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.

Evaluating Imputation Algorithms for Low-Depth Genotyping-By-Sequencing (GBS) Data.

Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordable, datasets derived from HTS methods suffer from sequencing error, alignment errors, and missing data, all of which introduce noise and uncertainty to variant discovery and genotype calling. Under such circumstances, meaningful analysis of the data is difficult. Our primary interest lies in the issue of how one can accurately infer or impute missing genotypes in HTS-derived datasets. Many of the existing genotype imputation algorithms and software packages were primarily developed by and optimized for the human genetics community, a field where a complete and accurate reference genome has been constructed and SNP arrays have, in large part, been the common genotyping platform. We set out to answer two questions: 1) can we use existing imputation methods developed by the human genetics community to impute missing genotypes in datasets derived from non-human species and 2) are these methods, which were developed and optimized to impute ascertained variants, amenable for imputation of missing genotypes at HTS-derived variants? We selected Beagle v.4, a widely used algorithm within the human genetics community with reportedly high accuracy, to serve as our imputation contender. We performed a series of cross-validation experiments, using GBS data collected from the species Manihot esculenta by the Next Generation (NEXTGEN) Cassava Breeding Project. NEXTGEN currently imputes missing genotypes in their datasets using a LASSO-penalized, linear regression method (denoted 'glmnet'). We selected glmnet to serve as a benchmark imputation method for this reason. We obtained estimates of imputation accuracy by masking a subset of observed genotypes, imputing, and calculating the sample Pearson correlation between observed and imputed genotype dosages at the site and individual level; computation time served as a second metric for comparison. We then set out to examine factors affecting imputation accuracy, such as levels of missing data, read depth, minor allele frequency (MAF), and reference panel composition.

Identification and distribution of the NBS-LRR gene family in the Cassava genome.

BACKGROUND: Plant resistance genes (R genes) exist in large families and usually contain both a nucleotide-binding site domain and a leucine-rich repeat domain, denoted NBS-LRR. The genome sequence of cassava (Manihot esculenta) is a valuable resource for analysing the genomic organization of resistance genes in this crop. RESULTS: With searches for Pfam domains and manual curation of the cassava gene annotations, we identified 228 NBS-LRR type genes and 99 partial NBS genes. These represent almost 1% of the total predicted genes and show high sequence similarity to proteins from other plant species. Furthermore, 34 contained an N-terminal toll/interleukin (TIR)-like domain, and 128 contained an N-terminal coiled-coil (CC) domain. 63% of the 327 R genes occurred in 39 clusters on the chromosomes. These clusters are mostly homogeneous, containing NBS-LRRs derived from a recent common ancestor. CONCLUSIONS: This study provides insight into the evolution of NBS-LRR genes in the cassava genome; the phylogenetic and mapping information may aid efforts to further characterize the function of these predicted R genes.

Association mapping provides insights into the origin and the fine structure of the sorghum aluminum tolerance locus, AltSB.

Root damage caused by aluminum (Al) toxicity is a major cause of grain yield reduction on acid soils, which are prevalent in tropical and subtropical regions of the world where food security is most tenuous. In sorghum, Al tolerance is conferred by SbMATE, an Al-activated root citrate efflux transporter that underlies the major Al tolerance locus, AltSB, on sorghum chromosome 3. We used association mapping to gain insights into the origin and evolution of Al tolerance in sorghum and to detect functional variants amenable to allele mining applications. Linkage disequilibrium across the AltSB locus decreased much faster than in previous reports in sorghum, and reached basal levels at approximately 1000 bp. Accordingly, intra-locus recombination events were found to be extensive. SNPs and indels highly associated with Al tolerance showed a narrow frequency range, between 0.06 and 0.1, suggesting a rather recent origin of Al tolerance mutations within AltSB. A haplotype network analysis suggested a single geographic and racial origin of causative mutations in primordial guinea domesticates in West Africa. Al tolerance assessment in accessions harboring recombinant haplotypes suggests that causative polymorphisms are localized to a ∼6 kb region including intronic polymorphisms and a transposon (MITE) insertion, whose size variation has been shown to be positively correlated with Al tolerance. The SNP with the strongest association signal, located in the second SbMATE intron, recovers 9 of the 14 highly Al tolerant accessions and 80% of all the Al tolerant and intermediately tolerant accessions in the association panel. Our results also demonstrate the pivotal importance of knowledge on the origin and evolution of Al tolerance mutations in molecular breeding applications. Allele mining strategies based on associated loci are expected to lead to the efficient identification, in diverse sorghum germplasm, of Al tolerant accessions able maintain grain yields under Al toxicity.

High-resolution mapping of resistance to cassava mosaic geminiviruses in cassava using genotyping-by-sequencing and its implications for breeding.

Cassava mosaic disease (CMD), caused by different species of cassava mosaic geminiviruses (CMGs), is the most important disease of cassava in Africa and the Indian sub-continent. The cultivated cassava species is protected from CMD by polygenic resistance introgressed from the wild species Manihot glaziovii and a dominant monogenic type of resistance, named CMD2, discovered in African landraces. The ability of the monogenic resistance to confer high levels of resistance in different genetic backgrounds has led recently to its extensive usage in breeding across Africa as well as pre-emptive breeding in Latin America. However, most of the landraces carrying the monogenic resistance are morphologically very similar and come from a geographically restricted area of West Africa, raising the possibility that the diversity of the single-gene resistance could be very limited, or even located at a single locus. Several mapping studies, employing bulk segregant analysis, in different genetic backgrounds have reported additional molecular markers linked to supposedly new resistance genes. However, it is not possible to tell if these are indeed new genes in the absence adequate genetic map framework or allelism tests. To address this important question, a high-density single nucleotide polymorphism (SNP) map of cassava was developed through genotyping-by-sequencing a bi-parental mapping population (N=180) that segregates for the dominant monogenic resistance to CMD. Virus screening using PCR showed that CMD symptoms and presence of virus were strongly correlated (r=0.98). Genome-wide scan and high-resolution composite interval mapping using 6756 SNPs uncovered a single locus with large effect (R(2)=0.74). Projection of the previously published resistance-linked microsatellite markers showed that they co-occurred in the same chromosomal location surrounding the presently mapped resistance locus. Moreover, their relative distance to the mapped resistance locus correlated with the reported degree of linkage with the resistance phenotype. Cluster analysis of the landraces first shown to have this type of resistance revealed that they are very closely related, if not identical. These findings suggest that there is a single source of monogenic resistance in the crop's genepool tracing back to a common ancestral clone. In the absence of further resistance diversification, the long-term effectiveness of the single gene resistance is known to be precarious, given the potential to be overcome by CMGs due to their fast-paced evolutionary rate. However, combining the quantitative with the qualitative type of resistance may ensure that this resistance gene continues to offer protection to cassava, a crop that is depended upon by millions of people in Africa against the devastating onslaught of CMGs.

A candidate gene based approach validates Md-PG1 as the main responsible for a QTL impacting fruit texture in apple (Malus x domestica Borkh).

BACKGROUND: Apple is a widely cultivated fruit crop for its quality properties and extended storability. Among the several quality factors, texture is the most important and appreciated, and within the apple variety panorama the cortex texture shows a broad range of variability. Anatomically these variations depend on degradation events occurring in both fruit primary cell wall and middle lamella. This physiological process is regulated by an enzymatic network generally encoded by large gene families, among which polygalacturonase is devoted to the depolymerization of pectin. In apple, Md-PG1, a key gene belonging to the polygalacturonase gene family, was mapped on chromosome 10 and co-localized within the statistical interval of a major hot spot QTL associated to several fruit texture sub-phenotypes. RESULTS: In this work, a QTL corresponding to the position of Md-PG1 was validated and new functional alleles associated to the fruit texture properties in 77 apple cultivars were discovered. 38 SNPs genotyped by gene full length resequencing and 2 SSR markers ad hoc targeted in the gene metacontig were employed. Out of this SNP set, eleven were used to define three significant haplotypes statistically associated to several texture components. The impact of Md-PG1 in the fruit cell wall disassembly was further confirmed by the cortex structure electron microscope scanning in two apple varieties characterized by opposite texture performance, such as 'Golden Delicious' and 'Granny Smith'. CONCLUSIONS: The results here presented step forward into the genetic dissection of fruit texture in apple. This new set of haplotypes, and microsatellite alleles, can represent a valuable toolbox for a more efficient parental selection as well as the identification of new apple accessions distinguished by superior fruit quality features.

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.

Population genetics of genomics-based crop improvement methods.

Many genome-wide association studies (GWAS) in humans are concluding that, even with very large sample sizes and high marker densities, most of the genetic basis of complex traits may remain unexplained. At the same time, recent research in plant GWAS is showing much greater success with fewer resources. Both GWAS and genomic selection (GS), a method for predicting phenotypes by the use of genome-wide marker data, are receiving considerable attention among plant breeders. In this review we explore how differences in population genetic histories, as well as past selection for traits of interest, have produced trait architectures and patterns of linkage disequilibrium (LD) that frequently differ dramatically between domesticated plants and humans, making detection of quantitative trait loci (QTL) effects in crops more rewarding and less costly than in humans.

Performance of single nucleotide polymorphisms versus haplotypes for genome-wide association analysis in barley.

Genome-wide association studies (GWAS) may benefit from utilizing haplotype information for making marker-phenotype associations. Several rationales for grouping single nucleotide polymorphisms (SNPs) into haplotype blocks exist, but any advantage may depend on such factors as genetic architecture of traits, patterns of linkage disequilibrium in the study population, and marker density. The objective of this study was to explore the utility of haplotypes for GWAS in barley (Hordeum vulgare) to offer a first detailed look at this approach for identifying agronomically important genes in crops. To accomplish this, we used genotype and phenotype data from the Barley Coordinated Agricultural Project and constructed haplotypes using three different methods. Marker-trait associations were tested by the efficient mixed-model association algorithm (EMMA). When QTL were simulated using single SNPs dropped from the marker dataset, a simple sliding window performed as well or better than single SNPs or the more sophisticated methods of blocking SNPs into haplotypes. Moreover, the haplotype analyses performed better 1) when QTL were simulated as polymorphisms that arose subsequent to marker variants, and 2) in analysis of empirical heading date data. These results demonstrate that the information content of haplotypes is dependent on the particular mutational and recombinational history of the QTL and nearby markers. Analysis of the empirical data also confirmed our intuition that the distribution of QTL alleles in nature is often unlike the distribution of marker variants, and hence utilizing haplotype information could capture associations that would elude single SNPs. We recommend routine use of both single SNP and haplotype markers for GWAS to take advantage of the full information content of the genotype data.

Positively selected disease response orthologous gene sets in the cereals identified using Sorghum bicolor L. Moench expression profiles and comparative genomics.

Disease response genes (DRGs) diverge under recurrent positive selection as a result of a molecular arms race between hosts and pathogens. Most of these studies were conducted in animals, and few defense genes have been shown to evolve adaptively in plants. To test for adaptation in the molecules mediating disease resistance in the cereals, we first combined information from the expression pattern of Sorghum bicolor genes and from divergence to the full genome of rice to identify candidate DRGs. We then used evolutionary analyses of orthologous gene sets from several grass species, to determine whether the DRGs show signals of positive selection and the residues targeted. We found 140 divergent genes upregulated under biotic stress in S. bicolor by evaluating the relative abundance of expressed sequence tags in different libraries and comparing them with rice genes. For 10 of these genes, we found sets of orthologs including sequences from rice and three other cereals; six genes showed a pattern of substitution that was consistent with positive selection. Three of these genes, a thaumatin, a peroxidase, and a barley mlo homolog, are known antifungal proteins. The other three genes with evidence of positive selection were a MCM-1 agamous deficiens SRF- (MADS) box transcription factor, an eIF5 translation initiation factor, and a gene of unknown function but with evidence of expression during stress. Permutation analyses, using different ortholog and paralog sequences, consistently identified five positively selected codons in the peroxidase, a member of a cluster of genes and a large gene family. We mapped the positively selected residues onto the structure of the peroxidase and thaumatin and found that all sites are on the surface of these proteins and several are close to biochemically determined active sites. Identifying new positively selected plant disease resistance genes and the critical amino acid sites provides a basis for functional studies that may increase our understanding of their underlying molecular mechanisms of action. Additionally, it may lead to the identification of individuals having variation at functionally important sites, as well as eventually using this information in the rational design and engineering of proteins involved in plant disease resistance.

Empirical comparison of Simple Sequence Repeats and single nucleotide polymorphisms in assessment of maize diversity and relatedness.

While Simple Sequence Repeats (SSRs) are extremely useful genetic markers, recent advances in technology have produced a shift toward use of single nucleotide polymorphisms (SNPs). The different mutational properties of these two classes of markers result in differences in heterozygosities and allele frequencies that may have implications for their use in assessing relatedness and evaluation of genetic diversity. We compared analyses based on 89 SSRs (primarily dinucleotide repeats) to analyses based on 847 SNPs in individuals from the same 259 inbred maize lines, which had been chosen to represent the diversity available among current and historic lines used in breeding. The SSRs performed better at clustering germplasm into populations than did a set of 847 SNPs or 554 SNP haplotypes, and SSRs provided more resolution in measuring genetic distance based on allele-sharing. Except for closely related pairs of individuals, measures of distance based on SSRs were only weakly correlated with measures of distance based on SNPs. Our results suggest that 1) large numbers of SNP loci will be required to replace highly polymorphic SSRs in studies of diversity and relatedness and 2) relatedness among highly-diverged maize lines is difficult to measure accurately regardless of the marker system.

Challenges of detecting directional selection after a bottleneck: lessons from Sorghum bicolor.

Multilocus surveys of sequence variation can be used to identify targets of directional selection, which are expected to have reduced levels of variation. Following a population bottleneck, the signal of directional selection may be hard to detect because many loci may have low variation by chance and the frequency spectrum of variation may be perturbed in ways that resemble the effects of selection. Cultivated Sorghum bicolor contains a subset of the genetic diversity found in its wild ancestor(s) due to the combined effects of a domestication bottleneck and human selection on traits associated with agriculture. As a framework for distinguishing between the effects of demography and selection, we sequenced 204 loci in a diverse panel of 17 cultivated S. bicolor accessions. Genomewide patterns of diversity depart strongly from equilibrium expectations with regard to the variance of the number of segregating sites, the site frequency spectrum, and haplotype configuration. Furthermore, gene genealogies of most loci with an excess of low frequency variants and/or an excess of segregating sites do not show the characteristic signatures of directional and diversifying selection, respectively. A simple bottleneck model provides an improved but inadequate fit to the data, suggesting the action of other population-level factors, such as population structure and migration. Despite a known history of recent selection, we find little evidence for directional selection, likely due to low statistical power and lack of an appropriate null model.

Equilibrium processes cannot explain high levels of short- and medium-range linkage disequilibrium in the domesticated grass Sorghum bicolor.

Patterns of linkage disequilibrium (LD) are of interest because they provide evidence of both equilibrium (e.g., mating system or long-term population structure) and nonequilibrium (e.g., demographic or selective) processes, as well as because of their importance in strategies for identifying the genetic basis of complex phenotypes. We report patterns of short and medium range (up to 100 kb) LD in six unlinked genomic regions in the partially selfing domesticated grass, Sorghum bicolor. The extent of allelic associations in S. bicolor, as assessed by pairwise measures of LD, is higher than in maize but lower than in Arabidopsis, in qualitative agreement with expectations based on mating system. Quantitative analyses of the population recombination parameter, rho, however, based on empirical estimates of rates of recombination, mutation, and self-pollination, show that LD is more extensive than expected under a neutral equilibrium model. The disparity between rho and the population mutation parameter, , is similar to that observed in other species whose population history appears to be complex. From a practical standpoint, these results suggest that S. bicolor is well suited for association studies using reasonable numbers of markers, since LD typically extends at least several kilobases but has largely decayed by 15 kb.

An analysis of genetic diversity across the maize genome using microsatellites.

How domestication bottlenecks and artificial selection shaped the amount and distribution of genetic variation in the genomes of modern crops is poorly understood. We analyzed diversity at 462 simple sequence repeats (SSRs) or microsatellites spread throughout the maize genome and compared the diversity observed at these SSRs in maize to that observed in its wild progenitor, teosinte. The results reveal a modest genome-wide deficit of diversity in maize relative to teosinte. The relative deficit of diversity is less for SSRs with dinucleotide repeat motifs than for SSRs with repeat motifs of more than two nucleotides, suggesting that the former with their higher mutation rate have partially recovered from the domestication bottleneck. We analyzed the relationship between SSR diversity and proximity to QTL for domestication traits and observed no relationship between these factors. However, we did observe a weak, although significant, spatial correlation for diversity statistics among SSRs within 2 cM of one another, suggesting that SSR diversity is weakly patterned across the genome. Twenty-four of 462 SSRs (5%) show some evidence of positive selection in maize under multiple tests. Overall, the pattern of genetic diversity at maize SSRs can be explained largely by a bottleneck effect with a smaller effect from selection.

Comparative population genetics of the panicoid grasses: sequence polymorphism, linkage disequilibrium and selection in a diverse sample of sorghum bicolor.

Levels of genetic variation and linkage disequilibrium (LD) are critical factors in association mapping methods as well as in identification of loci that have been targets of selection. Maize, an outcrosser, has a high level of sequence variation and a limited extent of LD. Sorghum, a closely related but largely self-pollinating panicoid grass, is expected to have higher levels of LD. As a first step in estimation of population genetic parameters in sorghum, we surveyed 27 diverse S. bicolor accessions for sequence variation at a total of 29,186 bp in 95 short regions derived from genetically mapped RFLPs located throughout the genome. Consistent with its higher level of inbreeding, the extent of LD is at least severalfold greater in sorghum than in maize. Total sequence variation in sorghum is about fourfold lower than that in maize, while synonymous variation is fivefold lower, suggesting a smaller effective population size in sorghum. Because we surveyed a species-wide sample, the mating system, which primarily affects population-level diversity, may not be primarily responsible for this difference. Comparisons of polymorphism and divergence suggest that both directional and diversifying selection have played important roles in shaping variation in the sorghum genome.

Molecular evolution of the phytochrome gene family in sorghum: changing rates of synonymous and replacement evolution.

The photoreceptor phytochromes, encoded by a small gene family, are responsible for controlling the expression of a number of light-responsive genes and photomorphogenic events, including agronomically important phenotypes such as flowering time and shade-avoidance behavior. The understanding and control of flowering time are particularly important goals in sorghum cultivar development for diverse environments, and naturally occurring variation in the phytochrome genes might prove useful in breeding programs. Also of interest is whether variation observed at the phytochrome loci in domesticated sorghum, or in particular races, is a result of human selection. Population genetic studies can reveal evidence of such selection in patterns of polymorphism and divergence. In this study we report a population genetic analysis of the PHY gene family in Sorghum bicolor (L.) Moench in a diverse panel including both cultivated and wild accessions. We show that the level of nucleotide variation in all gene family members is about half the average for this species, consistent with purifying selection acting on these loci. However, the rate of amino acid substitution is accelerated at PHYC compared to the other two loci. In comparisons to a closely related sorghum species, PHYC shows a pattern of intermediate frequency amino acid changes that differ from the patterns observed in comparisons across longer evolutionary distances. There is also a departure from expected patterns of polymorphism and divergence at synonymous sites in PHYC, although the data do not fit a simple model of directional or diversifying selection. Cultivated sorghum has a level of variation similar to that of wild relatives (ssp. verticilliflorum), but many polymorphisms are subspecies-specific, including several amino acid variants.

Complex signatures of natural selection at the Duffy blood group locus.

The Duffy blood group locus (FY) has long been considered a likely target of natural selection, because of the extreme pattern of geographic differentiation of its three major alleles (FY*B, FY*A, and FY*O). In the present study, we resequenced the FY region in samples of Hausa from Cameroon (fixed for FY*O), Han Chinese (fixed for FY*A), Italians, and Pakistanis. Our goals were to characterize the signature of directional selection on FY*O in sub-Saharan Africa and to understand the extent to which natural selection has also played a role in the extreme geographic differentiation of the other derived allele at this locus, FY*A. The data from the FY region are compared with the patterns of variation observed at 10 unlinked, putatively neutral loci from the same populations, as well as with theoretical expectations from the neutral-equilibrium model. The FY region in the Hausa shows evidence of directional selection in two independent properties of the data (i.e., level of sequence variation and frequency spectrum), observations that are consistent with the FY*O mutation being the target. The Italian and Chinese FY data show patterns of variation that are very unusual, particularly with regard to frequency spectrum and linkage disequilibrium, but do not fit the predictions of any simple model of selection. These patterns may represent a more complex and previously unrecognized signature of positive selection.

The vitellogenin gene family of Aedes aegypti

We have been interested in identifying genes that play a role in reproduction of the mosquito Aedes aegypti. Our interests are currently focused on the vitellogenin genes which in the mosquito are expressed only in the fat body in response to the insect steroid hormone, 20-hydroxyecdysone. Four of the five vitellogenin genes in the genome have been cloned. We have examined the relationships between these genes and find that they form a small gene family exhibiting different levels of relationship.