Identification and copy number profiling of sex chromosome specific gene fragments in crossbred pigs with numerically normal karyotype
Vandana Yadav A B , Nihar Ranjan Sahoo
A C E , Pushpendra Kumar A , G. K. Gaur A , A. P. Sahoo A C , G. V. P. P. S. Ravikumar A D and K. P. Singh A
+ Author Affiliations
- Author Affiliations
A ICAR, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243122, India.
B ICAR, National Dairy Research Institute, Karnal, Haryana, 132001, India.
C ICAR-PDFMD, Mukteshwar, Uttarakhand, 263138, India.
D National Institute of Animal Biotechnology, Hyderabad, Telangana, 500049, India.
E Corresponding author. Email: vet.nihar@gmail.com
Animal Production Science 60(9) 1145-1152 https://doi.org/10.1071/AN19287
Submitted: 2 August 2018 Accepted: 19 November 2019 Published: 10 March 2020
Abstract
We examined the copy number profile of sex chromosome specific genes in crossbred pigs with numerically normal karyotype. A total of 30 (15M + 15F) Landlly (Landrace crossbred) pigs from a research farm were cyto-screened by karyotyping to determine the number of chromosomes per diploid cell using short-term peripheral lymphocyte culture technique. All pigs had numerically normal karyotype with 38, XX (female) and 38, XY (male). Genomic DNA was extracted from cyto-screened pigs of both sexes. A set of sex chromosome (X and Y) specific single copy gene fragments along with an autosomal gene fragment were selected out of 12 pairs of primers on the basis of male specific PCR amplification, PCR and qPCR specificity. Quantitative real-time PCR was performed to study the relative copy number change of selected Phosphate repressible alkaline phosphatase X linked (PHOX) gene, using the Eukaryotic Translation Initiation Factor 1A Y-Linked (EIF1AY) gene as control and Breast Cancer Metastasis-Suppressor 1-Like (BRMS1 L- autosomal gene) as the reference gene. The relative copy number of the PHOX gene in females was found to be 1.873 times higher compared with the EIF1AY gene in males. The present study indicates that the real-time quantitative real-time PCR based copy number analysis can be helpful for the detection of sex chromosome ratio in pigs to aid as a preliminary screening for numerical sex chromosomal aneuploidies, adding to the rate of throughput of traditional cyto-screening.
Additional keywords: Landlly, porcine, ratio of sex chromosomal segments, quantitative real-time PCR, Y-chromosome specific gene fragment.
References
Backstrom L, Henricson B (1971) Intersexuality in the pig. Acta Veterinaria Scandinavica 12, 257–273.Barasc H, Ferchaud S, Mary N, Cucchi MA, Lucena AN, Raymond-Letron I, Calgaro A, Bonnet N, Dudez AM, Yerle M, Ducos A, Pinton A (2014) Cytogenetic analysis of somatic and germinal cells from 38, XX/38, XY phenotypically normal boars. Theriogenology 81, 368–372.
| Cytogenetic analysis of somatic and germinal cells from 38, XX/38, XY phenotypically normal boars.Crossref | GoogleScholarGoogle Scholar | 24200468PubMed |
Basrur PK, Stranzinger G (2008) Veterinary cytogenetics: past and perspective. Cytogenetic and Genome Research 120, 11–25.
| Veterinary cytogenetics: past and perspective.Crossref | GoogleScholarGoogle Scholar | 18467822PubMed |
Berletch JB, Yang F, Xu J, Carrel L, Disteche CM (2011) Genes that escape from X inactivation. Human Genetics 130, 237–245.
| Genes that escape from X inactivation.Crossref | GoogleScholarGoogle Scholar | 21614513PubMed |
Breeuwsma AJ (1968) A case of XXY sex chromosome constitution in an intersex pig. Journal of Reproduction and Fertility 16, 119–120.
| A case of XXY sex chromosome constitution in an intersex pig.Crossref | GoogleScholarGoogle Scholar | 5691159PubMed |
D’haene B, Vandesompele J, Hellemans J (2010) Accurate and objective copy number profiling using real-time quantitative PCR. Methods 50, 262–270.
| Accurate and objective copy number profiling using real-time quantitative PCR.Crossref | GoogleScholarGoogle Scholar | 20060046PubMed |
D’Hulst C, Parvanova I, Tomoiaga D, Sapar ML, Feinstein P (2013) Fast quantitative real-time PCR-based screening for common chromosomal aneuploidies in mouse embryonic stem cells. Stem Cell Reports 1, 350–359.
| Fast quantitative real-time PCR-based screening for common chromosomal aneuploidies in mouse embryonic stem cells.Crossref | GoogleScholarGoogle Scholar | 24319669PubMed |
Danielak-Czech B, Kozubska-Sobocinska A, Rejduch B (2016) Molecular cytogenetics in the diagnostics of balanced chromosome mutations in the pig (Sus scrofa) – a review. Annals of Animal Science 16, 679–699.
| Molecular cytogenetics in the diagnostics of balanced chromosome mutations in the pig (Sus scrofa) – a review.Crossref | GoogleScholarGoogle Scholar |
DiBerardino D, Hayes H, Fries R, Long S (1989) International System for Cytogenetic Nomenclature of Domestic Animals. In ‘2nd International Conference on Standardization of Domestic Animal Karyotypes’. Jouy-en-Josas, May 1989.
Ducos A, Berland HM, Bonnet N, Calgaro A, Billoux S, Mary N, Garnier-Bonnet A, Darré R, Pinton A (2007) Chromosomal control of pig populations in France: 2002–2006 survey. Genetics, Selection, Evolution. 39, 583–597.
| Chromosomal control of pig populations in France: 2002–2006 survey.Crossref | GoogleScholarGoogle Scholar | 17897598PubMed |
Ducos A, Revay T, Kovacs A, Hidas A, Pinton A, Bonnet-Garnier A, Molteni L, Slota E, Switonski M, Arruga MV, van Haeringen WA, Nicolae I, Chaves R, Guedes-Pinto H, Andersson M, Iannuzzi L (2008) Cytogenetic screening of livestock populations in Europe: an overview. Cytogenetic and Genome Research 120, 26–41.
| Cytogenetic screening of livestock populations in Europe: an overview.Crossref | GoogleScholarGoogle Scholar | 18467823PubMed |
Gustavsson I, Settergren I (1984) Reciprocal chromosome translocation with transfer of centromeric heterochromatin in the domestic pig karyotype. Hereditas 100, 1–5.
| Reciprocal chromosome translocation with transfer of centromeric heterochromatin in the domestic pig karyotype.Crossref | GoogleScholarGoogle Scholar | 6725001PubMed |
Hornak M, Oracova E, Hulinska P, Urbankova L, Rubes J (2012) Aneuploidy detection in pigs using comparative genomic hybridization: from the oocytes to blastocysts. PLoS One 7, e30335
| Aneuploidy detection in pigs using comparative genomic hybridization: from the oocytes to blastocysts.Crossref | GoogleScholarGoogle Scholar | 22291937PubMed |
Mangs HA, Morris BJ (2007) The human pseudoautosomal region (PAR): origin, function and future. Current Genomics 8, 129–136.
| The human pseudoautosomal region (PAR): origin, function and future.Crossref | GoogleScholarGoogle Scholar |
Ottesen AM, Garn ID, Aksglaede L, Juul A, Meyts RD (2007) A simple screening method for detection of Klinefelter syndrome and other X-chromosome aneuploidies based on copy number of the androgen receptor gene. Molecular Human Reproduction 13, 745–750.
Quach TA, Villagomez DAF, Coppola G, Pinton A, Hart EJ, Reyes ER, Basrur PK, King WA (2009) A cytogenetic study of breeding boars in Canada. Cytogenetic and Genome Research 126, 271–280.
| A cytogenetic study of breeding boars in Canada.Crossref | GoogleScholarGoogle Scholar | 20068298PubMed |
Quilter CR, Blott SC, Mileham AJ, Affara NA, Sargent CA, Griffin DK (2002) A mapping and evolutionary study of porcine sex chromosome gene. Mammalian Genome 13, 588–594.
| A mapping and evolutionary study of porcine sex chromosome gene.Crossref | GoogleScholarGoogle Scholar | 12420137PubMed |
Raudsepp T, Chowdhary BP (2011) Cytogenetics and chromosome maps. In ‘The genetics of the pig.’ 2nd edn. (Eds M Rothschild, A Ruvinsky) pp. 134–178. (CABI: Wallingford, UK)
Raudsepp T, Chowdhary BP (2016) Chromosome aberrations and fertility disorders in domestic animals. Annual Review of Animal Biosciences 4, 15–43.
| Chromosome aberrations and fertility disorders in domestic animals.Crossref | GoogleScholarGoogle Scholar | 26884101PubMed |
Raudsepp T, Das PJ, Avila F, Chowdhary BP (2012) The pseudoautosomal region and sex chromosome aneuploidies in domestic species. Sexual Development: Genetics, Molecular Biology, Evolution, Endocrinology, Embryology, and Pathology of Sex Determination and Differentiation 6, 72–83.
| The pseudoautosomal region and sex chromosome aneuploidies in domestic species.Crossref | GoogleScholarGoogle Scholar |
Rodriguez A, Sanz E, De Mercado E, Gomez E, Martin M, Carrascosa C, Gomez-Fidalgo E, Villagomez DA, Sanchez-Sanchez R (2010) Reproductive consequences of a reciprocal chromosomal translocation in two Duroc boars used to provide semen for artificial insemination. Theriogenology 74, 67–74.
| Reproductive consequences of a reciprocal chromosomal translocation in two Duroc boars used to provide semen for artificial insemination.Crossref | GoogleScholarGoogle Scholar | 20171726PubMed |
Rothschild MF (1996) Genetics and reproduction in the pig. Animal Reproduction Science 42, 143–151.
| Genetics and reproduction in the pig.Crossref | GoogleScholarGoogle Scholar |
Sambrook J, Russell DW (2001) ‘Molecular cloning, a laboratory manual.’ 3rd edn. (Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA)
Skinner BM, Lachani K, Sargent CA, Affara NA (2013) Regions of XY homology in the pig X chromosome and the boundary of the pseudoautosomal region. BMC Genetics 14, 3
| Regions of XY homology in the pig X chromosome and the boundary of the pseudoautosomal region.Crossref | GoogleScholarGoogle Scholar | 23320497PubMed |
Skinner BM, Sargent CA, Churcher C, Hunt T, Herrero J, Loveland JE, Gilbert J (2016) The pig X and Y chromosomes: structure, sequence, and evolution. Genome Research 26, 130–139.
| The pig X and Y chromosomes: structure, sequence, and evolution.Crossref | GoogleScholarGoogle Scholar | 26560630PubMed |
Szczerbal I, Switonski M (2016) Chromosome abnormalities in domestic animals as causes of disorders of sex development or impaired fertility. InTechOpen. Available at
Treff NR, Tao X, Ferry KM, Su J, Taylor D, Scott RT (2012) Development and validation of an accurate quantitative real-time polymerase chain reaction-based assay for human blastocyst comprehensive chromosomal aneuploidy screening. Fertility and Sterility 97, 819–824.
| Development and validation of an accurate quantitative real-time polymerase chain reaction-based assay for human blastocyst comprehensive chromosomal aneuploidy screening.Crossref | GoogleScholarGoogle Scholar | 22342859PubMed |
Tüttelmann F, Gromoll J (2010) Novel genetic aspects of Klinefelter’s syndrome. Molecular Human Reproduction 16, 386–395.
| Novel genetic aspects of Klinefelter’s syndrome.Crossref | GoogleScholarGoogle Scholar | 20228051PubMed |
Villagomez DAF, Parma P, Radi O, Di Meo G, Pinton A, Iannuzzi L, King WA (2009) Classical and molecular cytogenetics of disorders of sex development in domestic animals. Cytogenetic and Genome Research 126, 110–131.
| Classical and molecular cytogenetics of disorders of sex development in domestic animals.Crossref | GoogleScholarGoogle Scholar |
Visootsak J, Graham JM (2006) Klinefelter syndrome and other sex chromosomal aneuploidies. Orphanet Journal of Rare Diseases 1, 42
| Klinefelter syndrome and other sex chromosomal aneuploidies.Crossref | GoogleScholarGoogle Scholar | 17062147PubMed |
Weaver S, Dube S, Mir A, Qin J, Sun G, Ramakrishnan R, Jones RC, Livak KJ (2010) Taking qPCR to a higher level: analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution. Methods 50, 271–276.
| Taking qPCR to a higher level: analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution.Crossref | GoogleScholarGoogle Scholar | 20079846PubMed |
