Study of the copy number variation on the sex chromosome in some Iranian sheep breeds

Document Type : Research Paper

Authors

1 Department of Animal Science, Faculty of Animal Science and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

2 Department of Animal Science, Faculty of Animal Science and Fisheries, Sari Agricultural Sciences and Natural Resources University, SARI-IRAN, Iran

3 Department of Animal Science, Faculty of Animal Science and Fisheries, Sari Agricultural Sciences and Natural Resources University, Iran

4 Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak university, ARAK-IRAN, Iran

Abstract

Copy number variation (CNVs) is one of the most important structural variations in the genome which play an important role in the genetic variance of economic traits. In this study, CNVs and copy number variation regions (CNVRs) on the sex chromosome were investigated in three native Iranian sheep breeds, including fat-tailed Baluchi and Lori-Bakhtiari breeds and thin-tailed Zel breed. 50K genotyped samples were obtained and CNVs were identified for each individual using PennCNV software. After identifying CNVs in each individual, the quality control of CNVs was performed with different filters, and CNVRs were identified by using the overlapping regions of CNVs using CNVRuler software. In total, 37, 11 and 4 CNVs of gain events were identified on the X chromosome of Baluchi, Zel and Lori-Bakhtiari sheep, respectively. The minimum, maximum and average length of identified CNVs were 94477, 1293154 and 447694 bp in Baluchi breed, 271819, 906644 and 674854 bp in Zel breed and 99705, 306525 and 167913 bp in Lori Bakhtiari breed, respectively. After merging the CNVs, 30, 10 and 4 CNVRs were identified in these three breeds, respectively, all of which were of the gain events. The analysis of genes in CNVR regions showed that some of these genes (VEGF, VAM21, TRPC5, NDUFA1, APLN and TNMD) were related to fat metabolism. Annotations of genes for molecular function were significantly enriched in the pathway of arylsulfatase activity, which plays a role in reproduction. Further studies on these CNV regions can help identify genes affecting fat metabolism in sheep.

Keywords

Main Subjects


Extended Abstract

Introduction

Copy number variations (CNV), with a minimum size of 50 base-pairs, is one of the most important sources of genetic variation, which together with SNP data provides informative genomic structural data. Several studies have shown that some CNVs play significant role in the phenotypic diversity of economically important traits and the development of disease resistance or susceptibility in sheep. Genome-wide copy-number association studies provide new facilities to identify the genetic mechanisms underlying complex traits in sheep breeds. Since the X chromosome has a remarkable number of genes, it is a valuable study target for the identification of CNVs at the genome level. Several studies have reported the presence of CNVs and selection signatures on the sheep X chromosome. In this study, CNVs and copy number variation regions on the sex chromosome were investigated in three native Iranian sheep breeds, including fat-tailed Baluchi and Lori-Bakhtiari breeds and thin-tailed Zel breed.

 

Materials and Methods

To identify the copy number variation on the X chromosome, the genomic data of 96 Baluchi sheep, 47 Lori-Bakhtiari sheep and 47 Zel sheep, genotyped using Illumina Ovine SNP50 Bead Chip array, were used. The quality control steps were as follows: animals with more than 5% of missing genotypes, SNPs with MAF of less than 5% and SNPs with genotyping call rate of less than 5% and deviation from Hardy-Weinberg equilibrium (p<1×10-6) were discarded from the further study. To identify CNVs, signal intensity ratios (LRR) and B allele frequencies (BAF) files were obtained for each marker. The population B allele frequency was estimated based on the BAF of each marker in the population. The GC model file was created by considering the GC content of the regions surrounding one million base pairs (500 kb each side). Also, to compare and investigate the effect of considering the GC file, the analyzes were also performed without considering the gcmodel. Finally, CNVs were identified for each individual using PennCNV software. After identifying CNVs in each individual, the quality control and filtering of CNVs were performed: samples with standard deviation of log R ratio (LRR) less than 0.3, BAF drift less than 0.01, wave factor less than 0.05 and confidence score less than 15 (less than 10 for gain events) were excluded. Also, CNVs must had at least 3 consecutive SNPs. To identify CNVs on the X chromosome, lastchr --chrx argument in PennCNV was used. Identification of CNVRs was performed using the overlapping regions of CNVs in different animals using CNVRuler software. The UCSC genome browser tool (version 4) was used to identify the gene content located in CNVRs. Finally, gene ontology (GO) analysis was performed for the identified genes using the DAVID.

 

Results and discussion

In this study, X chromosome-linked CNVs and CNVRs were investigated in three Iranian breeds including two fat-tailed breeds (Baluchi and Lori-Bakhtiari) and one thin-tailed breed (Zel). The results showed that considering quality control for GC content, as in previous studies, led to a reduction in the number of CNVs. The minimum, maximum and average length of identified CNVs were 94477, 1293154 and 447694 bp in Baluchi breed, 271819, 906644 and 674854 bp in Zel breed and 99705, 306525 and 167913 bp in Lori Bakhtiari breed, respectively. After merging the CNVs, 30, 10 and 4 CNVRs were identified in these three breeds, respectively, all of which were of the gain events. The analysis of genes in CNVR regions showed that some of these genes (VEGF, VAM21, TRPC5, NDUFA1, APLN and TNMD) were related to fat metabolism. Annotations of genes for molecular function were significantly enriched in the pathway of arylsulfatase activity, which plays a role in reproduction. Further studies on these CNV regions can help identify genes affecting fat metabolism in sheep.

 

Conclusion

In general, more CNVs and CNVRs were identified in the thin- tailed Zell breed compared to the studied fat-tailed breeds. Several genes were identified in the CNVR regions, which were reported to be associated with fat metabolism in previous studies. Therefore, with more detailed studies of these genes, useful information regarding the underlying mechanism of fat metabolism in sheep can be obtained.

Bae, J. S., Cheong, H. S., Kim, L. H., NamGung, S., Park, T. J., Chun, J.-Y., Kim, J. Y., Pasaje, C. F. A., Lee, J. S., & Shin, H. D. (2010). Identification of copy number variations and common deletion polymorphisms in cattle. BMC Genomics, 11, 1–10.
Bekaert, M., & Conant, G. C. (2014). Gene Duplication and Phenotypic Changes in the Evolution of Mammalian Metabolic Networks. PLoS ONE, 9, e87115
Berletch, J. B., Deng, X., Nguyen, D. K., & Disteche, C. M. (2013). Female bias in Rhox6 and 9 regulation by the histone demethylase KDM6A. PLoS genetics, 9, e1003489.
Cannata Serio, M., Graham, L. A., Ashikov, A., Larsen, L. E., Raymond, K., Timal, S., & et al. (2020). Mutations in the V-ATPase Assembly Factor VMA21 Cause a Congenital Disorder of Glycosylation With Autophagic Liver Disease. Hepatology, 72, 1968–1986.
Conrad, D. F., Andrews, T. D., Carter, N. P., Hurles, M. E., & Pritchard, J. K.  A. (2006).  high-resolution survey of deletion polymorphism in the human genome. Nature Genetics, 38, 75–81.
Cui, Y., Yan, H., Wang, K., Xu, H., Zhang, X., Zhu, H., Liu, J., Qu, L., Lan, X., & Pan, C. (2018). Insertion/Deletion Within the KDM6A Gene Is Significantly Associated With Litter Size in Goat. Frontiers in Genetics, 9, 1664-8021
Di Gerlando, R., Mastrangelo, S., Tolone, M., Rizzuto, I., Sutera, A. M., Moscarelli, A., Portolano, B., & Sardina, M.T, (2022). Identification of Copy Number Variations and Genetic Diversity in Italian Insular Sheep Breeds. Animals, 12, 217.
Diskin, S. J., Li, M., Hou, C., Yang, S., Glessner, J., Hakonarson, H., Bucan, M., Maris, J. M., & Wang, K. (2008). Adjustment of genomic waves in signal intensities from whole-genome SNP genotyping platforms. Nucleic acids research, 36, e126–e126.
Fontanesi, L., Beretti, F., Martelli, P. L., Colombo, M., Dall’Olio, S., Occidente, M., Portolano, B., Casadio, R., Matassino, D., & Russo, V. (2011). A first comparative map of copy number variations in the sheep genome. Genomics, 97, 158–165
Gholizadeh, M., Rahimi-Mianji, G., Nejati-Javaremi, A., De Koning, D. J., & Jonas, E., (2014). Genomewide association study to detect QTL for twinning rate in Baluchi sheep. Journal of genetics, 93(2), 489-493.
Gustavson, K., & Hagberg, B. (1971). The incidence and genetics of metachromatic leucodystrophy in northern Sweden. Acta Paediatrica, 60, 585–590.
Hanson, S. R., Best, M. D., & Wong, C. (2004). Sulfatases: structure, mechanism, biological activity, inhibition, and synthetic utility. Angewandte Chemie International Edition, 43, 5736–5763.
Heyer, E., Segurel, L. (2010). Looking for signatures of sex-specific demography and local adaptation on the X chromosome. Genome biology, 11, 1–3.
Holmes, D. I. R., & Zachary, I. (2005). The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome biology, 6, 1–10.
Hou, Y., Liu, G. E., Bickhart, D. M., Cardone, M. F., Wang, K., Kim, E., Matukumalli, L. K., Ventura, M., Song, J., & VanRaden, P.M. (2011). Genomic characteristics of cattle copy number variations. BMC Genomics, 12, 1–11.
Karaman, S., Hollmén, M. Robciuc, M. R., Alitalo, A., Nurmi, H., Morf, B., Buschle, D., Alkan, H. F., Ochsenbein, A. M., Alitalo, K., Wolfrum, C., & Detmar, M. (2015). Blockade of VEGF-C and VEGF-D modulates adipose tissue inflammation and improves metabolic parameters under high-fat diet. Molecular Metabolism, 4, 93–105.
Kim, J. H., Hu, H. J., Yim, S. H., Bae, J.S., Kim, S. Y., & Chung, Y. J. (2012). CNVRuler: a copy number variation-based case–control association analysis tool. Bioinformatics, 28, 1790–1792.
Kolodny, E. H., Fluharty, A. L., Scriver, C. R., Beaudet, A. L., Sly, W. S., & Valle, D. (1995). The metabolic and molecular bases of inherited disease. 7th Ed. New York: McGraw-Hill; 1995. pp. 2693–2739
Kommadath, A., Grant, J. R., Krivushin, K., Butty, A. M., Baes, C.F., Carthy, T. R., Berry, D. P., & Stothard, P. (2019). A large interactive visual database of copy number variants discovered in taurine cattle. Gigascience, 8, giz073.
Laseca, N., Molina, A., Valera, M., Antonini, A., & Demyda-Peyrás, S. (2022). Copy Number Variation (CNV): A New Genomic Insight in Horses. Animals, 12, 1435.
Ladeira, G. C., Pilonetto, F., Fernandes, A. C., Bóscollo, P.P., Dauria, B. D., Titto, C. G., Coutinho, L. L., e Silva, F. F., Pinto, L. F. B., & Mourão, G. B. (2022). CNV detection and their association with growth, efficiency and carcass traits in Santa Inês sheep. Journal of Animal Breeding and Genetics, 139: 476-487.
Lai, F.N., Zhai, H.L., Cheng, M., Ma, J. Y., Cheng, S. F., Ge, W., Zhang, G. L., Wang, J. J., Zhang, R. Q., & Wang, X. (2016). Whole-genome scanning for the litter sizetrait associated genes and SNPs under selection in dairy goat (Capra hircus), Scientific Reports, 6, 38096.
Liu, J., Zhang, L., Xu, L., Ren, H., Lu, J., Zhang, X., Zhang, S., Zhou, X., Wei, C. & Zhao, F. (2013). Analysis of copy number variations in the sheep genome using 50K SNP BeadChip array. BMC Genomics 14, 1–11.
Ma, Q., Liu, X., Pan, J., Ma, L., Ma, Y., He, X., Zhao, Q., Pu, Y., Li, Y., &  Jiang, L. (2017). Genome-wide detection of copy number variation in Chinese indigenous sheep using an ovine high-density 600 K SNP array. Scientific Reports, 7, 912.
Ma, Y., Zhang, Q., Lu, Z., Zhao, X., & Zhang, Y. (2015). Analysis of copy number variations by SNP50 BeadChip array in Chinese sheep. Genomics, 106, 295–300.
Macé, A., Tuke, M.A., Deelen, P., Kristiansson, K., Mattsson, H., Nõukas, M., & et al. (2017). CNV-association meta-analysis in 191,161 European adults reveals new loci associated with anthropometric traits. Nature Communications, 8, 744.
Manolio, T. A., Collins, F. S., Cox, N.J., Goldstein, D. B., Hindorff, L. A., Hunter, D. J., McCarthy, M. I., Ramos, E. M., Cardon, L. R., & Chakravarti, A. (2009). Finding the missing heritability of complex diseases. Nature, 461, 747–753.
Mansour, A. A., Gafni, O., Weinberger, L., Zviran, A., Ayyash, M., Rais, Y., Krupalnik, V., Zerbib, M., Amann-Zalcenstein, D., & Maza, I. (2012). The H3K27 demethylase Utx regulates somatic and germ cell epigenetic reprogramming. Nature, 488, 409–413.
Mitsunaga-Nakatsubo, K., Akimoto, Y., Kawakami, H., & Akasaka, K., (2009). Sea urchin arylsulfatase, an extracellular matrix component, is involved in gastrulation during embryogenesis. Development genes and evolution, 219, 281–288.
Moradi, M. H., Nejati-Javaremi, A., Moradi-Shahrbabak, M., Dodds, K. G., & McEwan, J. C., (2012). Genomic scan of selective sweeps in thin and fat tail sheep breeds for identifying of candidate regions associated with fat deposition. BMC genetics, 13(1), 1-15.
Parenti, G., Meroni, G., & Ballabio, A. (1997). The sulfatase gene family. Current opinion in genetics & development, 7, 386–391.
Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M. A. R., Bender, D., Maller, J., Sklar, P., De Bakker, P. I. W., & Daly, M. J. (2007). PLINK: a tool set for whole-genome association and population-based linkage analyses. The American journal of human genetics 81, 559–575.
Redon, R., Ishikawa, S., Fitch, K.R., Feuk, L., & Perry, G.H., Andrews, T.D., Fiegler, H., Shapero, M.H., Carson, A.R., Chen, W. (2006). Global variation in copy number in the human genome. Nature, 444, 444–454.
Rode, B., Yuldasheva, N. Y., Baxter, P. D., Sedo, A., Ainscough, J.F., Shires, M., Kearney, M. T., Bailey, M. A., Wheatcroft, S.B., Beech, D. J. (2019). TRPC5 ion channel permeation promotes weight gain in hypercholesterolaemic mice. Scientific Reports, 9, 773.
Saiki, A., Olsson, M., Jernås, M., Gummesson, A., McTernan, P. G., Andersson, J., Jacobson, P., Sjöholm, K., Olsson, B., Yamamura, S., Walley, A., Froguel, P., Carlsson, B., Sjöström, L., Svensson, P. A., & Carlsson, L. M. S. (2009). Tenomodulin Is Highly Expressed in Adipose Tissue, Increased in Obesity, and Down-Regulated during Diet-Induced Weight Loss. The Journal of Clinical Endocrinology & Metabolism, 94, 3987–3994.
Shaikh, T. H. (2017). Copy number variation disorders. Current genetic medicine reports 5, 183–190.
Sole, M., Ablondi, M., Binzer-Panchal, A., Velie, B. D., Hollfelder, N., Buys, N., Ducro, B.J., Francois, L., Janssens, S., Schurink, A., & et al. (2019). Inter- and intra-breed genome-wide copy number diversity in a large cohort of European equine breeds. BMC Genomics, 20, 759.
Suriyaprom, K., Pheungruang, B., Tungtrongchitr, R., & Sroijit, O.Y. (2020). Relationships of apelin concentration and APLN T-1860C polymorphism with obesity in Thai children. BMC Pediatrics, 20, 455.
Taghizadeh, S., Gholizadeh, M., rahimi-Mianji, G., Moradi, M. H., Costilla, R., Moore, S., & Di Gerlando, R. (2022). Genome-wide identification of copy number variation and association with fat deposition in thin and fat-tailed sheep breeds. Scientific Reports, 12, 8834.
Tinahones, F. J., Coín-Aragüez, L., Mayas, M. D., Garcia-Fuentes, E., Hurtado-del-Pozo, C., Vendrell, J., Cardona, F., Calvo, R. M., Obregon, M. J., & El Bekay, R. (2012). Obesity-associated insulin resistance is correlated to adipose tissue vascular endothelial growth factors and metalloproteinase levels. BMC Physiology, 12, 4.
Uddin, M., Tammimies, K., Pellecchia, G., Alipanahi, B., Hu, P., Wang, Z., Pinto, D., Lau, L., Nalpathamkalam, T., & Marshall, C.R. (2014). Brain-expressed exons under purifying selection are enriched for de novo mutations in autism spectrum disorder. Nature Genetics, 46, 742–747
Viviano, B.L., Paine-Saunders, S., Gasiunas, N., Gallagher, J., & Saunders, S. (2004). Domain-specific modification of heparan sulfate by Qsulf1 modulates the binding of the bone morphogenetic protein antagonist Noggin. Journal of Biological Chemistry, 279, 5604–5611.
Wang, K., Li, M., Hadley, D., Liu, R., Glessner, J., Grant, S.F.A., Hakonarson, H., & Bucan, M. (2007). PennCNV: an integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data. Genome research, 17, 1665–1674.
Wang, Z., Guo, J., Guo, Y., Yang, Y., Teng, T., Yu, Q., Wang, T., Zhou, M., Zhu, Q., & Wang, W. (2020). Genome-wide detection of CNVs and association with body weight in sheep based on 600K SNP arrays. Frontiers in Genetics, 11, 558.
Whitman, M. C., Di Gioia, S. A., Chan, W.-M., Gelber, A., Pratt, B. M., Bell, J. L., et al. (2020). Recurrent Rare Copy Number Variants Increase Risk for Esotropia. Investigative Ophthalmology & Visual Science, 61, 22.
Wu, A., Anupriwan, A., Iamsaard, S., Chakrabandhu, K., Santos, D. C., Rupar, T., Tsang, B. K., Carmona, E., & Tanphaichitr, N. (2007). Sperm surface arylsulfatase A can disperse the cumulus matrix of cumulus oocyte complexes. Journal of cellular physiology, 213, 201–211.
Xu, K., Chen, X., Yang, H., Xu, Y., He, Y., Wang, C., Huang, H., Liu, B., Liu, W., & Li, J. (2017). Maternal Sall4 is indispensable for epigenetic maturation of mouse oocytes. Journal of Biological Chemistry, 292, 1798–1807.
Xu, L., Hou, Y., Bickhart, D. M., Song, J., & Liu, G.E. (2013). Comparative Analysis of CNV Calling Algorithms: Literature Survey and a Case Study Using Bovine High-Density SNP Data. Microarrays.
Yang, L., Xu, L., Zhou, Y., Liu, M., Wang, L., Kijas, J.W., Zhang, H., Li, L., & Liu, G. E. (2018). Diversity of copy number variation in a worldwide population of sheep. Genomics, 110, 143–148.
Yuan, C., Lu, Z., Guo, T., Yue, Y., Wang, X., Wang, T., Zhang, Y., Hou, F., Niu, C., & Sun, X. (2021). A global analysis of CNVs in Chinese indigenous fine-wool sheep populations using whole-genome resequencing. BMC Genomics, 22, 1–10.
Zhang, R., Hou, T., Cheng, H., & Wang, X. (2019). NDUFAB1 protects against obesity and insulin resistance by enhancing mitochondrial metabolism. The FASEB Journal, 33, 13310–13322.
Zhang, X., Yan, Q., Guo, X., Chen, C., Chen, R., Cai, Z., & Tang, A. (2016). Expression profile of SPACA5/Spaca5 in spermatogenesis and transitional cell carcinoma of the bladder. Oncology Letters, 12, 3731–3738.
Zhu, C., Fan, H., Yuan, Z., Hu, S., Ma, X., Xuan, J., Wang, H., Zhang, L., Wei, C., Zhang, Q., Zhao, F., & Du, L. (2016). Genome-wide detection of CNVs in Chinese indigenous sheep with different types of tails using ovine high-density 600K SNP arrays. Scientific Reports, 6, 27822.
Zhu, C., Li, M., Qin, S., Zhao, F., & Fang, S. (2020). Detection of copy number variation and selection signatures on the X chromosome in Chinese indigenous sheep with different types of tail. Asian-Australasian journal of animal sciences, 33, 1378.