Abstract
Resistance to fatal disease bovine spongiform encephalopathy (BSE), due to misfolded prion protein in cattle, is associated with a 23-bp indel polymorphism in the putative promoter and a 12-bp indel in intron 1 of the PRNP gene. Gayal (Bos frontalis) is an important semiwild bovid species and of great conservation concern, but till today these indel polymorphisms have not been evaluated in gayals. Therefore, we collected 225 samples of gayals and evaluated the genetic indel polymorphism in the two regions of this PRNP gene. The results revealed high allelic frequencies of insertions at these indel sites: 0.909 and 0.667 for, respectively, the 23 bp and 12 bp indels, both also with significant genotype frequencies (\(\chi ^{2}\): 9.81; 23 bp and \(\chi ^{2}\): 43.56; 12 bp). At the same time, the haplotype data showed indel polymorphisms with extremely low deletion (0.01) in both regions of the PRNP gene. We compared these data with those reported for healthy and BSE-affected cattle (Bos taurus) breeds from two European countries, Germany and Switzerland, and significant difference (\(P\,{<}\,0.001\)) was observed between BSE-affected as well as the healthy cattle. Further, our data were also extensively compared with previous reports on BSE and highly significant (\(P\,{<}\,0.001\)) outcomes were observed. This result suggested negligible genetic susceptibility to BSE in gayals. To the best of our knowledge, this study is the first comprehensive deciphering information about the PRNP indel polymorphisms of 23 bp and 12 bp in gayals, a semiwild species of China.
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Introduction
Bovine spongiform encephalopathy (BSE) diagnosed both clinically and pathologically as a neurodegenerative disorder that seriously affects the health of cattle. Its clinical features include aggressive behaviour and ataxia, while the pathological features are prion protein misfolding with aggregation followed by brain’s spongiform degeneration (Imran et al. 2012). Prion protein plays a fundamental role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). This protein is encoded by the prion protein gene (PRNP) which is a polymorphic gene associated with influencing BSE vulnerability in cattle. Among these, a number of polymorphisms and octapeptide repeats in coding sequence and two insertion/deletion (indel) polymorphisms characterized by a 23-bp indel in the putative promoter and a 12-bp indel in intron 1 were retrieved (Zhao et al. 2015). Previous studies revealed that these two traits emanated from the deletion of alleles either at a single or both 23 bp and 12 bp indel sites, associated with higher susceptibility to classical BSE (Sander et al. 2004; Juling et al. 2006). However, BSE has not been reported in gayal (Xi et al. 2011), a semiwild endangered bovine species with no defined background (Mei et al. 2016). The reports of gayal from Bangladesh, Bhutan, China, India and Myanmar have confirmed its possession of a different chromosome complement of \(2n=58\) (while cattle \(2n=60\) and gaur \(2n=56\); Xi et al. 2012).
There are no evidence of successful cross-breeding between gayal and other close relatives, thus the species still remains natural to date with morphological features such as bulging forehead ridge, wide and short ears and feet with white stockings (He et al. 2009; Qu et al. 2012). Gayal has been under studied possibly because it is not domestic cattle and because of its remote distributions (Mohan et al. 2007). Most of the plant species it feeds on are not suitable for stall feeding for domesticated cattle (Zhao et al. 2003; Deng et al. 2007; Xi et al. 2011; Mei et al. 2016). It feeds on leaves, grasses, bamboo, reeds and other locally available plant species (Mei et al. 2016; Memon et al. 2018). In contrast to its counterparts, gayal’s big body size and meat quality makes it suitable for hybrid breeding to improve meat and milk traits (Ge et al. 1996; Giasuddin et al. 2003).
Prion diseases are caused by mutation of host-encoded normal cellular prion protein \((\hbox {PrP}^{\mathrm{C}})\) into pathogenic misfolded scrapie prion protein \((\hbox {PrP}^{\mathrm{Sc}})\) (Prusiner et al. 1998; Collinge and Clarke 2007). The two noncoding polymorphisms, 23-bp indel in the putative promoter region and 12-bp indel in intron 1 of PRNP, regulate the variability of classical BSE in bovine (Sander et al. 2004). For instance, the 12-bp indel has been detected to influence resistance to classical BSE in UK Holsteins (Juling et al. 2006), while 23-bp indel effects mRNA, the levels of which are detected to be greater in the medulla oblongata of Japanese Black cattle (Msalya et al. 2011). However, there is evidence that the susceptibility of a typical BSE is not linked to either of 12-bp and 23-bp indel polymorphisms (Brunelle et al. 2007).
The objectives of this study were to identify the allele, genotype and haplotype frequencies of PRNP indel polymorphisms in gayal from the Yunnan province of China and compare them with the reported healthy and BSE-affected cattle for evaluation of potential resistance to BSE.
Materials and methods
Animals and sample collection
In total, 225 gayal samples were collected from the Gongshan, China. Physical appearances were considered during sample collection to minimize error of sampling crossbreds. Further, all the analysed samples were first investigated for the presence of mitochondrial lineage of gayal in the previous results (Gou et al. 2010; Xi et al. 2012). About 5 mL of blood sample from each animal was carefully collected from the jugular vein and were stored at \(-20{^{\circ }}\hbox {C}\).
Polymerase chain reaction (PCR) and sequencing
Total genomic DNA was extracted following a previous protocol (Sambrook et al. 1989). The PRNP region was amplified using previously described primers by Nakamitsu et al. (2006) – forward primer: \(5^\prime \)-CTTCTCTCTCGCAGAAGCAG-\(3^\prime \) and reverse primer: \(5^\prime \)-CCCTTGTTCTTCTGAGCTCC-\(3^\prime \) for the 12-bp indel and by Brunelle et al. (2008) – forward primer: \(5^\prime \)-AAGGCACTTCAATCAGTACAC-\(3^\prime \) and reverse primer: \(5^\prime \)-AAGAGTTGGACAGGCACAATG-\(3^\prime \) for the 23-bp indel, respectively. The PCR was carried out in a reaction volume of 25 \(\mu \hbox {L}\), containing 2.0 \(\mu \hbox {L}\) DNA (\(\sim \)50 ng), 2.0 \(\mu \hbox {L}\) 250 \(\mu \hbox {mol}/\hbox {L}\) dNTPs, 2.5 \(\mu \hbox {L}\) buffer (\(\hbox {MgCl}_{2}\) in the buffer provided by the manufacture), 2.0 \(\mu \hbox {L}\) 10 \(\mu \hbox {mol}/\hbox {L}\) forward primer, 2.0 \(\mu \hbox {L}\) 10 \(\mu \hbox {mol}/\hbox {L}\) reverse primer and \(0.2 \mu \hbox {L} 10 \times \) Taq DNA polymerase (5 \(\hbox {U}/\mu \hbox {L}\), Beijing TransGen Biotechnology, China). PCR cycling conditions were as follows: 5 min at \(94{^{\circ }}\hbox {C}\), 35 cycles of amplification (45 s at \(94{^{\circ }}\hbox {C}\), 45 s at \(54{^{\circ }}\hbox {C}\), 60 s at \(72{^{\circ }}\hbox {C}\)) and finally 6 min at \(72{^{\circ }}\hbox {C}\). All PCR products were electrophoresed on 2% agarose gel containing ethidium bromide for visualization. Electrophoresis of the PCR product conducted in either a 191 bp (+) or 168 bp (−) for 23-bp indel polymorphism and in either a 215 bp (+) or a 203 bp (−) for 12-bp indel polymorphism. PCR products were selected randomly and bidirectional sequencing was performed on an ABI 3730x Genetic Analyzer (Applied Biosystems, Foster City, USA).
Data analyses
Genotype and allele frequencies of the gayal PRNP promoter and intron 1 variants were analysed manually and deviation from the Hardy–Weinberg (H–W) equilibrium was calculated using the chi-squared test \((\chi ^{2})\) (Oztabak et al. 2009; Xi et al. 2011). The haplotype frequencies were analysed from the genotype data using Haploview 4.0 software (Barrett et al. 2005) and diversity indices were calculated following Nei and Li (1979).
Results
Allele genotype frequencies
We discovered that the allele frequencies of both 23-bp insertion in the promoter region and 12-bp insertion in intron 1 of PRNP were significantly high (0.91–23 bp insertion and 0.67–12 bp insertion). This results in significant chi-square values for genotype frequencies (++23 bp and \(+-\)12 bp indels) when tested for deviation from the H–W equilibrium (\(\chi ^{2}\): 9.81; \(P\,{<}\,0.01\) and \(\chi ^{2}\): 43.56; \(P\,{<}\,0.001\)) (table 1). Further, the allele and genotype frequencies from our data were compared with both healthy and affected German and Swiss cattle (table 2). The allele and genotype frequencies of 23-bp and 12-bp indel polymorphisms were significantly higher (\(P\,{<}\,0.001\)) from healthy as well as affected cattle of both breeds except that allele frequency of 12 bp of German healthy cattle was significantly different (\(P\,{<}\,0.05\)) less than (\(P\,{<}\,0.001\)) in table 2. Further, the allele frequency of gayal was compared extensively with previous data of cattle, buffalo and yak species (table 3) and we detected that the insertion was higher than the deletion.
Haplotype frequencies
We detected four different haplotypes (23+/12+, 23+/12−, 23−/12+ and 23−/12−) in the PRNP promoter and intron 1 of gayal species (table 1). The most frequent haplotype was 23+/12+ with a frequency of 0.79, which was higher than 23−/12+ (frequency=0.08), 23+/12− (frequency=0.12) and 23−/12− (frequency=0.01). The gayal haplotypes were further compared with healthy and affected German and Swiss cattle. The haplotypes of 23-bp and 12-bp indel polymorphisms were also significantly higher (\(P\,{<}\,0.001\)) compared with healthy as well as affected cattle of both breeds (table 4).
Discussion
Our study revealed that the allele and genotype frequencies of 23-bp and 12-bp indels were significantly different (\(P\,{<}\,0.05\)) from healthy as well as affected cattle of both breeds reported previously (Sander et al. 2004; Haase et al. 2007) in table 2. This finding is comparable with the previous study on Dehong cattle (Zhao et al. 2010). The PRNP indel polymorphism allele frequencies of the gayal data were further compared with different species of Bovidae family cattle, buffalo and yak (table 3). Invariant frequency of 12 bp polymorphism vary between 0.11 (Herefod cattle breed; Zhu et al. 2011) and 0.99/1.0 (Guangnan, Dehong, Chongqing and Guizhou buffalo breed; Zhao et al. 2015). The 12-bp indel polymorphisms in gayal and cattle populations were detected to be similar. In accordance with the previous reports, gayal showed variant of 23 bp polymorphism (frequency: 0.91) close to Anatolian water buffalo. From earlier studies, it is evident that insertion of 23 bp in the putative promoter and a 12 bp in intron 1 is strongly associated with BSE in cattle (Haase et al. 2007), buffalo (Oztabak et al. 2009) and yak (Zhao et al. 2009). However, a contradictory observation on the association of 23 bp in the putative promoter and 12 bp in intron 1 in Hereford cattle was presented by Zhu et al. (2011). Our results corroborated with the findings of the previous studies that were conducted on several cattle and buffaloes (Juling et al. 2006; Oztabak et al. 2009; Zhao et al. 2009, 2015) (table 3).
We further characterized and compared the frequency distributions of the 23-bp and 12-bp indel polymorphisms of gayal with healthy and BSE cattle. This includes extensively reported data of healthy cattle and BSE cattle (Sander et al. 2004; Seabury et al. 2004; Jeong et al. 2006; Juling et al. 2006; Nakamitsu et al. 2006; Brunelle et al. 2007, 2008; Czarnik et al. 2007, 2011; Haase et al. 2007; Kerber et al. 2008; Muramutsu et al. 2008; Un et al. 2008; Kim et al. 2009; Msalya et al. 2009; Murdoch et al. 2010; Shimogiri et al. 2010; Zhao et al. 2010; Qin et al. 2011; Zhu et al. 2011; Galvao et al. 2012; Gurgul et al. 2012; Uchida et al. 2014). The results showed that the 23 bp and 12 bp allele frequencies in BSE cattle were significantly higher (\(P\,{<}\,0.001\)) than gayal (figure 1, a&c). Moreover, the genotype frequencies of 23 and 12 bp were significantly different from healthy as well as affected cattle (figure 1, b&d). Further, the haplotype frequencies were also significant for both healthy and affected cattle (figure 1e). It was reported earlier that susceptibility to BSE was strongly associated with 12 bp deletion or D23–D12 haplotype (Juling et al. 2006). The low haplotype distribution of D23–D12 haplotypes indicated less susceptibility of Chinese cattle to BSE (Zhao et al. 2010). This is similar to our results with a lower haplotype D12–D23 frequency (0.01) showing lower tendency toward susceptibility.
Further, we compared our results with those reported for other species which have not been found with and probably never exposed to BSE. Our results showed that haplotype D23–D12 was lower than buffalo and yak (0.027 versus 0.05 and 0.813, respectively) (Oztabak et al. 2009; Zhao et al. 2009). More interestingly, the 23 bp or 12 bp insertion frequencies in our gayals of the Yunnan province of China were found even more favourable for protection against BSE infection than in yak and buffalo. However, only intracerebral and oral challenge studies with BSE in cattle or gayals could serve as real proof that the PRNP 23 bp/12 bp insertions are valid for application in breeding practice.
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Acknowledgements
The current study was funded by the National Nature Science Foundation of China (grant nos 31460583 and 31101640) and the Yunnan Provincial Key Laboratory of Animal Nutrition and Feed (grant no. DYCX2015004). We thank the two anonymous reviewers for invaluable comments on the manuscript and also appreciate the help of staff (especially Zhijun He and Baqi Su) from the Gongshan County Agriculture, Science and Technology Bureau during sample collection.
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Memon, S., Li, G., Xiong, H. et al. Deletion / insertion polymorphisms of the prion protein gene (PRNP) in gayal (Bos frontalis). J Genet 97, 1131–1138 (2018). https://doi.org/10.1007/s12041-018-1005-x
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DOI: https://doi.org/10.1007/s12041-018-1005-x