The Haemoglobin Subunits Alpha and Beta: Old and New Genetic Variants in the Italian Mediterranean Buffalo

Czech Journal of Animal Science, 64, 2019 (7): 279–290 Original Paper https://doi.org/10.17221/14/2018-CJAS The haemoglobin subunits alpha and beta: Old and new genetic variants in the Italian Mediterranean buffalo Rosario Rullo1, Aldo Di Luccia2, Elena Ciani3*, Elisa Pieragostini4 1Institute for the Animal Production Systems in the Mediterranean Environment, Napoli, Italy 2Department of Agricultural, Food and Environmental Sciences, University of Foggia, Foggia, Italy 3Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “Aldo Moro”, Bari, Italy 4Department of Emergency and Organ Transplantation, University of Bari “Aldo Moro”, Bari, Italy *Corresponding autor: [email protected] Citation: Rullo R., Di Luccia A., Ciani E., Pieragostini E. (2019): The haemoglobin subunits alpha and beta: Old and new genetic variants in the Italian Mediterranean buffalo. Czech J. Anim. Sci., 64, 279–290. Abstract: Haemoglobin (HB), the most widely distributed respiratory pigment in the animal kingdom, is among the best characterized oxygen-binding proteins, both at functional and molecular level. However, very little information is available about the genomic features of HB in river buffalo (Bubalus bubalis), even though there are reports in literature confirming the presence of interesting polymorphisms at the protein level in Mediterranean buffalo. We hence address the characterization of exonic as well as intronic nucleotide polymorphism in the haemoglobin subunit alpha and beta in a set of nine Italian Mediterranean buffaloes exhibiting different HB phenotypes. The nine buffaloes were selected from a random set of 398 samples, previously analysed for their HB protein polymorphism, in order to account for both globin variants and the evolution of intron variability within the most common domesticated species of the family Bovidae. All four sequenced clones of the subunit alpha were 1311 bp, whereas the length of the five different sequenced clones of the subunit beta ranged from 1841 to 1960 bp, due to an insertion of 119 nucleotides. Six polymorphic sites were detected in the four amplicons of alpha subunit. Among them, two variations concern exclusively haplotype A, while four sequence variations were found to be specific to haplotypeB . Several variations, both in exonic and intronic regions, were detected in the B. bubalis subunit beta. In conclusion, the nucleotide sequence variants observed in this work substantiate the known haemoglobin protein polymorphisms, and an updated protein nomenclature is provided here. In addition, we observed a high sequence similarity in the overall pattern of variation in the haemoglobin subunits, possibly the results of a concerted evolution, with relatively more extensive gene homogenization in river buffalo than in other ruminant species. Keywords: Bubalus bubalis; haemoglobin subunit variation; biodiversity; transposable elements; gene duplication The Italian Mediterranean buffalo (Bubalus into Italy probably in Roman times or with the bubalis), which belongs to the Bubalus genus Arab conquests, spreading across Southern Italy, of the Bovidae family, is generally classified as a where today it forms an economically significant river sub-type of water buffalo, similar to the buf- proportion of livestock. Widely used in the past as falo breeds of Hungary, Romania and the Balkan a draught animal, the Italian Mediterranean water countries (Bigi and Zanon 2008). It was introduced buffalo is now mainly kept for milk production to 279 Original Paper Czech Journal of Animal Science, 64, 2019 (7): 279–290 https://doi.org/10.17221/14/2018-CJAS make mozzarella cheese. A herd-book was opened Thus, our research moved toward the genetic in 1980, and the breed was officially recognised in characterization of exonic as well as intronic poly- 2000. Since then, as selection pressure has focused morphism in the subunits alpha and beta, in order mainly on milk production, it may be assumed to account for both globin variants and the evolu- that its genetic variability has been decreasing. tion of intron variability within the most common As is widely known, a decrease in genetic diversity domesticated species of the family Bovidae. can, in the long run, limit the ability of popula- tions to adapt to future environmental changes. In recent years, growing attention has focused on MATERIAL AND METHODS functional genetics, an area that is recurrently in- cluded amongst animal breeding research priorities. Nomenclature. The nucleotide sequences of all Thanks to its accessibility and obvious biological the buffalo’s haemoglobin alpha and beta subunits importance, haemoglobin has been one of the most have been deposited in the EMBL/GenBank DNA studied polymorphisms in vertebrate species since database and the accession numbers are listed the infancy of both population and evolutionary in Table 1. Parts A and B of Table 1 show the genetics. The functional effect of the haemoglobin gene names as well as the protein denominations phenotype on haematological patterns has been reported according to the National Center for demonstrated in humans as well as in mammalian Biotechnology Information guidelines (Murphy species (Pieragostini et al. 2010). Four statements et al. 2006). As shown in Figure 1, the subunits are crucial to our research mindset. First, being the in haplotype A and those in haplotype B encode most widely-distributed respiratory pigment in the four different globin chains in total, namely αS and animal kingdom, haemoglobin is the best charac- αN the former, and αI and αK the latter. Within terized oxygen-binding protein, both at functional subunits encoded by haplotype A, αS differs from and molecular level. Secondly, genetic diversity is αN at two amino acid positions (Leu130Phe and generally quantified by alleles and genotype fre- Ser132Asn). These replacements are not present quencies at one or more loci. Thirdly, though the in the alpha globin subunits encoded by haplo- structure and organization of haemoglobin subunit type B; in addition, both αS and αN differ from αI clusters has been reported in many species, very and αK at the amino acid residue 65 (Ala65Asn) little information is available about the genomic (Ferranti et al. 2001). As for the subunits encoded features of river buffalo. Finally, interesting protein by genes of haplotype B, αI and αK exhibit a single polymorphisms in the haemoglobin genetic system amino acid substitution each, namely Ile11Val and have been found in Mediterranean buffalo (Di Luc- Gln12Lys, respectively. Finally, as for the beta cia et al. 1991a; Iorio et al. 2004). globin cluster, only two adult beta globin chains Haplotype A HBA1 S HBA2 N 5’ // 3’ …Val-Gln-……………………-Ala-………………-Leu-…-Ser… …Val-Gln-……………………-Ala-………………-Phe-…-Asn… 11 12 65 130 132 11 12 65 130 132 αS αN Haplotype B HBA1 I HBA2 K 5’ // 3’ …Val-Gln-……………………-Ala-………………-Leu-…-Ser… …Val-Lys-……………………Asn-………………-Leu-…-Ser… 11 12 65 130 132 11 12 65 130 132 Figure 1. Schematic representationαI of the alpha subunits (haplotypes A and B) fromαK B. bubalis. Current name of gene, and the corresponding protein, are indicated on the top, and bottom, respectively. Only variant positions of the amino acid sequence are indicated 280 Table 1. Part Α shows the name and Αccession number of alpha and beta subunits. Part B compares new names of haemoglobin (HB) with the previous ones and indicates https://doi.org/10.17221/14/2018-CJAS Animal Science Czech Journalof the corresponding tetramers. Both gene and protein denominations are reported according to the National Center for Biotechnology Information guidelines (Murphy et al. 2006). Part C summarizes the tetramers observable depending on the haemoglobin electrophoretic phenotypes, which, in turn, depend on the genetic polymorphism at subunits alpha and beta. (The gene and protein variants have been named using as superscript letter the letter code indicating the substitution in the mature protein consequent to the mutation in the codons (Table 2). The superscript letter S assigned to the HB phenotype indicates the presence of the slow electrophoretic mobility of HB’s carrying T the β variant; the phenotypes are obviously named according to the homozygosity at HBB subunits and to the fact that HBΑ diplotypes exhibit equal or different subunits) Part Α (genes) Part B (proteins) Part C Subunit Αccession subunit Αccession HB previous HB new HB globin chain responsi- alpha HB HB tetramer alpha No. beta No. name1 name tetramer ble for the HB name diplotype phenotype per phenotype S Α S S Α N Α , 64,2019(7):279–290 HB1 HB S α 2 β 2 α ΑΑ α 2 β 2 , α 2 β 2 ΗΒΑ1 S ΑJ242731 HBB Α ΑM886147 N Α N S S Α N Α S T N T HB3 HB N α 2 β 2 α ΑΑ ΑΑ α 2β 2 , α 2β 2 , α 2β 2 , α 2β 2 I Α I S S S T N T HB2 HB I α 2β 2 α Α Α α 2β 2 , α 2β 2 ΗΒΑ2 Ν ΑJ242733 HBB T ΑM886148 K Α K S Α N Α I Α K Α HB4 HB K α 2 β 2 α ΑB α 2β 2 , α 2β 2 , α 2β 2 , α 2 β 2 HB1s HB ST αS βT αS and βT ΑSBS αS βT , αN βT , αI βT , αK βT 2 2 ΑB 2 2 2 2 2 2 2 2 S Α N Α I Α K Α ΗΒΑ1 Ι ΑJ242732 HBB G ΑM886151 N T N T S S α 2β 2 , α 2β 2 , α 2β 2 , α 2β 2 , HB3s HB NT α 2 β 2 α and β ΑB Α B S T N T I T K T α 2β 2 , α 2β 2 , α 2β 2 , α 2β 2 I T I T I Α K Α HB2s HB IT α 2β 2 α and β BB α 2β 2 , α 2 β 2 ΗΒΑ2 Κ ΑJ242734 HBB E ΑM886150 K T K T S I Α K Α I T K T HB4s HB KT α 2 β 2 α and β BB α 2β 2 , α 2β 2 , α 2β 2 , α 2β 2 BB S S S I T K T HBG HB G α 2 γ2 γ B B α 2β 2 , α 2β 2 HBB P ΑM886149 S HBE HB E α 2 ε2 ε 1Iorio et al.

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