Martha T. Hamblin

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.

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.

Diversity and selection in sorghum: simultaneous analyses using simple sequence repeats

Although molecular markers and DNA sequence data are now available for many crop species, our ability to identify genetic variation associated with functional or adaptive diversity is still limited. In this study, our aim was to quantify and characterize diversity in a panel of cultivated and wild sorghums (Sorghum bicolor), establish genetic relationships, and, simultaneously, identify selection signals that might be associated with sorghum domestication. We assayed 98 simple sequence repeat (SSR) loci distributed throughout the genome in a panel of 104 accessions comprising 73 landraces (i.e., cultivated lines) and 31 wild sorghums. Evaluation of SSR polymorphisms indicated that landraces retained 86% of the diversity observed in the wild sorghums. The landraces and wilds were moderately differentiated (F st=0.13), but there was little evidence of population differentiation among racial groups of cultivated sorghums (F st=0.06). Neighbor-joining analysis showed that wild sorghums generally formed a distinct group, and about half the landraces tended to cluster by race. Overall, bootstrap support was low, indicating a history of gene flow among the various cultivated types or recent common ancestry. Statistical methods (Ewens-Watterson test for allele excess, lnRH, and F st) for identifying genomic regions with patterns of variation consistent with selection gave significant results for 11 loci (approx. 15% of the SSRs used in the final analysis). Interestingly, seven of these loci mapped in or near genomic regions associated with domestication-related QTLs (i.e., shattering, seed weight, and rhizomatousness). We anticipate that such population genetics-based statistical approaches will be useful for re-evaluating extant SSR data for mining interesting genomic regions from germplasm collections.

Sweet Sorghum Genetic Diversity and Association Mapping for Brix and Height

Sweet sorghum [Sorghum bicolor (L.) Moench], like its close relative, sugarcane (Saccharum spp.), has been selected to accumulate high levels of edible sugars in the stem. Sweet sorghums are tall and produce high biomass in addition to sugar. Little has been documented about the genetic relationships and diversity within sweet sorghums and how sweet sorghums relate to grain sorghum racial types. In this study, a diverse panel of 125 sorghums (mostly sweet) was successfully genotyped with 47 simple sequence repeats (SSRs) and 322 single nucleotide polymorphisms (SNPs). Using both distance‐based and model‐based methods, we identified three main genetic groupings of sweet sorghums. Based on observed phenotypes and known origins we classified the three groups as historical and modern syrup, modern sugar/energy types, and amber types. Using SSR markers also scored in an available large grain sorghum germplasm panel, we found that these three sweet groupings clustered with kafir/bicolor, caudatum, and bicolor types, respectively. Using the information on population structure and relatedness, association mapping was performed for height and stem sugar (brix) traits. Three significant associations for height were detected. Two of these, on chromosomes 9 and 6, support published QTL studies. One significant association for brix, on chromosome 1, 12kb from a glucose‐6‐phosphate isomerase homolog, was detected.

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.

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

Background Acid soils comprise up to 50% of the worlds 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 AltSB 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 AltSB and SbMATE expression was undertaken to assess a possible role for AltSB 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. AltSB 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.

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 to100 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, ρ, 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 ρ and the population mutation parameter, θ, 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.

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 crops 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.

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.