Advantages of continuous genotype values over genotype classes for GWAS in higher polyploids: a comparative study in hexaploid chrysanthemum.

BACKGROUND: Association studies are an essential part of modern plant breeding, but are limited for polyploid crops. The increased number of possible genotype classes complicates the differentiation between them. Available methods are limited with respect to the ploidy level or data producing technologies. While genotype classification is an established noise reduction step in diploids, it gains complexity with increasing ploidy levels. Eventually, the errors produced by misclassifications exceed the benefits of genotype classes. Alternatively, continuous genotype values can be used for association analysis in higher polyploids. We associated continuous genotypes to three different traits and compared the results to the output of the genotype caller SuperMASSA. Linear, Bayesian and partial least squares regression were applied, to determine if the use of continuous genotypes is limited to a specific method. A disease, a flowering and a growth trait with h (2) of 0.51, 0.78 and 0.91 were associated with a hexaploid chrysanthemum genotypes. The data set consisted of 55,825 probes and 228 samples. RESULTS: We were able to detect associating probes using continuous genotypes for multiple traits, using different regression methods. The identified probe sets were overlapping, but not identical between the methods. Baysian regression was the most restrictive method, resulting in ten probes for one trait and none for the others. Linear and partial least squares regression led to numerous associating probes. Association based on genotype classes resulted in similar values, but missed several significant probes. A simulation study was used to successfully validate the number of associating markers. CONCLUSIONS: Association of various phenotypic traits with continuous genotypes is successful with both uni- and multivariate regression methods. Genotype calling does not improve the association and shows no advantages in this study. Instead, use of continuous genotypes simplifies the analysis, saves computational time and results more potential markers.

Epidemiology, presentation and population genetics of patent ductus arteriosus (PDA) in the Dutch Stabyhoun dog.

BACKGROUND: Patent ductus arteriosus (PDA) is one of the most common congenital heart defects in dogs and is considered to be a complex, polygenic threshold trait for which a female sex predisposition has been described. Histological studies in dogs suggest that smooth muscle hypoplasia and asymmetry of the ductus tissue is the major cause of PDA. The Stabyhoun population is small and a predisposition for PDA has been suggested. The aims of this study were to describe the incidence, presentation from a clinical and histopathological perspective, and the population genetics of PDA in the Dutch Stabyhoun population. RESULTS: Forty-six cases were identified between 2000 and 2013. Between 2009 and 2012 the birth incidence of PDA in the Stabyhoun breed was 1.05 %. We estimated this to be 7-13 times higher than expected in the general dog population. Twelve of the 46 cases were part of a litter in which more than one sibling was affected. There was no sex predilection in our case cohort. Dogs diagnosed in adulthood showed severe cardiomegaly. The mean inbreeding coefficient of the reference population of Stabyhoun dogs was 31.4 % and the actual and effective numbers of founders were 14 and 6.5, respectively. The heritability of PDA was 0.51 (±0.09) for the reference population and 0.41 (±0.10) for the phenotyped population. Histopathology of sections of the PDA from two dogs showed findings similar to those described in other breeds although the smooth muscle of the ductus adjacent to the pulmonary artery appeared more hypoplastic than that in the ductus adjacent to the aorta. CONCLUSIONS: The Stabyhoun breed shows a strong predisposition for PDA. Apart from the absence of a higher incidence in females, no other significant features distinguish PDA in Stabyhouns from the condition in other dog breeds. Heritability and the mean inbreeding coefficient are both very high making the Dutch Stabyhoun breed particularly suited to the study of inherited risk factors for PDA.

Genome-wide survey indicates involvement of loci on canine chromosomes 7 and 31 in patellar luxation in Flat-Coated Retrievers.

BACKGROUND: Patellar luxation is an orthopedic disorder in which the patella moves out of its normal location within the femoral trochlea of the knee and it can lead to osteoarthritis, lameness, and pain. In dogs it is a heritable trait, with both environmental and genetic factors contributing to the phenotype. The prevalence of patellar luxation in the Dutch Flat-Coated Retriever population is 24%. In this study, we investigated the molecular genetics of the disorder in this population. RESULTS: Genome-wide association analysis of 15,823 single nucleotide polymorphisms (SNPs) in 45 cases and 40 controls revealed that patellar luxation was significantly associated with a region on chromosome CFA07, and possibly with regions on CFA03, CFA31, and CFA36. The exons of the genes in these regions, 0.5 Mb combined, were analyzed further. These exons from 15 cases and a pooled sample from 15 controls were enriched using custom genomic hybridization arrays and analyzed by massive parallel DNA sequencing. In total 7257 variations were detected. Subsequently, a selection of 144 of these SNPs were genotyped in 95 Flat-Coated Retrievers. Nine SNPs, in eight genes on CFA07 and CFA31, were associated with patellar luxation (P <10-4). Genotyping of these SNPs in samples from a variety of breeds revealed that the disease-associated allele of one synonymous SNP in a pseudogene of FMO6 was unique to Flat-Coated Retrievers. CONCLUSION: Genome-wide association analysis followed by targeted DNA sequencing identified loci on chromosomes 7 and 31 as being involved in patellar luxation in the Flat-Coated Retriever breed.

Bovine Neonatal Pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the MHC haplotype.

Bovine Neonatal Pancytopenia (BNP), a bleeding syndrome of neonatal calves, is caused by alloantibodies absorbed from the colostrum of particular cows. A commercial BVD vaccine is the likely source of alloantigens eliciting BNP associated alloantibodies. We hypothesized that the rare occurrence of BNP in calves born to vaccinated dams could be associated with genetic differences within dams and calves. We found that the development of BNP within calves was a heritable trait for dams, not for calves and had a high heritability of 19%. To elucidate which genes play a role in the development of BNP we sequenced candidate genes and characterized BNP alloantibodies. Alloantigens present in the vaccine have to be presented to the dam's immune system via MHC class II, however sequencing of DRB3 showed no differences in MHC class II haplotype between BNP and non-BNP dams. MHC class I, a highly polymorphic alloantigen, is an important target of BNP alloantibodies. Using a novel sequence based MHC class I typing method, we found no association of BNP with MHC class I haplotype distribution in dams or calves. Alloantibodies were detected in both vaccinated BNP and non-BNP dams and we found no differences in alloantibody characteristics between these groups, but alloantibody levels were significantly higher in BNP dams. We concluded that the development of BNP in calves is a heritable trait of the dam rather than the calf and genetic differences between BNP and non-BNP dams are likely due to genes controlling the quantitative alloantibody response following vaccination.

Number and mode of inheritance of QTL influencing backfat thickness on SSC2p in Sino-European pig pedigrees.

BACKGROUND: In the pig, multiple QTL associated with growth and fatness traits have been mapped to chromosome 2 (SSC2) and among these, at least one shows paternal expression due to the IGF2-intron3-G3072A substitution. Previously published results on the position and imprinting status of this QTL disagree between analyses from French and Dutch F2 crossbred pig populations obtained with the same breeds (Meishan crossed with Large White or Landrace). METHODS: To study the role of paternal and maternal alleles at the IGF2 locus and to test the hypothesis of a second QTL affecting backfat thickness on the short arm of SSC2 (SSC2p), a QTL mapping analysis was carried out on a combined pedigree including both the French and Dutch F2 populations, on the progeny of F1 males that were heterozygous (A/G) and homozygous (G/G) at the IGF2 locus. Simulations were performed to clarify the relations between the two QTL and to understand to what extent they can explain the discrepancies previously reported. RESULTS: The QTL analyses showed the segregation of at least two QTL on chromosome 2 in both pedigrees, i.e. the IGF2 locus and a second QTL segregating at least in the G/G F1 males and located between positions 30 and 51 cM. Statistical analyses highlighted that the maternally inherited allele at the IGF2 locus had a significant effect but simulation studies showed that this is probably a spurious effect due to the segregation of the second QTL. CONCLUSIONS: Our results show that two QTL on SSC2p affect backfat thickness. Differences in the pedigree structures and in the number of heterozygous females at the IGF2 locus result in different imprinting statuses in the two pedigrees studied. The spurious effect observed when a maternally allele is present at the IGF2 locus, is in fact due to the presence of a second closely located QTL. This work confirms that pig chromosome 2 is a major region associated with fattening traits.

Combining two Meishan F2 crosses improves the detection of QTL on pig chromosomes 2, 4 and 6.

BACKGROUND: In pig, a number of experiments have been set up to identify QTL and a multitude of chromosomal regions harbouring genes influencing traits of interest have been identified. However, the mapping resolution remains limited in most cases and the detected QTL are rather inaccurately located. Mapping accuracy can be improved by increasing the number of phenotyped and genotyped individuals and/or the number of informative markers. An alternative approach to overcome the limited power of individual studies is to combine data from two or more independent designs. METHODS: In the present study we report a combined analysis of two independent design (a French and a Dutch F2 experimental designs), with 2000 F2 individuals. The purpose was to further map QTL for growth and fatness on pig chromosomes 2, 4 and 6. Using QTL-map software, uni- and multiple-QTL detection analyses were applied separately on the two pedigrees and then on the combination of the two pedigrees. RESULTS: Joint analyses of the combined pedigree provided (1) greater significance of shared QTL, (2) exclusion of false suggestive QTL and (3) greater mapping precision for shared QTL. CONCLUSIONS: Combining two Meishan x European breeds F2 pedigrees improved the mapping of QTL compared to analysing pedigrees separately. Our work was facilitated by the access to raw phenotypic data and DNA of animals from both pedigrees and the combination of the two designs with the addition of new markers allowed us to fine map QTL without phenotyping additional animals.

Application of massive parallel sequencing to whole genome SNP discovery in the porcine genome.

BACKGROUND: Although the Illumina 1 G Genome Analyzer generates billions of base pairs of sequence data, challenges arise in sequence selection due to the varying sequence quality. Therefore, in the framework of the International Porcine SNP Chip Consortium, this pilot study aimed to evaluate the impact of the quality level of the sequenced bases on mapping quality and identification of true SNPs on a large scale. RESULTS: DNA pooled from five animals from a commercial boar line was digested with DraI; 150-250-bp fragments were isolated and end-sequenced using the Illumina 1 G Genome Analyzer, yielding 70,348,064 sequences 36-bp long. Rules were developed to select sequences, which were then aligned to unique positions in a reference genome. Sequences were selected based on quality, and three thresholds of sequence quality (SQ) were compared. The highest threshold of SQ allowed identification of a larger number of SNPs (17,489), distributed widely across the pig genome. In total, 3,142 SNPs were validated with a success rate of 96%. The correlation between estimated minor allele frequency (MAF) and genotyped MAF was moderate, and SNPs were highly polymorphic in other pig breeds. Lowering the SQ threshold and maintaining the same criteria for SNP identification resulted in the discovery of fewer SNPs (16,768), of which 259 were not identified using higher SQ levels. Validation of SNPs found exclusively in the lower SQ threshold had a success rate of 94% and a low correlation between estimated MAF and genotyped MAF. Base change analysis suggested that the rate of transitions in the pig genome is likely to be similar to that observed in humans. Chromosome X showed reduced nucleotide diversity relative to autosomes, as observed for other species. CONCLUSION: Large numbers of SNPs can be identified reliably by creating strict rules for sequence selection, which simultaneously decreases sequence ambiguity. Selection of sequences using a higher SQ threshold leads to more reliable identification of SNPs. Lower SQ thresholds can be used to guarantee sufficient sequence coverage, resulting in high success rate but less reliable MAF estimation. Nucleotide diversity varies between porcine chromosomes, with the X chromosome showing less variation as observed in other species.

Linkage disequilibrium decay and haplotype block structure in the pig.

Linkage disequilibrium (LD) may reveal much about domestication and breed history. An investigation was conducted, to analyze the extent of LD, haploblock partitioning, and haplotype diversity within haploblocks across several pig breeds from China and Europe and in European wild boar. In total, 371 single-nucleotide-polymorphisms located in three genomic regions were genotyped. The extent of LD differed significantly between European and Chinese breeds, extending up to 2 cM in Europe and up to 0.05 cM in China. In European breeds, LD extended over large haploblocks up to 400 kb, whereas in Chinese breeds the extent of LD was smaller and generally did not exceed 10 kb. The European wild boar showed an intermediate level of LD between Chinese and European breeds. In Europe, the extent of LD also differed according to genomic region. Chinese breeds showed a higher level of haplotype diversity and shared high levels of frequent haplotypes with Large White, Landrace, and Duroc. The extent of LD differs between both centers of pig domestication, being higher in Europe. Two hypotheses can explain these findings. First, the European ancestral stock had a higher level of LD. Second, modern breeding programs increased the extent of LD in Europe and caused differences of LD between genomic regions. Large White, Landrace, and Duroc showed evidence of past introgression from Chinese breeds.

Haplotype inference in crossbred populations without pedigree information.

BACKGROUND: Current methods for haplotype inference without pedigree information assume random mating populations. In animal and plant breeding, however, mating is often not random. A particular form of nonrandom mating occurs when parental individuals of opposite sex originate from distinct populations. In animal breeding this is called crossbreeding and hybridization in plant breeding. In these situations, association between marker and putative gene alleles might differ between the founding populations and origin of alleles should be accounted for in studies which estimate breeding values with marker data. The sequence of alleles from one parent constitutes one haplotype of an individual. Haplotypes thus reveal allele origin in data of crossbred individuals. RESULTS: We introduce a new method for haplotype inference without pedigree that allows nonrandom mating and that can use genotype data of the parental populations and of a crossbred population. The aim of the method is to estimate line origin of alleles. The method has a Bayesian set up with a Dirichlet Process as prior for the haplotypes in the two parental populations. The basic idea is that only a subset of the complete set of possible haplotypes is present in the population. CONCLUSION: Line origin of approximately 95% of the alleles at heterozygous sites was assessed correctly in both simulated and real data. Comparing accuracy of haplotype frequencies inferred with the new algorithm to the accuracy of haplotype frequencies inferred with PHASE, an existing algorithm for haplotype inference, showed that the DP algorithm outperformed PHASE in situations of crossbreeding and that PHASE performed better in situations of random mating.

Mapping carcass and meat quality QTL on Sus Scrofa chromosome 2 in commercial finishing pigs.

Quantitative trait loci (QTL) affecting carcass and meat quality located on SSC2 were identified using variance component methods. A large number of traits involved in meat and carcass quality was detected in a commercial crossbred population: 1855 pigs sired by 17 boars from a synthetic line, which where homozygous (A/A) for IGF2. Using combined linkage and linkage disequilibrium mapping (LDLA), several QTL significantly affecting loin muscle mass, ham weight and ham muscles (outer ham and knuckle ham) and meat quality traits, such as Minolta-L* and -b*, ultimate pH and Japanese colour score were detected. These results agreed well with previous QTL-studies involving SSC2. Since our study is carried out on crossbreds, different QTL may be segregating in the parental lines. To address this question, we compared models with a single QTL-variance component with models allowing for separate sire and dam QTL-variance components. The same QTL were identified using a single QTL variance component model compared to a model allowing for separate variances with minor differences with respect to QTL location. However, the variance component method made it possible to detect QTL segregating in the paternal line (e.g. HAMB), the maternal lines (e.g. Ham) or in both (e.g. pHu). Combining association and linkage information among haplotypes improved slightly the significance of the QTL compared to an analysis using linkage information only.