Linseed oil (Linum usitatissimum L.) is valued for its food and non-food applications. Although Canada is the world’s largest linseed producer and exporter, linseed remains a minor crop in part because its yield has been stagnating over the last decade compared to other oilseeds. Narrow genetic base, absence of an efficient hybrid production system and limited genomic tools for linseed breeding are the main factors hindering yield and quality improvements. Here, we characterized the Canadian flax core collection of 407 accessions with 448 genome-wide simple sequence repeat markers and, using association mapping (AM), we demonstrated its potential for the improvement of seed quality and agronomic traits. Genetic structure analyses assigned all accessions to two major groups that were weakly differentiated (FST = 0.094). Genetic diversity was abundant in the total panel (5.32 alleles per locus) with weak familial relatedness (mean = 0.287) for most individual pairs. Linkage disequilibrium (LD) decayed relatively quickly with an average genome-wide LD of ~1 cM. AM for seven seed quality traits including oil content (OIL), palmitic acid (PAL), stearic acid (STE), oleic acid (OLE), linoleic acid (LIO), linolenic acid (LIN) and iodine value (IOD) identified nine stable candidate QTL. LIO and LIN QTL co-localized with previously identified QTL and some mapped in the vicinity of genes known to be involved in the fatty acid biosynthesis pathway. AM conducted for nine agronomic traits including yield, bolls per area (BPA), seeds per boll (SPB), thousand seed weight (TSW), start of flowering (FL5%), end of flowering (FL95%), plant height (PH), plant branching (PB) and lodging (LDG) identified twelve significant marker-trait associations for six of the traits. The associated markers explained between 0.5 to 18.5% of the phenotypic variation, with Lu526 and Lu2532 associated with TSW and Lu943 associated with flowering being the most promising for marker-assisted selection. Statistical simulation for five markers associated with TSW indicated that the favorable alleles have additive effects. None of the accessions carried the five favorable alleles but a few breeding lines had four, indicating that further improvement of TSW and yield could be achieved through marker assisted breeding

A pecuária de corte brasileira tem sofrido grande pressão devido às questões ambientais, às exigências dos mercados consumidores por carne de qualidade e rígidos padrões sanitários. Esses fatores aumentam a importância da melhoria da eficiência produtiva da pecuária de corte. A proposta do presente trabalho foi avaliar o efeito da inclusão dos valores genéticos moleculares, de um painel de marcadores genéticos comerciais (Perfil IGENITY® Nelore V3) na seleção de animais da raça Nelore. Foram utilizados dados de 9.749 animais da raça Nelore mensurados para área de olho de lombo, espessura de gordura subcutânea e espessura de gordura na picanha e 39.687 animais na matriz de parentesco. Dois modelos de análise foram utilizados. O modelo de análise uni-característica em que apenas os parâmetros genéticos do fenótipo foram estimados e o modelo bi-característica em que os valores genéticos moleculares de 3.033 animais foram incluídos como característica correlacionada. A inclusão dos valores genéticos moleculares no modelo aumentou as acurácias das estimativas dos valores genéticos preditos dos animais genotipados, principalmente dos machos jovens. As analises dos conflitos de seleção demonstraram maiores divergências nos touros e machos jovens que tiveram seus genótipos definidos. A taxa de ganho genético anual com a inclusão dos valores genéticos moleculares no modelo foi aumentada em 2,4% para área de olho de lombo, 0,9% para espessura de gordura subcutânea e 1,9% para espessura de gordura na picanha. Esses resultados demonstram que a utilização dos valores genéticos moleculares , mesmo quando oriundos de painéis de marcadores de DNA de baixa densidade, pode contribuir na seleção de animais superior mérito genético e, além de promover aumento nos ganhos genéticos dos programas de melhoramento genético da raça Nelore. / The Brazilian beef cattle chain is suffering a huge pressure due to environmental issues, the demand of consumer markets for meat quality and strict sanitary standards of the international market. These factors increase the importance of improving productive efficiency of beef cattle. The proposal of this study was to evaluate the effect of inclusion of the molecular breeding values of a commercial panel of genetic markers (Nellore Profile IGENITY® V3) in the genetic selection of Nellore cattle. Data of 9,749 animals measured for ribeye area, fat thickness and rump fat thickness were used in this study, with a relationship matrix compound of 39,687 animals. Two models of analysis were performed. Single trait model was performed only for each observed phenotypes and two-trait model was performed phenotypes and molecular breeding values of 3,033 animals as a correlated trait. The inclusion of molecular information in genetic evaluation provided increases on the accuracies of predicted breeding values of genotyped animals and, mainly, for replacement young bulls. The divergences of selection for 20% best animals classified by 1-trait breeding values and 2-trait breeding values demonstrated highest divergence for sires and replacement young bulls. The genetic change rate on the 2-trait model increased 2,4% for ribeye area, 0,9 for fat thickness and 1,9% for rump fat thickness. These results demonstrated that the inclusion of molecular breeding values, even when estimated from low density genetic markers panels, on animal breeding evaluations can contribute on the selection of best genetic merit animals and increase of genetic change rate on animal breeding programs for Nellore cattle.

A pecuária de corte brasileira tem sofrido grande pressão devido às questões ambientais, às exigências dos mercados consumidores por carne de qualidade e rígidos padrões sanitários. Esses fatores aumentam a importância da melhoria da eficiência produtiva da pecuária de corte. A proposta do presente trabalho foi avaliar o efeito da inclusão dos valores genéticos moleculares, de um painel de marcadores genéticos comerciais (Perfil IGENITY® Nelore V3) na seleção de animais da raça Nelore. Foram utilizados dados de 9.749 animais da raça Nelore mensurados para área de olho de lombo, espessura de gordura subcutânea e espessura de gordura na picanha e 39.687 animais na matriz de parentesco. Dois modelos de análise foram utilizados. O modelo de análise uni-característica em que apenas os parâmetros genéticos do fenótipo foram estimados e o modelo bi-característica em que os valores genéticos moleculares de 3.033 animais foram incluídos como característica correlacionada. A inclusão dos valores genéticos moleculares no modelo aumentou as acurácias das estimativas dos valores genéticos preditos dos animais genotipados, principalmente dos machos jovens. As analises dos conflitos de seleção demonstraram maiores divergências nos touros e machos jovens que tiveram seus genótipos definidos. A taxa de ganho genético anual com a inclusão dos valores genéticos moleculares no modelo foi aumentada em 2,4% para área de olho de lombo, 0,9% para espessura de gordura subcutânea e 1,9% para espessura de gordura na picanha. Esses resultados demonstram que a utilização dos valores genéticos moleculares , mesmo quando oriundos de painéis de marcadores de DNA de baixa densidade, pode contribuir na seleção de animais superior mérito genético e, além de promover aumento nos ganhos genéticos dos programas de melhoramento genético da raça Nelore. / The Brazilian beef cattle chain is suffering a huge pressure due to environmental issues, the demand of consumer markets for meat quality and strict sanitary standards of the international market. These factors increase the importance of improving productive efficiency of beef cattle. The proposal of this study was to evaluate the effect of inclusion of the molecular breeding values of a commercial panel of genetic markers (Nellore Profile IGENITY® V3) in the genetic selection of Nellore cattle. Data of 9,749 animals measured for ribeye area, fat thickness and rump fat thickness were used in this study, with a relationship matrix compound of 39,687 animals. Two models of analysis were performed. Single trait model was performed only for each observed phenotypes and two-trait model was performed phenotypes and molecular breeding values of 3,033 animals as a correlated trait. The inclusion of molecular information in genetic evaluation provided increases on the accuracies of predicted breeding values of genotyped animals and, mainly, for replacement young bulls. The divergences of selection for 20% best animals classified by 1-trait breeding values and 2-trait breeding values demonstrated highest divergence for sires and replacement young bulls. The genetic change rate on the 2-trait model increased 2,4% for ribeye area, 0,9 for fat thickness and 1,9% for rump fat thickness. These results demonstrated that the inclusion of molecular breeding values, even when estimated from low density genetic markers panels, on animal breeding evaluations can contribute on the selection of best genetic merit animals and increase of genetic change rate on animal breeding programs for Nellore cattle.

Porcine circovirus type 2 (PCV2) is a small, non-enveloped, single-stranded DNA virus that causes disease in pigs and is an economically important pathogen affecting pig populations worldwide. PCV2 contains two major open reading frames (ORF): ORF1 encodes two replicase proteins and ORF2 encodes the immunogenic capsid protein. There are three genotypes of PCV2 (PCV2a, PCV2b, and PCV2c), but vaccines available for PCV2 infection are only targeted against PCV2a. The objective of this thesis was to create viable chimeric PCV2 viruses with an ORF2 displaying genetic diversity from all PCV2 genotypes by synthetic DNA shuffling. Variation was identified at 55 amino acid positions in the ORF2 gene among 853 PCV2 capsid gene sequences available in the GenBank database. Degenerate oligonucleotide primers spanning ORF2 were synthesized to contain this naturally observed sequence diversity. Sets of overlapping oligonucleotide primers were fused together using overlap extension PCR to create full-length shuffled ORF2 sequences. The shuffled library of the ORF2 genes was subsequently cloned into the genomic backbone of a wildtype PCV2a infectious DNA clone and transfected into porcine kidney cells (PK-15). After transfection and infection of PK-15 cells, viability of chimeric viruses was screened by immunofluorescence assay (IFA) using anti-PCV2 Rep antibodies. PCR was used to amplify the genomes of viable shuffled viruses from infected cells. PCV2 viruses containing an ORF2 displaying genetic diversity from PCV2a, PCV2b, and PCV2c were isolated in vitro. These shuffled PCV2 viruses may be used as potential candidates for a broadly-protective PCV2 vaccine, although additional studies are warranted to determine in vivo infectivity and pathogenicity. / Master of Science

A disponibilidade de informações baseadas nos marcadores genéticos surgiu como oportunidade de aprimorar os programas de melhoramento animal pela incorporação desses efeitos nas avaliações genéticas. Nesse contexto, o presente estudo teve como objetivos comparar modelos que consideraram ou não os efeitos dos marcadores para a estimação dos valores genéticos dos animais, bem como estimar os efeitos de substituição alélica dos marcadores por seis metodologias distintas (regressão múltipla bayesiana, regressão de cumeeira bayesiana, Bayes A, Bayes B, Bayes Cπ e LASSO bayesiano) e avaliar o impacto da inclusão desses efeitos na acurácia das estimativas dos valores genéticos e os conflitos de seleção existentes aos serem comparadas as classificações dos animais com base nos valores genéticos clássicos e nos valores genéticos assistidos por marcadores. Dados de 83.404 animais pertencentes a um programa de seleção de animas da raça Nelore, mensurados para peso na desmama, ganho de peso pós-desmama, perímetro escrotal e escore de musculosidade, que corresponderam a 116.652 animais na matriz de parentesco, foram utilizados. Do total de animais com informações fenotípicas e genealógicas disponíveis, apenas 3.160 foram genotipados para 106 marcadores do tipo SNP. Os resultados obtidos para a comparação de modelos não demonstraram vantagens claras da inclusão conjunta dos efeitos poligênicos e dos marcadores nos modelos de avaliação genética, entretanto, os modelos que incluíram apenas o efeito dos marcadores tiveram os piores ajustes e desempenhos preditivos. As diferenças observadas entre as estimativas dos efeitos de substituição alélica dos marcadores pelas diferentes metodologias analisadas se devem à maneira como cada método regulariza esses efeitos. A incorporação das informações dos marcadores nas avaliações genéticas proporcionou, no geral, um aumento na acurácia das estimativas dos valores genéticos, especialmente para os tourinhos de reposição. Ao serem comparados os 20% melhores animais classificados com base no valor genético clássico e no valor genético assistido por marcadores, os maiores conflitos de seleção foram observados para os touros e tourinhos genotipados. Em suma, o presente projeto demonstrou que, embora a utilização de painéis de marcadores de muito baixa densidade não altere a capacidade preditiva dos modelos de avaliação genética, esses têm impacto na acurácia das estimativas dos valores genéticos. / The availability of molecular markers information turned out to be an opportunity to improve animal breeding programs, by the inclusion of those effects in the estimation of breeding values. Under that perspective, the aims of present research were to compare genetic evaluation models that assumed or not markers effects on the estimation of breeding values, as well estimate the allelic substitution effects of SNP markers applying six different methodologies (Bayesian multiple regression, Bayesian ridge regression, Bayes A, Bayes B, Bayes Cπ and Bayesian Lasso) and evaluate the impact of these effects on the reliability of breeding values and the divergences on animals classification based on classical breeding values and marker assisted breeding values. Data of 83,404 animals belonging to a Nellore beef cattle (Bos indicus) selection program, measured for post-weaning gain, scrotal circumference and muscle score, corresponding to 116,562 animals on the relationship matrix, were used. From those animals, a set of 3,160 animals with phenotypic and genealogy data available, was genotyped for a panel of 106 SNP markers. Model comparison results did not demonstrate clearly the advantage of assuming polygenic and markers effects together in genetic evaluation models, however, models that considered only markers effects presented the worst global fit and predictive ability. Differences observed on the markers effects estimates were due the shrinkage process applied by each method. The incorporation of markers information on genetic evaluations provided, in general, increases on the reliability of breeding values, mainly for replacement young animals. Comparing the 20% best animals classified by classical breeding value and marker assisted breeding value, the highest divergences were observed to sires and young bulls that were genotyped. Summarizing, although this research showed that the inclusion of very low density SNP chip information was not able to improve the predictive ability of genetic evaluation models, they increased the reliability of breeding values estimates.

Maize grain contains high levels of phytic acid which chelates iron, zinc and other micronutrients as it passes through the digestive systems of monogastric consumers reducing their bioavailability. Breeding for low phytic acid (LPA) content to improve micronutrient bioavailability is hampered by a tedious and destructive colorimetric method on the grain, low yields compared to the wild-types and reduced seed germination and vigour of LPA mutants. Breeding for LPA therefore should also incorporate breeding for improved germination and vigour in the mutants. Molecular markers to speed up the selection process and studies on gene action and combining ability for germination, vigour and yield parameters of the LPA mutants in combinations with other different maize germplasm will speed up breeding for this trait. The objectives of this study were: to develop a molecular marker linked to the lpa1-1 gene and apply this marker for foreground selection in a backcross breeding programme and to use amplified fragment length polymorphism (AFLP) markers for background selection to recover the recurrent parent genome to speed up the backcrossing process; to study gene action and combining ability for seed germination, vigour and yield from diallel crosses involving LPA mutants, QPM and normal endosperm maize inbred lines by replicated laboratory seed tests ( standard germination test and accelerated aging test) and field evaluations in South Africa and Zimbabwe. A co-dominant single nucleotide polymorphism (SNP) marker which detects the transition base change of C/T nucleotides was developed from the gene sequence to identify the lpa1-1 trait. The 150 bp lpa1-1 SNP marker was validated by forward and reverse DNA sequencing of the parental amplification products which confirmed the C to T base change resulting in the LPA phenotype. The lpa1-1 SNP marker was used for foreground selection in 250 BC(2)F(1) progenies of CM 32 (LPA) x P 16 as the recurrent parent. This SNP marker was used to genotype the lines into homozygous dominant (wild type) and homozygous recessive (LPA) genotypes by their melting profiles and heterozygous genotypes by the normalised difference plots using high resolution melt (HRM) analysis. Seventeen heterozygous and 11 homozygous recessive lines were identified for background selection by fingerprinting with AFLP markers to determine the amount of recurrent parent (P 16) genome present. There were six EcoRI/MseI primer combinations tested with 277 data points scored (84% polymorphism rate). The amount of recurrent parent (P 16) genome recovered ranged from 62% to 92% with 13 lines showing greater than 83% of the recurrent parent genome. The effects of diallel crosses generated between four LPA, three QPM and three normal endosperm maize lines were determined for seed germination and vigour using the standard germination and accelerated aging seed tests under laboratory conditions in accordance with the procedures of the International Seed Testing Association. The specific combining ability (SCA) effects and general combining ability (GCA) effects were significant for the seed germination and vigour traits, indicating that genes with non-additive and additive effects were important in controlling these traits. However, the SCA effects were greater than GCA effects suggesting that genes with non-additive effects were predominant. The LPA parents showed reduced vigour compared to the normal and QPM inbred lines under both conditions, with LPA lines CM 31 and CM 32 showing stress tolerance. There were some combinations involving LPA lines, such as LPA x normal, LPA x QPM and LPA x LPA that retained high vigour and high germination rates under accelerated aging conditions, suggesting that they could be stress-tolerant.. A 10 x 10 diallel involving four LPA, three QPM and three Nm inbred lines was evaluated in replicated trials across six environments. Results show that both additive and non-additive gene effects were significant for resistance to northern corn leaf blight (NCLB), grey leaf spot (GLS) and Phaeosphaeria leaf spot (PLS) diseases. The additive gene effects were predominant for the yield and associated secondary traits such as days to mid-pollen shed (DMP), days to mid-silking (DMS), ear per plant (EPP) and grain moisture content (GMC) and grain yield. The LPA lines were early flowering and had quick grain dry down rate but all showed undesirable negative and significant GCA effects for yield. The yield of the LPA x LPA, LPA x Nm and LPA x QPM group of crosses was lower than the check hybrids by about 32 to 67% showing the need for yield improvement of the LPA combinations. An eight x eight diallel involving two LPA and six normal endosperm lines was evaluated over two seasons in five locations with two replications for grain yield components and foliar diseases. There was significant additive and non-additive gene action for both seed germination and vigour traits with predominance of non-additive gene effects. Both additive and non-additive gene effects were significant for yield and associated traits such as anthesis dates and number of ears per plant. However, the additive gene action was predominant for yield and associated traits. Generally the LPA lines and their combinations showed lower germination and vigour. The LPA line, CM 32 showed stress tolerance under accelerated aging conditions. There were three LPA x Nm crosses that showed improvements to the means of seed germination and vigour and yield traits. Results indicated that there was not any significant correlation between yield and seed germination and between yield and vigour. Yield was, however, significantly and positively correlated with anthesis dates and GMC, indicating that higher yield was associated with longer growing cycles. This study was able to successfully develop and apply the lpa1-1 SNP marker for foreground selection and AFLP markers for background selection in a backcross breeding programme. Problems of low seed germination, seedling vigour and grain yield of LPA lines and their combinations were confirmed. However results also indicated some potential of combining the LPA and QPM traits in a single cultivar. In general, procedures such as reciprocal recurrent selection, that emphasise both GCA and SCA effects would be recommended to improve seed germination, seedling vigour and yield in developing varieties with LPA trait. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

A disponibilidade de informações baseadas nos marcadores genéticos surgiu como oportunidade de aprimorar os programas de melhoramento animal pela incorporação desses efeitos nas avaliações genéticas. Nesse contexto, o presente estudo teve como objetivos comparar modelos que consideraram ou não os efeitos dos marcadores para a estimação dos valores genéticos dos animais, bem como estimar os efeitos de substituição alélica dos marcadores por seis metodologias distintas (regressão múltipla bayesiana, regressão de cumeeira bayesiana, Bayes A, Bayes B, Bayes Cπ e LASSO bayesiano) e avaliar o impacto da inclusão desses efeitos na acurácia das estimativas dos valores genéticos e os conflitos de seleção existentes aos serem comparadas as classificações dos animais com base nos valores genéticos clássicos e nos valores genéticos assistidos por marcadores. Dados de 83.404 animais pertencentes a um programa de seleção de animas da raça Nelore, mensurados para peso na desmama, ganho de peso pós-desmama, perímetro escrotal e escore de musculosidade, que corresponderam a 116.652 animais na matriz de parentesco, foram utilizados. Do total de animais com informações fenotípicas e genealógicas disponíveis, apenas 3.160 foram genotipados para 106 marcadores do tipo SNP. Os resultados obtidos para a comparação de modelos não demonstraram vantagens claras da inclusão conjunta dos efeitos poligênicos e dos marcadores nos modelos de avaliação genética, entretanto, os modelos que incluíram apenas o efeito dos marcadores tiveram os piores ajustes e desempenhos preditivos. As diferenças observadas entre as estimativas dos efeitos de substituição alélica dos marcadores pelas diferentes metodologias analisadas se devem à maneira como cada método regulariza esses efeitos. A incorporação das informações dos marcadores nas avaliações genéticas proporcionou, no geral, um aumento na acurácia das estimativas dos valores genéticos, especialmente para os tourinhos de reposição. Ao serem comparados os 20% melhores animais classificados com base no valor genético clássico e no valor genético assistido por marcadores, os maiores conflitos de seleção foram observados para os touros e tourinhos genotipados. Em suma, o presente projeto demonstrou que, embora a utilização de painéis de marcadores de muito baixa densidade não altere a capacidade preditiva dos modelos de avaliação genética, esses têm impacto na acurácia das estimativas dos valores genéticos. / The availability of molecular markers information turned out to be an opportunity to improve animal breeding programs, by the inclusion of those effects in the estimation of breeding values. Under that perspective, the aims of present research were to compare genetic evaluation models that assumed or not markers effects on the estimation of breeding values, as well estimate the allelic substitution effects of SNP markers applying six different methodologies (Bayesian multiple regression, Bayesian ridge regression, Bayes A, Bayes B, Bayes Cπ and Bayesian Lasso) and evaluate the impact of these effects on the reliability of breeding values and the divergences on animals classification based on classical breeding values and marker assisted breeding values. Data of 83,404 animals belonging to a Nellore beef cattle (Bos indicus) selection program, measured for post-weaning gain, scrotal circumference and muscle score, corresponding to 116,562 animals on the relationship matrix, were used. From those animals, a set of 3,160 animals with phenotypic and genealogy data available, was genotyped for a panel of 106 SNP markers. Model comparison results did not demonstrate clearly the advantage of assuming polygenic and markers effects together in genetic evaluation models, however, models that considered only markers effects presented the worst global fit and predictive ability. Differences observed on the markers effects estimates were due the shrinkage process applied by each method. The incorporation of markers information on genetic evaluations provided, in general, increases on the reliability of breeding values, mainly for replacement young animals. Comparing the 20% best animals classified by classical breeding value and marker assisted breeding value, the highest divergences were observed to sires and young bulls that were genotyped. Summarizing, although this research showed that the inclusion of very low density SNP chip information was not able to improve the predictive ability of genetic evaluation models, they increased the reliability of breeding values estimates.