Phenotyping for resistance to pre-harvest sprouting in grain sorghum

Pre-harvest sprouting (PHS) is a common threat to cereal crops in which the grain maturation phase takes place under rainy, moist conditions. Susceptibility to PHS is higher in sorghum genotypes displaying low levels of seed dormancy before harvest maturity. Other attributes such as glume or panicle morphology may also affect susceptibility to PHS. Breeding for resistance to PHS in grain sorghum requires the identification of grain physiological and morphological attributes affecting this trait, and a protocol for phenotyping and rating genotypes according to their susceptibility to PHS. In this work, we tested germination under laboratory conditions using detached grains and intact panicles for a panel of 20 sorghum genotypes including 11 parental lines, 6 hybrids and 3 reference inbred lines with contrasting PHS response. Records for natural sprouting in the field for these genotypes were also included in the analysis. Multivariate analyses of germination data allowed separation of genotypes into two major categories (resistant and susceptible to PHS). Laboratory germination data correlated significantly with PHS in the field. In most genotypes, the glumes had a significant, inhibitory effect on germination. The low levels of grain dormancy were observed among high tannin backgrounds, and vice versa, indicating that a pigmented testa alone does not provide resistance to PHS. Altogether, the phenotyping protocol allowed the classification of sorghum genotypes according to their susceptibility to PHS and the identification of different attributes useful for breeding for PHS resistance in this crop.

Evaluation of the impact of heat on wheat dormancy, late maturity α-amylase and grain size under controlled conditions in diverse germplasm

In the Australian wheat belts, short episodes of high temperatures or hot spells during grain filling are becoming increasingly common and have an enormous impact on yield and quality, bringing multi-billion losses annually. This problem will become recurrent under the climate change scenario that forecast increasing extreme temperatures, but so far, no systematic analysis of the resistance to hot spells has yet been performed in a diverse genetic background. We developed a protocol to study the effects of heat on three important traits: grain size, grain dormancy and the presence of Late Maturity α-Amylase (LMA), and we validated it by analysing the phenotypes of 28 genetically diverse wheat landraces and exploring the potential variability existing in the responses to hot spells. Using controlled growth environments, the different genotypes were grown in our standard conditions until 20 days after anthesis, and then moved for 10 days into a heat chamber. Our study showed that our elevated temperature treatment during mid-late filling triggered multiple detrimental effects on yield and quality. We observed a reduction in grain size, a reduction in grain dormancy and increased LMA expression in most of the tested genotypes, but potential resistant lines were identified for each analyzed trait opening new perspectives for future genetic studies and breeding for heat-insensitive commercial lines.

Exome sequencing of bulked segregants identified a novel TaMKK3-A allele linked to the wheat ERA8 ABA-hypersensitive germination phenotype

Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat ‘Zak’. ERA8 was mapped to chromosome 4A in a Zak/‘ZakERA8’ backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA hypersensitive phenotype was strongly linked to a missense mutation TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an ‘Otis’/ZakERA8 but not in a ‘Louise’/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660S allele as Zak, whereas Louise carries the PHS tolerant MKK3-A-C660R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.Key MessageUsing bulked segregant analysis of exome sequence, we fine-mapped the ABA hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A.

A role for PM19-Like 1 in seed dormancy in Arabidopsis

The understanding of the genetic basis of grain dormancy in wheat has rapidly improved in the last few years, and a number of genes have been identified related to that trait. We recently identified the wheat genes TaPM19-A1 and -A2 and we have now taken the first step towards understanding the role of this class of genes in seeds. By investigating the Arabidopsis homologous PM19-Like 1 (PM19L1) we have found that it has a seed-specific expression pattern and, while its expression is higher in dormant than in non-dormant seeds, knock-out mutations produced seeds with increased dormancy. Not only primary dormancy, but also secondary dormancy in response to high temperature was increased by the loss-of-function. We have also examined the function of PM19L1 by localizing the PM19 protein primarily to the cotyledon cells in seeds, possibly in membranes. By investigating the co-expression network of this gene we have found that it is connected to a small group of abscisic acid (ABA)-induced seed maturation and storage-related genes. The function of PM19L1 represents a good opportunity to explore the interactions of key factors that can influence seed dormancy such as ABA, temperature and membrane properties.

Comparative Phosphoproteomic Analysis of Barley Embryos with Different Dormancy during Imbibition

Dormancy is the mechanism that allows seeds to become temporally quiescent in order to select the right time and place to germinate. Like in other species, in barley, grain dormancy is gradually reduced during after-ripening. Phosphosignaling networks in barley grains were investigated by a large-scale analysis of phosphoproteins to examine potential changes in response pathways to after-ripening. We used freshly harvested (FH) and after-ripened (AR) barley grains which showed different dormancy levels. The LC-MS/MS analysis identified 2346 phosphopeptides in barley embryos, with 269 and 97 of them being up- or downregulated during imbibition, respectively. A number of phosphopeptides were differentially regulated between FH and AR samples, suggesting that phosphoproteomic profiles were quite different between FH and AR grains. Motif analysis suggested multiple protein kinases including SnRK2 and MAPK could be involved in such a difference between FH and AR samples. Taken together, our results revealed phosphosignaling pathways in barley grains during the water imbibition process.