M. K. Morell

Genetic characterisation of dough rheological properties in a wheat doubled haploid population: additive genetic effects and epistatic interactions

Doubled haploid lines (n=160) from a cross between wheat cultivars ‘Cranbrook’ (high dough extensibility) and ‘Halberd’ (low dough extensibility) were grown at three Australian locations. The parents differ at all high- and low-molecular-weight glutenin loci. Dough rheological parameters were measured using small-scale testing procedures, and quantitative trait locus (QTL) mapping procedures were carried out using an existing well-saturated genetic linkage map for this cross. Genetic parameters were estimated using three software packages: QTLCartographer, Epistat and Genstat. Results indicated that environmental factors are a major determinant of dough extensibility across the three trial sites, whereas genotypic factors are the major determinants of dough strength. Composite interval mapping analysis across the 21 linkage groups revealed that as expected, the main additive QTLs for dough rheological properties are located at the high- and low-molecular-weight glutenin loci. A new QTL on chromosome 5A for M-extensibility (a mixograph-estimated measure of extensibility) was detected. Analysis of epistatic interactions revealed that there were significant conditional epistatic interactions related with the additive effects of glutenin loci on dough rheological properties. Therefore, the additive genetic effects of glutenins on dough rheological properties are conditional upon the genetic background of the wheat line. The molecular basis of the interactions with the glutenin loci may be via proteins that modify or alter the gluten protein matrix or variations in the expression level of the glutenin genes. Reverse-phase high performance liquid chromatography analysis of the molar number of individual glutenin subunits across the population showed that certain conditional epistases resulted in increased expression of the affected glutenin. The epistatic interactions detected in this study provide a possible explanation of the variable genetic effects of some glutenins on quality attributes in different genetic backgrounds and provide essential information for the accurate prediction of glutenin related variance in marker-assisted wheat breeding.

Cloning and characterization of a gene encoding wheat starch synthase I

Abstract A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene.

The low-molecular-weight glutenin subunit proteins of primitive wheats. III. The genes from D-genome species

Abstract Three accessions of T. boeoticum were selected for the cloning and sequencing of novel low-molecular-weight glutenin subunit (LMW-GS) genes, based on the results of SDS-PAGE and PCR analyses of the LMW-GS diversity in A-genome wheat (Lee et al. 1998 a). A comparison of the nucleotide and deduced amino-acid sequences of three cloned genes, LMWG-E2, LMWG-E4 and LMWG-AQ1, both to each other and to other known LMW-GS genes was carried out. The N-terminal domains showed one variable position; GAG (coding for a glutamic acid) for the E-type, and GAT (coding for an aspartic acid) for the Q-type. The comparisons of the LMW-GSs in the literature and this paper define three different types of N-terminal sequences; METSCIPGLERPW and MDTSCIPGLERPW from the durum and A-genome wheats, and METRCIPGLERPW from the hexaploid and D-genome wheats. The repetitive domains were AC-rich at the nucleotide level and coded for a large number of glutamine residues; this region showed 16 variable positions changing 12 amino-acid residues, three triple nucleotide deletions/additions, a large deletion of 18 nucleotides in LMWG-E4 and a deletion of 12 nucleotides in LMWG-E2. In the C-terminal domains 26 variable positions were found and 12 of these mutations changed amino-acid residues; no deletions/ additions were present in this region. It was shown that the LMWG-E2 and LMWG-E4 genes could be expressed in bacteria and this allowed the respective protein products to be related back to the proteins defined as LMW-GSs in vivo.

Detection and analysis systems for microsatellite markers in wheat

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.

Characterisation of a gene encoding wheat endosperm starch branching enzyme-I

Abstract A genomic DNA fragment from Triticum tauschii, the donor of the wheat D genome, contains a starch branching enzyme-I (SBE-I) gene spread over 6.5 kb. This gene (designated wSBE I-D4) encodes an amino acid sequence identical to that determined for the N-terminus of SBE-I from the hexaploid wheat (T. aestivum) endosperm. Cognate cDNA sequences for wSBE I-D4 were isolated from hexaploid wheat by hybridisation screening from an endosperm library and also by PCR. A contiguous sequence (D4 cDNA) was assembled from the sequence of five overlapping partial cDNAs which spanned wSBE I-D4. D4 cDNA encodes a mature polypeptide of 87 kDa that shows 90% identity to SBE-I amino acid sequences from rice and maize and contains all the residues considered essential for activity. D4 mRNA has been detected only in the endosperm and is at a maximum concentration mid-way through grain development. The wSBE I-D4 gene consists of 14 exons, similar to the structure for the equivalent gene in rice; the rice gene has a strikingly longer intron 2. The 3′ end of wSBE I-D4 was used to show that the gene is located on group 7 chromosomes. The sequence upstream of wSBE I-D4 was analysed with respect to conserved motifs.

Development of robust PCR-based DNA markers for each homoeo-allele of granule-bound starch synthase and their application in wheat breeding programs

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 DNAbased 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.

The low-molecular-weight glutenin subunit proteins of primitive wheats. IV. Functional properties of products from individual genes

Abstract Three genes encoding the low-molecular-weight glutenin subunits (LMW-GSs), LMWG-E2 and LMWG-E4, from A-genome diploid wheat species, and LMW-16/10 from a D-genome diploid wheat, were expressed in bacteria. The respective proteins were produced on a relatively large scale and compared with respect to their effects on flour-processing properties such as dough mixing, extensibility and maximum resistance; these are important features in the end-use of wheat for producing food products. The LMWG-E2 and LMWG-E4 proteins caused significant increases in peak resistance and mixing time, compared to the control, when incorporated into dough preparations. The LMWG-16/10 protein was qualitatively less effective in producing these changes. All three proteins also conferred varying degrees of decrease in dough breakdown. LMWG-E2 and LMWG-E4 caused significant increases in dough extensibility, and decreases in maximum resistance, relative to the control. LMW-16/10 did not show a significant effect on extensibility but showed a significant decrease in maximum resistance. The refinement of relating specific features of the structure of the LMW-GS genes to the functional properties of their respective proteins is discussed.

Genetic mapping of commercially significant starch characteristics in wheat crosses

Starch properties were measured on the doubled haploid progeny of 2 crosses, one between Cranbrook and Halberd and the other between CD87 and Katepwa. Properties studied included starch peak and final viscosity measured by Rapid Visco Analyser, starch granule size distribution measured by laser light scattering, starch gelatinisation temperature by differential scanning calorimetry, and flour swelling volume. In the Cranbrook × Halberd cross (samples from 2 environments), a highly significant quantitative trait locus (QTL) was located on chromosome 4A for both starch peak viscosity and starch/flour swelling volume, centred around the Wx-B1 locus. In previous studies, this locus has been found to be linked to Japanese noodle quality. The increases in starch peak viscosity and flour swelling volume are derived from the Halberd parent, consistent with the fact that Halberd is null for the Wx-B1 locus on chromosome 4A and is missing the respective granule-bound starch synthase protein, whereas Cranbrook is a wheat line carrying the normal 3 Wx loci. The final starch viscosity also showed an association with the Wx-B1 locus. In the CD87 × Katepwa cross, the progeny showed an association between peak viscosity and a marker on chromosome 7A. This appeared to be near the Wx-A1 locus. In some experiments, flour viscosity showed a highly significant QTL on chromosome 7B, apparently at the same locus as the late maturity α- amylase derived from the Cranbrook parent. Starch gelatinisation onset temperature indicated a significant QTL on chromosomes 2B and 7A (LOD = 2.58 and 3.66, respectively, in interval analyses). Starch gelatinisation peak temperatures indicated a QTL on chromosome 7A, which, although not in the significant (P = 0.05) class based on regression analyses, indicated a LOD score of 3.06 in interval analyses. Heat of gelatinisation (∆H) indicated a suggestive QTL (LRS = 14.5 with a threshold of 14.7 for P < 0.05, LOD = 2.65 for interval analysis), on chromosome 4A, at the Wx-B1 locus with an increased effect coming from the Halberd parent. The A:B granule ratio analysis indicated a significant QTL on chromosome 4B, but this was not observed in all environments and may be due to the fact that the QTL corresponded to the position of a major QTL controlling plant growth. Additional keywords: viscosity, gelatinisation, granule size, genetics, quantitative traits. I M S. h C. K. . A Genetic mapping ofstarch characteristics

Microsatellites as markers for Australian Wheat improvement

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.

Wheat starch biosynthesis

Starch biosynthesis in plants involves the concerted action of a numberof enzymes, including ADPglucose pyrophosphorylase, starch synthases,branching enzymes and debranching enzymes. We report on the cloningand characterisation of genes encoding these enzymes from wheat and ontheir chromosomal locations. The prospects for manipulating wheat starchstructure and functionality using these genes is discussed.