Improved pearl millet genomes representing the global heterotic pool offer a framework for molecular breeding applications.

High-quality reference genome assemblies, representative of global heterotic patterns, offer an ideal platform to accurately characterize and utilize genetic variation in the primary gene pool of hybrid crops. Here we report three platinum grade de-novo, near gap-free, chromosome-level reference genome assemblies from the active breeding germplasm in pearl millet with a high degree of contiguity, completeness, and accuracy. An improved Tift genome (Tift23D2B1-P1-P5) assembly has a contig N50 ~ 7,000-fold (126 Mb) compared to the previous version and better alignment in centromeric regions. Comparative genome analyses of these three lines clearly demonstrate a high level of collinearity and multiple structural variations, including inversions greater than 1 Mb. Differential genes in improved Tift genome are enriched for serine O-acetyltransferase and glycerol-3-phosphate metabolic process which play an important role in improving the nutritional quality of seed protein and disease resistance in plants, respectively. Multiple marker-trait associations are identified for a range of agronomic traits, including grain yield through genome-wide association study. Improved genome assemblies and marker resources developed in this study provide a comprehensive framework/platform for future applications such as marker-assisted selection of mono/oligogenic traits as well as whole-genome prediction and haplotype-based breeding of complex traits.

A population based expression atlas provides insights into disease resistance and other physiological traits in cassava (Manihot esculenta Crantz).

Cassava, a food security crop in Africa, is grown throughout the tropics and subtropics. Although cassava can provide high productivity in suboptimal conditions, the yield in Africa is substantially lower than in other geographies. The yield gap is attributable to many challenges faced by cassava in Africa, including susceptibility to diseases and poor soil conditions. In this study, we carried out 3'RNA sequencing on 150 accessions from the National Crops Resources Research Institute, Uganda for 5 tissue types, providing population-based transcriptomics resources to the research community in a web-based queryable cassava expression atlas. Differential expression and weighted gene co-expression network analysis were performed to detect 8820 significantly differentially expressed genes (DEGs), revealing similarity in expression patterns between tissue types and the clustering of detected DEGs into 18 gene modules. As a confirmation of data quality, differential expression and pathway analysis targeting cassava mosaic disease (CMD) identified 27 genes observed in the plant-pathogen interaction pathway, several previously identified CMD resistance genes, and two peroxidase family proteins different from the CMD2 gene. Present research work represents a novel resource towards understanding complex traits at expression and molecular levels for the development of resistant and high-yielding cassava varieties, as exemplified with CMD.

A sorghum practical haplotype graph facilitates genome-wide imputation and cost-effective genomic prediction.

Successful management and utilization of increasingly large genomic datasets is essential for breeding programs to accelerate cultivar development. To help with this, we developed a Sorghum bicolor Practical Haplotype Graph (PHG) pangenome database that stores haplotypes and variant information. We developed two PHGs in sorghum that were used to identify genome-wide variants for 24 founders of the Chibas sorghum breeding program from 0.01x sequence coverage. The PHG called single nucleotide polymorphisms (SNPs) with 5.9% error at 0.01x coverage-only 3% higher than PHG error when calling SNPs from 8x coverage sequence. Additionally, 207 progenies from the Chibas genomic selection (GS) training population were sequenced and processed through the PHG. Missing genotypes were imputed from PHG parental haplotypes and used for genomic prediction. Mean prediction accuracies with PHG SNP calls range from .57-.73 and are similar to prediction accuracies obtained with genotyping-by-sequencing or targeted amplicon sequencing (rhAmpSeq) markers. This study demonstrates the use of a sorghum PHG to impute SNPs from low-coverage sequence data and shows that the PHG can unify genotype calls across multiple sequencing platforms. By reducing input sequence requirements, the PHG can decrease the cost of genotyping, make GS more feasible, and facilitate larger breeding populations. Our results demonstrate that the PHG is a useful research and breeding tool that maintains variant information from a diverse group of taxa, stores sequence data in a condensed but readily accessible format, unifies genotypes across genotyping platforms, and provides a cost-effective option for genomic selection.

Genomic characterization of Ugandan smallholder farmer-preferred cassava varieties.

Understanding the genetic relationships among farmer-preferred cassava (Manihot esculenta Crantz) varieties is indispensable to genetic improvement efforts. In this study, we present a genetic analysis of 547 samples of cassava grown by 192 smallholder farmers, which were sampled at random within four districts in Uganda. We genotyped these samples at 287,952 single nucleotide polymorphisms using genotyping-by-sequencing and co-analyzed them with 349 cassava samples from the national breeding program in Uganda. The samples collected from smallholders consisted of 86 genetically unique varieties, as assessed using a genetic distance-based approach. Of these varieties, most were cultivated in only one district (30 in Kibaale, 19 in Masindi, 14 in Arua, and three in Apac), and only three were cultivated across all districts. The genetic differentiation we observed among farming districts in Uganda (mean fixation index [F ST] = .003) is similar to divergence observed within other countries. Despite the fact that none of the breeding lines were directly observed in farmer fields, genetic divergence between the populations was low (F ST = .020). Interestingly, we detected the presence of introgressions from the wild relative M. glaziovii Müll. Arg. on chromosomes 1 and 4, which implies ancestry with cassava breeding lines. Given the apparently similar pool of alleles in the breeding germplasm, it is likely that breeders have the raw genetic material they require to match the farmer-preferred trait combinations necessary for adoption. Our study highlights the importance of understanding the genetic makeup of cassava currently grown by smallholder farmers and relative to that of plant breeding germplasm.

Historical Introgressions from a Wild Relative of Modern Cassava Improved Important Traits and May Be Under Balancing Selection.

Introgression of alleles from wild relatives has often been adaptive in plant breeding. However, the significance of historical hybridization events in modern breeding is often not clear. Cassava (Manihot esculenta) is among the most important staple foods in the world, sustaining hundreds of millions of people in the tropics, especially in sub-Saharan Africa. Widespread genotyping makes cassava a model for clonally propagated root and tuber crops in the developing world, and provides an opportunity to study the modern benefits and consequences of historical introgression. We detected large introgressed Manihot glaziovii genome-segments in a collection of 2742 modern cassava landraces and elite germplasm, the legacy of a 1930s era breeding to combat disease epidemics. African landraces and improved varieties were, on average, 3.8% (max 13.6%) introgressed. Introgressions accounted for a significant (mean 20%, max 56%) portion of the heritability of tested traits. M. glaziovii alleles on the distal 10 Mb of chr. 1 increased dry matter and root number. On chr. 4, introgressions in a 20 Mb region improved harvest index and brown streak disease tolerance. We observed the introgression frequency on chr. 1 double over three cycles of selection, and that later stage trials selectively excluded homozygotes from consideration as varieties. This indicates a heterozygous advantage of introgressions. However, we also found that maintaining large recombination-suppressed introgressions in the heterozygous state allowed the accumulation of deleterious mutations. We conclude that targeted recombination of introgressions would increase the efficiency of cassava breeding by allowing simultaneous fixation of beneficial alleles and purging of genetic load.

Cassava haplotype map highlights fixation of deleterious mutations during clonal propagation.

Cassava (Manihot esculenta Crantz) is an important staple food crop in Africa and South America; however, ubiquitous deleterious mutations may severely decrease its fitness. To evaluate these deleterious mutations, we constructed a cassava haplotype map through deep sequencing 241 diverse accessions and identified >28 million segregating variants. We found that (i) although domestication has modified starch and ketone metabolism pathways to allow for human consumption, the concomitant bottleneck and clonal propagation have resulted in a large proportion of fixed deleterious amino acid changes, increased the number of deleterious alleles by 26%, and shifted the mutational burden toward common variants; (ii) deleterious mutations have been ineffectively purged, owing to limited recombination in the cassava genome; (iii) recent breeding efforts have maintained yield by masking the most damaging recessive mutations in the heterozygous state but have been unable to purge the mutation burden; such purging should be a key target in future cassava breeding.

Genome-Wide Association Mapping of Correlated Traits in Cassava: Dry Matter and Total Carotenoid Content.

Cassava is a starchy root crop cultivated in the tropics for fresh consumption and commercial processing. Primary selection objectives in cassava breeding include dry matter content and micronutrient density, particularly provitamin A carotenoids. These traits are negatively correlated in the African germplasm. This study aimed at identifying genetic markers associated with these traits and uncovering whether linkage and/or pleiotropy were responsible for observed negative correlation. A genome-wide association mapping using 672 clones genotyped at 72,279 single nucleotide polymorphism (SNP) loci was performed. Root yellowness was used indirectly to assess variation in carotenoid content. Two major loci for root yellowness were identified on chromosome 1 at positions 24.1 and 30.5 Mbp. A single locus for dry matter content that colocated with the 24.1 Mbp peak for carotenoids was identified. Haplotypes at these loci explained 70 and 37% of the phenotypic variability for root yellowness and dry matter content, respectively. Evidence of megabase-scale linkage disequilibrium (LD) around the major loci of the two traits and detection of the major dry matter locus in independent analysis for the white- and yellow-root subpopulations suggests that physical linkage rather that pleiotropy is more likely to be the cause of the negative correlation between the target traits. Moreover, candidate genes for carotenoid () and starch biosynthesis ( and ) occurred in the vicinity of the identified locus at 24.1 Mbp. These findings elucidate the genetic architecture of carotenoids and dry matter in cassava and provide an opportunity to accelerate breeding of these traits.

Genome-Wide Association and Prediction Reveals Genetic Architecture of Cassava Mosaic Disease Resistance and Prospects for Rapid Genetic Improvement.

C ( Crantz) is a crucial, under-researched crop feeding millions worldwide, especially in Africa. Cassava mosaic disease (CMD) has plagued production in Africa for over a century. Biparental mapping studies suggest primarily a single major gene mediates resistance. To investigate this genetic architecture, we conducted the first genome-wide association mapping study in cassava with up to 6128 genotyping-by-sequenced African breeding lines and 42,113 reference genome-mapped single-nucleotide polymorphism (SNP) markers. We found a single region on chromosome 8 that accounts for 30 to 66% of genetic resistance in the African cassava germplasm. Thirteen additional regions with small effects were also identified. Further dissection of the major quantitative trait locus (QTL) on chromosome 8 revealed the presence of two possibly epistatic loci and/or multiple resistance alleles, which may account for the difference between moderate and strong disease resistances in the germplasm. Search of potential candidate genes in the major QTL region identified two peroxidases and one thioredoxin. Finally, we found genomic prediction accuracy of 0.53 to 0.58 suggesting that genomic selection (GS) will be effective both for improving resistance in breeding populations and identifying highly resistant clones as varieties.

Genome-environment associations in sorghum landraces predict adaptive traits.

Improving environmental adaptation in crops is essential for food security under global change, but phenotyping adaptive traits remains a major bottleneck. If associations between single-nucleotide polymorphism (SNP) alleles and environment of origin in crop landraces reflect adaptation, then these could be used to predict phenotypic variation for adaptive traits. We tested this proposition in the global food crop Sorghum bicolor, characterizing 1943 georeferenced landraces at 404,627 SNPs and quantifying allelic associations with bioclimatic and soil gradients. Environment explained a substantial portion of SNP variation, independent of geographical distance, and genic SNPs were enriched for environmental associations. Further, environment-associated SNPs predicted genotype-by-environment interactions under experimental drought stress and aluminum toxicity. Our results suggest that genomic signatures of environmental adaptation may be useful for crop improvement, enhancing germplasm identification and marker-assisted selection. Together, genome-environment associations and phenotypic analyses may reveal the basis of environmental adaptation.

Tracking crop varieties using genotyping-by-sequencing markers: a case study using cassava (Manihot esculenta Crantz).

BACKGROUND: Accurate identification of crop cultivars is crucial in assessing the impact of crop improvement research outputs. Two commonly used identification approaches, elicitation of variety names from farmer interviews and morphological plant descriptors, have inherent uncertainty levels. Genotyping-by-sequencing (GBS) was used in a case study as an alternative method to track released varieties in farmers' fields, using cassava, a clonally propagated root crop widely grown in the tropics, and often disseminated through extension services and informal seed systems. A total of 917 accessions collected from 495 farming households across Ghana were genotyped at 56,489 SNP loci along with a "reference library" of 64 accessions of released varieties and popular landraces. RESULTS: Accurate cultivar identification and ancestry estimation was accomplished through two complementary clustering methods: (i) distance-based hierarchical clustering; and (ii) model-based maximum likelihood admixture analysis. Subsequently, 30% of the identified accessions from farmers' fields were matched to specific released varieties represented in the reference library. ADMIXTURE analysis revealed that the optimum number of major varieties was 11 and matched the hierarchical clustering results. The majority of the accessions (69%) belonged purely to one of the 11 groups, while the remaining accessions showed two or more ancestries. Further analysis using subsets of SNP markers reproduced results obtained from the full-set of markers, suggesting that GBS can be done at higher DNA multiplexing, thereby reducing the costs of variety fingerprinting. A large proportion of discrepancy between genetically unique cultivars as identified by markers and variety names as elicited from farmers were observed. Clustering results from ADMIXTURE analysis was validated using the assumption-free Discriminant Analysis of Principal Components (DAPC) method. CONCLUSION: We show that genome-wide SNP markers from increasingly affordable GBS methods coupled with complementary cluster analysis is a powerful tool for fine-scale population structure analysis and variety identification. Moreover, the ancestry estimation provides a framework for quantifying the contribution of exotic germplasm or older improved varieties to the genetic background of contemporary improved cultivars.

Population Genetics and Structure of a Global Foxtail Millet Germplasm Collection.

Foxtail millet [Setaria italica (L.) P. Beauv.] is one among the most ancient crops of dryland agriculture. It is the second most important crop among millets grown for grains or forage. Foxtail millet germplasm resources provide reservoirs of novel alleles and genes for crop improvement that have remained mostly unexplored. We genotyped a set of 190 foxtail millet germplasm accessions (including 155 accessions of the foxtail millet core collection) using genotyping-by-sequencing (GBS) for rapid single nucleotide polymorphisms (SNP) characterization to study population genetics and structure, which enable allele mining through association mapping approaches. After filtering a total 350,000 raw SNPs identified across 190 germplasm accessions for minor allele frequency (MAF), coverage for samples and coverage for sites, we retained 181 accessions with 17,714 high-quality SNPs with ≥5% MAF. Genetic structure analyses revealed that foxtail millet germplasm accessions are structured along both on the basis of races and geographic origin, and the maximum proportion of variation was due to among individuals within populations. Accessions of race indica were less diverse and are highly differentiated from those of maxima and moharia. Genome-wide linkage disequilibrium (LD) analysis showed on an average LD extends up to ∼150 kbp and varied with individual chromosomes. The utility of the data for performing genome-wide association studies (GWASs) was tested with plant pigmentation and days to flowering and identified significant marker-trait associations. This SNP data provides a foundation for exploration of foxtail millet diversity and for mining novel alleles and mapping genes for economically important traits.

The Genetic Makeup of a Global Barnyard Millet Germplasm Collection.

Barnyard millet (Echinochloa spp.) is an important crop for many smallholder farmers in southern and eastern Asia. It is valued for its drought tolerance, rapid maturation, and superior nutritional qualities. Despite these characteristics there are almost no genetic or genomic resources for this crop in either cultivated species [E. colona (L.) Link and E. crus-galli (L.) P. Beauv.]. Recently, a core collection of 89 barnyard millet accessions was developed at the genebank at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). To enhance the use of this germplasm and genomic research in barnyard millet improvement, we report the genetic characterization of this core collection using whole-genome genotyping-by-sequencing. We identified several thousand single-nucleotide polymorphisms segregating in the core collection, and we use them to show patterns of population structure and phylogenetic relationships among the accessions. We determine that there are probably four population clusters within the E. colona accessions and three such clusters within E. crus-galli. These clusters match phylogenetic relationships but by and large do not correspond to classification into individual races or clusters based on morphology. Geospatial data available for a subset of samples indicates that the clusters probably originate from geographic divisions. In all, these data will be useful to breeders working to improve this crop for smallholder farmers. This work also serves as a case study of how modern genomics can rapidly characterize crops, including ones with little to no prior genetic data.

Genome-wide association study of grain polyphenol concentrations in global sorghum [Sorghum bicolor (L.) Moench] germplasm.

Identifying natural variation of health-promoting compounds in staple crops and characterizing its genetic basis can help improve human nutrition through crop biofortification. Some varieties of sorghum, a staple cereal crop grown worldwide, have high concentrations of proanthocyanidins and 3-deoxyanthocyanidins, polyphenols with antioxidant and anti-inflammatory properties. We quantified total phenols, proanthocyanidins, and 3-deoxyanthocyanidins in a global sorghum diversity panel (n = 381) using near-infrared spectroscopy (NIRS), and characterized the patterns of variation with respect to geographic origin and botanical race. A genome-wide association study (GWAS) with 404,628 SNP markers identified novel quantitative trait loci for sorghum polyphenols, some of which colocalized with homologues of flavonoid pathway genes from other plants, including an orthologue of maize (Zea mays) Pr1 and a homologue of Arabidopsis (Arabidopsis thaliana) TT16. This survey of grain polyphenol variation in sorghum germplasm and catalog of flavonoid pathway loci may be useful to guide future enhancement of cereal polyphenols.

Dissecting genome-wide association signals for loss-of-function phenotypes in sorghum flavonoid pigmentation traits.

Genome-wide association studies are a powerful method to dissect the genetic basis of traits, although in practice the effects of complex genetic architecture and population structure remain poorly understood. To compare mapping strategies we dissected the genetic control of flavonoid pigmentation traits in the cereal grass sorghum by using high-resolution genotyping-by-sequencing single-nucleotide polymorphism markers. Studying the grain tannin trait, we find that general linear models (GLMs) are not able to precisely map tan1-a, a known loss-of-function allele of the Tannin1 gene, with either a small panel (n = 142) or large association panel (n = 336), and that indirect associations limit the mapping of the Tannin1 locus to Mb-resolution. A GLM that accounts for population structure (Q) or standard mixed linear model that accounts for kinship (K) can identify tan1-a, whereas a compressed mixed linear model performs worse than the naive GLM. Interestingly, a simple loss-of-function genome scan, for genotype-phenotype covariation only in the putative loss-of-function allele, is able to precisely identify the Tannin1 gene without considering relatedness. We also find that the tan1-a allele can be mapped with gene resolution in a biparental recombinant inbred line family (n = 263) using genotyping-by-sequencing markers but lower precision in the mapping of vegetative pigmentation traits suggest that consistent gene-level resolution will likely require larger families or multiple recombinant inbred lines. These findings highlight that complex association signals can emerge from even the simplest traits given epistasis and structured alleles, but that gene-resolution mapping of these traits is possible with high marker density and appropriate models.

Massive sorghum collection genotyped with SSR markers to enhance use of global genetic resources.

Large ex situ collections require approaches for sampling manageable amounts of germplasm for in-depth characterization and use. We present here a large diversity survey in sorghum with 3367 accessions and 41 reference nuclear SSR markers. Of 19 alleles on average per locus, the largest numbers of alleles were concentrated in central and eastern Africa. Cultivated sorghum appeared structured according to geographic regions and race within region. A total of 13 groups of variable size were distinguished. The peripheral groups in western Africa, southern Africa and eastern Asia were the most homogeneous and clearly differentiated. Except for Kafir, there was little correspondence between races and marker-based groups. Bicolor, Caudatum, Durra and Guinea types were each dispersed in three groups or more. Races should therefore better be referred to as morphotypes. Wild and weedy accessions were very diverse and scattered among cultivated samples, reinforcing the idea that large gene-flow exists between the different compartments. Our study provides an entry to global sorghum germplasm collections. Our reference marker kit can serve to aggregate additional studies and enhance international collaboration. We propose a core reference set in order to facilitate integrated phenotyping experiments towards refined functional understanding of sorghum diversity.

Population genomic and genome-wide association studies of agroclimatic traits in sorghum.

Accelerating crop improvement in sorghum, a staple food for people in semiarid regions across the developing world, is key to ensuring global food security in the context of climate change. To facilitate gene discovery and molecular breeding in sorghum, we have characterized ~265,000 single nucleotide polymorphisms (SNPs) in 971 worldwide accessions that have adapted to diverse agroclimatic conditions. Using this genome-wide SNP map, we have characterized population structure with respect to geographic origin and morphological type and identified patterns of ancient crop diffusion to diverse agroclimatic regions across Africa and Asia. To better understand the genomic patterns of diversification in sorghum, we quantified variation in nucleotide diversity, linkage disequilibrium, and recombination rates across the genome. Analyzing nucleotide diversity in landraces, we find evidence of selective sweeps around starch metabolism genes, whereas in landrace-derived introgression lines, we find introgressions around known height and maturity loci. To identify additional loci underlying variation in major agroclimatic traits, we performed genome-wide association studies (GWAS) on plant height components and inflorescence architecture. GWAS maps several classical loci for plant height, candidate genes for inflorescence architecture. Finally, we trace the independent spread of multiple haplotypes carrying alleles for short stature or long inflorescence branches. This genome-wide map of SNP variation in sorghum provides a basis for crop improvement through marker-assisted breeding and genomic selection.

Association analysis of photoperiodic flowering time genes in west and central African sorghum [Sorghum bicolor (L.) Moench].

BACKGROUND: Photoperiod-sensitive flowering is a key adaptive trait for sorghum (Sorghum bicolor) in West and Central Africa. In this study we performed an association analysis to investigate the effect of polymorphisms within the genes putatively related to variation in flowering time on photoperiod-sensitive flowering in sorghum. For this purpose a genetically characterized panel of 219 sorghum accessions from West and Central Africa was evaluated for their photoperiod response index (PRI) based on two sowing dates under field conditions. RESULTS: Sorghum accessions used in our study were genotyped for single nucleotide polymorphisms (SNPs) in six genes putatively involved in the photoperiodic control of flowering time. Applying a mixed model approach and previously-determined population structure parameters to these candidate genes, we found significant associations between several SNPs with PRI for the genes CRYPTOCHROME 1 (CRY1-b1) and GIGANTEA (GI). CONCLUSIONS: The negative values of Tajima's D, found for the genes of our study, suggested that purifying selection has acted on genes involved in photoperiodic control of flowering time in sorghum. The SNP markers of our study that showed significant associations with PRI can be used to create functional markers to serve as important tools for marker-assisted selection of photoperiod-sensitive cultivars in sorghum.

The relationship between population structure and aluminum tolerance in cultivated sorghum.

BACKGROUND: Acid soils comprise up to 50% of the world's arable lands and in these areas aluminum (Al) toxicity impairs root growth, strongly limiting crop yield. Food security is thereby compromised in many developing countries located in tropical and subtropical regions worldwide. In sorghum, SbMATE, an Al-activated citrate transporter, underlies the Alt(SB) locus on chromosome 3 and confers Al tolerance via Al-activated root citrate release. METHODOLOGY: Population structure was studied in 254 sorghum accessions representative of the diversity present in cultivated sorghums. Al tolerance was assessed as the degree of root growth inhibition in nutrient solution containing Al. A genetic analysis based on markers flanking Alt(SB) and SbMATE expression was undertaken to assess a possible role for Alt(SB) in Al tolerant accessions. In addition, the mode of gene action was estimated concerning the Al tolerance trait. Comparisons between models that include population structure were applied to assess the importance of each subpopulation to Al tolerance. CONCLUSION/SIGNIFICANCE: Six subpopulations were revealed featuring specific racial and geographic origins. Al tolerance was found to be rather rare and present primarily in guinea and to lesser extent in caudatum subpopulations. Alt(SB) was found to play a role in Al tolerance in most of the Al tolerant accessions. A striking variation was observed in the mode of gene action for the Al tolerance trait, which ranged from almost complete recessivity to near complete dominance, with a higher frequency of partially recessive sources of Al tolerance. A possible interpretation of our results concerning the origin and evolution of Al tolerance in cultivated sorghum is discussed. This study demonstrates the importance of deeply exploring the crop diversity reservoir both for a comprehensive view of the dynamics underlying the distribution and function of Al tolerance genes and to design efficient molecular breeding strategies aimed at enhancing Al tolerance.