Haydn Kuchel

Genetic and genomic tools to improve drought tolerance in wheat

Tolerance to drought is a quantitative trait, with a complex phenotype, often confounded by plant phenology. Breeding for drought tolerance is further complicated since several types of abiotic stress, such as high temperatures, high irradiance, and nutrient toxicities or deficiencies can challenge crop plants simultaneously. Although marker-assisted selection is now widely deployed in wheat, it has not contributed significantly to cultivar improvement for adaptation to low-yielding environments and breeding has relied largely on direct phenotypic selection for improved performance in these difficult environments. The limited success of the physiological and molecular breeding approaches now suggests that a careful rethink is needed of our strategies in order to understand better and breed for drought tolerance. A research programme for increasing drought tolerance of wheat should tackle the problem in a multi-disciplinary approach, considering interaction between multiple stresses and plant phenology, and integrating the physiological dissection of drought-tolerance traits and the genetic and genomics tools, such as quantitative trait loci (QTL), microarrays, and transgenic crops. In this paper, recent advances in the genetics and genomics of drought tolerance in wheat and barley are reviewed and used as a base for revisiting approaches to analyse drought tolerance in wheat. A strategy is then described where a specific environment is targeted and appropriate germplasm adapted to the chosen environment is selected, based on extensive definition of the morpho-physiological and molecular mechanisms of tolerance of the parents. This information was used to create structured populations and develop models for QTL analysis and positional cloning.

Genetic dissection of grain yield in bread wheat. I. QTL analysis

Grain yield forms one of the key economic drivers behind a successful wheat (Triticum aestivum L.) cropping enterprise and is consequently a major target for wheat breeding programmes. However, due to its complex nature, little is known regarding the genetic control of grain yield. A doubled-haploid population, comprising 182 individuals, produced from a cross between two cultivars ‘Trident’ and ‘Molineux’, was used to construct a linkage map based largely on microsatellite molecular makers. ‘Trident’ represents a lineage of wheat varieties from southern Australia that has achieved consistently high relative grain yield across a range of environments. In comparison, ‘Molineux’ would be rated as a variety with low to moderate grain yield. The doubled-haploid population was grown from 2002 to 2005 in replicated field experiments at a range of environments across the southern Australian wheat belt. In total, grain yield data were recorded for the population at 18 site-year combinations. Grain yield components were also measured at three of these environments. Many loci previously found to be involved in the control of plant height, rust resistance and ear-emergence were found to influence grain yield and grain yield components in this population. An additional nine QTL, apparently unrelated to these traits, were also associated with grain yield. A QTL associated with grain yield on chromosome 1B, with no significant relationship with plant height, ear-emergence or rust resistance, was detected (LOD ≥2) at eight of the 18 environments. The mean yield, across 18 environments, of individuals carrying the ‘Molineux’ allele at the 1B locus was 4.8% higher than the mean grain yield of those lines carrying the ‘Trident’ allele at this locus. Another QTL identified on chromosome 4D was also associated with overall gain yield at six of the 18 environments. Of the nine grain yield QTL not shown to be associated with plant height, phenology or rust resistance, two were located near QTL associated with grain yield components. A third QTL, associated with grain yield components at each of the environments used for testing, was located on chromosome 7D. However, this QTL was not associated with grain yield at any of the environments. The implications of these findings on marker-assisted selection for grain yield are discussed.

The genetic control of milling yield, dough rheology and baking quality of wheat

Improving the end-use quality of wheat is a key target for many breeding programmes. With the exception of the relationship between glutenin alleles and some dough rheological characters, knowledge concerning the genetic control of wheat quality traits is somewhat limited. A doubled haploid population produced from a cross between two Australian cultivars ‘Trident’ and ‘Molineux’ has been used to construct a linkage map based largely on microsatellite molecular makers. ‘Molineux’ is superior to ‘Trident’ for a number of milling, dough rheology and baking quality characteristics, although by international standards ‘Trident’ would still be regarded as possessing moderately good end-use quality. This population was therefore deemed useful for investigation of wheat end-use quality. A number of significant QTL identified for dough rheological traits mapped to HMW and LMW glutenin loci on chromosomes 1A and 1B. However, QTL associated with dough strength and loaf volume were also identified on chromosome 2A and a significant QTL associated with loaf volume and crumb quality was identified on chromosome 3A. A QTL for flour protein content and milling yield was identified on chromosome 6A and a QTL associated with flour colour reported previously on chromosome 7B was confirmed in this population. The detection of loci affecting dough strength, loaf volume and flour protein content may provide fresh opportunities for the application of marker-assisted selection to improve bread-making quality.

Joint modeling of additive and non-additive genetic line effects in single field trials

A statistical approach is presented for selection of best performing lines for commercial release and best parents for future breeding programs from standard agronomic trials. The method involves the partitioning of the genetic effect of a line into additive and non-additive effects using pedigree based inter-line relationships, in a similar manner to that used in animal breeding. A difference is the ability to estimate non-additive effects. Line performance can be assessed by an overall genetic line effect with greater accuracy than when ignoring pedigree information and the additive effects are predicted breeding values. A generalized definition of heritability is developed to account for the complex models presented.

The successful application of a marker-assisted wheat breeding strategy

A number of useful marker-trait associations have been reported for wheat. However the number of publications detailing the integrated and pragmatic use of molecular markers in wheat breeding is limited. A previous report by some of these authors showed how marker-assisted selection could increase the genetic gain and economic efficiency of a specific breeding strategy. Here, we present a practical validation of that study. The target of this breeding strategy was to produce wheat lines derived from an elite Australian cultivar ‘Stylet’, with superior dough properties and durable rust resistance donated from ‘Annuello’. Molecular markers were used to screen a BC1F1 population produced from a cross between the recurrent parent ‘Stylet’ and the donor parent ‘Annuello’ for the presence of rust resistance genes Lr34/Yr18 and Lr46/Yr29. Following this, marker-assisted selection was applied to haploid plants, prior to chromosome doubling with cochicine, for the rust resistance genes Lr24/Sr24, Lr34/Yr18, height reducing genes, and for the grain protein genes Glu-D1 and Glu-A3. In general, results from this study agreed with those of the simulation study. Genetic improvement for rust resistance was greatest when marker selection was applied on BC1F1 individuals. Introgression of both the Lr34/Yr18 and Lr46/Yr29 loci into the susceptible recurrent parent background resulted in substantial improvement in leaf rust and stripe rust resistance levels. Selection for favourable glutenin alleles significantly improved dough resistance and dough extensibility. Marker-assisted selection for improved grain yield, through the selection of recurrent parent genome using anonymous markers, only marginally improved grain yield at one of the five sites used for grain yield assessment. In summary, the integration of marker-assisted selection for specific target genes, particularly at the early stages of a breeding programme, is likely to substantially increase genetic improvement in wheat.

Genetic and economic analysis of a targeted marker-assisted wheat breeding strategy

The advent of molecular markers as a tool to aid selection has provided plant breeders with the opportunity to rapidly deliver superior genetic solutions to problems in agricultural production systems. However, a major constraint to the implementation of marker-assisted selection (MAS) in pragmatic breeding programs in the past has been the perceived high relative cost of MAS compared to conventional phenotypic selection. In this paper, computer simulation was used to design a genetically effective and economically efficient marker-assisted breeding strategy aimed at a specific outcome. Under investigation was a strategy involving the integration of both restricted backcrossing and doubled haploid (DH) technology. The point at which molecular markers are applied in a selection strategy can be critical to the effectiveness and cost efficiency of that strategy. The application of molecular markers was considered at three phases in the strategy: allele enrichment in the BC1F1 population, gene selection at the haploid stage and the selection for recurrent parent background of DHs prior to field testing. Overall, incorporating MAS at all three stages was the most effective, in terms of delivering a high frequency of desired outcomes and at combining the selected favourable rust resistance, end use quality and grain yield alleles. However, when costs were included in the model the combination of MAS at the BC1F1 and haploid stage was identified as the optimal strategy. A detailed economic analysis showed that incorporation of marker selection at these two stages not only increased genetic gain over the phenotypic alternative but actually reduced the over all cost by 40%.

Quantification of the effects of VRN1 and Ppd-D1 to predict spring wheat (Triticum aestivum) heading time across diverse environments

Heading time is a major determinant of the adaptation of wheat to different environments, and is critical in minimizing risks of frost, heat, and drought on reproductive development. Given that major developmental genes are known in wheat, a process-based model, APSIM, was modified to incorporate gene effects into estimation of heading time, while minimizing degradation in the predictive capability of the model. Model parameters describing environment responses were replaced with functions of the number of winter and photoperiod (PPD)-sensitive alleles at the three VRN1 loci and the Ppd-D1 locus, respectively. Two years of vernalization and PPD trials of 210 lines (spring wheats) at a single location were used to estimate the effects of the VRN1 and Ppd-D1 alleles, with validation against 190 trials (~4400 observations) across the Australian wheatbelt. Compared with spring genotypes, winter genotypes for Vrn-A1 (i.e. with two winter alleles) had a delay of 76.8 degree days (°Cd) in time to heading, which was double the effect of the Vrn-B1 or Vrn-D1 winter genotypes. Of the three VRN1 loci, winter alleles at Vrn-B1 had the strongest interaction with PPD, delaying heading time by 99.0 °Cd under long days. The gene-based model had root mean square error of 3.2 and 4.3 d for calibration and validation datasets, respectively. Virtual genotypes were created to examine heading time in comparison with frost and heat events and showed that new longer-season varieties could be heading later (with potential increased yield) when sown early in season. This gene-based model allows breeders to consider how to target gene combinations to current and future production environments using parameters determined from a small set of phenotyping treatments.

Genetic dissection of grain yield and physical grain quality in bread wheat (Triticum aestivum L.) under water-limited environments

In the water-limited bread wheat production environment of southern Australia, large advances in grain yield have previously been achieved through the introduction and improved understanding of agronomic traits controlled by major genes, such as the semi-dwarf plant stature and photoperiod insensitivity. However, more recent yield increases have been achieved through incremental genetic advances, of which, breeders and researchers do not fully understand the underlying mechanism(s). A doubled haploid population was utilised, derived from a cross between RAC875, a relatively drought-tolerant breeders’ line and Kukri, a locally adapted variety more intolerant of drought. Experiments were performed in 16 environments over four seasons in southern Australia, to physiologically dissect grain yield and to detect quantitative trait loci (QTL) for these traits. Two stage multi-environment trial analysis identified three main clusters of experiments (forming distinctive environments, ENVs), each with a distinctive growing season rainfall patterns. Kernels per square metre were positively correlated with grain yield and influenced by kernels per spikelet, a measure of fertility. QTL analysis detected nine loci for grain yield across these ENVs, individually accounting for between 3 and 18% of genetic variance within their respective ENVs, with the RAC875 allele conferring increased grain yield at seven of these loci. These loci were partially dissected by the detection of co-located QTL for other traits, namely kernels per square metre. While most loci for grain yield have previously been reported, their deployment and effect within local germplasm are now better understood. A number of novel loci can be further exploited to aid breeders’ efforts in improving grain yield in the southern Australian environment.

Identification of novel quantitative trait loci for days to ear emergence and flag leaf glaucousness in a bread wheat (Triticum aestivum L.) population adapted to southern Australian conditions

In southern Australia, where the climate is predominantly Mediterranean, achieving the correct flowering time in bread wheat minimizes the impact of in-season cyclical and terminal drought. Flag leaf glaucousness has been hypothesized as an important component of drought tolerance but its value and genetic basis in locally adapted germplasm is unknown. From a cross between Kukri and RAC875, a doubled-haploid (DH) population was developed. A genetic linkage map consisting of 456 DArT and SSR markers was used to detect QTL affecting time to ear emergence and Zadoks growth score in seven field experiments. While ear emergence time was similar between the parents, there was significant transgressive segregation in the population. This was the result of segregation for the previously characterized Ppd-D1a and Ppd-B1 photoperiod responsive alleles. QTL of smaller effect were also detected on chromosomes 1A, 4A, 4B, 5A, 5B, 7A and 7B. A novel QTL for flag leaf glaucousness of large, repeatable effect was detected in six field experiments, on chromosome 3A (QW.aww-3A) and accounted for up to 52 percent of genetic variance for this trait. QW.aww-3A was validated under glasshouse conditions in a recombinant inbred line population from the same cross. The genetic basis of time to ear emergence in this population will aid breeders’ understanding of phenological adaptation to the local environment. Novel loci identified for flag leaf glaucousness and the wide phenotypic variation within the DH population offers considerable scope to investigate the impact and value of this trait for bread wheat production in southern Australia.

Identification of genetic loci associated with ear-emergence in bread wheat

A doubled haploid population constructed from a cross between the South Australian wheat cultivars ‘Trident’ and ‘Molineux’ was grown under winter field conditions, under field conditions over summer and under artificial light both with and without vernalisation. The duration from planting to ear-emergence was recorded and QTL associated with heading date were detected using a previously constructed genetic linkage map. Associations were shown with chromosomal regions syntenous to previously identified photoperiod (Ppd-B1) and vernalisation (Vrn-A1) sensitive loci. Additional QTL associated with time to heading were also identified on chromosomes 1A, 2A, 2B, 6D, 7A and 7B. Comparisons between the genetic associations observed under the different growing conditions allowed the majority of these loci to be classified as having either photoperiod-sensitive, vernalisation-sensitive or earliness per se actions. The identification of a photoperiod-sensitive QTL on chromosome 1A provides evidence for a wheat gene possibly homoeologous to Ppd-H2 previously identified on chromosome 1H of barley. The occurrence of a putative major gene for photoperiod sensitivity observed on chromosome 7A is presented. The combined additive effects at these loci accounted for more than half the phenotypic variance in the duration from planting to ear-emergence in this population. The possible role of these loci on the adaptation of wheat in Australia is discussed.