An investigation of the role of Insulin-Like Growth Factor 1 (IGF-1) in determining adult height of the horse

IntroductionThe Insulin-Like Growth Factor-1 (IGF-1) gene is one of a number of determinants of adult height in several animals including the dog (Sutter et al., 2007). However, whilst causative polymorphisms can be tied to height variation between dog breeds, this is not true of other systems, such as humans. Welsh Pony and Cobs (a UK-native breed), are categorised into four distinct sections largely in accordance with their height: Section A, <122 cm; Section B, <137 cm; Section C, 122 to 137 cm, Section D, > 137 cm. Using the Welsh Pony and Cob as the example, the aim of this work was to assess whether height of the horse is influenced by single-nucleotide polymorphisms (SNP) of the IGF-1 gene. Material and method Hair follicle samples were collected from 6 individuals of each section (n = 24) and total DNA was extracted using Qiagen DNA MicroKit (Qiagen, UK). Amplification by PCR was performed using primers (sequences available on request) based upon the published canine, 1 Kb region of the IGF-1 sequence (Sutter et al., 2007) surrounding the primary causative SNP, re-designed for the equine genome and 0.4 of each forward and reverse primer was incubated with 10 ul (50 ng) template DNA, 20 ul Sensimix (Kapa Biosystems, UK) made up to a total volume of 40 ul with water. Reactions were incubatedat 95oC for 10 min, followed by 40 cycles of 95oC for 30 sec, 58oC for 45 sec and 72oC for 1 min, with a final extension of 72oC for 10 min. Products were size fractionated by gel electrophoresis, excised, transformed, cloned (TOPO TA Cloning Kit, Invitrogen, UK), extracted (QIAprep Minikit, Qiagen, UK) and sequenced (Geneservice, UK) to produce a comparative set of homologous sequences of the IGF1 gene. In addition, 3 individuals of each section were utilised to determine whether size variation existed between putative microsatellite markers (including those outside the 1 Kb region sequenced) within the IGF-1 gene. Briefly, 7 putative microsatellite primers (sequences available on request) within the IGF-1 gene were designed and 0.4 mM of each forward and reverse fluorescent markers was incubated with (50 ng) 3 ul template DNA, 10 ul Sensimix, made up to a total volume of 20 ul with water. Reactions were incubated at 95 oC for 10 min followed by 35 cycles of 95oC for 30 sec, 55 oC for 1 min, 72 oC for 2 min extension time, with a final extension of 72oC for 10 min. Amplified PCR products were subjected to capillary sequence analysis (ABI3730 capillary sequencer, Applied Biosystems) and compared using an internal size standard. Together, these data provided the nucleotide sequence for a portion of the IGF-1 gene surrounding the base position homologous to the causative SNP in the canine sequence for each of the four Welsh Pony and Cob sections and provided graphical evidence of minor shifts in the gene size between sections.Consensus sequences were aligned using ClustalW algorithm to identify base pair variation within the amplified portion of the IGF-1 gene acrossthe four sections. Differences in PCR fragment sizes of the microsatellite regions were identified using Gene-Mapper, version 3.7 (Applied Biosystems). Results There was no section-specific change in nucleotide sequence within the 1 Kb region of IGF-1 surveyed between the four distinct sections and therefore, height classifications of the Welsh Pony and Cob. However, for the 3 animals of each section selected for further putative microsatellite analysis that lay outside the 1 Kb region, differences between size of IGF-1 sequences were apparent; microsatellite SSR2 revealed 2 bp repeat at position 399-401 for Section A and section B individuals that was absent for Section C and D (Fig. 1 a and b); microsatellite SSR7 revealed short 4 bp repeat allele at position 438-442, present in 2 of 3 Section D individuals but were absent in Sections A, B and C (Figure 1, c and d).Conclusion The lack of section-specific allelic variationaround the causative locus of the dog in the Welsh Pony and Cobs suggest that IGF-1 is unlikely to have a major effect upon adult height of this breed of horse. Whilst allelic variation was observed in Section D animals for one microsatellite locus, no IGF-1 variation appeared to correlate with the smaller phenotype of Section As. However, the study only considered a portion of the IGF-1 gene and therefore, lends justification for further work to extend the investigation of surrounding genes that influence IGF-1 activity