August 2007
Volume 48, Issue 8
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Biochemistry and Molecular Biology  |   August 2007
Linkage Mapping of Ovine Microphthalmia to Chromosome 23, the Sheep Orthologue of Human Chromosome 18
Author Affiliations
  • Jens Tetens
    From the Institute of Animal Breeding and Genetics, the
  • Martin Ganter
    Clinic for Pigs and Small Ruminants, Forensic Medicine and Ambulatory Service, and the
  • Gundi Müller
    Institute of Pathology, University of Veterinary Medicine Hannover, Germany; and the
  • Cord Drögemüller
    From the Institute of Animal Breeding and Genetics, the
    Institute of Genetics, Vetsuisse Faculty, University of Berne, Switzerland.
Investigative Ophthalmology & Visual Science August 2007, Vol.48, 3506-3515. doi:10.1167/iovs.07-0041
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      Jens Tetens, Martin Ganter, Gundi Müller, Cord Drögemüller; Linkage Mapping of Ovine Microphthalmia to Chromosome 23, the Sheep Orthologue of Human Chromosome 18. Invest. Ophthalmol. Vis. Sci. 2007;48(8):3506-3515. doi: 10.1167/iovs.07-0041.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To characterize the phenotype and map the locus responsible for autosomal recessive inherited ovine microphthalmia (OMO) in sheep.

methods. Microphthalmia-affected lambs and their available relatives were collected in a field, and experimental matings were performed to obtain affected and normal lambs for detailed necropsy and histologic examinations. The matings resulted in 18 sheep families with 48 cases of microphthalmia. A comparative candidate gene approach was used to map the disease locus within the sheep genome. Initially, 27 loci responsible for the microphthalmia–anophthalmia phenotypes in humans or mice were selected to test for comparative linkage. Fifty flanking markers that were predicted from comparative genomic analysis to be closely linked to these genes were tested for linkage to the disease locus. After observation of statistical evidence for linkage, a confirmatory fine mapping strategy was applied by further genotyping of 43 microsatellites.

results. The clinical and pathologic examinations showed slightly variable expressivity of isolated bilateral microphthalmia. The anterior eye chamber was small or absent, and a white mass admixed with cystic spaces extended from the papilla to the anterior eye chamber, while no recognizable vitreous body or lens was found within the affected eyes. Significant linkage to a single candidate region was identified at sheep chromosome 23. Fine mapping and haplotype analysis assigned the candidate region to a critical interval of 12.4 cM. This ovine chromosome segment encompasses an ancestral chromosomal breakpoint corresponding to two orthologue segments of human chromosomes 18, short and long arms. For the examined animals, we excluded the complete coding region and adjacent intronic regions of ovine TGIF1 to harbor disease-causing mutations.

conclusions. This is the first genetic localization for hereditary ovine isolated microphthalmia. It seems unlikely that a mutation in the TGIF1 gene is responsible for this disorder. The studied sheep represent a valuable large animal model for similar human ocular phenotypes.

Congenital microphthalmia is characterized by an abnormally small eye in newborns that can be associated with other ocular abnormalities and represents a heterogeneous disorder occurring in various mammalian species. In humans, microphthalmia is highly variable, with anophthalmia in the most severe cases. Several genes harboring causative mutations for autosomal recessive and dominant syndromic and isolated microphthalmia have been found. 1 However, although clinical studies in these human defects revealed variable phenotypical expression, the establishment of precise genotype–phenotype correlations in the microphthalmia–anophthalmia spectrum may be difficult, and the molecular genetic basis of numerous human cases is still unknown. 1 Many inherited malformations of domestic animals are analogous to human hereditary anomalies and have been valuable animal models for the investigation of the pathogenesis of rare human phenotypes with identical molecular basis. 2 Naturally occurring large domestic animal models are particularly valuable because the eye size and structure in the animals is larger and more human-like than that of rodents, and their longer life expectancy allows for investigations or treatments over a longer time. In general, the clinical progression in larger animals more closely resembles that in humans. Domestic production animals have the additional advantages of being economic to maintain and of having been bred for easy management. Moreover, a high level of expertise in reproductive technology and veterinary care is available for them. 
In sheep, isolated hereditary bilateral microphthalmia is well known worldwide as a monogenic autosomal recessive inherited disorder with complete penetrance associated with the Texel breed. 3 4 5 6 7 8 9 Typically, animals affected by microphthalmia are entirely blind. 10 Macroscopically, the globes and optic nerves appear hypoplastic and histologically all embryonic components of the eye are present, but lens, ciliary body, iris, and retina are dysplastic. 10 An abnormal development of the lens vesicle was shown to be the primary event in hereditary ovine microphthalmia by comparative investigation of normal and affected eyes of sheep embryos at different gestational stages. 8  
The availability of sheep genome resources has expanded considerably in the past 2 years. The current ovine genetic map includes more than 1400 informative markers (see http://rubens.its.unimelb.edu.au/∼jillm/jill.htm). This powerful tool has been effectively used for linkage analyses in sheep, to identify genome regions associated with Mendelian disorders and economically important production traits. 11 Furthermore, public domain resources now include more than 375,000 genomic BAC end sequences (BES) representing one twelfth of the whole genome sequence (see http://www.ncbi.nlm.nih.gov/). Recently, an ovine radiation hybrid panel has been constructed to build integrated detailed physical maps of entire sheep chromosomes 12 and a genome-wide, sheep-specific single-nucleotide polymorphism (SNP) chip is under development (see http://www.sheepgenomics.com). In addition, the sequenced cattle genome (see http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9913) provides valuable data, because of the high interspecific sequence and genome similarity between ruminants 11 and the existence of high-resolution comparative maps between the bovine, human, and murine genomes. 13 14 Finally, these provide excellent tools for positional cloning of candidate genes or the identification of causal mutations in sheep. 15  
In this study we report the linkage mapping of recessive inherited ovine microphthalmia (OMO). Initially, several loci responsible for different microphthalmia–anophthalmia phenotypes in humans or mice were selected to test for comparative linkage in sheep with hereditary microphthalmia. Significant linkage to a single candidate region was identified at sheep chromosome (OAR) 23. The linked TGIF1 gene was excluded as a potential candidate for ovine microphthalmia. Fine mapping and haplotype analysis defined the critical interval of 12.4 cM at OAR23, the ovine orthologue of human chromosome (HSA) 18. 
Methods
Animals
The animals belonged to 17 different nonherdbook sheep farms with uncontrolled crossing of Texel or Texel/Whiteheaded mutton crossbred rams to Texel or Texel/Whiteheaded mutton crossbred ewes from a distinct region of northern Germany. For this study, we included only those litters of a single breeding ram in which affected progeny were produced. In general, each ram served at least 50 ewes within a single breeding season. Phenotypes were evaluated by visual inspection. Full blood samples were taken from the vena jugularis and collected in blood sampling tubes prepared with EDTA (Monovette; Sarstedt, Nümbrecht, Germany). 
A breeding experiment designed to augment the number of affected lambs for genetic and pathologic examination purposes was performed in 2004 and 2005 at the University of Veterinary Medicine Hannover, Germany. By mating of an affected ram to known disease carrier ewes, an additional nine affected and five normal lambs were obtained and integrated into the experimental pedigrees (Fig. 1 , families 16–18). Newborn offspring from experimental pedigrees were examined clinically and subsequently euthanatized within their second week of life. All procedures involving animals were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Lower Saxony (Germany) governmental animal rights protection authorities (Ref. No. 33-42502-04/851). 
In total, the study included 48 lambs with microphthalmia (28 male, 20 female) representing 56 relative affected pairs (9 full-sib pairs, 38 half-sib pairs, and 9 parent–offspring pairs) and 84 informative meioses for OMO. The 18 unrelated two-generation–spanning sheep families segregating for OMO consisted of 119 individuals with an average family size of 6.6 members, ranging from 4 to 18 animals per family (Fig. 1)
The entire animals were macroscopically examined, and the eyes were fixed in a solution of 4% paraformaldehyde, 2.5% glutaraldehyde, and 1% sodium cacodylate and sectioned in a median sagittal plane. Tissue samples from various other organs (brain, spinal cord, heart, lung, spleen, lymph nodes, thymus, thyroid, adrenal, pituitary gland, skeletal muscle, liver, kidney, urinary bladder, intestine, rumen, reticulum, omasum, abomasum, and reproductive system) were fixed in 10% formalin. All tissue samples were processed routinely for histopathology and stained with hematoxylin and eosin (HE). 
Linkage Mapping
Genomic DNA was extracted from blood (NucleoSpin 96 Blood Quick Pure Kit; Macherey-Nagel, Düren, Germany). In total, 50 microsatellite markers that were predicted from comparison of the ovine linkage map version 4.4 to human genome build 35.1 (see http://rubens.its.unimelb.edu.au/∼jillm/jill.htm) to be closely linked to the orthologues of putative candidate genes for microphthalmia represented 17 linkage groups (Table 1)
Genotyping of microsatellite markers was performed by PCR amplification and subsequent polyacrylamide gel electrophoresis. The PCR reaction with a final volume of 12 μL was performed using 20 ng of genomic ovine DNA, 100 μM dNTPs, 10 picomoles of each primer, and 0.5 U Taq polymerase in the reaction buffer supplied by the manufacturer (MP Biochemicals, Heidelberg, Germany). The thermocycler profile was 94°C for 4 minutes; 32 cycles of 94°C for 30 seconds; marker-specific annealing for 30 seconds and 72°C for 30 seconds; and a final cooling step at 4°C for 10 minutes. Infrared dye (IRD)-labeled PCR products were separated on 6% denaturing polyacrylamide gels by automated sequencer (Gene ReadIR 4200/4300 automated sequencer; LI-COR; Bad Homburg, Germany). Scoring and size determination of alleles was performed independently by two investigators by comparison to a size standard marker (LI-COR) and retyped in case of discrepancy. 
Genotypic data were initially analyzed by using CRI-MAP software, version 2.4. 16 Pair-wise linkage analysis was performed with the twopoint option to calculate the recombination fractions between OMO and marker loci and corresponding LOD scores. Subsequently, multipoint nonparametric linkage (NPL) and parametric linkage analyses were performed with MERLIN software, version 1.0.1. 17 We performed multipoint NPL analyses based on the degree of identity by descent allele-sharing among affected members, by using the pair-scoring function. In addition, we calculated multipoint LOD scores under both homogeneity and heterogeneity under the assumption that OMO segregates as a biallelic autosomal recessive trait, with complete penetrance. The frequency of the defect allele in the considered population is unknown, and there are no data available that would make it possible to estimate the frequency in a reliable manner. For the calculations, a frequency of 0.001 for the mutated allele was assumed. Under the assumption of locus heterogeneity, HLOD scores were maximized for varying fractions of linked families (alpha). 18  
Fine mapping was performed by additional genotyping of 43 microsatellites (Table 2)spanning the recent OAR23 linkage map. 12 Moreover, we estimated haplotypes by finding the most likely path of gene flow. To reconstruct likely haplotypes and identify recombinations, we applied the best option of MERLIN. 
Candidate Gene Analysis
An ovine BAC clone (CH243-13H3) assumed to contain TGIF1 was identified by BLASTN searches of ovine BES to the HSA18 sequence. 12 The BAC DNA was prepared with a plasmid kit (Midi; Qiagen, Hilden, Germany) according to the modified protocol for BAC clones (Qiagen). The human reference TGIF1 isoform-c mRNA (GenBank: NM_173208; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) was used as query in cross-species BLAST searches identifying five corresponding ovine EST entries (GenBank: CD288212, EE799622, EE830605, EE822729, and EE826815). A partial insert sequence of 11.8 kb of the CH243-13H3 BAC clone was completely determined by a primer-walking strategy until both strands were completely sequenced with dye termination chemistry (BigDye Terminator Sequencing Kit 3.1 on a 3730 capillary sequencer; Applied Biosystems [ABI] Rotkreuz, Switzerland). Initial sequencing primers were designed from the identified EST sequences to sequence the entire ovine TGIF1 gene. Sequence data were then analyzed (Sequencher 4.6; GeneCodes, Ann Arbor, MI). The exact ovine genomic structure was determined by alignment of ovine TGIF1 EST and human and murine TGIF1 mRNA to the determined ovine genomic sequence using Spidey (http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/index.html). Ovine TGIF1 gene sequence was submitted to EMBL (European Molecular Biology Laboratory, Heidelberg, Germany) under accession AM493688. 
The three ovine TGIF1 exons with flanking sequences were PCR amplified from genomic DNA (AmpliTaqGold; ABI) at standard conditions at 60°C annealing. Three sets of primer pairs designated I, II, and III, representing each individual TGIF1 exon were used: I, 5′-CCTCGCTCACTCTGACAGC-3′ and 5′-CTTTCAGACAAAGCGCCAAG-3′; II, 5′-GGAAGAACAGCGCTTGAAAC-3′ and 5′-TGACTGTCAAGTGTTTCTGTTTCTC-3′; and III, 5′-ACTGGGAGGAGTGGCTCTTT-3′ and 5′-GCTCTGAGTTTATAGTTCTTGGAATG-3′. For the resequencing, we used eight sheep: four unrelated affected lambs, two unrelated putative carrier sheep, and two unrelated rams from the Texel breed, respectively. The subsequent sequencing of the PCR products in both directions was performed after shrimp alkaline phosphatase (Roche, Basel, Switzerland) and exonuclease I (NEB; Axonlab, Baden, Switzerland) treatment by using the PCR primers with dye termination chemistry (BigDye Terminator Sequencing Kit 3.1 on the 3730 capillary sequencer; ABI). 
Results
Animals
Seventeen unrelated rams with at least two affected offspring were identified and, in total, 39 affected offspring and seven normal littermates were collected (Fig. 1) . Affected newborn lambs showed abnormally small eye balls (Fig. 2A) , ranging from 0.7 to 1.1 cm in diameter (Fig. 2B)compared with an average diameter of 2.25 cm in control animals. The anterior eye chamber was small or absent, and there was no recognizable vitreous body or lens (Fig. 2C) . The observed pathologic features are summarized in Table 3 . No lesions were found in the other investigated organs. The experimental breeding showed normal reproductive capacity of the affected ram at mating to unrelated carrier ewes producing nine affected among 14 offspring (Fig. 1) . The clinical examination of the nine affected animals from the breeding experiment showed that they were entirely blind, but did not show any further abnormality. 
Linkage Analysis
Linkage mapping was used to determine whether 27 known candidate loci influencing congenital isolated or syndromic microphthalmia–anophthalmia phenotypes in humans or mice were involved in the ovine microphthalmia phenotype in the examined sheep families. At least two polymorphic flanking microsatellite markers for each candidate locus were genotyped in all samples (Table 1) . As some of the candidate genes map to the same chromosome region, 17 linkage groups on 16 sheep chromosomes were studied (Table 1) . The twopoint and multipoint linkage results between OMO and ovine microsatellite markers are compiled in Table 1 . The twopoint linkage analysis yielded a maximum LOD score of 3.52 at θ = 0.03 with microsatellite marker ADCY1AP on OAR23, which was confirmed by nonparametric multipoint linkage analysis (Table 1 ; Fig. 3 ). Several positive linkage LOD or NPL scores on different chromosomes were mutually contradictory and therefore could be regarded as suggestive linkage results (Table 1) . The subsequent fine mapping and haplotype analysis firmly assigns OMO to a 12.4 cM interval of OAR23 between the markers DU274690 and DU259631 (Table 4) . Therefore, all 24 recombinant chromosomes were taken together to describe the most likely region of OMO on the chromosome map (Table 4)
This region of the OAR23 linkage map is compared with the corresponding sequence map for human and cattle (Fig. 4) . Thereby, the definitive marker order in sheep was confirmed by sequence alignments of the used ovine microsatellite markers to the corresponding bovine chromosome 24 sequence (Fig. 4)
Candidate Gene Analysis
The OMO linked region of OAR23 corresponds to segments of HSA18, based on the recent comparative sheep–human map. 12 At least one intrachromosomal rearrangement has occurred between sheep and human as the OMO linked segment of OAR23 is orthologue to the inverted centromeric region of HSA18q and the telomeric end of HSA18p, respectively (Fig. 4) . Besides this rearrangement, there is a perfect colinearity between the sheep and human maps. Thus, the human homologue for ovine microphthalmia is expected to be found in the two indicated HSA18 intervals (Fig. 4) . The candidate gene TGIF1 located on HSA18p11.3 appeared to be within the linked region. 12 We identified an ovine BAC clone containing the entire TGIF1 orthologue and experimentally confirmed the chromosome location by fluorescent in situ hybridization assignment of CH243-13H3 to OAR23q24 (data not shown). Genomic sequencing of the TGIF1 gene and mRNA to genomic sequence alignment using sheep TGIF1 EST sequences revealed that the ovine TGIF1 gene spans 7.7 kb and consists of three exons. All splice donor–splice acceptor sites conform to the GT/AG rule. The ovine TGIF1 transcript contains an open reading frame of 819 bp encoding a deduced protein of 272 amino acids which shows 99%, 91%, 90%, and 86% identity to the orthologous bovine, canine, human (isoform c), and murine TGIF1 protein, respectively. 
The resequencing of the three coding exons and flanking intron regions of TGIF1 using four affected as well as four healthy sheep revealed four SNPs which are located within TGIF1 intron 1 (c.16+356A→G and c.17-186T→G), exon 3 (c.420A→G), and the 3′UTR (*16T→C), respectively. The codon in exon 3 with the A/G transition codes for proline in both SNP variants and none of the three intronic polymorphisms affected splice sites in the TGIF1 gene. Two SNP (c.17-186T→G and c.420A→G) were observed only in comparisons to the established genomic DNA sequence derived from the ovine BAC clone. The eight sequenced sheep were all homozygous for the respective G allele at both positions. Assuming linkage disequilibrium between the two remaining polymorphic TGIF1 sites (c.16+356A→G and *16T→C) two different haplotypes (A-T and G-C) were constructed for the SNP genotypes in the examined sheep. Both haplotypes occurred in affected and unaffected sheep, respectively, in the homozygous and heterozygous states. 
Discussion
The observed segregation pattern of microphthalmia within the experimental breeding pedigrees can be explained by autosomal recessive inheritance. Both sexes were affected, and 9 of 14 offspring from the mating of an affected ram to known carrier females were reported to have the condition. Under the assumption of a biallelic recessive locus this does not fit perfectly to the theoretical expectation of 50% affected and 50% normal offspring, respectively, but it may be explained by the small number of animals used. In general, the total number of 39 recorded affected offspring from mating of 17 normal Texel or Texel/Whiteheaded mutton crossbred rams to approximately 850 normal Texel or Texel/Whiteheaded mutton crossbred ewes supports the assumption of a recessive deleterious allele. Taken together, these observations confirm the previously reported autosomal recessive mode of inheritance for this hereditary disorder in Texel sheep. 3 4 5 6 7 8 9 Because the ewes were collected from several unrelated sheep flocks, allelism of disease among the different familial origins was confirmed by test breeding. 
The clinical and pathologic examinations indicate a slightly variable expressivity of the fully penetrant phenotype, as previously reported. 8 10 The described pathology is consistent with prior reports. 8 10 Neither an anterior eye chamber nor a lens was found in any of the examined eyes, but several structures could be observed (e.g., lacrimal glands) that are of ectodermal origin, probably deriving from the lens vesicle. This supports the hypothesis of van der Linde-Sipmann et al. 8 assuming an abnormal development of the lens vesicle as that primary event leading to disintegration of the lens and subsequent secondary defects in other eye structures due to the overgrowth of mesenchymal tissue. This leads to regression of the embryonic globe that had reached at least the stage of the optic cup, which closely resembles autosomal recessive lens aplasia in mice. 19 Hereditary microphthalmia in sheep characterized by smaller eyes and lens dysplasia may be affected by the lens degeneration due to developmental arrest that causes collapse of the surrounding ocular tissue (reduction in eye size) and abnormal patterning of the anterior segment structures (cornea defects). Similar associations between lens defects, small eye, and malformal anterior chamber have been reported. 1  
The presented large animal model for isolated congenital microphthalmia was mapped to the sheep genome for the first time. We have successfully used a comparative candidate linkage mapping approach where we have applied information on established comparative maps between ruminants and human genomes. As the 12.4 cM interval identified on OAR23 shows conserved synteny with regions on HSA18 OMO is likely to represent a mutation in an orthologue gene. 12 The initially selected candidate gene TGIF1 encoding TGFB-induced factor, a homeodomain protein that acts as a transcription factor during early brain development, is located within the critical interval. Mutations within human TGIF1 causes holoprosencephaly. 20 Labs 11 concluded from her study of newborn lambs with microphthalmia that there was a primary anomaly of the lateral prosencephalon and the primary optic vesicle and that this would result in an abnormal interaction between the primary optic vesicle and the ectoderm, with the absence of lens formation. 
Direct-sequence screening analysis excluded the complete coding region of ovine TGIF1 to harbor disease-causing mutations in the examined sheep. Due to the assumption of a single recessive founder mutation within the Texel breed, we expect homozygosity in affected individuals. The presented data indicate that the detected SNP of ovine TGIF1 can be excluded as disease-harboring mutations in the examined sheep. Therefore, it seems to be likely that the entire TGIF1 gene can be excluded as a candidate for microphthalmia in this breed. 
The presented sequence-alignment–based sheep–human comparison helps to define the exact candidate gene regions on HSA18. The corresponding centromeric region of HSA18q between 0.00 and 6.95 Mb and the telomeric end of HSA18p between 16.78 and 22.75 Mb contains 51 and 49 annotated genes, respectively (human genome build 36.2). A comparison with cattle genome sequence suggests that the critical region in ruminants is smaller than 10 Mb (Fig. 4) . Up to the present, we could not identify any obvious candidate genes in the defined region assumed to harbor the human orthologue of OMO. Therefore, we expect the microphthalmia phenotype to be caused by a novel gene that has not previously been associated with ocular disorders or eye development in any species. It is therefore of considerable interest to reveal the nature of the microphthalmia mutation in sheep, because this could provide novel insights into the molecular mechanisms leading to similar phenotypes in human and mammalian eye development. Recently, a genetic linkage was reported to bovine chromosome 18 for cattle showing an inherited ocular defect characterized by microphthalmia, torsion of the eyes, and complete blindness. 21 The genomic evolutionary orthologue, HSA16, harbors several potential candidate genes for the bovine disorder 22 indicating another example for a possible valuable large animal model in this field. 
In conclusion, a further refinement of the candidate region may be necessary before a systematic mutation analysis of the genes located in the region can be undertaken. As there appears to be a fairly low recombination rate in the identified candidate region, it would be hard to achieve a high-resolution localization by linkage analysis. Therefore, we are now adopting a linkage disequilibrium strategy using SNP markers as the next step toward positional cloning of the OMO gene. Identifying the minimum shared haplotype associated with OMO could take advantage of the recombination events that have accumulated during the transmission of OMO from a putative common founder. 
 
Figure 1.
 
Experimental sheep pedigrees segregating for OMO. (*) The affected ram used for the experimental mating (families 16–18). The matings for each family represent matings of a single male to multiple unrelated carrier females.
Figure 1.
 
Experimental sheep pedigrees segregating for OMO. (*) The affected ram used for the experimental mating (families 16–18). The matings for each family represent matings of a single male to multiple unrelated carrier females.
Table 1.
 
List and Human Map Position of Candidate Loci and Map Information for Corresponding Ovine Microsatellite Markers Used in the Initial Linkage Analysis
Table 1.
 
List and Human Map Position of Candidate Loci and Map Information for Corresponding Ovine Microsatellite Markers Used in the Initial Linkage Analysis
Region Candidate Gene OMIM Human Chrom. Sheep Chrom. cM (SheepMap ver. 4.5) Microsatellite No. of Alleles PIC CRI-MAP Twopoint Linkage MERLIN Nonparametric Multipoint Linkage
Rec. Frac. LOD NPL Score P
1 EYA3 601655 1p36 1 16.8 INRA197 6 0.70 0.50 −∞ −0.17 0.6
FOXE3 107250 1p32 1 23.8 EPCDV21 7 0.41 0.50 −∞ −0.50 0.7
1 40.3 BMS835 11 0.78 0.50 −∞ −0.46 0.7
2 SIX3 603714 2p16-p21 3 66.3 FCB129 7 0.71 0.50 −∞ 0.70 0.2
OTX1 600036 2p13 3 80.6 BMS710 9 0.79 0.50 −∞ 0.90 0.2
3 100.2 ILSTS49 5 0.46 0.50 −∞ 0.13 0.4
3 MITF 156845 3p14.2-p14.1 19 31.7 MILVET8 7 0.65 0.50 −∞ −0.79 0.8
19 64.8 FCB304 6 0.55 0.50 −∞ −0.68 0.8
4 SOX2 206900 3q26.3-q27 1 229.5 BM6506 7 0.38 0.50 −∞ −0.61 0.7
1 235.3 LS6 11 0.80 0.50 −∞ 0.60 0.7
1 250.3 MCM130 9 0.72 0.50 −∞ 0.35 0.6
5 PITX2 601542 4q25-q27 6 16.0 INRA133 8 0.26 0.50 −∞ 1.20 0.12
6 29.7 MCM53 6 0.74 0.16 0.18 0.71 0.2
6 45.0 MCMA14 6 0.65 0.50 −∞ 0.35 0.4
6 FOXC1 601090 6p25 20 30.8 BM1258 6 0.47 0.50 −∞ −0.07 0.5
20 66.2 BM1818 5 0.65 0.50 −∞ 0.24 0.6
20 91.4 MCMA23 4 0.42 0.21 0.12 0.59 0.3
7 GJA1 164200 6q21-q23.2 8 61.4 BMS434 9 0.60 0.50 −∞ −0.78 0.8
8 88.6 BMS1724 7 0.46 0.50 −∞ −1.09 0.9
8 108.0 BMS1967 9 0.61 0.24 0.13 −0.06 0.5
8 SHH 120200 7q36 4 120.0 BMS648 6 0.65 0.50 −∞ −0.06 0.5
4 139.5 HH64 8 0.44 0.50 −∞ −0.29 0.6
9 EYA1 601653 8q13.3 9 60.1 ILSTS11 5 0.68 0.50 −∞ −0.26 0.6
9 78.4 MCM42 5 0.56 0.50 −∞ −0.49 0.7
10 PITX3 107250 10q25 22 34.5 BM1314 7 0.51 0.50 −∞ 0.50 0.3
22 57.2 MAF36 7 0.59 0.50 −∞ 0.31 0.4
11 PAX6 106210 11p13 15 46.5 MAF65 4 0.55 0.50 −∞ 0.27 0.4
NNO1 600165 11p 15 69.4 HBB2 7 0.72 0.50 −∞ 0.18 0.4
15 79.2 BMS2812 5 0.67 0.50 −∞ 0.05 0.5
15 92.5 SHP4 8 0.75 0.50 −∞ −0.03 0.5
15 96.0 BMS1660 5 0.69 0.50 −∞ 0.11 0.5
12 DACH1 603803 13q22 10 53.9 BMS975 7 0.74 0.50 −∞ −1.15 0.9
10 71.1 INRA5 6 0.65 0.50 −∞ −0.85 0.8
13 MFRP 605738 15q12-q15 7 75.0 INRA107 8 0.67 0.50 −∞ −0.56 0.7
BMP4 112262 14q22-q23 7 92.1 INRA71 3 0.53 0.50 −∞ −0.60 0.7
SIX6 606326 14q22.3-q23 7 105.5 BMS1620 7 0.37 0.50 −∞ −0.53 0.7
SIX4 606342 14q23 7 117.1 MCM149 11 0.78 0.50 −∞ −0.12 0.5
CHX10 142993 14q24 7 126.2 BMS2721 6 0.53 0.21 0.06 0.87 0.2
7 136.7 BMS2614 5 0.59 0.36 0.01 0.60 0.3
14 MCOP 251600 14q32 18 77.0 HH47 10 0.83 0.50 −∞ −1.14 0.9
18 88.7 TGLA122 11 0.82 0.50 −∞ −0.93 0.8
15 MAF 177075 16q22-q23 14 13.0 TGLA357 7 0.64 0.50 −∞ 0.21 0.4
14 26.6 CSRD47 7 0.56 0.50 −∞ −0.14 0.6
16 TGIF1 602630 18p11.3 23 23.5 BMS2270 6 0.66 0.11 0.84 3.20 0.0007
RAX 601881 18q21 23 43.1 ADCY1AP 8 0.76 0.03 3.52 4.18 0.00001
23 67.9 DIK2727 10 0.83 0.22 0.27 1.78 0.04
17 VSX1 122000 20p11.23- p11.22 13 65.0 HUJ616 7 0.56 0.50 −∞ 0.31 0.4
EYA2 601654 20q13.1 13 98.2 CSTBJ12 5 0.68 0.17 0.21 1.04 0.15
BMP7 112267 20q13 13 115.4 MMP9 7 0.70 0.50 −∞ −0.23 0.6
13 125.9 BMS995 6 0.66 0.50 −∞ 0.34 0.4
Table 2.
 
OAR23 Microsatellite Markers Used in the Fine Mapping Analysis
Table 2.
 
OAR23 Microsatellite Markers Used in the Fine Mapping Analysis
Microsatellite Sheep Chrom. 23 cM (SheepMap v4.7) Alleles (n) PIC Informative Meioses (n) CRI-MAP Twopoint Linkage MERLIN Parametric Multipoint Linkage MERLIN Nonparametric Multipoint Linkage
Rec. Frac. LOD LOD α HLOD NPL Score P
UW72A 0.0 2 0.23 13 0.47 −∞ −∞ 0.36 0.82 1.54 0.06
BL6 2.4 11 0.77 43 0.47 −∞ −∞ 0.35 0.87 1.73 0.04
MCM172 2.9 7 0.66 35 0.46 −∞ −∞ 0.35 0.86 1.74 0.04
BM226 7.6 8 0.70 19 0.48 −∞ −∞ 0.34 0.79 1.77 0.04
BMS2526 8.3 6 0.59 33 0.49 −∞ −∞ 0.34 0.78 1.77 0.04
DU280225 18.0 12 0.83 45 0.24 0.32 −∞ 0.31 0.92 2.16 0.02
DU330122 18.0 8 0.54 42 0.25 0.25 −∞ 0.32 0.98 2.93 0.002
CSRD148 18.6 10 0.83 26 0.39 −∞ −∞ 0.32 0.98 2.93 0.002
MCMA1 21.9 8 0.72 32 0.26 0.13 −∞ 0.42 1.49 3.45 0.0003
CABB12 22.6 6 0.70 24 0.23 0.23 −∞ 0.42 1.47 3.49 0.0002
BMS2270 23.5 6 0.66 32 0.11 0.84 −∞ 0.47 1.66 3.63 0.00014
CSSM31 30.3 10 0.51 16 0.07 1.13 −∞ 0.56 2.40 3.66 0.00012
ILSTS65 30.9 2 0.34 14 0.10 0.67 −∞ 0.55 2.40 3.68 0.00012
DIK4464 34.0 6 0.57 11 0.27 0.04 −∞ 0.47 2.11 3.55 0.0002
AGLA269 34.0 14 0.84 17 0.30 0.03 −∞ 0.47 2.11 3.55 0.0002
DU268178 9 0.80 48 0.18 1.12 −∞ 0.54 2.76 3.65 0.00013
DU216028 34.6 10 0.81 50 0.22 0.72 −∞ 0.57 3.00 4.38 0.00001
DU340520 35.1 6 0.67 39 0.25 0.20 −∞ 0.58 3.14 4.43 <0.00001
DU252884 35.4 2 0.37 19 0.17 0.51 −∞ 0.68 3.82 4.50 <0.00001
DU274690 35.7 4 0.42 19 0.37 0.01 −∞ 0.71 4.15 4.53 <0.00001
DU520666 37.7 4 0.27 20 0.00 0.60 6.51 0.91 6.85 4.78 <0.00001
DU416699 41.7 3 0.36 14 0.00 2.11 7.95 0.94 8.09 5.35 <0.00001
DU288059 41.7 4 0.55 16 0.00 0.60 8.32 0.95 8.40 5.47 <0.00001
CL638456 4 0.39 42 0.00 4.21 8.14 0.95 8.25 5.42 <0.00001
DU189183 7 0.60 42 0.04 2.67 8.79 1.00 8.79 5.48 <0.00001
DU377056 41.7 5 0.29 23 0.00 2.11 8.41 0.96 8.45 5.48 <0.00001
DU373930 41.7 8 0.63 19 0.06 1.64 8.35 0.96 8.42 5.47 <0.00001
ADCY1AP 42.7 8 0.76 43 0.03 3.52 9.62 1.00 9.62 5.49 <0.00001
DU239182 43.2 17 0.84 38 0.05 2.17 10.15 1.00 10.15 5.49 <0.00001
DU316110 44.7 6 0.69 33 0.05 1.92 10.70 1.00 10.70 5.50 <0.00001
DU441780 44.7 14 0.68 53 0.03 3.79 10.70 1.00 10.70 5.50 <0.00001
MAF35 44.7 5 0.52 43 0.09 1.53 10.70 1.00 10.70 5.50 <0.00001
DU291927 45.6 5 0.55 48 0.03 3.22 10.59 1.00 10.59 5.41 <0.00001
DU259631 48.1 6 0.59 43 0.04 2.70 −∞ 0.85 6.68 5.05 <0.00001
UW69A 48.1 6 0.42 41 0.08 1.46 −∞ 0.83 6.60 5.05 <0.00001
DU408420 48.1 3 0.64 16 0.00 2.11 −∞ 0.83 6.61 5.05 <0.00001
CSRD172 50.0 3 0.42 25 0.00 2.10 −∞ 0.80 5.43 4.70 <0.00001
DU171881 50.0 5 0.62 32 0.12 0.59 −∞ 0.79 5.32 4.69 <0.00001
BMS1332 50.0 6 0.56 21 0.00 1.51 −∞ 0.74 5.00 4.67 <0.00001
HEL6 51.7 3 0.37 2 0.00 0.30 −∞ 0.73 4.24 4.26 0.00001
MCM136 52.8 10 0.80 42 0.09 1.18 −∞ 0.63 3.14 4.06 0.00002
DIK5068 62.1 5 0.63 26 0.48 −∞ −∞ 0.30 0.79 2.36 0.009
CGBP 63.2 3 0.12 0 0.00 −∞ −7.98 0.32 0.83 2.34 0.01
DIK2727 67.5 10 0.83 17 0.22 0.27 −∞ 0.35 0.88 2.27 0.012
DU264615 74.5 5 0.55 34 0.16 0.47 −∞ 0.23 0.45 1.51 0.07
URB031 83.9 3 0.23 4 0.00 0.30 −0.51 0.41 0.54 1.17 0.12
Figure 2.
 
Gross appearance of microphthalmic eyes from an affected lamb (left) in comparison with an age-matched control globe (right). In vivo (A), isolated after necropsy (B), and sectioned in a median sagittal plane (C).
Figure 2.
 
Gross appearance of microphthalmic eyes from an affected lamb (left) in comparison with an age-matched control globe (right). In vivo (A), isolated after necropsy (B), and sectioned in a median sagittal plane (C).
Table 3.
 
Pathology Features of Ovine Microphthalmia
Table 3.
 
Pathology Features of Ovine Microphthalmia
Eye Structure Phenotype Description • Microscopic Details
Eye lids, conjunctiva, nictitating membrane Regular
Cornea Diffuse clouded showing distinct vascularization, pigmentation and a granulated surface
 • Variable lymphohistiocytic infiltration
 • Absence of the Descemet’s membrane and posterior epithelium
Anterior eye chamber Not visible
Lens, vitreous body Not recognizable
Iris and ciliary body  • Incorporated within or restricted to the periphery of the tissue mass
Posterior eye chamber, retina White mass composed of connective tissue with blood vessels, smooth muscle, cartilage and fat tissue
 • Anterior: islets of lacrimal glands and multiple cystic structures frequently lined by squamous epithelium and filled with keratin
 • Posterior: partly covered by detached retinal structures sometimes forming rosettes
Optic nerve Reduced diameter
 • Highly cellular
Figure 3.
 
Results of the parametric (LOD) and nonparametric (NPL) multipoint linkage analysis across OAR23 with 18 half- and full-sib families with the MERLIN software. The maximum parametric LOD score is 10.70 for three markers with relative positions at 44.7 cM. The markers ranging from 42.7 to 45.6 cM are significantly linked to OMO in all 18 families (α = 1). The highest NPL score is 5.50 for three markers with relative positions at 44.7 cM. The calculated nonparametric linkage is highly significant in the region between 18.0 and 63.2 cM with an error probability of P ≤ 0.01.
Figure 3.
 
Results of the parametric (LOD) and nonparametric (NPL) multipoint linkage analysis across OAR23 with 18 half- and full-sib families with the MERLIN software. The maximum parametric LOD score is 10.70 for three markers with relative positions at 44.7 cM. The markers ranging from 42.7 to 45.6 cM are significantly linked to OMO in all 18 families (α = 1). The highest NPL score is 5.50 for three markers with relative positions at 44.7 cM. The calculated nonparametric linkage is highly significant in the region between 18.0 and 63.2 cM with an error probability of P ≤ 0.01.
Table 4.
 
List of Recombinants Present in the Whole Set of Genotyped Sheep Families, Arrayed According to the Position of the Recombination Event*
Table 4.
 
List of Recombinants Present in the Whole Set of Genotyped Sheep Families, Arrayed According to the Position of the Recombination Event*
Marker cM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
UW72A 0.0 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 133 135 135 135 135 135 133 133
BL6 2.4 190 180 190 186 180 190 180 190 194 186 174 180 190 190 180 180 190 190 190 180 180 190 182 190
MCM172 2.9 143 135 143 141 135 135 135 143 143 135 143 135 135 135 135 135 149 135 147 135 135 135 143 149
BM226 7.6 126 126 140 126 126 124 126 126 124 126 126 126 124 126 126 126 132 126 140 126 140 126 126 132
BMS2526 8.3 150 156 158 150 150 152 150 150 156 150 154 150 154 154 150 150 150 154 150 150 150 150 154 150
DU280225 18.0 442 442 424 436 444 444 444 442 426 432 444 444 432 436 424 444 444 436 442 444 436 434 424 444
DU330122 18.0 291 289 289 289 289 289 289 291 277 303 289 289 303 289 289 289 289 289 289 289 293 287 289 289
CSRD148 18.6 391 397 401 375 401 393 401 391 401 391 397 291 375 375 401 401 401 375 397 291 393 377 401 401
MCMA1 21.9 126 136 140 124 136 124 136 126 150 130 126 136 136 136 136 136 136 136 136 136 126 144 140 136
CABB12 22.6 240 230 230 240 230 232 230 240 228 230 240 230 230 234 240 230 230 234 230 230 240 226 230 230
BMS2270 23.5 94 92 90 94 92 92 92 94 96 92 90 92 92 92 92 92 92 92 92 92 92 92 90 92
CSSM31 30.3 161 131 149 165 131 131 131 161 131 161 131 131 131 131 131 131 131 131 131 131 131 163 149 131
ILSTS65 30.9 115 115 115 115 115 117 115 115 117 115 115 115 115 115 115 115 115 115 117 115 117 115 115 115
DIK4464 34.0 226 224 218 224 224 216 224 226 226 226 230 226 226 226 226 226 226 224 216 226 224 226 218 226
AGLA269 34.0 238 266 272 300 270 300 270 238 266 300 266 270 272 266 266 270 270 266 300 270 300 238 272 270
DU268178 106 106 116 110 110 116 110 106 106 112 116 116 114 114 110 123 123 106 116 116 112 106 116 112
DU216028 34.6 221 213 221 217 213 225 213 221 219 193 193 225 217 217 213 213 193 213 225 193 217 221 221 193
DU340520 35.1 225 219 221 225 225 219 225 225 221 225 221 219 223 223 225 225 225 219 219 221 225 221 221 225
DU252884 35.4 324 324 324 324 324 326 324 324 324 326 326 326 324 324 324 324 326 324 326 326 326 324 324 326
DU274690 35.7 188 188 188 188 188 190 188 188 192 192 182 188 188 192 188 188 188 188 190 182 188 192 188 188
DU520666 37.7 226 226 226 226 224 226 224 226 226 226 226 226 226 226 226 226 226 226 226 224 226 226 226 226
DU416699 41.7 158 158 156 158 158 133 158 158 158 158 158 158 156 158 158 158 158 158 133 158 158 158 156 158
DU288059 110 110 120 110 120 115 120 110 115 115 110 115 120 120 110 110 110 110 115 115 110 120 120 110
CL638456 41.7 182 182 182 182 182 171 182 182 182 182 182 171 182 182 182 182 171 182 171 171 171 182 182 171
DU189183 41.7 213 199 213 205 199 199 199 213 213 213 199 198 199 199 199 199 199 199 199 198 215 199 213 199
DU377056 41.7 289 289 289 289 289 302 289 289 289 289 289 302 289 289 289 289 289 289 302 302 289 289 289 289
DU373930 315 315 315 307 307 307 307 315 315 315 315 307 307 307 315 315 315 315 307 307 318 307 315 315
ADCY1AP 42.7 106 108 87 116 87 108 87 106 114 106 106 108 87 87 106 106 106 108 108 108 114 87 87 106
DU239182 43.2 291 292 292 292 294 280 294 291 272 280 292 261 272 294 292 292 292 292 280 261 292 292 292 292
DU441780 44.7 146 146 210 179 161 146 161 146 196 148 210 146 146 161 210 210 210 146 146 146 210 210 210 210
DU316110 44.7 131 129 131 131 129 131 129 131 131 131 129 132 131 131 129 129 129 129 131 132 131 131 131 129
MAF35 44.7 106 104 106 110 104 106 104 106 104 104 106 106 106 104 106 106 106 104 106 106 106 106 106 106
DU291927 45.6 247 243 247 245 243 247 243 247 243 241 247 247 247 243 247 247 247 243 247 247 243 243 247 247
DU259631 48.1 154 154 158 152 152 154 152 154 154 154 154 154 154 152 152 152 152 154 154 154 154 154 158 152
UW69A 48.1 154 156 154 158 158 154 158 154 158 156 158 158 158 158 158 158 158 156 154 158 154 158 154 158
DU408420 48.1 288 288 286 288 288 286 288 288 290 288 296 294 294 288 288 288 288 288 286 294 288 294 286 288
CSRD2172 50.0 188 188 192 192 192 188 192 188 192 192 188 188 192 188 192 192 192 188 188 188 192 188 192 192
DU171881 50.0 164 178 164 178 162 164 162 164 176 164 164 162 164 162 164 164 162 178 164 162 164 162 164 162
BMS1332 50.0 136 138 142 142 142 140 142 138 142 140 142 140 142 140 142 142 140 138 140 140 140 140 142 140
HEL6 51.7 181 181 185 185 185 185 185 185 185 185 181 185 185 185 185 185 185 181 185 185 185 185 185 185
MCM136 52.8 148 142 138 146 164 164 138 148 144 164 142 142 164 142 140 140 140 142 164 142 164 142 146 140
DIK5068 62.1 90 92 92 90 92 92 92 90 98 90 90 90 92 90 90 90 92 90 94 90 94 90 92 92
CGBP 63.2 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265
DIK2727 67.5 208 194 194 194 202 212 202 208 226 208 206 212 208 212 208 208 184 212 212 212 212 212 194 184
DU264615 74.5 118 124 124 118 124 116 124 118 124 118 116 116 124 118 118 124 116 118 116 116 124 116 124 116
URB031 83.9 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234
Figure 4.
 
Comparative chromosome maps of the ovine microphthalmia critical interval. The human-sheep alignment is based on the OAR23 radiation hybrid map 12 indicating two different corresponding HSA18 blocks of conserved gene order. The sheep–cattle alignment is based by sequence alignments of microsatellite markers used within this study and the current bovine assembly (see http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9913). It indicates an approximate size of 9.5 Mb genomic chromosome segment for the critical interval between the markers DU274690 and DU259631, identified by haplotype analysis. #, two markers that were not mapped on the ovine linkage map. Their position was inferred from comparative sequence analysis between sheep and cattle. *, the single marker that could not be placed in the cattle genome sequence.
Figure 4.
 
Comparative chromosome maps of the ovine microphthalmia critical interval. The human-sheep alignment is based on the OAR23 radiation hybrid map 12 indicating two different corresponding HSA18 blocks of conserved gene order. The sheep–cattle alignment is based by sequence alignments of microsatellite markers used within this study and the current bovine assembly (see http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9913). It indicates an approximate size of 9.5 Mb genomic chromosome segment for the critical interval between the markers DU274690 and DU259631, identified by haplotype analysis. #, two markers that were not mapped on the ovine linkage map. Their position was inferred from comparative sequence analysis between sheep and cattle. *, the single marker that could not be placed in the cattle genome sequence.
The authors thank Hauke Peters for excellent assistance with collecting the blood samples. 
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Figure 1.
 
Experimental sheep pedigrees segregating for OMO. (*) The affected ram used for the experimental mating (families 16–18). The matings for each family represent matings of a single male to multiple unrelated carrier females.
Figure 1.
 
Experimental sheep pedigrees segregating for OMO. (*) The affected ram used for the experimental mating (families 16–18). The matings for each family represent matings of a single male to multiple unrelated carrier females.
Figure 2.
 
Gross appearance of microphthalmic eyes from an affected lamb (left) in comparison with an age-matched control globe (right). In vivo (A), isolated after necropsy (B), and sectioned in a median sagittal plane (C).
Figure 2.
 
Gross appearance of microphthalmic eyes from an affected lamb (left) in comparison with an age-matched control globe (right). In vivo (A), isolated after necropsy (B), and sectioned in a median sagittal plane (C).
Figure 3.
 
Results of the parametric (LOD) and nonparametric (NPL) multipoint linkage analysis across OAR23 with 18 half- and full-sib families with the MERLIN software. The maximum parametric LOD score is 10.70 for three markers with relative positions at 44.7 cM. The markers ranging from 42.7 to 45.6 cM are significantly linked to OMO in all 18 families (α = 1). The highest NPL score is 5.50 for three markers with relative positions at 44.7 cM. The calculated nonparametric linkage is highly significant in the region between 18.0 and 63.2 cM with an error probability of P ≤ 0.01.
Figure 3.
 
Results of the parametric (LOD) and nonparametric (NPL) multipoint linkage analysis across OAR23 with 18 half- and full-sib families with the MERLIN software. The maximum parametric LOD score is 10.70 for three markers with relative positions at 44.7 cM. The markers ranging from 42.7 to 45.6 cM are significantly linked to OMO in all 18 families (α = 1). The highest NPL score is 5.50 for three markers with relative positions at 44.7 cM. The calculated nonparametric linkage is highly significant in the region between 18.0 and 63.2 cM with an error probability of P ≤ 0.01.
Figure 4.
 
Comparative chromosome maps of the ovine microphthalmia critical interval. The human-sheep alignment is based on the OAR23 radiation hybrid map 12 indicating two different corresponding HSA18 blocks of conserved gene order. The sheep–cattle alignment is based by sequence alignments of microsatellite markers used within this study and the current bovine assembly (see http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9913). It indicates an approximate size of 9.5 Mb genomic chromosome segment for the critical interval between the markers DU274690 and DU259631, identified by haplotype analysis. #, two markers that were not mapped on the ovine linkage map. Their position was inferred from comparative sequence analysis between sheep and cattle. *, the single marker that could not be placed in the cattle genome sequence.
Figure 4.
 
Comparative chromosome maps of the ovine microphthalmia critical interval. The human-sheep alignment is based on the OAR23 radiation hybrid map 12 indicating two different corresponding HSA18 blocks of conserved gene order. The sheep–cattle alignment is based by sequence alignments of microsatellite markers used within this study and the current bovine assembly (see http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9913). It indicates an approximate size of 9.5 Mb genomic chromosome segment for the critical interval between the markers DU274690 and DU259631, identified by haplotype analysis. #, two markers that were not mapped on the ovine linkage map. Their position was inferred from comparative sequence analysis between sheep and cattle. *, the single marker that could not be placed in the cattle genome sequence.
Table 1.
 
List and Human Map Position of Candidate Loci and Map Information for Corresponding Ovine Microsatellite Markers Used in the Initial Linkage Analysis
Table 1.
 
List and Human Map Position of Candidate Loci and Map Information for Corresponding Ovine Microsatellite Markers Used in the Initial Linkage Analysis
Region Candidate Gene OMIM Human Chrom. Sheep Chrom. cM (SheepMap ver. 4.5) Microsatellite No. of Alleles PIC CRI-MAP Twopoint Linkage MERLIN Nonparametric Multipoint Linkage
Rec. Frac. LOD NPL Score P
1 EYA3 601655 1p36 1 16.8 INRA197 6 0.70 0.50 −∞ −0.17 0.6
FOXE3 107250 1p32 1 23.8 EPCDV21 7 0.41 0.50 −∞ −0.50 0.7
1 40.3 BMS835 11 0.78 0.50 −∞ −0.46 0.7
2 SIX3 603714 2p16-p21 3 66.3 FCB129 7 0.71 0.50 −∞ 0.70 0.2
OTX1 600036 2p13 3 80.6 BMS710 9 0.79 0.50 −∞ 0.90 0.2
3 100.2 ILSTS49 5 0.46 0.50 −∞ 0.13 0.4
3 MITF 156845 3p14.2-p14.1 19 31.7 MILVET8 7 0.65 0.50 −∞ −0.79 0.8
19 64.8 FCB304 6 0.55 0.50 −∞ −0.68 0.8
4 SOX2 206900 3q26.3-q27 1 229.5 BM6506 7 0.38 0.50 −∞ −0.61 0.7
1 235.3 LS6 11 0.80 0.50 −∞ 0.60 0.7
1 250.3 MCM130 9 0.72 0.50 −∞ 0.35 0.6
5 PITX2 601542 4q25-q27 6 16.0 INRA133 8 0.26 0.50 −∞ 1.20 0.12
6 29.7 MCM53 6 0.74 0.16 0.18 0.71 0.2
6 45.0 MCMA14 6 0.65 0.50 −∞ 0.35 0.4
6 FOXC1 601090 6p25 20 30.8 BM1258 6 0.47 0.50 −∞ −0.07 0.5
20 66.2 BM1818 5 0.65 0.50 −∞ 0.24 0.6
20 91.4 MCMA23 4 0.42 0.21 0.12 0.59 0.3
7 GJA1 164200 6q21-q23.2 8 61.4 BMS434 9 0.60 0.50 −∞ −0.78 0.8
8 88.6 BMS1724 7 0.46 0.50 −∞ −1.09 0.9
8 108.0 BMS1967 9 0.61 0.24 0.13 −0.06 0.5
8 SHH 120200 7q36 4 120.0 BMS648 6 0.65 0.50 −∞ −0.06 0.5
4 139.5 HH64 8 0.44 0.50 −∞ −0.29 0.6
9 EYA1 601653 8q13.3 9 60.1 ILSTS11 5 0.68 0.50 −∞ −0.26 0.6
9 78.4 MCM42 5 0.56 0.50 −∞ −0.49 0.7
10 PITX3 107250 10q25 22 34.5 BM1314 7 0.51 0.50 −∞ 0.50 0.3
22 57.2 MAF36 7 0.59 0.50 −∞ 0.31 0.4
11 PAX6 106210 11p13 15 46.5 MAF65 4 0.55 0.50 −∞ 0.27 0.4
NNO1 600165 11p 15 69.4 HBB2 7 0.72 0.50 −∞ 0.18 0.4
15 79.2 BMS2812 5 0.67 0.50 −∞ 0.05 0.5
15 92.5 SHP4 8 0.75 0.50 −∞ −0.03 0.5
15 96.0 BMS1660 5 0.69 0.50 −∞ 0.11 0.5
12 DACH1 603803 13q22 10 53.9 BMS975 7 0.74 0.50 −∞ −1.15 0.9
10 71.1 INRA5 6 0.65 0.50 −∞ −0.85 0.8
13 MFRP 605738 15q12-q15 7 75.0 INRA107 8 0.67 0.50 −∞ −0.56 0.7
BMP4 112262 14q22-q23 7 92.1 INRA71 3 0.53 0.50 −∞ −0.60 0.7
SIX6 606326 14q22.3-q23 7 105.5 BMS1620 7 0.37 0.50 −∞ −0.53 0.7
SIX4 606342 14q23 7 117.1 MCM149 11 0.78 0.50 −∞ −0.12 0.5
CHX10 142993 14q24 7 126.2 BMS2721 6 0.53 0.21 0.06 0.87 0.2
7 136.7 BMS2614 5 0.59 0.36 0.01 0.60 0.3
14 MCOP 251600 14q32 18 77.0 HH47 10 0.83 0.50 −∞ −1.14 0.9
18 88.7 TGLA122 11 0.82 0.50 −∞ −0.93 0.8
15 MAF 177075 16q22-q23 14 13.0 TGLA357 7 0.64 0.50 −∞ 0.21 0.4
14 26.6 CSRD47 7 0.56 0.50 −∞ −0.14 0.6
16 TGIF1 602630 18p11.3 23 23.5 BMS2270 6 0.66 0.11 0.84 3.20 0.0007
RAX 601881 18q21 23 43.1 ADCY1AP 8 0.76 0.03 3.52 4.18 0.00001
23 67.9 DIK2727 10 0.83 0.22 0.27 1.78 0.04
17 VSX1 122000 20p11.23- p11.22 13 65.0 HUJ616 7 0.56 0.50 −∞ 0.31 0.4
EYA2 601654 20q13.1 13 98.2 CSTBJ12 5 0.68 0.17 0.21 1.04 0.15
BMP7 112267 20q13 13 115.4 MMP9 7 0.70 0.50 −∞ −0.23 0.6
13 125.9 BMS995 6 0.66 0.50 −∞ 0.34 0.4
Table 2.
 
OAR23 Microsatellite Markers Used in the Fine Mapping Analysis
Table 2.
 
OAR23 Microsatellite Markers Used in the Fine Mapping Analysis
Microsatellite Sheep Chrom. 23 cM (SheepMap v4.7) Alleles (n) PIC Informative Meioses (n) CRI-MAP Twopoint Linkage MERLIN Parametric Multipoint Linkage MERLIN Nonparametric Multipoint Linkage
Rec. Frac. LOD LOD α HLOD NPL Score P
UW72A 0.0 2 0.23 13 0.47 −∞ −∞ 0.36 0.82 1.54 0.06
BL6 2.4 11 0.77 43 0.47 −∞ −∞ 0.35 0.87 1.73 0.04
MCM172 2.9 7 0.66 35 0.46 −∞ −∞ 0.35 0.86 1.74 0.04
BM226 7.6 8 0.70 19 0.48 −∞ −∞ 0.34 0.79 1.77 0.04
BMS2526 8.3 6 0.59 33 0.49 −∞ −∞ 0.34 0.78 1.77 0.04
DU280225 18.0 12 0.83 45 0.24 0.32 −∞ 0.31 0.92 2.16 0.02
DU330122 18.0 8 0.54 42 0.25 0.25 −∞ 0.32 0.98 2.93 0.002
CSRD148 18.6 10 0.83 26 0.39 −∞ −∞ 0.32 0.98 2.93 0.002
MCMA1 21.9 8 0.72 32 0.26 0.13 −∞ 0.42 1.49 3.45 0.0003
CABB12 22.6 6 0.70 24 0.23 0.23 −∞ 0.42 1.47 3.49 0.0002
BMS2270 23.5 6 0.66 32 0.11 0.84 −∞ 0.47 1.66 3.63 0.00014
CSSM31 30.3 10 0.51 16 0.07 1.13 −∞ 0.56 2.40 3.66 0.00012
ILSTS65 30.9 2 0.34 14 0.10 0.67 −∞ 0.55 2.40 3.68 0.00012
DIK4464 34.0 6 0.57 11 0.27 0.04 −∞ 0.47 2.11 3.55 0.0002
AGLA269 34.0 14 0.84 17 0.30 0.03 −∞ 0.47 2.11 3.55 0.0002
DU268178 9 0.80 48 0.18 1.12 −∞ 0.54 2.76 3.65 0.00013
DU216028 34.6 10 0.81 50 0.22 0.72 −∞ 0.57 3.00 4.38 0.00001
DU340520 35.1 6 0.67 39 0.25 0.20 −∞ 0.58 3.14 4.43 <0.00001
DU252884 35.4 2 0.37 19 0.17 0.51 −∞ 0.68 3.82 4.50 <0.00001
DU274690 35.7 4 0.42 19 0.37 0.01 −∞ 0.71 4.15 4.53 <0.00001
DU520666 37.7 4 0.27 20 0.00 0.60 6.51 0.91 6.85 4.78 <0.00001
DU416699 41.7 3 0.36 14 0.00 2.11 7.95 0.94 8.09 5.35 <0.00001
DU288059 41.7 4 0.55 16 0.00 0.60 8.32 0.95 8.40 5.47 <0.00001
CL638456 4 0.39 42 0.00 4.21 8.14 0.95 8.25 5.42 <0.00001
DU189183 7 0.60 42 0.04 2.67 8.79 1.00 8.79 5.48 <0.00001
DU377056 41.7 5 0.29 23 0.00 2.11 8.41 0.96 8.45 5.48 <0.00001
DU373930 41.7 8 0.63 19 0.06 1.64 8.35 0.96 8.42 5.47 <0.00001
ADCY1AP 42.7 8 0.76 43 0.03 3.52 9.62 1.00 9.62 5.49 <0.00001
DU239182 43.2 17 0.84 38 0.05 2.17 10.15 1.00 10.15 5.49 <0.00001
DU316110 44.7 6 0.69 33 0.05 1.92 10.70 1.00 10.70 5.50 <0.00001
DU441780 44.7 14 0.68 53 0.03 3.79 10.70 1.00 10.70 5.50 <0.00001
MAF35 44.7 5 0.52 43 0.09 1.53 10.70 1.00 10.70 5.50 <0.00001
DU291927 45.6 5 0.55 48 0.03 3.22 10.59 1.00 10.59 5.41 <0.00001
DU259631 48.1 6 0.59 43 0.04 2.70 −∞ 0.85 6.68 5.05 <0.00001
UW69A 48.1 6 0.42 41 0.08 1.46 −∞ 0.83 6.60 5.05 <0.00001
DU408420 48.1 3 0.64 16 0.00 2.11 −∞ 0.83 6.61 5.05 <0.00001
CSRD172 50.0 3 0.42 25 0.00 2.10 −∞ 0.80 5.43 4.70 <0.00001
DU171881 50.0 5 0.62 32 0.12 0.59 −∞ 0.79 5.32 4.69 <0.00001
BMS1332 50.0 6 0.56 21 0.00 1.51 −∞ 0.74 5.00 4.67 <0.00001
HEL6 51.7 3 0.37 2 0.00 0.30 −∞ 0.73 4.24 4.26 0.00001
MCM136 52.8 10 0.80 42 0.09 1.18 −∞ 0.63 3.14 4.06 0.00002
DIK5068 62.1 5 0.63 26 0.48 −∞ −∞ 0.30 0.79 2.36 0.009
CGBP 63.2 3 0.12 0 0.00 −∞ −7.98 0.32 0.83 2.34 0.01
DIK2727 67.5 10 0.83 17 0.22 0.27 −∞ 0.35 0.88 2.27 0.012
DU264615 74.5 5 0.55 34 0.16 0.47 −∞ 0.23 0.45 1.51 0.07
URB031 83.9 3 0.23 4 0.00 0.30 −0.51 0.41 0.54 1.17 0.12
Table 3.
 
Pathology Features of Ovine Microphthalmia
Table 3.
 
Pathology Features of Ovine Microphthalmia
Eye Structure Phenotype Description • Microscopic Details
Eye lids, conjunctiva, nictitating membrane Regular
Cornea Diffuse clouded showing distinct vascularization, pigmentation and a granulated surface
 • Variable lymphohistiocytic infiltration
 • Absence of the Descemet’s membrane and posterior epithelium
Anterior eye chamber Not visible
Lens, vitreous body Not recognizable
Iris and ciliary body  • Incorporated within or restricted to the periphery of the tissue mass
Posterior eye chamber, retina White mass composed of connective tissue with blood vessels, smooth muscle, cartilage and fat tissue
 • Anterior: islets of lacrimal glands and multiple cystic structures frequently lined by squamous epithelium and filled with keratin
 • Posterior: partly covered by detached retinal structures sometimes forming rosettes
Optic nerve Reduced diameter
 • Highly cellular
Table 4.
 
List of Recombinants Present in the Whole Set of Genotyped Sheep Families, Arrayed According to the Position of the Recombination Event*
Table 4.
 
List of Recombinants Present in the Whole Set of Genotyped Sheep Families, Arrayed According to the Position of the Recombination Event*
Marker cM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
UW72A 0.0 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 133 135 135 135 135 135 133 133
BL6 2.4 190 180 190 186 180 190 180 190 194 186 174 180 190 190 180 180 190 190 190 180 180 190 182 190
MCM172 2.9 143 135 143 141 135 135 135 143 143 135 143 135 135 135 135 135 149 135 147 135 135 135 143 149
BM226 7.6 126 126 140 126 126 124 126 126 124 126 126 126 124 126 126 126 132 126 140 126 140 126 126 132
BMS2526 8.3 150 156 158 150 150 152 150 150 156 150 154 150 154 154 150 150 150 154 150 150 150 150 154 150
DU280225 18.0 442 442 424 436 444 444 444 442 426 432 444 444 432 436 424 444 444 436 442 444 436 434 424 444
DU330122 18.0 291 289 289 289 289 289 289 291 277 303 289 289 303 289 289 289 289 289 289 289 293 287 289 289
CSRD148 18.6 391 397 401 375 401 393 401 391 401 391 397 291 375 375 401 401 401 375 397 291 393 377 401 401
MCMA1 21.9 126 136 140 124 136 124 136 126 150 130 126 136 136 136 136 136 136 136 136 136 126 144 140 136
CABB12 22.6 240 230 230 240 230 232 230 240 228 230 240 230 230 234 240 230 230 234 230 230 240 226 230 230
BMS2270 23.5 94 92 90 94 92 92 92 94 96 92 90 92 92 92 92 92 92 92 92 92 92 92 90 92
CSSM31 30.3 161 131 149 165 131 131 131 161 131 161 131 131 131 131 131 131 131 131 131 131 131 163 149 131
ILSTS65 30.9 115 115 115 115 115 117 115 115 117 115 115 115 115 115 115 115 115 115 117 115 117 115 115 115
DIK4464 34.0 226 224 218 224 224 216 224 226 226 226 230 226 226 226 226 226 226 224 216 226 224 226 218 226
AGLA269 34.0 238 266 272 300 270 300 270 238 266 300 266 270 272 266 266 270 270 266 300 270 300 238 272 270
DU268178 106 106 116 110 110 116 110 106 106 112 116 116 114 114 110 123 123 106 116 116 112 106 116 112
DU216028 34.6 221 213 221 217 213 225 213 221 219 193 193 225 217 217 213 213 193 213 225 193 217 221 221 193
DU340520 35.1 225 219 221 225 225 219 225 225 221 225 221 219 223 223 225 225 225 219 219 221 225 221 221 225
DU252884 35.4 324 324 324 324 324 326 324 324 324 326 326 326 324 324 324 324 326 324 326 326 326 324 324 326
DU274690 35.7 188 188 188 188 188 190 188 188 192 192 182 188 188 192 188 188 188 188 190 182 188 192 188 188
DU520666 37.7 226 226 226 226 224 226 224 226 226 226 226 226 226 226 226 226 226 226 226 224 226 226 226 226
DU416699 41.7 158 158 156 158 158 133 158 158 158 158 158 158 156 158 158 158 158 158 133 158 158 158 156 158
DU288059 110 110 120 110 120 115 120 110 115 115 110 115 120 120 110 110 110 110 115 115 110 120 120 110
CL638456 41.7 182 182 182 182 182 171 182 182 182 182 182 171 182 182 182 182 171 182 171 171 171 182 182 171
DU189183 41.7 213 199 213 205 199 199 199 213 213 213 199 198 199 199 199 199 199 199 199 198 215 199 213 199
DU377056 41.7 289 289 289 289 289 302 289 289 289 289 289 302 289 289 289 289 289 289 302 302 289 289 289 289
DU373930 315 315 315 307 307 307 307 315 315 315 315 307 307 307 315 315 315 315 307 307 318 307 315 315
ADCY1AP 42.7 106 108 87 116 87 108 87 106 114 106 106 108 87 87 106 106 106 108 108 108 114 87 87 106
DU239182 43.2 291 292 292 292 294 280 294 291 272 280 292 261 272 294 292 292 292 292 280 261 292 292 292 292
DU441780 44.7 146 146 210 179 161 146 161 146 196 148 210 146 146 161 210 210 210 146 146 146 210 210 210 210
DU316110 44.7 131 129 131 131 129 131 129 131 131 131 129 132 131 131 129 129 129 129 131 132 131 131 131 129
MAF35 44.7 106 104 106 110 104 106 104 106 104 104 106 106 106 104 106 106 106 104 106 106 106 106 106 106
DU291927 45.6 247 243 247 245 243 247 243 247 243 241 247 247 247 243 247 247 247 243 247 247 243 243 247 247
DU259631 48.1 154 154 158 152 152 154 152 154 154 154 154 154 154 152 152 152 152 154 154 154 154 154 158 152
UW69A 48.1 154 156 154 158 158 154 158 154 158 156 158 158 158 158 158 158 158 156 154 158 154 158 154 158
DU408420 48.1 288 288 286 288 288 286 288 288 290 288 296 294 294 288 288 288 288 288 286 294 288 294 286 288
CSRD2172 50.0 188 188 192 192 192 188 192 188 192 192 188 188 192 188 192 192 192 188 188 188 192 188 192 192
DU171881 50.0 164 178 164 178 162 164 162 164 176 164 164 162 164 162 164 164 162 178 164 162 164 162 164 162
BMS1332 50.0 136 138 142 142 142 140 142 138 142 140 142 140 142 140 142 142 140 138 140 140 140 140 142 140
HEL6 51.7 181 181 185 185 185 185 185 185 185 185 181 185 185 185 185 185 185 181 185 185 185 185 185 185
MCM136 52.8 148 142 138 146 164 164 138 148 144 164 142 142 164 142 140 140 140 142 164 142 164 142 146 140
DIK5068 62.1 90 92 92 90 92 92 92 90 98 90 90 90 92 90 90 90 92 90 94 90 94 90 92 92
CGBP 63.2 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265 265
DIK2727 67.5 208 194 194 194 202 212 202 208 226 208 206 212 208 212 208 208 184 212 212 212 212 212 194 184
DU264615 74.5 118 124 124 118 124 116 124 118 124 118 116 116 124 118 118 124 116 118 116 116 124 116 124 116
URB031 83.9 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234 234
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