Animal Genetics 1992, 23, 537-541
SHORT COMMUNICATION
Characterization of five new bovine dinucleotide repeats D. VAIMAN, R. OSTA, D . MERCIER, C. GROHS & H. LEVEZIEL Laboratoire de Gkne'tigue Biochimique, INRA-CRJ, 78350 Jouy-en-Josas, France
Summary. Five new bovine (TG)n microsatellite sequences were characterized. During the screening procedure, one-third of the clones had essentially the same flanking sequences. A fast technique was devised to eliminate these clones. Keywords: microsatellites, genetic map
Using 'classical' (TG)n repeats detection procedure, 21 microsatellite sequences were isolated from two size selected (between 400 and 800 base pairs) pGEM4Z genomic libraries obtained with Tag1 and Sau3A. Among these sequences, five were fully characterized. We also used a panel of hamster-bovine somatic cell hybrids to assign these microsatellites to syntenic groups. One-third of the 21 (TG)n repeats identified were found to display essentially the same flanking sequence. A GenBank database search using the GCG software package (Devereux et af. 1984) for this microsatellite sequence revealed a strong similarity with part of an already described bovine satellite DNA, bovine satellite 1.709, accession number X00979 (Skowronski et al. 1984). Finding this sequence frequently was perturbing our efforts to isolate new microsatellites. We present here a simple procedure that we have designed to overcome such a problem (ASPS: anti-satellite polymerase chain reaction [PCR] screening). DNA to be used as a template was prepared from (TG)n positive bacterial colonies. 100pl from 5 h 3 ml bacterial culture was pelleted by 5 min centrifugation at 5000 rpm. The pellet was resuspended in 5Opl of distilled water, and the bacteria were incubated for 10 min in boiling water. Bacterial debris was then centrifuged at 15000 rpm at 4°C. The position of the Sau3A site did not allow us to synthesize a primer in one of the flanking sequences. Consequently, two PCR reactions were carried out on 5 p1 from the supernatant, the first one with a primer specific to the repeated sequence (5' TACCTATCAGTGATAGTAGC 3') and the M13 reverse primer and the second one with the same microsatellite specific primer and the M13 universal primer (Fig. 1). Southern blotting with increasing concentration of DNA (result not shown) showed that the satellite sequence could be present at a copy number ranging Correspondence: Dr D. Vaiman, Laboratoire de GCnCtique Biochimique, INRA-CRJ, 78350 Jouy-en-Josas, France. Accepted 22 June I992
537
538 D. Vaiman et al.
A microsatellite 2990
3160
SadAfra
3260
3625
ent
B 19 gcccctcccc tccccacaca
IIIIIIIIII IIIII GCCCCTCCCC TCCCC. . . . . qacacqcaca cacacacaca cacacacaca cacacacaca cacacacaca c c a a q a . . a t
I I I I ~ I I CACGCACACA CACACACACA CACACACACA CACACACAC. CCAAGATCAG
i i i III IIIIIIIIII IIIIII I III IIII IIIIII IIIIIIIII
....CGCACG
166 taagaaacca aacaaaaaaa a t t t g . t t t c t t a c .
..... ....................
I IIII I IIIIIIIIII I Ill 1 / 1 1 Ill1 AACCAAACAA AACAAAAAAA AATTGCTTTC TTACGTTTCA CATGTCAGGA TGGTGCCAGA 3299
........................................
....................
CACTCTTATT GGATAGTCAA AAATCTCTTT TGTTTCTGTG TAAAAGGGAG GTCCTTTCAA 167
. . . . . . .agc
tttcagagct t g a a t t t a t t tggtta.gtc
IIIIIII II IIIIIIIIII 1 1 1 1
ccagctcttc agtacactgt
I I I IIIIIIIIII
IIIIIIIIII GGCGTGAATG TTTCAGAACT TGAATTTATT TGGAAATGAC CCAGCTCTTC AGTACACTGT I
3394 c q t c a c q t t a q t t t a q c a c a qqataqaaac t c q q q t a a c c a a a a c a c c t q qaqaaacqat
i i i r i i - i t i i i i r i i i r i i i i i i i i i t t i I I ~ ~ ~ I I I I II I I I I I I ~ i i~ i i i i r i i r CGTCAC.TTA GTTTAGCACA GGATAGAAAC TCGGGTAACC AAAACACCTG GAGAAACGAT t g t a t g t t c a g t t c a g t t c a g t t c a g t g g c t c a g t c g t g t c t g a c t c t t t gggaccccgt
IIIII IIIIIIIIII IIIIIIIIII IIIIIIIIII IIIIII Ill IIIIIIIIII TGTAT.... . GTTCAGTTCA GTTCAGTGGC TCAGTCGTGT CTGACTGTTT GGGACCCCGT 368 ggcctgcagg acgctgggct t c c g t g t c c
llllllllll
llllllllll
IIIIIIIII
GGCCTGCAGG ACGCTGGGCT TCCGTGTCC 3589 Figure 1. Position of the (TG)n repeat on bovine 1.709 satellite DNA. The size-selected genomic fragments cut with Taq 1 (465 bp) and Sau 3A (270bp) are shown in A. Part B of the figure shows alignment between the 1.709 satellite sequence and the Taq 1 fragment. The primer used during ASPS is framed (see text).
Five new bovine microsatellites 539
between 15000 and 30000, and so present a major problem in finding new bovine microsatellite sequences. Testing clones by ASPS before DNA purification and sequencing considerably improved the screening conditions and allowed us to sequence only potentially interesting clones. Among the 14 remaining microsatellites, five were thoroughly studied. We evaluated allelic frequencies on a panel of 40 unrelated animals from eight different French breeds (Tarentaise, MontbCliarde, Normande, Abondance, Franqaise
Table 1.
Accession Local number name
Primer sequences (5'-3') and average size of the repeat
Number Allele Allele of alleles size frequency 174 172 170 168 164
0.04
3
232 230 228
0.36 0.47 0.17
5
115 111 109 105 101
CTGGAGGTGTGTGAGCCCCATTTA
X63794
pSat3
CTAAGAGTCGAAGGTGTGACTAGG (AC)20
5
CCITI'CA AAAACACGGAAAmCGGGGG X63793
pSat5
CTTCAGGCATACCCTACACCACATG
(AC)13 AGGAATATCTGTATCAACCTCAGTC X63795
pSat6
CTGAGCTGGGGTGGGAGCTATAAATA
(AC)13
CGAGTITCllTCCTCGTGGTAGGC X63792
@at1 1
GCTCGGCACATCITCCITAGCAAC
8
(AC)8 AT (AC)9
GCACAGTGACCTCTCAATAAATGC X63796
pSatl3
CCACTAlTCITGCCTGAAGAATCC
(AC)20
10
0.15 0.49 0.15 0.07
Observed heterozygosity
0.51
0.50
0.02 0.05
0.21 0.06
0.46
0.66
216 214 210 206 204 200 196 194
0.01 0.15 0.12 0.01
199 197 195 193 191 189 187 185 181 177
0.02 0.08 0.31 0.23 0.08 0.19 0.02 0.02 0.02 0.01
0.01
0.40
0.65 0.04 0.01
0.74
540 D. Vairnan et al.
Figure 2. Autoradiographies of denaturing polyacrylamide gels illustrating Mendelian inheritance of PCR-amplified bovine microsatellite alleles. (A) PSat 3 (Accession number: X63794); (B) @at 6 (Accession number: X63795); (C) @at 5 (Accession number: X63793); (D) @at 11 (Accession number: X63792); (E) FSat 13 (Accession number: X63796).
Five new bovine microsatellites 541
Frisonne Pie Noire, Charolaise, Brune des Alpes, Limousine). Results are summarized on Table 1. Mendelian segregation is presented in Fig. 2 for the five microsatellite systems in half-sib families derived from a male heterozygous at the studied microsatellite locus. Labelling of the fragments was done either by end-labelling of one primer, or by direct incorporation of [35S]dATPduring the PCR. Conditions for the amplification reaction were 15s at 95"C, 15s at 58°C and 15s at 72"C, 30 cycles. Chromosomal assignment of the five sequences was addressed by using a panel of 36 somatic cell hybrids (Heuertz & Horst-Cayla 1978). Twenty-four enzymes were tested in our laboratory for this panel, thus allowing us to allocate syntenic groups to our hybrids by reference to other panels (Fries et al. 1989; Georges et al. 1991). DNAs of these hybrids was used as PCR templates and revealed that two microsatellites (pSat3 and pSat6) are syntenic, while pSatll belongs to the syntenic group U10. We have not yet been able to allocate the other microsatellites to syntenic groups even though they clearly appear only in some hybrids and not in others.
Acknowledgements
Excellent technical assistance from D. Bruneau is greatly acknowledged. We thank M. Nocart for preparing somatic cell hybrids DNA and P. Martin for critical reading of the manuscript, and constant interest in this work.
References Devereux J., Haeberli P. & Smithies 0.(1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12,387-95. Fries R., Beckmann J.S., Georges M., Soller M. & Womack J. (1989) The bovine gene map. Animal Genetics 20, 3-29. Georges M., Gunawardana A,, Threadgill D., Lathrop M., Olsaker I., Mishra A., Sargeant L.L. etal. (1991) Characterization of a set of variable number of tandem repeats conserved in Bovidae. Genomics 11,2432. Heuertz S . & Horst-Cayla M.C. (1978) Carte gtnttique des bovins par la technique d'hybridation cellulaire. Localisation sur le chromosome X de la glucose-6-phosphate dtshydrogtnase, de la phosphoglyctrate kinase, de I'alpha-galactosidase, et de I'hypoxanthine guanine phosphoribosyl tranftrase. Annales de Gtnttique 21, 197-202. Skowronski J., Plucienniczak A., Bednarek A. & Jaworski J. (1984) Bovine 1.709 satellite: recombination hotspots and dispersed repeated sequence. Journal of Molecular Biology 177,399-416.
SHORT COMMUNICATION
Characterization of five new bovine dinucleotide repeats D. VAIMAN, R. OSTA, D . MERCIER, C. GROHS & H. LEVEZIEL Laboratoire de Gkne'tigue Biochimique, INRA-CRJ, 78350 Jouy-en-Josas, France
Summary. Five new bovine (TG)n microsatellite sequences were characterized. During the screening procedure, one-third of the clones had essentially the same flanking sequences. A fast technique was devised to eliminate these clones. Keywords: microsatellites, genetic map
Using 'classical' (TG)n repeats detection procedure, 21 microsatellite sequences were isolated from two size selected (between 400 and 800 base pairs) pGEM4Z genomic libraries obtained with Tag1 and Sau3A. Among these sequences, five were fully characterized. We also used a panel of hamster-bovine somatic cell hybrids to assign these microsatellites to syntenic groups. One-third of the 21 (TG)n repeats identified were found to display essentially the same flanking sequence. A GenBank database search using the GCG software package (Devereux et af. 1984) for this microsatellite sequence revealed a strong similarity with part of an already described bovine satellite DNA, bovine satellite 1.709, accession number X00979 (Skowronski et al. 1984). Finding this sequence frequently was perturbing our efforts to isolate new microsatellites. We present here a simple procedure that we have designed to overcome such a problem (ASPS: anti-satellite polymerase chain reaction [PCR] screening). DNA to be used as a template was prepared from (TG)n positive bacterial colonies. 100pl from 5 h 3 ml bacterial culture was pelleted by 5 min centrifugation at 5000 rpm. The pellet was resuspended in 5Opl of distilled water, and the bacteria were incubated for 10 min in boiling water. Bacterial debris was then centrifuged at 15000 rpm at 4°C. The position of the Sau3A site did not allow us to synthesize a primer in one of the flanking sequences. Consequently, two PCR reactions were carried out on 5 p1 from the supernatant, the first one with a primer specific to the repeated sequence (5' TACCTATCAGTGATAGTAGC 3') and the M13 reverse primer and the second one with the same microsatellite specific primer and the M13 universal primer (Fig. 1). Southern blotting with increasing concentration of DNA (result not shown) showed that the satellite sequence could be present at a copy number ranging Correspondence: Dr D. Vaiman, Laboratoire de GCnCtique Biochimique, INRA-CRJ, 78350 Jouy-en-Josas, France. Accepted 22 June I992
537
538 D. Vaiman et al.
A microsatellite 2990
3160
SadAfra
3260
3625
ent
B 19 gcccctcccc tccccacaca
IIIIIIIIII IIIII GCCCCTCCCC TCCCC. . . . . qacacqcaca cacacacaca cacacacaca cacacacaca cacacacaca c c a a q a . . a t
I I I I ~ I I CACGCACACA CACACACACA CACACACACA CACACACAC. CCAAGATCAG
i i i III IIIIIIIIII IIIIII I III IIII IIIIII IIIIIIIII
....CGCACG
166 taagaaacca aacaaaaaaa a t t t g . t t t c t t a c .
..... ....................
I IIII I IIIIIIIIII I Ill 1 / 1 1 Ill1 AACCAAACAA AACAAAAAAA AATTGCTTTC TTACGTTTCA CATGTCAGGA TGGTGCCAGA 3299
........................................
....................
CACTCTTATT GGATAGTCAA AAATCTCTTT TGTTTCTGTG TAAAAGGGAG GTCCTTTCAA 167
. . . . . . .agc
tttcagagct t g a a t t t a t t tggtta.gtc
IIIIIII II IIIIIIIIII 1 1 1 1
ccagctcttc agtacactgt
I I I IIIIIIIIII
IIIIIIIIII GGCGTGAATG TTTCAGAACT TGAATTTATT TGGAAATGAC CCAGCTCTTC AGTACACTGT I
3394 c q t c a c q t t a q t t t a q c a c a qqataqaaac t c q q q t a a c c a a a a c a c c t q qaqaaacqat
i i i r i i - i t i i i i r i i i r i i i i i i i i i t t i I I ~ ~ ~ I I I I II I I I I I I ~ i i~ i i i i r i i r CGTCAC.TTA GTTTAGCACA GGATAGAAAC TCGGGTAACC AAAACACCTG GAGAAACGAT t g t a t g t t c a g t t c a g t t c a g t t c a g t g g c t c a g t c g t g t c t g a c t c t t t gggaccccgt
IIIII IIIIIIIIII IIIIIIIIII IIIIIIIIII IIIIII Ill IIIIIIIIII TGTAT.... . GTTCAGTTCA GTTCAGTGGC TCAGTCGTGT CTGACTGTTT GGGACCCCGT 368 ggcctgcagg acgctgggct t c c g t g t c c
llllllllll
llllllllll
IIIIIIIII
GGCCTGCAGG ACGCTGGGCT TCCGTGTCC 3589 Figure 1. Position of the (TG)n repeat on bovine 1.709 satellite DNA. The size-selected genomic fragments cut with Taq 1 (465 bp) and Sau 3A (270bp) are shown in A. Part B of the figure shows alignment between the 1.709 satellite sequence and the Taq 1 fragment. The primer used during ASPS is framed (see text).
Five new bovine microsatellites 539
between 15000 and 30000, and so present a major problem in finding new bovine microsatellite sequences. Testing clones by ASPS before DNA purification and sequencing considerably improved the screening conditions and allowed us to sequence only potentially interesting clones. Among the 14 remaining microsatellites, five were thoroughly studied. We evaluated allelic frequencies on a panel of 40 unrelated animals from eight different French breeds (Tarentaise, MontbCliarde, Normande, Abondance, Franqaise
Table 1.
Accession Local number name
Primer sequences (5'-3') and average size of the repeat
Number Allele Allele of alleles size frequency 174 172 170 168 164
0.04
3
232 230 228
0.36 0.47 0.17
5
115 111 109 105 101
CTGGAGGTGTGTGAGCCCCATTTA
X63794
pSat3
CTAAGAGTCGAAGGTGTGACTAGG (AC)20
5
CCITI'CA AAAACACGGAAAmCGGGGG X63793
pSat5
CTTCAGGCATACCCTACACCACATG
(AC)13 AGGAATATCTGTATCAACCTCAGTC X63795
pSat6
CTGAGCTGGGGTGGGAGCTATAAATA
(AC)13
CGAGTITCllTCCTCGTGGTAGGC X63792
@at1 1
GCTCGGCACATCITCCITAGCAAC
8
(AC)8 AT (AC)9
GCACAGTGACCTCTCAATAAATGC X63796
pSatl3
CCACTAlTCITGCCTGAAGAATCC
(AC)20
10
0.15 0.49 0.15 0.07
Observed heterozygosity
0.51
0.50
0.02 0.05
0.21 0.06
0.46
0.66
216 214 210 206 204 200 196 194
0.01 0.15 0.12 0.01
199 197 195 193 191 189 187 185 181 177
0.02 0.08 0.31 0.23 0.08 0.19 0.02 0.02 0.02 0.01
0.01
0.40
0.65 0.04 0.01
0.74
540 D. Vairnan et al.
Figure 2. Autoradiographies of denaturing polyacrylamide gels illustrating Mendelian inheritance of PCR-amplified bovine microsatellite alleles. (A) PSat 3 (Accession number: X63794); (B) @at 6 (Accession number: X63795); (C) @at 5 (Accession number: X63793); (D) @at 11 (Accession number: X63792); (E) FSat 13 (Accession number: X63796).
Five new bovine microsatellites 541
Frisonne Pie Noire, Charolaise, Brune des Alpes, Limousine). Results are summarized on Table 1. Mendelian segregation is presented in Fig. 2 for the five microsatellite systems in half-sib families derived from a male heterozygous at the studied microsatellite locus. Labelling of the fragments was done either by end-labelling of one primer, or by direct incorporation of [35S]dATPduring the PCR. Conditions for the amplification reaction were 15s at 95"C, 15s at 58°C and 15s at 72"C, 30 cycles. Chromosomal assignment of the five sequences was addressed by using a panel of 36 somatic cell hybrids (Heuertz & Horst-Cayla 1978). Twenty-four enzymes were tested in our laboratory for this panel, thus allowing us to allocate syntenic groups to our hybrids by reference to other panels (Fries et al. 1989; Georges et al. 1991). DNAs of these hybrids was used as PCR templates and revealed that two microsatellites (pSat3 and pSat6) are syntenic, while pSatll belongs to the syntenic group U10. We have not yet been able to allocate the other microsatellites to syntenic groups even though they clearly appear only in some hybrids and not in others.
Acknowledgements
Excellent technical assistance from D. Bruneau is greatly acknowledged. We thank M. Nocart for preparing somatic cell hybrids DNA and P. Martin for critical reading of the manuscript, and constant interest in this work.
References Devereux J., Haeberli P. & Smithies 0.(1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12,387-95. Fries R., Beckmann J.S., Georges M., Soller M. & Womack J. (1989) The bovine gene map. Animal Genetics 20, 3-29. Georges M., Gunawardana A,, Threadgill D., Lathrop M., Olsaker I., Mishra A., Sargeant L.L. etal. (1991) Characterization of a set of variable number of tandem repeats conserved in Bovidae. Genomics 11,2432. Heuertz S . & Horst-Cayla M.C. (1978) Carte gtnttique des bovins par la technique d'hybridation cellulaire. Localisation sur le chromosome X de la glucose-6-phosphate dtshydrogtnase, de la phosphoglyctrate kinase, de I'alpha-galactosidase, et de I'hypoxanthine guanine phosphoribosyl tranftrase. Annales de Gtnttique 21, 197-202. Skowronski J., Plucienniczak A., Bednarek A. & Jaworski J. (1984) Bovine 1.709 satellite: recombination hotspots and dispersed repeated sequence. Journal of Molecular Biology 177,399-416.
