Cold tolerance in sorghum is largely stage- and environment-specific yet partially shared genetic control allows improvement across developmental stages with mostly modest, trait-specific trade-offs. Cold tolerance is a major constraint to sorghum production in temperate environments, and its improvement is therefore a key breeding objective. However, cold tolerance varies across developmental stages and environments, and selection based on stage-specific phenotypes may overlook shared genetic components. We analysed a diversity panel of 394 sorghum genotypes evaluated across 10 environments representing three developmental stages using multi-environment mixed models, genome-wide haplotype block analysis, and in silico haplotype stacking. Substantial genotype-by-environment interaction was detected, with the strength of GEI varying markedly across traits. Genetic correlations were high within developmental stages but lower and more variable across stages, indicating that the genetic architecture of cold tolerance-relevant traits is largely stage-dependent. Haplotype block analyses identified both stage- and environment-specific regions, as well as regions shared across stages and environments. In silico stacking of favourable haplotypes for early-stage cold tolerance resulted in nonlinear predicted gains, with strong initial responses at low stacking levels followed by diminishing returns. Correlated responses in yield-related traits were generally positive, whereas flowering time was consistently delayed, indicating a potential trait-specific trade-off in cold tolerance improvement. Sorghum cold tolerance can therefore not be treated as a single uniform trait but rather as a combination of stage-specific and shared genetic components, with implications for multi-stage breeding strategies.

The loss of yield due to cold stress during the early reproductive phase poses challenges to the expansion of sorghum cultivation into temperate regions. A better understanding of the physiological mechanisms is crucial for rapid progress in breeding cold-tolerant sorghum varieties. To identify the floral phytohormones responsible for reproductive cold tolerance, a cold-tolerant and a cold-sensitive genotype were subjected to cold stress at various developmental stages during the early reproductive phase. In addition to abscisic acid and its derivatives, including abscisic acid glucose ester, dihydrophaseic acid, and phaseic acid, various gibberellins as well as jasmonic acid and its bioactive form jasmonic acid isoleucine were examined. We found that cold-tolerant sorghum is capable of downregulating abscisic acid concentration under cold stress. While existing literature primarily attributes increased abscisic acid concentration, combined with an insufficient pool of bioactive gibberellins, in sensitive plants as a result of abnormal pollen development, this study shows that this is not the case in sorghum. Additionally, an antagonistic interaction between gibberellins and jasmonic acid was observed regardless of genotype and environmental conditions. These findings contribute to a better understanding of the physiological mechanisms behind cold tolerance in sorghum and could provide important insights for future breeding efforts aiming to accelerate the expansion of cold-tolerant sorghum varieties into temperate climates.

The ability to withstand periods of light frost during juvenile development is an important adaptive trait for sorghum (Sorghum bicolor L. Moench) production in high-latitude temperate areas. Frost tolerance would allow for earlier sowing and prolongation of growing season and maturity, thereby increasing yield potential. Using a broad diversity set (n = 285), a field cold emergence trial and two semi-controlled frost survival experiments were conducted to investigate the phenotypic diversity and genetic architecture of frost survival traits. The results show high phenotypic diversity for frost survival, including superior genotypes combining high frost tolerance and cold emergence levels via quantitative trait inheritance. Tolerance to temperatures above and below 0 °C was not correlated, suggesting separate genetic control. The found QTL do not overlap for the traits of frost survival and plant emergence under cold sowing conditions. Genome-wide association studies revealed four significant associations for frost survival, where the QTLs are located on chromosomes Sb02, Sb07, and Sb08. For the trait of emergence under cold sowing conditions, a total of 12 QTLs were found on chromosomes Sb01, Sb03, Sb04, Sb05, and Sb06. A good overview regarding the suitability of the tested genotypes for early sowing was obtained due to the large diversity investigated in this study, with surprisingly high survival rates of numerous genotypes after frost stress. These results will contribute to the breeding of cultivars with improved cold and frost tolerance, facilitating better adaptation for production in high-latitude temperate areas, as well as earlier sowing to avoid summer drought.

The influence of cold stress during the reproductive phase can lead to substantial yield losses in sorghum. In order to extend cultivation into temperate regions, a better understanding of reproductive cold tolerance is essential for breeding progress. To further elucidate the mechanisms responsible for cold tolerance, a cold-tolerant and a cold-sensitive parental line, along with their reciprocal F1 hybrids, were subjected to cold stress at various stages of reproductive development, with a focus on pollen fertility and receptivity of female floral organs. For this purpose, pollen measurements were conducted using impedance flow cytometry, and the panicle harvest index was determined post-maturation. While existing literature primarily attributes reduced pollen fertility as the cause of decreased seed set, this study provides evidence that female floral organs might be more affected than previously assumed. We found that the onset of generative tissue formation until BBCH39 (flag leaf visible) is the most cold-sensitive developmental stage and that there is no predominance of maternal or paternal effects associated with the inheritance of cold tolerance in reciprocal F1 hybrids. These findings offer valuable insights for the development of cold-tolerant sorghum varieties to enable cultivation in colder regions and enhance yield stability in temperate climates. Further studies should aim at validating and expanding these findings from the limited number of representative genotypes analyzed in the present manuscript to global sorghum diversity.

Understanding sorghum response to water stress at different developmental stages is important for developing sorghum varieties with improved tolerance to drought. This study set out to finely characterize key agro-physiological responses and water use of sorghum subjected to different drought scenarios. A greenhouse experiment was conducted using a DroughtSpotter facility that enables real-time quantification of water use by gravimetric tracking. Four different water treatments were assessed: (i) early vegetative drought stress (DS1), (ii) DS1 plus recovery (DS1R), (iii) late vegetative drought stress (DS2), and (iv) well-watered (WW). Plant pheno-morphology and yield data were recorded. Leaf fluorescence and photosynthesis were continuously recorded throughout the experiment. Our results showed that green leaf number and plant leaf area were more affected by DS2 than DS1 and delayed plant flowering. Nevertheless, plants in DS2 were taller and produced higher stem and leaf biomass compared to DS1. No significant difference was recorded in grain yield between DS1 and DS2 but were outperformed by DS1R and WW. The transpiration and photosynthetic rates were shown to decrease at the same time as the stomatal conductance. This can therefore be assimilated to a stomatal down-regulation limiting CO2 uptake. However, the increase in intercellular CO2 concentration is likely to indicate the presence of CO2 in the substomatal cavity that was not conveyed to the carboxylation sites. This suggests a non-stomatal limitation of the photosynthesis. Moreover, the plants recovered quite well from DS1, and this was more prominent for physiological parameters than morphological ones. Globally, water use efficiency (WUE) for DS2 was higher compared to WW and DS1 treatments, confirming the growing point differentiation as a critical stage where drought stress should be avoided to ensure yield and better WUE. Adaptation responses were related to the reduction of transpiration through plant leaf area reduction, the reduction of stomatal conductance, and the increase of intercellular CO2 limiting photosynthesis. Further studies focusing on the biomarkers of stress and transcriptomic analyses are needed to provide further insight into the drought adaptation mechanisms of this line.

Abstract Background The Plant Genetic Resources Centre at the Uganda National Gene Bank houses has over 3000 genetically diverse landraces and wild relatives of Sorghum bicolor accessions. This genetic diversity resource is untapped, under-utilized, and has not been systematically incorporated into sorghum breeding programs. In this study, we characterized the germplasm collection using whole-genome SNP markers (DArTseq). Discriminant analysis of principal components (DAPC) was implemented to study the racial ancestry of the accessions in comparison to a global sorghum diversity set and characterize the sub-groups present in the Ugandan (UG) germplasm. Results Population structure and phylogenetic analysis revealed the presence of five subgroups among the Ugandan accessions. The samples from the highlands of the southwestern region were genetically distinct as compared to the rest of the population. This subset was predominated by the caudatum race and unique in comparison to the other sub-populations. In this study, we detected QTL for juvenile cold tolerance by genome-wide association studies (GWAS) resulting in the identification of 4 markers associated (−log10p > 3) to survival under cold stress under both field and climate chamber conditions, located on 3 chromosomes (02, 06, 09). To our best knowledge, the QTL on Sb09 with the strongest association was discovered for the first time. Conclusion This study demonstrates how genebank genomics can potentially facilitate effective and efficient usage of valuable, untapped germplasm collections for agronomic trait evaluation and subsequent allele mining. In face of adverse climate change, identification of genomic regions potentially involved in the adaptation of Ugandan sorghum accessions to cooler climatic conditions would be of interest for the expansion of sorghum production into temperate latitudes.

The National Genebank of Uganda houses diverse and rich Sorghum bicolor germplasm collection. This genetic diversity resource is untapped, under-utilized and has not been systematically incorporated into sorghum breeding programs. In this study, we characterized the germplasm collection using whole genome SNP markers. Discriminant analysis of principal components (DAPC) was implemented to study racial ancestry of the accessions in comparison to a global sorghum diversity set and characterize sub-groups and admixture in the Ugandan germplasm. Genetic structure and phylogenetic analysis was conducted to identify distinct genotypes in the Ugandan collection and relationships among groups. Furthermore, in a case study for identification of potentially useful adaptive trait variation for breeding, we performed genome-wide association studies for juvenile cold tolerance. Genomic regions potentially involved in adaptation of Ugandan sorghum varieties to cooler climatic conditions were identified that could be of interest for expansion of sorghum production into temperate latitudes. The study demonstrates how genebank genomics can potentially facilitate effective and efficient usage of valuable, untapped germplasm collections for agronomic trait evaluation and subsequent allele mining.

This chapter reviews germplasm sources for and new developments in the identification and implementation of useful genetic diversity for temperate climate adaptation, along with genomics-based methods for breeding of complex, low-heritability traits like abiotic stress tolerance in sorghum.

Abundance and diversity of pollinating insects are decreasing. Intensification of agricultural bioenergy production is presumed to accelerate the decline of pollinators. Sorghum (Sorghum bicolor L. Moench) is a promising bioenergy crop. Enhanced dual‐purpose type cultivars have been developed and tested for suitability for bioenergy cropping in Germany. Sorghum is assumed to be a nutritional resource for pollen‐collecting insects. To evaluate this assumption, we studied the foraging strategy of A. mellifera colonies, which were migrated to sorghum fields in Germany. The bee hives were equipped with bottom fixed pollen traps. The pollen loads of the colonies contained variable shares of sorghum pollen ranging between approx. 10% and 50% (weight/weight). Sorghum pollen occurred frequently in more than 50% of all pollen samples. Experimental mini colonies were placed in plots which were grown with two varieties of sorghum, phacelia (Phacelia tanacetifolia), maize (Zea mays) and a control plot without any vegetation. All plots were encased with flight tents. Significant effects of the crop were found for the productivity parameters brood rearing and pollen collection. The sorghum and maize variants performed significantly better than the controls but significantly poorer than phacelia (p < 0.05). The parameters number of dead bees and colony sizes were not affected by the crops (p < 0.05). Pollen of sorghum is a valuable food for bees which supports nursing of bee brood, but its availability proved to be inferior to phacelia as the pollen shedding of sorghum lasted a considerably shorter time. Pollen collection by honeybees did not negatively affect seed yield of sorghum in any case. Under unfavourable weather conditions, flower visiting bees enhanced seed yield of sorghum.

Enhancements in reproductive cold tolerance of sorghum are essential to expand growing areas into both high-latitude temperate areas and tropical high-altitude environments. Here we present first insights into the genetic architecture of this trait via genome-wide association studies in a broad genetic diversity set (n = 330) phenotyped in multi-location field trials including high-altitude tropical (Mexico) and high-latitude temperate (Germany) environments. We observed a high degree of phenotypic variation and identified several novel, temperate-adapted accessions with superior and environmentally stable cold tolerance. Good heritability indicates strong potential for implementation of reproductive cold tolerance in breeding. Although the trait was found to be strongly quantitative, promising genomic regions with multiple-trait associations were found, including hotspots on chromosomes 3 and 10 which contain candidate genes implicated in different developmental and survival processes under abiotic stress conditions.

Sorghum (Sorghum bicolor L. Moench) is a promising novel crop for Central Europe. However, enhancements in cold tolerance and early maturity are essential for a successful adaptation to cooler climates. We scored a broad sorghum diversity set (n = 338) for early chilling tolerance, high-latitude adaptation, and bioenergy related agronomical traits in multi-environment trials. Our results show a high phenotypic variation and medium to high heritabilities for most traits, indicating that a robust breeding progress is feasible. Several public accessions with a good adaptation to cooler climates were identified, which can serve as valuable base material for sorghum breeding in temperate areas. Genome-wide association studies reveal a polygenic (quantitative) character for most of the traits, confirming previous studies. Hence, for practical breeding, it will be difficult to conduct efficient marker-assisted selection for temperate-adaptation traits in genetically diverse material.

The sensitivity of sorghum to pre-flowering cold stress, resulting in reduced pollen viability and poor seed set, is a major constraint for expanding growing areas into higher altitudes and latitudes. Nevertheless, compared to juvenile cold tolerance, reproductive cold tolerance in sorghum has received much less attention so far, and very little is known about its inheritance in F-1-hybrids. We have composed a representative factorial (n = 49 experimental F-1-hybrids) for a comprehensive study on heterosis and combining ability for crucial tolerance traits as spikelet fertility (panicle harvest index), seed yield and pollen viability, using field trials in stress- and control environments in Germany and Mexico as well as climate chamber experiments. Our results indicate a heterotic and rather dominant inheritance of reproductive cold tolerance in sorghum, with strong effects of female general combining ability (GCA) on F-1-hybrid performance in our material. These findings, together with the comparatively low contribution of specific combining ability (SCA) effects and high heritability estimates, suggest that robust and efficient enhancement of reproductive cold tolerance is feasible via hybrid breeding.

Sorghum (Sorghum bicolor L. Moench) is regarded a drought-tolerant alternative to maize as a bioenergy and fodder crop, but its early-stage chilling sensitivity is obstructing a successful implementation in temperate areas. While several studies have identified quantitative trait loci (QTL) underlying chilling tolerance-related traits in sorghum lines, little is known about the inheritance of these traits in F-1 hybrids. We have conducted a comprehensive approach to analyse heterosis, combining ability and the relation between line per se and hybrid performance for emergence and early shoot and root development comprising both field trials and controlled environment experiments including chilling tests. To our best knowledge, this is the first study analysing heterosis for sorghum root parameters under chilling. Our results show that most traits are heterotic and that the mid-parent values are rather poor predictors of hybrid performance. Hybrid breeding programmes should focus on efficient GCA tests and the establishment of genetically diverse pools to maximise heterosis rather than on a too strict selection among lines based on their per se performance. The medium-to-high heritabilities estimated for seedling emergence and juvenile biomass suggest that a robust breeding progress for these complex traits is feasible.

Sorghum is regarded a promising bioenergy crop and may be an alternative to maize for biogas use in temperate Europe in the near future. Presently, the principal shortcoming compared to maize besides chilling sensitivity during juvenile development is that current sorghum varieties fail to combine a high dry matter yield with adequate dry matter content for silage and satisfying methane yield. Our major goal was to evaluate whether early-maturing silage type Sorghum bicolor hybrids with a higher contribution of panicles and grains to total dry matter may allow an improvement of methane yield, representing a novel, alternative variety type for biogas use in temperate Europe. Our results indicate that under adequate conditions, they reach higher dry matter contents. Due to enhanced methane yields per dry matter unit which offset slightly lower dry matter yields, their methane yield per area unit is similar or superior to existing biomass type varieties. Since panicles account for up to 50% of total dry matter yield, seed set and maturity in cool environments represent critical factors for yield stability. Breeding efforts focus on chilling tolerance during flowering to avoid male sterility and on the development of appropriate parental lines combining earliness and adequate height. While selection for maturity can be reliably conducted on per se performance of parental lines, hybrid yield is predominantly determined by general combining ability.

Sorghum is a promising alternative to maize for bioenergy production in Europe; however, its use is currently limited by poor adaptation to low temperatures during and after germination. We collected multi-trait phenotype data under optimal and suboptimal temperatures in a genetically diverse recombinant inbred line (RIL) mapping population showing contrasting segregation patterns for pre- and post-emergence chilling tolerance. Germination, emergence, seedling development, root architecture and seedling survival were assessed in two different seedlots. Emergence and root establishment were found to be the key determinants of development and survival under chilling stress. Highly interactive epistatic quantitative trait loci (QTL) hotspots, including a previously unknown QTL on Sb06 with a significant effect on prolonged chilling survival, were found to regulate different physiological mechanisms contributing to maintenance of growth and development despite the chilling temperatures. The major QTL regions harbour promising candidate genes with known roles in abiotic stress tolerance. Identification of loci in the QTL hotspot regions conferring maintenance of cell division and growth under early chilling stress represents a promising step towards breeding for successful establishment of sorghum in temperate climates.