Browsing by Person "Melchinger, Albrecht E."

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Application of Near-Infrared Spectroscopy in Plant Breeding Programs
(2006) Montes, Juan Manuel; Melchinger, Albrecht E.
The success of plant breeding programs depends on the availability of genetic variation and efficient data collection processes that allow large-scale screenings of genotypes. When genetic variation is present, the goal is to identify those genotypes that are closest to the breeding objectives. In this context, the evaluation of a large number of genotypes requires optimization of the data collection process in order to provide reliable information for making selection decisions. The process of data collection must yield an accurate and precise assessment of genotypes timely because the information is needed to plan the next generation for breeding and cultivar development. Laboratory NIRS is routinely used in the data collection process of many breeding programs, but it requires the withdrawal of field plot samples and involves manual work. Applications of the near-infrared spectroscopy on choppers (NOC) and near-infrared spectroscopy on combine harvester (NOCH) are a step forward to the automation of data collection processes, by which sampling, labor, and sources of error in the data can be reduced. The objective of this thesis research was to assess the potential of NOC and NOCH for application in breeding programs of grain maize, rapeseed, and silage maize. Plot combine harvesters and choppers were equipped with diode-array spectrometers for collection of near-infrared plot spectra, and used to harvest experimental varieties of breeding programs in Central Europe. Two alternative sample presentation designs (conveyor belt and spout) were used for the NOC systems. The NOCH systems used the conveyor belt as sample presentation design. NOCH showed a high potential for determination of dry matter (DM), crude protein (CP), and starch (ST) contents of maize grain. NOCH calibration models yielded standard errors of prediction (SEP) and coefficients of determination of validation (R2V) of 1.2% and 0.95 for DM, 0.3% and 0.88 for CP, and 1.0% and 0.79 for ST, respectively. The potential of NOCH for determination of DM, CP, oil and glucosinolate contents of rapeseed was also high. NOCH calibration models yielded standard errors of cross validation (SECV) and coefficients of determination of cross validation (R2CV) of 0.3% and 0.96 for DM, 0.6% and 0.69 for CP, 0.9% and 0.71 for oil, and 2.2 μmol/g and 0.40 for glucosinolate, respectively. The NOC systems showed high potential for the determination of DM, ST, and soluble sugars (SS) content of silage maize hybrids. The NOC system equipped with a conveyor belt design yielded calibration models with SEP and R2V of 0.9% and 0.93 for DM, and 2.1% and 0.78 for ST, respectively. For the NOC system equipped with the spout design, the SEP and R2V amounted to 1.4% and 0.84 for DM, 2.3% and 0.75 for ST, and 0.9% and 0.81 for SS. The potential of both NOC systems for determination of fiber contents (CF, ADF, and NDF), digestibility and energy-related traits was lower than for DM, ST, and SS. The precision of NOCH for the determination of DM content in maize grain was higher than by traditional drying-oven method. A higher precision of NOCH is also expected for other traits and may also be extended to the NOC systems because the sampling error associated with traditional processes of data collection is reduced drastically by NOC and NOCH. The investigation of the effects caused by the calibration technique, mathematical transformation of the near-infrared spectra, and scatter correction on the development of NOCH calibration models for the prediction of DM, CP, and ST content in maize grain revealed that calibration technique was the most important factor affecting the prediction ability, whereas the importance of mathematical transformation and scatter correction depended on the particular constituent considered. Presently, there exists high uncertainty about the optimal NOC and NOCH sample presentation designs for agricultural harvesters. The dynamic signal range, i.e., the range of spectral values on which predictions are based, and the amount of plot material measured were identified as guide parameters for optimization of sample presentation designs. In addition, calibration transferability between NOC systems with different sample presentation designs proved to be feasible after merging spectra from both NOC systems in the calibration set. In conclusion, NOC and NOCH show high potential for replacing laboratory NIRS analysis of several traits in a plant breeding context and yield a more accurate and precise evaluation of field plot characteristics. Therefore, technological applications of the electromagnetic radiation is predicted to have a high impact in plant breeding, precision farming, and agriculture.
Assessing the Genetic Diversity in Crops with Molecular Markers: Theory and Experimental Results with CIMMYT Wheat and Maize Elite Germplasm and Genetic Resources
(2004) Reif, Jochen Christoph; Melchinger, Albrecht E.
Genetic diversity is a valuable natural resource and plays a key role in future breeding progress. Germplasm collections as a source of genetic diversity must be well-characterized for an efficient management and effective exploitation. The advent of PCR-based molecular markers such as sim-ple sequence repeats (SSRs) has created an opportunity for fine-scale genetic characterization of germplasm collections. The objective of this research was to optimize the utilization of genetic re-sources conserved at the International Wheat and Maize Improvement Center (CIMMYT), with the aid of DNA markers. Choice of suitable dissimilarity measures is important to facilitate the interpretation of findings from DNA marker studies on a theoretically sound basis. The objective of a theoretical study was to examine 10 dissimilarity coefficients widely used in germplasm surveys, with special focus on applications in plant breeding and seed banks. The distance and Euclidean properties of the dissimi-larity coefficients were investigated as well as the underlying genetic models. Application areas for different coefficients were suggested on the basis of the theoretical findings. It has been claimed that plant breeding reduces genetic diversity in elite germplasm, which could seriously jeopardize the continued ability to improve crops. The objectives of the presented ex-perimental study with wheat were to examine the loss of genetic diversity during (i) domestication of the species, (ii) change from traditional landrace cultivars (LC) to modern breeding varieties, and (iii) intensive selection over 50 years of international breeding. A sample of 253 CIMMYT or CIMMYT-related modern wheat cultivars, LC, and Triticum tauschii accessions were characterized with up to 90 SSR markers covering the entire wheat genome. A loss of genetic diversity was ob-served from T. tauschii to LC and from LC to the elite breeding germplasm. Wheat genetic diver-sity was narrowed from 1950 to 1989, but was enhanced from 1990 to 1997. The results indicate that breeders averted the narrowing of the wheat germplasm base and subsequently increased the genetic diversity through the introgression of novel materials. The LC and T. tauschii contain nu-merous unique alleles that were absent in modern wheat cultivars. Consequently, both LC and T. tauschii represent useful sources for broadening the genetic base of elite wheat breeding germ-plasm. In the 1980's, CIMMYT generated more than 100 maize populations and pools but little is known about the genetic diversity of this germplasm. The objective of the study with 23 CIMMYT maize populations was to characterize their population genetic structure with SSRs. The populations adapted to tropical, subtropical intermediate-maturity, subtropical early-maturity, and temperate mega-environments (ME) were fingerprinted with 83 SSR markers. Estimates of genetic differen-tiation between populations revealed that most of the molecular variation was found within the populations. Principal coordinate analysis based on allele frequencies of the populations revealed that populations adapted to the same ME clustered together and, thus, supported clearly the ME structure. Novel strategies were suggested to optimize the conservation of the genetic diversity within and among the populations. Heterotic groups and patterns are of fundamental importance in hybrid breeding. The objective of the presented study with a subset of 20 out of the 23 maize populations was to investigate the rela-tionship between heterosis and genetic distance determined with SSR markers. The published data of three diallels and one factorial trial evaluated for grain yield were re-analyzed to calculate het-erosis in population hybrids. Correlations of squared modified Rogers distance and heterosis were mostly positive and significant, but adaption problems caused deviations in some cases. For popu-lations adapted to the target regions, genetic distance can be used as a further criterion in the search for promising heterotic patterns and groups. For intermediate- and early-maturity subtropical germ-plasm, two heterotic groups were suggested, consisting of a flint and dent composite. For the tropi-cal germplasm, it was possible to assign population (Pop29) to the established heterotic group A and propose new heterotic groups (Pop25, Pop43). Our experimental results corroborate that SSRs are a powerful tool to (i) detect relationships among different germplasm, (ii) assess the level of genetic diversity present in germplasm pools and its flux over time, and (iii) search for promising heterotic groups for hybrid breeding in complementa-tion to field trials.
Association analysis of genes controlling variation of flowering time in West and Central African sorghum
(2012) Bhosale, Sankalp; Melchinger, Albrecht E.
Sorghum is extremely important for the food security in the arid to semi-arid regions of West and Central Africa (WCA). A serious constraint to the sorghum production in WCA is the scattered beginning but relatively fixed end of the rainy season among years, forcing farmers to adjust their individual sowing dates according to the start of the rains. Owing to the delayed sowing and fixed end of the rainy season, farmers require varieties that flower at the end of the rainy season, regardless of the sowing date. Photoperiod sensitivity of sorghum accessions is an important adaptation trait that allows flowering or synchronized flowering of the accessions at the end of the rainy season. This is also particularly important in avoiding grain mold, insect and bird damages for early maturing varieties, and incomplete grain filling due to soil water shortage occurring at the end of the season in late maturing varieties. Cultivars with photoperiod sensitivity may have the potential to increase yield and yield stability. Unfortunately, in WCA most of the present day cultivars are photoperiod insensitive. Furthermore, unavailability of simple screening methods in selecting photoperiod sensitive cultivars complicates the situation. Breeding techniques such as marker assisted selection (MAS) by employment of molecular markers would greatly enhance the selection efficiency for this major adaptation trait. Candidate-gene (CG) based association studies can assist in investigating the effect of polymorphisms in flowering time genes on phenotypic variation. Allele-specific molecular markers can be developed after a significant marker-phenotype association is identified. These markers can effectively be used in MAS of photoperiod sensitive sorghum cultivars. In this study we carried out a CG based association analysis to investigate the association between variation for photoperiodic sensitivity of flowering time in sorghum and polymorphisms in six partially amplified genes putatively related to variation in flowering time. Five out of six CGs were known to be involved in photoperiod pathway of flowering time [CRYPTOCHROME 1 (CRY1-b1), CRYPTOCHROME 2 (CRY2), LATE ELONGATED HYPOCOTYL (LHY), GIGANTEA (GI), HEADING DATE 6 (HD6)], and the gene SbD8 was involved in the gibberellic acid (GA) pathway of flowering time. In the first part of the study we determined the presence, the expression and the molecular diversity of genes homologous to the important flowering time gene D8 in maize on a set of 26 sorghum and 20 pearl millet accessions. Homologs of D8 were successfully amplified and tested for their expression in sorghum (SbD8) and pearl millet (PgD8). Pearl millet, because of its autogamous nature, showed higher nucleotide diversity than sorghum, which is an allogamous species. In maize, a 6 bp deletion flanking the SH2-like domain of D8 was found to be significantly associated with flowering by Thornsberry et al. (2001). We found in the PgD8 gene a 3 bp insertion or deletion (Indel) flanking the SH2 domain in the region, which was only conserved between D8 and PgD8. Cluster analysis performed for the D8, SbD8, and PgD8 indicated that maize is more closely related to pearl millet than sorghum. These findings suggest that, similar to maize, the indel in PgD8 flanking the SH2 domain might play an important role in determination of flowering. It is advisable to carry out an association study to reveal the potential role of PgD8 in flowering time control in pearl millet. After successfully amplifying and confirming the expression of SbD8 and PgD8, we carried out the association analysis on the selected CGs. A panel of 219 mostly inbred accessions of sorghum from major sorghum growing areas in WCA was complied. In the second part of the study the association analysis panel of accessions was phenotyped for their flowering response in the field in 2007 in Mali. The entire panel was sown twice (June and July), photoperiod response index (PRI) was estimated as the difference between DFL50% of the two sowing dates of the accessions. The PRI of the accessions showed a wide range from close to zero (photoperiod-insensitive) up to values close to 30 or above (highly-photoperiod sensitive). This result confirmed that the range of response based on the choice of the accessions was appropriate for an association analysis. The plant height reduction observed in accessions sown in July compared to the once sown in June was in accordance with previous studies performed in West African sorghum varieties. The sorghum accessions were genotyped using 27 simple sequence repeat markers. Population structure analysis using software STRUCTURE was carried out to control the false positives in the association analysis. The results showed existence of two subgroups in our sorghum accessions. The first subgroup included mainly race guinea (83%) originating from western West African countries such as Mali and Bukina Faso and the second subgroup included accessions mainly from Nigeria and Niger and also accessions originating from other countries and other major races. The race guinea could clearly be distinguished from the other races. Fisher's exact test for the presence of earliness among subgroups showed that there are significantly (p = 0.06) more early maturing accessions in subgroup one than subgroup two. But there was an absence of a clear structuring pattern. The study suggests that the race, the geographical origin, and maturity of the accessions are the most likely forces behind the observed structuring pattern of the accessions. We found a high level of genetic diversity among the sorghum accessions. Race guinea was found to be the most diverse and race kaura was the least diverse. In general, the estimates of the gene diversity were comparable to previous studies. The results showed that clustering of early-intermediate maturing guinea varieties may have increased the linkage disequilibrium (LD) in subgroup one compared to subgroup two. The differences in the extent of LD between our study and those in the previous studies can be due to the differences in the molecular markers used as well as differences in the racial composition of the accessions studied. In the final part of the study the association analysis was carried out using a mixed-model method. This method takes both population structure and kinship information into account. The candidate genes polymorphism data were obtained by amplifying and sequencing of the chosen genes. The association analysis for the polymorphism found within the CGs was carried out using values of PRI for each accession. From the six genes studied, genes CRY1-b1 and GI had several polymorphic sites which were significantly (p < 0.005) associated with PRI variation in the sorghum panel. The most important polymorphism in the gene CRY1-b1 showed an effect on PRI value of up to -4.2 days. This single nucleotide polymorphism (SNP) at position 722 in CRY1-b1 was located in the flavin adenine dinucleotide binding domain (N-terminal domain) of SbCRY1; hence, this domain appears to be important in photomorphogenesis in sorghum. In the case of the GI gene homolog, SNP888 had the largest effect on PRI of about +8 days. Similar to the studies in rice, the GI gene delayed flowering under June sowing (long-day conditons) and shortened the time to flower in sorghum under July sowing (short-day conditons). Therefore, the action of the GI gene homolog in sorghum might be revealed by a detailed investigation of GI by comparison of sorghum accessions grown under short-day and long-day conditions. In the case of gene SbD8, no significant association with PRI could be found; hence, the potential involvement of this gene in flowering time control of sorghum was not confirmed. Negative Tajima?s D values, of CGs indicated that the genes may have been subjected to adaptive selection as variation in flowering time may confer adaptive advantages in sorghum. The results showed that CG-based association analysis using a mixed model approach can be successfully applied to unravel the genetic variation related to phenotypic variation in flowering time. The polymorphisms significantly associated with PRI can be used to develop cleaved amplified polymorphic sequence markers. Functional markers could also be created directly from the significant SNPs. These molecular markers can serve as powerful tools in MAS for sorghum to identify cultivars sensitive to photoperiod.
Biometrical Analyses of Epistasis and the Relationship between Line per se and Testcross Performance of Agronomic Traits in Elite Populations of European Maize (Zea mays L.)
(2005) Mihaljevic, Renata; Melchinger, Albrecht E.
Relations of yield and other important agronomic traits of inbred lines to the same traits in hybrids have been studied from the time of initiation of hybrid breeding to the present. Because crossing lines to a tester and conducting yield trials are expensive and time-consuming, reliable information on inbred lines that is indicative of their testcross performance is crucial for optimum testing schemes in hybrid breeding as well as simultaneous improvement of commercial hybrids and their inbred parents. It has therefore been of great importance to determine the magnitude of correlation between line per se performance (LP) and testcross performance (TP) and investigate if epistasis influences this correlation. The comprehensive study on hand was performed with five populations (F3 to F6 lines) differing in size (ranging from 71 to 344), level of inbreeding, and the number of common parents. The populations employed were derived from three biparental crosses within the heterotic pool of European elite flint maize (Zea mays L.). All five populations were evaluated for TP (using an unrelated dent tester inbred) of five agronomically important quantitative traits: grain yield, grain moisture, kernel weight, protein concentration, and plant height. Four of these populations were also evaluated for LP of the same five traits. The objectives were to (i) estimate phenotypic and genotypic correlations between LP and TP within four populations for all five traits, (ii) map quantitative trait loci (QTL) for LP and TP in four and five populations, respectively, for all five traits, (iii) validate estimated QTL effects and positions for TP by assessing QTL congruency among testcross populations differing in size and genetic background, (iv) determine the value of LP-QTL for the prediction of TP, (v) estimate the importance of epistatic effects for LP and TP of grain yield and grain moisture by generation means analysis as well as genome-wide testing for epistatic marker pairs, and (vi) draw conclusions regarding the prospects of marker-assisted selection (MAS). Genotypic correlations between LP and TP, rg(LP, TP), estimated herein were comparable with those obtained for European flint or U.S. dent material. The magnitude of rg(LP, TP) was trait-specific: for traits of high heritability, i.e. grain moisture, kernel weight, protein concentration, and plant height, estimates were generally larger than 0.7 across all four populations, whereas for grain yield, estimates were consistently lower and did not exceed the intermediate level of 0.5. For grain yield, lowest rg(LP, TP) were estimated with lowest precision (largest confidence intervals). This requires testing for both LP and TP and/or combining the data in a selection index to ensure sufficient inbred performance (seed production) and yield improvement. However, combined selection for LP and TP proved less efficient than sole selection for TP unless unadapted material was employed. For kernel weight, protein concentration, and plant height, we detected "large" congruent QTL across testcross populations derived from the same cross, which individually explained up to 46% of the validated genotypic variance p. However, as the p values estimated from validation were still below the corresponding heritability estimates, MAS will be superior to phenotypic selection only if it is more cost-efficient. For the above traits, similar numbers of QTL for LP and TP were detected across populations. More than half of the QTL regions detected for LP were in common for LP and TP in the largest population (N = 280). To assess the value of QTL identified for LP in predicting TP, we calculated the genotypic correlation rg(MLP, YTP). This parameter assesses QTL congruency for LP and TP quantitatively and is thus the key parameter for assessing the prospects of MAS. The number of common QTL for LP and TP (qualitative QTL congruency) was generally not indicative of the magnitude of rg(MLP, YTP) due to the differences in the effect size of the respective QTL detected for LP and used for the prediction of TP. For all traits, rg(MLP, YTP) were smaller than rg(LP, TP). This is because rg(MLP, YTP) is only predictive for the validated proportion of genotypic variance explained by the QTL for LP, which was generally below 50% because of the limited power of QTL detection, in particular with small sample sizes below 100. Only if QTL detected for LP explain a substantial proportion of the genotypic variance, MAS based on these QTL can be applied, provided it is more cost-efficient than an indirect phenotypic selection for TP based on LP. QTL detection power was drastically reduced for the complex trait grain yield with a presumably large number of small QTL underlying its genetic architecture. Thus, the number of common QTL for LP and TP as well as the QTL congruency across testcross populations was much lower for grain yield than the other four traits. Estimated gene action of QTL detected for LP was primarily additive for grain yield. Evidence for dominance and/or epistasis, which may be a reason for the low rg(LP, TP) and the low number of common QTL for LP and TP was generally weak. Both generation means analysis for LP and TP and genome-wide search for epistatic marker pairs yielded no evidence for epistasis. This is not only because the detected epistatic effects could not be validated, but also because there is low chance to find epistasis unless the generation examined displays the full epistatic variance such as expected from doubled haploids produced from an F1 cross. Thus, it is anticipated that the relative importance of epistatic effects in hybrid maize breeding may strongly increase with the currently happening shift in line development from recurrent selfing towards the production of doubled haploids.
Can we abandon phosphorus starter fertilizer in maize? Results from a diverse panel of elite and doubled haploid landrace lines of maize (Zea mays L.)
(2022) Roller, Sandra; Weiß, Thea M.; Li, Dongdong; Liu, Wenxin; Schipprack, Wolfgang; Melchinger, Albrecht E.; Hahn, Volker; Leiser, Willmar L.; Würschum, Tobias
The importance of phosphorus (P) in agriculture contrasts with the negative environmental impact and the limited resources worldwide. Reducing P fertilizer application by utilizing more efficient genotypes is a promising way to address these issues. To approach this, a large panel of maize (Zea mays L.) comprising each 100 Flint and Dent elite lines and 199 doubled haploid lines from six landraces was assessed in multi-environment field trials with and without the application of P starter fertilizer. The treatment comparison showed that omitting the starter fertilizer can significantly affect traits in early plant development but had no effect on grain yield. Young maize plants provided with additional P showed an increased biomass, faster growth and superior vigor, which, however, was only the case under environmental conditions considered stressful for maize cultivation. Importantly, though the genotype-by-treatment interaction variance was comparably small, there is genotypic variation for this response that can be utilized in breeding. The comparison of elite and doubled haploid landrace lines revealed a superior agronomic performance of elite material but also potentially valuable variation for early traits in the landrace doubled haploid lines. In conclusion, our results illustrate that breeding for P efficient maize cultivars is possible towards a reduction of P fertilizer in a more sustainable agriculture.
Characterization and management of Jatropha curcas L. germplasm
(2018) Senger, Elisa; Melchinger, Albrecht E.
Jatropha curcas L. (jatropha) is a perennial plant of the Euphorbiaceae family that grows in the tropics and subtropics worldwide. Jatropha is targeted to be grown in marginal environments. The seeds are used mainly for production of food products and bioenergy, amongst others. Jatropha breeding is at an early stage. The first obstacle is to generate competitive cultivars for economically feasible cultivation. Mayor breeding objectives are to increase seed yield and yield stability, to decrease production costs, and to improve product quality adapted to specific markets. Jatropha breeding needs to be optimized in several research areas, such as methods and tools for germplasm characterization and breeding techniques, while considering requirements of the agronomic management and product processing. The germplasm can be separated into two naturally occurring germplasm pools that differ in the presence of phorbol esters (PE). These chemical compounds have antinutritional effects on humans and animals and cannot be inactivated or eliminated from the plant material on an industrial scale yet. Therefore, food production is based on cultivars lacking PE, while bioenergy production is less affected from PE presence. The germplasm needs to be characterized and grouped depending on breeding objectives and strategies. Tools for identification of plants that synthesize PE exist, but bear decisive disadvantages or need to be advanced. These tools are exploited for germplasm management and food safety strategies. The objectives of this study were to i) examine the variation of relevant traits among genotypes and between germplasm pools, ii) estimate phenotypic and genotypic trait correlations, iii) investigate location effects and genotype by environment interactions, iv) investigate parental and heterotic effects of genotypes from different germplasm pools as well as the effect of the mating type on expression of relevant traits, and v) develop recommendations for implementation of the findings in jatropha breeding programs. In the first two publications, stress response was investigated. Leaf chlorophyll content (SPAD) was used as a dynamic trait that can be influenced by e.g. water stress and nutrient deficiency. Different genotypes were screened at several locations and at different time points. High genetic diversity was found not only in stress response but also in SPAD value. The fast and non-destructive method is highly promising to be applied in further screenings or stress response studies. In the second publication, genotypic differences in aluminum tolerance were found among seedlings in a greenhouse trial. The rapid test method is applicable in further screenings. However, it needs to be proven that aluminum tolerance at the seedling stage observed under greenhouse conditions is expressed also at later plant developmental stages in the field. In the consecutive three publications, several traits were assessed on seeds and seedlings to detect significant differences between genotypes and/or between germplasm pools. Such traits would be highly valuable for germplasm management. We found that random variation is a disadvantage of quantitative traits and hinders clear assignment of each experimental unit to the respective germplasm pool. Thus, qualitative traits might be favored, such as the “silver shimmer inside the seed testa” that differentiated toxic from non-toxic seeds with a low error rate. However, these results need to be validated. Another application area of the investigated traits is the identification of self-fertilized material within hybrid progeny. In our study, self-fertilized seeds could be differentiated from cross-fertilized ones in specific genotype combinations. Similarly, many seedling traits showed heterotic effects. In the sixth publication, genotype by environment interactions were investigated and recommendations for breeding programs elaborated. A large set of genotypes was grown for four years at three different locations. We showed that selection at only one testing location is highly risky because cultivars with low yield stability could be selected. Therefore, it is indispensable for breeders to work in a network of testing locations that differ in edapho-climatic conditions and apply appropriate experimental designs and statistical tools. In the final publication, several parameters related to the nutritional value of kernels of non-toxic genotypes grown at two locations were assessed. The high nutritional value of this material was presented and compared to soybean, peanut, hazelnut, and corn. Furthermore, preliminary conclusions related to location effects and product processing were drawn. The findings of this thesis contribute to characterization of this novel crop with regard to stress tolerances, seed and seedling characteristics as well as food quality, and help to increase breeding efficiency by presenting simple methods for fast genotype screening as well as grouping of germplasm and by efficient exploitation of testing facilities.
Comparison of omics technologies for hybrid prediction
(2019) Westhues, Matthias; Melchinger, Albrecht E.
One of the great challenges for plant breeders is dealing with the vast number of putative candidates, which cannot be tested exhaustively in multi-environment field trials. Using pedigree records helped breeders narrowing down the number of candidates substantially. With pedigree information, only a subset of candidates need to be subjected to exhaustive tests of their phenotype whereas the phenotype of the majority of untested relatives is inferred from their common pedigree. A caveat of pedigree information is its inability to capture Mendelian sampling and to accurately reflect relationships among individuals. This shortcoming was mitigated with the advent of marker assays covering regions harboring causal quantitative trait loci. Today, the prediction of untested candidates using information from genomic markers, called genomic prediction, is a routine procedure in larger plant breeding companies. Genomic prediction has revolutionized the prediction of traits with complex genetic architecture but, just as pedigree, cannot properly capture physiological epistasis, referring to complex interactions among genes and endophenotypes, such as RNA, proteins and metabolites. Given their intermediate position in the genotype-phenotype cascade, endophenotypes are expected to represent some of the information missing from the genome, thereby potentially improving predictive abilities. In a first study we explored the ability of several predictor types to forecast genetic values for complex agronomic traits recorded on maize hybrids. Pedigree and genomic information were included as the benchmark for evaluating the merit of metabolites and gene expression data in genetic value prediction. Metabolites, sampled from maize plants grown in field trials, were poor predictors for all traits. Conversely, root-metabolites, grown under controlled conditions, were moderate to competitive predictors for the traits fat as well as dry matter yield. Gene expression data outperformed other individual predictors for the prediction of genetic values for protein and the economically most relevant trait dry matter yield. A genome-wide association study suggested that gene expression data integrated SNP interactions. This might explain the superior performance of this predictor type in the prediction of protein and dry matter yield. Small RNAs were probed for their potential as predictors, given their involvement in transcriptional, post-transcriptional and post-translational regulation. Regardless of the trait, small RNAs could not outperform other predictors. Combinations of predictors did not considerably improve the predictive ability of the best single predictor for any trait but improved the stability of their performance across traits. By assigning different weights to each predictor, we evaluated each predictors optimal contribution for attaining maximum predictive ability. This approach revealed that pedigree, genomic information and gene expression data contribute equally when maximizing predictive ability for grain dry matter content. When attempting to maximize predictive ability for grain yield, pedigree information was superfluous. For genotypes having only genomic information, gene expression data were imputed by using genotypes having both, genomic as well as gene expression data. Previously, this single-step prediction framework was only used for qualitative predictors. Our study revealed that this framework can be employed for improving the cost-effectiveness of quantitative endophenotypes in hybrid prediction. We hope that these studies will further promote exploring endophenotypes as additional predictor types in breeding.
Design of breeding strategies for energy maize in Central Europe
(2012) Grieder, Christoph; Melchinger, Albrecht E.
The area of maize (Zea mays L.) grown for production of biogas has tremendously increased in Germany during the past decade. Thus, breeding companies have a keen interest to develop special varieties for this new market segment. A high methane yield per area (MY), which depends multiplicatively on dry matter yield (DMY) and methane fermentation yield (MFY), is required to ensure the efficiency of biogas maize cultivation. However, information on the targeted biogas maize ideotype is still missing and estimates of relevant quantitative genetic parameters for representative material are required to design optimum breeding strategies. We conducted a large field experiment to assess the relevant traits in biogas maize, their variation, and associations among them. In detail, our objectives were to (1) determine MFY and its production kinetics as well as the chemical composition, (2) examine the relationship of MFY and traits related to its kinetics with plant chemical composition and silage quality traits like in vitro digestible organic matter (IVDOM) and metabolizable energy concentration (MEC); (3) examine the potential of near infrared spectroscopy (NIRS) for prediction of traits related to methane production; (4) evaluate a large population of inbred lines and their testcrosses under field conditions for agronomic and quality traits; (5) estimate variance components and heritabilities (h2) of traits relevant to biogas production; (6) study correlations among traits as well as between inbred line per se (LP) and testcross performance (TP); and (7) draw conclusions for breeding maize as a substrate for biogas production. For this purpose, a representative set of 285 dent inbred lines from diverse origins and their 570 testcross progenies with two adapted flint testers was produced. Both material groups were evaluated in field experiments conducted in six environments (three locations, two years) in Germany. For analysis of MFY, samples of a diverse core set of 16 inbred lines and their 32 testcrosses were analyzed using the Hohenheim Biogas Yield Test, a discontinuous, laboratory fermentation assay. The kinetics of methane production was assessed by non-linear regression. Estimates of h2 for MFY measured after short fermentation time (3 days) were high, but genotypic variance and, therefore, also h2 decreased towards the end of the fermentation period (35 days). This was presumably the consequence of a nearly complete degradation of all chemical components during the long fermentation period. This interpretation was supported by strong correlations of MFY with chemical components, IVDOM and MEC for the early, but not the late fermentation stages. Based on the samples in the core set, NIRS calibrations were developed for MFY, parameters related to the kinetics of methane production, and chemical composition. With a coefficient of determination from validation (R2V) of 0.82, accuracy of prediction was sufficiently high for the maximum methane production rate, which is related to the early fermentation phase, but not satisfactory for the time needed to reach 95% of a sample?s final MFY (R2V = 0.51). In agreement with the trend of h2, performance of NIRS to predict MFY on day 35 (R2V = 0.77) was lower than for MFY on day 3 (R2V = 0.85), but still at a satisfactory level, as was the case for concentrations of different chemical components. Hence, NIRS proved to be a powerful tool for prediction of MFY and chemical composition in the main experiment. For TP, estimates of variance components from the main experiments revealed that general combining ability (GCA) was the major source of variation. The very tight correlation of MY with DMY but not with MFY indicated that variation in MY was primarily attributable to differences in DMY. Compared to MEC, MFY showed a weaker association with chemical composition. Genotypic correlation (rg) of MFY was strongest with non-degradable lignin (-0.58). Correlation of MFY with starch was not significant and indicated a lower importance of high cob proportions for biogas maize than for forage maize. Hence, to improve MY, selection should primarily focus on increasing DMY. Results for LP in the main experiment largely confirmed results from testcrosses and favor selection for high dry matter yielding genotypes with less emphasis on ear proportion. Estimates of rg between LP and GCA were highest (> 0.94) for maturity traits (days to silking, dry matter concentration) and moderate (> 0.65) for DMY and MY. Indirect selection for GCA on basis of LP looks promising for maturity traits, plant height, and to some extent also for DMY.
Development and applications of Plabsoft : a computer program for population genetic data analyses and simulations in plant breeding
(2008) Maurer, Hans Peter; Melchinger, Albrecht E.
Marker-assisted breeding approaches are promising tools for enhancement of the conventional plant breeding process. They have been successfully applied in many areas such as plant variety protection, classification of germplasm, assessment of genetic diversity, mapping of genes underlying important agronomic traits, and using the mapping information for selection decisions. Powerful and flexible bioinformatic tools are urgently required for a better integration of molecular marker applications and classical plant breeding methods. The objective of my thesis work was to develop and apply Plabsoft, a computer program for population genetic data analyses and simulations in plant breeding. The assumption of Hardy-Weinberg equilibrium is a cornerstone of many concepts in population and quantitative genetics. Therefore, tests for Hardy-Weinberg equilibrium are of crucial importance, but the assumptions underlying asymptotic chi-square tests are often not met in datasets from plant breeding programs. I developed and implemented in Plabsoft a new algorithm for exact tests of Hardy-Weinberg equilibrium with multiple alleles. The newly derived algorithm has considerable computational advantages over previously described algorithms and extends substantially the range of problems that can be tested. Knowledge about the amount and distribution of linkage disequilibrium (LD) in breeding populations is of fundamental importance to assess the prospects for gene mapping with whole-genome association studies. To analyze LD in breeding populations, I implemented various LD measures in Plabsoft and developed a new significance test for these LD measures. The routines were employed to analyze LD in 497 elite maize lines from a commercial hybrid breeding program, which were fingerprinted by 81 simple sequence repeat (SSR) markers covering the entire genome. Strong LD was detected and, therefore, whole-genome association studies were recommended as promising. However, LD between unlinked loci will most likely result in a high rate of false positives. The prediction of hybrid performance with DNA markers facilitates the identification of superior hybrids. The single marker models used so far do not take into account the correlation between allele frequencies at linked markers. To overcome this problem, the concept of haplotype blocks was proposed. I developed and implemented in Plabsoft three alternative algorithms for haplotype block detection suitable for plant breeding. The algorithms were applied for the haplotype-based prediction of the hybrid performance of 270 hybrids, the parents of which were fingerprinted with 20 amplified fragment length polymorphism (AFLP) primer combinations. Employing haplotypes resulted in an improved prediction of hybrid performance compared with single marker models. Consequently, haplotype-based prediction methods have a high potential to improve substantially the efficiency of hybrid breeding programs. Computer simulations can be employed to solve population genetic problems in plant breeding, for which the simplifying assumptions underlying the classical population genetic theory do not hold true. However, before the start of my thesis no flexible simulation software was available. I developed algorithms for simulation of single breeding steps and entire plant breeding programs and implemented these in Plabsoft. The routines allow the simulation of plant breeding programs as they are conducted in practice. The simulation routines of Plabsoft were validated by simulating two marker-assisted backcross programs in rice conducted by the International Rice Research Institute (IRRI). In the simulations, the frequency distributions of the proportion of recurrent parent genome in the backcross populations were assessed. The simulation results were in good agreement with the experimental data. Therefore, computer simulations are a useful tool for pre-test estimation of selection response in marker-assisted backcrossing. The application of Plabsoft was exemplified by two studies in maize. In the first study, the expected LD decay in the intermating generations of two recurrent selections programs was determined with simulations. This application demonstrates the use of Plabsoft to solve problems for which analytical results are not available. In the second study, the forces generating and maintaining LD in a hybrid maize breeding program were investigated with computer simulations. This application demonstrates the capability of modeling complex long-term breeding programs as performed in practice. The studies of my thesis provide an example for the broad range of possible applications of Plabsoft. In addition to the presented studies, Plabsoft has so far been employed in about 40 further studies, which corroborates the usefulness of Plabsoft for integrating new genomic tools in applied plant breeding programs.
Development and fine mapping of markers closely linked to the SCMV resistance loci Scmv1 and Scmv2 in European maize (Zea mays L.)
(2002) Dußle, Christina M.; Melchinger, Albrecht E.
Sugarcane mosaic virus (SCMV) is an important disease in European maize cultivars (Zea mays L.). Because of its non-persistent transmission by aphid vectors, it is not possible to control SCMV directly. Therefore, cultivation of resistant maize varieties is an efficient way to control SCMV infections. The overall objectives of this study were the genetic analysis of SCMV resistance in cross F7 x FAP1360A and the identification of closely linked markers to the SCMV resistance genes Scmv1 on chromosome 6 and Scmv2 on chromosome 3 for map-based cloning and marker-assisted selection (MAS). The technical objectives were to (1) identify in particular the location of Scmv1 and Scmv2 on chromosomes 3 and 6 in cross F7 x FAP1360A, (2) estimate the gene action of the alleles present at these loci, (3) enrich the SCMV resistance regions surrounding Scmv1 and Scmv2 with amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers by applying a modified targeted bulked segregant analysis, tBSA, (4) convert AFLP markers into codominant, simple PCR-based markers as a tool for MAS and map-based cloning of Scmv1 and Scmv2 and, (5) assess resistance gene analogues (RGAs) as potential candidate genes for Scmv1 and Scmv2. Quantitative trait loci (QTL) mapping SSR markers revealed the presence of two QTL on chromosome 6 (Scmv1a and Scmv1b) and one QTL on chromosome 3 (Scmv2). tBSA identified 24 AFLP and 25 SSR markers adjacent to either Scmv1 or Scmv2. AFLP marker E35M62-1, closely linked to Scmv1 on chromosome 6, was successfully converted into an indel marker. For chromosome 3, AFLP marker E33M61-2 was converted into a CAPS marker. Both converted AFLP markers mapped to the same chromosome region as their original AFLP markers. Development of CAPS of the RGAs and mapping in relation to SCMV resistance genes Scmv1 and Scmv2 identified pic19 and pic13 as potential candidates for these resistance genes. In this study, useful markers were developed for applications in MAS. Because inheritance of SCMV resistance is strongly affected by the environment, MAS enables the selection of resistant individuals independently of field experiments. Furthermore, MAS can assist breeders to identify resistant individuals before flowering and to pyramid resistance genes in elite inbred lines. Another benefit of these closely linked markers is their application for map-based cloning. Final evidence, whether there are one or more genes clustered on chromosomes 3 and 6, conferring resistance against SCMV, can only be solved after cloning these genes.
Dissection of the genetic architecture of stalk mechanical strength and in vivo haploid induction in maize
(2016) Hu, Haixiao; Melchinger, Albrecht E.
Stalk lodging causes yield losses in maize cultivation ranging from 5 to 20% annually worldwide and stalk mechanical strength is widely accepted as an indirect indicator for its measurement. QTL mapping can reveal the genetic basis of stalk strength and provide information about markers suitable for marker-assisted selection (MAS). Constantly increasing market demands urge maize geneticists and breeders not only to enhance the field performance of new hybrids, but also to improve the breeding process. During the last decade, advances in the double haploid (DH) technology based on in vivo haploid induction (HI) shifted the breeding paradigm and greatly accelerated the breeding process in maize. Further spread of DH technology urgently demands a simple but efficient way for developing new inducers, which could be achieved by introducing the mandatory QTL/gene(s) of HI to advanced breeding lines. Therefore, the main goal of my thesis was to dissect the genetic architecture of stalk strength and detect the mandatory genomic region(s) of HI using genome-wide molecular markers. Several methods have been developed and applied in the literature to evaluate stalk mechanical strength, among which the rind penetrometer resistance (RPR) is a simple, rapid and non-destructive measurement during data collection, whereas stalk bending strength (SBS) is more closely associated with stalk lodging in the field. According to common knowledge in the mechanics of materials, SBS is reflected by the maximum load exerted to breaking (Fmax), the breaking moment (Mmax) and the critical stress (σmax). Thus, to have a complete understanding of the genetic architecture of stalk strength in maize, RPR and SBS (measured by Fmax, Mmax and σmax) were used to characterize stalk strength in our study. Utilizing a segregating population with 216 recombinant inbred lines, our analysis showed that stalk strength traits, RPR and SBS, have high heritability, ranging from 0.75 to 0.91. Nine QTL and one epistatic interaction between QTL were detected for RPR. Two, three and two QTL were detected for Fmax, Mmax and σmax, respectively. All QTL showed minor effects and only one QTL on chromosome 10 had overlapping support intervals between RPR and SBS. Co-locations of QTL and high positive correlations between stalk strength traits and other stalk traits suggested presence of pleiotropism and a complex genetic architecture of stalk strength. Owing to lack of major QTL, MAS solely based on molecular markers was found to be less effective than classical phenotypic selection for stalk strength. However, for SBS we observed considerably higher proportions of genetic variance explained by a genomic selection approach than obtained in QTL mapping with cross validation. Therefore, genomic selection might be a promising tool to improve the efficiency of breeding for stalk strength. All QTL mapping studies conducted hitherto for unraveling the genetic architecture of HI rate detected a major QTL, termed qhir1, in bin 1.04. Dong et al. (2013) further narrowed down this QTL to a 243 kb region. Considering the complex genetic architecture of HI and genetic background noise possibly affecting fine mapping of qhir1, we attempted to validate these results with an alternative approach before embarking on map-based gene isolation. Utilizing 51 maize haploid inducers and 1,482 non-inducers collected worldwide, we were able to investigate the genetic diversity between inducers and non-inducers and detect genomic regions mandatory for HI. The genetic diversity analyses indicated that the inducer group was clearly separated from other germplasm groups and had high familial relatedness. Analyzing our data by a case-control association approach failed because the segregation of HI was heavily confounded with population structure. Moreover, selective sweep approaches commonly used in the literature that are designed for capturing selective sweeps in a long-term evolutionary context failed due to high familial relatedness among inducers. To solve this problem, we developed a novel genome scan approach to detect fixed segments among inducers. With this approach, we detected a segment, termed qhir12, 4.0 Mb in length, within the support interval of the qhir1. This segment was the longest genomic segment detected by our novel approach and was entirely absent in all non-inducers analyzed. However, qhir12 has no overlap with the fine mapping region of Dong et al. (2013), termed qhir11. This indicates that the genomic region harboring the mandatory gene of HI should be confirmed by further experiments to corroborate its existence and identify its location in the maize genome.
Extensions of genomic prediction methods and approaches for plant breeding
(2013) Technow, Frank; Melchinger, Albrecht E.
Marker assisted selection (MAS) was a first attempt to exploit molecular marker information for selection purposes in plant breeding. The MAS approach rested on the identification of quantitative trait loci (QTL). Because of inherent shortcomings of this approach, MAS failed as a tool for improving polygenic traits, in most instances. By shifting focus from QTL identification to prediction of genetic values, a novel approach called 'genomic selection', originally suggested for breeding of dairy cattle, presents a solution to the shortcomings of MAS. In genomic selection, a training population of phenotyped and genotyped individuals is used for building the prediction model. This model uses the whole marker information simultaneously, without a preceding QTL identification step. Genetic values of selection candidates, which are only genotyped, are then predicted based on that model. Finally, the candidates are selected according their predicted genetic values. Because of its success, genomic selection completely revolutionized dairy cattle breeding. It is now on the verge of revolutionizing plant breeding, too. However, several features set apart plant breeding programs from dairy cattle breeding. Thus, the methodology has to be extended to cover typical scenarios in plant breeding. Providing such extensions to important aspects of plant breeding are the main objectives of this thesis. Single-cross hybrids are the predominant type of cultivar in maize and many other crops. Prediction of hybrid performance is of tremendous importance for identification of superior hybrids. Using genomic prediction approaches for this purpose is therefore of great interest to breeders. The conventional genomic prediction models estimate a single additive effect per marker. This was not appropriate for prediction of hybrid performance because of two reasons. (1) The parental inbred lines of single-cross hybrids are usually taken from genetically very distant germplasm groups. For example, in hybrid maize breeding in Central Europe, these are the Dent and Flint heterotic groups, separated for more than 500 years. Because of the strong divergence between the heterotic groups, it seemed necessary to estimate heterotic group specific marker effects. (2) Dominance effects are an important component of hybrid performance. They had to be included into the prediction models to capture the genetic variance between hybrids maximally. The use of different heterotic groups in hybrid breeding requires parallel breeding programs for inbred line development in each heterotic group. Increasing the training population size with lines from the opposite heterotic group was not attempted previously. Thus, a further objective of this thesis was to investigate whether an increase in the accuracy of genomic prediction can be achieved by using combined training sets. Important traits in plant breeding are characterized by binomially distributed phenotypes. Examples are germination rate, fertility rates, haploid induction rate and spontaneous chromosome doubling rate. No genomic prediction methods for such traits were available. Therefore, another objective was to provide methodological extensions for such traits. We found that incorporation of dominance effects for genomic prediction of maize hybrid performance led to considerable gains in prediction accuracy when the variance attributable to dominance effects was substantial compared to additive genetic variance. Estimation of marker effects specific to the Dent and Flint heterotic group was of less importance, at least not under the high marker densities available today. The main reason for this was the surprisingly high linkage phase consistency between Dent and Flint heterotic groups. Furthermore, combining individuals from different heterotic groups (Flint and Dent) into a single training population can result in considerable increases in prediction accuracy. Our extensions of the prediction methods to binomially distributed data yielded considerably higher prediction accuracies than approximate Gaussian methods. In conclusion, the developed extensions of prediction methods (to hybrid prediction and binomially distributed data) and approaches (training populations combining heterotic groups) can lead to considerable, cost free gains in prediction accuracy. They are therefore valuable tools for exploiting the full potential of genomic selection in plant breeding.
Factors influencing the accuracy of genomic prediction in plant breeding
(2017) Schopp, Pascal; Melchinger, Albrecht E.
Genomic prediction (GP) is a novel statistical tool to estimate breeding values of selection candidates without the necessity to evaluate them phenotypically. The method calibrates a prediction model based on data of phenotyped individuals that were also genotyped with genome-wide molecular markers. The renunciation of an explicit identification of causal polymorphisms in the DNA sequence allows GP to explain significantly larger amounts of the genetic variance of complex traits than previous mapping-based approaches employed for marker-assisted selection. For these reasons, GP rapidly revolutionized dairy cattle breeding, where the method was originally developed and first implemented. By comparison, plant breeding is characterized by often intensively structured populations and more restricted resources routinely available for model calibration. This thesis addresses important issues related to these peculiarities to further promote an efficient integration of GP into plant breeding.
Gene mining in doubled haploid lines from European maize landraces with association mapping
(2014) Strigens, Alexander Carl Georg; Melchinger, Albrecht E.
Since the introduction of maize into Europe, open-pollinated varieties of flint maize were cultivated across the continent. Natural selection promoted adaptation to the climatic conditions prevailing in the different regions. With the advent of hybrid breeding in Europe during the 1950’s, some of the genes responsible for the specific adaptations of the landraces to abiotic and biotic stress were captured in the first developed inbred lines, but most of their genetic diversity is still untapped. Development of inbred lines out of this material by recurrent selfing is very tedious due to strong inbreeding depression. In contrast, the doubled-haploid (DH) technology allows producing fully homozygous lines out of landraces in only one step. This allows their precise characterization in replicated trials and identification of new genes by genome wide association (GWA) mapping. In this study we genotyped a set of 132 DH lines derived from European Flint landraces and 364 elite European flint (EU-F), European dent (EU-D) and North-American dent (NA-D) inbred lines with 56,110 single nucleotide polymorphism (SNP) markers. The lines were evaluated in field trials for morphologic and agronomic traits and GWA mapping was performed to identify underlying quantitative trait loci (QTL). In particular, our objectives were to (1) develop a robust method for quantifying early growth with a non-destructive remote-sensing platform, (2) evaluate the importance of early growth performance of inbred lines with regard to their testcross performance, (3) determine the potential of GWA mapping to identify genes underlying early growth and cold tolerance related traits, (4) evaluate the phenotypic and genotypic diversity recovered in the DH lines derived from the landraces, (5) estimate the effect of the DH method on the recovered genetic diversity, (6) identify new genes by GWA mapping in the DH lines derived from landraces, and (8) discuss the potential of DH lines derived from landraces to improve the genetic diversity and performance of elite maize germplasm. A phenotyping platform using spectral reflectance and light curtains was used to perform repeated measurements of biomass and estimate relative growth rates (RGR) of the DH and inbred lines, as well as of two testcrosses of 300 dent inbred lines. The DH lines derived from the landraces Schindelmeiser and Gelber Badischer had the highest RGR followed by EU-F lines, DH lines derived from Bugard, EU-D lines and, finally, NA-D lines. For inbred lines, whole plant dry matter yield (DMY) was positively correlated with RGR (r = 0.49), whereas this relation was weaker in the testcrosses (r = 0.29). RGR of the inbred lines correlated with RGR of their testcrosses (r = 0.42), but it had no influence on testcross DMY. A set of 375 EU-F, EU-D and NA-D lines were further evaluated in growth chambers under chilling (16/13°C) and optimal (27/25°C) temperatures. Photosynthetic and early growth performance were estimated for each treatment and an adaptation index (AI) built as the chilling to optimal performance ratio. Nineteen QTL were identified by GWA mapping for trait performance and AI. Candidate genes involved in ethylene signaling, brassinolide, and lignin biosynthesis were found in their vicinity. Several QTL for photosynthetic performance co-located with previously reported QTL and the QTL identified for shoot dry wieght under optimal conditions co-located with a QTL for RGR. Comparison of the DH lines derived from landraces with the EU-F lines showed that genotypic variances in single DH populations were greater than in the EU-F breeding population. A high average genetic distance among the DH lines derived from the same landrace as well as a rapid decay of linkage disequilibrium suggests a high effective population size of the landraces. Because no systematic phenotypic differences were observed between the landraces and synthetic landraces obtained by intermating the corresponding DH lines, the expected purge of lethal recessive alleles during the DH production did neither improve grain yield performance nor affect the recovered genetic diversity. Performing GWA in the DH lines derived from landraces as well as the EU-F, and EU-D lines allowed the identification of 49 QTL for 27 traits. A larger set of DH lines derived from more landraces might solve problems arising from population structure and allow a much higher power for the detection of new alleles. In conclusion, the introgression of DH lines derived from landraces into the elite breeding material would strongly broaden its genetic base. However, grain yield performance was 22% higher in EU-F lines than in the DH lines derived from landraces. Selection of the best DH lines would allow partially bridging this yield gap and marker-assisted selection may allow introgression of positive QTL without introducing negative features by linkage drag.
Genetic analysis of resistance to ear rot and mycotoxin contamination caused by Fusarium graminearum in European maize
(2012) Martin, Matthias; Melchinger, Albrecht E.
Maize is affected by a number of diseases. Among the various ear rots of maize, Gibberella ear rot (GER) caused by Fusarium graminearum is prevalent in Central Europe. This fungal pathogen produces secondary metabolites (mycotoxins), which adversely affect the health of humans and animals. Two important mycotoxins are the immunosuppressant deoxynivalenol (DON) and the mycoestrogen zearalenone (ZEA). The most efficient method to reduce mycotoxin contamination in maize is cultivation of resistant varieties. However, resistance breeding using classical phenotypic selection is laborious and time-consuming. Therefore, marker-assisted selection (MAS) may be a promising alternative to classical selection. Furthermore, for setting up a breeding program, knowledge about the relevance of the different modes of gene action and genotypic correlations among resistance and agronomic traits is required. The objectives of this study were to (1) estimate quantitative genetic parameters for GER severity and mycotoxin concentration in connected populations of doubled haploid (DH) lines, (2) map quantitative trait loci (QTL) for GER resistance and reduced mycotoxin contamination in these populations, (3) examine the congruency of QTL in these populations, (4) evaluate the prospects of using MAS to breed for GER resistance and reduced mycotoxin contamination, (5) estimate the genotypic correlation between the resistance of DH lines per se and the resistance of their testcrosses, (6) evaluate the influence of selection for increased resistance on agronomic performance of hybrids and (7) examine the relevance of different modes of gene action involved in the expression of the resistance in flint maize. Three field experiments were conducted, each of which comprised a different set of plant material. Experiment I comprised five DH line populations derived from the following F1 crosses among elite flint inbred lines: D152×UH006, D152×UH007, UH007×UH006, UH009×UH006 and UH009×UH007. Experiment II comprised testcross progenies of 94 DH lines and a dent single cross tester. Experiment III comprised the five F1 crosses, from which the DH populations had been derived, the F2 and the first backcross generations to the parents (BC1-P1, BC1-P2) as well as the two parent lines of each cross. Plants were artificially infected with spores of F. graminearum shortly after mid-silking using the silk channel inoculation technique. The DH lines were genotyped with simple sequence repeat (SSR) DNA markers, genetic linkage maps were constructed and QTL analyses were performed for resistance to GER, DON and ZEA contamination. Estimates of genotypic and genotype-by-environment interaction variances in Experiment I for GER severity and mycotoxin concentration were significant and heritabilities were moderately high to high in all populations. Thus, differences among DH lines for the resistance traits were mainly caused genetically and the resistance response varied depending on the environment. Owing to the effectiveness of artificial inoculation, the prospects are good to improve line resistance using a small number of test environments. QTL were detected in the four largest populations. Depending on the population, the mapped QTL together explained 21-51% of the genotypic variance for GER severity and 19-45% for DON concentration and 52% for ZEA concentration. Additive gene action was more important than digenic interactions of QTL, as indicated by the number of QTL having significant additive effects, their relative contributions to the total genotypic variance explained and the magnitude of their effects. Colocalized QTL for resistance to GER and mycotoxin contamination were identified in each mapping population. This was in agreement with strong genotypic correlations among these traits. QTL located at similar positions were detected across three populations in two chromosomal regions and across two populations in additional two regions. The results of this study indicated that a combination of classical phenotypic selection and MAS is a promising strategy for resistance breeding. In Experiment II, significant genotypic variation for resistance in lines and testcrosses showed that selection will be successful in both groups. Owing to low genotypic correlations between lines and testcrosses, however, resources should be mainly allocated to the evaluation of GER in testcrosses. Correlations of resistance with agronomic traits were weak or not significant. Therefore, selection for resistance and better agronomic performance can be carried out simultaneously. In Experiment III, generation means analysis indicated a prevalence of additive gene action for resistance. Significant dominance effects were found in only one cross for resistance to GER, but in four crosses for resistance to DON contamination. Owing to prevalence of additive gene action, the prospects are good to improve the resistance of the flint germplasm and to accumulate more favorable gene combinations in future breeding lines. Comparing the hybrid performance of flint×flint crosses of Experiment II and flint×dent crosses of Experiment III with their corresponding mid-parent performances indicated mid-parent heterosis for resistance. Therefore, prediction of hybrid performance based on performance of their parents will be possible only to a very limited extent. Future research should focus on fine mapping and validating of the detected QTL. For an efficient use of the QTL in a marker-assisted breeding program, knowledge about their effects in different genetic backgrounds is needed. Of particular importance are thereby the QTL effects in flint×dent crosses, which represent the preferred type of hybrid in Central European maize breeding programs.
Genetic diversity in elite lines and landraces of CIMMYT spring bread wheat and hybrid performance of crosses among elite germplasm
(2005) Dreisigacker, Susanne; Melchinger, Albrecht E.
Wheat (Triticum aestivum) is one of the major cereals in the world. During the past years, the world consumption of wheat increased up to nearly 600 million tones, whereas wheat production continuously decreased. Due to land limitations, new production gains must be achieved from improved plant management systems as well as from the development of high yielding varieties. The International Maize and Wheat Improvement Center (CIMMYT) employs different strategies to enhance yield potential in wheat especially for developing countries. For instance, the wheat breeding program focuses on defined mega-environments (MEs), assuming similar growing conditions in certain countries. In the search for useful alleles, breeders often turn back to wild relatives of wheat stored in the CIMMYT gene bank. With the production of synthetic hexaploid bread wheat (SHWs), characteristics from T. durum and T. tauschii can be combined and via backcrossing incorporated into modern breeding materials. Wheat landraces (LCs) are an additional reservoir of resistances to pests and diseases as well as for environmental adaptation. The production of wheat hybrids is seen as a further option to improve yield potential. A considerable amount of genetic diversity among the materials is a prerequisite for all strategies. Due to the worldwide importance of CIMMYT wheat varieties, they represent a suitable source to examine different breeding strategies in wheat. The main objective of our research was to determine the genetic diversity in modern wheat breeding materials and genetic resources at CIMMYT. Specific research questions were: (i) Is the systematic breeding targeted for different MEs reflected in the genetic diversity among breeding lines (Experiment 1)? (ii) Does the production of SHWs (Experiment 2) and the use of LCs (Experiment 3) enhance the genetic variation in modern breeding materials? (iii) Does the development of hybrids represent an option to improve yield potential in wheat? (iv) Is it possible to predict levels of heterosis with the determination of genetic distance (GD) among hybrid parents? (v) Do genomic and EST- derived SSRs differ in the measurement of genetic diversity (Experiments 1 and 3)? (vi) Are GD values based on SSRs correlated with the coefficient of parentage (COP) (Experiments 1 to 4)? In Experiment 1, a total of 68 CIMMYT advanced breeding lines was analyzed with 99 SSRs, of which 51 were EST- and 46 genomic derived SSRs. A high level of genetic diversity (GD = 0.41) was observed among the breeding lines. The majority of variation (91%) was detected among lines targeted to one specific ME, which indicates a broad genetic base of the current CIMMYT breeding materials. Principal coordinate analysis (PCoA) could clearly separate the lines, but they clustered independently from their target MEs. Main explanations are: (i) alleles were selected that provide fitness to several MEs, (ii) adaptation depends only on a small number of genes that were not detected with the SSRs applied, or (iii) too few cycles of selection were considered to separate the germplasm. In Experiment 2, a total of 11 SHWs, 7 recurrent parent lines, and 13 families of backcross-derived lines (SBLs) were analyzed with 90 SSRs. The SHWs clustered far from the SBLs and the recurrent parents in the cluster analyses and PCoA, and formed a distinct germplasm pool with high allelic variation. Two families of SBLs were tested for a selective advantage of the SHW alleles. Six SSRs revealed non-Mendelian inheritance, indicating that the genomic region of SHWs was actively selected for. Thus, the production of SHWs provides a promising approach for the enhancement of genetic variation in modern breeding materials. In Experiment 3, gene bank accessions of 36 LCs from different countries and a total of 119 accessions from nine LCs populations collected in Turkey and Mexico were analysed with 44 and 76 SSRs, respectively. Both LC materials revealed high allelic variation (GD = 0.69 and 0.54). The 36 LC accessions could not be separated according to their continent of origin. An unexpected relationship was observed between the Chilean LC ?Trigo africano? and the Nigerian LCs ?Dikwa?. All of the nine LC populations could be discriminated except for two Turkish LCs collected from the same location. In accordance with previous studies, considerable genetic variation was observed within the LC populations. Our results contributed a lot to the characterisation of the LCs and generated important knowledge for the management of seed bank accessions. In Experiment 4, a total of 112 wheat hybrids and their 22 parental lines were evaluated at two locations in Mexico for grain yield, plant height, days to flowering and maturity. The level of heterosis varied between -15.3% and 14.1%, but was generally too low to compensate for the high costs of hybrid seed production. The correlations between mid-parent values and hybrid performance, as well as between parental line per se performance and general combining ability were significant (P < 0.01) for all traits, and particularly high for grain yield (r = 0.86 and 0.91). PCoA based on 113 SSR markers revealed three groups of parents. However, the correlations of GDs and COPs with the values of heterosis were negative and not significant. Thus, the prospects of large-scale cultivation of hybrid wheat in developing countries are low. The correlations between GDs and COP in Experiments 1 and 3 were generally significant but low. This can be explained by unrealistic assumptions in the calculation of COPs, which ignore the effects of selection and genetic drift. Similarly to genomic SSRs, EST-SSRs did not reflect functional diversity. The latter revealed lower degrees of polymorphism than genomic SSRs in all experiments, but the allele designation was simpler and more reliable. Across all experiments, our study demonstrates that plant breeding does not inevitably lead to a loss of genetic diversity. We confirmed that CIMMYT?s breeding strategies contributed to a successful increase in genetic variation. These results provide useful information to wheat breeders in CIMMYT and other national programs, regarding the use of wild relatives and landraces for the enhancement of the genetic base of wheat germplasm. In addition, our research provides a base of knowledge for future association studies, identification of useful alleles, and their use in marker-assisted selection.
Genetic diversity in germplasm of cornsalad (Valerianella locusta L.), radish (Raphanus sativus L.), and celeriac (Apium graveolens L. var. rapaceum), investigated with PCR-based molecular markers
(2004) Muminovic, Jasmina; Melchinger, Albrecht E.
During the last couple of decades, production and economic importance of cornsalad (Valerianella locusta L.; fam. Valerianaceae), radish (Raphanus sativus L. var. sativus convar. radicula; fam. Brassicaceae), and celeriac (Apium graveolens L. var. rapaceum; fam. Apiaceae) have been considerably increasing in Europe. Nevertheless, genetic diversity currently utilized for breeding cornsalad, radish, and celeriac is narrow, whereas their germplasm collections in gene banks are relatively poor. Assessment of genetic diversity among breeding materials and genetic resources is an important consideration for the optimal design of further breeding programs. The major objective of this study was to investigate genetic diversity in germplasm of cornsalad, radish, and celeriac, applying amplified fragment length polymorphisms (AFLPs) and inter simple sequence repeats (ISSRs) molecular markers. In particular, the objectives were to (i) analyze relationships among breeding materials of the three vegetable crops (referred to as elite germplasm), as well as among their formerly grown varieties, gene bank and botanical garden accessions (referred to as exotic germplasm), (ii) reveal genetic structure of radish germplasm to establish heterotic pools for hybrid breeding, and (iii) evaluate the usefulness of introducing exotic materials for broadening of the elite germplasm in cornsalad, radish, and celeriac. Average genetic similarity in 34 elite varieties of cornsalad was very high (GS = 0.90), which is comparable with other autogamous crops. The majority of elite varieties clustered closely applying the UPGMA analysis because of a narrow-based germplasm in cornsalad breeding. A substantial level of genetic diversity (GS = 0.47) was detected in 30 cornsalad varieties representing exotic germplasm. Exotic varieties that interspersed the sub-clusters of the elite may serve as a direct genetic resource for broadening the elite cornsalad germplasm base, whereas Valerianella locusta-related species that were distinct from cultivated germplasm can contribute to the introgression of new (resistance) genes. Sixty-eight varieties of cultivated radish (garden and Black radish) created sub-clusters with GS estimates higher than 0.70, thus supporting the assumption that the currently used radish germplasm in Europe relies on a narrow genetic base. Owing to a high degree of heterogeneity and heterozygosity within radish varieties, the detected between-variety diversity was low, but there still was a substantial overall diversity in available radish germplasm. Applying both UPGMA and principal coordinate analyses, Black radish varieties were distinct from garden radish. A further unambiguous division within garden radish germplasm was revealed with the model-based clustering approach. These sub-groups can be employed for establishment of heterotic pools within European modern cultivars of garden radish. In addition, ISSRs can substantially reduce hybrid radish production costs by an early detection of two closely related weed species (R. raphanistrum and R. sativus L. var. sativus convar. sinensis). AFLPs and the evaluation of morphological traits were used to investigate genetic diversity in 34 varieties of elite celeriac germplasm and 35 accessions of exotic germplasm. Only two morphological traits supported the clustering pattern obtained with UPGMA analysis of morphological distance estimates. AFLP-based GS estimates offered a clearer view of diversity present in elite (GS = 0.68-0.95) and exotic germplasm (GS = 0.05-0.95), and clustered the two sets in distinct UPGMA-based sub-clusters. This indicated that only a small fraction of available genetic diversity is exploited for current breeding of celeriac. Exotic celeriac germplasm as well as varieties of celery and leaf celery might substantially improve commercial celeriac breeding. Wild relatives of Apium graveolens are valuable resources for the introgression of resistance genes. Regarding the generally high level of GS in celeriac germplasm conserved in the German gene bank, a broadening of the germplasm collection was suggested. This study demonstrated the capacity of molecular markers to be highly discriminating among varieties of cornsalad, radish, and celeriac. AFLP-based genetic similarity estimates in the three vegetable crops (i) allowed the first insight into the genetic diversity and structure present in the germplasm, (ii) offered suggestions for germplasm broadening, and (iii) proposed a way of rationalization and utilization of available germplasm resources.
Genetic diversity, population structure, and linkage disequilibrium in the context of genome-wide association mapping of northern corn leaf blight resistance
(2012) van Inghelandt, Delphine; Melchinger, Albrecht E.
Besides linkage mapping, association mapping (AM) has become a powerful complement for understanding the genetic basis of complex traits. AM utilizes the natural genetic diversity and the linkage disequilibrium (LD) present in a diverse germplasm set. Setosphaeria turcica is a fungal pathogen that causes northern corn leaf blight (NCLB) in maize. The objective of this thesis research was to set the stage for and perform AM in elite maize breeding populations for NCLB resistance. Information about the genetic diversity and population structure in elite breeding material is of fundamental importance for the improvement of crops. The objectives of my study were to (i) examine the population structure and the genetic diversity in elite maize germplasm based on simple sequence repeat (SSR) markers, (ii) compare these results with those obtained from single nucleotide polymorphism (SNP) markers, and (iii) compare the coancestry coefficient calculated from pedigree records with genetic distance estimates calculated from SSR and SNP markers. The study was based on 1 537 elite maize inbred lines genotyped with 359 SSR and 8 244 SNP markers. My results indicated that both SSR and SNP markers are suitable for uncovering population structure. The same conclusions regarding the structure and the diversity of heterotic pools can be drawn from both markers types. However, fewer SSRs as SNPs are required for this goal, which facilitates the computations, for instance by the STRUCTURE software. Finally, the findings indicated that under the assumption of a fixed budget, modified Roger?s distances and gene diversity could be more precisely estimated with SNPs than with SSRs, and we proposed that between 7 and 11 times more SNPs than SSRs should be used for analyzing population structure and genetic diversity. Association mapping is based on LD shaped by historical recombinations. Many factors affect LD and, therefore, it must be determined empirically in the germplasm under investigation to examine the prospects of genomewide association mapping studies. The objectives of my study were to (i) examine the extent of LD with SSR and SNP markers in 1 537 commercial maize inbred lines belonging to four heterotic pools, (ii) compare the LD patterns determined by these two marker types, (iii) evaluate the number of SNP markers needed to perform genome-wide association analyses, and (iv) investigate temporal trends of LD. The results suggested that SNP markers of the examined density, unlike SSR markers, can be used effectively for association studies in commercial maize germplasm. Based on the decay of LD in the various heterotic pools, between 4 000 and 65 000 SNP markers would be needed to detect with a reasonable power associations with rather large quantitative trait loci (QTL). The 60 K SNP chip currently available for maize seems appropriate to identify QTLs that explain at least 10% of the phenotypic variance. However, to identify QTLs with smaller effects, which is a realistic situation for most traits of interest to maize breeders, a much higher marker density is required. NCLB 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 1 487 European maize inbred lines was used to (i) identify chromosomal regions affecting flowering time (FT) and 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. We observed for FT, a trait for which already various genetic analyses have been performed in maize, a very well interpretable pattern of SNP associations, suggesting that data from practical plant breeding programs can be used to dissect polygenic traits. Furthermore, we described SNPs associated with NCLB and NCLB corrected for FT resistance that are located in genes for which a direct link to the trait is discernable or which are located in bins of the maize genome for which previously QTLs have been reported. Some of the SNPs showed significant epistatic interactions with markers from the genetic background. The observation that the listed SNPs and their epistatic interactions explained in the entire germplasm set about 10% and in some individual heterotic pools up to 30% of the genetic variance suggests that significant progress towards improving the resistance of maize against NCLB by marker-assisted selection is possible with these markers, without much compromising on late flowering time. Furthermore, these regions are interesting for further research to understand the mechanisms of resistance against NCLB and diseases in general, because some of the genes identified have not been annotated so far for these functions.
Genetic variation for cold tolerance in two nested association mapping populations
(2023) Revilla, Pedro; Butrón, Ana; Rodriguez, Víctor Manuel; Rincent, Renaud; Charcosset, Alain; Giauffret, Catherine; Melchinger, Albrecht E.; Schön, Chris-Carolin; Bauer, Eva; Altmann, Thomas; Brunel, Dominique; Moreno-González, Jesús; Campo, Laura; Ouzunova, Milena; Álvarez, Ángel; Ruíz de Galarreta, José Ignacio; Laborde, Jacques; Malvar, Rosa Ana
Cold reduces maize (Zea mays L.) production and delays sowings. Cold tolerance in maize is very limited, and breeding maize for cold tolerance is still a major challenge. Our objective was to detect QTL for cold tolerance at germination and seedling stages. We evaluated, under cold and control conditions, 919 Dent and 1009 Flint inbred lines from two nested association mapping designs consisting in 24 double-haploid populations, genotyped with 56,110 SNPs. We found a large diversity of maize cold tolerance within these NAM populations. We detected one QTL for plant weight and four for fluorescence under cold conditions, as well as one for plant weight and two for chlorophyll content under control conditions in the Dent-NAM. There were fewer significant QTL under control conditions than under cold conditions, and half of the QTL were for quantum efficiency of photosystem II. Our results supported the large genetic discrepancy between optimal and low temperatures, as the quantity and the position of the QTL were very variable between control and cold conditions. Furthermore, as we have not found alleles with significant effects on these NAM designs, further studies are needed with other experimental designs to find favorable alleles with important effects for improving cold tolerance in maize.
Genetic variation in early maturing European maize germplasm for resistance to ear rots and mycotoxin contamination caused by Fusarium spp.
(2010) Bolduan, Christof; Melchinger, Albrecht E.
Ear rots of maize, caused by Fusarium spp., are of major concern because they lead to losses in grain yield and contamination with mycotoxins which harm animals and humans. In the absence of other strategies, breeding maize for genetic resistance is currently the most promising avenue to control these rots and mycotoxin accumulation. The predominant pathogens in Central Europe are F. graminearum, the causative agent of Gibberella ear rot (GER), and F. verticillioides, the causative agent of Fusarium ear rot (FER). GER causes contamination with deoxynivalenol (DON), nivalenol and zearalenone (ZEA), whereas FER causes contamination with fumonisins (FUM). Information on the resistance to GER and FER and mycotoxin contamination is lacking for maize adapted to the cooler climatic conditions of Central Europe. In this study we investigated (1) the resistance of early maturing European elite inbred lines against GER and FER and contamination of mycotoxins, (2) the genetic variances and heritabilities for ear rot ratings and mycotoxin concentrations, (3) the correlations of ear rot ratings with mycotoxin concentrations, (4) the correlations between line per se (LP) and testcross performance (TP) for GER rating and DON concentration, (5) the aggressiveness of and mycotoxins produced by different isolates of F. graminearum and F. verticillioides, and (6) the potential of near infrared spectroscopy (NIRS) to estimate concentrations of DON and FUM in maize grains under artificial inoculation. Significant genotypic variances and moderate to high heritabilities were found for GER, DON and ZEA among the inbred lines and for GER and DON among the testcrosses, as well as for FER and FUM among the inbred lines. Further, genotype x environment interaction variances were significant for all traits except FUM. Thus, the results underlined the presence of ample genotypic variation and the need to conduct multi-environment tests for reliable identification of resistant genotypes. Ear rot ratings and mycotoxin production of eight isolates each of F. graminearum and F. verticillioides differed significantly. Even though, isolate x inbred interactions were significant only in the case of F. graminearum, and no rank reversals occurred among the tested inbred lines. Most isolates differentiated the susceptible inbreds from the resistant ones for severity ratings. However, the differences between the two groups were smaller for the less aggressive isolates. Therefore, we recommend using a single, environmentally stable and sufficiently aggressive isolate for resistance screenings under artificial inoculation. Strong correlations between ear rot severity and mycotoxin concentrations indicated that selection for low ear rot severity under artificial inoculation will result in high correlated selection response for low mycotoxin concentration, particularly for GER and DON. Selection for ear rot severity is less resource-demanding and quicker than selection for mycotoxin concentration. Thus, it enables the breeder to maximize selection gain for a given budget. However, the selected elite material should be evaluated for mycotoxin concentrations in order to avoid ?false positives?. In this regard, NIRS showed high potential to predict DON concentrations in grain obtained from artificially inoculated maize. Compared to the commonly employed ELISA assay, NIRS assays are considerably cheaper, because no mycotoxin extractions and test kits are needed. We observed moderate positive correlations between GER and FER, and identified inbreds combining resistance to both ear rots. Therefore, selection for resistance to one pathogen is expected to result in indirect response to the other. Nevertheless, in advanced stages of each breeding cycle, lines preselected for other agronomically important traits should be evaluated for resistance to both pathogens. Genotypic variances for GER and DON were generally higher in LP than TP. Thus, assuming identical selection intensities for each scheme, the expected response to selection for LP should be higher than for TP. However, owing to moderate correlations between LP and TP for GER and DON, selection based on LP is not sufficient, because the ultimate goal is to develop resistant hybrids. Therefore, a multi-stage selection procedure is recommended with evaluation of agronomically promising lines for GER in only one environment in order to eliminate highly susceptible lines, followed by evaluation of TP of the selected lines for GER with one tester of moderate to high resistance level from the opposite heterotic pool in two to three environments.