Characterization and linkage map assignments for 61 new horse microsatellite loci (AHT49-109).

This research focuses on the development and characterization of 61 new horse microsatellite markers, achieved through the use of a genomic horse DNA library, screening for dinucleotide repeats, sequencing and polymerase chain reaction (PCR) techniques. These markers were confirmed to follow Mendelian inheritance patterns across three generations of different horse breeds, with most markers being autosomal in origin and a few segregating as X chromosome markers.

Construction of Horse DNA Library

• Genomic horse DNA was digested using the restriction enzyme Sau3A and the fragmented DNA was cloned into the M13 vector to create a short insert library.
• The library was then screened for dinucleotide repeats using a highly sensitive 32P-labelled probe, with positive regions identified and stored for further investigation.
• In order to identify isolated positive plaques for each primary pick, secondary screenings were performed.

Sequencing and Genotyping

• DNA from each of the positively identified plaques was sequenced using standard forward and reverse primers, along with unique internal primers in some instances.
• The sequencing reactions were processed using an Applied Biosystems automated sequencer for accurate analysis.
• Primers suitable for genotyping were designed using a tool called ‘Primer’. The sequences of these primers were listed in Table 1 of the study.

PCR Conditions

• Polymerase Chain Reactions (PCRs) were conducted using a specific PCR buffer, dNTPS, Amplitaq GOLD, and specific concentrations of MgCl2 and primers in a given volume.
• The PCR conditions involved heating cycles at different temperatures to facilitate the different stages of the PCR process.

Mendelian Inheritance and Nature of Markers

• The inheritance pattern of these markers was observed across three generations of two full-sibling families, including different breeds such as Arab, Thoroughbred, Welsh Cob, Polish Warmblood and Icelandic breeds.
• The inheritance pattern and segregation of the alleles suggested an autosomal origin for most markers while a few markers segregated like those on the X chromosome.