Andrew Milgate

Molecular diversity and genetic structure of modern and traditional landrace cultivars of wheat (Triticum aestivum L.)

Wheatisoneofthemostimportantcerealcropsoftheworld.Inordertoachievecontinuedgeneticgaininwheat improvement programs, an assessment and utilisation of genetic diversity in a wide range of germplasm are required. The Australian Winter Cereal Collection (AWCC, Tamworth) holds over 33 000 accessions of wheat. In this study, we scanned the genome of 1057 accessions of hexaploid common wheat (Triticum aestivum L.) originating from different geographic regions of the world, with 178 polymorphic DArT markers. These accessions comprised modern cultivars (MCs), advanced breeding lines (BLs), and landrace cultivars (LCs). Our results indicate that the LCs had higher polymorphic informationcontent(PICvalues)thantheMCsandBLs.Clusterandprincipalcoordinateanalysisbasedongeneticdistance matrices enabled classification of the 1057 accessions into 12 subgroups. The structure of subgroups appeared to be geographically determined and was generally consistent with pedigrees. Molecular analyses revealed that LCs have unique alleles compared with MCs and BLs, which may be useful for the genetic improvement of wheat.

Durum wheat quality in high-input irrigation systems in south-eastern Australia

Abstract. To extend the production base of durum wheat in Australia, field trials were conducted on seven registered durum varieties across four seasons and six sites in locations where irrigation was supplied during crop growth. The purpose was to determine if the quality of the grain produced met the requirements for good milling and pasta-making quality and to understand the genotype, environment and their interaction in affecting yield and technological quality of the grain and derived pasta. High grain yields and grain protein were obtained, producing large grain weights, low screenings and low percentage of hard vitreous kernels. Yellow colour of semolina and pasta was reduced marginally but dough and other pasta technological characteristics were similar to typical dryland durum production, with some exceptions. Varieties were identified with potential for production under irrigation.

The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana

Bipolaris sorokiniana is the causal agent of multiple diseases on wheat and barley and is the primary constraint to cereal production throughout South Asia. Despite its significance, the molecular basis of disease is poorly understood. To address this, the genomes of three Australian isolates of B. sorokiniana were sequenced and screened for known pathogenicity genes. Sequence analysis revealed that the isolate BRIP10943 harboured the ToxA gene, which has been associated previously with disease in the wheat pathogens Parastagonospora nodorum and Pyrenophora tritici-repentis. Analysis of the regions flanking ToxA within B. sorokiniana revealed that it was embedded within a 12-kb genomic element nearly identical to the corresponding regions in P. nodorum and P. tritici-repentis. A screen of 35 Australian B. sorokiniana isolates confirmed that ToxA was present in 12 isolates. Sequencing of the ToxA genes within these isolates revealed two haplotypes, which differed by a single non-synonymous nucleotide substitution. Pathogenicity assays showed that a B. sorokiniana isolate harbouring ToxA was more virulent on wheat lines that contained the sensitivity gene when compared with a non-ToxA isolate. This work demonstrates that proteins that confer host-specific virulence can be horizontally acquired across multiple species. This acquisition can dramatically increase the virulence of pathogenic strains on susceptible cultivars, which, in an agricultural setting, can have devastating economic and social impacts.

Indirect selection using reference and probe genotype performance in multi-environment trials

There is a substantial challenge in identifying appropriate cultivars from databases for introduction into a breedingprogram.Weproposeanindirectselectionprocedurethatillustrateshowstrategicallydesignedmulti-environment trials, linked to historical performance databases, can identify germplasm to meet objectives of plant breeding programs. Two strategies for indirect selection of germplasm from the International Wheat and Maize Improvement Centers (CIMMYT) trial database were developed based on reference and probe genotype sets included in the International Adaptation Trial (IAT). The IAT was designed to improve the understanding of relationships among global spring wheat (Triticumspp.)locations.Grainyield(t/ha)datawerecollatedfrom183IATtrialsgrownin40countries(includingAustralia) between 2001 and 2004. ThereferencegenotypesetstrategyusedthegeneticcorrelationsamonglocationsintheIATtoidentifylocationssimilar to a target environment. For a key southern Australian breeding location, Roseworthy, the number of cultivars targeted for selection was reduced to 35% of the original 1252. The Irrigated Winter Cereals Trials (2008-09) aimed to identify highyieldpotentiallinesinsouth-easternAustralianirrigatedenvironments.Thirty-fiveCIMMYTcultivarsidentifiedusing the reference genotype selection strategy were grown in this trial series. In all trials, the proportion of CIMMYT cultivars in the top 20% yielding lines exceeded the expected proportion, 0.20. The probe genotype strategy utilised contrasting line yield responses to assess the occurrence of soil-borne stresses such as root lesion nematode (Pratylenchus thorneii) and boron toxicity. For these stresses, the number of targeted cultivars was reduced to 25% and 83% of the original 1252, respectively.

Utilizing Gene Tree Variation to Identify Candidate Effector Genes in Zymoseptoria tritici

Zymoseptoria tritici is a host-specific, necrotrophic pathogen of wheat. Infection by Z. tritici is characterized by its extended latent period, which typically lasts 2 wks, and is followed by extensive host cell death, and rapid proliferation of fungal biomass. This work characterizes the level of genomic variation in 13 isolates, for which we have measured virulence on 11 wheat cultivars with differential resistance genes. Between the reference isolate, IPO323, and the 13 Australian isolates we identified over 800,000 single nucleotide polymorphisms, of which ∼10% had an effect on the coding regions of the genome. Furthermore, we identified over 1700 probable presence/absence polymorphisms in genes across the Australian isolates using de novo assembly. Finally, we developed a gene tree sorting method that quickly identifies groups of isolates within a single gene alignment whose sequence haplotypes correspond with virulence scores on a single wheat cultivar. Using this method, we have identified < 100 candidate effector genes whose gene sequence correlates with virulence toward a wheat cultivar carrying a major resistance gene.

Next-generation re-sequencing as a tool for rapid bioinformatic screening of presence and absence of genes and accessory chromosomes across isolates of Zymoseptoria tritici

The wheat pathogen Zymoseptoria tritici possesses a large number of accessory chromosomes that may be present or absent in its genome. The genome of the reference isolate IPO323 has been assembled to a very high standard and contains 21 full length chromosome sequences, 8 of which represent accessory chromosomes. The IPO323 reference, when combined with low-cost next-generation sequencing and bioinformatics, can be used as a powerful tool to assess the presence or absence of accessory chromosomes. We present an outline of a range of bioinformatics techniques that can be applied to the analysis of presence-absence variation among accessory chromosomes across 13 novel isolates of Z. tritici.

Genome-wide associations for water-soluble carbohydrate concentration and relative maturity in wheat using SNP and DArT marker arrays

Improving water-use efficiency by incorporating drought avoidance traits into new wheat varieties is an important objective for wheat breeding in water-limited environments. This study uses genome wide association studies (GWAS) to identify candidate loci for water-soluble carbohydrate accumulation—an important drought-avoidance characteristic in wheat. Phenotypes from a multi-environment trial with experiments differing in water availability and separate single nucleotide polymorphism (SNP) and diversity arrays technology (DArT) marker sets were used to perform the analyses. Significant associations for water-soluble carbohydrate accumulation were identified on chromosomes 1A, 1B, 1D, 2D, and 4A. Notably, these loci did not collocate with the major loci identified for relative maturity. Loci on chromosome 1D collocated with markers previously associated with the high molecular weight glutenin Glu-D1 locus. Genetic × environmental interactions impacted the results strongly, with significant associations for carbohydrate accumulation identified only in the water-deficit experiments. The markers associated with carbohydrate accumulation may be useful for marker-assisted selection of drought tolerance in wheat.

First Report of Resistance to DMI Fungicides in Australian Populations of the Wheat Pathogen Zymoseptoria tritici

The erosion of demethylation inhibitor (DMI) fungicides effectiveness over time in Europe has been attributed to mutation sites in the 14α-demethylase encoded by the nuclear gene CYP51 (Cools and Fraaije 2013). These mutations first appeared in Europe in the 1990s, became widespread over the next 20 years, and were also recently reported in North America (Estep et al. 2015; Lucas et al. 2015). Zymoseptoria tritici has been present and causing disease on wheat (Triticum aestivum) in Australia for many decades and the use of fungicides for its control has become more common over the past 15 years. However, no significant changes in the field performance of fungicides have been noted by growers. To investigate possible undetected changes, we sequenced the CYP51 gene of 18 isolates cultured from wheat leaves collected in a commercial field on 1 July 2012 at Inverleigh, Victoria (–38.086743° S; 143.935783° E), and 3 isolates cultured from wheat leaves collected from trial plots on 22 August 2002 at Wagga Wagga,NSW (–35.044544° S; 147.316318° E). The nucleotide sequence of Z. tritici strain ST1 eburicol 14 alpha-demethylase (CYP51) gene (GenBank Accession No. AY730587.1) was used to design a set of three forward and three reverse sequencing primers across the known mutations. The sequence reads were assembled into contigs and checked for complete sequence. To generate an alignment of mature protein sequences, the mature protein sequence of ST1 (CYP51) gene was downloaded from NCBI to use as a reference. CYP51 nucleotide sequences were translated into amino acid sequences using all six possible frame shifts. These were aligned to the mature protein sequence of ST1 (CYP51) to identify the correct frame-shift sequence, enable the removal of intron sequences, and finally generate an amino acid alignment. Nucleotide and amino acid sequences are deposited in GenBank with Accession Nos. KT201543 to KT201563. Sixteen of the isolates from Victoria were carrying the Y137F mutation, one isolate carried the L50S-Y461S mutation, and one the L50S-S188N-N513K. The three isolates from NSW collected in 2002 contained no mutations compared with the reference accession. Fungicide sensitivities for propiconazole were determined at 50% effective concentrations (EC50) using rates of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 mg/liter, and the resistance factor (RF) of each isolate was calculated as fold change in the EC50 compared with that of the wild-type strains as per Cools et al. (2011), four replicates per isolate were performed. Results of the phenotypic assay of a subset of nine isolates (four Y137F mutants, one L50S-Y461S, one L50S-S188N-N513K, and three Wild-type) confirmed the elevated EC50 values for the mutations known to cause reduced sensitivity. The Y137F and L50S-Y461S mutants showed EC50 values of 1.08 and 1.03 mg/liter and RF levels in the range of 3.07 and 2.93. respectively, while the wild-type and L50S-S188N-N513K mutant had EC50 of 0.352 and 0.367 mg/liter, respectively. The EC50 values observed for the isolates appear higher than those found by Cools et al. (2011); however, the RF values are lower for both mutations. Further exploration of CYP51 mutations occurring in Z. tritici within the Australian cropping regions will be necessary to establish how widespread the Y137F and L50S-Y461S mutants are, and if others such as the S524T or the V136A, which are associated with higher levels of resistance to DMIs, are also present.

Genetic improvement of triticale for irrigated systems in south-eastern Australia: a study of genotype and genotype × environment interactions

Abstract. Research into winter cereal breeding in Australia has focused primarily on studying the effects of rainfed environments. These studies typically show large genotype × environment (GE) interactions, and the complexity of these interactions acts as an impediment to the efficient selection of improved varieties. Wheat has been studied extensively; however, there are no published studies on the GE interactions of triticale in Australia under irrigated production systems. We conducted trials on 101 triticale genotypes at two locations over 4 years under intensive irrigated management practices and measured the yield potential, GE interactions, heritability and estimated genetic gain of yield, lodging resistance and several other traits important for breeding triticale. We found that high yield potential exceeding 10 t ha–1 exists in the Australian germplasm tested and that, in these irrigated trials, genotype accounted for a high proportion of the variability in all measured traits. All genetic parameters such as heritability and estimated genetic gain were high compared with rainfed studies. Breeding of triticale with improved yield and lodging resistance for irrigated environments is achievable and can be pursued with confidence in breeding programs.

Accounting for Genotype-by-Environment Interactions and Residual Genetic Variation in Genomic Selection for Water-Soluble Carbohydrate Concentration in Wheat

Abiotic stress tolerance traits are often complex and recalcitrant targets for conventional breeding improvement in many crop species. This study evaluated the potential of genomic selection to predict water-soluble carbohydrate concentration (WSCC), an important drought tolerance trait, in wheat under field conditions. A panel of 358 varieties and breeding lines constrained for maturity was evaluated under rainfed and irrigated treatments across two locations and two years. Whole-genome marker profiles and factor analytic mixed models were used to generate genomic estimated breeding values (GEBVs) for specific environments and environment groups. Additive genetic variance was smaller than residual genetic variance for WSCC, such that genotypic values were dominated by residual genetic effects rather than additive breeding values. As a result, GEBVs were not accurate predictors of genotypic values of the extant lines, but GEBVs should be reliable selection criteria to choose parents for intermating to produce new populations. The accuracy of GEBVs for untested lines was sufficient to increase predicted genetic gain from genomic selection per unit time compared to phenotypic selection if the breeding cycle is reduced by half by the use of GEBVs in off-season generations. Further, genomic prediction accuracy depended on having phenotypic data from environments with strong correlations with target production environments to build prediction models. By combining high-density marker genotypes, stress-managed field evaluations, and mixed models that model simultaneously covariances among genotypes and covariances of complex trait performance between pairs of environments, we were able to train models with good accuracy to facilitate genetic gain from genomic selection.