A set of 99 cattle microsatellites: characterization, synteny mapping, and polymorphism. D. Vaiman, 1 D. Mercier, 1 K. Moazami-Goudarzi, 1 A. Eggen, 1 R.
Mammalian Genome5,288-297 (1994). e 9 Springer-VerlagNew York Inc, 1994
A set of 99 cattle microsatellites: characterization, synteny mapping, and polymorphism D. Vaiman, 1 D. Mercier, 1 K. Moazami-Goudarzi, 1 A. Eggen, 1 R. Ciampolini, 1 A. L6pingle, 1 R. Velmala, 2 J. Kaukinen, 3 S.L. Varvio, 3 P. Martin, 1 H. Lev6ziel, 1 G. Gu~rin 1
~Laboratoirede Gdn6tique biochimique et de Cytogdn6tique, INRA-CRJ,Domaine de Vilvert, 78352 Jouy-en-Josas,France 2Agricultural Research Centre, Institute of Animal Production, SF-31600, Jokioinen, Finland 3Department of Genetics, P.O. Box 17 (Arkadiankatu 7), SF-00014, Universityof Helsinki, Finland Received: 1 November 1993 / Accepted: 14 January 1994
Abstract. Cattle microsatellite clones (136) were isolated from cosmid (10) and plasmid (126) libraries and sequenced. The dinucleotide repeats were studied in each of these sequences and compared with dinucleotide repeats found in other vertebrate species where information was available. The distribution in cattle was similar to that described for other mammals, such as rat, mouse, pig, or human. A major difference resides in the number of sequences present in the bovine genome, which seemed at best one-third as large as in other species. Oligonucleotide primers (117 pairs) were synthesized, and a PCR product of expected size was obtained for 88 microsatellite sequences (75%). Synteny or chromosome assignment was searched for each locus with PCR amplification on a panel of 36 hamster/bovine somatic cell hybrids. Of our bovine microsatellites, eighty-six could be assigned to synteny groups of chromosomes. In addition, 10 other microsatellites--HEL 5, 6, 9, 11, 12, 13 (Kaukinen and Varvio 1993), HEL 4, 7, 14, 15--as well as the microsatellite found in the N-casein gene (Fries et al. 1990) were mapped on the hybrids. Microsatellite polymorphism was checked on at least 30 unrelated animals of different breeds. Almost all the autosomal and X Chr microsatellites displayed polymorphism, with the number of alleles varying between two and 44. We assume that these microsatellites could be very helpful in the construction of a primary public linkage map of the bovine genome, with an aim of finding markers for Economic Trait Loci (ETL) in cattle.
Introduction
Microsatellites which are mainly of the (dG. dT) n type have been identified in all eukaryotic species studied so far. On average, their number is estimated at 100,000 in mam-
Correspondence to: D. Vaiman
mals. Evenly distributed throughout the euchromatin, they display high polymorphism, with a median Polymorphic Information Content (PIC) of 0.60. Furthermore, the mutation rate of microsatellites was recently estimated at 4.7 10 . 4 in mice, which positions them between coding sequences and other categories of repetitive DNA (Dallas 1992). This relatively low mutation rate enables the segregation of microsatellite alleles in pedigrees to be followed unambiguously. Technically, microsatellites are relatively easy to isolate from partial plasmid libraries, and the PCRbased typing of the alleles can be readily automated. As a result of these qualities, microsatellites are becoming increasingly important markers for genetic mapping (Todd 1992). In human (Weissenbach et al. 1992) and mouse (Dietrich et al. 1992), anonymous microsatellite sequences were used to construct maps with an average distance of 5 cM between consecutive markers. In rats, a map has alr e a d y been p u b l i s h e d c o m p o s e d e x c l u s i v e l y of microsatellite polymorphisms associated to coding sequences (Serikawa et al. 1992). This allows for a clear synergism between physical and genetic cartography of the genome. A greater number of microsatellite sequences have been characterized in these species than for domestic mammalian species such as bovids, pig, dog, or cat. A saturated map of polymorphic markers is the first requirement for detection of linkages with economically valuable traits in domestic animal families (Womack 1992). In cattle, considerable success has already been obtained with the characterization of a microsatellite marker closely linked to the weaver disease locus (Georges et al. 1993a) and another microsatellite linked, albeit less closely, to the Poll (hornless) gene (Georges et al. 1993b). A first obvious prerequisite to increase the precision of the bovine linkage map is the production of more microsatellite markers. Because the number of microsatellites on maps of domestic animals is still relatively low, an approach of random production of microsatellite markers is appropriate. In this report, we describe the isolation, analysis, and characterization at the level of structure, genomic repartition, and polymorphism
D. Vaiman et al.: Characterization of 99 cattle microsatellites
of microsatellite sequences from bovine genomic DNA ]mostly of the (TG) n type].
289
Sequence analysis Screening of GenBank-EMBL database was carried out with the GCG software package (Devereux et al. 1984).
Materials and methods
PCR amplification of hybrid cell DNA Animals A minimum of 36 unrelated animals from 10 different breeds [Bretonne Pie Noire (2), Brune des Alpes (2), Charolaise (1), Creole (2), Friesian (1), Limousine (2), Montb61iarde (8), Normande (14), Pie Rouge des Plaines (2), and Tarentaise (2)] were used to estimate microsatellite polymorphism.
Synteny mapping was carried out by PCR analysis of hamster/bovine somatic hybrid cell line DNA. PCR was carried out in a Cetus 9600 thermocycler with bOO ng of DNA and a Promega PCR kit, in 10 gl of reaction volume with 1.5 mM MgCt 2. Samples were preheated for 5 min at 92~ and then subjected to cycling at 94~ for 15 s, 55-65~ (see Table 1) for 15 s, and 72~ for 20 s for 30 cycles. Reactions were analyzed by agarose gel electrophoresis.
Somatic cell cultures Polymorphism evaluation
Thirty-six previously described (Gudrin et al. 1994; Heuertz and HorsCayla 1981; Hors-Cayla and Heuertz 1978; Vaiman et al. 1994) hamster-bovine somatic hybrid cell lines were used to assign microsatellites to international synteny groups. Definition of reference synteny groups or chromosomes in the panel was obtained with loci previously mapped on these hybrids so that only one pattern was retained to describe each cluster. All microsatellite patterns were then compared with one another and with the reference clusters. Correlation coefficients were estimated, and synteny assignation was determined according to the rules of Chevalet and Corpet (1986).
Nomenclature
Leukocyte DNA extraction
International locus names were given to the microsatellites according to their assignation to synteny groups or chromosomes (for example, D1SI5 for INRA049; see Table 1).
DNA was extracted from l0 ml of peripheral blood according to the following protocol: erythrocytes were lysed in NE (NaC1 10 raM, EDTA l0 mM, pH 7.5), and leukocytes were pelleted at 3000 g for 30 rain (Jeanpierre 1987). Cultured cells were washed with PBS and collected by centrifugation prior to lysis. The DNA was recovered by ethanol precipitation, washed three times with ethanol 70%, dried under vacuum, and resuspended in TE 10-1, and the concentration of DNA was adjusted to 200 ng/gl.
Microsatellite isolation and library screening Construction of the plasmid library. DNA (30 pg) from a male Bos taurus was digested to completion by either Taql or Sau3AI (Boehringer), dephosphorylated with calf intestinal alkaline phosphatase (Boehringer) to prevent concatemer formation in the subsequent ligation step, and fractionated by agarose gel electrophoresis. Fragments ranging from 300 to 500 bp or 500 to 800 bp were isolated from the slices by the deep-freeze method and ligated into the Accl- or BamHl-(Boehringer) digested pGEM4Z, in the presence of T4 DNA ligase (BRL). Bovine Taql and Sau3AI restriction fragments were cloned in the Accl or BamHI site of pGEM4Z (Promega), respectively. Ligated plasmids were transformed into DH5c~-competent cells (BRL) following the manufacturer's protocol, and bacteria were plated on LB-agar plates. Duplicate filters were lifted from the plates, pre-hybridized, and hybridized at 58~ with a mix of (TC)~ 0 and (TG)I 0 probes 5' end-labeled with y [32p] dATP (Amersham) and T4 polynucleotide kinase (Boehringer), according to standard protocols (Sambrook et al. 1989). Positive clones were picked onto a fresh grid and re-screened. Plasmid DNA was prepared from a 3-ml overnight culture and sequenced with an Applied Biosystems 373A automatic sequencer.
Cosmid library. A cosmid library was obtained from Clontech (average insert size = 38 kb). Cosmids were plated and screened in the same way as described above. Positive cosmids were digested to completion with Sau3AI; the fragments were dephosphorylated and cloned from every individual cosmid into the BamHI site of pGEM4Z. After transformation, bacteria were plated and screened as previously described Ibr plasmid libraries.
Polymorphism was evaluated by PCR performed in the presence of 1 pCi of ~x[35S] dATP (Amersham, 6000 Ci/mmol) as described above except that the concentration of unlabeled deoxynucleotides was lowered to 25 {I.M for dCTP, dTTP, and dGTP, and to 2.5 [.tM for dATP. PCR products were analyzed by polyacrylamide/urea gel electrophoresis.
Results
Characteristics of the isolated sequences The screening for microsatellite sequences was performed with two oligonucleotides, (TG)I 0 and (TC)10, which possess the same calculated melting temperature (60~ One hundred thirty-six microsatellite sequences were obtained, of which 91% were of the TG type, and the remainder were TC and AT microsatellites. The latter TC and AT microsatellites were often composed of compound repeats containing a stretch of TG dinucleotides. The microsatellites could be categorized in three classes according to their structure (Weber 1990): perfect (uninterrupted run of dinucleotides), imperfect (one or more interrupted runs of the same dinucleotide), and compound (successive runs of different dinucleotide repeats). Perfect repeats constituted 68% of the sequences. The average size of the microsatellites was 14.2 repeats (Fig. 1). A sample of 45 microsatellite sequences was used to screen the Genbank/EMBL database. Similarity with one or more bovine repetitive elements was found in 18 of the microsatellite sequences (40%). The most frequent repetitive sequences were the M26330 SINE found 12 times (30%), a bovine lysozyme gene (M95097) also in 12 cases (30%), regions of caseins and globins genes, 21 hydroxylase genes, and the Bov tA element (X64124) in 7 occurrences (17.5%) (Lenstra et al. 1993). Alignments between the M26330 SINE and microsatellite sequences showed that only one flanking sequence of the microsatellite could be aligned with the SINE, with more than 80% similarity.
290
D. Vaiman et al.: Characterization of 99 cattle microsatellites
4.=
t
nd g.
I
25
Choice of PCR primers Of the 136 microsatellite sequences that were determined, 5 were excluded because of their similarity to the bovine satellite 1.709 (Skowronski et al. 1984). This sequence was found in about one-third of the microsatellite clones from the first library (Taql fragments). We first screened against the bovine 1.709 using ASPS (Vaiman et al. 1992), which eliminated the satellite-containing clones prior to sequencing. Construction of a library containing inserts with an average size of 600 bp instead of 400 bp eliminated the bovine 1.709 sequence fi'om all our subsequent screenings experiments. Of the 131 sequences left, 14 were eliminated because of the proximity of the microsatellite to the cloning site or because the sequence was difficult to read. All the remaining sequences (117) were tested for PCR amplification. PCR primers were designed with or without the help of the Oligo 4.0 software, in order to amplify fragments in the range of 80 to 270 bp. The annealing temperature chosen for the PCR primers was 58~176 allowing for a simultaneous amplification of most of the loci, with standardized PCR conditions (that is, 58~ for the annealing temperature; exceptions are indicated in Table 1). We obtained a PCR product of the expected size in 88 of the cases (75%). The list of all the PCR primers is given in Table 1.
Assignments to synteny groups or chromosomes PCR primers derived from the different microsatellite sequences were used to amplify the DNA prepared from 36 previously described hamster/bovine somatic hybrid cell lines. This allowed the assignment of most of the microsatellites to bovine synteny groups (Table 2). Two groups of microsatellites were called A (seven microsatellites) and B (three microsatellites) because no correlation could be found with known synteny groups, even
~V
~o
28
more
than 30
Fig. 1. 3-D histogram plotting the number of microsatellite sequences of each category (perfect, imperfect, compound) against the size of the repeats. For compound or imperfect repeats, only the longest continuous stretch of dinucleotide repeats was taken into account and plotted.
though the pattern of response on the hybrids was characteristic and unambiguous. One microsatellite, INRA090, gave a very clear PCR product of the expected size on 12 hybrids out of the 36 of the panel, but its pattern did not match any of the previously defined synteny group patterns. For another microsatellite, INRA085, polymorphism was easily detectable with radioactive labeling, but no amplification was visible from the somatic cell hybrid DNA.
Polymorphism Polymorphism was evaluated on a panel of unrelated animals from different breeds (see Materials and methods). In Fig. 2, the number of alleles is plotted against the PIC value for the different microsatellites studied in a semi-logarithmic system of coordinates. This showed a good fit with the equation PIC = 0.295 • LN (Nb alleles) - 0.0028, which was estimated by the least-squares method (r 2 = 0.70). As expected, a good correlation was also found between the number of alleles and the size of the dinucleotide repeat (correlation coefficient -- 0.79). Allelic frequencies and PIC were calculated. All these data are reported in Table 1. The average number of alleles was evaluated for the different microsatellite structures (Table 3). No polymorphism could be found for the markers situated on the Y chromosome, and two autosomal microsatellites were monomorphic (INRA039 and INRA055). For the others the number of alleles was between 2 and 44 with an average of 6.4 (_+5.4).
Discussion
Up to now, a total of 60 microsatellite loci have been localized on the bovine gene map (Fries et al. 1993). In this paper, 97 microsatellite loci, of which 84 are new, have
291
D. V a i m a n et al.: C h a r a c t e r i z a t i o n o f 99 cattle m i c r o s a t e l l i t e s
Table 1. PCR primers. Locus name
Lab name
Accession number
D3S8
INRA003
X63794
DU27S4
INRA005
X63793
D3S9
INRO006
X63795
DYS3
INRA008
X73126
D1S6
INRA0I 1
X63792
DI6SIO
INRA013
X63796
B
INRA016
X67828
DIOSll
INRA018
X67826
D3SIO
INRA023
X67830
D17S6
INRA025
X67824
DUl2S4
INRA026
X67832
B
INRA027
X67829
D1853
INRA028
X67826
DXS8
INRA030
X67822
D21S12
INRA031
X73132
DI/S9
INRA032
X67823
Dl6Sll
INRA035
X68049
D20S3
1NRA036
X71554
DIOSI2
[NRA037
X7151
D18S4
[NRA038
X7155
D20S4
INRA039
X71556
D2S15
INRA040
X71558
$3S11
INRA041
X71559
DI1SIO
INRA044
X7158
D15S5
INRA046
X71495
D16S12
INRA048
X71591
D1S15
INRA049
X7158
D15S6
1NRA050
X71494
DU27S5
INRA051
X71587
D13S7
INRA052
X71496
D7S6
INRA053
X71497
D1S16
INRA054
X71498
DI1Sll
INRA055
X71999
DYS4
INRA057
X71501
A
INRA059
X71503
D21S13
INRA060
X71504
D28S6
INRA061
X71505
DYS5
INRA062
X71506
D18S5
INRA063
X71507
D23S15
INRA064
X71508
Primers CT G G A G G T GT GT G A G C C C C A T T T A CT A A G A G T C G A A G G T GT GAC T AGG C A A T CT G C A T G A A G T AT A A A T AT CTTCAGGCATACCCTACACC AGGAATATCTGTATCAACCT CAGTC CT G A G C T GGGGT G G G A G C T A T AAAT A G A G C C T G T G T GT GT A T A C A C GGCACTTTCCTCTCCTGTCGCG C G A G T T T C T T T C C T C G T G G T GGC GCTCGGGCACATCTTCCTTAGCAAC G C A C A G T G A C C T CT C A A T AA AT GC CCACT ATTCTTGCCTGAAGAATCC A C G C A G A C C T T AGC AT AGGA GA GT C G C A A T GAGT T G G A C A C A AC TCTAAT AGTATCATTAAGGT G TTTATGGTTTAAAATTACCC G GAGT A G A G C T A C A A G A T AAA CT T C Y A A C T A C A G G G T G T T AGAT G AACT CA GGT T T C C T GT AT G A A C C C A G GAG GC T AC GGT C C AT A G G G T T GC AAA AGGAAAGAGT AGGAAGAACT AGTC AACT A A A G A G C C C T G T G C T G T A A C CTCCCCACTTAGGAACTCTGTATC C A C T G C A T C C C T C C C C A C T AAC C A G G G T T C A G T C A G A A A A A G AAG C ACT GAT AAT T GT GGGT GGT T C A ATGCAAATGTGCT ACATCAC CTAT TGGCCCAACTCTCACATCCAGATC A C A T AAAGT AGGAT AAAT AC T A G C A C A A T T AAAGT A C A T AT C AA A A A C T G T A T T C T C T A A T A G C AC G C A A G A C A T A T C T C C A T T CC T T T ATCCTTTGCAGCCTCCACAT TG TTGTGCTTTATGACACTATCCG C A G A A A G A A A T A G A A T GGAC AG AAAAGATGTGAGCTGGTTCT TG G A T C C T G C T T A T A T T T A A C C AC A A A A T T CC AT G G A G A G A G A A AC GGGACTCCACAGTCATGGTGTC C T T C T C T A G C C C C A A A C T GC CC GCTCTCTGGCTCAATAGCTGG GC CT G G A G A A T C C CC AT GGC A T C A G T CT G G A G G A G A G A A A A C CTCTGCCCTGGGGATGATTG T G C A A A A T T C T T C T A A G A T A C T T T AA A A C A T T T T AT GT AGT T T AAT T T GAAAC G A G T T AGAC AT GACT GAGC A AC G A A G T GGT AGC AGGT C A G C C T CAGCTCATGTGTTTACATGGC AGTCTCTGGAACTTCTCCTAC C T G T C C C T C A G T A A A C A A G T CG A A G C T AAAGT AGC A G G G A A G G T G T A T T A G T T T G T G T T C T T T GGC TTGGCTTCCACAATCACACA ACAGGCT ACAGT CCAT GGGGTT T AT AGAAC A G A A A A A T GAC T AC AC G C AT C A A A T T T AGT G A A A A G G G CCT AAAAGAACACAT G C C T G T T G GAT C A A A T T T AGT G A A A A G G G CCT AAAAGAACACAT GCCT G T T G A A A G T C A G A T A C A A C T G A G T GAC A A T C A C C A G A A A T T C A C T T C ACC GAAT C A G A C A T GACT T A G C A AC T T C A T C A G G A G G T A C A T G T T GT CAT A T G A G C C A C C A G T G A A G T CT GT G A C T A G G A A G A A C C A G CCTAGCGACTGTCCAAGCG CACGGGCTGAGAATTCAAA C GGATGCTTCT AGACTGACAC C GGGAT GAAAT T T A T G C T CT G T G T T C C A T A T A G C A T T T A G A A T AGC A G A C A C T C C C A A A T AAAAT C CC CCATGTACAGAGGACCCT G A C A T G C A T G T G C T T G T G T CG TGTGCAGCACCTTGTCTCC ACATGCATGTGCTTGTGTCG A T T T G C A C A A G C T AAAT CT A A C C A A A C C A C A G A A A T G C T T GGA AG GCCCACAGCGCTCTCTAC C T G A A A G C AGAAT G A G G T GC
Number of alleles*
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D. V a i m a n et al.: Characterization o f 99 cattle microsatellites
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Table 1. Continued. Locus name
Lab name
Accession number
DIOS13
INRA069
X71513
D10S14
INRA071
X71514
D4Sll
INRA072
X71515
D1S,!8
INRA073
X71516
D14S6
INRA079
X71522
A
INRA080
X71523
D26S5
INRA081
X71524
D4S12
INRA082
X71525
DU2S4
INRA084
X71527
?
INRA085
X71528
D3S12
INRA088
X71566
D6S5
INRAK
X14908
?
INRA090
X71563
D15S8
INRA091
X71565
DI4SIO
INRA092
X71573
D3S13
INRA093
X71572
D14S7
INRA094
X71571
DIOS17
INRA096
X71570
A
INRA097
X71561
D14S8
INRAI00
X71562
DIOS15
INRA101
X71574
D21S14
INRA103
X71531
D5S9
INRAI04
X71532
D13S8
INRAI05
X71579
D]OS16
INRA107
X71577
DllSt2
INRAI08
X71581
D17S9
INRA110
X71580
DllS13
INRA111
X71590
D7S7
INRAll2
X71589
DIISI6
INRA115
X71538
D1S20
INRA117
X71540
D3S14
INRA118
X71543
D1S]9
INRAlI9
Z71542
DXS9
INRA120
X71544
D18S6
INRA121
X71545
A
INRA122
XY1547
D3S15
INRA123
X71533
DYS6
INRA124
X71546
DYS7
INRA126
X71533
DU2S5
INRA127
X71550
Primers A G A G C C C C A T A A T A G G C A A CC C AT T T AC AGAGC CT AGT GAT AGG GCCTAGCATCCACAATACCAC G G C A G G A C C T G A A G T G TGGT C CTTAACTCATTCACCTCAACTG AGT G A T T G A G C A C A T T GCGC AT ACTGAGGAACTAAGCACCGC GAAAAGCAAGGCTGTCCGAC G G G T T T T A G G A G C T C T G T A CCAG GTGTTTCTCAACCGTGCTG GACAGAGGAGCCTGGTTGGC G T T T C T T A G T C G G G T A T A A T GG CGGCTCACGGTCTCTATCGG G C G A A C C C A A G A A T C A G A C TC CT A G A G G A T C A G C A A C T T C A A T C C T C A C T G A T T T G A C T T G A C T CC CTAAAGCTTTCCTCCATCTC CCTGGTGATGTTTGGATGTC C C C A T T T C C A G T C C T C T C A T CC AT A C A A T GT A C C A C A T GACA GG CATTCCTTTGATGTAGGAG CT GT C A C A A A G A G A T A C A G C ATGCACCTTAACCTAATCC A C A A T A C A C A A G C A T A C TAC G G T C A T T T T C C A T T AT G A C A G C A G GGTGTTACCTTTTTTAGTCTCC AACTCAGCAGGCGG A G A G T T G G A C A C A G C TGAGG G G T T T G A C T T C A A C T T C A A CC G G G A G T C T G T G G A G A A TGCA AG CTACAACAAGGGAATTTGTG C A G A G T C G A A C A T G A C T A A G TG CT GT A C C A G A A A C A G A C A G AG T GT C AGGC T AC AGT C C AT GG G G C T G A T T C A C T T C G C T A T A C AG C AGT AC AT GAGGT T GC AAAG AG GCTACAGTCTGTAAGGTTCC CCGTAAGATATCGTGGAATC ACAGGCTCTTTTATGACTTG AAGTGGGTGTGAGCCAAGCC CTAAGCAACCAACACTTTAGG A T A C A C A T G T G C A A G C A T G TG TTGTCCAGCCCAGCATTTAGC GGAGAAGACTTATGGGAGC C T GT C A G A C A T G A C T G A G T G A C A A A A G A G A T T G A A A G C G C C TG G G T T A C A A G A G T C GGACAT GAC G G G A A C T A T A C T T A G T A T C TT G T A T C C C A G A T A C A G A T G C A A C AG GGAGAGCCGAGGGCTTCAGC AGT CT GT G G G G T T C C A G A G T T G TTACAGTTAACTCACCCTCC T ACAGT CCGT G G G G T CACAG AG A A C A G A A A T TCCT T CAT GAG C T T GT CGGT GT G G A G A A G C A C C GTTTCCCGGTAACCAATTCC GCCTCTCAAAGCCACCTGC GAT CT AACT A G A G C T T T CC GACT A A C A C A A A A T GACT C G T G G G A G G G G G A C T A C T T C T C A TGC GTTTCTAGTAACATATTGAC T T AGAC AT GACT GAAGCAAC G T C T T T G G C A A G C C A G C T C T CC AGAGT C A G G A T T T G A A T C C A GG GGAACTAAGCACCGCACAGC T GT C AAAC AGAGC AGT GAGA GC TGCAGCAACGAGGACCCACG AACCGCATTTGCTCCTCTG G G A A A C C C A T T G G A G G A T T TG CTTCACTATTCCCCACAAAGC G T A A A T A A A A T T A T A T A C C TACC A A A G G G T C A G T TA A T T T T AC AC TCTAGAGGATCCCCGCTGAC AGAGAGCAACTCCACTGTGC GATCTTTGCAACTGGTTTG C A G G A C A C A G G TCT GACAAY G TCTAGAGGATCAAGGATTTGTG A A T C C A T G G A A A G A T G C A C TG CT A C A G C T C T G A T G A G A A C C CGTTTTCTCAAACTTCATTG
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8
132
0.68
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6
173
0.60
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5
168
0.64
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7
129
0.54
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8
178
0,74
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2
122
0.05
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4
157
0.60
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5
125
0.62
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6
183
0.61
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8
179
0.69
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3
100
0.22
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5
135
0.66
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3
99
0.38
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3
120
0,22
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4
166
0.33
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5
154
0.39
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4
213
0.34
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7
137
0.47
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6
171
0.77
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5
167
0.49
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4
97
0.13
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2
217
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5
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4
171
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159
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D. V a i m a n et al.: Characterization o f 99 cattle microsatellites
293
Table 1. Continued. Locus name
Lab name
Accession number
DIS17
INRA128
X71534
A
INRA129
X71535
D17SIO
INRA130
X71536
DIISI4
1NRAI31
X71548
D23S16
INRA132
X71552
B
INRA134
X73125
D17S7
INRA135
X73128
DU2S
INRA136
X73129
DllSI7
INRA177
X74201
DIS2t
HEL6
X65206
DIlS15
HELl3
X65207
A
HEL4
X65203
A
HEL9
X65214
D26S6
HEL 11
X65216
D20S5
HELl2
X65211
D21S15
HEL5
X65204
D23S17
HELl5
X65208
DXSIO
HELl4
X65209
D13S9
HEL7
X65210
Number of alleles*
Primers T A A G C A C C G C A C A G C A G A TG C AGACTAGTCAGGCTTCCTAC GGGT AGCCT GT T A A A A T GCA G CAGT GCT GACCT CT G A A G T A AG GATCCCCGCTGACCACTCAG A A G A G A G C A A C TC C A C T GT G C GGT AAAAT CCT GCAAAACAC AG T G A C T G T A T A G A C TGAAGCA AC A A C A T T T CAGCT GAT GGT GGC TTCTGTTTTGAGTGGTAAGC TG GCCCATCGGTCCCAGGTGG TTCCATCTATCTTGGGAGC GGAGACTTCACTGGAAAGG AATCCCATGGACAGAGGAGG TGGCCTAAATATTTATCAGT G GAGACCTTTCCTCTTTCTCAGC C A G C A G T A G T C A C C T AAAAC C A A G G A A A C T C C A A A A C A C C A GG GGACACGACTGAGCAAGTAA AGGC AGAT AC AT T AC C ACT A TAAGGACTTGAGATAAGGAG CCATCTACCTCCATCTTAAC AGT T GGAC AT GAC T GAGT GC GTAACTTAGTGGGTGCAGCT CCCATTCAGTCTTCAGAGGT CACATCCATGTTCTCACCAC C T T T GT GGAAGGCT AAGAT G TCCCACATGATCTATGGTGC S C A T T A G G T T CT C C A G A G A A CAGACTT GTCAGACTCCATA GCAGGAT CACTT GTTAGGGA A G A C G T T A G T GT A C A T T A A C A G A G A A G T CT G G T G G G C T A T T C A G T T GT CT A G A T T AAGCA CC AACC A G G G T T T G A A C T GA TGGTTATGTGTT ATGTGGCA T T AT GACT GAGT AGT AT T T C T AGAT AT GACT G A G A G A C T A
* T m fixed at 58~ excepted when noted: (a) 55~
(b) 65~
(c)60~
(e) 52~
Average size
PIC
Reference
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Kaakinen and Varvio 1993 Kaukinen and Varvio 1993 This paper
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Varvio Varvio Varvio Varvio
(f) 50~
been assigned to bovine synteny groups. Although a much larger number of bovine microsatellites have been isolated by others (Georges et al. 1993b), the set of PCR primers developed here considerably increases the possibilities of building a public domain bovine genetic map.
Sequence characteristics and analyses Because the screening was done together with (TG)l 0 and (TC)j0, the chances were a priori equivalent to selection of (TG) n or (TC)n clones. The high level of incidence of (TG) n microsatellites in the bovine genome (91%) indicates that these sequences could be ten times more frequent in this species than (TC)n microsatellites. This proportion is considerably higher than the one found in other mammalian species, such as rat or human, where the ratio is only three (TG) n to one (TC) n (Beckmann and Weber 1992). In the horse, the ratio of (TG)u to (TC) n has been previously reported to be 7:1 by Ellegren and coworkers (1992). In this study, we found a lower ratio of 5:1 for the horse (unpublished data). In the dog, we could observe a ratio of 3:1 (unpublished data). Differences in the average size of microsatellites between human [12% of (TG)n microsatellites longer than 20 repeats] and rodents (43%) have previously been described (Beckmann and Weber 1992). In cattle, the average size of the dinucleotide repeats was comparable to the range ob-
served in pig (Wintero et al. 1992; mean - 14.89; SD = 6.32) or human. Compared with other species, the repartition of the different microsatellite types in cattle was similar for perfect and imperfect repeats, whereas the average size of compound repeats was larger (Beckmann and Weber 1992; Wintero et al. 1992). Average distances between neighboring microsatellites is classically estimated by dividing the total length of screened D N A sequences by the number of microsatellites found in these sequences. The validity of this approach is limited by the repartition of the microsatellites and thus is dependent on the distribution of the dinucleotide repeats throughout the genome. Furthermore, the sample of small fragments present in the genomic library needs to be representative of the whole genomic DNA. Assuming that these two conditions are fulfilled, we were able to calculate that a total of 15 Mbp of bovine genomic D N A (25,000 clones • 600 bp on average) was screened for (TG)n and (TC) n sequences. 126 microsatellite clones were isolated from the plasmid library, suggesting that about one microsatellite of the TG type was present every 120 kb of bovine genomic DNA, and one of the TC type about every 1 Mbp of DNA. This estimation is consistent with the calculations of Steffen (Steffen et al. 1993): one (TG)n every 188 kb. This density of microsatellites is much smaller than has been calculated in other mammalian species, such as human, one (TG) n every 28 kbp; mouse, one every 18 kbp; rat, one every 21 kbp (Stallings et al. 1991); pig, one every
294
D. Vaiman et al.: Characterization of 99 cattle microsatellites
Table 2. Assignment of microsatellite loci to synteny groups or chromosomes. Synteny group Chr UI U2 U3 U4 U5 U6 U7 U8 U9 UI0 UII U12 U13 U14 U15 U16 U17 U18 UI9 U20 U21 U22 U23 U24 U25 U26 U27 U28 U29 X Y
16 9 5 2I 10 3
INRA013(0.8) INRA084(0.87) INRAI04(0.8) [NRA031(0.93) INRA018(0.7) INRA003(1)
18 1 13
INRA028(0.86) INRA038(0.86) INRA011(0.94) INRA049(0.83) 1NRA052(0.86) INRAI05(0.84) INRA026(0.85) INRA072(I) INRA082(0.86) nd INRAK(I) INRA032(1) INRA044(0.88) INRA040(0.85) INRA135(1) nd INRA046(0.75) INRA050(0,74) INRA064(0.89) INRA132(0.83)
4 6 1I 2 15 23 19 7 17 14 26 24 28 20
A B Isolated
INRA035(0.87) INRA048(0.93) INRA127(0.93) INRA136(0.79) INRAO60(I) INRAI03(0.86) HEL5(0.87) INRA037(0.79) INRA069(0.85) INRA071(0.85) INRA101(0.77) INRA107(1) INRA006(1) INRA023(0.91) INRA04I (0.8) INRA088(0.9) INRA093(0.9)
INRA053(0.88) INRA112(0.81) INRA025(0.88) INRA110(0.8) INRA079(0.77) INRA092(0.82) nd INRA0891 (0.74) HELl 1(0.66) INRA005(0.89) INRA051(0.83) nd INRA06[(0.94) I N R A 0 3 0 ( 0 . 9 3INRA120(0.93) ) INRA008(I) INRA057(0.79) INRA036(0.94) INRA039(0.88) lNRA059(0.93) INRA080(1) INRA016(1) INRA027(0.7) INRA085 INRA090
INRA063(0.82) INRA121(0.86) INRA054(0.89) INRA073(0.78) INRAI17(0.78) HELT(0.74)
INRAl19(0.73)
INRA096(0.77) INRAI I8(0.9) INRA123(0.79a)
INRA128(0.95) HEL6(0.78)
INRA055(0.86) INRA108(0.71) INRAlll(0.86) INRA115(0.71) INRA131(0.93) INRA177(0.93)
HELl 3(1)
INRA091(0.74) HELl5(0.89) INRA130(0.7) INRA094(0.82) [NRAI00(0.94)
HELl4(0.94) INRA062(1) INRAI24(I) HELl2(0.94) INRA097(0.8) INRA 122(1) INRA134(0.88)
[NRA126(1) INRA129(1)
HEL4(I)
HEL9(I)
Correlation coefficients are indicated in parentheses. ~'INRA 123 displayed also a correlation coefficient of 0.72 with U 19 (see text).
47 kbp (Wintero et al. 1992). With small fragment libraries made from horse and dog Sau3A-digested DNA, we could detect 10 times and 4 times more (TG) n sequences in horse and dog than in bovine DNA respectively (data not shown), although a report on horse (TG) n microsatellites indicates a frequency of only one (TG) n repeat every 100 kbp (E1legren et al. 1992). Since the construction of the library and the screening procedures were carried out under exactly the same conditions for the three species, the probability of an experimental bias is low, suggesting that microsatellites are less abundant in the bovine genome. The number of microsatellites isolated from bovine cosmid libraries was very low: both in the Clontech library and in a library constructed in our laboratory, the proportion of microsatellite-carrying cosmids ranged from one to three out of 100 cosmids. This number is considerably lower than that which was described for humans, where 40 cosmids out of 95 (42%; Litt and Luty 1989) and 3833 in 7546 (51%; Stallings et al. 1992) were shown to contain a (TG)n repeat. Assuming an average size of 35,000 bp for the genomic DNA contained in a cosmid, calculation suggests the presence of only one microsatellite every 1-3 Mbp of bovine genomic DNA. Similar values have been reported by other workers for similar independent cattle cosmid libraries (Steffen 1992). The disagreement between the density calculated from the cosmid library on the one hand and from the plasmid library on the other hand could be explained, at least partially, by a higher content of microsatellites in small-size restriction fragments. On the
whole, our results suggest that bovine genomic DNA possesses less (TG) n sequences than other m a m m a l i a n genomes studied so far. The total number of microsatellites in bovine DNA probably ranges from a few thousand, for the most pessimistic evaluation, to 30,000. Similarity between bovine SINE elements and cloned microsatellites has previously been found in 45% of the cases studied (Kaukinen and Varvio 1992). This figure is comparable to what we observed here as 18 occurrences among 45 sequences analyzed (40%). Presence or absence of repetitive sequences could not be used as a criterion to select useful microsatellites, since the proportion of microsatellites that yielded clear amplification products by PCR was comparable whether or not they contained SINE sequences. Actually, one of the flanking regions of some well-described bovine microsatellites (for example, at the loci CYP-21, 21-hydroxylase, or CASK, K-casein), belong to a family of repetitive elements, and such SINE elements have been proposed as an aid for cloning more efficiently new microsatellite sequences (Kaukinen and Varvio 1992), as well as for direct study of their polymorphism (Miller and Archibald 1993). The fact that microsatellites similar to the M26330 SINE were found in different synteny groups favors the hypothesis of a mobile DNA element that was inserted at many different loci in bovine chromosomes. The presence of a (TG) n repeat in the M26330 SINE could explain the high ratio of (TG)n:(TC) n microsatellites in the bovine genome as compared with other species.
D. Vaiman et al.: Characterization of 99 cattle microsatellites
295
100
10 .Q
9
E 9
:
:
: ~.
Z 9
9
9
,~
9
__ :
0,00
m
m 9
~
:::
~-=
: ~.--
~
~
~
9
|
H
:
_-
9
9
9
0,10
0,20
0,30
0,40
0,50
0,60
0,70
0,80
0,90
RC Fig. 2. For 60 microsatellites, the number of alleles was plotted against Polymorphic Information Content (PIC) in a semi-log system of coordinates. This allowed estimation of parameters of a linear regression (see text). Table 3. Alleles for different microsatellite structures.
Total Perfect Imperfect Compound
Mean number of aIleles
SD
Number of clones
Average size of the longest repeat
6.40 5.84 8.31 5.57
5.38 3.05 9.71 1.72
82 54 19 9
14.51 14.67 14.00 14,44
Synteny mapping Most of the microsatellites used in this study could be assigned to synteny groups or chromosomes provided that several factors were taken into consideration. First, two significant correlations were found between U08 and U24 (0.72) and between U16 and U27 (0.78). The first correlation, however, did not interfere with our interpretation, but the second introduced significant correlations between U16, U27, and five microsatellite loci: INRA005 (0.75 with U16/0.89 with U27), INRA032 (1/0.78), INRA131 (0.93/0.72), INRA177 (0.93/0.72), and H E L l 3 (1/0.78). In these cases, the highest correlation coefficient was retained for assignation. Three other microsatellites were assigned with relative accuracy: INRA055 to U16 (0.86) and to U12 (0.75), INRA053 to U22 (0.88) and to U16 (0.72). For INRA123, the situation is more ambiguous between U06 (0.79), and U19 (0.72). Secondly, synteny groups U08 and U26 were less well characterized than other groups because their pattern of response was defined by only one enzymatic locus on 26 hybrid cell lines
instead of 36 for the other synteny groups. This may explain why no microsatellite was assigned to U08 and why the identification to U26 is only significant for INRA081 (0.74), HELl1 being syntenic with INRA081 (0.81), and possibly to U26 (0.66). Assignation of microsatellites to the sexual chromosomes was achieved with the following considerations. First, each of the hybrid cell lines should contain one bovine X Chr, or at least the chromosomal segment encompassing the HPRT gene, which is indispensable for the survival of the cells. Despite our inability to detect PGK in 12 hybrids out of 36, we still admitted it as reference locus. Three microsatellite loci were highly correlated with PGK, and thus we assumed they belonged to the X Chr. The Y Chr was characterized by the assignation of five microsatellite loci. These loci (INRA008, INRA057, INRA062, INRA124, and INRA 126) could be amplified only from male bovine DNA. Furthermore, the five microsatellites could be amplified on hybrid cells derived from a fusion with calf fibroblast (in 4 out of t0 hybrids). No amplification product was detectable with the hybrids derived from the fusion with heifer lymphocytes (0 hybrids out of 26). Only two microsatellite loci, INRA085 and INRA090, could not be assigned to any synteny group in our panel, although for different reasons. Even in the presence of a radiolabeled nucleotide, amplification of INRA085 was relatively weak, and it probably could not be detected on the hybrids for technical reasons. INRA090 displayed a very clear pattern, different from the one of Chr 20, A
296
group and B group. It could probably account for one of the unidentified or ill-defined synteny groups of the panel (U14, U18, U25, or U28). Actually, recent data (Gu6rin et al. 1994) suggest that group A corresponds to U18. We have no evidence that a synteny group is absent from our panel, which makes it a valuable tool for mapping and a useful complement to other existing panels. It is worth noting that in all the cases where linkage studies have been performed, that is, 4 markers on U06 (Vaiman et al. 1994), 4 markers on U19 (Vaiman et al. 1993), 6 markers on U21, INRA018 and INRA037 on U05, INRA016 and INRA027 on the B synteny group (unpublished results), significant genetic linkages were detected, proving the validity of the present assignation. Similarly, the synteny assignation of INRA046 to U19 was confirmed later on by fluorescent in situ hybridization on Chr 15 of the cosmid from which INRA046 was derived (Vaiman et al. 1993). The randomness of the microsatellite distribution on the different synteny groups was checked. On the basis of cytogenetic measurements, we assumed that the number of microsatellites on a given chromosome would be proportional to the physical chromosome size, as was already done in mouse (Dietrich et al. 1992) and in human (Hudson et al. 1992). Bovine chromosomes are very similar in size and morphology, the difference between two consecutive pairs of autosomes being very small. Relative lengths of bovine autosomes were taken from Cribiu and Popescu (1974). An expected number of microsatellites could be inferred from these data and compared with the actual number obtained. Small discrepancies were observed for Chrs 3, 5, and 11. These differences can probably be accounted for by the sampling error, because the total number of microsatellites per chromosome is still relatively limited. Further results with more microsatellite loci will be necessary to ascertain the randomness of their chromosomal repartition. A special mention should be made about sex chromosomes. On the X Chr only three microsatellites were found, compared with the eight microsatellites present on the similar-sized Chr 1. This result can probably be explained by the fact that the plasmid library was built from male DNA, resulting in a twofold under-representation of microsatellite sequences from the X Chr. The expected size for the Y Chr in cattle is comparable to that of Chr 22-26. For these chromosomes, the expected (or observed) number of microsatellites ranges between 1 and 2. As only one Y Chr was used in the bovine library, the anticipated number of microsatellites on this chromosome is t/2 • 0.0234 (relative estimated size of the Y Chr) • 83 = 0.97. Five dinucleotide repeats were actually found on Chr Y. This difference is probably explained by the richness of this chromosome in repetitive sequences that contain a high number of (TG) n repeats (Kashi et al. 1990; Perret et al. 1990).
Conclusion In this work, 81 polymorphic bovine microsatellites were characterized, and 97 were assigned to international syntenic groups. With greater international effort, production of a bovine primary map of polymorphic microsatellite markers spaced by distances lower than 20 cM will be
D. Vaiman et al.: Characterization of 99 cattle microsatellites
achieved in only a few years. Such a map is an essential prerequisite for more efficient marker-assisted selection, molecular-based genetic introgression, and ultimately molecular characterization of genes of economic interest.
Acknowledgments. R. Ciampolini was supported by a fellowship of the
Italian Ministry of Research. This work was supported by the French GREG (Groupment de Recherches et d'Etudes sur les G6nomes). Excellent technical work by M. Nocart is greatly acknowledged. We thank Dr. P.J. L'Huillier and Dr. M. Vaiman for critical reading of the manuscript and for fruitful suggestions, and Dr. Ruedi Fries for attributing locus symbols to the new microsatellite loci.
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