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• Locus Chromosome Reference ASB17 2 (18) LEX33 4 (22) HMS2 10 (19) VHL20 30 (23) LEX3 X (20) HTG6 15 (24) ASB23 3 (21) AHT4 24 (25) ASB2 15 (18) AHT5 8 (25) HMS7 1 (19) HTG4 9 (24) HMS3 9 (19) HMS6 4 (19) HTG10 21 (14) HTG7 4 (14) Results The PCR amplicons ranged between 93 bp and 211 bp in size. As shown in Table 2, the total Na was 91 in the registered group, with a mean of 5.69 per locus, and 123 alleles in the nonregistered group, with a mean of 7.69. The Na per locus ranged from 3 for HTG6 and HTG7 to 8 for ASB2 in the registered group, while in the nonregistered group, the Na ranged from 4 for HTG7 to 14 for ASB17. The Ne varied from 1.86 for HTG7 to 5.464 for ASB17 in the registered group and from 2.141 for HTG7 to 988 for ASB17 in the nonregistered group. The mean Ne was 3.747 in the registered group and 4.476 in the nonregistered group. The observed heterozygosity per locus in the registered group varied from 0.362 for HTG7 to 0.915 for LEX33 and from 0.469 for HMS2 to 0.878 for ASB17 for the nonregistered group, with means of 0.694 and 0.711, respectively. The lowest PIC value for both groups was for HTG7 (0.385 in the registered group and 0.454 in the nonregistered group), while the highest value was for ASB17 (0.781 in the registered group and 0.803 in the nonregistered group). The mean PIC was 0.657 in the registered group and 0.715 in the nonregistered group. The individual PE ranged from 32% at the HTG7 locus to 80% at ASB17 for the registered group and 41% at the HTG7 locus to 84% at ASB17 for the nonregistered group. The CPEs for all of the loci were more than 99.999% in each group. The Figure shows the CPE values for both groups as a function of the number of microsatellite loci. Discussion How informative a locus is depends upon the Na exhibited by the locus and the frequency distribution of these alleles in the population (8). The mean Na in the present study was higher than that seen in Sorraia, a breed that has a high level of inbreeding (29). Values of diversity statistics similar to those observed here for the Arabian breed have been Table Number of alleles (Na), number of effective alleles (Ne), observed (Ho) and expected (He) heterozygosity, polymorphic information content (PIC), probability of exclusion (PE), and combined probabilities of exclusion (CPE) for registered and nonregistered Arabian horses. Group Registered Nonregistered Locus Na Ne Ho He PIC PE Na Ne Ho He PIC PE ASB17 7 4 0.872 0.817 0.781 0.800 14 9 0.878 0.833 0.803 0.839 HMS2 7 2 0.872 0.809 0.771 0.789 9 8 0.469 0.830 0.799 0.831 LEX3 7 7 0.426 0.790 0.748 0.755 9 7 0.837 0.825 0.791 0.814 ASB23 6 2 0.702 0.767 0.723 0.731 8 0 0.735 0.803 0.766 0.785 ASB2 8 0 0.766 0.754 0.707 0.713 9 0 0.837 0.800 0.763 0.784 HMS7 6 2 0.787 0.766 0.717 0.709 8 1 0.673 0.805 0.766 0.781 HMS3 6 0 0.809 0.751 0.701 0.697 7 0 0.714 0.803 0.762 0.769 HTG10 5 9 0.766 0.749 0.698 0.689 8 5 0.694 0.782 0.743 0.762 LEX33 5 8 0.915 0.742 0.690 0.675 9 5 0.796 0.780 0.737 0.747 VHL20 5 4 0.745 0.709 0.657 0.650 8 4 0.714 0.774 0.732 0.742 HMS6 5 2 0.702 0.696 0.639 0.626 6 2 0.776 0.765 0.720 0.726 AHT4 6 3 0.596 0.698 0.634 0.604 6 1 0.735 0.760 0.712 0.711 AHT5 6 0 0.660 0.675 0.610 0.594 7 5 0.673 0.720 0.666 0.657 HTG4 6 5 0.553 0.607 0.560 0.559 5 8 0.633 0.648 0.595 0.581 HTG6 3 4 0.574 0.594 0.499 0.434 6 2 0.653 0.690 0.621 0.587 HTG7 3 8 0.362 0.463 0.385 0.329 4 1 0.551 0.533 0.454 0.408 Mean 69 7 0.694 0.712 0.657 CPE >0.9999 69 4 0.711 0.759 0.715 CPE >0.9999 recorded for a number of equine populations, including the Thoroughbred (30), Lipizzaner (16), Lithuanian native horse breeds (15), and Pantaneiro horses (9). However, the individual PE for 13 of the 16 loci used in this study was higher than that reported in the Thoroughbred (30).
• Furthermore, all 15 loci tested by Monies et al. (31) had lower PE values than we found using the same loci; this may be due to the different Arabian population that was used. Furthermore, some problems in genotyping the HMS3 locus were indicated by Monies et al. (31). Similar problems in HMS3 were noticed in the Potoka horse breed (32), which may be a result of nonamplification due to a base substitution in the sequence flanking the M allele priming site of the HMS3 (33). Such a problem was not noticed in our results as well in different horse breeds (9,10,13). In this study, for the registered group, 7 microsatellite markers (ASB17, HMS2, LEX3, ASB23, ASB2, HMS7, and HMS3) had high PIC values (>7). A very high level of CPE (>0.99999) can be reached using only 6 of the 16 loci
• (Figure), which makes these markers highly valuable for use in parentage testing for these Arabian horses. Ellegren et al. (24) suggested that at least 10 microsatellite loci should be used to achieve maximum exclusion in horses, but our results show that fewer loci can give a relatively high exclusion power, similar to the results found by Sereno et al. (9). Two markers, HTG6 and HTG7, were found to have a PIC value of less than 0.5 for the registered group. As these markers are considered uninformative (28) and they are in the less efficient 3plex, these 2 loci plus HMS2 can easily be excluded from routine parentage testing for the Arabian horses with no significant loss of exclusion power. The nonregistered group had a mean PIC of 0.715, which was significantly higher than the mean PIC in the registered group (P < 0.0001). This value reflects a higher level of variation in the nonregistered group compared to the registered horses. The difference of variation between the registered and nonregistered horses may be due to the restricted mating in the registered group, where registered horses must be mated within the same RASAN, while the nonregistered horses can be crossed with any horse. The higher Na indicates that horses other than those within the RASAN may have been introduced into this group or that the nonregistered group has retained alleles lost in the registered horses.
• The heterozygosity in both groups was within the range of heterozygosity in different horse breeds (9,34). The heterozygosity levels were consistent with the high Na per locus seen in our study and indicate no serious loss of variability due to the breeding method employed by the local breeders in Syria compared to other horse breeds (34). Comparing the genetic variability of Syrian populations tested here with some other modern Arabian populations, we can recognize substantive disagreements in variability. In the Polish Arabian population, an increase in the inbreeding coefficient and reduction in the genetic diversity were found (35), and the same situation was reported in Spanish Arabian horses (36). The reduction in variability in these modern Arabian populations was due to the founder effect caused by a breeding policy that included mating between relatives. However, the mean heterozygosity for Syrian horses was a little bit higher than what was reported in a similar recent study about a different population of Arabian horses with some reservations on HMS3 locus (31). The International Stud Book Committee (ISBC) has required that the CPE value for parentage verification and an individual identification in a horse be higher than 0.9995 (37). Here, we found that the CPE using 12 autosomal loci was greater than the value required by the ISBC. Based on these results, we confirm that the loci of the 8plex and 5plex PCR can be used in parentage testing with high efficiency for the Arabian horses from Syria. The data presented here will help solve the problems related to registration issues and will provide the breeders with an effective tool for confirming lineages. Genetic testing will also generate valuable data necessary for the conservation of this breed. Acknowledgments
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