Synergy Between MC1R And ASIP For Coat Color In Horses And Development Of 13-plex PCR Systems For Commonly Used STRs In Horses

In recent years,China’s horse industry has developed rapidly.Horses with high-quality pedigree are imported to China for participating in competitions and offspring.Pure pedigree and different coat colors directly affect the value of horses.Several genes have been suggested to influence coat color in horses.Melanocortin-1 receptor(MC1R)and agouti signaling protein(ASIP)which is the significant genes for regulating coat color in mammals.Mutations at the C901T position of MC1R lead to chestnut coat color,while the 11 bp deletion at the second exon of ASIP causes the black coat color,however,how the combination of the two important genes correlate with the horse coat color has not been studied.We collected 709 horses from 15 breeds with seven different coat color(black,brown,dark bay,bay,chestnut,gray and white),and examined the frequencies of each genotype for both gene combinations in all phenotypes.We characterize the possible synergistic effects of these two genes on the variation of horse coat color by analyzing the distribution of genotypes.National horse associations register and maintain breeding records through individual identification and paternity testing techniques to prove the pedigree of foals.ISAG(International society for animal genetics)has recommended 17 microsatellites for horse individual identification and paternity testing,contains 12 core loci(AHT4,AHT5,ASB2,ASB17,ASB23,HMS2,HMS3,HMS6,HMS7,HTG4,HTG10,VHL20)and 5 non-core loci(CA425,HMS1,HTG6,HTG7 and LEX3).Previous methods had disadvantages,that decreased the accuracy of the results(primer specificity issue),lacked the inclusion of all commonly used STRs,or increased the experimental cost and time.We aimed to develop two multiplex STR typing system to resolve the above issues.The sensitivity,species-specificity,repeatability and reliability of the two systems were verified by subsequent experiments.The main results are as follows:(1)We found that the phenotypes of horse coat colors were significantly related with the genotypes of MC1R and ASIP.The E~EE~E genotype frequency at MC1R decreased from dark to light colors(black=64.5%,brown=67.5%,dark bay=47.0%,bay=16.5%and chestnut=0.0%),whereas the A~AA~A genotype frequency at ASIP increased as coat color lightened(black=0.0%,brown=22.9%,dark bay=69.2%and bay=83.0%).When combined genotypes at MC1R and ASIP were examined,different advantage genotype combinations were found for each color:black E~EE~E-A~aA~a(64.5%),brown E~EE~E-A~AA~a(47.0%),dark bay E~EE~E-A~AA~A and E~EE~e-A~AA~A(36.2%and 33.0%,totally 69.2%),bay E~EE~e-A~AA~A(69.6%)and chestnut E~eE~e-A~AA~A(62.6%).These results indicated that MC1R and ASIP may synergistically affect the levels of melanin in equine coat colors through the different genotype combinations.(2)Development of a 13-plex STR typing system for horses:Through the redesigning of microsatellite primers and optimization of multiplex PCR system,a 13-plex STR typing systems for horses were developmented.The genotyping profile of 12 recommended loci can be generated by one PCR reaction,and identify the gender of tested horse.Optimized volume of the 13plex system was 10 ul.The time of PCR reaction was 55 minutes.Full profiles were easily generated in one fast PCR reaction using a low-cost polymerase,as little as 1 ng of horse DNA template.The system was sensitivity,species-specificity,repeatability and reliability.The combined probability of paternity exclusion for this system were 0.994659935(CPEduo)and 0.999854032(CPEtrio).This system can be used for individual identification and paternity identification of horses,and also for genetic diversity research of horses based on microsatellite markers.(3)Development of a 5-plex STR typing system for horses:A5-plex STR typing system was established for the detection of five non-core loci.The system was sensitivity,species-specificity,repeatability and reliability.Optimized volume of the5-plex system was 10 ul.The PCR time was 55 minutes.Full profiles were easily generated in one fast PCR reaction using a low-cost polymerase,as little as 0.5 ng of horse DNA template.The CPEduo and CPEtrio of this system were 0.778582831 and 0.935585818,respectively.