Comparison of ovine β-globin haplotype sequences and a new multiplex PCR for identification

• Published in: Veterinary parasitology Vol. 300; p. 109592
• Main Authors: Okino, Cintia Hiromi, Caminotto, Lara Cristina Frizoni, Kapritchkoff, Rafaela Tami Ikeda, Niciura, Simone Cristina Méo, Esteves, Sérgio Novita, Santos, Isabella Barbosa dos, Toscano, João Henrique Barbosa, Oliveira, M.C.S., Chagas, A.C.S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2021
• Subjects: Allele; Animals; B haplotype; Beta globin C; beta-Globins - genetics; Haemonchiasis - veterinary; Haemonchus - genetics; Haplotypes; Morada Nova; Multiplex Polymerase Chain Reaction - veterinary; Resistance; Sheep; Sheep Diseases - diagnosis; Resistance; Sheep; Allele; Beta globin C; Morada Nova; B haplotype
• ISSN: 0304-4017; 1873-2550
• DOI: 10.1016/j.vetpar.2021.109592

•New multiplex conventional PCR for ovine β-globin haplotype identification.•100 % of agreement between qPCR and new multiplex PCR assays.•New oligonucleotides for ovine β-globin sequencing.•Alignment of β-globin haplotype sequences.•Unique nucleotide deletion in AA haplotype of Morada Nova breed. Deletion of pre-adult βC-globin in sheep harboring BB haplotype of β-globin was associated to decreased tolerance to anemia and hypoxia, and consequently, reduced resistance to Haemonchus contortus infection, which is closely related to severe anemia. Recently, a qPCR using hydrolysis probe was successfully developed for β-globin haplotype identification, and association between resistance against H. contortus and presence of βA allele was observed in Morada Nova sheep. Thus, this study aimed to better investigate the differences between β-globin haplotypes and to develop a conventional multiplex PCR, as an alternative to qPCR assay for β-globin haplotype identification. A total of 333 Morada Nova lambs had their blood collected and tested by both qPCR and new multiplex PCR, and 100 % of agreement was observed between the results. Since different primers were designed for such assay development, including different target genes, high specificity of both methods may be also highlighted. Three A haplotype samples were submitted to DNA Sanger sequencing of β-globin gene and compared to sequences previously deposited in Genbank. One nucleotide deletion in intronic region was observed only in AA haplotype of Morada Nova animals, while in BB animals the nucleotide remained present. To the best of our knowledge, this is the first report of multiplex conventional PCR for ovine β-globin haplotype identification. The advantages of the developed conventional PCR are reduced reagents costs (less than a half price) and wider reachability, since even labs without real time PCR thermocyclers are able to offer this assay. Therefore, it may become an important tool for sheep producers to improve genetic selection of parasite resistant animals.