Variation in mitogenome structural conformation in wild and cultivated lineages of sorghum corresponds with domestication history and plastome evolution.

BACKGROUND: Mitochondria are organelles within eukaryotic cells that are central to the metabolic processes of cellular respiration and ATP production. However, the evolution of mitochondrial genomes (mitogenomes) in plants is virtually unknown compared to animal mitogenomes or plant plastids, due to complex structural variation and long stretches of repetitive DNA making accurate genome assembly more challenging. Comparing the structural and sequence differences of organellar genomes within and between sorghum species is an essential step in understanding evolutionary processes such as organellar sequence transfer to the nuclear genome as well as improving agronomic traits in sorghum related to cellular metabolism. RESULTS: Here, we assembled seven sorghum mitochondrial and plastid genomes and resolved reticulated mitogenome structures with multilinked relationships that could be grouped into three structural conformations that differ in the content of repeats and genes by contig. The grouping of these mitogenome structural types reflects the two domestication events for sorghum in east and west Africa. CONCLUSIONS: We report seven mitogenomes of sorghum from different cultivars and wild sources. The assembly method used here will be helpful in resolving complex genomic structures in other plant species. Our findings give new insights into the structure of sorghum mitogenomes that provides an important foundation for future research into the improvement of sorghum traits related to cellular respiration, cytonuclear incompatibly, and disease resistance.

Genetic basis of sorghum leaf width and its potential as a surrogate for transpiration efficiency.

KEY MESSAGE: Leaf width was correlated with plant-level transpiration efficiency and associated with 19 QTL in sorghum, suggesting it could be a surrogate for transpiration efficiency in large breeding program. Enhancing plant transpiration efficiency (TE) by reducing transpiration without compromising photosynthesis and yield is a desirable selection target in crop improvement programs. While narrow individual leaf width has been correlated with greater intrinsic water use efficiency in C4 species, the extent to which this translates to greater plant TE has not been investigated. The aims of this study were to evaluate the correlation of leaf width with TE at the whole-plant scale and investigate the genetic control of leaf width in sorghum. Two lysimetry experiments using 16 genotypes varying for stomatal conductance and three field trials using a large sorghum diversity panel (n = 701 lines) were conducted. Negative associations of leaf width with plant TE were found in the lysimetry experiments, suggesting narrow leaves may result in reduced plant transpiration without trade-offs in biomass accumulation. A wide range in width of the largest leaf was found in the sorghum diversity panel with consistent ranking among sorghum races, suggesting that environmental adaptation may have a role in modifying leaf width. Nineteen QTL were identified by genome-wide association studies on leaf width adjusted for flowering time. The QTL identified showed high levels of correspondence with those in maize and rice, suggesting similarities in the genetic control of leaf width across cereals. Three a priori candidate genes for leaf width, previously found to regulate dorsoventrality, were identified based on a 1-cM threshold. This study provides useful physiological and genetic insights for potential manipulation of leaf width to improve plant adaptation to diverse environments.

Genetic control of leaf angle in sorghum and its effect on light interception.

Developing sorghum genotypes adapted to different light environments requires understanding of a plant's ability to capture light, determined through leaf angle specifically. This study dissected the genetic basis of leaf angle in 3 year field trials at two sites, using a sorghum diversity panel (729 accessions). A wide range of variation in leaf angle with medium heritability was observed. Leaf angle explained 36% variation in canopy light extinction coefficient, highlighting the extent to which variation in leaf angle influences light interception at the whole-canopy level. This study also found that the sorghum races of Guinea and Durra consistently having the largest and smallest leaf angle, respectively, highlighting the potential role of leaf angle in adaptation to distinct environments. The genome-wide association study detected 33 quantitative trait loci (QTLs) associated with leaf angle. Strong synteny was observed with previously detected leaf angle QTLs in maize (70%) and rice (40%) within 10 cM, among which the overlap was significantly enriched according to χ2 tests, suggesting a highly consistent genetic control in grasses. A priori leaf angle candidate genes identified in maize and rice were found to be enriched within a 1-cM window around the sorghum leaf angle QTLs. Additionally, protein domain analysis identified the WD40 protein domain as being enriched within a 1-cM window around the QTLs. These outcomes show that there is sufficient heritability and natural variation in the angle of upper leaves in sorghum which may be exploited to change light interception and optimize crop canopies for different contexts.

Manipulating assimilate availability provides insight into the genes controlling grain size in sorghum.

Variation in grain size, a major determinant of grain yield and quality in cereal crops, is determined by both the plant's genetic potential and the available assimilate to fill the grain in the absence of stress. This study investigated grain size variation in response to variation in assimilate supply in sorghum using a diversity panel (n = 837) and a backcross-nested association mapping population (n = 1421) across four experiments. To explore the effects of genetic potential and assimilate availability on grain size, the top half of selected panicles was removed at anthesis. Results showed substantial variation in five grain size parameters with high heritability. Artificial reduction in grain number resulted in a general increase in grain weight, with the extent of the increase varying across genotypes. Genome-wide association studies identified 44 grain size quantitative trait locus (QTL) that were likely to act on assimilate availability and 50 QTL that were likely to act on genetic potential. This finding was further supported by functional enrichment analysis and co-location analysis with known grain number QTL and candidate genes. RNA interference and overexpression experiments were conducted to validate the function of one of the identified gene, SbDEP1, showing that SbDEP1 positively regulates grain number and negatively regulates grain size by controlling primary branching in sorghum. Haplotype analysis of SbDEP1 suggested a possible role in racial differentiation. The enhanced understanding of grain size variation in relation to assimilate availability presented in this study will benefit sorghum improvement and have implications for other cereal crops.

Extensive variation within the pan-genome of cultivated and wild sorghum.

Sorghum is a drought-tolerant staple crop for half a billion people in Africa and Asia, an important source of animal feed throughout the world and a biofuel feedstock of growing importance. Cultivated sorghum and its inter-fertile wild relatives constitute the primary gene pool for sorghum. Understanding and characterizing the diversity within this valuable resource is fundamental for its effective utilization in crop improvement. Here, we report analysis of a sorghum pan-genome to explore genetic diversity within the sorghum primary gene pool. We assembled 13 genomes representing cultivated sorghum and its wild relatives, and integrated them with 3 other published genomes to generate a pan-genome of 44,079 gene families with 222.6 Mb of new sequence identified. The pan-genome displays substantial gene-content variation, with 64% of gene families showing presence/absence variation among genomes. Comparisons between core genes and dispensable genes suggest that dispensable genes are important for sorghum adaptation. Extensive genetic variation was uncovered within the pan-genome, and the distribution of these variations was influenced by variation of recombination rate and transposable element content across the genome. We identified presence/absence variants that were under selection during sorghum domestication and improvement, and demonstrated that such variation had important phenotypic outcomes that could contribute to crop improvement. The constructed sorghum pan-genome represents an important resource for sorghum improvement and gene discovery.

Tall 3-dwarfs: oxymoron or opportunity to increase grain yield in sorghum?

MAIN CONCLUSION: Plant height was positively correlated with grain yield across a large set of 3-dwarf sorghum hybrids and production environments in north-eastern Australia. In industrialised countries, plant breeders tend to select for short plant stature in cereals like wheat, barley and rice, but also grain sorghum. This is mainly to prevent stalk lodging and to allow for machine harvesting. However, this counteracts an intrinsic positive relationship between plant height and yield potential often observed in cereals. We used data from multi-environment breeding trials comprising large sets of female sorghum lines from a range of pedigrees in hybrid combination with five different male testers. The hybrids were grown in 22 different rainfed environments in north-eastern Australia, which allowed us to thoroughly examine the relationship between plant height and yield across a range of productivity levels. Covariate analysis showed that in 38 out of the 90 tested relationships, grain yield was significantly (p < 0.05) positively and in only one case significantly negatively associated with plant height. This strong positive association between the traits was supported by the observation that 87% of the effects were either positive or zero. The effects of height on yield ranged from a decrease of 0.015 t ha-1 to an increase of 0.057 t ha-1 cm-1. The majority of the negative effects were observed in low-yielding trials and the positive effect of height tended to increase with increasing mean trial yield. Opportunities to increase yield potential by selecting for slightly taller sorghum hybrids therefore need to be explored in context with the target environments and in combination with other means to control the risks of lodging.

Large-scale genome-wide association study reveals that drought-induced lodging in grain sorghum is associated with plant height and traits linked to carbon remobilisation.

KEY MESSAGE: We detected 213 lodging QTLs and demonstrated that drought-induced stem lodging in grain sorghum is substantially associated with stay-green and plant height suggesting a critical role of carbon remobilisation. Sorghum is generally grown in water limited conditions and often lodges under post-anthesis drought, which reduces yield and quality. Due to its complexity, our understanding on the genetic control of lodging is very limited. We dissected the genetic architecture of lodging in grain sorghum through genome-wide association study (GWAS) on 2308 unique hybrids grown in 17 Australian sorghum trials over 3 years. The GWAS detected 213 QTLs, the majority of which showed a significant association with leaf senescence and plant height (72% and 71%, respectively). Only 16 lodging QTLs were not associated with either leaf senescence or plant height. The high incidence of multi-trait association for the lodging QTLs indicates that lodging in grain sorghum is mainly associated with plant height and traits linked to carbohydrate remobilisation. This result supported the selection for stay-green (delayed leaf senescence) to reduce lodging susceptibility, rather than selection for short stature and lodging resistance per se, which likely reduces yield. Additionally, our data suggested a protective effect of stay-green on weakening the association between lodging susceptibility and plant height. Our study also showed that lodging resistance might be improved by selection for stem composition but was unlikely to be improved by selection for classical resistance to stalk rots.

Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.).

C4 photosynthesis has evolved in over 60 different plant taxa and is an excellent example of convergent evolution. Plants using the C4 photosynthetic pathway have an efficiency advantage, particularly in hot and dry environments. They account for 23% of global primary production and include some of our most productive cereals. While previous genetic studies comparing phylogenetically related C3 and C4 species have elucidated the genetic diversity underpinning the C4 photosynthetic pathway, no previous studies have described the genetic diversity of the genes involved in this pathway within a C4 crop species. Enhanced understanding of the allelic diversity and selection signatures of genes in this pathway may present opportunities to improve photosynthetic efficiency, and ultimately yield, by exploiting natural variation. Here, we present the first genetic diversity survey of 8 known C4 gene families in an important C4 crop, Sorghum bicolor (L.) Moench, using sequence data of 48 genotypes covering wild and domesticated sorghum accessions. Average nucleotide diversity of C4 gene families varied more than 20-fold from the NADP-malate dehydrogenase (MDH) gene family (θπ = 0.2 × 10-3) to the pyruvate orthophosphate dikinase (PPDK) gene family (θπ = 5.21 × 10-3). Genetic diversity of C4 genes was reduced by 22.43% in cultivated sorghum compared to wild and weedy sorghum, indicating that the group of wild and weedy sorghum may constitute an untapped reservoir for alleles related to the C4 photosynthetic pathway. A SNP-level analysis identified purifying selection signals on C4 PPDK and carbonic anhydrase (CA) genes, and balancing selection signals on C4 PPDK-regulatory protein (RP) and phosphoenolpyruvate carboxylase (PEPC) genes. Allelic distribution of these C4 genes was consistent with selection signals detected. A better understanding of the genetic diversity of C4 pathway in sorghum paves the way for mining the natural allelic variation for the improvement of photosynthesis.

Large-scale GWAS in sorghum reveals common genetic control of grain size among cereals.

Grain size is a key yield component of cereal crops and a major quality attribute. It is determined by a genotype's genetic potential and its capacity to fill the grains. This study aims to dissect the genetic architecture of grain size in sorghum. An integrated genome-wide association study (GWAS) was conducted using a diversity panel (n = 837) and a BC-NAM population (n = 1421). To isolate genetic effects associated with genetic potential of grain size, rather than the genotype's capacity to fill the grains, a treatment of removing half of the panicle was imposed during flowering. Extensive and highly heritable variation in grain size was observed in both populations in 5 field trials, and 81 grain size QTL were identified in subsequent GWAS. These QTL were enriched for orthologues of known grain size genes in rice and maize, and had significant overlap with SNPs associated with grain size in rice and maize, supporting common genetic control of this trait among cereals. Grain size genes with opposite effect on grain number were less likely to overlap with the grain size QTL from this study, indicating the treatment facilitated identification of genetic regions related to the genetic potential of grain size. These results enhance understanding of the genetic architecture of grain size in cereal, and pave the way for exploration of underlying molecular mechanisms and manipulation of this trait in breeding practices.

Novel Grain Weight Loci Revealed in a Cross between Cultivated and Wild Sorghum.

Grain weight has increased during domestication of cereals. Together with grain number it determines yield, but the two are often negatively correlated. Understanding the genetic architecture of grain weight and its relationship with grain number is critical to enhance crop yield. Sorghum is an important food, feed, and biofuel crop well-known for its adaptation to drought and heat. This study aimed to dissect the genetic basis of thousand grain weight (TGW) in a BCF population between a domesticated sorghum accession and its wild progenitor, subsp. and investigate its relationship with grain number. Thousand grain weight, grain number, and yield were measured in field trials in two successive years. A strong negative correlation between TGW and grain number was observed in both trials. In total, 17 TGW quantitative trait loci (QTL) were identified, with 11 of them exhibiting an opposing effect on grain number, implying the correlation between TGW and grain number is due to pleiotropy. Nine grain size candidate genes were identified within 6 TGW QTL, and of these 5 showed signatures of selection during sorghum domestication. Large-effect QTL in this study that have not been identified previously in cultivated sorghum were found to contain candidate genes with domestication signal, indicating that these QTL were affected during sorghum domestication. This study sheds new light on the genetic basis of TGW, its relationship with grain number, and sorghum domestication.

Whole-Genome Analysis of Candidate genes Associated with Seed Size and Weight in Sorghum bicolor Reveals Signatures of Artificial Selection and Insights into Parallel Domestication in Cereal Crops.

Seed size and seed weight are major quality attributes and important determinants of yield that have been strongly selected for during crop domestication. Limited information is available about the genetic control and genes associated with seed size and weight in sorghum. This study identified sorghum orthologs of genes with proven effects on seed size and weight in other plant species and searched for evidence of selection during domestication by utilizing resequencing data from a diversity panel. In total, 114 seed size candidate genes were identified in sorghum, 63 of which exhibited signals of purifying selection during domestication. A significant number of these genes also had domestication signatures in maize and rice, consistent with the parallel domestication of seed size in cereals. Seed size candidate genes that exhibited differentially high expression levels in seed were also found more likely to be under selection during domestication, supporting the hypothesis that modification to seed size during domestication preferentially targeted genes for intrinsic seed size rather than genes associated with physiological factors involved in the carbohydrate supply and transport. Our results provide improved understanding of the complex genetic control of seed size and weight and the impact of domestication on these genes.

A physiological framework to explain genetic and environmental regulation of tillering in sorghum.

Tillering determines the plant size of sorghum (Sorghum bicolor) and an understanding of its regulation is important to match genotypes to prevalent growing conditions in target production environments. The aim of this study was to determine the physiological and environmental regulation of variability in tillering among sorghum genotypes, and to develop a framework for this regulation. Diverse sorghum genotypes were grown in three experiments with contrasting temperature, radiation and plant density to create variation in tillering. Data on phenology, tillering, and leaf and plant size were collected. A carbohydrate supply/demand (S/D) index that incorporated environmental and genotypic parameters was developed to represent the effects of assimilate availability on tillering. Genotypic differences in tillering not explained by this index were defined as propensity to tiller (PTT) and probably represented hormonal effects. Genotypic variation in tillering was associated with differences in leaf width, stem diameter and PTT. The S/D index captured most of the environmental effects on tillering and PTT most of the genotypic effects. A framework that captures genetic and environmental regulation of tillering through assimilate availability and PTT was developed, and provides a basis for the development of a model that connects genetic control of tillering to its phenotypic consequences.

Two distinct classes of QTL determine rust resistance in sorghum.

BACKGROUND: Agriculture is facing enormous challenges to feed a growing population in the face of rapidly evolving pests and pathogens. The rusts, in particular, are a major pathogen of cereal crops with the potential to cause large reductions in yield. Improving stable disease resistance is an on-going major and challenging focus for many plant breeding programs, due to the rapidly evolving nature of the pathogen. Sorghum is a major summer cereal crop that is also a host for a rust pathogen Puccinia purpurea, which occurs in almost all sorghum growing areas of the world, causing direct and indirect yield losses in sorghum worldwide, however knowledge about its genetic control is still limited. In order to further investigate this issue, QTL and association mapping methods were implemented to study rust resistance in three bi-parental populations and an association mapping set of elite breeding lines in different environments. RESULTS: In total, 64 significant or highly significant QTL and 21 suggestive rust resistance QTL were identified representing 55 unique genomic regions. Comparisons across populations within the current study and with rust QTL identified previously in both sorghum and maize revealed a high degree of correspondence in QTL location. Negative phenotypic correlations were observed between rust, maturity and height, indicating a trend for both early maturing and shorter genotypes to be more susceptible to rust. CONCLUSIONS: The significant amount of QTL co-location across traits, in addition to the consistency in the direction of QTL allele effects, has provided evidence to support pleiotropic QTL action across rust, height, maturity and stay-green, supporting the role of carbon stress in susceptibility to rust. Classical rust resistance QTL regions that did not co-locate with height, maturity or stay-green QTL were found to be significantly enriched for the defence-related NBS-encoding gene family, in contrast to the lack of defence-related gene enrichment in multi-trait effect rust resistance QTL. The distinction of disease resistance QTL hot-spots, enriched with defence-related gene families from QTL which impact on development and partitioning, provides plant breeders with knowledge which will allow for fast-tracking varieties with both durable pathogen resistance and appropriate adaptive traits.

The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes.

BACKGROUND: Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. RESULTS: In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. CONCLUSIONS: NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the "arms race" with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

Whole-genome sequencing reveals untapped genetic potential in Africa's indigenous cereal crop sorghum.

Sorghum is a food and feed cereal crop adapted to heat and drought and a staple for 500 million of the world's poorest people. Its small diploid genome and phenotypic diversity make it an ideal C4 grass model as a complement to C3 rice. Here we present high coverage (16-45 × ) resequenced genomes of 44 sorghum lines representing the primary gene pool and spanning dimensions of geographic origin, end-use and taxonomic group. We also report the first resequenced genome of S. propinquum, identifying 8 M high-quality SNPs, 1.9 M indels and specific gene loss and gain events in S. bicolor. We observe strong racial structure and a complex domestication history involving at least two distinct domestication events. These assembled genomes enable the leveraging of existing cereal functional genomics data against the novel diversity available in sorghum, providing an unmatched resource for the genetic improvement of sorghum and other grass species.

Allelic variation at a single gene increases food value in a drought-tolerant staple cereal.

The production of adequate agricultural outputs to support the growing human population places great demands on agriculture, especially in light of ever-greater restrictions on input resources. Sorghum is a drought-adapted cereal capable of reliable production where other cereals fail, and thus represents a good candidate to address food security as agricultural inputs of water and arable land grow scarce. A long-standing issue with sorghum grain is that it has an inherently lower digestibility. Here we show that a low-frequency allele type in the starch metabolic gene, pullulanase, is associated with increased digestibility, regardless of genotypic background. We also provide evidence that the beneficial allele type is not associated with deleterious pleiotropic effects in the modern field environment. We argue that increasing the digestibility of an adapted crop is a viable way forward towards addressing food security while maximizing water and land-use efficiency.

Lack of low frequency variants masks patterns of non-neutral evolution following domestication.

Detecting artificial selection in the genome of domesticated species can not only shed light on human history but can also be beneficial to future breeding strategies. Evidence for selection has been documented in domesticated species including maize and rice, but few studies have to date detected signals of artificial selection in the Sorghum bicolor genome. Based on evidence that domesticated S. bicolor and its wild relatives show significant differences in endosperm structure and quality, we sequenced three candidate seed storage protein (kafirin) loci and three candidate starch biosynthesis loci to test whether these genes show non-neutral evolution resulting from the domestication process. We found strong evidence of non-neutral selection at the starch synthase IIa gene, while both starch branching enzyme I and the beta kafirin gene showed weaker evidence of non-neutral selection. We argue that the power to detect consistent signals of non-neutral selection in our dataset is confounded by the absence of low frequency variants at four of the six candidate genes. A future challenge in the detection of positive selection associated with domestication in sorghum is to develop models that can accommodate for skewed frequency spectrums.

Peanut stripe potyvirus resistance in peanut (Arachis hypogaea L.) plants carrying viral coat protein gene sequences.

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardment with the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.