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-Casein Gene Revealed by Resequencing and Haplotype Inference Analysis
* Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO), Universidade do Porto, Campus Agrário de Vairão, 4485-661 Vairão, Portugal
Department of Zoology, Government Vidarbha Institute of Science and Humanities, Amravati, India
Reproduction Laboratory, Research Center of Sheep Breeding, Mynbaevo village, Almaty Oblast, 040622 Kazakhstan
Institute of Animal Breeding and Genetics, Justus-Liebig University, 35390 Giessen, Germany
1 Corresponding author: albanobp{at}fc.up.pt
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ABSTRACT |
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)-casein gene (CSN3) for a panel of samples of domestic cattle from 8 countries and a close relative species, the gayal (Bos frontalis). Six single nucleotide polymorphisms were identified. Haplotype inference revealed 12 haplotypes, of which 8 were newly discovered. Among these 8 new haplotypes, 5 differed by one nonsynonymous mutation and 3 differed by one silent mutation from previously well-characterized CSN3 alleles. From those, one was shared by the gayal and Zebu, was different from CSN3*B at position Ile136Thr, and showed a close phylogenetic relationship with the banteng, gaur, and yak. The other 7 new haplotypes were detected in our panel of worldwide local cattle breeds but were absent from previously reported commercial breeds. These results support the hypothesis that genetic diversity at the coding region of CSN3 has been underestimated. This study also highlights how important it is to resequence functionally important genes in worldwide local cattle breeds, many of which are threatened by extinction or replacement by commercial breeds.
Key Words: -casein • single nucleotide polymorphism • haplotype inference
The bovine -casein protein plays an important role in cheese production technology (e.g., stabilization of casein micelles), and in physiological processes such as cytotoxic and antibacterial activities important for disease resistance (Dziuba and Minkiewicz, 1996; Matin and Otani, 2002). For example, previous studies demonstrated that genetic variants A and B of this protein show different biological activities (e.g., Yvon et al., 1994) and are associated with milk production traits (e.g., Ng-Kwai-Hang et al., 1984). Because different variants of this protein might have different functional activities or associations with milk yield traits, it is of longstanding interest to identify and characterize the diversity harbored in the -casein gene (CSN3) in the bovine species (Bos spp.).
Until now, researchers have reported 10 well-characterized genetic variants (A, A1, B, C, E, F, G, H, I, and J) in domestic cattle (Bos taurus and Bos indicus) (Prinzenberg et al., 2008) and 4 genetic variants found in the domestic yak (Bos grunniens; Bai et al., 2008; Prinzenberg et al., 2008). So far, most of these variants have been genotyped by using PCR-single strand conformation polymorphism analyses (e.g., Prinzenberg et al., 1999, 2008). This routine genotyping system is cost-effective and sufficiently sensitive for both the genotyping of known mutations and the detection of new mutations in DNA fragments. However, its efficiency is poor for detecting sequence variation for larger DNA fragments (e.g., >300 bp; Sheffield et al., 1993; Jaeckel et al., 1998).
Characterization of variation in functional genes (such as the milk protein genes) with potentially adaptive variation is increasingly urgent because numerous local breeds and wild relative taxa are being lost at an accelerating rate (Food and Agriculture Organization of the United Nations, 2007). However, most studies on functional genes include only a few selected cattle breeds from Europe (Prinzenberg et al., 1999). Apart from the few studies in the most widely distributed breeds of B. indicus (e.g., Sahiwal, Gir), there is a great lack of information on genetic diversity in native Asiatic cattle at functional important genes, such as the CSN3. For example, the few existing reports on the genetic diversity of CSN3 in native Asiatic cattle were limited to the genotyping of the major genetic variants (alleles A and B) by using techniques such as PCR-RFLP, which does not allow detection of new variants (Mitra et al., 1998). Thus, it is important to screen for genetic diversity at the CSN3 (as well as in the other milk protein and functional genes) in cattle from Asia to have a more accurate worldwide estimation of the genetic diversity patterns for this gene.
Because very few molecular-based data exist on the genetic origin of cattle from Central and South Asia, it is important first to use a more efficient approach to detect new variants in the CSN3 gene. For that task, resequencing of the entire exon IV, which harbors all the functionally important mutations, is highly recommended. Indeed, technological advances have contributed to a significant reduction of the resequencing cost; therefore, resequencing has become an affordable alternative to other techniques such as PCR-single strand conformation polymorphism.
The aim of this study was to use resequencing to search for new CSN3 alleles in a panel of native Asian and Northern African cattle. Because limited information exists on the genetic diversity in this gene for other species in the Bos genus, we also sequenced samples from the supposed Zebu x gaur (Bos gaurus) hybrid, the gayal (Bos frontalis).
Tissue samples were collected from local cattle in Kazakhstan (B. taurus, n = 3), Kyrgyzstan (B. taurus, n = 4), Mongolia (B. taurus, n = 1), Turkmenistan (B. taurus x B. indicus, n = 7), India (B. indicus, n = 29), Yemen (B. indicus, n = 8), Egypt (B. taurus, n = 2), and Morocco (B. taurus, n = 10). In addition, 7 samples from Myanmar gayal (B. frontalis) were included in this study. Because most of the countries in Central and Southern Asia represent a contact zone between taurine- and indicine-like cattle (Felius, 1995), some individual samples from Kazakhstan, Kyrgyzstan, Yemen, and Turkmenistan, might in fact be crosses between B. taurus and B. indicus. Therefore, we expected to observe sequence variants originating in both the taurine and indicine lineages. Efforts were made to avoid sampling related individuals. We sampled only from small remote villages and excluded research centers, large cities, and coastal harbors where recent international shipping of cattle was possible. Genomic DNA was extracted by a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA).
The CSN3 exon IV was PCR amplified by using primers ExIV#1 and 551#2 (Prinzenberg et al., 1999). Polymerase chain reactions were performed in a 20-µL volume containing 1x PCR buffer, 2.5 mM MgCl2, 0.2 mM deoxynucleotide 5’-triphosphates, 0.4 µM each primer, 0.6 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA), and approximately 30 ng of genomic DNA. The PCR mixture underwent 10 min at 95°C, 40 cycles of 30 s at 95°C, 30 s at 52°C, 45 s at 72°C, and a final 5 min at 72°C on a GeneAmp PCR System 9700 (Applied Biosystems). Polymerase chain reaction products were purified and sequenced in the High-Throughput Genomics Unit, Department of Genome Sciences, University of Washington. Sequence trace files were checked and aligned by using DNAStar 6.0 (DNAStar Inc., Madison, WI). All new haplotype sequences were submitted to GenBank (accession numbers EU595506 to 595513).
A Bayesian model-based algorithm (Phase 2.1; Stephens et al., 2001; Stephens and Scheet, 2005) permitted us to infer haplotypes with a high confidence level (probability 0.97 to 1.00) from unphased genotypes and to estimate their frequencies. Several independent runs were performed to check the consistency between different runs. A neighbor-joining tree of newly inferred haplotypes and known bovine CSN3 alleles was constructed under uncorrected p distance by using MEGA4 (Tamura et al., 2007).
In our small panel of samples, 13 different genotypes were defined by 6 SNP, including 5 phase-known genotypes (G03, G04, G06, G10, and G11) and 8 unphased genotypes (Table 1
). Notably, 5 genotypes (G01 to G05) are shared by gayal samples and domestic cattle. From these 13 genotypes, 12 haplotypes (Table 1) were reconstructed and their frequencies were estimated. Among the 12 haplotypes, H02, H04, H05, and H06 corresponded to the known cattle alleles CSN3*A, CSN3*A1, CSN3*B, and CSN3*H, respectively. The new haplotypes, H08 and H10, differed from CSN3*A by one nonsynonymous mutation (T5110C–Ser70Pro and T5309C–Ile136Thr), whereas the new haplotypes H01, H11, and H12 were differentiated from CSN3*B by one nonsynonymous mutation (T5309C–Ile136Thr, A5345C–Ala148Asp, and T5110C–Ser70Pro). Finally, the new haplotypes H03, H07, and H09 were different by one silent mutation from CSN3*A, CSN3*H, and CSN3*B, respectively (Table 1). Interestingly, H01 was observed in 3 gayal and 3 Zebu individuals (Table 1) and had a close phylogenetic relationship with the banteng (Bos javanicus), gaur, and yak (Figure 1). Our results support the hypothesis that genetic diversity in exon IV of CSN3 has been underestimated in domestic cattle; it also highlights the urgent need to resequence functionally important genes in locally adapted cattle populations. This study shows the advantages of reporting the haplotypes instead of single sequences. Notably, a search in GenBank for the number of unique sequences (haplotypes) from cattle CSN3 exon IV returned only 9 different haplotypes (representing 7 polymorphic sites). This demonstrates that genetic diversity is highly underestimated, because this study alone contributed 8 new haplotypes.
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ACKNOWLEDGEMENTS |
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Received for publication March 29, 2008. Accepted for publication May 26, 2008.
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-casein genetic variant in the Chinese yak, Bos grunniens.J. Dairy Sci. 91:1204–1208.-casein. Int. Dairy J. 6:1017–1044.[CrossRef]-casecidin and its partial peptide fragments. J. Dairy Res. 69:329–334.[Medline]-Casein polymorphisms in Indian dairy cattle and buffalo: A new genetic variant in buffalo. Anim. Biotechnol. 9:81–87.[Medline]-casein gene (CSN3) of Chinese yak (Bos grunniens) and phylogenetic analysis of CSN3 sequences in the genus Bos.J. Dairy Sci. 91:1198–1203.This article has been cited by other articles:
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  A. M. Caroli, S. Chessa, and G. J. Erhardt Invited review: Milk protein polymorphisms in cattle: Effect on animal breeding and human nutrition J Dairy Sci, November 1, 2009; 92(11): 5335 - 5352. [Abstract] [Full Text] [PDF]
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