Porcine gene discovery by normalized cDNA-library sequencing
and EST cluster assembly
Scott C. Fahrenkrug,
* Timothy P.L. Smith,
Brad A. Freking,
John W. Keele
USDA, ARS, U.S. Meat Animal Research Center, P.O. Box 166, Clay Center, Nebraska 68933, USA
The Institute of Genomic Research (TIGR), Rockville, MD, USA
Received: 4 April 2001 / Accepted: 5 April 2002
Abstract. Genetic and environmental factors aect the e-
ciency of pork production by in¯uencing gene expression
during porcine reproduction, tissue development, and growth.
The identi®cation and functional analysis of gene products
important to these processes would be greatly enhanced by the
development of a database of expressed porcine gene sequence.
Two normalized porcine cDNA libraries (MARC 1PIG and
MARC 2PIG), derived respectively from embryonic and re-
productive tissues, were constructed, sequenced, and analyzed.
A total of 66,245 clones from these two libraries were 5¢-end
sequenced and deposited in GenBank. Cluster analysis re-
vealed that within-library redundancy is low, and comparison
of all porcine ESTs with the human database suggests that the
sequences from these two libraries represent portions of a
signi®cant number of independent pig genes. A Porcine Gene
Index (PGI), comprising 15,616 tentative consensus sequences
and 31,466 singletons, includes all sequences in public reposi-
tories and has been developed to facilitate further comparative
map development and characterization of porcine genes
(http://www.tigr.org/tdb/ssgi/). The clones and sequences from
these libraries provide a catalog of expressed porcine genes and
a resource for development of high-density hybridization ar-
rays for transcriptional pro®ling of porcine tissues. In addi-
tion, comparison of porcine ESTs with sequences from other
species serves as a valuable resource for comparative map
development. Both arrayed cDNA libraries are available for
unrestricted public use.
Livestock genomics has made signi®cant progress in recent
years with the development of comprehensive genetic linkage
maps (Kappes et al. 1997; Rohrer et al. 1996). These maps
have been instrumental in genome scans to detect quantitative
trait loci (QTL) for production characteristics in several live-
stock species (Andersson-Eklund et al. 1998; de Koning et al.
1999; Kirkpatrick et al. 2000; Knott et al. 1998; Rohrer and
Keele 1998a, 1998b; Stone et al. 1999; Zhang et al. 1998).
However, identi®cation of genetic determinants in¯uencing
production trait variation has been constrained by a lack of
livestock sequence data. Information about the sequence and
location of expressed sequences in the porcine genome will
enhance the selection and evaluation of candidate genes.
Two porcine cDNA libraries were developed and analyzed
in the current study. Library MARC 1PIG was produced from
pig embryos at several developmental stages selected to cor-
respond with early muscle development and organogenesis
(Butler and Juurlink 1987), which are associated with signi®-
cant embryo mortality in U.S. pig breeds (Ford 1997; Vallet
2000). Library MARC 2PIG was produced with pooled RNA
from tissues with important roles in porcine reproductive
physiology. Over 30,000 clones from each library have been
sequenced, representing a signi®cant contribution to the
number of porcine expressed sequences in public databases.
Cluster analysis of all porcine sequences suggests the discovery
of up to 34,000 expressed pig genes that have been assembled
and annotated in a Porcine Gene-Index (PGI).
Material and methods
To produce the tissues for the MARC 1PIG em-
bryonic library, Landrace-Yorkshire boars were mated to gilts having
at least one estrous cycle of normal length (17±23 days). Gilts were
slaughtered at 11, 13, 15, 20, and 30 days, and the uterus was recov-
ered. On days 11±15, each uterine horn was ¯ushed with 20 ml 0.9%
saline, and the complete conceptus for each embryo (including
trophectoderm) was recovered from the ¯ushings and frozen in liquid
nitrogen. On days 20 and 30, the uterus was opened along the an-
timesometrial border, and embryos were dissected from the placentas
and snap frozen in liquid nitrogen. To produce MARC 2PIG, tissues
were collected from multiple Landrace-Yorkshire female animals at
various stages of sexual development. Pituitary and hypothalamus
were collected from d10 and d15 of the midluteal phase of adult ani-
mals. Ovaries were collected from the following physiological stages:
prepubertal; d10 and d15 midluteal; undergoing estrous; and anestrous
animals. Animals were de®ned as anestrous if they did not display
estrous within 14 days postweaning. In addition, endometrium and
placenta (days 20 and 30 of pregnancy) were collected from the ani-
mals that provided embryos for MARC 1PIG. Testes (110 days of
gestation and adult) were collected from Meishan ´ Chester White-
Landrace-Yorkshire boars. For both libraries, matched tissues from
several individuals were pooled before mRNA isolation to provide a
potential resource for polymorphism discovery.
RNA extraction and pooling.
Tissues were shipped to Life Tech-
nologies, Inc. (Rockville, Md.) for contracted primary library pro-
duction essentially as described (Smith et al. 2001). To construct the
pooled-tissue libraries, poly(A) + RNA was puri®ed from each source
tissue and pooled by weight to produce the desired proportional rep-
resentation. MARC 1PIG was produced with RNA from embryos at
11, 13, 15, 20, and 30 days post coitum (dpc), with 20% of the total
weight of RNA from each stage. MARC 2PIG was 25% ovary (equal
parts prepubertal, d10, d15 cycle, estrous, and anestrous), 21% hypo-
thalamus (equal parts d10, d15 cycle, estrous, and anestrous), 21%
pituitary (equal parts d10, d15 cycle, estrous, and anestrous), 11%
placenta (equal parts 20 & 30 dpc), 11% endometrium (equal parts days
15 and 30 dpc), 11% testis (equal parts day 110 gestation and adult).
Mammalian Genome 13, 475±478(2002)
*Present address: University of Minnesota, Department of Animal
Science, 1988 Fitch Ave., St. Paul, MN 55108-60143, USA.
Correspondence to: T.P.L. Smith; E-mail: Smith@email.marc.usda.gov