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.

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29

Linkage and physical

mapping of prolactin

ã 1998 International Society for Animal Genetics, Animal Genetics 29, 27±29