Mammalian Genome 5,288-297 (1994).

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�9 Springer-Verlag New 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

~Laboratoire de 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, University of Helsinki, Finland

Received: 1 November 1993 / Accepted: 14 January 1994

Abstract. Cattle microsatellite clones (136) were isolat- ed from cosmid (10) and plasmid (126) libraries and se- quenced. 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 de- scribed for other mammals, such as rat, mouse, pig, or hu- man. A major difference resides in the number of se- quences 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 se- quences (75%). Synteny or chromosome assignment was searched for each locus with PCR amplification on a pan- el 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 microsatel- l i tes--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 hy- brids. Microsatellite polymorphism was checked on at least 30 unrelated animals of different breeds. Almost all the au- tosomal and X Chr microsatellites displayed polymor- phism, 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 mark- ers 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 mu- tation rate of microsatellites was recently estimated at 4.7 10 . 4 in mice, which positions them between coding se- quences and other categories of repetitive DNA (Dallas 1992). This relatively low mutation rate enables the seg- regation of microsatellite alleles in pedigrees to be followed unambiguously. Technically, microsatellites are relatively easy to isolate from partial plasmid libraries, and the PCR- based typing of the alleles can be readily automated. As a result of these qualities, microsatellites are becoming in- creasingly important markers for genetic mapping (Todd 1992). In human (Weissenbach et al. 1992) and mouse (Di- etrich 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 al- ready been publ i shed composed exc lus ive ly of mi- crosatellite 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 mam- malian species such as bovids, pig, dog, or cat. A saturat- ed map of polymorphic markers is the first requirement for detection of linkages with economically valuable traits in domestic animal families (Womack 1992). In cattle, con- siderable success has already been obtained with the char- acterization 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 (horn- less) gene (Georges et al. 1993b). A first obvious prere- quisite 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 289

of microsatellite sequences from bovine genomic DNA ]mostly of the (TG) n type].

Materials and methods

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 poly- morphism.

Somatic cell cultures

Thirty-six previously described (Gudrin et al. 1994; Heuertz and Hors- Cayla 1981; Hors-Cayla and Heuertz 1978; Vaiman et al. 1994) ham- ster-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 anoth- er and with the reference clusters. Correlation coefficients were estimat- ed, and synteny assignation was determined according to the rules of Chevalet and Corpet (1986).

Leukocyte DNA extraction

DNA was extracted from l0 ml of peripheral blood according to the fol- lowing 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 (Jean- pierre 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 tau- rus 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 in- to 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 pro- tocols (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 se- quencer.

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 in- dividual cosmid into the BamHI site of pGEM4Z. After transformation, bacteria were plated and screened as previously described Ibr plasmid li- braries.

Sequence analysis

Screening of GenBank-EMBL database was carried out with the GCG software package (Devereux et al. 1984).

PCR amplification of hybrid cell DNA

Synteny mapping was carried out by PCR analysis of hamster/bovine so- matic hybrid cell line DNA. PCR was carried out in a Cetus 9600 ther- mocycler with bOO ng of DNA and a Promega PCR kit, in 10 gl of reac- tion 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.

Polymorphism evaluation

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.

Nomenclature

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).

Results

Characteristics of the isolated sequences

The screening for microsatellite sequences was performed with two oligonucleotides, (TG)I 0 and (TC)10, which pos- sess 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 mi- crosatellites were often composed of compound repeats containing a stretch of TG dinucleotides.

The microsatellites could be categorized in three class- es according to their structure (Weber 1990): perfect (un- interrupted run of dinucleotides), imperfect (one or more interrupted runs of the same dinucleotide), and compound (successive runs of different dinucleotide repeats). Per- fect repeats constituted 68% of the sequences. The aver- age 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 repet- itive sequences were the M26330 SINE found 12 times (30%), a bovine lysozyme gene (M95097) also in 12 cas- es (30%), regions of caseins and globins genes, 21 hy- droxylase genes, and the Bov tA element (X64124) in 7 oc- currences (17.5%) (Lenstra et al. 1993). Alignments be- tween the M26330 SINE and microsatellite sequences showed that only one f lanking sequence of the mi- crosatellite could be aligned with the SINE, with more than 80% similarity.

290 D. Vaiman et al.: Characterization of 99 cattle microsatellites

4.=

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 se- quencing. 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 screen- ings experiments. Of the 131 sequences left, 14 were elim- inated 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 am- plification.

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 simul- taneous amplification of most of the loci, with standardized PCR conditions (that is, 58~ for the annealing tempera- ture; 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 se- quences 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 mi- crosatellites to bovine synteny groups (Table 2). Two groups of microsatel l i tes were called A (seven mi- crosatellites) and B (three microsatellites) because no cor- relation could be found with known synteny groups, even

~ o ~V

28 more

than 30

t

nd

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 charac- teristic 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 pat- terns. For another microsatellite, INRA085, polymorphism was easily detectable with radioactive labeling, but no am- plification was visible from the somatic cell hybrid DNA.

Polymorphism

Polymorphism was evaluated on a panel of unrelated ani- mals from different breeds (see Materials and methods). In Fig. 2, the number of alleles is plotted against the PIC val- ue for the different microsatellites studied in a semi-loga- rithmic 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 be- tween 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 sit- uated on the Y chromosome, and two autosomal mi- crosatellites 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 lo- calized on the bovine gene map (Fries et al. 1993). In this paper, 97 microsatellite loci, of which 84 are new, have

D. V a i m a n et al.: Character iza t ion of 99 cattle microsatel l i tes

Table 1. PCR primers.

Locus Lab Accession Number Average name name number Primers of alleles* size PIC Refernece

D3S8 INRA003 X63794 CT GGAGGT GT GT G A G C C C C A T T T A 10 200 0.77 CT AAGAGT CGAAGGT GT GAC T AGG

DU27S4 INRA005 X63793 CAAT CT G C A T G A A G T AT AAAT AT 3 150 0.51 C T T C A G G C A T A C C C T A C A C C

D3S9 INRO006 X63795 A G G A A T A T C T G T A T C A A C C T CAGTC 7 105 0.54 CT GAGCT GGGGT GGGAGCT A T AAAT A

DYS3 INRA008 X73126 GAGCCT GTGT GT GT ATACAC nd 140 G G C A C T T T C C T C T C C T G T C G C G

D1S6 INRA0I 1 X63792 C G A G T T T C T T T C C T C G T G G T GGC 8 200 0.41 G C T C G G G C A C A T C T T C C T T A G C A A C

DI6SIO INRA013 X63796 GCACAGT GACCT CT CAAT AA AT GC i i 193 0.71 CCACT A T T C T T G C C T G A A G A A T C C

B INRA016 X67828 A C G C A G A C C T T AGC AT AGGA GA 9 143 0.73 GT CGCAAT GAGT T GGACACA AC

DIOSll INRA018 X67826 T C T A A T A G T A T C A T T A A G G T G 7 170 0.29 T T T A T G G T T T A A A A T T A C C C G

D3SIO INRA023 X67830 GAGT AGAGCT ACAAGAT AAA CT T C I2 220 0.77 Y AACT ACAGGGT GTT AGAT G AACT CA

D17S6 INRA025 X67824 GGT TT CCT GT AT GAACCCAG GAG 12 220 0.77 GC T AC GGT C C AT AGGGT T GC AAA

DUl2S4 INRA026 X67832 AGGAAAGAGT AGGAAGAACT AGTC 7 200 0.67 AACT A A A G A G C C C T G T G C T G T A A C

B INRA027 X67829 C T C C C C A C T T A G G A A C T C T G T A T C 8 160 0.71 C A C T G C A T C C C T C C C C A C T AAC

D1853 INRA028 X67826 CAGGGT T CAGT CAGAAAAAG AAG 3 255 0.16 C ACT GAT AAT T GT GGGT GGT T C A

DXS8 INRA030 X67822 A T G C A A A T G T G C T ACATCAC CTAT 3 160 0.22 T G G C C C A A C T C T C A C A T C C A G A T C

D21S12 INRA031 X73132 ACAT AAAGT AGGAT AAAT AC T A 10 200 0.71 G C A C A A T T AAAGT ACAT AT C AA

DI/S9 INRA032 X67823 A A A C T G T A T T C T C T AATAGC AC 6 190 0.54 G C A A G A C A T A T C T C C A T T CC T T T

Dl6Sl l INRA035 X68049 A T C C T T T G C A G C C T C C A C A T TG 7 120 0.38 T T G T G C T T T A T G A C A C T A T C C G

D20S3 1NRA036 X71554 CAGAAAGAAAT AGAAT GGAC AG 4 170 0.47 A A A A G A T G T G A G C T G G T T C T TG

DIOSI2 [NRA037 X7151 G A T C C T G C T T A T A T T T A A C C AC 11 110 0.77 AAAAT T CC AT GGAGAGAGAA AC

D18S4 [NRA038 X7155 G G G A C T C C A C A G T C A T G G T G T C 3 115 0.25 C T T C T C T A G C C C C A A A C T GC CC

D20S4 INRA039 X71556 G C T C T C T G G C T C A A T A G C T G G 1 150 0 GC CT GGAGAAT C C CC AT GGC A

D2S15 INRA040 X71558 T CAGT CT GGAGGAGAGAAAA C 44 205 0.87 C T C T G C C C T G G G G A T G A T T G b

$3S11 INRA041 X71559 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 4 170 0.42 AACAT TT T AT GT AGT TT AAT TT GAAAC

DI1SIO INRA044 X7158 GAGTT AGAC AT GACT GAGC A AC 8 90 0.49 GAAGT GGT AGC AGGT CAGCC T c

D15S5 INRA046 X71495 C A G C T C A T G T G T T T A C A T G G C 3 115 0.28 A G T C T C T G G A A C T T C T C C T A C

D16S12 INRA048 X71591 C T G T C C C T C A G T A A A C A A G T CG 8 279 0.77 AAGCT AAAGT AGC AGGGAAG G

D1S15 INRA049 X7158 T G T A T T A G T T T G T G T T C T T T GGC 8 i60 0.62 T T G G C T T C C A C A A T C A C A C A

D15S6 1NRA050 X71494 ACAGGCT ACAGT CCAT G G G G T T 7 140 0.66 T AT AGAAC AGAAAAAT GAC T AC AC G

DU27S5 INRA051 X71587 C AT C A A A T T T AGT GAAAAGG G 6 230 0.47 CCT AAAAGAACACAT G C C T G T T G

D13S7 INRA052 X71496 GAT C A A A T T T AGT GAAAAGGG 6 130 0.65 CCT AAAAGAACACAT GCCT G T T G

D7S6 INRA053 X71497 A A A G T C A G A T A C A A C T G A G T GAC 5 100 0.69 A A T C A C C A G A A A T T C A C T T C ACC

D1S16 INRA054 X71498 GAAT C AGACAT GACT T AGCA AC 2 180 0 T T C A T C A G G A G G T A C A T GTT GT

DI1Sl l INRA055 X71999 CAT A T G A G C C A C C A G T G A A G 1 130 0 T CT GT GACT AGGAAGAAC C A G

DYS4 INRA057 X71501 C C T A G C G A C T G T C C A A G C G nd 125 C A C G G G C T G A G A A T T C A A A C

A INRA059 X71503 G G A T G C T T C T AGACTGACAC C 2 105 0 GGGAT GAAAT TT A T G C T CT G T G a

D21S13 INRA060 X71504 T T C C A T A T A G C A T T T A G A A T AGC 3 80 0.41 AGACACT CCCAAAT AAAAT C CC

D28S6 INRA061 X71505 C C A T G T A C A G A G G A C C C T G 6 170 0.67 A C A T G C A T G T G C T T G T G T CG

DYS5 INRA062 X71506 T G T G C A G C A C C T T G T C T C C nd 150 A C A T G C A T G T G C T T G T G T C G

D18S5 INRA063 X71507 ATT T GCACAAGCT AAAT CT AACC 7 180 0.46 AAACCACAGAAAT G C T T GGA AG

D23S15 INRA064 X71508 G C C C A C A G C G C T C T C T A C 5 175 0.57 C T GAAAGC AGAAT GAGGT GC

291

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Goudarzi et ah 1993

Vaiman et al, 1994

Vaiman et al. 1994

This paper

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Ciampolini et al. 1993

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Vaiman et al. 1994

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Vaiman et al. 1993

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292

Table 1. Continued.

D. Vaiman et al.: Characterizat ion of 99 cattle microsatell i tes

Locus Lab Accession Number Average name name number Primers of alleles* size PIC Refernece

DIOS13 INRA069 X71513 AGAGCCCCATAATAGGCAAC C 4 130 0.56 This paper C AT T T AC AGAGC CT AGT GAT AGG

D10S14 INRA071 X71514 G C C T A G C A T C C A C A A T A C C A C 6 220 0.68 This paper G G C A G G A C C T G A A G T G T GGT C a

D4Sll INRA072 X71515 C T T A A C T C A T T C A C C T C A A C TG 11 140 0.57 This paper AGT GATT GAGCACAT T GCGC AT

D1S,!8 INRA073 X71516 ACTGAGGAACTAAGCACCGC 5 160 0.63 This paper GAAAAGCAAGGCTGTCCGAC d

D14S6 INRA079 X71522 G G G T T T T A G G A G C T C T G T A C CAG 4 147 0.53 This paper G T G T T T C T C A A C C G T G C T G

A INRA080 X71523 G A C A G A G G A G C C T G G T T G G C 4 I74 0.49 This paper G T T T C T T A G T C G G G T A T A A T GG

D26S5 INRA081 X71524 C G G C T C A C G G T C T C T A T C G G 6 170 0.70 This paper GCGAACCCAAGAATCAGACT C

D4S12 INRA082 X71525 CT AGAGGAT CAGCAACT T C A A T C 3 124 0.37 This paper C T C A C T G A T T T G A C T T G A C T CC

DU2S4 INRA084 X71527 C T A A A G C T T T C C T C C A T C T C 8 t l0 0,70 This paper C C T G G T G A T G T T T G G A T G T C

? INRA085 X71528 C C C A T T T C C A G T C C T C T C A T CC 3 211 0.19 This paper AT ACAAT GT ACCACAT GACA GG a

D3S12 INRA088 X71566 C A T T C C T T T G A T G T A G G A G 8 132 0.68 This paper CT GT CACAAAGAGATACAGC

D6S5 INRAK X14908 A T G C A C C T T A A C C T A A T C C 6 173 0.60 This paper A C A A T A C A C A A G C A T A C T AC

? INRA090 X71563 G G T C A T T T T C CATT AT GACAGCAG 5 168 0.64 This paper G G T G T T A C C T T T T T T A G T C T C C

D15S8 INRA091 X71565 AACTCAGCAGGCGG 7 129 0.54 This paper A G A G T T G G A C A C A G C T GAGG

DI4SIO INRA092 X71573 G G T T T G A C T T C A A C T T C A A C C 8 178 0,74 This paper GGGAGTCT GTGGAGAAT GCA AG

D3S13 INRA093 X71572 C T A C A A C A A G G G A A T T T G T G 2 122 0.05 This paper CAGAGT C G A A C A T G A C T A A G T G

D14S7 INRA094 X71571 CT GT ACCAGAAACAGACAGA G 4 157 0.60 This paper T GT C AGGC T AC AGT C C AT GG G

DIOS17 INRA096 X71570 G C T G A T T C A C T T C G C T A T A C AG 5 125 0.62 This paper C AGT AC AT GAGGT T GC AAAG AG d

A INRA097 X71561 G C T A C A G T C T G T A A G G T T C C 6 183 0.61 This paper C C G T A A G A T A T C G T G G A A T C

D14S8 INRAI00 X71562 A C A G G C T C T T T T A T G A C T T G 8 179 0.69 This paper A A G T G G G T G T G A G C C A A G C C

DIOS15 INRA101 X71574 C T A A G C A A C C A A C A C T T T A G G 3 100 0.22 This paper A T A C A C A T G T G C A A G C A T G T G

D21S14 INRA103 X71531 T T G T C C A G C C C A G C A T T T A G C 5 135 0.66 This paper G G A G A A G A C T T A T G G G A G C C

D5S9 INRAI04 X71532 T GT CAGACATGACT GAGTGAC 3 99 0.38 This paper A A A A G A G A T T G A A A G C G C C T G

D13S8 INRAI05 X71579 GGTT ACAAGAGT C GGACAT GAC 3 120 0,22 This paper G G G A A C T A T A C T T A G T A T C T T G T A

D]OS16 INRA107 X71577 T CCCAGAT ACAGATGCAAC AG 4 166 0.33 This paper GGAGAGCCGAGGGCTTCAGC

Dl lS t2 INRAI08 X71581 AGT CT GT GGGGTT CCAGAGT T G 5 154 0.39 This paper T T A C A G T T A A C T C A C C C T C C

D17S9 INRA110 X71580 T ACAGT CCGT GGGGT CACAG AG 4 213 0.34 This paper AACAGAAATT CCT T CAT GAG C

DllS13 INRA111 X71590 T T GT CGGT GT GGAGAAGCAC C 7 137 0.47 This paper G T T T C C C G G T A A C C A A T T C C

D7S7 INRAll2 X71589 G C C T C T C A A A G C C A C C T G C 6 171 0.77 This paper GAT CT AACT AGAGCT T T CC

DIISI6 INRA115 X71538 GACT AACACAAAAT GACT C G T G 5 167 0.49 This paper G G A G G G G G A C T A C T T C T C A T GC e

D1S20 INRA117 X71540 G T T T C T A G T A A C A T A T T G A C 4 97 0.13 This paper T T AGAC AT GACT GAAGCAAC

D3S14 INRA118 X71543 G T C T T T G G C A A G C C A G C T C T CC 2 217 0.03 This paper AGAGT C A G G A T T T GAATCC A GG

D1S]9 INRAlI9 Z71542 GGAACTAAGCACCGCACAGC 5 137 0.67 This paper T GT C AAAC AGAGC AGT GAGA GC

DXS9 INRA120 X71544 TGCAGCAACGAGGACCCACG 4 171 0,60 This paper A A C C G C A T T T G C T C C T C T G

D18S6 INRA121 X71545 G G A A A C C C A T T G G A G G A T T T G 12 i28 0.79 This paper C T T C A C T A T T C C C C A C A A A G C f

A INRA122 XY1547 G T A A A T A A A A T T A T A T A C C T ACC 5 159 0.70 This paper A A A G G G T C A G T T A A T T T T AC AC

D3S15 INRA123 X71533 T C T A G A G G A T C C C C G C T G A C 5 106 0.5l This paper A G A G A G C A A C T C C A C T G T G C

DYS6 INRA124 X71546 G A T C T T T G C A A C T G G T T T G nd 132 This paper CAGGACACAGGT CT GACAAY G

DYS7 INRA126 X71533 T C T A G A G G A T C A A G G A T T T G T G nd 250 This paper A A T C C A T G G A A A G A T G C A C T G

DU2S5 INRA127 X71550 CT ACAGCTCT GATGAGAACC 2 179 0.19 This paper C G T T T T C T C A A A C T T C A T T G

D. Vaiman et al.: Characterization o f 99 cattle microsatel l i tes 293

Table 1. Continued.

Locus Lab Accession Number Average name name number Primers of alleles* size PIC Reference

DIS17 INRA128 X71534 T AAGCACCGCACAGCAGAT G C 5 182 0.62 This paper A G A C T A G T C A G G C T T C C T A C

A INRA129 X71535 GGGT AGCCT GT T AAAAT GCA G 4 157 0.49 CAGT GCT GACCT CT GAAGT A AG

D17SIO INRA130 X71536 G A T C C C C G C T G A C C A C T C A G 2 100 0 AAGAGAGCAACT CCACT GT G C

DIISI4 1NRAI31 X71548 GGT AAAAT CCT GCAAAACAC AG 6 125 0.65 T G A C T G T A T A G A C T GAAGCA AC

D23S16 INRA132 X71552 AACAT T T CAGCT GAT GGT GGC 5 163 0.50 T T C T G T T T T G A G T G G T A A G C TG

B INRA134 X73125 G C C C A T C G G T C C C A G G T G G 10 163 0.57 T T C C A T C T A T C T T G G G A G C

D17S7 INRA135 X73128 G G A G A C T T C A C T G G A A A G G 8 174 0.76 A A T C C C A T G G A C A G A G G A G G

DU2S INRA136 X73129 T G G C C T A A A T A T T T A T C A G T G 5 128 0.67 G A G A C C T T T C C T C T T T C T C A G C

D l l S I 7 INRA177 X74201 CAGCAGT AGTCACCT AAAAC C 9 195 0.70 AAGGAAACT CCAAAACACCA GG

DIS2t HEL6 X65206 G G A C A C G A C T G A G C A A G T A A 10 257 0.80 AGGC AGAT AC AT T AC C ACT A

DIlS15 HELl3 X65207 T A A G G A C T T G A G A T A A G G A G 4 198 0.61 C C A T C T A C C T C C A T C T T A A C

A HEL4 X65203 AGT T GGAC AT GAC T GAGT GC nd 140 nd G T A A C T T A G T G G G T G C A G C T

A HEL9 X65214 C C C A T T C A G T C T T C A G A G G T 11 169 0.73 C A C A T C C A T G T T C T C A C C A C

D26S6 HEL 11 X65216 C T T T GT GGAAGGC T AAGAT G 7 191 0.69 T C C C A C A T G A T C T A T G G T G C

D20S5 HELl2 X65211 SCAT T AGGTT CT CCAGAGAA 5 156 0.66 CAGACTT G T C A G A C T C C A T A d

D21S15 HEL5 X65204 GCAGGAT CACTT G T T A G G G A 6 161 0.70 A G A C G T T A G T GT ACATTAAC

D23S17 HELl5 X65208 AGAGAAGT CT G G T G G G C T A T 5 126 0.38 T C AGTT GT CT AGATT AAGCA

DXSIO HELl4 X65209 CC AACC AGGGTT T GAACT GA nd 216 nd T G G T T A T G T G T T A T G T G G C A

D13S9 HEL7 X65210 TT AT GACT GAGT AGT AT TT C nd 101 nd T AGAT AT GACT GAGAGACT A

This paper

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Kaakinen and Varvio 1993

Kaukinen and Varvio 1993

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Kaukinen and Varvio i993

Kaukinen and Varvio 1993

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Kaukinen and Varvio 1993

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*Tm fixed at 58~ excepted when noted: (a) 55~ (b) 65~ (c)60~ (e) 52~ (f) 50~

been assigned to bovine synteny groups. Although a much larger number of bovine microsatellites have been isolat- ed 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 o f 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 mam- malian species, such as rat or human, where the ratio is on- ly 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 (unpub- lished data). In the dog, we could observe a ratio of 3:1 (un- published data).

Differences in the average size of microsatellites be- tween 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 repar- tition of the different microsatellite types in cattle was similar for perfect and imperfect repeats, whereas the av- erage 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 DNA 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 rep- resentative of the whole genomic DNA. Assuming that these two conditions are fulfilled, we were able to calcu- late that a total of 15 Mbp of bovine genomic DNA (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 mi- crosatellite 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 cal- culations 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 16 INRA013(0.8) INRA035(0.87) INRA048(0.93) U2 9 INRA084(0.87) INRA127(0.93) INRA136(0.79) U3 5 INRAI04(0.8) U4 2I [NRA031(0.93) INRAO60(I) INRAI03(0.86) HEL5(0.87) U5 10 INRA018(0.7) INRA037(0.79) INRA069(0.85) INRA071(0.85) INRA101(0.77) INRA107(1) U6 3 INRA003(1) INRA006(1) INRA023(0.91) INRA04I (0.8) INRA088(0.9) INRA093(0.9) U7 U8 U9 18 INRA028(0.86) INRA038(0.86) INRA063(0.82) INRA121(0.86) UI0 1 INRA011(0.94) INRA049(0.83) INRA054(0.89) INRA073(0.78) INRAI17(0.78) INRAl19(0.73) UII 13 1NRA052(0.86) INRAI05(0.84) HELT(0.74) U12 INRA026(0.85) U13 4 INRA072(I) INRA082(0.86) U14 nd U15 6 INRAK(I) U16 1I INRA032(1) INRA044(0.88) INRA055(0.86) INRA108(0.71) INRAlll(0.86) INRA115(0.71) U17 2 INRA040(0.85) INRA135(1) U18 nd UI9 15 INRA046(0.75) INRA050(0,74) INRA091(0.74) U20 23 INRA064(0.89) INRA132(0.83) HELl5(0.89) U21 19 U22 7 INRA053(0.88) INRA112(0.81) U23 17 INRA025(0.88) INRA110(0.8) INRA130(0.7) U24 14 INRA079(0.77) INRA092(0.82) INRA094(0.82) [NRAI00(0.94) U25 nd U26 26 INRA08�91 (0.74) HELl 1(0.66) U27 INRA005(0.89) INRA051 (0.83) U28 24 nd U29 28 INRA06[(0.94) X INRA030(0 .93) INRA120(0.93) HELl4(0.94) Y INRA008(I) INRA057(0.79) INRA062(1) INRAI24(I) [NRA126(1)

20 INRA036(0.94) INRA039(0.88) HELl2(0.94) A lNRA059(0.93) INRA080(1) INRA097(0.8) INRA 122(1) INRA129(1) HEL4(I) B INRA016(1) INRA027(0.7) INRA134(0.88) Isolated I N R A 0 8 5 INRA090

INRA096(0.77) INRAI I8(0.9) INRA123(0.79 a )

INRA128(0.95) HEL6(0.78)

INRA131(0.93) INRA177(0.93) HELl 3(1)

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 (E1- legren 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 propor- tion 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 sug- gests 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 mi- crosatellites in small-size restriction fragments. On the

whole, our results suggest that bovine genomic DNA pos- sesses less (TG) n sequences than other mammal i an 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 mi- crosatellites 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 ele- ments have been proposed as an aid for cloning more ef- ficiently new microsatellite sequences (Kaukinen and Varvio 1992), as well as for direct study of their poly- morphism (Miller and Archibald 1993). The fact that mi- crosatellites similar to the M26330 SINE were found in dif- ferent 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 oth- er species.

D. Vaiman et al.: Characterization of 99 cattle microsatellites 295

100

.Q E Z

10

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__ : �9

�9 ~-=

�9 : : : : m

~. ~.-- �9 | _-

�9 �9 m ~ H :

�9 �9 ~ ~ ~ �9

,~ ::: �9

0,00 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.

Mean number Number Average size of of aIleles SD of clones the longest repeat

Total 6.40 5.38 82 14.51 Perfect 5.84 3.05 54 14.67 Imperfect 8.31 9.71 19 14.00 Compound 5.57 1.72 9 14,44

Synteny mapping

Most of the microsatellites used in this study could be as- signed to synteny groups or chromosomes provided that several factors were taken into consideration. First, two sig- nificant correlations were found between U08 and U24 (0.72) and between U16 and U27 (0.78). The first corre- lation, 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 HELl3 (1/0.78). In these cases, the highest correlation coefficient was re- tained 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 be- tween 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 de- fined by only one enzymatic locus on 26 hybrid cell lines

instead of 36 for the other synteny groups. This may ex- plain 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 chromo- somes 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 en- compassing 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 lo- cus. 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 mi- crosatellite loci. These loci (INRA008, INRA057, IN- RA062, INRA124, and INRA 126) could be amplified on- ly from male bovine DNA. Furthermore, the five mi- crosatellites 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 D. Vaiman et al.: Characterization of 99 cattle microsatellites

group and B group. It could probably account for one of the unidentified or ill-defined synteny groups of the pan- el (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 va- lidity 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 cy- togenetic measurements, we assumed that the number of microsatellites on a given chromosome would be propor- tional to the physical chromosome size, as was already done in mouse (Dietrich et al. 1992) and in human (Hud- son et al. 1992). Bovine chromosomes are very similar in size and morphology, the difference between two consec- utive 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 in- ferred from these data and compared with the actual num- ber obtained. Small discrepancies were observed for Chrs 3, 5, and 11. These differences can probably be account- ed 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 nec- essary to ascertain the randomness of their chromosomal repartition.

A special mention should be made about sex chromo- somes. 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 mi- crosatellite 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) num- ber of microsatellites ranges between 1 and 2. As only one Y Chr was used in the bovine library, the anticipated num- ber 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 syn- tenic groups. With greater international effort, production of a bovine primary map of polymorphic microsatellite markers spaced by distances lower than 20 cM will be

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 mol- ecular 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). Excel- lent 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 sym- bols to the new microsatellite loci.

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