Meeting future demands of food security will require enhanced rice production that is more environmentally sustainable. To achieve this it is important to know the genetic and molecular mechanism controlling the root traits. High throughput phenotyping which can keep pace with genotyping is needed, but for many researchers this needs to be cheap as well as meaningful. Here a very simple, low cost and reliable method of assessing root depth of seedling using a layer of diuron-soaked filter paper buried 25 cm deep in a soil-filled box has been developed which is suitable for screening of hundreds of accessions. The assumption is that deep-rooting plants die quicker. This method was then used to screen five established rice panels. Deep rooted cultivars were screened from a panel of an aus panel from IRRI and a panel of Brazilian and Japanese cultivars by using this method. Root QTLs were detected by using bi-parental mapping population and GWA study was performed in two panels, the rice diversity panel (RDP-1) and Bengal Assam Association Population. Assessing 139 RILs from Bala x Azucena bi-parental population revealed heritability of 55% for herbicide symptoms where eleven QTLs were detected, many of which were co-localised with previously reported root QTLs in this population. A GWA study was performed using RDP1) of 356 accessions with 44k SNP markers. Analysis revealed 17% of phenotypic variation of herbicide score was attributable to rice sub-population where the aus showed the deepest rooting systems. A number of QTLs have been identified and a number of positional candidate gene lists were produced. A further 298 cultivars from Bengal and Assam were screened and GWA was performed using 2 M SNP database available from sequencing. ANOVA revealed 37% variation for herbicide score explained by genotype. Soil-filled rhizotron were used to assess 12 of these cultivars, revealing strong xx correlations between deep root traits and herbicide score, confirming the reliability of this method. GWA revealed a number of significant SNPs associated with the traits in this population. Finally a set of mutant gene (LOC_Os09g31478, LOC_Os05g40330, LOC_Os11g34140) which are functional candidate gene for root growth QTLs were studied. Here hydroponic phenotypic screening approach were used to identify the T-DNA mutant lines. However, no convincing mutants were revealed. The herbicide screening method has been shown to be a quick and robust system for the assessment of deep rooting rice plants in soil. This method can now be used for screening large number of cultivars and the identification of QTLs and candidate genes.

The World Health Organization estimates that vector-borne diseases account for 17% of the global burden of all infectious diseases and has identified the mosquito as the most dangerous of all disease-transmitting insects, being responsible for several million deaths and hundreds of millions of cases each year. The study of mosquito genomics provides a deeper understanding of the molecular mechanisms involved in every aspect of vector biology, such as sex determination, host-parasite interaction, ecology, feeding behavior, immunity and evolutionary trends and can be used for the development of new strategies for vector control. We developed the first map of the mitotic chromosomes of the major vector for West Nile fever and lymphatic filariasis, Culex quinquefasciatus. The map was then successfully utilized for mapping of approximately 90% of available genetic markers to their precise positions on the chromosomes. Idiograms were integrated with 140 genetic supercontigs representing 26.5% of the genome. A linear regression analysis demonstrated good overall correlation between the positioning of markers on physical and genetic linkage maps. This will improve gene annotation and help in distinguishing potential haplotype scaffolds and regions of segmental duplications. It will also facilitate identification of epidemiologically important genes that can be used as targets for the vector control and provide a better framework for comparative genomics that will help understanding of the evolution of epidemiologically important traits. In another study, we confirmed the presence of the newly described species, Anopheles daciae, in regions of Russia using molecular data. Although sympatric with its sibling species, Anopheles messeae, five nucleotide substitutions in the internal transcribed spacer 2 of ribosomal DNA can be used to distinguish the morphologically similar species. Chromosome rearrangements have a significant impact on mosquito adaptation and speciation. Using sequencing data in combination with karyotyping, we demonstrated that significant differences in inversion frequencies distinguish An. messeae from An. daciae, suggesting that these inversions are actively involved in adaptation and speciation. It is essential to have reliable toolbox for correct identification of these species and to know their range for future possible malaria outbreaks prevention. / Ph. D. / The more you study, the more you know The more you know, the more you forget The more you forget, the less you know So why study? According to the World Health Organization, mosquitoes are one of the deadliest animals in the world. They spread disease to humans resulting in hundreds of millions of illnesses and several million deaths every year. Study of the mosquito genome can help us understand vector biology and speciation and can be used to develop new strategies for vector control. Culex quinquefasciatus, the southern house mosquito, is one of the major vectors for the West Nile virus in the U.S. and for lymphatic filariasis, a disabling and disfiguring disease, worldwide. The traditional methods of control are of limited effectiveness because of high insecticide resistance in many populations of the mosquito. To enhance our resources for the control strategies, we developed physical maps of the chromosomes for this mosquito and effectively integrated it with available genetic linkage map. This work will help to identify epidemiologically important genes that can be used as targets for the vector control. Malaria vectors, mosquitoes from the genus Anopheles, are known for their ecological plasticity, which can be partially explained by chromosome rearrangements called inversion. A global malaria eradication program significantly reduced the number of deaths related to malaria, especially in Europe and the U.S. However, malaria outbreaks can occur anywhere competent vectors occur. We studied Anopheles messeae, one of the major European malaria vectors and its closely related species, Anopheles danciae. We report for the first time the presence of An. daciae in Russia and demonstrate that its distribution overlaps with that of An. messeae. Using genetic sequence data in combination with chromosome structure, we demonstrated that significant differences in inversion frequencies reliably distinguish An. messeae from An. daciae. These inversions may be involved in adaptation and speciation of these two species. It is essential to have reliable toolbox for correct identification of these species and to know their range for future possible malaria outbreaks prevention.

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Bibliography: leaves 114-141. / vi, 141, [42] leaves, [19] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Using RFLP, STS-PCR and isoenzyme techniques, maps the structural genes of hydrolytic enzymes important in seed germination processes, and determines the contribution of each gene to the activity of the enzyme. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1998

The gene encoding chicken growth hormone (GH) was isolated from a chicken genomic library. The size of the gene was 4 kb. It was digested with PstI and subcloned into pUC18. Three of the PstI fragments were used for restriction fragment length polymorphisms (RFLPs) analysis at the GH locus in two chicken strains (fat and lean line). Four polymorphic sites were detected using a PstI fragment (PII) as a probe. One polymorphism was located at a SacI restriction site (PS1), and three at MspI sites (PM1, PM2 and PM3). A method based on polymerase chain reaction (PCR) was developed for detecting polymorphisms at PM3 site. A fragment of 823 base pairs which contained the PM3 polymorphic site was amplified. Three genotypes (+/+,$-$/$-$ and +/$-$) were distinguished by examining the MspI digested PCR products in either agarose or polyacrylamide gel. / Ten anonymous cDNA clones were also isolated from a chicken liver cDNA library and used for RFLPs analysis. Three of these clones were found to be able to detected RFLPs at MspI sites in chicken strains (strain 7, 8, 9, 8R, S and K) indicating that a high frequency of genes are polymorphic and can be used as markers in mapping experiments. One of the three clones was present on a haploid genetic element. Segregation analysis showed that the inheritance of this haploid gene was determined by the genotype of the female parent.

We report here the use of RAPD-PCR (Random Amplified Polymorphic DNA - Polymerase Chain Reaction) to identify segregating loci in the haploid progeny of an Armillaria ostoyae basidiocarp and the construction of the first genetic linkage map of this fungus, one of the causal species of Armillaria Root Disease. Upon screening 75 RAPD primers, 18 were found to identify a total of 43 loci segregating with a 1 : 1 Mendelian ratio. These loci were analysed for linkage among 58 monospore progeny. The map constructed with Mapmaker (LOD = 3.0, r = 0.38) was confirmed by GMendel (LOD = 1.5, r = 0.38). This map arranged 30 loci into 6 linkage groups and 4 linkage pairs. Thirteen markers remained unlinked. Using the Kosambi mapping function the linked loci accounted for approximately 450 cM and the genome was estimated to be 1600 cM. This preliminary map covers approximately 28% of the A. ostoyae genome.

Due to the importance of insulin-like growth factor-I (IGF-I) in regulating many physiological and metabolic processes, the IGF-I gene was chosen as a candidate gene to study trait associated polymorphisms in chickens. A PstI restriction fragment length polymorphism (RFLP) was detected at the 5' region of the gene and mapped to about 7 Kb upstream of the published promoter sequence. Analysis for association of the marker with traits of economic importance in an unselected, random-bred population of 359 White Leghorns revealed a significant association with egg weight (P ≤ 0.05) and specific gravity (P ≤ 0.05). There was also a trend for association with juvenile body weight (P = 0.08) but not adult body weight. For egg weight the PstI (-/-) genotype was associated with lower egg weight as compared to the heterozygote or the PstI (+/+) genotype. The PstI marker also was found to be significantly associated with differences in trait correlations. A regulatory loop that co-ordinated feed consumption, body weight, egg weight and rate of egg laying was detected, and this regulatory loop differed among the IGF-I genotypic classes. In the PstI (+/-) genotype, the degree of correlation between some of the traits was time dependent, while in the PstI (+/+) genotype it remained constant through the different periods of measurement. Since IGF-I is known to play an important role in immune functions, the association of the IGF-I genotypes with immune traits was also investigated. A significant association was found for delayed type hypersensitivity, interferon production and T-cell count (P ≤ 0.05). Individuals belonging to the PstI (+/-) genotypic class exhibited higher immune response, reflected by the delayed type hypersensitivity reaction and antibody the interactive effects of marker genotypes in the GH, GH-receptor and IGF-I genes on traits and trait correlations indicated that the three are part of an epistatic pathway, wherein the phenotypic consequences of

Errata slip inserted. / Bibliography: leaves 68-93. / viii, 93, [42] leaves, [15] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Rye chromosome 6R in triticale contains a gene useful for resistance to cereal cyst nematode. This study shows that the complex structure of this chromosome may prevent render its use impracticle in introgression of this resistance into wheat. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1998