Suitability of microsatellites BM 1329 and OarAElOl as markers for the introgression of the Fec B locus into different sheep breeds

Litter size in sheep can be improved by introgression of the Fec gene. Genetic markers closely linked to the Fec locus can be used to detect gene carriers. The efficiency of introgression can be increased by using marker assisted selection. Individuals ofthe prolific breeds Tyrolean Mountain sheep (n=41), Romanov (n=43), East Friesian Milksheep (n=35) and Merinoland sheep (n=265) were genotyped with the microsatellite markers OarAElOl and BMI 329 linked to the Fec gene to estimate allele frequencies of both microsatellites in these breeds. The frequencies ofthe microsatellite alleles linked to the Fec gene in Merinoland sheep were 0.07 and 0.1 respectively. The alleles occured also in the other breeds (Tyrolean Mountain sheep, Romanov and East Friesian Milksheep) but with mostly higher frequencies. Furthermore the segregation ofthe microsatellite alleles was studied in families produced by mating Merinoland sheep ewes (Fec+/Fec+) with two heterozygous Booroola Merino rams (Fec+/Fec) in order to use these markers within an indirect gene test. The recombination rate between BM 1329 and OarAElOl was 13 cM (LOD score 14.6).


Introduction
Litter size in sheep has a low heritability coefficient and a large variability between and within breeds (ELSEN et al., 1994).Attempts to increase litter size by selection within breeds have been hindered by a slow selective gain.For identifying major genes with effects on Ovulation rate and litter size, a single autosomal segregation was discovered in the Booroola strain (DAVIS et al., 1982).The Booroola gene (Fec B gene) is known for its large impact on prolificacy.One single copy ofthe gene resulted in +1.3 extra lambs in 2-year-old ewes (NIEUWHOF et al., 1998).Introgression of the Fee mutation in other breeds requires extensive progeny testing of the rams or measuring the Ovulation rate of the ewes by laparoscopy over three to five cycles.The use of genetic markers linked to the Fec B locus would allow an early identification of Fec B carriers and is independent from sex.Further on the use of marker assisted selection costs less time and money than the use of laparoscopy.Linkage to Fee was first demonstrated with two microsatellite markers (OarAElOl and OarHH55) and epidermal growth factor (EGF) from human chromosome 4 (MONTGOMERY et al., 1993).Comparison ofthe human gene map with the gene maps of cattle and sheep, Fec D could be mapped to sheep chromosome 6 (MONTGOMERY et al., 1994).BLATTMANN et al. (1996) assumed linkage between the microsatellite BMI329 and the Fec B gene.That was confirmed by linkage mapping of this microsatellite between OarAElOl and EGF on sheep chromosome 6 (LORD et al.,1996).Actually, Fec B is localized in a region of 10 cM between the two microsatellite markers BM1329 and OarAElOl (LORD et al., 1998).The aim of this study was to determine the allele frequencies of microsatellites BM1329 and OarAElOl in four sheep breeds in order to test the suitability of these two microsatellites as markers for the introgression of the Fec B locus into the Merinoland sheep.Furtheron the segregation of microsatellites alleles BM1329 and OarAElOl should be demonstrated in families of German Merinoland sheep in order to improve litter size and the recombination rate between BM1329 and OarAElOl was calculated.

Material and Methods
For the estimation of allele frequencies of microsatellites BM1329 and OarAElOl samples were collected from Merinoland sheep (n=265), Romanov (n=43), Tyrolean Mountain sheep (n=41) and East Friesian Milksheep (n=35).The Booroola Merino breeding program at the Research Station 'Oberer Hardthof of the University in Giessen has been started in 1989.Heterozygous F, Booroola Merino rams were produced by inseminating eight Merinoland ewes with semen from a homozygous Fee carrier ram.The F 2 generation was obtained by mating three heterozygous Fec B carrier rams with 107 Merinoland ewes.The Ovulation rates ofthe F 2 daughters were recorded by repeated laparoscopy.In the last two years two further heterozygous Fec B carrier rams were mated with 125 Merinoland ewes for segregation analysis.35 heterozygous Fec B carrying daughters ofthe produced F, generation were mated with non-Fec B carriers.For the twopoint linkage analysis six halfsib families of crossbreds between Merinoland sheep breed and Texel (n = 390) were used.DNA was isolated from white blood cells aecording to MONTGOMERY & SISE (1990).The PCR (15pl final volume) for amplifying the microsatellites BM1329 and OarAElOl was performed using 50 to 100 ng ovine genomic DNA and a Perkin Eimer Gene Amp PCR system 9600 cycler.The forward primer of each microsatellite was Cy5 fluorescense end-labelled (Amersham Pharmacia, Freiburg).The microsatellite OarAElOl was amplified aecording to the conditions described by MONTGOMERY et al. (1993).The PCR for amplifying the microsatellite BM1329 was performed using 10 pM of each primer, 200 uM of each dNTP, 0.4 units of Taq polymerase (Hybaid-AGS, Heidelberg), 1.5 mM MgCl 2 , and PCR buffer (20 mMTris-HCl, pH 8.55; 1.6 mMNH 4 S0 4 ).Thermal cycling began with an initial cycle of 94°C (1.5 min), followed by 30 cycles of 94°C (1 min), 58°C (1 min), and 72°C (1 min), and concluded with a final extension at 72°C (5 min).Microsatellite typing was done using an automated laser detection system (A.L.F.express, Amersham Pharmacia, Freiburg).The PCR products were run on a 5.5% Long Ranger gel (0,5 mm, 6M UREA).The fragment analysis were done using AlleleLinks vi.00 (Amersham Pharmacia, Freiburg).Twopoint linkage analysis between BMI329 and OarAElOl were done using CRI-MAP v.2.4 (GREEN et al., 1990).Allele frequencies were estimated by counting the alleles of unrelated individuals of each breed.

Results
Between the four breeds great differences in the oecurence and frequency of the different alleles of microsatellites BM1329 and OarAElOl were observed.Six alleles were identified with microsatellite BM1329, but only three of them were found in all breeds (Table 1).In the microsatellite BMI329 allele 162 and in OarAElOl allele 97 was linked to the Fec B allele which was shown in the Booroola/Merinoland families.The marker analyses were validated by the Ovulation rate data of the F 2 daughters.Both microsatellites alleles linked to the Fec B gene were rare in the Merinoland sheep population.The twopoint linkage analyses in six half sib families showed a genetic distance (sex averaged) of 13 cM with a LOD score of 14.6.The number of informative meioses were 233 for OarAElOl and 203 for BM1329 respectively.

Discussion
The aim of this study was to establish a genotyping system to identify Fec B carriers in sheep using the microsatellite markers BM1329 and OarAElOl.Both markers are mapped on ovine chromosome 6 flanking the Fec B locus (http://www.ri.bbsrc.ac.uk).In former investigations it was difficult to identify heterozygous Fec B gene carriers clearly: for example VISSCHER et al. (1998) compared results from laparoscopy and genotyping and found a descrepancy of 4 %.In the studies of LORD et al. (1998) the control of laparoscopy results with three Fec B flanking markers showed, that 6-13 % of the heterozygous Fee gene carriers were not identified by laparoscopy.As the alleles 162 and 97 ofthe two markers BM 1329 and OarAElOl occur also in non Fec B carriers, genotyping of the informative ancestors was also done in the present study.So the origin ofthe alleles linked to the Fec B gene could be followed up to the Fee /Fcc + -father or -grandfather of the ewes.Therefore only 1.9 % of the animals could not be genotyped clearly conceming their Fec B Status.The genetic distance (sex averaged) between BM1329 and OarAElOl calculated in this study was 13 cM.LORD et al. (1998) described the region between BMI 329 and OarAElOl with a genetic distance of 10 cM in a male map.The two microsatellites BM1329 and OarAElOl represent an efficient and robust genotyping system, but the alleles of both markers linked to the Fec B allele occur also in other sheep breeds (Romanov, Tyrolean Mountain Sheep, East Friesian Milksheep) than the booroola merinos but with different frequencies.Because the conceming alleles are rare in the Merinoland sheep population, this system can be used for identifying the introgression of the Fee gene when the parents and if need the grandparents are genotyped too.Until a direct gene test is available at the Fec B gene locus the two microsatellites BM1329 and OarAElOl can be used for an indirect genetest to identify Fec B gene carriers.
The Romanov and East Friesian Milksheep showed a high oecurence ofthe allele 164, whereas in the breeds Merinoland sheep and Tyrolean Mountain sheep the allele 166 showed the highest frequency.Allele 180 occured in the Merinoland sheep breed only at a low frequency.With microsatellite marker OarAElOl 10 alleles were obtained (Table2).The two alleles 97 and 109 were present in all four breeds whereas the other 8 alleles showed breed specific alleles, for example allele 107 occurs only in Tyrolean Mountain Sheep.Allele 97 had a very high frequency in the Romanov breed and a very low one in the Merinoland sheep breed.Allele 101 was not present in Tyrolean Mountain Sheep and had very low frequency in Romanov whereas it was very frequent in the Merinoland sheep.Allele 109 had the highest frequency in Tyrolean Mountain Sheep and lower frequencies in the other breeds.The observed distribution of microsatellites alleles within breeds follows Hardy-Weinberg expectation of equilibrium.

Table 2
Allele frequencies of the OarAElOl microsatellite in different breeds (Allelfrequenzen des Mikrosatelliten OarAElOl in verschiedenen Schafrassen)