions, MII (Bruere and Mills 1971) and
MI11 (Bruere et al. 1972).
Bruere and Chapman (1974) renamed the translocation chromosomes tl,tz and t3, respectively. As this
renaming preceded the frrst standardization of the sheep karyotype at Reading (Ford et al. 1980), the
numbers of the chromosomes involved in the Robertsonian n-an&cations were subsequently changed by
Ansari et al. (1993), using the Reading karyotype, to tl = (6;24), t2 = (9;10), and t3 = (7;25).
Two new Robertsonian translocations, to = (5;8), and ts = (8;22) (Pearce et al. 1994) have been discovered.
Unfortunately the ewe carrying b died and this fusion chromosome is no longer present in the translocation
flock, which now has animals with tl, t2, and t3, either singly (in a homozygous state) or in combination
with ts (in a homozygous but more often in a heterozygous state). Thus, the number of chromosomes in
this flock ranges from 2n = 52 to 2n = 48, the normal karyotype being 2n = 54.
Fibroblast cultures of each of these five translocations have been established, and are in frozen storage
2. SheeD x hamster cell hvbrids
A most productive, long-standing, and on-going collaboration with Dr Carol Jones and her colleagues,
Eleanor Roosevelt Institute, Denver, USA, has led to the creation of a very useful panel of sheep x hamster
cell hybrids. Produced initially by Jones et al. (1985) and later, more extensively, by Burkin et al. (1993),
their utility derives from their intact and extensively characterized complement of sheep chromosomes,
many of which are present in a mono-chromosomal state.
The initial mini-panel of hybrid cells produced by Burkin et al. (1993), which is being continually enlarged
by successive rounds of cell fusions, selection and chraracterization,
was produced using buffy-coat
leukocytes from sheep carrying the Robertsonian chromosomes tl, t2, and t3 described above. The strategy
of using different auxotrophic mutant hamster cells for these fusions has enabled some selectiveness about
which sheep chromosomes to “capture” in different cell fusion experiments (see Burkin et al. 1993).
Frequently, only the selected chromosome is retained, but generally a small number of non-selected
chromosomes are also retained after the fusion. These can be eliminated by selection as outlined in Burkin
et al. (1993), by sub-cloning, or by extended growth in non-selective medium. Thus, using this strategy of
directed cell fusions, cell hybrids have been produced retaining sheep chromosomes 1, lq, 2, 3, 3q, 6 (also
from tlq), 7, t2 (ie. 9 and lo), 18, 24 (also from tl), 25, 26, and X.
Selected cell hybrids have also been used to generate chromosome-specific
regions of the sheep genome of particular interest.
libraries for fine mapping
3. International MaoDing Flock
This resource flock of nine, three-generation pedigrees which have 7-17 offspring in the third generation,
was established using multiple ovulation and embryo transfer techniques. Five breeds have contributed to
the pedigrees: Texel, Coopworth, Perendale, Romney, and Merino. The four Fl sires all had a common
Texel sire but different Coopworth dams. The nine Fl dams all had different Perendale-Coopworth aoss
dams, but had one of three Merino-Romney aoss sires.
DNA from the pedigrees has been sent to a consortium of nine laboratories contributing markers and
genotyping information to the linkage map of the sheep genome. These laboratories are: in Australia;
Centre for Animal Biotechnology, University of Melbourne; Victorian Institute of Animal Science,
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Melbourne; and CSIRO M&laster Laboratory, Glebe, NSW: in USA, USDA Meat Animal Research
Center (MARC), Clay Center, Nebraska; College of Veterinary Medicine, University of Georgia, Athens,
Georgia; and USDA Utah State University, Logan, Utah: and in Europe; Laboratoire des Groupes
Sanguins, INRA, Jouy-en-Josas, France; Molecular Human Genetics, Ruhr-University, Bochum, Germany;
and Justus-Liebig University, Giessen, Germauy.
As part of the consortium’s resources, further full-sib pedigrees are being developed for fine-mapping
individual sheep chromosomes. Dr Craig Beattie, USDA MARC, has generated 40, three-generation fullsib pedigrees with about 8 individuals in the F2 generation of each pedigree; Dr Jean-Michel Elsen, INRAToulouse, has contributed 2 large full-sib pedigrees each of about 50 individuals; and Dr Margaret
Matthews, Victorian Institute of Animal Science, Melbourne, has also contributed a full-sib pedigree.
4. Lame-insert sheen YAC librarv
Broom and Hill (1994) reported the development of a sheep yeast artificial chromosome library resource of
13,400 clones with a mean insert size of 680 kb. Based on an estimated length of the sheep genome at 3 x
10’ base pairs, the library provides a three-fold coverage of the sheep genome. The size of the library has
been recently increased to 29,000 clones to provide a seven-fold coverage of the sheep genome. This will
minimise complications arising Tom chiieric YACs being used for the assembly of contiguous clones, and
will augment the coverage of the more unstable regions of the sheep genome.
The YAC library is screened by robotics high-density gridding and FCR. Access to the library is available
is available, on request, to interested parties.
5. Comnutine and databases
Databases have been developed for the Booroola, Inverdale, and International Mapping Flocks 0
to
relate and manage pedigree, phenotype, and genotype information.
To process genotyping data, these relational databases have been designed to allow double-blind data entry,
data checking for pedigree mismatches, and preliminary 2-point linkage analysis. Data from the JMF
database can be exported into CBI-MAP (Lauder and Green 1987). which is the package used for
multipoint linkage analysis. Other relational databases designed to handle QTL information are currently
under development.
All published data for mapped loci in sheep have been compiled with the help of Dr AL Hillyard (USA) in
the SheepBase database using GBase (mouse) software. Data entry aud status will be managed by an
editorial panel of: Drs GW Montgomery (NZ), FW Nicholas (Australia), NE Cockett (USA) and TE Broad
(NZ). The database will be publicly accessible on World Wide Web in the next 2 months.
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REFERENcEs
ANDERSSON,L., HALEY, C.S., ELLEGREN, H., KNOTT, S.A., JOHANSSON, M., AMIERSSON, K.,
ANDERSSON-EKLUND, L., EDFORS-LILJA, I., FREDHOLM, M., HANSSON I., HAKANSSON, J.
and LUNDSTROM, K. (1994) Science 263: 177 1.
A.A. and BROAD, T.E.
ANSARI, H.A., PEARCE,
P.D., MAHER, D.W., MALCOLM,
(1993)chromosom
102: 340.
ANSARI, H.A., BOSMA, A.A.. BUNCH, T.D., LONG, S.E. (COORDINATOR) and POPESCU, C.P.
(1994) Cytogenet. Cell Gene?. 67: 114.
BROAD, T.E. and HILL, D.F. (1994) Br. Vet. J, 150: 237.
BROOM, M.F. and HILL, DF. (1994) Muma. Gemme 5: 817.
BRUERE, A.N. (1969) Qtogenet. 8: 209.
BRUERE, A.N. and CHAPMAN, H.M.(1974) Cytogenet. Cell Genet. 13: 342.
BRUERE, A.N. and MILLS, R.A.(1971) Cytogenet. 10: 260.
BRUERE, A.N., CHAPMAN, H.M. and WYLLIE, DR. (1972) Cytogenet. 11: 233.
BURKIN, D.J., MORSE, H.G., BROAD, T.E., PEARCE, P.D., ANSARI, H.A., LEWIS, P.E. and JONES,
C. (1993) Gemtics 16: 466.
CHAPMAN, H.M. andBRUERE, A.N. (1977) Cunud. J.Gemt. QtoL 19: 93.
CRAWFORD, A.M., DODDS, K.G., PIERSON, C.A., EDE, A.J., MONTGOMERY,
G.W.,
GARMONSWAY, H.G., BEATTIE, A.E., DAVIES, K., MADDOX, J.F. KAPPES, S.W.,
STONE,
R.T., NGUYEN, T.C., PENTY, J.M.,LORD, E.A., BROOM, J.E., BUITKAMP,
J., SCHWAIGER
BEH, K.J., MCGRAW,
W., EPPLEN, J.T., MATTHEW, P., MA-ITHFWS, M.E., HULME, D.J.,
R.A. and BEATTIE, C.W. (1995) Genetics (in press).
DAVIS, G.H., MCEWAN, J.C., FENNESSY, P.F., DODDS, K.G. and FARQUHAR, P.A. (1991) Biol.
Reprod. 44:620.
FORD, C.E., POLLOCK, D.L. and GUSTAVSSSON, I. (1970) Hereditm 92: 145.
JONES, C., MORSE, H.G.. GEYER, D., SCOTT, LAS. and BROAD, T.E. (1985) Cytogenet. Cell Gem?.
40: 662.
LANDER, E.S. and GREEN, P. (1987) Proc. NatL Acad. Sci., U.S.A. 84: 2263.
MONTGOMERY, G.W., CRAWFORD, A.M., PENTY, J.M., DODDS, K.G., EDE, A.J., HENRY, H.M.,
PIERSON, C.A., LORD, E.A., GALLOWAY, S.M., SCHMACK, A.E., SISE, J.A., SWARBRICK, P.A.,
HANRAHAN, V, V., BUCHANAN, F.C. and HILL, D.F. (1993) Nature Genet. 4: 410.
MONTGOMERY, G.W., LORD, E.A., PENTY, J.M., DODDS, K.G., BROAD, T-E., CAMBRIDGE,
L.M., SUNDEN S.L.F., ST0NER.T. and CRAWFORD, A.M. (1994) Gemmics 22: 148.
MORRIS, CA., TOWERS, N.R., CAMPBELL, A.G., MEYER, H.H., WESSELINK, C., and WHEELER
M. (1989) N.Z J. Agr. Rex 32: 379.
O’BRIEN, S.J., WOMACK, J.E., LYONS, L.A., MOORE, K.J., JENKINS, N.A. and COPELAND, N.G.
(1993) Nature Genet. 3: 103.
PEARCE, P.D., ANSARI, H.A., MAHER, D.W., MALCOLM, A.A., STEWART-SCOTT, I.A. and
BROAD, T.E. (1994) Cytogenet. Cell Genet. 67: 137.
TOWERS, N.R., and STRATTON, G.C. (1978) N.Z Vet. J. 26: 109.
WOMACK, J.E. (1992) Nature 360: 108.
ZHANG, Y., PROENCA, R., MAFFEI, M., BARONE M., LEOPOLD, L. and FRIEDMAN, J.M. (1994)
Nature 372: 425.
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