Michael G. K. Jones

Molecular markers make the pyramiding of rust genes possible in adapted elite lines. In this study we report the microsatellite tagging of leaf rust genes Lr13 and Lr28 in the Leichardt/WAWHT2071 and Sunland/Arrino populations, respectively. Leichardt and Sunland were the sources of resistance genes for Lr13 and Lr28, respectively. F2 and F2:3 populations were used for microsatellite tagging of the genes. Closely linked SSR markers were identified for Lr13 and Lr28 on chromosomes 2BS, 4AL, respectively. Molecular markers for a range of other rust resistance genes (Lr9, Lr19/Sr25, Lr24/Sr24, Lr34/Yr18 Lr46/Yr29, Lr47, Sr26, Sr32, Sr33 and Sr36) are currently being implemented for variety development and germplasm enhancement.

Molecular markers are being increasingly used as a selection tool in wheat breeding programs around the world. The deployment of multiple genes for disease, quality and agronomic traits into breeding lines is the major aim of a breeding program. To effectively utilise molecular markers, breeders require timely delivered accurate and reliable information, and lots of them! This presentation aims to outline; (1) Development of molecular markers for the breeders’ traits and an informative gene map for breeders that includes traits of immediate interest, and (2) discussions about the high-throughput and logistics of MAS applications in a large breeding program. Molecular marker development is fully integrated with the breeding operations, and activities are focused on the traits that breeders prioritise in the program. As the new markers were identified from our work and/or literature these were then integrated into marker cassettes formed for different group of traits (i.e. quality, disease) for their effective use in MAS. Based on the traits that are required by the breeding program we have established a trait based map that serve as a guide to breeders in their daily activities including crossing decisions. The program currently is using marker-assisted selection for 40 traits/genes, for which more than one marker are used for the quantitative traits. To define the needs of three sub-programs and for the timely delivery of the outputs by the molecular lab, we have developed a simple excel based database. This DB has allowed us to keep track the requirements of each of the three sub-programs to make sure that marker laboratory spends its resources equally. Data analysis is one of the limitations of high-throughput marker applications and to overcome this additional analysis tools are being developed.

The potential for lupins as an agricultural crop has been recognized worldwide. Despite work going on in several different laboratories around the world, the efficiency of stable gene transfer by Agrobacterium remains at approximately 1 % for modern varieties. Here we investigate particle bombardment as an alternative method of gene transfer. Preliminary studies were carried out by using tungsten particles ( ~ 1 μm in diameter) coated carrying DNA encoding the gus reporter gene and the bar gene for selection of transformants. Particles were accelerated into embryonic axes by helium inflow. A number of parameters were studied including plasmid preparation methods, pre-treatment of target tissue, particle density, helium pressure, DNA concentration, and the distance between the injector and target tissue (target distance). Expression as determined by blue spots of GUS staining has been increased significantly, but stable transformation, as determined by survival of shoots in selection medium (containing 10 mg/ml PPT), has not been achieved. Analysis of GUS expression seven days post-bombardment, and penetration depth of the tungsten particles in target tissue have suggested that the particles did not penetrate to the LII and LIII tissue layers of embryogenic axes, which give rise to organs including inflorescence of grown plants.

Cucumber Mosaic Virus (CMV) i s a major viral pathogen affecting lupin crops in Western Australia. Infection causes yield losses of up 50% and can persist through seed transmission. Current methods of control rely on the use of virus- free seed, the control of insect vectors through pesticides and breeding for resistance. Natural resistance to CMV has been identified in a cultivar of L. luteus. Resistance is manifested as a hyper sensitive response and is controlled by a single, dominant gene Ncm-1. The aims of this research are to identify molecular markers linked to CMV resistance, for use in marker assisted selection, and to isolate the Ncm-1 resistance gene. Molecular markers linked to Ncm-1 have been identified using fluorescent AFLPs. A number of polymorphic fragments segregating with resistance together with five other characterised traits have been isolated using a preliminary mapping population. A further population consisting of at least 200 individuals has been developed and will be used to confirm these putative markers and evaluate their use in the lupin breeding program. In order to further characterise and isolate Ncm-1, degenerate primers based on alignments of resistance gene analogues (RGAs) have been used to generate resistance genes candidates. Amplicons have been cloned and sequenced and one fragment showing high identity with the Leucine Rich Repeat motif has so far been identified. Further examination of the resistance gene candidate obtained is in progress.