Linkage and physical mapping of the porcinethyroglobulin (TG ) gene

A. M. Ramos*, J. M. Helm†, Y. D. Zhang†,T. Rangel-Figueiredo* and M. F. Rothschild†

*Departamento de Zootecnia, Universidade de Tras-os-Montes e

Alto Douro, Apartado 202, 5000-911 Vila Real, Portugal.†Department of Animal Science, Iowa State University, 2255 Kildee

Hall, Ames, IA 50011, USA.

Accepted 29 November 2001

Source/description: Thyroglobulin (TG) is the predominant

glycoprotein of the thyroid gland and is the biosynthetic pre-

cursor of the thyroid hormones, tri-iodothyronine (T3) and

thyroxine (T4)1. Consensus primers were designed from human

and bovine TG exon 1 (TG1F) and exon 2 (TG1R) sequences

(GenBank accession nos. X06059, X05380, X06060 and

X05381, respectively). The sequence of the porcine PCR prod-

ucts revealed 78 and 80% nucleotide identity to the corres-

ponding human and cattle sequences, respectively, including

the promoter region and exon 1 of the TG gene. The porcine TG

sequence has been submitted to GenBank (accession no.

AY063124). Using this sequence, pig specific primers, TG2F

and TG2R, were designed to amplify a 278-bp product.

Sequence analysis of the PCR products from the pooled DNAs of

individual pigs of several different breeds detected a nucleotide

substitution within intron 1, situated at an HhaI restriction

enzyme recognition site.

Primer sequences:

TG1F: Forward primer 5¢-TTG ACC AGC AGA GAA AAC AGG-3¢TG1R: Reverse primer 5¢-GTT TCC CTC TGC AGC TCA CA-3¢TG2F: Forward primer 5¢-CAG CGG CTC CTA CCC CTT C-3¢TG2R: Reverse primer 5¢-TGC CTC TCA GAC CCT CGG-3¢

PCR conditions: PCR reactions were performed using 12.5 ng of

porcine genomic DNA, 1· PCR buffer, 1.5 mM MgCl2, 0.125 mM

dNTP, 0.3 lM of each primer, and 0.5 U Taq DNA polymerase

(Promega, Madison, WI, USA) in a 10-ll final volume. The PCR

profile included 5 min at 94 �C; 35 cycles of 45 s at 94 �C, 30 s

at 61 �C, 30 s at 72 �C; and a final 3 min extension at 72 �C in a

PTC200 (MJ Research, Inc., Watertown, MA, USA).

Polymorphisms: The HhaI digestion of the 278-bp PCR product

produced allelic fragments of 278-bp (allele 1), or 237 and

41-bp (allele 2). This PCR restriction fragment length poly-

morphism (RFLP) was used to genotype animals from the

PiGMaP reference families2 and from several breeds in the Iowa

State University swine breeding herd. The PCR-RFLP fragments

were separated by electrophoresis on 4% agarose gels (Fig. 1).

Mendelian inheritance/allele frequencies: Mendelian segregation

of the HhaI PCR-RFLP was observed in six three-generation

PiGMaP families. The allele frequencies of the polymorphism

were determined by genotyping 118 unrelated animals from

several breeds in the Iowa State University swine breeding herd

and the PiGMaP grandparents. Allele 2 was the most common

allele, observed with a frequency of 1.00 in Duroc (n ¼ 20),

0.91 in Berkshire (n ¼ 22), 0.85 in Hampshire (n ¼ 20), 0.60

in Large White (n ¼ 21) and 0.68 in Landrace (n ¼ 22).

However, in Meishans allele 1 was the most common allele

with a frequency of 0.92 (n ¼ 13).

Chromosomal location/physical: The porcine TG gene was

assigned to chromosome 4p13 (P ¼ 0.88) by PCR analysis of a

pig–rodent somatic cell hybrid panel3. Two sets of intronic

primers were used to physically map the TG gene.

Chromosomal location/linkage: Two-point and multipoint link-

age analyses were performed using the genotypes of the PiGMaP

families and the CRIMAP program4. The TG gene was signifi-

cantly linked with several markers on porcine chromosome 4.

Two-point linkage analysis determined that the two most closely

linked markers (recombination fraction and LOD score) were

S0011 (0.06, 13.09) and S0301 (0.11, 12.01). The best map

order of the TG gene produced by multipoint linkage analysis

with other linked markers was (with distance in Kosambi cM):

S0011 – 3.0 – S0227 – 6.2 – TG – 12.1 – S0301.

Comments: The TG gene was previously assigned to SSC4p15

using fluorescent in-situ hybridization5. This is in accordance

with our results from the linkage analysis, because the two

microsatellites flanking the TG gene, S0227 and S0301, had

been physically mapped to 4p14–p156 and 4p157, respectively.

However, our results showed that the TG gene physically

mapped to SSC4p13, which was confirmed by PCR analysis of

the pig–rodent somatic cell hybrid panel with two different sets

of intronic primers. This suggests it is near the boundary of the

p13 and p14 regions. Once additional markers are physically

and linkage mapped to this region, the chromosomal locations

for the TG gene will likely be resolved.

Acknowledgements This work is supported by the PIC Inter-

national Group and the Iowa Agriculture and Home Economics

Experimental Station, Ames, paper no. J-19613, project no.

3600, as well as by Hatch Act and State of Iowa funds. Support

by the EU for the PiGMaP DNA and bioinformatics support by

A. Archibald and associates of the Roslin Institute is greatly

appreciated.

References1 van de Graaf, S. et al. (1999) Journal of Clinical Endocrino-

logical Metabolism 84, 2537–42.

2 Archibald, A. et al. (1995) Mammalian Genome 6, 157–75.

3 Yerle, M. et al. (1996) Cytogenet Cell Genetics 73, 194–202.

4 Green, P. et al. (1990) Documentation for CRIMAP, Version

2.4. Washington University, School of Medicine, St Louis, MO.

Figure 1 Porcine thyroglobulin HhaI PCR-RFLP resulted in allelic

fragments of 278 bp for allele 1 and 237 bp and 41 bp (not shown) for

allele 2. Genotype patterns are illustrated.

228 Brief notes

� 2002 International Society for Animal Genetics, Animal Genetics, 33, 224–248

5 Pinton, P. et al. (2000) Mamm Genome 11, 306–15.

6 Robic, A. et al. (1996) Mamm Genome 7, 438–45.

7 Høyheim, B. et al. (1994) Animal Genetics 25, 432.

Correspondence: M.F. Rothschild, (mfrothsc@iastate.edu)

An insertion/deletion polymorphismin the fourth intron of POP5is used for linkage mapping in sheep

C. Diez-Tascon�, K. G. Dodds† and A. M. Crawford�

*AgResearch Molecular Biology Unit, Department of Biochemistry,

University of Otago, PO Box 56, Dunedin, New Zealand.†AgResearch Invermay Agricultural Centre, Private Bag 50034,

Mosgiel, New Zealand

Accepted 11 December 2001

Source/description: The POP5 is an RNA component associated

with both endoribonucleases mitochondrial RNA processing

(MRP) and RNAse P1. It is alternatively known as HSPC004

because of its expression in CD34+ hematopoietic stem/pro-

genitor cells (HSPCs) and has been mapped in human chro-

mosome 122. Using Targeted Intronic Polymorphic Sequence

(TIPS) identification3, we designed polymerase chain reaction

(PCR) primers flanking the fourth intron of POP5 in sheep. The

sequence of the 199 base pair (bp) amplified product was tested

for similarity with non-redundant (nr) sequences in GenBank

using the BLASTN algorithm4. The coding sequences flanking

the ovine intron were unequivocally aligned to their homo-

logues in human (GenBank accession number AJ306296). The

DNA sequence was deposited with the GenBank nucleotide

sequence database under accession number AF435572. By

sequencing different individuals we identified a 2-bp insertion/

deletion polymorphism within the intron. The variation is

caused by the presence or absence of a ‘GA’ sequence at posi-

tion 134 in AF435572. This polymorphism was used to map

POP5 on sheep chromosome 17.

Primer sequences: Forward primer: 5¢-AACATGCCAGAAGTTC-

CTG-3¢ Reverse primer: 5¢-CAGACTTCTGGATAGCCTCCC-3¢

PCR conditions and analysis: The fragment containing the POP5

intron was amplified using 50 ng of DNA in a final volume of

20 ll. The constituents of the reaction were: 1 lM of each pri-

mer, dNTPs (200 lM), MgCl2 (2 mM), 1· PCR Buffer supplied by

the manufacturer and 1 U of Red Hot DNA polymerase (ABgene,

Surrey, UK). Amplification was performed in a Robocycler ma-

chine (Stratagene, La Jolla, CA, USA) using the following con-

ditions: one cycle of 95 �C for 2 min; 35 cycles at 95 �C for 30 s,

54 �C for 45 s and 72 �C for 1 min; final extension step at 72 �C

for 10 min. The PCR product was sequenced using an ABI377

DNA Sequencer (Perkin-Elmer, Applied Biosystems Interna-

tional, Melbourne, Australia). Genotyping of the insertion/

deletion polymorphism was carried out by end-labelling the

forward primer with [c 33P] adenosine triphosphate (ATP) and

T4 polynucleotide kinase (New England Biolabs Inc., Beverly,

MA, USA). The DNA samples were amplified using 8 nM of the

labelled primer in the PCR reaction. The amplified product was

electrophoresed on 6% denaturing sequencing gels and the al-

leles were visualized by autoradiography.

Mendelian inheritance: Codominant segregation of the poly-

morphism was verified in the AgResearch International Map-

ping Flock (IMF), comprising nine three generation families

with a total of 127 animals5.

Chromosomal location: The POP5 was mapped against the

markers on the latest version of the sheep framework map6.

Multipoint linkage analysis of the IMF pedigrees using CRI-

MAP7 localized POP5 in the distal part of ovine chromosome

17, between ILSTS058 and EPCDV009, with its most likely

position between markers BM1862 and EPCDV009 (Fig. 1).

Figure 1 Linkage map position of POP5 (on sheep chromosome 17).

The vertical bar to the right of the chromosome represents the lod-3

support interval. The most likely position within this interval is indicated.

229Brief notes

� 2002 International Society for Animal Genetics, Animal Genetics, 33, 224–248