Molecular genetic diversity of donkey (Equus asinus) in South Korea

The research on domestic donkey has little information and criteria comparing to other livestock, companion animal in South Korea. We analyzed genetic database of domestic donkey using microsatellite marker to clarify domestic donkey identification and paternity test. It is the first microsatellite marker analysis on domestic donkey in South Korea. We studied 179 horse samples from 50 Thoroughbred, 50 Jeju Halla horse, including 79 donkeys then analyzed 15 microsatellite marker (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG10, LEX3 and VHL20). We observed genetic diversity from biostatic analysis of them. The number of alleles on total average is 6.08 observed from 1 (ASB17), 2 (HMS1) to 14 (AHT5). The observed heterozygosity (OHet) is from 0.0000 (ASB17, HMS1) to 0.8608 (ASB23) which is mean value of 0.4861, the expected heterozygosity (EHet) is from 0.0000 (CA425) to 0.9104 (AHT5) with mean value of 0.5915, and the Polymorphism Information Content (PIC) on each group of microsatellite marker is from 0.0000 (ASB17) to 0.8968 (AHT5) observed as a mean value of 0.5374. Among 15 markers AHT4, AHT5, ASB23, CA425, HMS2, HMS3, HTG4, HTG10, LEX3 is observed above 5.000. The results on 15 microsatellite marker analysis of 3 horse groups is that the donkey had 0.5915 EHet, 0.4861 OHet on average, Thoroughbred had 0.6721 EHet, 0.6587 OHet on average, and the Jeju halla horse had 0.7898 EHet, 0.7093 OHet on average observed. Furthermore the mean alleles value is observed as 6.08, 4.83, 8.00 in donkey, Thoroughbred, Jeju halla horse breed in each.

object level as well as in the group, so it is used as a useful tool for genetic mapping as well 1 as information about the hereditary diversity of animals, including human and plants [8, 10, 2 13, 14, 24]. 3 Polymorphism of microsatellite is formed by the number of repeats of the basic repeating unit, 4 which is inherited according to Mendel's law of inheritance of a half from parents. individual [14]. In parentage diagnosis, microsatellite and minisatellite can be used 1 4 simultaneously, but using microsatellite used as a standard for international genetic mapping   It is judged that in South Korea, donkey will be in the limelight as a high-value genetic 2 1 resource in the future. In addition, it may be disguised as horse meat. To monitor this, it is 2 2 necessary to protect breeding farms through a donkey meat traceability system. This study 2 3 aims to secure basic data to increase the protection and utilization of genetic resources of 2 4 domestic donkey, getting ready for the traceability system of domestic donkey using 2 5 microsatellite markers and investigating the hereditary diversity of donkey. For the quantification of the separated DNA, the absorbance was measured at the 260 nm and 5 280 nm wavelengths using Nanodrop TM 8000 Spectrophotometer (Thermo, USA), and DNA 6 extracted based on absorbance at 260 nm at the value of 1.0 (Path length = 10.0 mm) was 7 diluted, and the concentration was adjusted to 50 ng/ul. In addition, samples with too high or 8 too low A260/A280 ratio based on 1.8 purity was judged to be low, and DNA was re-9 extracted from the blood to use in the experiment. Electrophoresis Cell (TaKara, Japan).  conducted. As for composition for PCR, template DNA 2 μ l, 10 Pmol forward and reverse 2 0 primer 2 ul, respectively and sterile distilled water 2 ul were mixed on PCR Premix buffer 2 1 (Qiagen, Germany), adjusted to 15 μ l in total, and then, amplified by GeneAmp PCR system 2 2 9700 (Applied Biosystems, USA).

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In the PCR process, heating at 95℃ for 10 min. to induce degeneration and three steps of denaturation at 95℃ for 30 sec., annealing at 60℃ for 30 sec., and extension at 72℃ for 60 2 5 sec. were repeated 30 times in total, and lastly, the final extension process was made at 72℃ 2 6 for 60 min. For the single PCR, template DNA 2 μ l, 10 Pmol forward and reverse primer 2 ul, 1 respectively and sterile distilled water 6.5 ul were mixed on PCR Premix buffer (Qiagen, 2 Germany), adjusted to 25 μ l in total, and then, amplified by GeneAmp PCR system 9700 3 (Applied Biosystems, USA). 4 In the PCR process, heating at 95℃ for 5 min. to induce degeneration and three steps of 5 denaturation at 95℃ for 30 sec., annealing at 60℃ for 30 sec., and extension at 72℃ for 60 6 sec. were repeated 35 times in total, and lastly, the final extension process was made at 72℃ 7 for 60 min.

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With the amplified DNA, dielectrolysis was made with 2.5% agarose gel, and by comparing 9 the amplification and concentration indirectly, it was tested for genotype determination.

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The genotype analysis after the PCR was as follows: Mixing the amplified fragment 0.5 ul,  [16].

Analysis of the genetic diversity of donkey 1 3
As shown in Table 1, it was observed that the number of allelic genes was 1 (ASB17) and 2 Halla horse, allelic genes and frequency are as shown in Table 2  horse.

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Based on the result of an analysis of microsatellite markers, dendrograms of groups for the 2 8 standard genetic distance and the minimum genetic distance were drawn, using Unweighted 2 9 Pair Group Method with Arithmetic mean (UPGMA) and Neighbor Joining (NJ) clustering 1 method, based on the genetic matrix and presented in Figure 1. To compare the dendrograms, 2 in three breeds 179 horses, donkey and Thoroughbred breed formed a clearly different group, 3 but it was observed that Jeju Halla horse formed a group, mixed with a Thoroughbred horse.  Genotype is a stable unit at the object level but becomes an unstable unit through generations.

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In Mendel's the law of segregation, "Genotype is the combination of two allelic genes, and 1 2 one side of the allelic genes, only, is delivered to the next generation in the same probability,"  In a shift in generations, that only one of the allelic genes is delivered at the same probability 1 6 means that, since individual's genotype is the combination of two allelic genes, the 1 7 probability at which one allelic gene is delivered is 1/2. At the chromosome level, allelic  As compared to genotype frequency, gene frequency is very stable beyond the generation, so what is directly related to the phenotype of individual is genotype [7, 20].

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As a results of an analysis of genotype distribution using microsatellite DNA markers with 0.7093 in Jeju Halla horse. Also, it was observed that the average number of allelic genes was 1 6.00, 4.83 and 8.00 in donkey, Thoroughbred and Jeju Halla horse, respectively. It was noted 2 that Equus asinus or Thoroughbred was fixed into a single breed. However, it is assumed that 3 Jeju Halla horse had high OHet, EHet and number of allelic genes because they are cross-4 bred horses, in which the genes of various breeds were mixed. In addition, it was observed 5 that microsatellite markers AHT4, AHT5, ASB23, CA425, HMS2, HMS3, HTG4, HTG10 6 and LEX3 were Polymorphism Information Content (PIC) over 5.000, so it is expected that 7 they can be utilized in the differentiation of individuals of Equus asinus or paternity test.

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Based on PIC value of each marker, the validity and reliability of the marker can be estimated, 9 and if PIC value is higher than 0.5000, it is judged that the reliability of the marker is valid 1 0 for blood analysis. If it is higher than 0.7000, it is known that it has universal validity for 1 1 analysis and can get a result of high reliability.

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In a single gene locus, the indicator that expresses the diversity of a group is heterozygosity.

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Heterozygosity is defined as "the probability that, when randomly two allelic genes are 1 4 extracted from a group, the two may differ." In an association analysis or linkage 1 5 disequilibrium analysis, to be used as a marker, the higher the heterozygosity, the more horse. In addition, as a result of drawing up and analyzing a dendrogram of groups about the 2 standard genetic distance and minimum genetic distance, in three breeds, 179 horses, donkey 3 and Thoroughbred breed formed a clearly different group, but it was observed that Jeju Halla 4 horse formed a group, mixed with Thoroughbred horse. Heterozygosity of domestically bred 5 donkey was lower than two species and formed a group clearly differentiated like 6 Thoroughbred horse. It is assumed that heterozygosity is low because of few breeding heads 7 of domestically bred donkey and inbreeding by limited male horses. observed that the number of allelic genes was 1 (ASB17) and 2 (HMS1) to 14 (AHT5), 6.00