Whole-genome resequencing shows numerous genes with nonsynonymous SNPs in the Japanese native cattle Kuchinoshima-Ushi
- Ryouka Kawahara-Miki†1,
- Kaoru Tsuda†1,
- Yuh Shiwa1,
- Yuko Arai-Kichise1,
- Takashi Matsumoto1,
- Yu Kanesaki1,
- Sen-ichi Oda2,
- Shizufumi Ebihara2,
- Shunsuke Yajima1, 3,
- Hirofumi Yoshikawa1, 3 and
- Tomohiro Kono1, 3Email author
© Kawahara-Miki et al; licensee BioMed Central Ltd. 2011
Received: 20 May 2010
Accepted: 10 February 2011
Published: 10 February 2011
Because the Japanese native cattle Kuchinoshima-Ushi have been isolated in a small island and their lineage has been intensely protected, it has been assumed to date that numerous and valuable genomic variations are conserved in this cattle breed.
In this study, we evaluated genetic features of this breed, including single nucleotide polymorphism (SNP) information, by whole-genome sequencing using a Genome Analyzer II. A total of 64.2 Gb of sequence was generated, of which 86% of the obtained reads were successfully mapped to the reference sequence (Btau 4.0) with BWA. On an average, 93% of the genome was covered by the reads and the number of mapped reads corresponded to 15.8-fold coverage across the covered region. From these data, we identified 6.3 million SNPs, of which more than 5.5 million (87%) were found to be new. Out of the SNPs annotated in the bovine sequence assembly, 20,432 were found in protein-coding regions containing 11,713 nonsynonymous SNPs in 4,643 genes. Furthermore, phylogenetic analysis using sequence data from 10 genes (more than 10 kbp) showed that Kuchinoshima-Ushi is clearly distinct from European domestic breeds of cattle.
These results provide a framework for further genetic studies in the Kuchinoshima-Ushi population and research on functions of SNP-containing genes, which would aid in understanding the molecular basis underlying phenotypic variation of economically important traits in cattle and in improving intrinsic defects in domestic cattle breeds.
Since the first whole-genome assembly of the human genome in 2001 , the sequencing and assembly of mammalian genomes have quickly progressed. The bovine genome was assembled by the international bovine whole-genome sequencing project through a combination of shotgun and bacterial artificial chromosome (BAC) sequencing of an inbred Hereford cow and her sire by using capillary sequencing . Recently, Van Tassell et al. (2008) contributed more than 23,000 single nucleotide polymorphisms (SNPs) to the bovine SNP database (dbSNP) by next-generation sequencing using a dairy breed (Holstein) and seven major breeds of beef cattle , and more than 2 million SNPs have been submitted to the bovine dbSNP to date. Although the array approach with these SNPs is useful, the underlying SNP resource is far from complete for understanding genome structure [4, 5]. Eck et al. (2009) generated 24 gigabase (Gb) of sequence with an average 7.4-fold sequence depth from a single Fleckvieh bull by next-generation sequencing  and identified more than 2 million previously unknown SNPs and 115,000 small insertions and deletions (indels) in comparison with the reference sequence. Although the bovine genome and HapMap projects have progressed [2, 7], the sequences of Japanese cattle have not been utilized in the respective projects. Therefore, we conducted a whole-genome analysis to examine the genetic features of the Japanese native cattle Kuchinoshima-Ushi and to gain a better understanding of the genetic relationship between domestic cattle breeds and Kuchinoshima-Ushi. This study provides detailed genetic information of this breed of cattle based on 64.2 Gb of sequence data generated by next-generation sequencing.
Results and Discussion
Sequencing, mapping, and SNP/indel detection
On an average, 93% of the genome was covered by reads (Additional file 2), and the number of mapped reads corresponds to 15.8-fold coverage across the covered region. However, the previous study describing whole-genome sequencing of a Fleckvieh bull reported that 98% of the genome was covered by the reads at a relatively low read depth (7.4-fold) . The relatively lower genome coverage in our study is probably due to the fact that compared to the Fleckvieh breed, the Kuchinoshima-Ushi breed is evolutionarily more distantly related to the breed of the reference genome (Hereford). The difference in coverage can also be due to the number of prepared libraries (three different libraries in the Fleckvieh breed, whereas only one library in the Kuchinoshima-Ushi breed).
Sequence data were deposited in the DDBJ Read Archive (DRA) [Accession #: DRA000172] and identified SNPs excluding ambiguous bases and those mapping to multiple locations unless providing more than 5 kbp flanking sequences were submitted to the single nucleotide polymorphism database (dbSNP) at NCBI under the handle TUANGRC. In addition to submitting data to the standard repositories, the positions of the SNPs for Kuchinoshima-Ushi can be viewed in a customized installation of the UCSC Genome Browser http://www.nodai-genome.org/btau/cgi-bin/hgGateway, along with supporting evidence (the number of reads for each allele and the density of the SNPs).
Functional annotation of nonsynonymous SNPs (nsSNPs) and nsSNP-containing genes
The SNPs in gene regions were annotated using the RefSeq and GenBank gene sets (16,635 genes). We found 1,004,496 SNPs in introns, 17,684 in untranslated regions (UTRs), 801 SNPs in splice-sites, and 20,432 coding SNPs leading to 11,713 nonsynonymous nucleotide substitutions (Figure 3A). The percentage of nonsynonymous changes in the coding region was 57.3%, which was higher than that of any other previous studies of whole-genome resequencing in humans and cattle [6, 10–15]. This finding would indicate on a possible occurrence of pseudogenes, the existence of proteins whose functions have been severely affected by extensive amino acid substitution, or significant differences in segmental duplications or copy number variants. Alternatively, the number of nsSNPs might have increased due to the false positives created by incomplete annotation information.
We also examined whether nsSNP-containing genes were reportedly associated with economically important traits such as meat/milk production, growth rate, and domestication in other breeds of cattle. In order to extract information from studies reporting relationships between SNPs/genes and traits, we performed a PubMed search using economical traits such as growth rate or meat production as the query and limited the search to studies of Bos Taurus. From the result of the search, GenBank/EMBL/DDBJ nucleotide records reported in the obtained articles as well as sequences referred to in the articles as references (RefSeqs) were extracted and compared with the list of nsSNP-containing genes in our analysis of Kuchinoshima-Ushi.
Of the 4,643 nsSNP-containing genes, 708 matched to genes that have been reported to be potentially associated with economically important traits such as meat/milk production, growth rate, and domestication in other cattle breeds [18–22] (the list of genes containing nsSNPs that were reported as trait-associated in other cattle breeds is shown in Additional file 8). Some of the genes were also found to reside at positions of significant quantitative trait loci (QTL; data from Cattle QTLdb http://www.animalgenome.org/cgi-bin/QTLdb/BT/index); for example, body growth-associated genes such as genes for growth hormone (GH), growth hormone receptor (GHR), and leptin receptor (LEPR) [18–20]; lactation-related genes like prolactin receptor (PRLR) and caseins (CSN); and genes encoding immune-related proteins in milk, such as toll-like receptors (TLR s) [21, 22].
Recently, associations between SNPs and phenotypes have been reported in many studies, including cattle [23, 24]. Some SNP sites identified in Kuchinoshima-Ushi have already been reported to be associated with phenotypes in other cattle breeds (Figure 4B). For example, a mutation found in the fatty acid binding protein 4 (FABP4) was reported to be associated with palmitoleic acid (C16:1) content in intramuscular fat in Japanese Black cattle . A mutation of the urotensin 2 receptor (UTS2R) was associated with skeletal muscle fat accumulation in Wagyu × Limousin population  and a mutation in calpain 1 (CAPN1) was associated with meat tenderness in Aberdeen Angus-cross cattle . The nsSNPs found in the nucleotide-binding oligomerization domain containing 2 (NOD2/CARD15) gene were associated with the estimated breeding values for somatic cell score in Canadian Holstein cattle . Identified SNP sites in the LEP and LEPR genes were associated with fat content in carcass meat of other cattle breeds including Nellore, Holstein-Friesian, Angus, Charolais, Hereford, and Simmental [19, 29, 30].
Nevertheless, population genetic studies in the Kuchinoshima-Ushi breed are required to validate the identified nsSNPs and to examine their association with relevant phenotypic traits in this population. Also, most nsSNPs identified in Kuchinoshima-Ushi have yet to be reported, nor have their functions or associations been investigated; new nsSNPs identified in this study will be valuable for future research.
Phylogeny of bovine-related species
In the present study, Japanese native cattle Kuchinoshima-Ushi, which have retained their phenotype over time without being affected genetically by European domestic breeds, were used for whole-genome resequencing with a next-generation sequencer. We identified 6.3 million SNPs, of which more than 5.5 million (87%) were found to be new. Among the identified SNPs, some nonsynonymous SNPs were found in genes that have been reported as candidate genes for QTL affecting economically important traits in other cattle breeds. The results of phylogenetic analysis showed the phylogenetic position of Kuchinoshima-Ushi. In the current status of domestic cattle, repeated selection for specific traits such as weight gain as well as milk and meat production has progressed, but problems have emerged with unconsidered traits such as reproduction and disease resistance. Our results provide a framework for future investigations aiming at the understanding of gene functions and identifying the molecular basis underlying phenotypic variation of economically important traits in domestic cattle breeds. It would contribute to the development of a new, more efficient breeding system preferentially for East Asian domestic cattle breeds including Japanese one. Furthermore, genome information for Kuchinoshima-Ushi is expected to be important not only in revealing the genetic structure of a geographically isolated breed originating from ancient, native Japanese cattle but also in identifying genetic traits that distinguish domestic breeds from original, undomesticated species.
Breeding conditions for Kuchinoshima-Ushi
For our experiments we used genomic DNA from a horned 119-month-old male Kuchinoshima-Ushi (#8915) (Figure 1B) that weighed 485 kg and was black with large white spots. The cattle in the wide field of Nagoya University are bred under good and relaxed conditions. In 1990, one male and three female Kuchinoshima-Ushi were captured on Kuchinoshima Island and transferred to the stock farm at Shitara Field, Nagoya University. In 1993, two males and three females were brought to the field from the Nagoya Animal Science Foundation. By 2004, the group had increased to 23 males and 22 females with inbred conditions in a closed colony.
DNA library construction and sequencing
Blood sampling was carried out according to the 'Regulations for Animal Experiments in Nagoya University' and the 'Guidelines for the Care and Use of Laboratory Animals', as specified by the Tokyo University of Agriculture.
Genomic DNA was extracted from blood using standard phenol/chloroform extraction methods . A genomic DNA library was prepared using a paired-end DNA sample prep kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer's instructions (Preparing Samples for Sequencing Genomic DNA, Part # 1003806 Rev.B, Illumina) with slight modification as follows: 2 μg of DNA were fragmented to a median fragment size of 200 bp using Adaptive Focused Acoustics (Covaris, Inc., Woburn, MA, USA). After size selection on a 2% agarose gel, 10 μl of the DNA fragments was enriched by 12 cycles of PCR. For quality control, an aliquot of the library was cloned into a sequencing vector (TOPO TA Cloning® Kit for Sequencing, Invitrogen Corp., Carlsbad, CA, USA) and 96 clones were sequenced by sanger sequencing. We found that all sequences were unique, and duplicates were not detected in the analyzed sample. BLAST search in the bovine genome sequence database using these sequences revealed high (90%) similarities to respective bovine sequences. DNA concentration was measured using UV-1800 UV-VIS Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The genomic DNA library was diluted to 10 nM, and a 2 μl aliquot was used to generate clusters on the Illumina Cluster Station using the Paired-End Cluster Generation Kit v2 (Illumina) and sequenced on the GAII with the SBS 36-cycle Sequencing Kit v3 following the manufacturer's instructions (Using the Paired-End Cluster Generation Kit v2 on the Cluster Station and Paired-End Module, Part #: 1005629 Rev.C; Using the SBS Sequencing Kit v3 on the Genome Analyzer, Part #: 1005637 Rev.A, Illumina) as 75-bp and 36-bp reads (28 and 6 paired-end lanes, respectively).
For alignment and annotation of the sequence reads, we used the bovine genome assembly Btau 4.0 ftp://hgdownload.cse.ucsc.edu/goldenPath/bosTau4/chromosomes/ as a reference source. In this study, sequence scaffolds not yet assigned to specific chromosomes were excluded and no repeat masker was applied to the assembly.
Image analysis and ELAND alignment were performed with Illumina's Pipeline Analysis software ver. 1.4. Sequences passing through the standard Illumina GA pipeline filters (i.e., clusters with intensities greater than 0.6-times the average of the highest and the sum of the two highest intensities for the first 25 cycles) were retained for further analysis.
For short-read alignment and consensus assembly, we used a recently developed algorithm, BWA ver. 0.5.0 (the outline of the algorithm is available online http://bio-bwa.sourceforge.net/) . The BWA default values for mapping were as follows: maximum edit distance (maxDiff) = 0.04, maximum number of gap opens (maxGapO) = 1, maximum number of gap extensions (maxGapE) = -1, disallow a long deletion within bp (nDelTail) = 16, disallow an indel within bp (nIndelEnd) = 5, take the first subsequence as seed (seedLen) = infinite, maximum edit distance in the seed (maxSeedDiff) = 2, number of threads (nThrds) = 1, mismatch penalty (misMsc) = 3, gap open penalty (gapOsc) = 11, gap extension penalty (gapEsc) = 4, and parameter for read trimming (trimQual) = 0. After read mapping, we discarded the reads mapped to multiple chromosomal positions and unmapped reads. Only reads mapped to a unique position on the reference genome sequence were used for SNP calling.
To call SNPs, we used SAMtools  and applied additional filters as follows: minimum read depth = 3, minimum read depth calling the SNP = 2, and a 30% cutoff of percent aligned reads calling the SNP per total mapped reads at the SNP sites. We also filtered these identified SNPs with more stringent parameters (i.e., minimum depth = 4, minimum SNP = 2, and 30% or higher aligned reads calling the SNP; and minimum depth = 5, minimum SNP = 3, and 30% or higher aligned reads calling the SNP), but the difference in the number of SNPs was small (Additional file 9). Heterozygous and homozygous SNPs were distinguished using an 80% cutoff of percent aligned reads calling the SNP. We also used BWA to estimate the sequence read depth, which influences the coverage and accuracy of SNP calling. After SNP calling, we annotated the SNPs using the GenBank and RefSeq gene sets (16,635 genes; the gene set is available from the UCSC download site ftp://hgdownload.cse.ucsc.edu/goldenPath/bosTau4/database).
We used 15 species from the Bovinae subfamily (9 species from subtribe Bovina, 5 species from subtribe Bubalina, and one species from tribe Tragelaphini as an outgroup) for the phylogenetic analysis (Additional file 10). On the basis of a previous study , we used 10 nuclear genes (listed in Additional file 11). All of the sequence data except for that of Kuchinoshima-Ushi were previously determined and published (accession numbers can be seen in Additional file 11). Sequences of the genes from Kuchinoshima-Ushi were determined reflecting its SNP information in the reference sequence data. We concatenated newly-determined sequences of Kuchinoshima-Ushi and the published sequences and aligned them for the phylogenetic analysis. Phylogenetic trees were estimated using partitioned ML and partitioned Bayesian methods using RAxML ver. 7.0.3  and MrBayes ver. 3.1.2 , respectively.
We thank Yutaka Yoshida, affiliated to the Tokyo University of Agriculture, for his help in preparation of the sample, Satoshi Sano and Misaki Imai for technical supports, and Yoshiaki Kikkawa, from the Tokyo University of Agriculture, Takahisa Yamada, from Niigata University, and Tetsuo Kunieda, from Okayama University, for their valuable discussions and suggestions. We also thank Hiroshi Ando, Nobue Tsuichihara, and Fumitaka Yoshimura for technical support of breeding and maintenance of this breed in the Field Science Center of Nagoya University. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (S0801025).
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