SHORT COMMUNICATION
Linkage and physical mapping of prolactin toporcine chromosome 7A Vincent, L Wang, C K Tuggle, M F Rothschild
Summary
Comparative mapping studies between human
and pig have shown that there is conserved
synteny between human chromosome 6 and pig
chromosomes 1 and 7, but some gene locations
are not well established. Prolactin (PRL), an
anterior pituitary hormone, has been mapped to
human chromosome 6, and has tentatively
mapped to pig chromosome 7 using Southern-
RFLP analysis with a limited number of
meioses. To confirm the assignment of prolactin
to porcine chromosome 7 by physical and
linkage analysis, pig cDNA and human genomic
DNA sequences were used to design pig-specific
PCR primers. The primers amplified a fragment
of »2z8 kb. Two polymorphic restriction sites were
identified within this fragment with the restriction
endonuclease BstUI. Prolactin was significantly
linked to six markers on the published PiGMaP
map of pig chromosome 7. Prolactin was
physically mapped using a pig ´ rodent
somatic cell hybrid panel. An analysis of these
data placed PRL on pig 7p1z1±p1z2 with 100%
concordance and was in complete agreement with
the linkage data. Both mapping techniques placed
PRL in a conserved order with the loci in the
syntenic region of human chromosome 6.
Keywords: PCR-RFLP, porcine, prolactin
Prolactin (PRL) is a peptide hormone primarily
secreted by the anterior pituitary in response to
factors such as oestrogen. Prolactin has numer-
ous actions in mammalian tissue and is essen-
tial for reproductive success. The role of PRL in
the synthesis of milk proteins in mammals has
been well characterized, and has been shown to
activate transcription of genes such as b-casein
and b-lactalbumen through interaction with its
receptor (Kelly et al. 1991). Prolactin is thought
to play a role in the maintenance of pregnancy
in the pig by acting on corpora lutea cells and
possibly initiating production of progesterone
(Yuan & Lucy 1996). Prolactin has been shown
to participate in the maintenance of relaxin after
its preprogrammed release (Felder et al. 1988; Li
et al. 1989) in sows.
Prolactin had been previously mapped to pig
chromosome 7 (SSC7) using a Southern-RFLP,
but this marker had limited informativeness,
and allowed only a tentative assignment
between S0102 and ANPEP (Archibald et al.
1995) in the SSC7q region (Marklund et al.
1996). This weak linkage placement was com-
pounded by the lack of physical mapping data.
Comparative mapping has demonstrated a rear-
rangement of the loci on human chromosome 6
(HSA6) to pig chromosome 1 (SSC1) and SSC7
through bidirectional chromosome painting
(Goureau et al. 1996). However, only a limited
number of genes have been placed on the
linkage map of SSC7, and further mapping is
required to determine the order and breakpoints
of these loci. The previous linkage placement of
PRL indicated that it was near the breakpoint
between the HSA6 and human chromosome 15
(HSA15) syntenic groups on SSC7q, and a more
precise localization could be helpful in defining
the rearrangement.
Sequence information from human genomic
DNA (Truong et al. 1984) and porcine cDNA
(Schulz-Aellen et al. 1989) was compared to
locate likely intron±exon boundaries in the
porcine cDNA. Pig-specific primers were
designed to span the second intron: (forward)
59-ACC TCT CTT CGG AAA TGT TCA-39;
(reverse) 59-CTG TTG GGC TTG CTC TTT
GTC-39. PCR was performed using these primers
in 25 ml reactions in the following conditions:
1z3 mm MgCl2, 0.35 mm dNTPs, 0z3 mm forward
and reverse primers, 30 ng DNA template, 1´Taq Extender buffer (Stratagene, La Jolla, CA), 1
U Taq Extender (Stratagene), and 1 unit Taq
Polymerase (Promega, Madison, WI). The PCR
profile was 92 °C for 2 min, followed by 35 cycles of
92 °C for 30 s, 56 °C for 30 s and 72 °C for 3 min,
and a final extension at 72 °C for 7 min. The 2z8-kb
product was purified and »600 bp from the 59 and
39 ends were sequenced to confirm that it was PRL.
The exonic regions of the fragment had 100%
identity with the corresponding regions of the
published porcine cDNA sequence. Grandpar-
Animal Genetics,
1998, 29, 27±29
A Vincent, L Wang, C KTuggle, M F Rothschild225 Kildee Hall, Depart-
ment of Animal Science,
Iowa State University,
Ames IA 50011±3150,USA
ã 1998 International Society for Animal Genetics 27
Correspondence: M F Rothschild.
Accepted 22 September 1997
ent animals from the PiGMaP mapping families
(Archibald et al. 1995) were screened to identify
an RFLP. Two polymorphic sites were found
using the restriction enzyme BstUI. The two
polymorphic sites were combined into alleles to
give more power to the linkage analysis. The
alleles observed are described as follows: (1)
1350, 1020 and 410 bp; (2) 2370 and 410 bp;
and (3) 1430 and 1350 bp. A fourth possible
allele (2800 bp, representing the undigested
PCR product) has not been observed in any
animals tested thus far. These alleles can be
identified by a unique fragment: (1) 1020 bp;
(2:) 2370 bp; and (3) 1430 bp.
Individuals from the three-generation PiG-
MaP families (Large White by Meishan crosses
or Large White by European wild boar cross)
were genotyped, and the genotypes were inher-
ited in an autosomal Mendelian pattern. These
data were analysed using the software package
CRIMAP version 2z4 with data from PiGMaP
ResPig Database (Archibald et al. 1995). Pair-
wise linkage analysis with the 121 informative
meioses was performed for all loci with LOD
scores of three or greater being considered
significant. Prolactin was significantly linked
to six markers on the published PiGMaP SSC7
map (two point LOD score in parentheses):
S0064 (5z84); S0013 (11z35); TNFB (10z54);
S0047 (4z19); S0078 (3z76); and S0066 (3z65). A
multi-point analysis was then performed to
construct a SSC7 map (LOD: ±116z07) including
significantly linked loci (Fig. 1).
DNA samples from unrelated animals from
seven breeds were also genotyped. The frequen-
cies for the PRL BstUI alleles were calculated
within breeds (alleles 1, 2 and 3, respectively):
Chester White (n = 9) 0z83, 0z17 and 0; Duroc
(n = 10) 1z0, 0 and 0; Hampshire (n = 11) 1z0, 0
and 0; Landrace (n = 10) 1z0, 0 and 0; Large
White (n = 11) 0z41, 0z23 and 0z36; Yorkshire
(n = 10) 0z60, 0z25 and 0z15; and Meishan (n = 9)
0z78, 0z11 and 0z11.
To assign PRL to a cytogenetic region, the
pig primers were used to amplify DNA from
a pig ´ rodent somatic cell hybrid panel
(Yerle et al. 1996) using the PCR conditions
described above. A product of expected size was
amplified in clones 7, 10, 11, 16, 19, 21, 23, 24,
25 and 27. The data was submitted via the INRA
Cellular Genetics Laboratory web page (http://
www.toulouse.inra.fr/lgc/pig/hybrid.htm). Pro-
lactin was localized to SSC7p1z1±p1z2 with
100% concordance (Fig. 2). The physical loca-
lization placed PRL proximal to S0102, and in
the same region as S0064 and TNFB (Robic et al.
1996), and confirmed the genetic linkage place-
ment.
Both the physical and genetic linkage data
presented for PRL provide evidence that the p
arm of HSA6 is syntenic to SSC7p±q1z4. Since
the earlier mapping of PRL was of poor resolu-
tion, a more defined location of PRL in the pig
genome was needed. The current mapping
placed PRL on the short arm of SSC7, which is
contradictory to earlier work, and moves this
gene away from the area anticipated to contain
the breakpoint between the HSA6 and HSA15
syntenic groups. The mapping of other genes
from HSA6 and HSA15 must be done to
elucidate the rearrangement and breakpoint
locations on this pig chromosome.
Fig. 1. Multiple-point map of prolactin and
significantly linked markers on SSC7. Multiple-point
analysis was done using CriMap to produce a sex-
averaged map with all significantly linked loci.
Fig. 2. Schematic diagram representing presence of fragments of SSC7 in each
hybrid clone. Each clone is labelled numerically and the vertical solid bars below
represent the presence of fragments contained within that clone: (+) a positive PCR
amplification; and (±) no PCR amplification of the clone DNA. PRL is assigned to
region B.
ã 1998 International Society for Animal Genetics, Animal Genetics 29, 27±29
28
Vincent, Wang,
Tuggle, Rothschild
Acknowledgements
The authors wish to thank J. Helm, H. Sun and
C. Ernst for technical assistance, and M. Yerle
for use of the somatic cell hybrid panel. Partial
financial assistance from PIC Group is greatly
appreciated. This work was supported in part
by the Iowa Agriculture and Home Economics
Experiment Station, Ames, IA, USA, and by
Hatch Act and State of Iowa funds, Journal
Paper No. J-17418, Project no. 3043. This work
is part of the PiGMaP international genetic
mapping collaboration supported by the E. C.
Bridges programme.
References
Archibald A., Haley C., Brown J., Couperwhite S.,
McQueen H., Nicholson D., Coppieters W., Van de
Weghe A., Stratil A., Wintero A., Fredholm M.,
Larsen N., Nielsen V., Milan D., Woloszyn N., Robic
A., Dalens M., Riquet J., Gellin J., Caritez J.-C.,
Burgaud G., Ollivier L., Bidanel J.-P., Vaiman M.,
Renard C., Geldermann H., Davoli R., Ruyter D.,
Verstege E., Groenen M., Davies W., Hoyheim B.,
Keiserud A., Andersson L., Ellegren H., Johansson
M., Marklund L., Miller J., Anderson Dear D., Signer
E., Jeffreys A., Moran C., Le Tissier P., Muladno,
Rothschild M., Tuggle C., Vaske D., Helm J., Liu H.-
C., Rahman A., Yu T.-P., Larson R.G. & Schmitz C.
(1995) The PiGMaP consortium linkage map of the
pig (Sus scrofa). Mammalian Genome 6, 157±75.
Felder K.J., Klindt J., Bolt D.J. & Anderson L.L. (1988)
Relaxin and progesterone secretion as affected by
luteinizing hormone and prolactin after hysterect-
omy in the pig. Endocrinology 122, 1751±60.
Goureau A., Yerle M., Schmitz A., Riquet J., Milan D.,
Pinton P., Frelat G. & Gellin J. (1996) Human and
porcine correspondence of chromosome segments
using bidirectional chromosome painting. Geno-
mics 36, 252±62.
Kelly P., Djiane J., Postel-Vinay M. & Edery M. (1991)
The Prolactin/Growth Hormone Receptor Family.
Endocrinology Review 12, 235±51.
Li Y., Molina J.R., Klindt J., Bolt D.J. & Anderson L.L.
(1989) Prolactin maintains relaxin and progesterone
secretion by aging corpora lutea after hypophysial
stalk transection or hypophysectomy in the pig.
Endocrinology 124, 1294±304.
Marklund L., Johansson Moller M., Hoyheim B.,
Davies W., Fredholm M., Juneja R.K., Mariani P.,
Coppieters W., Ellegren H. & Andersson L. (1996) A
comprehensive linkage map of the pig based on a
wild pig-Large White intercross. Animal Genetics
27, 255±69.
Robic A., Riquet J., Yerle M., Milan D., Lahbib-
Mansais Y., Dubut-Fontana C. & Gellin J. (1996)
Porcine linkage and cytogenetic maps integrated by
regional mapping of 100 microsatellites on somatic
cell hybrid panel. Mammalian Genome 7, 438±45.
Schulz-Aellen M.F., Schmid E. & Movva R. (1989)
Nucleotide sequence of porcine preprolactin cDNA.
Nucleic Acids Research 17, 3295.
Truong A.T., Duez C., Belayew A., Renard A., Pictet
R., Bell G. & Martial J. (1984) Isolation and
characterization of the human prolactin gene.
EMBO Journal 3, 429±37.
Yerle M., Echard G., Robic A., Mairal A., Dubut-
Fontana C., Riquet J., Pinton P., Milan D., Lahbib-
Mansais Y. & Gellin J. (1996) A somatic cell hybrid
panel for pig regional gene mapping characterized
by molecular cytogenetics. Cytogenetics and Cellu-
lar Genetics 73, 194±202.
Yuan W. & Lucy M. (1996) Effects of growth hormone,
prolactin, insulin-like growth factors, and gonado-
tropins on progesterone secretion by porcine luteal
cells. Journal of Animal Science 74, 866±2.
29
Linkage and physical
mapping of prolactin
ã 1998 International Society for Animal Genetics, Animal Genetics 29, 27±29