Parentage Exclusion Probability Research Articles

Parentage exclusion of close relatives in haplodiploid species

Parentage exclusion probability is usually calculated to evaluate the informativeness of a set of markers for, and the statistical power of, a parentage analysis. Equations for parentage exclusion probability have been derived in various scenarios such as paternity exclusion when maternity is known or unknown or when candidate males are unrelated or loosely related (being from the same subpopulation) to the father. All previous work assumes a diploid species. Although marker-based parentage analyses have been conducted in haploidiploid species (such as ants, bees and wasps) for diploid offspring at the individual level or haploid offspring at the class level, rigorously derived formulations of parentage exclusion probability for haploid offspring at the individual level are lacking, which prevents the precise evaluation of the informativeness for and the statistical power of a parentage analysis. In this study we derive equations for the exclusion probability of maternity of a haploid male when multiple mother candidates (workers or queens) are unrelated or fullsibs to the mother. The usefulness of the equations is exemplified by numerical examples, and the results are discussed in the context of the study of worker reproductivity in eusocial haplodiploid species. The results are especially valuable for an optimal experimental design in determining sampling intensities (e.g. number of markers and number of individuals) to achieve satisfactory statistical power of a parentage analysis in investigating workers’ reproductivity in eusocial haplodiploid species.

SNP panel for evaluation of genetic variability and relationship in roe deer (Capreolus capreolus).

Blood samples from forty-six roe deer ( Capreolus capreolus) acquired during officially approved hunting in six hunting divisions throughout Poland were used to isolate the genomic DNA. All individuals were genotyped by MD_Bovine BeadChip (Illumina) for 46.750 Single Nucleotide Polymorphism (SNP) markers. SNPs of inappropriate clusters, with a marker call rate lower than 90% and with a minor allele frequency (MAF) lower than 0.01, located on sex chromosomes and mitochondrial DNA, were removed. Altogether, 21.033 SNP markers were included for further analysis. Observed and expected heterozygosity amounted to 0.098 and 0.119, respectively. Among 21.033 markers, a panel of 148 SNPs were selected for relationship analysis. They were unlinked and had a MAF higher than 0.2. This set of SNPs showed a probability of parentage exclusion of 1.29x10 -6 and 2.37x10 -19 for one, and two known parents, respectively. The probability of identity was estimated at 1.8x10 -40. The probabilities obtained in this study are sufficient for the monitoring and effective management of the genetic diversity of roe deer in Poland and is a cost-effective complementary tool for forensic applications.

Development of polymorphic microsatellite markers for the Trichoglossus haematodus and cross-species amplification in Trichoglossus moluccanus.

Trichoglossus haematodus is the most popular parrots globally and one of the most bred species in Korea's zoos. However, despite its popularity, there are limited studies on the population genetics of this species. In this study, 10 polymorphic microsatellite markers were developed for T. haematodus. The number of alleles ranged from 6 to 9 (mean 7.9). Null alleles were present in two loci (TH-07 and TH-08). The observed heterozygosity ranged from 0.4444 to 1.0000 (mean 0.7000). One locus (TH-08) indicated a significant deviation from the Hardy-Weinberg equilibrium after Bonferroni correction (p < 0.005). The mean inbreeding coefficient (FIS) of the 10 loci was positive, suggesting that there is inbreeding in the population. Since the polymorphism information content (PIC) values were more than 0.7 in all loci, all markers developed in this study were classified as informative. The parentage exclusion probabilities considering all loci were higher than 0.99 in all three cases (P1, P2, and P3). The cross-species amplification of the 10 markers was tested in T. moluccanus, a close relative species of T. haematodus. These markers were also informative for T. moluccanus with PIC values higher than 0.7 in all loci. Additionally, the parentage exclusion probabilities (P1, P2 and P3) for T. moluccanus were above 0.99. However, due to the small number of T. haematodus and T. moluccanus investigated in this study, the 10 microsatellite markers should be analyzed with more individuals of these two species in future studies. The markers developed in this study might be helpful for investigations of genetic diversity and parentage analysis of T. haematodus and T. moluccanus.

Evaluation of the International Society forAnimal Genetics bovine single nucleotide polymorphism parentage panel in South African Bonsmara and Drakensberger cattle.

A panel of 200 single nucleotide polymorphisms (SNPs) have been recommended by the International Society for Animal Genetics (ISAG) for use in parentage verification of cattle. While the SNPs included on the ISAG panel are segregating in European Bos taurus and Bos indicus breeds, their applicability in South African (SA) Sanga cattle has never been evaluated. This study, therefore, assessed the usefulness of the ISAG panel in SA Bonsmara (BON) and Drakensberger (DRB) cattle. Genotypes of 185 ISAG SNPs from 64 BON and 97 DRB sire-offspring pairs were available, all of which were validated with 119,375 SNPs. Of the 185 ISAG SNPs, 14 and 18 in the BON and DRB, respectively (9 in common to both breeds), were either monomorphic, exhibited at least one discordance between validated sire-offspring pairs, or had poor call rate or clustering issue. The mean minor allele frequency of the 185 ISAG SNPs was 0.331 in the BON and 0.359 in the DRB. The combined probability of parentage exclusion (PE) was the same (99.46%) for both breeds, while the probability of identity varied from 1.61 × 10-48 (BON) to 1.11 × 10-54 (DRB). Fifteen (23.4%) and 32 (33%) of the already validated sire-offspring pairs for the BON and DRB, respectively, were determined by the ISAG panel to be false-negatives based on a threshold of having at least two discordant SNPs. In comparison to sire discovery using the 119,375 SNPs, sire discovery using only the ISAG panel identified correctly 44 (out of 64 identified using the 119,375 SNPs) unique sire-offspring BON pairs and 62 (out of 97 identified using the 119,375 SNPs) unique sire-offspring DRB when all sires were masked. Five BON and three DRB offspring had > 1 sire nominated. This study demonstrated that the use of the ISAG panel may result in incorrect exclusions and multiple candidate sires for a given animal. Selection of more informative SNPs is, therefore, necessary in the pursuit of a low-cost and effective SNP panel for indigenous cattle breeds in SA.

Genetic Diversity and Population Structure of Polish Konik Horse Based on Individuals from All the Male Founder Lines and Microsatellite Markers.

Simple SummaryWe investigated the genetic diversity of Polish Konik in sire lines. The Polish Konik horse is a Polish native horse breed of a primitive type and is one of the breeds managed via a conservation program. Due to the small number of breed founders (six male lines) and paternal lineages with unknown representation, the genetic diversity and structure of the population should be monitored regularly. This is the first characterization of the genetic diversity of Polish Konik paternal lines based on microsatellite markers (STRs). The fixation index (FST), which measures population differentiation, was low for all the studied markers and its mean value was 0.0305. Our analysis also revealed that there has been no inbreeding in the Polish Konik population for the studied group. This monitoring of genetic diversity could be helpful for the Polish Konik breeding and conservation programs and also for making informed decisions in the management of paternal lineages.The Polish Konik horse is a primitive native breed included in the genetic resource conservation program in Poland. After World War II, intensive breeding work began, aimed at rebuilding this breed. Now, the whole Polish Konik population is represented by six male founder lines (Wicek, Myszak, Glejt I, Goraj, Chochlik and Liliput). Individuals representing all six paternal lineages were selected based on their breeding documentation. We performed a fragment analysis with 17 microsatellite markers (STRs) recommended by the International Society for Animal Genetics (ISAG). The genetic diversity and structure within the paternal lineages and the whole of the studied group were investigated. The average allelic richness was 6.497 for the whole studied group. The fixation index (FST; measure of population differentiation) was low (about 3%), the mean inbreeding coefficient (FIT) was low and close to 0, and the mean inbreeding index value (FIS) was negative. The mean expected heterozygosity was established at 0.7046 and was lower than the observed heterozygosity. The power of discrimination and power of exclusion were 99.9999%. The cumulative parentage exclusion probability equaled 99.9269% when one parental genotype was known and 99.9996% with both parents’ genotypic information was available. About 3% of the genetic variation was caused by differences in the breed origin and about 97% was attributed to differences among individuals. Our analysis revealed that there has been no inbreeding in the Polish Konik breed for the studied population. The genetic diversity was high, and its parameters were similar to those calculated for native breeds from other countries reported in the literature. However, due to the small number of breed founders and paternal lineages with unknown representation, the population’s genetic diversity and structure should be monitored regularly.

Evaluation of microsatellite markers for use during genotyping and parentage exclusion for dromedary camels in qatar

This study was aimed to evaluate the efficiency of 20 previously published microsatellite markers for the determination of parentage within the racing camel population in Qatar, using multiplex polymerase chain reaction (PCR), capillary electrophoresis, and genotyping. These markers amplified 127 alleles, and 15 out of 20 loci were polymorphic among the dromedary camels in Qatar, with an average of 8.13 alleles per locus. The mean expected heterozygosity (He) among the studied population was 0.562 (range 0.114–0.867). The polymorphic information content (PIC) ranged from 0.107 to 0.852, with an average value of 0.516. These results indicated a low probability of identity (2.10E−11), with a high parentage exclusion probability if either one (0.99959) or both parents (0.99999) were putative. In those study cases with parentage assignment, the 15 microsatellite loci successfully assigned 135 young calves to the correct parents, with 95% confidence. Our results demonstrated that a set of nine microsatellite DNA markers could provide highly precise individual identification and paternity assignment within the studied camel population.

Highly promiscuous paternity in mainland and island populations of the endangered Northern Quoll

AbstractPolyandry is a commonly utilized strategy in mammalian reproductive systems, where females engage in mating with multiple males to increase either the genetic diversity or quality of their offspring. Some species also exhibit male semelparity, a peculiar life‐history trait where all or most males within a population begin to die following mating. The northern quoll (Dasyurus hallucatus) possesses both of these reproductive adaptations, and females of this species may produce up to eight young in a single litter. Here we aimed to quantify whether there was any difference in female quolls’ choice of male sires between two genetically distinct populations located in the Pilbara region of Western Australia (one mainland‐dwelling and one island‐dwelling population). The total parentage exclusion probability was 0.9999 for excluding a candidate parent from parentage, given the genotype of a known mother. Overall, we found that every litter had young resulting from multiple males. In some litters, a different male fathered every offspring. Thus, northern quolls demonstrated a greater level of polyandry than has previously been detected in marsupials. Furthermore, females from the less genetically diverse island population exercised mate choice and preferentially bred with males that were, on average, 20% smaller than a different male randomly sampled from the island. There was no detectable difference with regard to male sire body size in the mainland population, indicating a difference of selective pressures for the two populations.

Parentage assignment using microsatellite DNA typing for the endangered numbat (Myrmecobius fasciatus)

The numbat (Myrmecobius fasciatus) is an endangered and peculiar marsupial with a diet that consists almost exclusively of termites. This study developed a parentage-testing system for numbats using microsatellite markers. Nineteen loci detected 143 alleles, with 4–13 alleles/locus and average expected heterozygosity of 77% (range 0.665–0.855). The total parentage exclusion probability was &amp;gt;0.9999 (given only the genotype of the offspring), &amp;gt;0.9999 for excluding a candidate parent from the parentage of an arbitrary offspring (given the genotype of the offspring and parent) and the probability of identity for full-sibs was 4.6×10–9. Overall, these microsatellites offer a simple and highly informative marker-set for parentage identification in numbats.

Identification of microsatellites and parentage testing development of bactrian camel (Camelus bactrianus)

This report is aimed to identify polymorphic microsatellites from Bactrian camel transcriptome sequences and apply microsatellite multiplex systems for parentage testing. 1, 820 simple sequence repeats (SSRs) were identified from 23, 624 transcriptome sequences, representing an average density of SSR/26.57 kb. The most abundant SSR, the trinucleotide repeat motif, accounts for 30% of all SSRs. Tetra-, penta-, di-and hexanucleotide repeats account for 29%, 22%, 17% and 2% of SSRs, respectively. Based on sequence redundancy and PCR amplification, we selected 14 polymorphic microsatellites for Bactrian camel parentage testing analysis in a population of 117 unrelated camels from China and Mongolia. We identified a total of 149 alleles, with 5–23 alleles per locus (10.64 ± 5.472) and an average heterozygosity (HE) of 0.676 (range: 0.380–0.888). Based on only the genotype of the offspring, a parentage exclusion probability of 0.9999 was calculated when excluding a candidate parent from parentage of an arbitrary offspring. When excluding a candidate parent from parentage of an arbitrary offspring and based on both the genotype of the offspring and the other parent, an exclusion probability of 0.9999 was calculated. We selected 15 baby camels, 20 sires and 20 dams for parentage testing. According to the parentage assignment, the microsatellite panel assigned all 15 offspring with high confidence. This core set of 14 microsatellites represent a powerful and efficient method for determining parentage in domestic Bactrian camels.

Evaluation of efficiency of controlled pollination based parentage analysis in a Larix gmelinii var. principis-rupprechtii Mayr. seed orchard.

Controlled pollination (CP) is an important tool for breeding programs to improve seed quality, as it rapidly generates desirable genotypes and maximizes genetic gains. However, few studies have evaluated the success rate of CP, especially in Larix gmelinii var. principis-rupprechtii Mayr. seed orchards. In this study, we estimated the rate of correct parentage in 257 CP progeny in an L. gmelinii var. principis-rupprechtii seed orchard from ten candidate parents using 13 microsatellites. The parentage exclusion probabilities of all combined loci in the single parent and parent pair tests were > 0.99, which was sufficient to distinguish the relatedness of the sampled individuals. Comparing the maximum likelihood-based parentage analysis results with breeding records revealed that the percentages of correctly identified maternal and paternal parents were 22.6% and 35.0% at 95% CL, respectively, suggestive of parent mislabeling and pollen contamination in the CP population. We conducted a pedigree reconstruction by identifying the expected parents and assigned maternity, paternity, and parent pairs to 176 (68.5%), 199 (77.4%), and 132 (51.4%) progeny, respectively. This study provides a reference for future selection of elite genotypes for commercial production. To increase the efficiency of CP, molecular markers should be used to correctly identify individuals in seed orchards before conducting CP.

Analysis of microsatellite DNA polymorphism in the Tatra Shepherd Dog

ABSTRACTThe Tatra Shepherd Dog is one of five Polish native breeds of dogs originating from the Polish Tatra Mountains. The objective of the study was to determine genetic variation in a population of 60 dogs of this breed, based on polymorphism of 18 microsatellite (STR) markers, recommended by ISAG for canine parentage testing. The analysis showed considerable genetic variability in the studied loci. The 100 alleles identified in the test material were used to determine the polymorphism of the discussed markers. The highest polymorphism was found in the locus AHT171, in which 8 alleles were identified and PIC and HO values exceeded 0.8. The lowest polymorphism was detected for AHTk211, in which 3 alleles were determined, and PIC and HO values were 0.233 and 0.281, respectively. Average FIS had a low negative value, which suggests zero inbreeding of the studied breed. The probability of parentage exclusion estimated for the 18 markers totalled 99.996%.

Polymorphic DNA microsatellite markers for forensic individual identification and parentage analyses of seven threatened species of parrots (family Psittacidae).

The parrot family represents one of the bird group with the largest number of endangered species, as a result of habitat destruction and illegal trade. This illicit traffic involves the smuggling of eggs and animals, and the laundering through captive breeding facilities of wild-caught animals. Despite the huge potential of wildlife DNA forensics to determine with conclusive evidence illegal trade, current usage of DNA profiling approaches in parrots has been limited by the lack of suitable molecular markers specifically developed for the focal species and by low cross-species polymorphism. In this study, we isolated DNA microsatellite markers in seven parrot species threatened with extinction (Amazona brasiliensis, A. oratrix, A. pretrei, A. rhodocorytha, Anodorhynchus leari, Ara rubrogenys and Primolius couloni). From an enriched genomic library followed by 454 pyrosequencing, we characterized a total of 106 polymorphic microsatellite markers (mostly tetranucleotides) in the seven species and tested them across an average number of 19 individuals per species. The mean number of alleles per species and across loci varied from 6.4 to 8.3, with the mean observed heterozygosities ranging from 0.65 to 0.84. Identity and parentage exclusion probabilities were highly discriminatory. The high variability displayed by these microsatellite loci demonstrates their potential utility to perform individual genotyping and parentage analyses, in order to develop a DNA testing framework to determine illegal traffic in these threatened species.

Performance of microsatellites for parentage assignment following mass controlled pollination in a clonal seed orchard of loblolly pine (Pinus taeda L.)

Mass controlled pollination (MCP), involving large-scale application of pollen on physically isolated female reproductive organs, has been a lower cost alternative to controlled pollination for the commercial production of genetically improved seeds. Nevertheless, rare are the studies that examined the efficacy of operational MCP and no such assessment has been done in loblolly pine to date. The success of MCP was assessed by a microsatellite-based investigation of the realized versus expected parentage of a set of 300 Pinus taeda offspring in 19 families generated in two subsequent rounds of MCP in 2005 and 2006 in a clonal seed orchard in Brazil. Multi-locus combined probability of parentage exclusion both theoretical and realized from actual testing was >99 % for single parent and parent pair testing when using nine or ten markers. Parentage assignments carried out under a maximum likelihood framework revealed a significantly higher success rate of MCP in 2006 (84 %) following technical improvements adopted to minimize pollen contamination and maximize male reproductive success, although significant variability in the correct maternity and full parentage was seen among individual families. The observed patterns of unexpected parentage indicated that this variability likely resulted from mislabeling of clonal ramets of the parents used in the crosses which impacted both maternity and paternity. Preventing pollen contamination will not be sufficient for successful MCP if inaccuracies exist in the identity of the clonal plants that ultimately provide pollen and female strobili, showing that DNA marker auditing and correction of identity of all ramets in a clonal seed orchard should be a standard practice in the operational implementation of MCP.

Isolation and characterization of microsatellite loci for the large-seeded tree Protorhus deflexa (Anacardiaceae).

• Premise of the study: Protorhus deflexa is an endemic large-seeded tree in Madagascar that depends heavily on insects for cross-pollination and on large-bodied frugivores for seed dispersal. Because such mutualistic relationships are vulnerable to human disturbance, the development of microsatellite markers will enhance analyses of gene flow in this tree species in degraded forests.• Methods and Results: Nineteen microsatellite markers were developed for P. deflexa using 454 pyrosequencing. The number of alleles ranged from two to nine, and the ranges of observed and expected heterozygosities were 0.200–0.800 and 0.303–0.821, respectively. The parentage exclusion probability by the 19 loci reached 0.98583 for the first parent and 0.99971 for the second parent.• Conclusions: These markers will be useful for studying gene flow via pollination and seed dispersal by animals and the genetic structure of P. deflexa in protected and degraded forests in Madagascar.

Null alleles in microsatellite markers

Microsatellite markers are widely used in genetic analysis, but the common occurrence of null alleles, is an unfortunate drawback, and one which has not been paid enough attention. In this review, we confirm the concept of null alleles and their characteristics, detail methods for estimating the frequency of null alleles and discuss potential causes. We also consider the effect of null alleles on genetic studies, such as measurement of population genetic diversity, parentage analysis and assignment tests. The review has a certain reference value for future studies. One main cause of microsatellite null alleles is poor primer annealing due to nucleotide sequence divergence (point mutations or indels) in one or both flanking primers. Microsatellite null alleles are not a natural characteristic of a specific gene, and are essentially different from isozyme null alleles. Although microsatellite null alleles have the characteristics of universality, complexity and invisibility, we can test and estimate their frequency through multiplex approaches, using Hardy-Weinberg equilibrium tests, parent-offspring genotype analysis, and new primer design. Null alleles have significant effects on the results of genetic analysis, potentially decreasing population genetic diversity and increasing genetic differentiation among populations. Moreover, null alleles may decrease the probability of parentage exclusion, even leading to errors or confusion in parentage analysis. Null alleles should be paid more atten118 生 物 多 样 性 Biodiversity Science 第 21 卷 tion and demand caution in future studies. Through a variety of methods, such as locus selection, null allele data adjustment, and the design of new primers, we can escape the adverse effects of null alleles and achieve greater accuracy in genetic analysis.

Preliminary studies of polymorphism of selected microsatellite sequences in different colour types of raccoon dog (Nyctereutes Procyonoides) / Wstępne badania polimorfizmu wybranych sekwencji mikrosatelitarnych różnie umaszczonych jenotów (Nyctereutes procyonoides)

Summary. Primary sequences designed for microsatellite loci in the domestic dog genome (FH2054, CPH3, FH2168, FH2164, FH2097), which were used in this study, also allowed for the amplification of the corresponding loci in white and standard Raccoon dogs. In these groups the frequency of alleles, observed and expected heterozygosity, the polymorphic information content and the probability of parentage exclusion were analyzed. The studies allowed to define the expected degree of polymorphism of five microsatellite loci in the two color varieties of Raccoon dogs. All of the five selected microsatellite sequences may be used for checking the origin of Raccoon dogs and the construction of genetic maps within the canids. The obtained values of genetic similarity and genetic distance shows that there is a high genetic similarity between the groups of white and standard Raccoon dog genetic, and thus the small genetic distance. This suggests only small genetic differences between white and standard Raccoon dogs.

Microsatellite markers for Aspidosperma polyneuron (Apocynaceae), an endangered tropical tree species

This study was designed to develop and characterize a preliminary set of nuclear microsatellite markers for Aspidosperma polyneuron, an endangered tropical tree species. Leaf samples were collected from a group of mature A. polyneuron trees in a large area along the Pardo and Mogi-Guaçu river basins in São Paulo State in the southeastern region of Brazil. Twenty-five simple sequence repeat (SSR) markers were developed and 16 of 30 samples had polymorphic loci. The number of alleles per locus ranged from two to 17 (average = 7.06). A high diversity (H(e) = 0.65) and parentage exclusion probability for the first (0.9995) and second (0.9999) parents were measured. These 16 polymorphic markers suggest a high potential for genetic diversity, gene flow, and mating system analyses in A. polyneuron.

Parentage testing of racing camels (Camelus dromedarius) using microsatellite DNA typing

The camel racing industry would have added value in being able to assign parentage with high certainty. This study was aimed at assessing and applying microsatellite multiplexes to construct a parentage testing system for camels. An efficient system of 17 loci from 700 camel samples was used to construct a database of unrelated adults. Based on this, we estimated measures of polymorphism among the markers. In three multiplex reactions, we detected a total of 224 alleles, with 5–23 alleles/locus (mean = 13.18 ± 6.95 SD) and an average heterozygosity (HE) of 0.54 (range 0.032–0.905). The total parentage exclusion probability was 0.99999 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.9999 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. We used 15 juveniles for parentage testing, as well as 17 sires (bull camels) and 21 dams (cows). In the case of parentage assignment, the microsatellite panel assigned all 15 offspring parentage with high confidence. Overall, these findings offer a set of microsatellite markers that are easy, simple and highly informative for parentage testing in camels.

Development of novel SNP system for individual and pedigree control in a Japanese Black cattle population using whole‐genome genotyping assay

Individual identification and parentage analysis using DNA markers are essential for assuring food identity and managing livestock population. The objective of this study was to develop a single nucleotide polymorphism (SNP) panel system for individual effective identification and parentage testing in a Japanese Black cattle population using BovineSNP50 BeadChip. On the basis of SNP frequencies, 60 unlinked informative SNPs were finally selected as candidate markers. The allelic frequencies for each SNP were estimated using additional individuals by PCR-RFLP (restriction fragment length polymorphism). A total of 87 SNP markers added in conjunction with previously developed 27 SNPs were evaluated to assess the utility of the test. The estimated identity power was 2.01 x 10(-34). Parentage exclusion probabilities, when both suspected parents' genotypes were known and when only one suspected parent was genotyped, were estimated as 0.99999997 and 0.99998010, respectively. This developed SNP panel was quite powerful and could successfully exclude false sires with a probability of >0.9999 even if the dam's genotype information was not obtained. The SNP system would contribute to management of the beef industry in Japan.

Development of SNP markers for individual identification and parentage test in a Japanese Black cattle population

This study describes the development of efficient single nucleotide polymorphism (SNP) markers for individual identification and parentage tests in a Japanese Black cattle population. An amplified fragment length polymorphism method was employed to detect informative candidate markers, and yielded 44 SNP markers from 220 primer combinations. 29 unlinked SNPs were finally selected as diagnostic markers. The allelic frequencies for each marker were estimated by using PCR-RFLP in the Japanese Black population. Based on the frequency data, the estimated identity power of these markers was 2.73 x 10(-12). Parentage exclusion probabilities, when both suspected parents' genotypes were known and when only one suspected parent was genotyped, were estimated as 0.96929 and 0.99693, respectively. This panel of SNP markers is theoretically sufficient for individual identification, and would also be a powerful tool for a parentage test in Japanese Black cattle. The markers could contribute to the management of the beef industry in Japan.