Introduction

Short tandem repeats (STRs), or microsatellite markers, are co-dominant polymorphic DNA loci that contain repeated nucleotide sequences, with the number of repeats often differing between alleles, and individuals, resulting in alleles with varying lengths of nucleotide sequence1,2. Differences in genotype at a certain number of STRs can clearly distinguish individuals3,4. Based on Mendelian genetic laws, parentage testing can be performed by comparing the typing results of parents and offspring5,6. With the development of sequencing technologies, SNP markers have also been utilized for individual identification and parentage testing7,8,9. Accurate animal identification and parentage verification can be used to pedigree populations, support selective breeding and be applied forensic investigations10,11,12,13.

STR markers for the accurate identification of individuals and verification of parentage have been developed for many species such as human14, dog15,16, cat17, pig18, goat19, sheep19, donkey20, horse21, zebra fish6, and panda3. Multiplex PCR technology22 has improved the experimental efficiencies of using these STR. In general, more than 10 microsatellites are required to identify a specific individual, which, in the past, required separate PCR reactions for each microsatellite followed by polyacrylamide gel electrophoresis3,23,24. The combination of the individual PCR reactions through multiplex PCR technology greatly improved efficiency, where differentially labeled fluorescent primers and capillary electrophoresis can be used to obtain typing results from multiple STRs in one reaction14,25. Multiplex PCR is widely used for the identification of individual humans and dogs and cats, with commercial kits have been developed for the detection of more than 20 microsatellite markers in a single reaction26,27.

In 2019 the International Society for Animal Genetics (ISAG) has recommended a panel of 16 STRs for the identification of individual cattle and the verification of their parentage28, while twelve of these STRs were suggested for a cattle STR comparison test for rank calculation in 20218 and 20239. The commercial StockMarks™ Cattle Bovine Parenting Typing Kit (Thermo Fisher Scientific, USA) was developed for cattle STR genotyping and contains 11 of the 16 ISAG recommend STRs. Some other multiplex systems have also been used in cattle breeding studies29, but no method that obtains results for all STRs recommended by ISAG, as well as the ability to determine sex, has been described.

Here, we develop a 17-plex STR typing system that contains all 16 ISAG recommend STRs and the sex-determining marker Amelogenin (AMEL)30. The typing results of these 16 STRs can be obtained simultaneously in one single multiplex PCR reaction. The sensitivity, species specificity, stutter analysis and population genetic parameters of this 17-plex system were examined. Application of this system will improve the efficiency of the identification of individual cattle and their paternity, which will support population genetic research and the selective breeding of cattle.

Results

Multiplex PCR

Primer pairs for the 16 ISAG recommended STRs and AMEL were redesigned to establish our 17-plex typing system (Table 1). The forward primers were labeled with one of three fluorescent dyes (FAM, HEX and ROX) (Table 1). After multiplex PCR optimization, a successful 17-plex typing system was obtained. The PCR reaction had a final volume of 15 µl, and the reaction procedure could be completed in 1 h and 37 min. Details of the reaction system and procedures are described in the “Methods” section. DNA from Charolais, Holstein, Simmental, and Liaoyu white cattle were tested and obtained effective typing results. A genotype profile of a cattle is shown in Fig. 1, where the genotypes at the HAUT27, BM2113, CSSM66, ILSTS006, WTH225, BM1818, ETH3, and TGLA122 loci were heterozygous and the genotypes at the BM1824, INRA023, ETH10, TGLA227, TGLA126, TGLA53, CSRM60, and SPS115 loci were homozygous. The genotype of this individual at the AMEL loci is female (only one peak of X), which is consistent with the sampling record. The numbers below the peaks are the genotype results of the corresponding STRs.

Table 1 STR and primer information for the 17-plex typing system.
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Fig. 1
figure 1

Genotype profile of a cattle generated by the 17-plex typing system.

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17-plex STR sensitivity

To determine the sensitivity of our 17-plex STR system we tested DNA template concentrations that ranged from 20 to 0.125 ng/µL. Complete genotype profiles were obtained with template DNA amounts ranging from 1 to 20 ng/µL (Fig. 2). When the concentration of the DNA template was 0.5 ng/µL, the peaks for the BM1824, TGLA227, TGLA53, TGLA122, and TGLA126 STR loci were below 200 relative fluorescence units (rfu), which reduces the accuracy of correctly typing these sites. When the amount of template DNA was reduced to 0.25 ng/µL, the peak heights of thirteen STRs were below 200 rfu and three of them had non-specific amplification results (Fig. 2). Based on the above results, the minimum addition of DNA needed for complete genotyping is 1 ng.

Fig. 2
figure 2

Sensitivity testing for the 17-plex PCR system using a range of DNA templates. Black arrows indicate allele peaks that were below the 200 rfu threshold and red arrows indicate nonspecific peaks using low DNA templates.

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Species specificity of the 17-plex STR system

To examine the species-specificity of our multiplex STR amplification, we tested DNA from 12 commonly found species as template. No amplification for any STR was found when DNA from human, donkey, dog, cat, rat, chick, goose and duck were used as template. When horse DNA was used, three non-specific amplification peaks, of fixed product size, were generated, with 2 peaks for the AMEL primers and 1 peak with the CSSM66 primers. Alleles for the BM2113, CSRM60, and ETH225 loci were detected when deer DNA was used as template. When DNA from sheep and goats, the tested species most closely related to cattle, were used, alleles were detected for four loci in sheep (TGLA53, CSRM60, SPS115, and AMEL) and seven loci in goats (TGLA53, BM2113, CSRM60, ETH225, ETH10, TGLA122, and AMEL).

Stutter analysis of the STRs

Since STR stutters affect the ability to accurately genotype alleles, we assessed the stutters for our STRs. For this analysis the average ratio of stutters for the 16 STRs were assessed. The mean value of minus stutter ranged from 0.1086 to 0.4736, with the lowest value found at the TGLA122 locus and the highest value at the CSRM60 locus (Table S1). The mean value of the plus stutter ranged from 0.0060 to 0.2136, with the lowest value at the CSSM66 locus, and the highest value at the CSRM60 locus. The lower allele represents the ratio of the second allele peak to the first allele peak, with the mean ranging from 0.5494 to 0.8661.

Population genetic data analysis

To examine the diversity of alleles identified by our 17-plex STR system, we examined results obtained from the Liaoyu white cattle, our cattle population with the largest sample size. A total of 176 alleles were identified in the 140 Liaoyu white cattle examined here (Table 2). The number of alleles (Na) for the STRs ranged between 6 and 20, with the TGLA53 locus having the highest number and the BM1824 and ETH10 loci have the fewest (Table 3). Comparison with the published alleles identified by ISAG (https://strbase.nist.gov//cattleSTRs.htm), we found 24 new alleles in the Liaoyu white cattle, which were distributed to the BM1824, CSRM60, CSSM66, ETH3, ETH225, HAUT27, INRA023, SPS115, TGLA53, and TGLA126 loci (Table S2). Allele frequencies at the STRs ranged from 0.0031to 0.9338, with an allele at the ETH10 locus having the highest allele frequency, and allele at the TGLA53 locus having the lowest allele frequency is (Table 2). The results of the population structure statistic test for 140 Liaoyu white cattle is shown in Table 3. The range for observed heterozygosity (Ho) was from 0.1192 to 0.8344, the range for expected heterozygosity (He) was 0.1272 to 0.8802. The power of matching (PM) of the 16 STRs ranged from 0.1213 to 0.8783; the power of discrimination (PD) ranged from 0.1267 to 0.8787, with the minimum value at the loci ETH10 and the maximum value at the loci 0.8787. The PEduo ranged from 0.0082 to 0.5593, and CPEduo was 0.999804697. The range of PEtrio was from 0.0665 to 0.7307, and CPEtrio is 0.9999999260. These results mean that the 17-plex system is suitable for the identification of specific cattle individuals and for parentage testing.

Table 2 Allele frequencies of 140 Liaoyu white cattle.
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Table 3 Population structure statistics of 140 Liaoyu white cattle.
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Discussion

We developed a 17-plex multiplex typing system for the identification of individual cattle and the verification of their parentage. This 17-plex multiplex typing system includes the 16 ISAG recommend STRs along with a sex-determining marker AMEL. Our results show that genotyping of the 17 markers could be conducted in a single PCR reaction and simultaneously allow gender discrimination. In our 17-plex typing system, four reagents (PCR Buffer, MgCl2, dNTP mix and DNA polymerase) found in most typing kits were replaced by a single regent, Easy Taq PCR Supermix. This simplified the PCR procedure, which has been shortened from 4.5 h to 1 h 37 min. Moreover, the new system has flexibility, allowing researchers to modify the number of STR markers used by the system according to the needs of the experiment, with the removed STR components replaced with sterilized water. This type of modification cannot be made with commercial kits.

Stutters are frequently found in PCR reactions for STR markers, especially for di-nucleotide STRs, which have higher peak ratios than tetranucleotide and pentanucleotide STRs26,31. Ratio data for the minus and plus stutter peaks to the allele peak are critical for accurately genotyping the results of di-nucleotide STR based genotyping systems. Analysis of our system shows that the minus ratio was below 0.4736 but the ratio of the second allele peak to the first allele peak was higher than 0.5495 for the 16 cattle STRs. Accurate typing results can be obtained by referring to the stutter data. We also tested twelve commonly found species in our species-specificity test. No alleles were amplified for most of the species, with the exception of the closely related species deer, sheep and goats. In these three species, alleles were amplified in three to seven loci, likely due to their close evolutionary relationship with cattle. Similar observations were made during the development of a typing system for horses, where donkey DNA could generate some alleles32. Our 17-plex typing system was used to examine allele diversity in 140 Liaoyu white cattle from Liaoning province, China. The breed is a cross between Charolais and local Chinese cattle breeds. Within this population we identified 24 new alleles, from 10 STRs, compared to alleles identified previously in twenty breeds of cattle, suggesting that these new alleles came from the local Chinese cattle genomes33. The CPEduo and CPEtrio results demonstrate that our 17-plex typing system can be used to identify individual cattle and be used as a parentage test in cattle to generate pedigree identification.

As a commonly used molecular marker, STRs can be used to study the genetic diversity and population structure of species34,35,36. The International Society for Animal Genetics (ISAG) and Food and Agriculture Organization of the United Nations (FAO) have co-recommended thirty STRs29. Sixteen or fewer STRs in our 17-plex system can be selected for research in cattle, with the use of multiplex PCR technology, and the freely selectable number of STRs can significantly improve the convenience and efficiency of such studies. Moreover, the 17-plex system may also be applied for forensic purposes, although the 16 STRs recommended by ISAG are dinucleotide STRs, thus, the sensitivity is lower than for systems develop to use tetranucleotide and pentanucleotide STRs, however, to date, there are no reports of individual identification based on tetranucleotide and pentanucleotide STRs in cattle6,14,37. With the development of sequencing technologies, SNP markers have been used for individual identification and parentage testing38. The ISAG recommends the 100 core SNP and 100 back up SNP for cattle. Compared with STR markers8,9, SNPs have the advantage of larger quantities, lower mutation rate and more suitable for degraded DNA39, they also cover a much larger portion of the genome and can be genotyped using high-throughput sequencing40, but the cost of commercial chips is still not conducive to large-scale application. Therefore, STR marker-based parentage testing is still the most widely used technology at present, even for human applications.

Methods

Animal experimentation and ethics approval

Animal experimentation was conducted following proper veterinary practices and in accordance with the guidelines41 and regulations of the Animal Care and Use Committee of Shenyang Agricultural University. The use of animal samples was approved by the Animal Care and Use Committee of Shenyang Agricultural University (approval number: 2023103001).

The animal experimentation used in this study was compliance with the ARRIVE guidelines (https://arriveguidelines.org/about).

Human experimentation and ethics approval

Three human DNA samples were extracted from the authors’ hair roots after obtaining informed consent. Experimentation was conducted in accordance with the guidelines and regulations of the Committee of Shenyang Agricultural University.

The method using human DNA samples was reviewed and approved by the Animal Care and Use Committee of Shenyang Agricultural University (approval number: 2023103001).

Sample collection and DNA extraction

A total of 158 blood, sperm or hair samples were collected from cattle, including 10 Charolais, 4 Holstein, 4 Simmental, and 140 Liaoyu white cattle (a cross between Charolais and a local Chinese cattle breed). Genomic DNA was extracted from blood, seminal fluid, and hair samples using the TIANamp Blood DNA Kit (Tiangen Biotech, China), the Biosentech Sperm Genomic DNA Kit (Biosntech, China), and the TIANamp Genomic DNA Kit (Tiangen Biotech, China), respectively. Concentrations of the DNA samples were qualified using Tecan Infinite 200 (Tecan, Switzerland).

STR selection

The 16 ISAG recommended STRs (BM1818, BM1824, BM2113, CSRM60, CSSM66, ETH3, ETH10, ETH225, HAUT27, INRA023, ILSTS006, SPS115, TGLA53, TGLA122, TGLA126 and TGLA227)28, and a sex determine loci AMEL were chosen to establish our 17-plex multiple STR typing system for cattle.

Multiplex PCR

To develop the 17-plex PCR system, primers for the 16 STRs and the AMEL loci were redesigned, and a fluorescent FAM, HEX or ROX label was added to the forward primer for each marker. The principle for primer redesign followed our earliest research21. The final total volume of the reaction system was 15 µL, which included 7.5 µL 2×Easy Taq PCR Supermix, 1 µL DNA template and 6.5 µL primer mix. The PCR procedure is 95 °C for 5 min followed by 30 cycles of 95 °C for 30 s, 58 °C for 35 s and 72 °C for 1 min, followed by a final extension at 72 °C for 30 min. Information on primer sequences, product sizes, concentrations, and label fluorescent dyes for each STR and the AMEL loci are shown in Table 1. As an option, if researchers want to use a subset of the 17 molecular markers, they can replace the discarded marker primers with sterile water in the primer mix.

Capillary electrophoresis and data analysis

PCR amplification products were separated by capillary electrophoresis (CE). The specific method is as follows: mix 0.5 µL of the multiplex PCR amplification product with sterile water at a 1:1 ratio to yield a final volume of 1 µL; add 9 µL of Hi-Di formamide and GeneScan 500 LIZ Size Standard mixture (mix ratio is 250:1) and thoroughly mix. The samples were then examined using a 3730XL DNA Analyzer. The CE results were then genotyped using GeneMapper V4.0 software with comparison to the genotypes of positive controls.

Positive controls

DNA from one bull and two cows that had homozygous genotypes for most of their 16 STRs were selected as positive controls. The sequences and the repeat unit numbers of the STRs were determined by cloning and sequencing the products of single-plex PCR for each STR. Sequencing results were confirmed by the CE analysis of the products with fluorescently labelled primers. The genotype results for the other samples were obtained by comparison to these positive controls.

Sensitivity

To evaluate the sensitivity of the 17-plex typing system, DNA from one cattle with a heterozygous genotype at most markers was selected. The DNA was diluted using sterilized water to yield a concentration gradient of 20, 10, 5, 2, 1, 0.5, 0.25 and 0.125 ng /µL. The diluted DNA templates were then amplified using the 17-plex multiplex PCR system. The sensitivity was determined as the minimum concentration of DNA template that obtained a complete and accurate genotype result for the 16 STRs and the AMEL loci.

Species-specificity testing

To test species specificity of the system, three DNA samples from each of 12 species (human, sheep, goat, horse, donkey, deer, dog, cat, rat, chicken, goose, and duck) were used as template for the 17-plex system under the same PCR amplification conditions used in cattle to test for potential interference. Of these, deer, dog, cat, goose and duck DNA were extracted from blood; sheep, goat, rat and chicken DNA were extracted from muscles; and DNA of human, horse, and donkey DNA were extracted from hairs.

Stutter analysis

Stutter is the slip product of STRs under PCR reactions, which is a common phenomenon of di-nucleotide STR markers. Compared to tetranucleotide and pentanucleotide STRs, di-nucleotide STRs have higher stutters that can cause difficulties in automated genotyping. Two types of stutters: “plus stutter” and “minus stutter” were analyzed by calculating the average ratio of the stutter peaks compared with the allele peaks in each STR. Stutters occurrence and the ratio standard deviation (SD) were calculated21.

Data analysis

Population statistics were calculated based on the genotypes from the 140 Liaoyu white cattle. The number of alleles (Na), observed and expected heterozygosity (Ho and He) for the STRs were calculated using Genepop V4.2. We used published formulas to calculate the Power of matching (PM), power of discrimination (PD), probability of paternity exclusion (PE) and combined probabilities of paternity exclusion (CPEduo and CPEtrio)14.