Stephen Kresovich

A unified mixed-model method for association mapping that accounts for multiple levels of relatedness

As population structure can result in spurious associations, it has constrained the use of association studies in human and plant genetics. Association mapping, however, holds great promise if true signals of functional association can be separated from the vast number of false signals generated by population structure. We have developed a unified mixed-model approach to account for multiple levels of relatedness simultaneously as detected by random genetic markers. We applied this new approach to two samples: a family-based sample of 14 human families, for quantitative gene expression dissection, and a sample of 277 diverse maize inbred lines with complex familial relationships and population structure, for quantitative trait dissection. Our method demonstrates improved control of both type I and type II error rates over other methods. As this new method crosses the boundary between family-based and structured association samples, it provides a powerful complement to currently available methods for association mapping.

The Sorghum bicolor genome and the diversification of grasses

Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the ∼730-megabase Sorghum bicolor (L.) Moench genome, placing ∼98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the ∼75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization ∼70 million years ago, most duplicated gene sets lost one member before the sorghum–rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum’s drought tolerance.

Structure of linkage disequilibrium and phenotypic associations in the maize genome

Association studies based on linkage disequilibrium (LD) can provide high resolution for identifying genes that may contribute to phenotypic variation. We report patterns of local and genome-wide LD in 102 maize inbred lines representing much of the worldwide genetic diversity used in maize breeding, and address its implications for association studies in maize. In a survey of six genes, we found that intragenic LD generally declined rapidly with distance (r2 < 0.1 within 1500 bp), but rates of decline were highly variable among genes. This rapid decline probably reflects large effective population sizes in maize during its evolution and high levels of recombination within genes. A set of 47 simple sequence repeat (SSR) loci showed stronger evidence of genome-wide LD than did single-nucleotide polymorphisms (SNPs) in candidate genes. LD was greatly reduced but not eliminated by grouping lines into three empirically determined subpopulations. SSR data also supplied evidence that divergent artificial selection on flowering time may have played a role in generating population structure. Provided the effects of population structure are effectively controlled, this research suggests that association studies show great promise for identifying the genetic basis of important traits in maize with very high resolution.

An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPS and pedigree

Abstract The utility of 131 simple sequence repeat (SSR) loci to characterize and identify maize inbred lines, validate pedigree, and show associations among inbred lines was evaluated using a set of 58 inbred lines and four hybrids. Thirteen sets of inbred parent-progeny triplet pedigrees together with four hybrids and their parental lines were used to quantify incidences of scoring that departed from expectations based upon simple Mendelian inheritance. Results were compared to those obtained using 80 restriction fragment length polymorphism (RFLP) probes. Over all inbred triplets, 2.2% of SSRs and 3.6% of RFLP loci resulted in profiles that were scored as having segregated in a non-Mendelian fashion. Polymorphic index content (PIC, a measure of discrimination ability) values ranged from 0.06 to 0.91 for SSRs and from 0.10 to 0.84 for RFLPs. Mean values for PIC for SSRs and RFLPs were similar, approximately 0.62. However, PIC values for nine SSRs exceeded the maximum PIC for RFLPs. Di-repeats gave the highest mean PIC scores for SSRs but this class of repeats can result in “stutter” bands that complicate accurate genotyping. Associations among inbreds were similar for SSR and RFLP data, closely approximating expectations from known pedigrees. SSR technology presents the potential advantages of reliability, reproducibility, discrimination, standardization and cost effectiveness over RFLPs. SSR profiles can be readily interpreted in terms of alleles at mapped loci across a broad range of maize germ plasm. Consequently, SSRs represent the optimum approach for the identification and pedigree validation of maize genotypes compared to other currently available methods.

Genetic Properties of the Maize Nested Association Mapping Population

Codifying Maize Modifications Maize, one of our most important crop species, has been the target of genetic investigation and experimentation for more than 100 years. Crossing two inbred lines tends to result in “better” offspring, in a process known as heterosis. Attempts to map the genetic loci that control traits important for farming have been made, but few have been successful (see the Perspective by Mackay). Buckler et al. (p. 714) and McMullen et al. (p. 737) produced a genomic map of maize that relates recombination to genome structure. Even tremendous adaptations in very diverse species were produced by numerous, small additive steps. Differences in flowering time in maize among inbred lines were not caused by a few genes with large effects, but by the cumulative effects of numerous quantitative trait loci—each of which has only a small impact on the trait. Outcrossing vigor in maize is most likely due to retained variability in regions around the centromeres. Maize genetic diversity has been used to understand the molecular basis of phenotypic variation and to improve agricultural efficiency and sustainability. We crossed 25 diverse inbred maize lines to the B73 reference line, capturing a total of 136,000 recombination events. Variation for recombination frequencies was observed among families, influenced by local (cis) genetic variation. We identified evidence for numerous minor single-locus effects but little two-locus linkage disequilibrium or segregation distortion, which indicated a limited role for genes with large effects and epistatic interactions on fitness. We observed excess residual heterozygosity in pericentromeric regions, which suggested that selection in inbred lines has been less efficient in these regions because of reduced recombination frequency. This implies that pericentromeric regions may contribute disproportionally to heterosis.

Genome-wide association study of quantitative resistance to southern leaf blight in the maize nested association mapping population

Nested association mapping (NAM) offers power to resolve complex, quantitative traits to their causal loci. The maize NAM population, consisting of 5,000 recombinant inbred lines (RILs) from 25 families representing the global diversity of maize, was evaluated for resistance to southern leaf blight (SLB) disease. Joint-linkage analysis identified 32 quantitative trait loci (QTLs) with predominantly small, additive effects on SLB resistance. Genome-wide association tests of maize HapMap SNPs were conducted by imputing founder SNP genotypes onto the NAM RILs. SNPs both within and outside of QTL intervals were associated with variation for SLB resistance. Many of these SNPs were within or near sequences homologous to genes previously shown to be involved in plant disease resistance. Limited linkage disequilibrium was observed around some SNPs associated with SLB resistance, indicating that the maize NAM population enables high-resolution mapping of some genome regions.

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.

Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]

Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.

Identifying genes of agronomic importance in maize by screening microsatellites for evidence of selection during domestication.

Crop species experienced strong selective pressure directed at genes controlling traits of agronomic importance during their domestication and subsequent episodes of selective breeding. Consequently, these genes are expected to exhibit the signature of selection. We screened 501 maize genes for the signature of selection using microsatellites or simple sequence repeats (SSRs). We applied the Ewens–Watterson test, which can reveal deviations from a neutral-equilibrium model, as well as two nonequilibrium tests that incorporate the domestication bottleneck. We investigated two classes of SSRs: those known to be polymorphic in maize (Class I) and those previously classified as monomorphic in maize (Class II). Fifteen SSRs exhibited some evidence for selection in maize and 10 showed evidence under stringent criteria. The genes containing nonneutral SSRs are candidates for agronomically important genes. Because demographic factors can bias our tests, further independent tests of these candidates are necessary. We applied such an additional test to one candidate, which encodes a MADS box transcriptional regulator, and confirmed that this gene experienced a selective sweep during maize domestication. Genomic scans for the signature of selection offer a means of identifying new genes of agronomic importance even when gene function and the phenotype of interest are unknown.

Microsatellites in Zea - variability, patterns of mutations, and use for evolutionary studies.

Abstract To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees.