PROJECT OVERVIEW AND OBJECTIVES

This study aims to develop new approaches for genomics and transcriptomics through the use of major resources developed in model species for research in crop species, using oil palm and Bambara groundnut as two exemplar species. In the present study DNA from oil palm was cross-hybridised onto heterologous Affymetrix microarrays (Arabidopsis and rice) in order to identify potential SFPs for traits (focusing on the shell thickness genes initially) and thereby generating molecular markers for crop breeding and an understanding of important agronomic traits. The use of XSpecies microarray approach has been demonstrated in many crop species (Hammond et al., 2005; Moore et al., 2005; Bagnaresi et al., 2008; Davey et al., 2009). In Bambara groundnut, a combination of XSpecies and genetical genomics approaches were employed to evaluate Bambara groundnut at both genetics and transcriptomics level. Firstly, a F5segregating population derived from the cross between DipC

and Tiga Nicuru in Bambara groundnut was subjected to a mild drought condition in a controlled glasshouse, allowing the early responses of Bambara groundnut to drought stress to be studied and also providing the phenotypic traits for QTL mapping. Secondly, RNA from Bambara groundnut was cross hybridised with soybean GeneChip, to develop gene expression markers (GEMs) based on differential hybridisation signals of RNA to individual oligonucleotide probes. These GEMs were used in the construction of a genetic linkage map (GEM map) as well as QTL mapping. In addition, a genetic linkage map (DArTseq map) was also created by combining dominant DArT and SNPs markers (developed using DArT Seq technology) with pre-existing microarray-based DArT and SSR markers using the F3segregating population of the the same cross (DipC x Tiga Nicuru),

followed by the integration of DArTseq and GEM maps. Thirdly, an attempt was made to overlay Bambara groundnut genetic linkage maps with the pseudo physical map in soybean in order to identify the location of genes on the genetic maps of the two species. The advanced genomic tools provide an insight into

the efficiency of using major resources in model species to study crop species, leading to exploitation of agricultural biodiversity which is potentially important to address food security challenges.

The objectives of the study are:

- To identify potential SFPs, from XSpecies microarray analysis, that are linked to the gene(s) controlling shell thickness in oil palm using a newly developed bioinformatics tool, PIGEONS software.

- To evaluate the effect of drought and changes in gene expression of Bambara groundnut segregating population subjected to mild stress. - To develop and characterise DArTseq (both dominant DArT and SNPs)

markers, and utilise DArTseq markers to construct a high density genetic linkage map using F3 segregating populations.

- To develop GEM markers for use in the construction of an expression based map using F5 segregating population.

- To construct an integrated genetic linkage map using DArTseq and GEM markers derived from two different generational populations of Bambara groundnut, the F3and F5segregating populations.

- To perform QTL analysis of agronomic and drought-related traits for the mapped populations.

- To provide a framework for identification of candidate genes in Bambara groundnut using soybean pseudo physical map.

Thesis outline:

Chapter 1: The introduction of the oil palm and Bambara groundnut, reviews on modern technologies such as XSpecies microarray approach, molecular markers, genetic linkage map, genetical genomics approach and their impacts are presented. In addition, project overview and the objectives of study are also stated in this chapter.

Chapter 2: Material and methods that generally used throughout the study are described, including list of standard solutions, preparation and quantitation of nucleic acids, polymerase chain reactions (PCR), gel electrophoresis and XSpecies microarrays analysis.

Chapter 3: The use of XSpecies microarray analysis on oil palm using Affymetrix Arabidopsis GeneChip and rice GeneChip is reported in this chapter. The

development of molecular markers using dataset generated from microarray is focused. In addition, the use of new bioinformatics software, PIGEONS, is also exploited to examine the probe sets and probe pairs that differentially expressed from individual palms with different fruit types.

Chapter 4: A mild drought stress experiment on a F5 segregating population

derived from a cross between DipC and Tiga Nicuru in Bambara groundnut is reported. The distribution of population, morpho-physiological studies and responses of Bambara groundnut plants to early drought stress are focused. Due to the variation between two parental lines, individual plants from segregating population with high-yielding characters and drought tolerance behaviour are reviewed.

Chapter 5: The construction of genetic map of a F3 segregating population in

Bambara groundnut using dominant DArT and SNPs markers, which are developed from DArT Seq technology, is described.

Chapter 6: Cross-hybridisation of Bambara groundnut RNA samples subjected under drought conditions onto Affymetrix soybean GeneChip is described in order to produce GEM markers. Three rounds of analyses for GEM markers development as well as the construction of genetic map using GEM markers for F5segregating population are focused. Furthermore, the attempt of integrating

DArTseq and GEM map is also reported.

Chapter 7: This chapter focuses on the QTL analysis of agronomically important traits using DArTseq map generated from dominant DArT and SNP markers and GEM map with GEMs, respectively.

Chapter 8: An attempt of identifying the location of genes of the markers represent in Bambara groundnut genetic map using major resources developed in soybean is reported in this chapter.

Chapter 9: General discussion on the study is reported, including potential problem in terms of food security, importance of agricultural biodiversity, review on the application of advanced genomics tools (XSpecies microarray approach combined with genetical genomics approach) in breeding programme, impacts of the findings and also future works.