A radiation hybrid map of bovine Chromosome 7 and comparative
mapping with human Chromosome 19 p arm
James E. Womack,
Brian W. Kirkpatrick
Department of Animal Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA
Department of Veterinary Pathology, Texas A&M University, College Station, Texas 77843, USA
Received: 23 April 1999 / Accepted: 20 July 1999
Radiation hybrid mapping is expected to be an efficient and pow-
erful strategy in livestock genome mapping. A 5000-rad bovine
whole-genome radiation hybrid panel has recently been con-
structed (Womack et al. 1997), and radiation hybrid maps of bo-
vine Chromosomes (Chrs) 13, 19, and 23 have been reported with
this panel (Schläpfer et al. 1997; Yang et al. 1998; Band et al.
1998). Such RH maps demonstrate that radiation hybrid mapping
is a powerful tool for determining gene order, for facilitating com-
parative studies between cattle and other species, for bovine EST
mapping, and for integration of existing linkage maps (Yang and
Womack 1998; Yang et al. 1998). The goal of the current project
was to develop a radiation hybrid map of bovine Chr 7 (BTA7) as
well as to study the conservation of gene order between the BTA7
centromeric region and human Chr 19 (HSA19) p arm.
The radiation hybrid panel was kindly provided by James E.
Womack and his colleagues at Texas A&M University. Radiation
hybrids were produced by fusion of Chinese hamster A23 cells
with a male diploid bovine fibroblast cell line from an Angus bull
JEW38. The bovine fibroblast culture was exposed to 5000 rads of
gamma irradiation, and the hybrids were selected with HAT (hy-
poxanthine, aminopterine, thymidine) medium (Womack et al.
1997). Of the original 101 radiation hybrid lines, 90 were used to
constitute the BovRH5 panel.
A total of 34 markers were typed. Four of them were mono-
morphic STSs, which were developed within genes (Gu et al.
1997). They are cartilage oligomeric matrix protein (COMP), lym-
phoblastic leukemia derived sequence 1 (LYL1), lysosomal alpha-
mannosidase (MANB), and RAS oncogene family member
RAB3A (RAB3A). IL4 and RASA are polymorphic STS markers
associated with interleukin-4 and RAS p21 protein activator
(GTPase activing protein) genes respectively. The remaining 28
markers were polymorphic type II microsatellite markers.
PCR reactions were performed on an MJ Research PTC-100
thermal cycler in 13- to 15-l reaction volumes with1×Mg
PCR buffer (Promega), 0.125 m
0.035 U/l Taq polymerase (Promega), and magnesium with con-
centrations ranging from 1.5 to 2.5 m
. PCR reactions were pre-
heated at 95°C for 2 min and followed by 35 cycles at 94°C for 30
s, 52–66°C for 30–50 s, and 72°C for 30–60 s. The annealing
temperature was according to the published conditions for each
marker. Approximately 14–20 nanogram of radiation hybrid DNA
was used per reaction.
Markers with similar annealing temperature and different size
of PCR products were grouped together to perform duplex PCR.
Markers were typed with duplex PCR when possible to make
efficient use of the radiation hybrid DNA. Each of the 34 markers
was typed twice to reduce typing error. Markers were retyped if
there was a discrepancy between results from the first two runs.
For markers that were quite difficult to score (that is, nonspe-
cific amplification of many weak bands), a duplex PCR with a pair
of Chinese Hamster PCR primers was carried out. One of two
alternative pairs of Chinese Hamster primers yielding products of
different size were used in this duplex PCR approach. One Chinese
Hamster primer pair was designed from metallothionein I gene
(MET-1), and the other was developed from phospholipase C-delta
1 gene (PHOSC). The MET-1 5Ј and 3Ј primers were 5ЈAGCTC-
CAGGCACTACTAAA3Ј and 5ЈCGGAGGAGAAAGAAGACA3Ј
and amplified a product of 214 bp. The PHOSC 5Ј and 3Ј primers
were 5ЈGTTTCCTTCTCTGAGCCTAAATTGT3Ј and 5ЈTCCTC-
TGCCCTCCTTTTC3Ј and amplified a product of 512 bp. The
Chinese Hamster PCR primers produced a specific product from
Chinese Hamster DNA and lessened nonspecific amplification of
Chinese hamster DNA by the bovine primers. This strategy was
helpful in reducing the background problem in some cases.
The PCR products were size-separated on a 3% agarose gels
made up of 1.5% Nusieve (FMC Biotech) and 1.5% regular aga-
rose (FMC Biotech). The data were scored visually as positive,
negative, and unknown. A hybrid was considered positive for a
STS when the PCR product was easily observed and had the same
size as the positive control (bovine DNA). When no product was
seen, the hybrid was scored as negative for that specific STS. If the
amount of PCR product was low and the DNA band on the gel was
weak, it was scored as unknown.
The panel of 90 radiation hybrids was tested for the presence
or absence of 34 markers. Two pairs of markers, BM7160– SRC40
and RAB3A–RM012 were found to have identical retention pat-
terns and were treated as one locus in the subsequent ordering. The
marker retention frequency ranged from 8.9% to 24.4% (Fig. 1).
The average retention frequency for all 34 markers was 14.5%.
The 34 markers were grouped into four linkage groups. The first
linkage group contained SRC40, BM7160, RAB3A, RM012,
COMP, ILSTS001, TGLA176, BM9289, LYL, MANB, IDVGA-
90. The second linkage group consisted of IL4, BM6105,
TGLA303, BM741, UWCA20, BM6117 and CSSM29. The third
group included BMS2840, BMS904, INRA112, BMS2258,
BMS792, INRA192, BM1557, BMS1853, OARAE129 and
BMS1331. Finally, RASA, ILSTS006, BMS522, BMS1979,
Correspondence to: B.W. Kirkpatrick
Mammalian Genome 10, 1112–1114 (1999).
© Springer-Verlag New York Inc. 1999