The difference between the allelic expression of a gene is known as allele-specific expression (ASE), and it is a common event in mammal?s transcriptome. ASE is named genomic imprinting when guided by epigenetics mechanisms regarding the allele parental origin

AFONSO, J.

CESAR, A. S. M

COUTINHO, L. L.

DINIZ, W. J.

LIMA, A. O.

NICIURA, S. C. M.

OLIVEIRA, P. S. N. de

REGITANO, L. C. de A.

ROCHA, M. I. P.

SOUZA, M. M. de

TIZIOTO, P. C.

ZERLOTINI NETO, A.

English

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Proceedings of the World Congress on Genetics Applied to LivestockProduction, 11.892Genome-wide distribution of allele-specific expression in Nelore steersmuscleM. M. Souza1, A. Zerlotini2, S.C.M. Niciura1, M.I.P. Rocha, W.J.S. Diniz3, P.C. Tizioto4, P.S.N.Oliveira1, A.O. Lima3, J. Afonso3, A.S.M. Cesar2, L.L. Coutinho2 & L. C. A. Regitano11Embrapa Pecuária Sudeste, Washington Luiz Highway, Km 234, 13560-970, São Carlos,Brazilmm.rissato@gmail.com (Corresponding Author)2Embrapa Informática Agropecuária, AV. Dr. André Tosello, 209 – Cidade Universitária,Campinas, 13083-886, São Paulo, Brazil3Federal University of São Carlos - UFSCar, Department of Genetic and Evolution, SãoCarlos, Washington Luiz Highway, Km 235, 13565-905, São PauloBrazil4University of São Paulo - ESALQ, Department of Animal Science, Pádua Dias Ave.,Piracicaba, 13418-900, São Paulo, BrazilSummaryThe difference between the allelic expression of a gene is known as allele-specific expression(ASE), and it is a common event in mammal’s transcriptome. ASE is named genomicimprinting when guided by epigenetics mechanisms regarding the allele parental origin.Although these events are an important sources of phenotypic variation, knowledge thereofregarding cattle and effects on economically important traits are still incipient. Therefore,analysis and characterization of ASE profile in the muscle of adult Nelore animals wereperformed, and besides that, the ASE pattern and gene imprinting occurrence were checked ingenes previously associated with meat tenderness. For this, RNA sequencing data of 146Nelore steer muscles were used, and these animals were also genotyped for approximately770,000 SNPs using Illumina BovineHD BeadChip. For each animal, a diploid genome wasbuilt based on their genotypes, and afterward, RNA-Seq reads were mapped to this genome.After counting the reads considering each SNP allele, a binomial test was applied to identifywhether the allele contribution to total expression significantly diverged from 50%, followedby false discovery rate (FDR) test (0.05). Among 519 analyzed SNPs, 402 showed at leastone sample with ASE, and they were spread among all autosomal chromosomes. SNPsshowing ASE in more than five samples were tested regarding ASE extension, and mostpresented an incomplete pattern. Four ASE genes were previously identified in a Genome-Wide Association Study (GWAS) and five were found differentially expressed betweenextreme phenotype animals for meat tenderness. Therefore, the results will complement thebovine imprinted genes database, further they can provide new information to genetic effectsprediction in animal breeding increasing the accuracy of meat tenderness selection.Keywords: allelic, ASE, gene expression, sequencing, transcriptomeIntroductionAccording to Mendelian inheritance model, the equal contribution of the parental alleleto gene expression is expected (Saupe, 2012). However, much evidence of unequal expressionof the alleles of a gene has been found in mammals’ genome (Tycko, 2010). Allele-specificexpression (ASE) may be the consequence of genetics or epigenetic events, which couldresult in phenotypic variance among individuals (Muráni et al., 2009; Tuch et al., 2010) andbias in the predicted additive effect of molecular markers associated with production traits.Polymorphisms in cis-regulatory sites are the principal cause of ASE. These loci affectonly the DNA molecule in which they are inserted (Lagarrigue et al., 2013). In addition, ASEalso can be governed by epigenetic events, as genomic imprinting, in which a DNAmethylation distinguishes the chromosomes inherited maternal and paternally resulting in apattern of expression depending of the parental origin of the allele (Ruvinsky, 1999).Even complex traits and diseases can be affected by genes showing allele-specificexpression pattern, only one study was developed in the bovine whole-transcriptome(Chamberlain et al., 2015). By RNA sequencing, Chamberlain et al. (2015) built the EAEprofile of a dairy cow in 18 tissues. Although these authors could answer some questionsabout tissue-specific expression, other questions about repeatability and individual-specificpatterns remain unresolved.The large difference of available data and knowledge between human and bovineimprinting (http://igc.otago.ac.nz/) reinforces the need for the development of new studiesaiming to describe the expression patterns of interest for animal production sinceeconomically important traits can be affected by variation in allelic expression.Considering this scenario, the present study aimed to build the EAE profile of bovineLongissimus dorsi, to check the variation of EAE among individuals contributing to theelaboration of more accurate strategies of inclusion of genomic information in animalbreeding programs.Material and methodsThe Institutional Animal Care and Use Committee Guidelines from Embrapa (BrazilianAgricultural Research Corporation) approved all the experimental procedures involvinganimals in this study.AnimalsOne hundred forty-six Nelore steers, sired by 34 unrelated sires, were produced byEmbrapa, Brazil. The steers were raised in feedlot system, receiving similar nutritional andsanitary management. After about 90 days of feeding the animals were slaughtered andLongissimus dorsi (LD) muscle samples were collected for RNA extraction.DNA Genotyping and RNA sequencingDNA was extracted from blood and genotyped by using Illumina BovineHD BeadChip(Illumina Inc, San Diego, CA, EUA), in two laboratories: USDA ARS Bovine FunctionalGenomics (Beltsville, MD, EUA) and ESALQ genomic center (Piracicaba, São Paulo,Brazil). Quality control was accessed by PLINK (Purcell et al., 2007) and the linkage phaseof the SNPs was obtained by Beagle 4.0 (Browning & Browning, 2007). All heterozygousSNPs of each animal were identified to analyze the ASE.Expression profiles of mRNA were measured with RNA-Seq in LD muscle samples (n= 146). RNA extraction, library preparation, and sequencing were realized at ESALQgenomic center (Piracicaba, São Paulo, Brazil). RNA library was prepared using the TruSeqRNA Sample Preparation Kit (Illumina, San Diego, CA, EUA). Clustering and sequencingwere done on Illumina HiSeq 2500® (Illumina, San Diego, CA, EUA).Allele-Specific expression analysis (ASE)We built an individual diploid genome in silico from individual haplotypes for eachanimal. For this, ALEA software (YOUNESY et al., 2014) was used using the bovinereference genome (Bos taurus UMD3.1, http://www.ensembl.org/Bos_taurus/Info/Index/) andindividual genotypes obtained from the Illumina BovineHD BeadChip.For each animal, reads were mapped to the individual diploid genome. We performedthe reads mapping counting considering only the reads mapping specific to one allele of eachSNP. Then, we selected SNPs for which the total reads counting was higher than 20. Thebinomial test was applied for each SNP of each animal to check whether the reads countingwas statistical different between alleles. The null hypothesis was the half of the reads mappedin each SNP allele. Also, multiple test correction was performed by applying False DiscoveryRate test (FDR, Benjamini-Hochberg (P < 0,05)).Results and discussionFrom 777,000 SNPs contained on BovineHD BeadChip, 447,437 SNPs passed onquality filter after genotyping. Around 3.6 billion of reads were produced in the sequencing,and 24.7 million of reads was the reads average total per sample.Considering all SNPs heterozygous in all animals that were expressed in muscle, weperformed 6,384 ASE tests. We found 519 SNPs showing at least one heterozygous animalanalyzed. The results revealed that most SNPs tested here (77.45%; n = 402) presented ASEin at least one animal (n = 402) which are in agreement with the literature. Crowley et al.(2015) found 89% of tested genes in mouse showing ASE and Chamberlain et al. (2015)found from 74 to 89% in 18 bovine tissue. However, the results diverged when taking accountonly the analysis in leg muscle performed by Chamberlain et al. (2015). They found only18% of analyzed SNPs showing ASE, but the proportion increased considerably when thesampling was raised to 20 sample of white blood cells and liver.The discrepancy of ASE percentage among the results obtained by Chamberlain et al.(2015), when they tested one or 18 tissues, and one or 20 animals were also found in ourresults. We found animals showing 0.03% of tested heterozygous SNPs showing ASEmeanwhile others showing 100% (n = 88 SNPs). So, the divergence of ASE among samplesmay be governed by specific environmental events or genotype (Chamberlain et al., 2015).Therefore, as previously described by Lagarrigue et al. (2013), the comparison of the ASEresults from one bovine muscle (Chamberlain et al., 2015) with the present study in which146 bovine muscle were analyzed, suggests the need of biological replicates in ASE studies.The ASE observed here were well distributed throughout Nelore muscle transcriptome,with the average of 13.83 SNPs showing ASE per chromosome. Chromosome 3 showed thehighest number of ASE observed (n = 31), while the opposite was found on chromosome 24(n = 4).ASE studies have been shown important for animal breeding programs. For example,SNPs identified here showing ASE were identified to increase the accuracy of birds selectionregarding the infection by Marek disease virus (Cheng et al., 2015; Perumbakkam et al.,2013). Also, ASE may contribute to gene imprinting identification, which, in turn, maycontribute to increasing the animal breeding programs accuracy. For example, IGF2R is awell-known imprinted gene associated with growth traits (Berkowicz et al., 2011). Othersimprinted genes, PEG3 and GNAS, were related to milk production and fertility, respectively(Magee et al., 2014; Sikora et al., 2011).Despite a large number of imprinted validated genes in human and mouse, it isimportant to study specific genes of livestock animals since the conservation of the allelicexpression pattern between primates and rodents has been lower than expected (Khatib, 2007;Monk et al., 2006), and the same may be possible to occur between human and productionanimal.ConclusionThe results presented here show that ASE is present and widely distributed in thebovine transcriptome. The first step was done to provide the ASE profile as a basis forsignificant advances in the future that will enhance information on regulatory elements.Literature CitedBerkowicz, E.W., Magee, D. a, Sikora, K.M., Berry, D.P., Howard, D.J., Mullen, M.P.,Evans, R.D., Spillane, C., MacHugh, D.E., 2011. 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Genome-wide distribution of allele-specific expression in Nelore steers muscle.

http://www.alice.cnptia.embrapa.br/bitstream/doc/1100405/1/GenomeWideDistribution.pdf

Genome-wide distribution of allele-specific expression in Nelore steers muscle.

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