Martin Heckenberger

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.

Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties.

Genetic distances (GDs) based on molecular markers are important parameters for identifying essentially derived varieties (EDVs). In this context information about the variability of molecular markers within maize inbred lines is essential. Our objectives were to (1) determine the variation in the size of simple sequence repeat (SSR) fragments among different accessions of maize inbreds and doubled haploid (DH) lines, (2) attribute the observed variation to genetic and marker system-specific sources, and (3) investigate the effect of SSR fragment size differences within maize lines on the GD between maize lines and their consequences for the identification of essentially derived varieties. Two to five accessions from nine inbred lines and five DH lines were taken from different sources or drawn as independent samples from the same seed lot. Each accession was genotyped with 100 SSR markers that evenly covered the whole maize genome. In total, 437 SSR fragments were identified, with a mean of 4.4 alleles per locus. The average polymorphic information content (PIC) was 0.58. GD estimates between two accessions of the same genotype ranged from 0.00 to 0.12 with an average of 0.029 for inbred lines and 0.001 for DH lines. An average of 11.1 SSRs was polymorphic between accessions of the same inbred line due to non-amplification (8.1 SSRs), heterogeneity (4.0 SSRs) or unknown alleles (2.6 SSRs). In contrast to lab errors, heterogeneity contributed considerably to the observed variation for GD. In order to decrease the probability to be suited for infringing an EDV threshold by chance, we recommend to increase the level of homogeneity of inbred lines before applying for plant variety protection.

Trends in genetic diversity among European maize cultivars and their parental components during the past 50 years

It has been claimed that the system that delivers the products of plant breeding reduces the diversity of cultivated varieties leading to an increased genetic vulnerability. The main goal of our study was to monitor the temporal trends in genetic diversity over the past five decades among maize cultivars with the largest acreage in Central Europe. Our objectives were to (1) investigate how much of the genetic diversity present in important adapted open-pollinated varieties (OPVs) has been captured in the elite flint germplasm pool, (2) examine changes in the genetic diversity among the most important commercial hybrids as well as in their dent and flint parents, (3) analyze temporal changes in allele frequencies between the dent and flint parental inbreds, and (4) investigate linkage disequilibrium (LD) trends between pairs of loci within the set of parental dent and flint lines. We examined 30 individuals of five prominent OPVs from Central Europe, 85 maize hybrids of economic importance, and their dent and flint parental components with 55 SSRs. LD was significant at probability level P=0.01 for 20.2% of the SSR marker pairs in the 82 dent lines and for 17.2% in the 66 flint lines. The dent and flint heterotic groups were clearly separated already at the beginning of hybrid breeding in Central Europe. Furthermore, the genetic variation within and among varieties decreased significantly during the five decades. The five OPVs contain numerous unique alleles that were absent in the elite flint pool. Consequently, OPVs could present useful sources for broadening the genetic base of elite maize breeding germplasm.

Genetic structure and diversity of European flint maize populations determined with SSR analyses of individuals and bulks

Landraces of maize represent a valuable genetic resource for breeding and genetic studies. Using simple sequence repeat (SSR) markers, we analysed five flint maize populations from Central Europe that had played an important role in the pre-hybrid era in Germany. Our objectives were to (1) investigate the molecular genetic diversity within and among the populations based on the SSR analysis of individuals, (2) compare these results of the SSR analysis based on individuals with those based on bulks, (3) examine genotype frequencies for deviations from Hardy–Weinberg equilibrium (HWE) at individual loci, and (4) test for linkage disequilibrium (LD) between pairs of loci within populations. Thirty individuals and their bulked DNA per population were fingerprinted with 55 SSR markers. Across all populations, 46.7% of the SSR markers deviated significantly from HWE, with an excess of homozygosity in 97% of the cases. This excess of homozygosity can largely be explained by experimental errors during the amplification of SSRs apart from genuine genetic causes. Allele frequencies of the SSR analyses of individuals and bulks were significantly correlated (r=0.85, P< 0.01), suggesting that SSR analysis of bulks is very cost-effective for large-scale molecular characterisation of germplasm collections. No evidence for genome-wide LD among pairs of loci was observed, indicating that the populations are well suited for high resolution association mapping studies.

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.

Analysis of a triple testcross design with recombinant inbred lines reveals a significant role of epistasis in heterosis for biomass-related traits in Arabidopsis

Primary causes of heterosis are still unknown. Our goal was to investigate the extent and underlying genetic causes of heterosis for five biomass-related traits in Arabidopsis thaliana. We (i) investigated the relative contribution of dominance and epistatic effects to heterosis in the hybrid C24 × Col-0 by generation means analysis and estimates of variance components based on a triple testcross (TTC) design with recombinant inbred lines (RILs), (ii) estimated the average degree of dominance, and (iii) examined the importance of reciprocal and maternal effects in this cross. In total, 234 RILs were crossed to parental lines and their F1s. Midparent heterosis (MPH) was high for rosette diameter at 22 days after sowing (DAS) and 29 DAS, growth rate (GR), and biomass yield (BY). Using the F2-metric, directional dominance prevailed for the majority of traits studied but reciprocal and maternal effects were not significant. Additive and dominance variances were significant for all traits. Additive × additive and dominance × dominance variances were significant for all traits but GR. We conclude that dominance as well as digenic and possibly higher-order epistatic effects play an important role in heterosis for biomass-related traits. Our results encourage the use of Arabidopsis hybrid C24 × Col-0 for identification and description of quantitative trait loci (QTL) for heterosis for biomass-related traits and further genomic studies.

Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties: II. Genetic and technical sources of variation in AFLP data and comparison with SSR data

Accuracy and reproducibility of genetic distances (GDs) based on molecular markers are crucial issues for identification of essentially derived varieties (EDVs). Our objectives were to investigate (1) the amount of variation for amplified fragment length polymorphism (AFLP) markers found among different accessions within maize inbreds and doubled haploid (DH) lines, (2) the proportion attributable to genetic and technical components and marker system specific sources, (3) its effect on GDs between maize lines and implications for identification of EDVs, and (4) the comparison to published SSR data from the same plant materials. Two to five accessions from nine inbred lines and five DH lines were taken from different sources of maintenance breeding or drawn as independent samples from the same seed lot. Each of the 41 accessions was genotyped with 20 AFLP primer combinations revealing 988 AFLP markers. Map positions were available for 605 AFLPs covering all maize chromosomes. On average, six (0.6%) AFLP bands were polymorphic between different accessions of the same line. GDs between two accessions of the same line averaged 0.013 for inbreds and 0.006 for DH lines. The correlation of GDs based on AFLPs and SSRs was tight (r = 0.97**) across all 946 pairs of accessions but decreased (r = 0.55**) for 43 pairs of accessions originating from the same line. On the basis of our results, we recommend specific EDV thresholds for marker systems with different degree of polymorphism. In addition, precautions should be taken to warrant a high level of homogeneity for DNA markers within maize lines before applying for plant variety protection.

Identification of Essentially Derived Varieties Obtained from Biparental Crosses of Homozygous Lines. III. AFLP Data from Maize Inbreds and Comparison with SSR Data

Genetic distance (GD) estimates based on molecular markers are a preferred approach to estimate genetic conformity between putative essentially derived varieties (EDVs) and their initial varieties (IVs). The objectives of our study were to (1) estimate the variation in the marker-estimated parental contribution (p) to the genome of the progeny, (2) investigate the power of AFLP-based GD estimates for discriminating between homozygous lines with F2, BC1, and BC2 relationships, and (3) compare AFLP data with SSR data from a companion study, as well as with theoretical and simulated results. In total, 104 European maize inbred lines comprising 66 triplets were analyzed with 20 AFLP primer combinations and 100 SSR primer pairs. Each triplet consisted of one F2-, or BC1-derived progeny line and both parental lines. For lines with both F2 and BC1 relationship, mean estimates of p based on AFLP (pF2 = 0.46; pBC1 = 0.70) or SSR data (pF2 = 0.45; pBC1 = 0.67) were lower than the respective expected values of 0.50 and 0.75. Considerable differences were detected between AFLP- and SSR-based mean GD estimates for unrelated inbred lines due to the lower degree of polymorphism of AFLPs compared with SSRs. With each marker system, the GD between progeny lines and parents was little affected by the variation in GD between the parents, particularly for lines with BC1 relationship. Substantial differences in Type I (α) and Type II (β) errors were detected between flint and dent germplasm pools with different marker systems and when fixed EDV thresholds were considered. To counterbalance advantages and disadvantages of AFLP and SSRs, a complementary application of the two marker systems is suggested for discrimination between EDVs and independently derived varieties (IDVs).

Identification of essentially derived varieties with molecular markers: an approach based on statistical test theory and computer simulations.

Genetic similarities (GS) based on molecular markers have been proposed as a tool for identification of essentially derived varieties (EDVs). Nevertheless, scientifically reliable criteria for discrimination of EDVs and independently derived varieties with GS estimates are scanty, and implementation into practical breeding has not yet taken place. Our objectives were to (1) assess the influence of chromosome number and length, marker density, and distribution, as well as the degree of polymorphism between the parental inbreds on the distribution of GS between parental inbreds and their progenies [GS(P1,O)] derived from F2 and different backcross populations and (2) evaluate these factors with regard to the power for distinguishing F2- versus BC1- and BC1- versus BC2-derived lines with molecular markers. We developed an approach based on statistical test theory for the identification of EDVs with molecular markers. Standard deviations and overlaps of distributions of GS(P1,O) of F2-, BC1-, and BC2-derived lines were smaller with (1) increasing chromosome number and length, (2) increasing marker density, and (3) uniformly instead of randomly distributed markers, approaching a lower boundary determined by the genetic parameters. The degree of polymorphism between the parental inbreds influenced the power only if the remaining number of polymorphic markers was low. Furthermore, suggestions are made for (1) determining the number of markers required to ascertain a given power and (2) EDV identification procedures.