Benjamin Stich

Comparison of Mixed-Model Approaches for Association Mapping

Association-mapping methods promise to overcome the limitations of linkage-mapping methods. The main objectives of this study were to (i) evaluate various methods for association mapping in the autogamous species wheat using an empirical data set, (ii) determine a marker-based kinship matrix using a restricted maximum-likelihood (REML) estimate of the probability of two alleles at the same locus being identical in state but not identical by descent, and (iii) compare the results of association-mapping approaches based on adjusted entry means (two-step approaches) with the results of approaches in which the phenotypic data analysis and the association analysis were performed in one step (one-step approaches). On the basis of the phenotypic and genotypic data of 303 soft winter wheat (Triticum aestivum L.) inbreds, various association-mapping methods were evaluated. Spearmans rank correlation between P-values calculated on the basis of one- and two-stage association-mapping methods ranged from 0.63 to 0.93. The mixed-model association-mapping approaches using a kinship matrix estimated by REML are more appropriate for association mapping than the recently proposed QK method with respect to (i) the adherence to the nominal α-level and (ii) the adjusted power for detection of quantitative trait loci. Furthermore, we showed that our data set could be analyzed by using two-step approaches of the proposed association-mapping method without substantially increasing the empirical type I error rate in comparison to the corresponding one-step approaches.

Linkage disequilibrium in European elite maize germplasm investigated with SSRs

Information about the extent and genomic distribution of linkage disequilibrium (LD) is of fundamental importance for association mapping. The main objectives of this study were to (1) investigate genetic diversity within germplasm groups of elite European maize (Zea mays L.) inbred lines, (2) examine the population structure of elite European maize germplasm, and (3) determine the extent and genomic distribution of LD between pairs of simple sequence repeat (SSR) markers. We examined genetic diversity and LD in a cross section of European and US elite breeding material comprising 147 inbred lines genotyped with 100 SSR markers. For gene diversity within each group, significant (P<0.05) differences existed among the groups. The LD was significant (P<0.05) for 49% of the SSR marker pairs in the 80 flint lines and for 56% of the SSR marker pairs in the 57 dent lines. The ratio of linked to unlinked loci in LD was 1.1 for both germplasm groups. The high incidence of LD suggests that the extent of LD between SSR markers should allow the detection of marker-phenotype associations in a genome scan. However, our results also indicate that a high proportion of the observed LD is generated by forces, such as relatedness, population stratification, and genetic drift, which cause a high risk of detecting false positives in association mapping.

Genome-wide association mapping of iron homeostasis in the maize association population

BackgroundIron (Fe) deficiency in plants is the result of low Fe soil availability affecting 30% of cultivated soils worldwide. To improve our understanding on Fe-efficiency this study aimed to (i) evaluate the influence of two different Fe regimes on morphological and physiological trait formation, (ii) identify polymorphisms statistically associated with morphological and physiological traits, and (iii) dissect the correlation between morphological and physiological traits using an association mapping population.ResultsThe fine-mapping analyses on quantitative trait loci (QTL) confidence intervals of the intermated B73 × Mo17 (IBM) population provided a total of 13 and 2 single nucleotide polymorphisms (SNPs) under limited and adequate Fe regimes, respectively, which were significantly (FDR = 0.05) associated with cytochrome P450 94A1, invertase beta-fructofuranosidase insoluble isoenzyme 6, and a low-temperature-induced 65 kDa protein. The genome-wide association (GWA) analyses under limited and adequate Fe regimes provided in total 18 and 17 significant SNPs, respectively.ConclusionsSignificantly associated SNPs on a genome-wide level under both Fe regimes for the traits leaf necrosis (NEC), root weight (RW), shoot dry weight (SDW), water (H 2O), and SPAD value of leaf 3 (SP3) were located in genes or recognition sites of transcriptional regulators, which indicates a direct impact on the phenotype. SNPs which were significantly associated on a genome-wide level under both Fe regimes with the traits NEC, RW, SDW, H 2O, and SP3 might be attractive targets for marker assisted selection as well as interesting objects for future functional analyses.

High-throughput polymorphism detection and genotyping in Brassica napus using next-generation RAD sequencing

BackgroundThe complex genome of rapeseed (Brassica napus) is not well understood despite the economic importance of the species. Good knowledge of sequence variation is needed for genetics approaches and breeding purposes. We used a diversity set of B. napus representing eight different germplasm types to sequence genome-wide distributed restriction-site associated DNA (RAD) fragments for polymorphism detection and genotyping.ResultsMore than 113,000 RAD clusters with more than 20,000 single nucleotide polymorphisms (SNPs) and 125 insertions/deletions were detected and characterized. About one third of the RAD clusters and polymorphisms mapped to the Brassica rapa reference sequence. An even distribution of RAD clusters and polymorphisms was observed across the B. rapa chromosomes, which suggests that there might be an equal distribution over the Brassica oleracea chromosomes, too. The representation of Gene Ontology (GO) terms for unigenes with RAD clusters and polymorphisms revealed no signature of selection with respect to the distribution of polymorphisms within genes belonging to a specific GO category.ConclusionsConsidering the decreasing costs for next-generation sequencing, the results of our study suggest that RAD sequencing is not only a simple and cost-effective method for high-density polymorphism detection but also an alternative to SNP genotyping from transcriptome sequencing or SNP arrays, even for species with complex genomes such as B. napus.

Comparison of mixed-model approaches for association mapping in rapeseed, potato, sugar beet, maize, and Arabidopsis.

BackgroundIn recent years, several attempts have been made in plant genetics to detect QTL by using association mapping methods. The objectives of this study were to (i) evaluate various methods for association mapping in five plant species and (ii) for three traits in each of the plant species compare the Topt, the restricted maximum likelihood (REML) estimate of the conditional probability that two genotypes carry at the same locus alleles that are identical in state but not identical by descent. In order to compare the association mapping methods based on scenarios with realistic estimates of population structure and familial relatedness, we analyzed phenotypic and genotypic data of rapeseed, potato, sugar beet, maize, and Arabidopsis. For the same reason, QTL effects were simulated on top of the observed phenotypic values when examining the adjusted power for QTL detection.ResultsThe correlation between the Toptvalues identified using REML deviance profiles and profiles of the mean of squared difference between observed and expected P values was 0.83.ConclusionThe mixed-model association mapping approaches using a kinship matrix, which was based on Topt, were more appropriate for association mapping than the recently proposed QK method with respect to the adherence to the nominal α level and the adjusted power for QTL detection. Furthermore, we showed that Toptdiffers considerably among the five plant species but only marginally among different traits.

Co-Variation between Seed Dormancy, Growth Rate and Flowering Time Changes with Latitude in Arabidopsis thaliana

Life-history traits controlling the duration and timing of developmental phases in the life cycle jointly determine fitness. Therefore, life-history traits studied in isolation provide an incomplete view on the relevance of life-cycle variation for adaptation. In this study, we examine genetic variation in traits covering the major life history events of the annual species Arabidopsis thaliana: seed dormancy, vegetative growth rate and flowering time. In a sample of 112 genotypes collected throughout the European range of the species, both seed dormancy and flowering time follow a latitudinal gradient independent of the major population structure gradient. This finding confirms previous studies reporting the adaptive evolution of these two traits. Here, however, we further analyze patterns of co-variation among traits. We observe that co-variation between primary dormancy, vegetative growth rate and flowering time also follows a latitudinal cline. At higher latitudes, vegetative growth rate is positively correlated with primary dormancy and negatively with flowering time. In the South, this trend disappears. Patterns of trait co-variation change, presumably because major environmental gradients shift with latitude. This pattern appears unrelated to population structure, suggesting that changes in the coordinated evolution of major life history traits is adaptive. Our data suggest that A. thaliana provides a good model for the evolution of trade-offs and their genetic basis.

Comparison of Linkage Disequilibrium in Elite European Maize Inbred Lines using AFLP and SSR Markers

Application of association mapping to plant breeding populations has the potential to revolutionize plant genetics. The main objectives of this study were to (i) investigate the extent and genomic distribution of linkage disequilibrium (LD) between pairs of amplified fragment length polymorphism (AFLP) markers, (ii) compare these results with those obtained with simple sequence repeat (SSR) markers, and (iii) compare the usefulness of AFLP and SSR markers for genomewide association mapping in plant breeding populations. We examined LD in a cross-section of 72 European elite inbred lines genotyped with 452 AFLP and 93 SSR markers. LD was significant (p < 0.05) for about 15% of the AFLP marker pairs and for about 49% of the SSR marker pairs in each of the two germplasm groups, flint and dent. In both germplasm groups the ratio of linked to unlinked loci pairs in LD was higher for AFLPs than for SSRs. The observation of LD due to linkage for both marker types suggested that genome-wide association mapping should be possible using either AFLPs or SSRs. The results of our study indicated that SSRs should be favored over AFLPs but the opposite applies to populations with a long history of recombination.

Single Nucleotide Polymorphisms in the Allene Oxide Synthase 2 Gene Are Associated With Field Resistance to Late Blight in Populations of Tetraploid Potato Cultivars

The oomycete Phytophthora infestans causes late blight, the most relevant disease of potato (Solanum tuberosum) worldwide. Field resistance to late blight is a complex trait. When potatoes are cultivated under long day conditions in temperate climates, this resistance is correlated with late plant maturity, an undesirable characteristic. Identification of natural gene variation underlying late blight resistance not compromised by late maturity will facilitate the selection of resistant cultivars and give new insight in the mechanisms controlling quantitative pathogen resistance. We tested 24 candidate loci for association with field resistance to late blight and plant maturity in a population of 184 tetraploid potato individuals. The individuals were genotyped for 230 single nucleotide polymorphisms (SNPs) and 166 microsatellite alleles. For association analysis we used a mixed model, taking into account population structure, kinship, allele substitution and interaction effects of the marker alleles at a locus with four allele doses. Nine SNPs were associated with maturity corrected resistance (P < 0.001), which collectively explained 50% of the genetic variance of this trait. A major association was found at the StAOS2 locus encoding allene oxide synthase 2, a key enzyme in the biosynthesis of jasmonates, plant hormones that function in defense signaling. This finding supports StAOS2 as being one of the factors controlling natural variation of pathogen resistance.

Power to Detect Higher-Order Epistatic Interactions in a Metabolic Pathway Using a New Mapping Strategy

Epistatic interactions among quantitative trait loci (QTL) contribute substantially to the variation in complex traits. The main objectives of this study were to (i) compare three- vs. four-step genome scans to identify three-way epistatic interactions among QTL belonging to a metabolic pathway, (ii) investigate by computer simulations the power and proportion of false positives (PFP) for detecting three-way interactions among QTL in recombinant inbred line (RIL) populations derived from a nested mating design, and (iii) compare these estimates to those obtained for detecting three-way interactions among QTL in RIL populations derived from diallel and different partial diallel mating designs. The single-nucleotide polymorphism haplotype data of B73 and 25 diverse maize inbreds were used to simulate the production of various RIL populations. Compared to the three-step genome scan, the power to detect three-way interactions was higher with the four-step genome scan. Higher power to detect three-way interactions was observed for RILs derived from optimally allocated distance-based designs than from nested designs or diallel designs. The power and PFP to detect three-way interactions using a nested design with 5000 RILs were for both the 4-QTL and the 12-QTL scenario of a magnitude that seems promising for their identification.

Genome-wide association mapping of flowering time and northern corn leaf blight (Setosphaeria turcica) resistance in a vast commercial maize germplasm set

BackgroundSetosphaeria turcica is a fungal pathogen that causes northern corn leaf blight (NCLB) which is a serious foliar disease in maize. In order to unravel the genetic architecture of the resistance against this disease, a vast association mapping panel comprising 1487 European maize inbred lines was used to (i) identify chromosomal regions affecting flowering time (FT) and northern corn leaf blight (NCLB) resistance, (ii) examine the epistatic interactions of the identified chromosomal regions with the genetic background on an individual molecular marker basis, and (iii) dissect the correlation between NCLB resistance and FT.ResultsThe single marker analyses performed for 8 244 single nucleotide polymorphism (SNP) markers revealed seven, four, and four SNP markers significantly (α=0.05, amplicon wise Bonferroni correction) associated with FT, NCLB, and NCLB resistance corrected for FT, respectively. These markers explained individually between 0.36 and 14.29% of the genetic variance of the corresponding trait.ConclusionsThe very well interpretable pattern of SNP associations observed for FT suggested that data from applied plant breeding programs can be used to dissect polygenic traits. This in turn indicates that the associations identified for NCLB resistance might be successfully used in marker-assisted selection programs. Furthermore, the associated genes are also of interest for further research concerning the mechanism of resistance to NCLB and plant diseases in general, because some of the associated genes have not been mentioned in this context so far.