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Description:

  1. Top of page
  2. Description:
  3. Genotyping:
  4. SNP Association with LP and CSNB:
  5. Acknowledgements:
  6. Conflicts of interest
  7. References

Leopard complex spotting occurs in several breeds of horses and is caused by a single incompletely dominant autosomal locus, LP.1 In the Appaloosa breed, homozygosity for LP has been associated with congenital stationary night blindness (CSNB).2 Decreased expression of TRPM1 has been implicated as the cause for both LP and CSNB.3LP and CSNB were fine-mapped to a 173 -kb haplotype on ECA1 and Illumina sequencing identified six SNPs for further investigation (Table 1).4 In this study, we investigate these six SNPs for association with CSNB in the Appaloosa and for association with LP in several breeds of horses.

Table 1.   Candidate SNPs investigated for LP and CSNB. PCR conditions and results of SNP association with LP genotype in 10 animals from five different breeds are presented.
SNPs testedForward primer sequenceReverse primer sequenceAnnealing temp. (°C)PCR product size (bp)Associationwith LPgenotype insmall panel
  1. 1SNPs with complete association were investigated further and were completely associated in a panel of 541 horses.

  2. CSNB, congenital stationary night blindness.

  3. LP, leopara complex spotting.

ECA1 g.108281765T>C5′-ACTGGGGAGTCTGTCCACTG-3′5′-CTTAGCTCAAGCCCCTTCCT-3′6532610/10 associated1
ECA1 g.108288853C>T5′-CAGTGAACCGGGCTCTTAAA-3′5′-GCAGCCTCCAAAACTGACTC-3′5822310/10 associated1
ECA1 g.108337089T>G5′-CTTAGGTGAGGCGAGGTCAG-3′5′-ACCTGGCAGGCCTATCTTCT-3′6548510/10 associated1
ECA1 g.108489901G>A5′-TGTCCTCGGCCACGCCC-3′5′-GTCAGCCCCCAGCCACAT-3′65153 8/10 associated
ECA1 g.108497669C>A5′-CCTGCACCGTGACTTCTATG-3′5′-GCCAAGCTGGAGAGAACACT-3′60246 9/10 associated
ECA1 g.108497990C>T5′-TTCTCTCCAGCTTGGCAGTT-3′5′-GATGCTCAGTTCAGCCACAA-3′62306 8/10 associated

Genotyping:

  1. Top of page
  2. Description:
  3. Genotyping:
  4. SNP Association with LP and CSNB:
  5. Acknowledgements:
  6. Conflicts of interest
  7. References

DNA was isolated from blood or hair of unrelated horses from breeds segregating for LP: Appaloosa (N = 205), Knabstrupper (N = 66), Noriker (N = 112), American Miniature (N = 63), Pony of the America (N = 20), British Spotted Pony (N = 25), and Australian Spotted Pony (N = 10). DNA was also isolated from the Thoroughbred (N = 37) and American Quarter Horse (N = 3), which are breeds not segregating for LP. LP genotype and CSNB status were determined as previously described.3 Initially, all six SNPs were investigated by PCR amplification and sequencing DNA from 10 individuals (Table 1). Three of these SNPs did not show complete association with LP genotype in this panel and were excluded as the causative mutation and thus not investigated further. The other three SNPs showed complete association and were either genotyped by direct sequencing or by custom TaqMan genotyping assays in 531 additional individuals (Assay IDs AHS0Q19, AHRRSV1, and AHT9O8H, Applied Biosystems) (Table 1). All TaqMan assays were performed in 5 -μL reactions on a Mastercyler® ep realplex thermocycler (Eppendorf).

SNP Association with LP and CSNB:

  1. Top of page
  2. Description:
  3. Genotyping:
  4. SNP Association with LP and CSNB:
  5. Acknowledgements:
  6. Conflicts of interest
  7. References

ECA1 g.108281765T>C, ECA1 g.108288853C>T, and ECA1 g.108337089T>G genotypes were analysed for association with LP genotype and CSNB status by chi-squared analysis. All three SNPs were completely associated with LP (N = 513, X2 = 1026, P ≪ 0.0005) and CSNB (N = 28, X2 = 28, P ≪ 0.0005). It is possible that one of these SNPs is the causative mutation for LP and CSNB. However, none of these are located in exonic regions that have previously been characterized, and thus further investigation is warranted.4 It is also likely that these SNPs may simply be associated and are not causative. Nevertheless, any of these SNPs could be used as a DNA test for LP and CSNB until the causative mutation has been identified or confirmed.

Acknowledgements:

  1. Top of page
  2. Description:
  3. Genotyping:
  4. SNP Association with LP and CSNB:
  5. Acknowledgements:
  6. Conflicts of interest
  7. References

The authors thank all of the horse owners who provided samples. We thank Karla Brown, Scott Lawson, Elizabeth Kowalski and Janelle Nelson for their assistance in DNA extraction and Dominic Trillizio for his technical assistance. We acknowledge Eppendorf for use of equipment. This study was supported by the Heather Ryan and L. David Dube′ Veterinary Health and Research Fund and a Dana Faculty Development Grant from the University of Tampa.

References

  1. Top of page
  2. Description:
  3. Genotyping:
  4. SNP Association with LP and CSNB:
  5. Acknowledgements:
  6. Conflicts of interest
  7. References