Copy number variation within CLEC4E locus as a molecular tool for distinguishing DNA from domestic pigs and wild boars

Natonek-Wiśniewska, Małgorzata; Koseniuk, Anna; Ropka-Molik, Katarzyna; Kmita, Bogumiła; Zalewski, Dariusz; Radko, Anna

doi:10.2478/aspr-2025-0012

Abstract

Wild boar, the primary wild ancestor of the domestic pig, frequently interbreeds with its domesticated counterpart. Distinguishing between the domestic pig (Sus scrofa domestica), wild boar (Sus scrofa), and their hybrids is ambitious due to their close evolutionary relationship and the various genetic markers they have in common. The aim of the study was to develop a reliable and straightforward method to distinguish pig and wild boar DNA based on CNV in the CLEC4E gene, preventing food fraud and ensuring product safety.

The study population consisted of domestic pigs (n=42), wild boars (n=40) and their hybrids (n=6). The domestic pig group is comprised of four breeds: Polish Large White, Duroc, Polish Landrace and Puławska. Wild boars were sampled from the South West region of Poland. The pair of primers (F/R) designed for the CLEC4E gene were used for analysis. A quantitative-comparative CT reaction with a melting curve (SybrGreen) was performed for the tested samples.

The obtained results indicate different CT values for pigs and wild boars. The CT values below 18.70 cycles and melting temperature equal or above 90°C indicate wild origin, while the CT below 17.67, and melting temperature lower than 89.20°C additionally supported by the shape of the melting curve, indicate the presence of a domestic pig or hybrid. The analysis of 85 out of 88 tested samples, supported the assumptions based on the CT value, while for the remaining 3 the result was doubtful due to inconsistent interpretation for CT and / or Tm.

References

ABDURIYIM S., NISHITA Y., ABRAMOV A., SOLOVYEV V., SAVELJEV A., KOSINTSEV P., KRYUKOV A., RAICHEV E., VAINOLA R., KANEKO Y, MASUDA R., 2019 - Variation in pancreatic amylase gene copy number among Eurasian badgers (Carnivora, Mustelidae, Meles) and its relationship to diet. Journal of Zoology 308(1), 28-36.
Search in Google Scholar Back to article
AXELSSON E., RATNAKUMAR A., ARENDT M., MAQBOOL K., WEBSTER M., PERLOSKI M., LIBERG O., ARNEMO J., HEDHAMMAR A., LINDBLAD-TOH K., 2013 - The genomic signature of dog domestication reveals adaptation to a starch-rich diet. Nature 495, 360-4.
Search in Google Scholar Back to article
BEHLER F., STEINWEDE K., BALBOA L., UEBERBERG B., MAUS R., KIRCHHOF G., MAUS U. A., 2012 - Role of Mincle in alveolar macrophage-dependent innate immunity against mycobacterial infections in mice. The Journal of Immunology 189(6), 3121-3129.
Search in Google Scholar Back to article
CHUNG H., LEE K., JANG G, CHOI J., HONG J., KIM T., 2015 - A genome-wide analysis of the ultimate pH in swine. Genetics and Molecular Research 14(4), 15668–15682.
Search in Google Scholar Back to article
FAJARDO V., GONZÁLEZ I., MARTÍN I., ROJAS M., HERNÁNDEZ P., GARCIA T., MARTÍN R., 2008 - Differentiation of European wild boar (Sus scrofa scrofa) and domestic swine (Sus scrofa domestica) meats by PCR analysis targeting the mitochondrial D-loop and the nuclear melanocortin receptor 1 (MC1R) genes. Meat Science 78, 314-322.
Search in Google Scholar Back to article
FOWLER K.E., PONG-WONG R., BAUER J., CLEMENTE E. J., REITTER C. P., AFFARA N. A., GRIFFIN D. K., 2013 - Genome wide analysis reveals single nucleotide polymorphisms associated with fatness and putative novel copy number variants in three pig breeds. BMC Genomics 14, 1-15.
Search in Google Scholar Back to article
GORLA E., COZZI M., ROMÁN-PONCE S., RUIZ LÓPEZ F., VEGA-MURILLO V., CEROLINI S., BAGNATO A., STRILLACCI M. 2017 - Genomic variability in Mexican chicken population using copy number variants. BMC Genetics 18, 1-11.
Search in Google Scholar Back to article
GROENEN M.A.M., ARCHIBALD A.L., UENISHI H., TUGGLE C.K., TAKEUCHI Y., ROTHSCHILD M.F., ROGEL-GAILLARD C., PARK C., MILAN D., MEGENS H.J., LI S., LARKIN D.M., KIM H., FRANTZ L.A.F., CACCAMO M., AHN H., AKEN B.L., ANSELMO A., ANTHON C., AUVIL L., BADAOUI B., BEATTIE C.W., BENDIXEN C., BERMAN D., BLECHA F., BLOMBERG J., BOLUND L., BOSSE M., BOTTI S., BUJIE Z., BYSTROM M., CAPITANU B., CARVALHO-SILVA D., CHARDON P., CHEN C., CHENG R., CHOI S.H., CHOW W., CLARK R.C., CLEE C., CROOIJMANS R.P.M.A., DAWSON H.D., DEHAIS P., DE SAPIO F., DIBBITS B., DROU N., DU Z.Q., EVERSOLE K., FADISTA J., FAIRLEY S., FARAUT T., FAULKNER G.J., FOWLER K.E., FREDHOLM M., FRITZ E., GILBERT J.G.R., GIUFFRA E., GORODKIN J., GRIFFIN D.K., HARROW J.L., HAYWARD A., HOWE K., HU Z.L., HUMPHRAY S.J., HUNT T., HORNSHOØJ H., JEON J.T., JERN P., JONES M., JURKA J., KANAMORI H., KAPETANOVIC R., KIM J.H., KIM J.H., KIM K.W., KIM T.H., LARSON G., LEE K.T.K., LEE K.T.K., LEGGETT R., LEWIN H.A., LI Y., LIU W., LOVELAND J.E., LU Y., LUNNEY J.K., MA J., MADSEN O., MANN K., MATTHEWS L., MCLAREN S., MOROZUMI T., MURTAUGH M.P., NARAYAN J., NGUYEN D.T., NI P., OH S.J., ONTERU S., PANITZ F., PARK E.W., PARK H.S., PASCAL G., PAUDEL Y., PEREZ-ENCISO M., RAMIREZ- GONZALEZ R., REECY J.M., RODRIGUEZ-ZAS S., ROHRER G.A., RUND L., SANG Y., SCHACHTSCHNEIDER K., SCHRAIBER J.G., SCHWARTZ J., SCOBIE L., SCOTT C., SEARLE S., SERVIN B., SOUTHEY B.R., SPERBER G., STADLER P., SWEEDLER J. V., TAFER H., THOMSEN B., WALI R., WANG J.J., WANG J.J., WHITE S., XU X., YERLE M., ZHANG G., ZHANG J.J., ZHANG J.J., ZHAO S., ROGERS J., CHURCHER C., SCHOOK L.B., 2012 - Analyses of pig genomes provide insight into porcine demography and evolution. Nature 491, 393-398.
Search in Google Scholar Back to article
HEID C., STEVENS J., LIVAK K., WILLIAMS P., 1996 - Real time quantitative PCR. Genome Research 6, 986-994.
Search in Google Scholar Back to article
HIGUCHI R., FOCKLER C., DOLLINGER G., WATSON R., 1993 - “Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions”. Biotechnology 11, 1026-1030.
Search in Google Scholar Back to article
JANG J., KIM B., JHANG S. Y., AHN B., KANG M., PARK C., KIM, H., 2023 - Population differentiated copy number variation between Eurasian wild boar and domesticated pig populations. Scientific Reports 13(1), 1115.
Search in Google Scholar Back to article
JANIAK MC., 2016 - Digestive enzymes of human and nonhuman primates. Evolutionary Anthropology 25(5), 253-66.
Search in Google Scholar Back to article
IACOLINA L., PERTOLDI C., AMILLS M.; KUSZA S., MEGENS H.J., BÂLTEANU V.A., BAKAN J., CUBRIC-CURIC V., OJA R., SAARMA U., SCANDURA M, ŠPREM N., STRONEN A.V., 2018 - Hotspots of recent hybridization between pigs and wild boars in Europe Scientific Reports 8, DOI:10.1038/s41598-018-35865-8
Search in Google Scholar Back to article
KOSENIUK A., SMOŁUCHA G., GURGUL A., SZMATOŁA T., OCZKOWICZ M., RADKO A., 2023 - Differentiation of the domestic pig and wild boar using genotyping-by-sequencing. Folia Biologica (Kraków), 71(1), 1-11.
Search in Google Scholar Back to article
KOUTSOGIANNOULI E., MOUTOU K., SARAFIDOU T., STAMATIS C., MAMURIS Z., 2010 - Detection of hybrids between wild boars (Sus scrofa scrofa) and domestic pigs (Sus scrofa f. domestica) in Greece, using the PCR-RFLP method on melanocortin-1 receptor (MC1R) mutations. Mammalian Biology 75, 69-73
Search in Google Scholar Back to article
LORENZINI R., FANELLI R., TANCREDI F., SICLARI A., GAROFALO, L., 2020 - Matching STR and SNP genotyping to discriminate between wild boar, domestic pigs and their recent hybrids for forensic purposes. Scientific Reports 10(1), 3188.
Search in Google Scholar Back to article
MIKAWA S., MOROZUMI T., SHIMANUKI S., HAYASHI T, UENISHI, H., DOMUKAI M., OKUMURA N., AWATA T., 2007 – Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1). Genome Research 17, 586-593.
Search in Google Scholar Back to article
NATONEK-WIŚNIEWSKA M., KRZYŚCIN P., PIESTRZYŃSKA-KAJTOCH A., 2013 - The species identification of bovine, porcine, ovine and chicken components in animal meals, feeds and their ingredients, based on COX I analysis and ribosomal DNA sequences. Food Control 34 (1), 69-78.
Search in Google Scholar Back to article
PARK Y., LIM S., LIM B., KIM J., 2022 - Time series ovarian transcriptome analyses of the porcine estrous cycle reveals gene expression changes during steroid metabolism and corpus luteum development. Animals 12(3), 376.
Search in Google Scholar Back to article
PATIN E., ORR S., SCHAIBLE U., 2017 - Macrophage inducible C-type lectin as a multifunctional player in immunity. Frontiers in Immunology 8, 861.
Search in Google Scholar Back to article
PERRY G., DOMINY N., CLAW K., LEE A., FIEGLER H., REDON R., WERNER J., VILLANEA F., MOUNTAIN J., MISRA R., CARTER N., LEE C., STONE A., 2007 - Diet and the evolution of human amylase gene copy number variation. Nature Genetics 39(10),1256-60.
Search in Google Scholar Back to article
RADVANSKY J., RESKO P., SUROVY M., MINARIK G., FICEK A., KADASI L., 2010 - High-resolution melting analysis for genotyping of the myotonic dystrophy type 1 associated Alu insertion/deletion polymorphism. Analytical Biochemistry 398, 126-128.
Search in Google Scholar Back to article
RINKER D., SPECIAN N., ZHAO S., GIBBONS J., 2019 - Polar bear evolution is marked by rapid changes in gene copy number in response to dietary shift. Proceedings of the National Academy of Sciences of the United States of America 116, 13446-13451.
Search in Google Scholar Back to article
RUBIN C.J., MEGENS H.J., MARTINEZ B.A., MAQBOOL K., SAYYAB S., SCHWOCHOW D., WANG C., CARLBORG O. JERN P., JORGENSEN C.B., ARCHIBALD A., FREDHOLM M., GROENEN M., ANDERSSON L., 2012 - Strong signatures of selection in the domestic pig genome. Proceedings of the National Academy of Sciences USA 109, 19529-19536.
Search in Google Scholar Back to article
SAFDAR M., JUNEJO Y., ARMAN K., ABASIYANIK M. 2014 - A highly sensitive and specific tetraplex PCR assay for soybean, poultry, horse and pork species identification in sausages: Development and validation. Meat Science 98, 296-300.
Search in Google Scholar Back to article
SAMBORSKI A., KREBS S., KESSLER B. BAUERSACHS S. 2013 - Deep sequencing of the porcine endometrial transcript on day 14 of pregnancy. Biology of Reproduction 88(4), 84-1.
Search in Google Scholar Back to article
SUDEEP SAHADEVAN S., THOLEN E., GROSSE-BRINKHAUS C., SCHELLANDER K., TESFAYE D., HOFMANN-APITIUS M., NEUHOFF C., 2015 - Identification of gene co-expression clusters in liver tissues from multiple porcine populations with high and low backfat androstenone phenotype. BMC Genetics 16(1), 1-18.
Search in Google Scholar Back to article
YOSHIDOMI T., HIROSE K., KUGE T., OKADA Y., KUROSAWA Y., TAKIZAWA T., TANAKA K.. 2021 - Detection of copy number variation of alpha amylase genes in domestic pigs (Sus scrofa domesticus) and wild boars (Sus scrofa). Czech Journal of Animal Science 66(03), 73-77.
Search in Google Scholar Back to article
ZHENG X., ZHAO P., YANG K. NING C., WANG H., ZHOU L., LIU J., 2020 - CNV analysis of Meishan pig by next-generation sequencing and effects of AHR gene CNV on pig reproductive traits. Journal of Animal Science and Biotechnology 11, 1-11.
Search in Google Scholar Back to article

Copy number variation within CLEC4E locus as a molecular tool for distinguishing DNA from domestic pigs and wild boars

Abstract

Paradigm

My account