Czech J. Anim. Sci., 61, 2016 (7): 317–325 Original Paper doi: 10.17221/30/2015-CJAS Identification of missense mutations in the PCP4 and CD109 genes to validate the effect of neutral genetic markers B. Moioli1, L. De Grossi2, R. Steri1, F. Pilla3 1Council for Agricultural Research and Analysis of Agricultural Economics, Monterotondo, Italy 2Istituto Zooprofilattico Sperimentale, Viterbo, Italy 3Department of Animal, Vegetable and Environmental Sciences, University of Molise, Campobasso, Italy ABSTRACT: The marker-assisted selection exploits anonymous genetic markers that have been associated with measurable differences on complex traits. Because it is based on the linkage disequilibrium (LD) between the polymorphic markers and the polymorphisms which code for the trait, its success is limited to the popula- tion in which the association has been assessed. The identification of the gene with effect on the target trait and the detection of the functional mutations will allow selection in independent populations, while encour- aging studies on gene expression. In a flock of sheep infected with M. paratuberculosis, a genome-wide scan, performed with the Illumina OvineSNP50 BeadChip, had identified two candidate genes, the PCP4 and the CD109, located in proximity of the markers with significant allele substitution effect on the positivity level at paratuberculosis serological assessment. The coding region of the two genes was directly sequenced. Three missense mutations were detected: two in the PCP4 gene and one in the second exon of the CD109 gene, the latter showing a strong LD with the anonymous marker. Direct sequencing of the DNA of sheep of different populations showed that disequilibrium was maintained. Allele frequency at the hypothesized marker associ- ated to immune response, calculated for other breeds of sheep, showed that the marker allele potentially asso- ciated to disease resistance is more frequent in the local breeds and in breeds that have not been submitted to selection programs. Keywords: immune response; local sheep; genomics; genome-wide association INTRODUCTION possibility to detect the direct markers (i.e. poly- morphisms that code for the functional mutations) The aim of marker-assisted selection (MAS) is opens the way to wider fields of applications, on to select specific DNA variations that have been one side because selection could be performed associated with a measurable difference on complex in independent populations, with no care of the traits. It is based on the linkage disequilibrium LD extent; on the other side, the identification (LD) between the polymorphic markers and the of the gene having major effect on the trait will polymorphisms which code for the trait. There- allow a deeper knowledge of the function and the fore the marker must by necessity be close to the expression of the gene, as well as of the related functional mutation for sufficient population- gene network, and such knowlege might be of use wide LD between the marker and the gene. Also also to other species and different target traits. for this reason, success of MAS has been limited Paratuberculosis, or Johne’s disease, is a chronic in livestock and few studies reported substantial granulomatous enteritis caused by Mycobacterium gains with real populations (Dekkers 2004). The avium subsp. paratuberculosis (MAP), which af- 317 Original Paper Czech J. Anim. Sci., 61, 2016 (7): 317–325 doi: 10.17221/30/2015-CJAS fects ruminants. It causes substantially reduced sheep were chosen so to be equally distributed production, higher susceptibility to acquire other between positive and negative. diseases and infertility (Kennedy and Benedictus The genomic scaffolds encoding the PCP4 2001). In a flock of MAP-infected sheep, Moioli et and the CD109 genes were obtained from the al. (2015) performed a Genome Wide Association NCBI database (http://www.ncbi.nlm.nih.gov/ Analysis (GWAS) with the OvineSNP50 BeadChip genome/?term=ovis+aries). The size and the po- on 100 ewes representing the extreme divergent sition of the coding regions of the PCP4 and the animals for the optical density (S/P) ratio obtained CD109 genes on the corresponding chromosomes from MAP serological assay, assessed on about – OAR1 and OAR8 – were identified by performing 700 sheep. The authors hypothesized the pres- a standard nucleotide blast (Altschul et al. 1990) ence of 30 putative candidate genes of disease of the genomic scaffolds against the expressed susceptibility, these genes being in close proximity sequence of the gene under investigation in the of the polymorphic markers which showed a sig- NCBI (www.ncbi.nlm.nih.gov/nuccore). nificant effect on the obtained serological value. Because Moioli et al. (2015) had hypothesized that While the majority of these genes did not appear OAR1_278980576.1 and OAR8_270360, located in to have a straightforward and recognizable effect proximity of the PCP4 and the CD109 genes, were on disease resistance, being responsible for basic potential markers of MAP resistance, the genotyp- cellular processes, two of them, the Purkinje Cell ing results of the 100 Sarda sheep of the previous Protein 4 (PCP4), and the CD109 genes appeared study were used to perform a χ2 test of significance worth to be studied in more depth for their po- of differences of the allele frequencies between the tential role in influencing the immune system. In 50 serologically positive and 50 negative sheep. The fact, Jacobson et al. (2009), in the murine B cell χ2 test was performed on the genomic scaffolds co-receptor complex, demonstrated an increased encompassing the PCP4 and the CD109 genes, in expression of PCP4 in wild type mice compared to order to restrain the genomic region to be submit- complement-deficient animals, and correlated the ted to direct DNA sequencing, because the aver- increased expression of PCP4 with B cell matura- age distance between markers of the OvineSNP50 tion into end stage phenotypes. On the other hand, BeadChip ranges from 60 to 200K. The χ2 test values the CD109 belongs to the complement gene family are deemed to provide solid indication of the region and is a co-receptor of the Transforming growth in which LD had been maintained (Lewis 2002). factor beta (TGF-β), a type of cytokine which Primer pairs to amplify the expressed regions plays a role in immunity (Hashimoto et al. 2004). of the genes were designed on the ovine sequence The present study was conceived to search for the by using Primer3Plus software, Version 3.0, 2007 presence of further polymorphisms in the coding (www.bioinformatics.nl/cgi-bin/primer3plus/prim- or regulatory region of PCP4 and CD109 genes er3plus.cgi). that might explain and corroborate the effect of In a first step, DNA of 10 individuals (5 serologi- the two anonymous markers identified by Moioli cally positive and 5 negative) selected from the et al. (2015) in influencing disease resistance. 42 Sarda sheep was amplified at each amplicon, under the assumption that this number was suffi- MATERIAL AND METHODS cient to detect the presence of further SNPs. Once the novel SNP had been detected, direct sequencing Genotyping results at the OvineSNP50 BeadChip of the amplicon was performed on all 42 sheep. for 100 sheep of the Sarda breed were available Direct sequencing of the coding regions of the from the previous study (Moioli et al. 2015); the putative candidate genes was performed on the sheep represented the two tails of the distribution 3500 Genetic Analyzer (Applied Biosystems, Foster of the serological assessment for MAP diagnose, City, USA). Allele frequencies and LD measures, that had been performed on 759 sheep of the same for both the anonymous markers and the novel flock using the ELISA test. DNA of 42 genotyped detected polymorphisms, were estimated using Sarda sheep for the present study was provided the Allele Procedure in SAS software. by the Animal Health Institute of Viterbo, Italy. Because all the sheep assessed for MAP diagnose Because the ELISA kit for MAP diagnosis attests belonged to the Sarda breed, to verify whether positive results when S/P values > 70, the 42 Sarda LD between the novel detected SNPs and the 318 Czech J. Anim. Sci., 61, 2016 (7): 317–325 Original Paper doi: 10.17221/30/2015-CJAS Table 1. The χ2 test of differences between 50 serologically positive and 50 negative sheep at the markers falling in the region around the PCP4 gene (genomic scaffold NW_004080164.1) Marker Gene Position on OARv3.1 χ2 P (χ2) Position in the gene s49212.1 OAR1 258029257 1.2 ns 5’ UTR PCP4 start OAR1 258032168 OAR1_278884883.1 OAR1 258068271 6.0 0.01 intron 1 OAR1_278927044.1 OAR1 258105475 0.7 ns intron 2 PCP4 stop OAR1 258118984 OAR1_278980576.1 OAR1 258148544 15.2 0.0001 3’ UTR CD109 start OAR8 273740 OAR8_270360.1 OAR8 274469 17.5 0.00003 intron 2 OAR8_360354.1 OAR8 342710 13.0 0.0003 intron 8 OAR8_409032.1 OAR8 387749 3.6 ns intron 26 OAR8_423752.1 OAR8 403376 0.6 ns intron 31 CD109 stop OAR8 408954 ns = non-significant OvineSNP50 BeadChip anonymous markers were blast (Altschul et al. 1990) of the genomic scaffold maintained also in different breeds, DNA of 33 NW_004080164.1 against the expressed sequence more sheep of two Italian breeds – Altamurana of PCP4 (XM_004003899.1) showed that the gene and Comisana – was directly sequenced at the is composed of 3 exons (Figure 1). The two mark- amplicons encompassing the coding regions of ers, with significant difference in allele frequency, the putative candidate genes. These breeds and OAR1_278884883.1 and OAR1_278980576.1, were individuals were chosen because their DNA was located, respectively, in the first intron and 29K available in the laboratory performing the present downstream the stop codon of the gene. Therefore, study, and because their genotyping results at the hypothesizing that putative mutations influencing OvineSNP50 BeadChip were also available, thanks the target trait be located either in the first part, to a previous biodiversity project (Ciani et al.