A gene is imprinted when its expression is dependent on the sex of the parent from which it was inherited. An increasing number of studies are suggesting that imprinted genes have a major influence on medically, agriculturally and evolutionarily important traits, such as disease severity and livestock production traits. While some genes have a large effect on the traits of an individual, quantitative characters such as height are influenced by many genes and by the environment, including maternal effects. The interaction between these genes and the environment produces variation in the characteristics of individuals. Many quantitative characters are likely to be influenced by a small number of imprinted genes, but at present there is no general theoretical model of the quantitative genetics of imprinting incorporating multiple loci, environmental effects and maternal effects. This research develops models for the quantitative genetics of imprinting incorporating these effects, including deriving expressions for genetic variation and resemblances between relatives. Imprinting introduces both parent-of-origin and generation dependent differences in the derivation of standard quantitative genetic models that are generally equivalent under Mendelian expression. Further, factors such as epistasis, maternal effects and interactions between genotype and environment may mask the effect of imprinting in a quantitative trait. Maternal effects may also mimic a number of signatures in variance and covariance components that are expected in a population with genomic imprinting. This research allows a more comprehensive understanding of the processes influencing an individual�s characteristics.

For many years, natural products have been a primary source of new molecules for the treatment of disease, and microorganisms have been a prolific source of these molecules. Recent studies have indicated, however, that many biosynthetic pathways are present in organisms for which no natural product can be associated, and only a small fraction of the microbial life present in the environment can be grown in culture. This indicates that if methods could be developed for the isolation of these pathways and production of their target molecules in heterologous hosts, great numbers of potentially valuable compounds might be discovered. In these investigations, large insert libraries of two microorganisms were constructed, one a bacterial artificial chromosome (BAC) library, the other a fosmid library, and two large insert fosmid libraries were constructed with DNA isolated from marine environmental samples. A mathematical formula was derived to estimate probabilities of cloning intact biosynthetic pathways with large insert genomic libraries and tested with a computer simulation. This indicated that even large pathways could be cloned intact in large insert libraries, provided there was an adequate size difference between the target pathway and the library inserts, and there was a concomitant increase in the size of the library with the targeting of these larger pathways. In addition, an investigation into a mixed marine culture sample lead to the identification of an unusual relationship between two bacteria for which extended co-culture leads to the production of pyocyanin. However, no useful biosynthetic pathways were located within the genomic libraries. It is concluded that significant improvements would be required to make this approach feasible for larger scale investigations. It is further concluded, on the basis of recent developments in the field, including a reduction in the cost of sequencing, improvements in techniques of whole-genome shotgun sequencing, and the development of recombination based cloning, that the employment of mass sequencing efforts and sequence-driven, recombinationbased cloning, might prove to be a more fruitful and efficient alternative to large-insert library construction for the isolation and expression of these pathways. A possible paradigm for the cloning of pathways on the basis of this technology is proposed.

A gene is imprinted when its expression is dependent on the sex of the parent from which it was inherited. An increasing number of studies are suggesting that imprinted genes have a major influence on medically, agriculturally and evolutionarily important traits, such as disease severity and livestock production traits. While some genes have a large effect on the traits of an individual, quantitative characters such as height are influenced by many genes and by the environment, including maternal effects. The interaction between these genes and the environment produces variation in the characteristics of individuals. Many quantitative characters are likely to be influenced by a small number of imprinted genes, but at present there is no general theoretical model of the quantitative genetics of imprinting incorporating multiple loci, environmental effects and maternal effects. This research develops models for the quantitative genetics of imprinting incorporating these effects, including deriving expressions for genetic variation and resemblances between relatives. Imprinting introduces both parent-of-origin and generation dependent differences in the derivation of standard quantitative genetic models that are generally equivalent under Mendelian expression. Further, factors such as epistasis, maternal effects and interactions between genotype and environment may mask the effect of imprinting in a quantitative trait. Maternal effects may also mimic a number of signatures in variance and covariance components that are expected in a population with genomic imprinting. This research allows a more comprehensive understanding of the processes influencing an individual�s characteristics.

Thesis (Ph.D.) -- Australian National University, 2008.

No description available.

Doctor of Philosophy / Department of Agronomy / Allan Fritz / Wheat breeders are constantly working to develop new wheat varieties with improved performance for agronomically important traits such as yield and disease resistance. Identifying better ways of phenotyping germplasm, developing methods for predicting performance based on genetic information, and identifying novel sources of genetic disease resistance can all improve the efficiency of breeding efforts. Three studies relating to these research interests were conducted. Synthetic hexaploid wheat lines were screened for resistance to root-lesion nematodes, an economically important pest of wheat. This resulted in the identification of three lines resistant to the root-lesion nematode species Pratylenchus thornei. Grain yield data from multi-location yield trials and average yields for counties in Kansas were used to identify wheat production areas in Kansas. Knowledge obtained from this study is useful for both interpreting data from yield trials and deciding where to place them in order to identify new higher yielding varieties. These data also aided the final research study, developing a genomic selection (GS) model for yield in the Kansas State University wheat breeding program. This model was used to assess the accuracy of GS in conditions experienced in a breeding project. Available measurements of GS have been constructed using simulations or using conditions not typical of those experienced in a wheat breeding program. The estimate of accuracy determined in this study was less than many of the reported measurements. This measure of accuracy will aid in determining if GS is a cost efficient tool for use in wheat breeding.

Genomic selection (GS) has been adopted by the dairy cattle breeding industry and the opportunity exists to implement this technology in UK beef and sheep breeding. However, these sectors do not appear so readily predisposed to GS implementation. Following an introduction to GS in Chapter 1, Chapter 2 investigated the structure of the little-studied UK beef breeding sector. This provided estimates of key commercial and pedigree population parameters, for use in modelling genetic gain from GS. Terminal traits were found to be the dominant selection goals, with 85% of beef-sired commercial matings resulting in progeny being slaughtered at a prime age. Pedigree bulls disseminated the majority of genes in the sector via natural service. The correlation between the terminal selection index (TI) and the sale price of breeding bulls was moderate, suggesting a modest uptake of genetic technology in the sector. Chapter 3 estimated selection intensity for TI, generation interval and the dissemination rate of improved genes in the pedigree Limousin population. In order to predict the genetic gain achievable in using GS in beef and sheep breeding, Chapters 4 to 6 undertook deterministic selection index simulations, incorporating genomic information as correlated traits. In Chapter 4, GS was modelled for terminal beef traits, when incorporating carcass information and accounting for likely genotype by environment interaction. Using a training population of 2000 sires, this concept was predicted to offer 40% greater genetic gain than existing BLUP selection using pedigree phenotypes. Gene flow methodology projected the commercial value of this gain to offer a substantial return net of genotyping costs. Chapter 5 explored GS for maternal beef traits within the concept of a nucleus breeding scheme. Whilst greater genetic gain was predicted with GS than with conventional BLUP, the economic value of this gain was projected to be too low to justify such a scheme in the UK. Chapter 6 proposed a synergy between computer tomography (CT) phenotypes and GS in sheep breeding. Developing a genomic predictor from male selection candidates with CT phenotypes and conventional performance records was predicted to increase genetic gain by 55% over BLUP selection without CT traits. Introducing GBV contributed most of the accuracy in this scenario, suggesting that the existing performance recording structure in UK sheep breeding could in the future be replaced by GS using CT. In the general discussion, the potential for GS in other beef and sheep traits was considered in the light of the outcomes of these simulations. Given the lack of vertical integration in UK beef and sheep sectors, the drivers for implementation of GS are examined. Finally, the options for international cooperation and the possibilities offered by future genotyping technology are considered. It was concluded that implementation of GS incorporating beef carcass phenotypes was merited and could provide a platform for future GS implementation in other novel traits. Sheep GS with CT traits was considered a higher risk strategy, due to the lack of evidence for uptake of existing EBV technology.

Doctor of Philosophy / Genetics Interdepartmental Program - Plant Pathology / Jesse A. Poland / A major challenge for 21st century plant geneticists is to predict plant performance based on genetic information. This is a daunting challenge, especially when there are thousands of genes that control complex traits as well as the extreme variation that results from the environment where plants are grown. Rapid advances in technology are assisting in overcoming the obstacle of connecting the genotype to phenotype. Next generation sequencing has provided a wealth of genomic information resulting in numerous completely sequenced genomes and the ability to quickly genotype thousands of individuals. The ability to pair the dense genotypic data with phenotypic data, the observed plant performance, will culminate in successfully predicting cultivar performance. While genomics has advanced rapidly, phenomics, the science and ability to measure plant phenotypes, has slowly progressed, resulting in an imbalance of genotypic to phenotypic data. The disproportion of high-throughput phenotyping (HTP) data is a bottleneck to many genetic and association mapping studies as well as genomic selection (GS). To alleviate the phenomics bottleneck, an affordable and portable phenotyping platform, Phenocart, was developed and evaluated. The Phenocart was capable of taking multiple types of georeferenced measurements including normalized difference vegetation index and canopy temperature, throughout the growing season. The Phenocart performed as well as existing manual measurements while increasing the amount of data exponentially. The deluge of phenotypic data offered opportunities to evaluate lines at specific time points, as well as combining data throughout the season to assess for genotypic differences. Finally in an effort to predict crop performance, the phenotypic data was used in GS models. The models combined molecular marker data from genotyping-by-sequencing with high-throughput phenotyping for plant phenotypic characterization. Utilizing HTP data, rather than just the often measured yield, increased the accuracy of GS models. Achieving the goal of connecting genotype to phenotype has direct impact on plant breeding by allowing selection of higher yielding crops as well as selecting crops that are adapted to local environments. This will allow for a faster rate of improvement in crops, which is imperative to meet the growing global population demand for plant products.

The two human pathogenic sub-species T.b.gambiense and T.b.rhodesiense can be distinguished from the morphologically identical T.b.brucei by their ability to infect humans, enabling them to cause sleeping sickness. This is because they are resistant to lysis by the lytic factor (APOL-I) present in normal human serum (NHS). In T.b.rhodesiense resistance to this lytic factor is due to a truncated VSG gene termed SRA which blocks lysis by interacting with APOL-I in the lysosome. SRA does not exist in T.b.gambiense. The search for a similar truncated VSG gene lead to the identification of a T.b.gambiense specific glycoprotein termed TGSGP. TGSGP transfected alone into the sensitive T.b.brucei is unable to confer resistance to this sub-species. This is either due to incorrect processing of this gene is this sub-species or because TGSGP requires a partner to confer resistance. In the search for a partner, the genomic locus of TGSGP was cloned and sequenced. We found that TGSGP is linked to a truncated gene homologous to the S.cerevisiae AUT1 gene, a gene implicated in autophagy and more specifically in membrane expansion. Southern blot hybridization and PCR analysis on genomic DNA from several isolates demonstrated that this feature was a specific to T.b.gambiense. In addition, we observed a correlation between the aut1 allele size and the geographical origin of the isolate. Since in trypanosomes lysis by NHS is due to an uncontrolled expansion of the lysosome, we speculated that the truncation of the aut1 allele could be implication in the resistance to human serum. We characterized the genomic organisation of the AUT1 locus. T.b.brucei possesses two native AUT1 alleles whilst T.b.gambiense possesses a truncated aut1 allele, as well as a native AUT1 allele. We showed that in the T.b.gambiense LiTAR isolate (aut1/AUT1), despite the presence of a wild-type allele this gene is no longer expressed at the mRNA and protein level. Our complimentary results by run-on transcription assay showed that the AUT1 region is transcribed but that the messenger is unstable. LiTAR is a functional knock-out for AUT1, but Northern blot analysis on several T.b.gambiense isolates showed that this is not a generalised T.b.gambiense characteristic. We explored the role of AUT1 in trypanosomes by invalidation of the AUT1 gene in T.b.brucei and by the over-expression of the AUT1 and aut1 alleles in T.b.brucei. By functional analysis of AUT1 knocked-down cells we showed that AUT1 is not essential in trypanosomes. By recreating in T.b.brucei the T.b.gambiense AUT1/aut1 genotype we were able to show that the expression of the aut1 UTR down-regulated the expression of the wild-type AUT1 allele. We speculated that this may be due to a natural RNAi mechanism. Par northern blot, using probes covering the potential target region of AUT1, we detected a 50nt small RNA specific to T.b.gambiense. In addition, we showed that in a LiTAR strain in which the RNAi pathway was abolished AUT1 expression is restored. We continued to investigate TGSGP’s role in the resistance to human serum by invalidation of TGSGP in T.b.gambiense and by expressing TGSGP in the NHS-sensitive T.b.brucei. Because T.b.gambiense cannot be cultured in vitro we established a new in vivo transfection technique and as the knock-out of TGSGP is most probably lethal, we created an inducible RNAi T.b.gambiense cell strain. These indispensable tools will be used to test whether invalidation TGSGP is sufficient to confer resistance to NHS. Many strategies were tested in order to correctly expressing TGSGP in T.b.brucei; in none of these transfectants was TGSGP correctly located in the flagellar pocket as is the case in T.b.gambiense and only partial resistance was ever obtained. In order to identify the factors in human serum that could interacts with TGSGP, we subjected NHS to affinity chromatography using TGSGP as bait. We showed that TGSGP interacts with APOA-I, a major component of HDLs.

No description available.