BRIEF NOTES 287

Table 1. Characterization of microsatellite loci LEX071–LEX078

No ofPrimersSize range Heterozygosity Primer sequencealleles SyntenyLocus IEGMW families

LEX071 7 185–204 0.48 Not tested8–10F CTTTATTCTACTCTTTGGTCC ECA208–10R CCGATATTTCACTGATTATT

LEX072 3 222–230 0.30 8–4F AGGGTAGAGAAAACATTAAGAT ECA4 A, B, F, G, J, K8–4R GTCCTTGCTTTTCCTATG

LEX073 14 234–264 0.64 8–13F CCCTAGAGCCATCTCTTTACA ECA19 B, C, D, E, F, G, H, L, N8–13R CAGATCCAGACTCAGGACAG

LEX074 10 148–168 0.77 8–21F AAGAGTGCTCCCGTGTG ECA24 B, C, D, F, G, H, J, K, L, N, QGACAATGCAGAACTGGGTAA8–21R

ECA30TGAAAAGTTGCAGTTTGAGA8–25F0.72 Not tested152–1605LEX0758–25R CAACCTCTTGCTACCAGAATA

8–32F TTACCTCAGCCACAATCTTCTT ECA17LEX076 Not tested3 222–226 0.23TTGCCAGACACAGAGAAGTG8–32R

A, B, D, E, G, H, J, K, L, N, QECA1LEX077 8 181–199 0.59 8–33F GGTCTCTATGGATCTCTTTGTG

8–33R GACTGATAAAACATGGAAGGTT

8–35F AATGTGCGCATTTAACCACTGTG ECA14LEX078 C, D, G, K5 156–166 0.49CAAGCCATGCTGTGGAAACG8–35R

Chromosomal location: Synteny mapping was performed with asomatic cell hybrid panel2. Chromosomal locations of the markersbased on synteny mapping are shown in Table 1.

Other comments: The sequence for LEX078 includes the ERE-2repetitive element on one side of the microsatellite repeat3. TheDNA sequences for these repeats and the flanking regions can befound in the Genbank database AF213357 through AF213364.

Acknowledgements: This work was supported by a grant from theGrayson-Jockey Club Research Foundation, Inc. and is published inconnection with a project of the University of Kentucky Agricul-tural Experiment Station as paper number 99-14-171.

References1 Guerin G. et al. (1999) Animal Genet 30, 341–54.2 Shiue Y.-L. et al. (1999) Animal Genet 30, 1–9.3 Gallagher P.C. et al. (1999) Mamm Genome 10, 140–4.

Correspondence: E Bailey (e-mail: ebailey@ca.uky.edu)

Linkage mapping of the a-B-crystallin (CRYAB)gene to porcine chromosome 9L Fontanesi, R Davoli, J Milc, V RussoDIPROVAL—Sezione di Allevamenti Zootecnici, Faculty ofAgriculture, University of Bologna, Via F.lli Rosselli 107, VillaLevi—Coviolo, 42100 Reggio Emilia, Italy

Accepted 6 April 2000Source/description: An expressed sequence tag (EST) of 224 bpwas isolated from a porcine skeletal muscle cDNA library (EMBLaccession number Z98816)1. BLASTN2 analysis showed homologywith human (M28638) a-B-crystallin (CRYAB) gene sequence (E-value=2e-81, 93% identity, 203/218 nt). Alignment of the 224 bpporcine sequence with the human CRYAB sequence identifiedputative regions of the isolated EST: 78 bp were of the 3%-end of thecoding sequence, 139 bp were of the 3%-untranslated region and 7bp were of the poly(A) tail. From the 3%-untranslated region of theporcine CRYAB sequence, PCR primers were designed using Oligoversion 2·0 (MedProbe) to give a 115-bp amplification product.

Primer sequences:Forward: 5%-GCC CTT CCT CTA ACT GCA TTT-3%Reverse: 5%-AGC TTA ATA ATT TGG GCC TGC-3%

PCR conditions and SSCP analysis : PCR was performed in a totalvolume of 20 ml using 100 ng porcine genomic DNA, 10 pmol eachprimer, 250 mM each dNTP, 1·0 mM MgCl2 and 1 U Taq polymerase(Sigma Aldrich). Amplification was performed using a 9600 PerkinElmer DNA Thermal Cycler. After an initial denaturation step of5 min at 95 °C, 35 cycles were performed as follows: 30 s at 95 °C,30 s at 57 °C and 30 s at 72 °C. A final extension step at 72 °C for5 min followed the PCR cycles. The size of the PCR product wasverified on 10% polyacrylamide gel stained with ethidium bro-mide. Aliquots (1·5 ml) of each PCR product were added to 18·5 mldenaturing sample buffer (95% formamide, 20 mM EDTA, 0·05%bromophenol blue). Samples were boiled for 15 min, chilled on iceand loaded on 0·5X MDE (FMC Bioproducts), 6% glycerol, 0·5XTBE gels. Gels were run at 30 W for 5 h at 20 °C on a D-CodeUniversal Mutation Detection System (Bio-Rad Laboratories) in0·5X TBE. Single strand DNA was revealed by silver staining.

Polymorphism, Mendelian inheritance and allele frequencies : Twoalleles were identified in the 115-bp fragment of the porcineCRYAB 3%-untranslated region (data not shown). Co-dominant seg-regation was confirmed in three-generation families of the PiGMaPConsortium3. Allele frequencies were studied in a sample of 86unrelated pigs from seven different breeds (Table 1).

Linkage mapping: DNA samples belonging to four three-generationfamilies of the PiGMaP Consortium were genotyped at the CRYABlocus by SSCP analysis. Twopoint and multipoint procedures ofthe CRI-MAP package version 2·44 were performed by merging theCRYAB data with the PiGMaP Consortium ResPig database at the

Table 1. Frequencies of the SSCP alleles of the porcine a-B-crystallin(CRYAB) gene

Allele frequencies

Breeds No. of animals Allele1 Allele 2

0·8727Large White 0·130·00 1·00Landrace 9

0·960·0414Duroc0·05 0·95Belgian Landrace 90·00 1·00Hampshire 7

11 0·82 0·18Meishan

© 2000 International Society for Animal Genetics, Animal Genetics 31, 280–291

BRIEF NOTES288

Roslin Institute3. Twopoint sex-specific analysis, with 77 informa-tive meiosis, revealed association of the new SSCP marker with thefollowing loci already identified on porcine chromosome 93:APOA1 (uf=0·30, um=0·00, LOD=6·08); PPP2ARB (uf=0·00,um=0·39, LOD=5·74); OTF11 (uf=0·10, um=0·10, LOD=4·33);and S0095 (uf=0·25, um=0·15, LOD=3·24). A multipoint sex-av-eraged map of porcine chromosome 9, constructed using optionsALL, BUILD, CHROMPIC and FLIPS2-6, placed CRYAB betweenmarkers PPP2ARB and APOA1 (PPP2ARB�16·9 CM�CRYAB�5·9CM�APOA1–). The human CRYAB gene maps to chromosome11q22·1-q23·25. The localization of CRYAB on porcine chromo-some 9 is in agreement with the conservation of synteny as revealedby chromosome painting between humans and pigs6.

Acknowledgements: This work was supported by the EC contractBIO4-CT98-0237 (GENETPIG), by the Italian MURST ex 40% funds‘Projects of relevant national interest, 1997’ and was associatedwith the PiGMaP international genetic mapping collaboration.

References1 Davoli R. et al. (1999) Gene 233, 181–8.2 Altschul S.F. et al. (1997) Nucleic Acids Res 25, 3389–402.3 Archibald A.L. et al. (1995) Mamm Genome 6, 157–75.4 Green P. et al. (1990) Documentation for CRI-MAP, version 2·4,

Washington University School of Medicine, St. Louis, MO.5 Jeanpierre C. et al. (1993) Mamm Genome 4, 104–8.6 Goureau A. et al. (1996) Genomics 36, 252–62.

Correspondence: Luca Fontanesi (e-mail: luca.fontanesi@stpa.unibo.it).

A highly polymorphic repetitivepolypyrimidine/polypurine (CCTTT)n sequencein the 5% untranslated sequence of the porcineandrogen receptor geneN Trakooljul, S Ponsuksili, K Schellander, K WimmersInstitute of Animal Breeding Science, University of Bonn,Endenicher Allee 15, 53115 Bonn, Germany

Accepted 19 April 2000Source/description: The complete cDNA sequence of the porcineandrogen receptor (AR) gene was obtained starting with het-

erologous primers designed from conserved regions of the humanand rat AR and subsequent 5% and 3%-RACE using homologousprimers (SMARTTM RACE cDNA Amplification Kit, CLONTECH)(GenBank accession no.: AF161717). Primers were designed fromthe sequence flanking every 350–450 bp over the entire genesequence to screen for polymorphism. A primer pair located in the5% untranslated sequence was successfully used to amplify a 381 bpPCR product from cDNA and 1152 bp from genomic DNA. Thefragment amplified from genomic DNA was sequenced. The se-quence was submitted to GenBank (accession no.: AF218781). Apentanucleotide repeat (CCTTT)n was recognised in the porcinesequence and its polymorphism was evaluated.

Locus-specific-primer sequences : Forward primer (5% IRD700 la-belled): ARms1: 5%-TGCATTCAGAAGGCGGAAG-3%Reverse primer: ARms2: 5%-GGTTCACAATATGTTCCTG-3%

PCR condition: Each 10 ml of PCR reactions contained 25 nggenomic DNA, 0.2 mM of each primer, 50 mM of each dNTP, 0.5 U ofTaq polymerase (Pharmacia), and 1.5 mM MgCl2. PCR were per-formed in thermal cycler PTC100 (MJ Research) at 94 °C for 3 min,followed by 34 cycles of 94 °C 30 sec, 59 °C 45 sec, 70 °C 1 min andfinal extension at 72 °C for 5 min. PCR products were run on 6%denaturing polyacrylamide gels using a LI-COR DNA sequencermodel 4200.

Polymorphism: Seven alleles were found in five pig breeds. Allalleles were sequenced from homozygous/hemizygous pigs. Struc-ture of polymorphic repetitive polypyrimidine/polypurine (PPY/PPU) sequences of all alleles are shown in Table 1. Allelefrequencies were determined in 124 unrelated pigs, 44 GermanLandrace, 30 Large White, 43 Pietran, five Berlin-miniature pig andtwo Duroc (Table 1).

Chromosomal location: Porcine AR gene has been assigned to the Xchromosome q13. A microsatellite in intron 7 of the gene and itslinked markers have been reported3. No recombination was foundbetween the microsatellite in intron 7 and our new microsatellite inthe 5% untranslated region of the porcine AR in 350 pigs from 20families of the Berlin–Bonn resource population (LOD score193·93, CRI-MAP version 2·4)5.

Mendelian inheritance: X-chromosomal segregation of the alleleswas observed in families of Berlin–Bonn resource population (Fig.1).

Comments: The androgen receptor mediates hormonal responsive-ness to androgens, which play important roles in sexual differentia-

Table 1. Patterns of polymorphic repetitive polypyrimidine/polypurine sequences in 5% untranslated region of the porcine AR gene and frequencies ofalleles observed in five pig breeds

Structure of repetitive GermanBerlin-miniaturePietrainpolypyrimidine/polypurine sequence Large White Landrace Durocpig n=5Allele size (bp) n=30 n=43n=44 n=2(5%–3%)

0·150 0·062 0·046172 GTTTTCC (T)8 ( CCTTT) 7

TTCCTTCTGT (CTTT)2 C(T) 14

0·1390·047173 GTTTTCC (T)8 ( CCTTT) 7 0·283TTCCTTCTGT (CTTT)2 C (T) 15

0.127 NC*186 GTTTTCC (T)8 ( CCTTT) 10* 0·533 0·625TTCCTTCTGT (CTTT)2 C (T) 13

0·016193 GTTTTCC (T)8 ( CCTTT) 11

TTCCTTCTGT (CTTT)2 C (T) 15

0·686196 GTTTTCC (T)8 ( CCTTT) 12* 0·033 0·250TTCCTTCTGTCTCT (CTTT) C (T)13

NC* NC*203 GTTTTCC (T)4 C (T) 3 ( CCTTT) 13

TTCCTGT (CTTT)2 CTTTTTC (T) 13

228 GTTTTCC (T)4 C (T) 3 ( CCTTT) 18* NC*TTCCTGT (CTTT)2 CTTTTTC (T) 13

Note that one repeat of alleles 186, 196, and 228 were replaced by (CCTTC), (CCTCT), and (CCCTT), respectively.NC*, Allele was detected within the group of pigs but allele frequency was not calculated.

© 2000 International Society for Animal Genetics, Animal Genetics 31, 280–291