PCR was used to amplify low-molecular-weight (LMW) glutenin genes from the Glu-A3 loci of hexaploid wheat cultivars containing different Glu-A3 alleles. The complete coding sequence of one LMW glutenin gene was obtained for each of the seven alleles Glu-A3a to Glu-A3g. Chromosome assignment of PCR products using Chinese Spring nulli-tetrasomic lines confirmed the amplified products were from chromosome 1A. All sequences were classified as LMW-i-type genes based on the presence of an N-terminal isoleucine residue and eight cysteine residues located within the C-terminal domain of the predicted, mature amino acid sequence. All genes contained a single uninterrupted open reading frame, including the sequence from the Glu-A3e allele, for which no protein product has been identified. Comparison of LMW glutenin gene sequences obtained from different alleles showed a wide range of sequence identity between the genes, with between 1 and 37 single nucleotide polymorphisms and between one and five insertion/deletion events between genes from different alleles. Allele-specific PCR markers were designed based on the DNA polymorphisms identified between the LMW glutenin genes, and these markers were validated against a panel of cultivars containing different Glu-A3 alleles. This collection of markers represents a valuable resource for use in marker-assisted breeding to select for specific alleles of this important quality-determining locus in bread wheat.

Microsatellite markers have been shown to be highly polymorphic and simple to use in hexaploid wheat. This study aimed to establish microsatellites as informative markers for Australian wheat improvement. By screening microsatellites developed as part of the Wheat Microsatellite Consortium and other available microsatellite sources, 257 informative microsatellites for Australian wheat varieties were identified and reported in the Australian National Wheat Molecular Marker Program microsatellite database (http://www.scu.edu.au/research/cpcg/). Of these, 151 microsatellites identifying 172 loci were scored on at least 1 of 4 double haploid mapping populations and were then integrated, where possible, into existing genetic maps. Polymorphism information content values were calculated for most microsatellites to establish a reference for their value for future investigations. The mapping of available microsatellites enhances the quality of the genetic maps and may provide useful genetic markers for traits of interest to the Australian wheat breeding programs.

This paper describes and discusses strategies for screening microsatellites for use in plant genetic research and illustrates how a subset of useful microsatellites can be optimised for implementation on breeding and research using a range of techniques. Beginning with the initial screening of microsatellites for potential polymorphisms in a core set of potential parental lines, through to scaling up for mapping or breeding purposes, we present a time- and cost-effective approach to microsatellite analysis in wheat lines of interest. Each stage of this process benefits from a fresh examination of the techniques applied in order to increase the efficiency with which key markers can be identified and implemented. For the primary screening we use primers without modification to prime PCRs in the presence of f-dNTP (fluorescently labelled nucleotide) to provide the basis for high resolution screening for polymorphisms. As markers are defined for use in a breeding program, the focus changes to a smaller set of primer pairs that will be used to screen large numbers of DNA samples either from the analysis of progeny from a cross or the routine checking of cultivar identity in the industry. We then examine appropriate analysis platforms and refinement of PCR primers and conditions in order to identify procedures that can be implemented widely, not just in specialised well-equipped laboratories. In many cases we are able to use lower cost agarose analysis for identified polymorphisms. Where this is not feasable we examine primers for potential redesign to optimise their application either by altering the sequence of the primer itself, based on available sequence information, or by adding tails to the primers. The latter is shown to alter the ‘stutter’ pattern that is commonly observed with wheat microsatellites so that a single band is prominent and thus allows size polymorphisms to be more readily scored. The addition of a generic 5′ tail also provides a method of using a generic fluorescent primer that can be applied to multiple tagged markers in a costeffective fashion. The potential of alternative analytical systems and further refinement of primers to show plus/minus reactions with wheat lines in order to produce simple tests for use in breeding programs are also discussed.

Genetic maps were compiled from the analysis of 160–180 doubled haploid lines derived from 3 crosses: Cranbrook Halberd, CD87 Katepwa, and Sunco Tasman. The parental wheat lines covered a wide range of the germplasm used in Australian wheat breeding. The linkage maps were constructed with RFLP, AFLP, microsatellite markers, known genes, and proteins. The numbers of markers placed on each map were 902 for Cranbrook Halberd, 505 for CD87 Katepwa, and 355 for Sunco Tasman. Most of the expected linkage groups could be determined, but 10–20% of markers could not be assigned to a specific linkage group. Homologous chromosomes could be aligned between the populations described here and linkage groups reported in the literature, based around the RFLP, protein, and microsatellite markers. For most chromosomes, colinearity of markers was found for the maps reported here and those recorded on published physical maps of wheat. AFLP markers proved to be effective in filling gaps in the maps. In addition, it was found that many AFLP markers defined specific genetic loci in wheat across all 3 populations. The quality of the maps and the density of markers differs for each population. Some chromosomes, particularly D genome chromosomes, are poorly covered. There was also evidence of segregation distortion in some regions, and the distribution of recombination events was uneven, with substantial numbers of doubled haploid lines in each population displaying one or more parental chromosomes. These features will affect the reliability of the maps in localising loci controlling some traits, particularly complex quantitative traits and traits of low heritability. The parents used to develop the mapping populations were selected based on their quality characteristics and the maps provide a basis for the analysis of the genetic control of components of processing quality. However, the parents also differ in resistance to several important diseases, in a range of physiological traits, and in tolerance to some abiotic stresses.

The absence of expression of the granule-bound starch synthase I (GBSSI) allele from chromosome 4A of wheat is associated with improved starch quality for making Udon noodles. Several PCR-based methods for the analysis of GBSS alleles have been developed for application in wheat. A widely applied approach has involved a simple PCR followed by electrophoretic separation of DNA products on agarose gels. The PCR amplifies one band from each of the loci on chromosomes 4A (Wx-B1), 7A (Wx-A1), and 7D (Wx-D1), and the band from the Wx-B1 locus is diagnostic for the occurrence of the null Wx-B1 allele that is associated with improved starch quality. The reliable detection of the null Wx-B1 allele has been important in identifying wheat breeding lines. Allele-specific PCR has also been used to successfully detect the occurrence of the null Wx-B1 allele. In the present paper the various protocols were evaluated by testing a segregating double haploid population from a cross between Cranbrook and Halberd and the tests gave good agreement in different laboratories. The application of the DNA-based tests applied in wheat breeding programs provides one of the first examples of a molecular marker selection for a grain quality trait being successfully applied in an Australian wheat breeding program.