Landessaatzuchtanstalt

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Molecular and phenotypic analyses of pathogenicity, aggressiveness, mycotoxin production, and colonization in the wheat-Gibberella zeae pathosystem
(2004) Cumagun, Christian Joseph R.; Miedaner, Thomas
Fusarium head blight (FHB), caused by Gibberella zeae (Schwein.) Petch (anamorph: Fusarium graminearum Schwabe), is one of the principal diseases responsible for extensive damage in wheat fields and contamination of grain with the mycotoxins deoxynivalenol (DON) and nivalenol (NIV), rendering the harvest unsafe for human and animal consumption. Control of FHB is difficult because of the complex nature of host-pathogen-environment interaction and the nonavailability of highly effective fungicides. Agronomic practices and resistance breeding, therefore, offer the best strategies for disease management. Mapping by molecular markers provides an accurate approach for genetic analyses of simple and complex traits particularly pathogenicity, aggressiveness, and mycotoxin production. Pathogenicity, as defined here, is the ability to cause disease whereas aggressiveness is the quantity of disease induced by a pathogenic isolate on a susceptible host in which isolates do not interact differentially with host cultivars. The project aims to (1) map pathogenicity and aggressiveness of G. zeae based on a published genetic map (2) estimate genetic diversity of four parent isolates by PCR-based markers (3) examine the inheritance of pathogenicity, aggressiveness, mycotoxin type (DON/NIV), and DON production on a phenotypic basis, (4) analyse genetic covariation among aggressiveness, DON, and fungal colonization, (5) and compare aggressiveness of 42 isolates in greenhouse and field environments. Two crosses of G. zeae using nit (nitrate nonutilizing) marker technique were performed: (1) pathogenic DON-producing Z-3639 (Kansas, USA) x nonpathogenic NIV-producing R-5470 (Japan) belonging to lineage 7 and 6, respectively, and (2) DON-producing FG24 (Hungary) x FG3211 (Germany), both aggressive lineage 7 isolates. For the first cross, 99 progeny segregated in a consistent 61:38 for pathogenicity: nonpathogenicity in a two-year greenhouse experiment. Among the 61 pathogenic progeny, disease severity, measured as percentage infected spikelets, varied significantly (P = 0.01). Heritability for aggressiveness was high. Pathogenicity locus was mapped on linkage group IV near loci PIG1 (red pigment production), TOX1 (trichothecene toxin amount), and PER1 (perithecial production) explaining 60%, 43%, and 51% of the phenotypic variation, respectively. Two large aggressiveness QTLs were mapped on linkage group I linked to the locus TRI5 (trichodiene synthase in the trichothecene gene cluster) and an amplified fragment length polymorphism (AFLP) marker (EAAMTG0655K), explaining 51% and 29% of the observed phenotypic variation, respectively. These unlinked loci suggest that genetic basis between pathogenicity and aggressiveness were different. TRI5 is located in the same gene cluster as a previously identified gene known as TRI13, which determines whether DON or NIV will be produced. DON-producing progeny were, on average, twice as aggressive as were those producing NIV. Loci were only detected in the two linkage groups mentioned from the nine linkage groups present in the map. For the second cross FG24 x FG3211 with 153 progeny, head blight rating and relative plot yield were used as aggressiveness traits. DON production was measured by a commercial kit enzyme immunoassay. These three traits were quantitatively inherited among 153 progeny across three environments. Repeatabilities within each environment were medium to high but heritabilities across environments were medium only due to high progeny-environment interaction. DON was a less environmentally stable trait than aggressiveness. Transgressive segregants were detected frequently. This implies that even a cross within a lineage could lead to an increase in aggressiveness. Mapping of this cross was not initiated because the parents were not polymorphic enough to construct a genetic map. Instead, the parents were analysed for polymorphism in comparison to the parents of the first cross using 31 AFLP primer combinations and 56 random amplified polymorphic DNA (RAPD) primers. Polymorphism between Z-3639 and R-5470 was about three to four times higher than between FG24 and FG3211. Cluster analysis revealed that R-5470 was genetically separated from the other three parents, thus confirming the lineage assignments. Among preselected 50 progeny from the same field experiments that showed normal distribution for aggressiveness - head blight rating, fungal colonization, and DON production were correlated (r = 0.7, P = 0.01). Fungal colonization measured as Fusarium exoantigen (ExAg) content using enzyme-linked immunosorbent assay (ELISA) varied also quantitatively, but heritability was lower due to high progeny-environment interaction and error. Strong correlations among all traits indicate control by similar genes or gene complexes. No significant variation was observed for DON/ExAg ratio. Aggressiveness traits and DON production were more environmentally stable compared to Fusarium ExAg content. Our findings imply that aggressiveness may have other components apart from mycotoxin production. Genotypic variation for aggressiveness among the 42 progeny in one greenhouse and three field environments was significant and their correlation was moderate (r = 0.7, P = 0.01). High heritability in both environments again indicates that aggressiveness was a relatively stable trait, although methods of inoculation differed, i.e., injection for greenhouse and spraying for field experiments. Greenhouse aggressiveness could predict aggressiveness in the field, and thereby should reduce costs for resistance and phytopathological studies. In conclusion, we consider G. zeae as medium-risk pathogen with the potential to evolve to a higher level of aggressiveness due to sexual recombination. Erosion of quantitative resistance in FHB cannot be ignored, especially if host resistances with oligogenic inheritance, e.g. Sumai 3 from China, are used on a large acreage. Consequently, the rather simple inheritance of pathogenicity and aggressiveness in G. zeae could lead to a gradual increase of aggressiveness. These results should enhance efforts of plant breeders to use several, genetic distinct sources of resistance in order to avoid possible FHB outbreaks in the future.
Wirkung rassenspezifischer Braunrostresistenzen in genetisch diversifizierten Roggenpopulationen
(2004) Wilde, Katinka; Miedaner, Thomas
Leaf rust (Puccinia recondita f.sp. secalis) is the most frequently occuring leaf disease in German winter rye (Secale cereale L.). To test the usefulness and potentially prolong the durability of race-specific resistance genes, we increased host complexity and diversity by producing synthetic (Syn) populations segregating for one to four resistance sources as a model system for hybrid varieties. Thirty synthetics were grown together with three highly susceptible synthetics as checks, two Russian full-sib families (FSF), a leaf-rust-resistant-hybrid variety, a set of 17 differential genotypes and ten parental lines at six locations in three years (17 environments) in Germany under natural infection. The parental lines were only sown in 11 environments. Disease severity of about 120 plants for each synthetic, FSF, hybrid and about 25 plants for each differential genotype and parental line was estimated as percentage of infected leaf area on the leaf below the flag leaf at the mid and the end of the natural epidemic. Susceptible synthetics showed that disease severity was similarly high in all environments. Only two out of 30 synthetics and both FSF were resistant across 17 environments. Low resistance of the synthetic was caused by the low resistance of the parental lines. Nonetheless, some of the synthetics were more resistant than the tested commercial hybrid variety. Even though the resistance was not used in practical breeding programs to date we detected virulence for each resistance gene tested. In conclusion, the German leaf rust population seems to be highly diverse and none of the self-fertile resistance sources used provided full protection. New race-specific resistances or highly effective quantitative resistances are therefore needed and should be combined.
Quantitativ-genetische Untersuchungen zur Vererbung der Resistenz gegen Ährenfusarium bei Triticale (x Triticosecale Wittmack)
(2004) Heinrich, Nicole; Miedaner, Thomas
Fusarium head blight (FHB), caused by Fusarium culmorum (W.G. Smith) Sacc. and F. graminearum Schwabe, is recognized as one of the most destructive diseases of small-grain cereals. Fusarium infection can cause substantial yield losses. Infected grain may also be contaminated by mycotoxins that are harmful to humans and livestock. Agronomical measures and fungicides are only partly effective in controlling FHB. The development of disease-resistant cultivars together with appropriate crop management practices are effective strategies to control FHB. In this study, seven triticale cultivars and three breeding strains, representing a range of FHB resistances, their 45 diallel F1 crosses, progenies of 15 F2s from a six-parent diallel and their 30 backcrosses (BC, 15 to each parent), and five F2:3 bulks were investigated. Parents and their progenies were grown in several environments (years, locations) and tested for FHB resistance after artificial inoculation with Fusarium culmorum. Within the scope of this study, three experiments were conducted to estimate various quantitative-genetic parameters of several traits. In Experiment 1, the influence of FHB on yield-related traits of the ten parents was assessed. Compared to a non-inoculated variant, Fusarium reduced 1000-grain weight by 10.0%, spike weight by 9.3%, the number of kernels per spike by 4.3%, and test weight by 7.4%. Inoculation also increased deoxynivalenol (DON, 26.4 mg kg-1) and exoantigen (1.34 OD). content of the kernels. Genotypic variation and genotype-environment interaction were significant for all traits. The correlation between symptom ratings (spikes, kernels) and yield traits and between spike weight and kernels per spike were negative and high. The aim of Experiment 2 was to estimate combining ability, hybrid performance and heterosis for FHB ratings, DON and exoantigen content. Heterosis of FHB for spike and kernel rating was small. Across environments, the DON content in F1 crosses, however, was 15.5% higher than their mid-parent value. A high and significant (P = 0.01) correlation of r = 0.8 was found for both spike and kernel FHB symptom ratings between mid-parent and F1 performance. Except for exoantigen content, the general combining ability (GCA) was the main source of variation, suggesting additive gene effects for FHB resistance. Significant specific combining ability variance implies non-additive types of allelic interaction also. Therefore, in some crosses dominant effects can play an important role. The relationship between the GCA effect of a parent and its per se performance was close. In Experiment 3, genetic variation and effects for FHB resistance were estimated in segregating generations. The resistance level of the parents and their F2 progenies were similar. In contrast, the resistance of the BC progenies to the resistant parent was considerably higher than that of the backcrosses to the susceptible parent. Significant differences between the means of the 15 crosses and a high genetic variation within crosses were observed. Transgression could not be detected. F2:3 bulks and their parents had a comparable resistance level. For F2 and BC progenies, the additive effect was more important than the dominant effect. In contrast, the F1 crosses had a higher dominant effect, but with a large error. The study revealed considerable genetic variation in all generations for FHB resistance that can be exploited in a breeding programme. The mainly additive genetic effect makes it possible to select crossing parents on the basis of their per se performance. Due to the importance of genotype-environment interaction, resistance tests in various environments are strongly recommended. Screening for FHB resistance can best be accomplished by assessing symptom ratings of spikes and/or the spike weight relative to a non-inoculated variant. The high cross-environment interaction variance in the F2 generation points to the problem of selecting in unreplicated segregating material. Selection should be postponed to the F3 or later generations. The large genetic variation of FHB resistance and the preponderance of additive gene effects are encouraging to further increase resistance in triticale by recurrent selection.
Genetische Variation für Resistenz gegen Mutterkorn (Claviceps purpurea [Fr.] Tul.) bei selbstinkompatiblen und selbstfertilen Roggenpopulationen
(2006) Mirdita, Vilson; Miedaner, Thomas
Ergot (Claviceps purpurea [Fr.] Tul.) is one of the most important diseases in rye. Infection during flowering results in the production of black, overwintering organs (sclerotia) instead of kernels, which contain harmful alcaloids. Three experiments were conducted to estimate quantitative-genetic parameters of the resistance of rye to ergot under the conditions of organic farming. The general aim was the estimation of genetic variation among and within self-incompatible rye populations and among CMS lines and their male-sterile testcrosses. In 2002 and 2004, genetic variation in resistance to ergot was tested among 65 rye populations at each of two locations (Experiment 1). Thirteen populations were registered rye varieties and the remaining 52 were genetic resources. To assess genetic variation within populations, 50 full-sib families (FSF) from each of five rye populations were developed and tested at four locations (Experiment 2). To test genetic differences in the susceptibility of ovaries towards fungal penetration in the absence of pollen, (i) 64 currently available CMS lines and (ii) their male-sterile crosses with three testers (=sets) were tested in 2003 and 2004, and in 2004, respectively. Inoculation was performed by spraying an aggressive mixture of isolates of Claviceps purpurea three times during the flowering period. The micro-plots were grown in a chess-board design separated by wheat plots to reduce the neighbouring effects. Traits of resistance were the proportion of infected spikes relative to the total number of spikes per plot, and the percentage by weight of ergot sclerotia in the grain. In Experiment 3, the weight of slcerotia per spike and per pair of spikelet were measured due to the absence of grain. Amount of pollen shedding was rated on the basis of the anther size and extrusion. Highly significant genotypic and genotype-environment interaction variances were found among rye populations in the percentage of ergot sclerotia in the grain. All genotypes were infected by ergot. No differences in mean among the registered rye varieties and genetic resources were detected. Because all populations were highly pollen shedding, the results indicate the existence of genetically determined resistance to ergot within the self-incompatible rye. Correlation between both resistance traits was significant (rp = 0.92). Genetic variation within populations was highly significant for all five populations. Individual progenies with resistance higher than the population mean were observed. The mean resistance of initial populations hardly differed from the mean of their progeny indicating a predominantly additive inheritance. Highly significant genetic variation in resistance to ergot was also detected among the currently available 64 CMS lines. Corresponding testcrosses mostly had a higher weight of sclerotia per spike than the lines. Considerable differences in the level of resistance were observed among testcrosses. Crosses with tester line 1 were substantially more susceptible, whereas those with tester 2 were hardly over the mean of the parental lines. The material showed a quantitative distribution of ergot resistance. Weak to medium-sized correlations (0.33 ? 0.47) between locations were detected among lines. The correlation between locations was even weaker in testcrosses. Weak correlations in ergot weight per spike were observed between CMS lines and their testcrosses in sets 2 and 3. In set 1, the estimated phenotypic correlation was higher (rp = 0.65). Estimates of error-corrected correlations were always higher than phenotypic correlations. No genetic difference was detected among the CMS lines for the amount of alkaloids in their sclerotia. This study shows that incompatible rye populations as well as self-fertile hybrid populations contain a substantial genetic variation for resistance to ergot that is inherited quantitatively. In both materials, mainly additive genetic variance was found. Because of a significant genotype-environment interaction, multi-environment trials are necessary to select for resistance. The results of this study nevertheless indicate good prospects to improve resistance to ergot in rye breeding in the long term.
Genetics of resistance to ear diseases and mycotoxin accumulation in the pathosystems maize/Fusarium and wheat/Fusarium
(2010) Messerschmidt, Martin; Miedaner, Thomas
Infection of ears of maize with Fusarium graminearum (FG) reduces yield and, more important, contaminate the harvest with mycotoxins. F. verticillioides (FV) is an economically important cause of ear rot. Among other mycotoxins, FV produces the fumonisins (FUM) and FG produces deoxynivalenol (DON) and zearalenone (ZEA). All three mycotoxins are harmful to humans and animals. Therefore, the European Union released legally enforceable limits. One alternative to reduce ear rot severity and mycotoxin concentrations is breeding and growing varieties resistant to Fusarium infections. However, few is known about breeding parameters for resistance to Fusarium infections and mycotoxin accumulation in European maize breeding material. The main objective of this thesis was to draw conclusions for breeding of resistance to ear rot and mycotoxin accumulation with special attention on three European maize maturity groups. We investigated methodical aspects like (1) the comparison of natural and artificial inoculation to evaluate ear rot resistance and (2) the necessity of separate testing of FV and FG. Furthermore, quantitative-genetic parameters like heritabilities and correlations were estimated to draw conclusions about (3a) genetic variation in line and testcross performance and the relationships (3b) between ear rot severity and mycotoxin concentrations in lines and testcrosses and (3c) between line and testcross performance. Three maturity groups (early, mid-late, late) each comprising about 150 maize inbred lines were evaluated for ear rot resistance to FV. The same genotypes of the early maturity group were additionally evaluated for resistance to FG in separate, but adjacent trials. Field evaluation was conducted in two to six environments with silk channel inoculation and natural infection, respectively. In the late maturity group kernel inoculation was conducted additionally. Out of the 150 lines, 50 to 60 lines per maturity group were crossed with two unrelated testers of the opposite heterotic group. The concentrations of toxins FUM, DON and ZEA of the chosen lines and their testcrosses were analyzed by immunotests. Despite significant genotypic differences among the inbred lines after inoculation or natural infections, inoculation was found to be superior due to easier visual differentiation and increased accuracy. Therefore, inoculation should be conducted. In the late maturity group silk channel inoculation (simulating infection over the silks) and kernel inoculation (simulating secondary infection after wounding) were appropriate since both caused similar ear rot severity. However, both inoculation methods should be tested separately due to only moderate correlations between them. In the early maturity group resistance to FG or FV should be tested separately due to moderate correlations. Significant genotypic variances in large sets and subsets of lines and also in testcrosses revealed that there is genetic variation in all maturity groups and also within heterotic groups. In the flint group less lines were resistant to FV and FG than in dents indicating that resistance needs improvement, i.e. by introgression of resistance alleles followed by recurrent selection. Significant genotype x environment interactions may complicate selection and, therefore, multi-environmental trials are required for an accurate selection. High genotypic correlations between ear rot rating and mycotoxin concentrations were found among lines and testcrosses. The cost efficient indirect selection for mycotoxin concentrations based on ear rot rating could increase response to selection by testing more genotypes and/or in more test environments assuming a fixed budget. This should increase selection intensity and/or heritability. Moderate genotypic correlations between line and testcross performance were. One moderately to highly susceptible tester is sufficient due to high genotypic correlations between testcrosses of different testers. Both indicates a mainly additive gene action, but also non-additive gene action may play a role in some crosses. Selection for testcross performance based on line performance was less effective when calculating relative efficiencies. Different scenarios have been identified: (1) In Central Europe mainly resistance to ear rot in lines needs to be tested to ensure high seed quality, whereas resistance in testcrosses is not important due to low natural infection. (2) In Southern Europe, where high natural infections occur regularly, parallel selection for resistance to ear rot in lines and testcrosses is important. One susceptible tester should be used for creation of testcrosses. For selection in lines all parental lines should be inoculated but only lines selected out of testcrosses for agronomic traits would be rated afterwards saving resources. This is feasible due to later harvest date of lines than of testcrosses.
Effects of non-adapted quantitative trait loci (QTL) for Fusarium head blight resistance on European winter wheat and Fusarium isolates
(2010) Ohe, Christiane von der; Miedaner, Thomas
Fusarium head blight (FHB), caused by Fusarium graminearum and F. culmorum, is a devastating disease responsible for tremendous damage in wheat fields and contamination of grain with mycotoxins deoxynivalenol (DON) and nivalenol (NIV), rendering the harvest unsafe for human and animal consumption. The variability of Fusarium populations is high and changes in aggressiveness, chemotypes or species within and among Fusarium populations are known. Stable FHB resistance combined with high yield is one main target in wheat breeding programs. Mapping studies detected several quantitative trait loci (QTL) for FHB resistance in non-adapted sources, such as Sumai3 from China. The two most important and commonly used major QTL are located on chromosome 3BS (Fhb1) und 5A (Qfhs.ifa-5A). However, negative side effects of non-adapted resistance sources introgressed in elite winter wheat material are feared in Europe. Furthermore, the stability of the QTL effect against changing Fusarium populations is unknown. The objectives of this research were to analyze whether (1) the QTL Fhb1 and Qfhs.ifa-5A introgressed from a non-adapted resistance source into two winter wheat varieties have possible side effects on agronomic and quality performance, (2) 3-ADON and 15-ADON chemotypes are significantly different in their aggressiveness and DON production, (3) competition among Fusarium isolates in mixtures exists, and if so, how the resistant host will influence this competition. In conclusion, both resistance QTL are effective and stable in elite spring and winter wheat backgrounds. For improvement of FHB resistance both QTL are valuable, but Qfhs.ifa-5A would suffice for European breeding programs. Due to chemotype shifts, 3-ADON isolates could pose a greater risk to food safety than 15-ADON but breeding and use of highly resistant lines can reduce the risks associated with DON in wheat. Accordingly, resistant spring wheat lines were less affected by the tested Fusarium isolates and mixtures and, therefore, confirmed a high stability of these QTL. Directed selection of highly aggressive isolates due to the resistance QTL seems to be unlikely in the short term.
Inheritance of quantitative resistance and aggressiveness in the wheat/Fusarium pathosystem with emphasis on Rht dwarfing genes
(2010) Voß, Hans-Henning; Miedaner, Thomas
Fusarium head blight (FHB), or scab, is one of the most devastating fungal diseases affecting small-grain cereals and maize, causing severe yield losses and contamination of grain with mycotoxins such as deoxynivalenol (DON) worldwide. Fusarium graminearum (teleomorph Gibberella zeae) and Fusarium culmorum are the most prevalent Fusarium species in wheat production in Central and Northern Europe. Breeding for increased resistance to FHB in wheat is considered the most effective strategy for large scale disease management and mycotoxin reduction. Height reducing Rht genes are extensively used in wheat breeding programmes worldwide in order to improve lodging resistance and yield potential, with Rht-D1b being the most important Rht allele in Northern Europe. However, their individual effects on FHB resistance are yet unclear. Due to the incremental approach to increase host resistance the question arises whether the Fusarium pathogen has the capability to adapt by increased aggressiveness and/or increased mycotoxin production. Therefore, the objectives of the present study were to investigate the effects on FHB resistance of Rht-D1b and additional Rht alleles, the segregation variance for FHB resistance and identification of FHB resistance QTL in subsequent mapping analyses in three crossing populations segregating for the semi-dwarfing Rht-D1b allele and two sets of isogenic wheat lines. Regarding the pathogen, the study aims to determine the segregation variance in two F. graminearum crosses of highly aggressive parental isolates and to examine the stability of host FHB resistance, pathogen aggressiveness and the complex host-pathogen-environment interactions in a factorial field trial. All experiments were conducted on the basis of multienvironmental field trials including artificial inoculation of spores. The presence of Rht-D1b resulted in 7-18% reduction in plant height, but considerably increased FHB severity by 22-53% within progenies from three tested European elite winter wheat crosses. In the following QTL mapping analyses the QTL with the strongest additive effects was located at the Rht-D1 locus on chromosome arm 4DS and accordingly coincided with a major QTL for plant height in all three wheat populations. On total, a high number of 8 to 14 minor QTL for FHB reaction that were found in the three populations which emphasised the quantitative inheritance of FHB resistance in European winter wheat. The detected QTL mostly showed significant QTL-by-environment interactions and often coincided with QTL for plant height. By means of isogenic lines in the genetic background of the variety Mercia, Rht-D1b and Rht-B1d significantly increased mean FHB severity by 52 and 35%, respectively, compared to the wild-type (rht). Among the Maris Huntsman data set, the Rht alleles increased mean FHB severity by 22 up to 83%, but only the very short lines carrying Rht-B1c or Rht-B1b+Rht-D1b showed significance. The analyses of 120 progenies of the crosses from each of the highly aggressive parental F. graminearum isolates revealed significant genetic variation for aggressiveness, DON and fungal mycelium production following sexual recombination. This variation resulted in stable transgressive segregants towards increased aggressiveness in one of the two progeny. The factorial field trial, including eleven F. graminearum and F. culmorum isolates varying in aggressiveness and seven European elite winter wheat varieties, varying in their FHB resistance level, displayed no significant wheat variety × isolate interaction. Nevertheless, isolates possessing increased aggressiveness significantly increased FHB severity and DON production at a progressive rate on varieties with reduced FHB resistance. In conclusion, the analysed Rht alleles led to differently pronounced negative effects on FHB resistance that strongly depended on the genetic background. However, significant genetic variation for FHB resistance exists for selection and, thus, to largely counteract these effects by accumulating major and minor FHB resistance QTL. Significant genetic variation for aggressiveness among F. graminearum and the capability to increase its level of aggressiveness beyond yet known levels simply by sexual recombination may lead to long term erosion of FHB resistance. The rate at which increased aggressiveness develops will depend on the selection intensity and whether it is of constant, episodic or balanced nature. Consequently, the selection pressure imposed on the pathogen should be minimized by creating and maintaining a broad genetic base of FHB resistance that relies on more than one genetically unrelated resistance source by combining phenotypic and marker-assisted selection to achieve a sustainably improved FHB resistance in wheat breeding.
Mapping of quantitative-trait loci (QTL) for adult-plant resistance to Septoria tritici in five wheat populations (Triticum aestivum L.)
(2010) Risser, Peter; Miedaner, Thomas
Septoria tritici blotch (STB), caused by Septoria tritici (teleomorph Mycosphaerella graminicola), is one of the most important diseases in wheat varieties worldwide, responsible for severe damage of the leaves causing yield losses between 30 and 40 %. Control of STB includes crop rotation, soil tillage, fungicide application, and cultivation of resistant varieties. Profit-making wheat growers are forced to apply narrow crop rotations under reduced tillage. Some fungicides including widely-used strobilurins are no longer effective due to mutations in the highly variable pathogen population of S. tritici. Therefore, resistance breeding using genetic mapping to identify quantitative-trait loci (QTL) associated with STB resistance provides a promising strategy for controlling the disease. The main goal of this study was to detect chromosomal regions for quantitative adult-plant resistance of winter wheat to STB. Besides this, we analyzed the genetic diversity of 24 European varieties after inoculation with four different isolates of S. tritici. Multienvironmental field trials inoculated with S. tritici were applied to test isolates and varieties and to phenotype mapping populations. In detail, the objectives were to (1) compare natural infection and inoculation, (2) evaluate genotypic variation of adult-plant resistance to STB in European varieties, (3) analyze genotype x environment (G x E) interaction, (4) evaluate and analyze phenotypic data including STB severity, heading date (HED), and plant height (PLH) of five mapping populations, (5) construct genetic linkage maps of these populations using AFLP, DArT, and SSR markers, (6) determine number, positions, and genetic effects of QTL for evaluated traits, and (7) reveal QTL regions for multiple-disease resistance within mapping populations using QTL meta-analysis. In all trials, inoculation with one to four preselected isolates was performed and STB severity was visually scored plotwise as percentage coverage of flag leaves with lesions bearing pycnidia. 24 winter wheat varieties were chosen with maximal differentiation in resistance to STB and evaluated across three years including nine environments. Five mapping populations, Florett/Biscay, Tuareg/Biscay, History/Rubens, Arina/Forno, and Solitär/Bussard, each comprising a cross of a resistant and a susceptible variety, with population sizes ranging from 81 to 316, were phenotyped across four to six environments. In parallel, 221 to 491 polymorphic genetic markers were assigned to linkage groups covering 1,314 to 3,305 cM of the genome. Based on these linkage maps, the number, positions, and genetic effects of QTL could be determined by composite interval mapping. Furthermore, raw data of different experiments evaluated for resistance to two other pathogens, Fusarium head blight and Stagnospora glume blotch, were used to reveal multiple-disease resistance QTL within Arina/Forno and History/Rubens populations by the software package PLABMQTL. Results of inoculated field trials coincided with not inoculated trials showing natural infection (r = 0.84 to 0.99, P < 0.01), thus inoculation method was accurate to evaluate STB severity in the field. Genotypic variation between 24 varieties ranged from 8 % (Solitär) to 63 % (Rubens) flag leaf area infected. In the analysis of variance, genotypic variance had highest impact followed by G x E interaction (P < 0.01). Therefore, environmental stability of varieties should be a major breeding goal. The varieties Solitär, History, and Florett were most stable, as revealed by a regression approach. In contrast, disease symptoms of Biscay ranged from 19 to 72 % within the three experimental years. Phenotypic data revealed significant (P < 0.01) genotypic differentiation for STB, HED, and PLH within all five mapping populations and between the parents. Entry-mean heritabilities (h²) ranged from 0.69 to 0.87 for STB, the only exception was Tuareg/Biscay (h² = 0.38). For HED (h² = 0.78 to 0.93) and PLH (h² = 0.92 to 0.98) heritabilities were high. All correlations between STB and HED (r = -0.18 to -0.33) as well as between STB and PLH (r = -0.13 to -0.45) were negative and moderate. The exception was History/Rubens which is segregating at the Rht-D1 locus showing considerably higher correlation between STB and PLH (r = -0.55, P < 0.01). The five mapping populations showed a wide and continuous distribution of mean STB severity averaged across three to six environments in field trials at adult-plant stage. In QTL analysis, one to nine, zero to nine, and four to eleven QTL were detected for STB, HED, and PLH, respectively, across five wheat populations using composite interval mapping. One to two major QTL for resistance to STB were detected consistently across environments in each population (QStb.lsa_fb-3B, QStb.lsa_fb-6D, QStb.lsa_tb-4B, QStb.lsa_tb-6B, QStb.lsa_hr-4D, QStb.lsa_hr-5B.1, QStb.lsa_af-3B, QStb.lsa_bs-7A) explaining more than 10 % of normalized adjusted phenotypic variance. Altogether, resistance QTL explained 14 to 55 % of adjusted phenotypic variance. Both parents contributed resistant alleles. Major QTL, however, were all from the resistant parent. QTL meta-analysis revealed each of four loci for multiple-disease resistance located on chromosomes 3B, 4B, 5B, and 6D in Arina/Forno, and on chromosomes 2B, 4D, 5B, and 7B in History/Rubens. The most effective meta QTL was on chromosome 4D in History/Rubens closely linked to Rht-D1. The resistance allele from History reduced disease severity by 9.8 % for STB and 6.3 % for FHB, thus explaining 47 % and 60 % of partial phenotypic variance. In general, European wheat varieties showed a wide range of genotypic variation for STB resistance useful for breeding. Although the influence of environment and G x E interaction was high, some resistant varieties which were stable across multiple environments were found (Solitär, History, Florett). Genomic regions associated with STB resistance were mapped across 13 out of 21 wheat chromosomes. Together with the continuous distribution of five segregating populations for flag leaf infection, it can be concluded that the adult-plant resistance to S. tritici was inherited quantitatively depending on several loci explaining part of phenotypic variance. QTL meta-analysis across three severe pathogens, including Fusarium head blight, Stagnospora glume blotch, and STB, within two populations revealed eight loci for multiple-disease resistance with closely linked markers applicable in resistance breeding. Combining detected major QTL as well as meta QTL in present breeding material by applying marker-assisted selection seems a promising approach to the breeding of varieties with improved resistance to Septoria tritici blotch, Fusarium head blight, and Stagnospora glume blotch.
Molecular and genetic analyses of aggressiveness in Fusarium graminearum populations and variation for Fusarium head blight resistance in durum wheat
(2011) Talas, Firas; Miedaner, Thomas
Fusarium head blight (FHB) is a devastating disease of wheat, barley and other cereals, which affects all wheat-growing areas of the world. The most prevalent species are Fusarium graminearum Schwabe (teleomorph: Gibberella zeae (Schweinitz) Petch) and Fusarium culmorum (W. G. Smith) Saccardo. Wheat breeding for FHB resistance has become the most effective and cost efficient strategy to combat this disease. Assisting long term stable breeding programs need a better understanding of the biology and dynamic changes of the population structure. Deoxyninalenol (DON) has the most economical impact among the other mycotoxin secreted by this fungus. Several chemotypes characterizes F. graminearum isolates. All chemotypes (3-ADON, 15-ADON, and NIV) were detected in Europe. The prevalent chemotype in Germany and UK is 15-ADON. Population structure is the result of evolutionary forces acting on the population in time and space together with mutation, recombination, and migration enhancing the genetic variance of a population, random drift and the selection reducing it. Aggressiveness in F. graminearum denotes the quantity of disease induced by a pathogenic isolate on a susceptible host in a non-race specific pathosystem, and is measured quantitatively. The quantitative traits such as aggressiveness and DON production mirror both the environmental changes and the genetic variation. Several genes are responsible for DON production; majority of these genes are grouped in TRI5 cluster. Few genes are known to be associated with F. graminearum aggressiveness such as MAP kinase genes, RAS2, and TRI14. Association between single nucleotide polymorphism and genetic variation of aggressiveness and DON production traits provide a clear identification of quantitative participation of different SNPs in expressing the trait. Also, this approach provides a good method to test the association between candidate genes and the traits. The objectives of this research were to (1) screen some durum wheat landraces for FHB resistance; (2) determine the genetic and chemotypic structure of natural population of F. graminearum in Germany; (3) determine the phenotypic variation in Aggressiveness and DON production, which come out one farmer wheat field; (4) compare the phenotypic variation and genetic variation occurring in one wheat field; and (5) associate the phenotypic traits with SNPs in candidate genes. Screening for FHB resistance was performed on sixty-eight entries form the Syrian landraces. The main characters of selection for resisting FHB disease are low mean value of infection and stability in different environments. Four genotypes (ICDW95842, ICDW92330, ICDW96165, Chahba) had small mean FHB value, small value of deviation form regression, and regression coefficient close to zero. These genotypes were considered as candidate resistant sources of FHB for further agronomic performance analysis through backcrossing generation. The causal agent of FHB in Germany is F. graminearum s.s. with a dominating rate of 64.9 % (out of 521 Fusarium spp. isolates). Nonetheless, the three chemotypes were detected in Germany and some times within one wheat field. The 15-ADON chemotype dominated the populations of F. graminearum s.s. in Germany followed by 3-ADON then NIV chemotype (92, 6.8, and 1.2%, respectively). High genetic diversity (Nei?s gene diversity ranged form 0.30 to 0.58) was detected on a single wheat field scale. Analysis of molecular variance (AMOVA) revealed a higher variance within populations (71.2%) than among populations (28.8%). Populations of F. graminearum s.s. in Germany display a tremendous genetic variation on a local scale with a restricted diversity among populations. Surprisingly the phenotypic variation of aggressiveness and DON production revealed a similar partitioning scale as the genetic variation. In other words, analyses of variance (ANOVA) revealed a higher variance within populations (72%) than between (28%) populations. The wide spectrum of aggressiveness (i.e., from 18 to 39%) and DON production (from 0.3 to 23 mg kg-1) within single wheat field simulate the global variation in both traits. Consequently, associating the observed variation of aggressiveness and DON production with detected single nucleotide polymorphism (SNPs) in some candidate genes revealed few but significant associations. According to Bonferroni-Holm adjustment, three SNPs were associated significantly with the aggressiveness, two in MetAP1 and one in Erf2 with explained proportions of genotypic variance (pG) of 25.6%, 0.5%, and 13.1%, respectively. One SNP in TRI1 was significantly associated with DON content on TRI1 (pG=4.4). The rapid decay of the LD facilitate a better high resolution of the association approach and is in turn suggest the need of higher number of SNP marker to facilitate a genome wide association study. The linkage disequilibrium between unlinked genes suggests the involvement of these genes in the same biosynthesis network. In conclusion, building wheat breeding program for FHB resistance depend initially on identifying sources of resistance among wheat varieties or wild relatives. Moreover, understanding the population structure of the pathogen and the selection forces causing genetic alteration of the population structure enable us employ a sufficient increase of the host resistance. Keeping such a balanced equilibrium between increasing host resistance and changes occur in genetic structure of F. graminearum population would insure no application of additional selection pressure. Further association of candidate genes with aggressiveness can provide effective information of the population development. Continuous observation of Fusarium population?s development is needed to insure a stable management of Fusarium head blight disease.
Quantitative trait loci (QTL) mapping in multi-line crosses of European maize
(2012) Steinhoff, Jana; Reif, Jochen Christoph
Multiple-line crosses (MC) have been proposed as promising mapping resource for quantitative trait loci (QTL) detection for agronomic important traits. In contrast to mapping populations derived from a single biparental population, MC can increase the statistical power of QTL detection, the accuracy of QTL location and of QTL effect estimates. Additionally, MC-QTL mapping has the advantage of using data routinely collected in plant breeding programs. The objectives of this study were to (i) assess the reliability of the maize genetic consensus map by comparing it to its six single population linkage maps, (ii) exploit the benefits of a combined analysis by applying two MC-QTL mapping models and to compare the results to single-population analyses, and (iii) investigate the genetic architecture of grain yield, grain moisture, adaptation, and flowering time in elite maize. The experiment comprised six populations with 109 to 150 individuals, resulting from crosses of elite maize breeding material. The germplasm was provided by Syngenta Seeds, Bad Salzuflen, Germany. The 788 genotypes were genotyped with 857 SNP markers. After constructing genetic linkage maps of the six single populations, the genotypic information of the single populations was integrated in a consensus map and its reliability was tested for QTL studies. The average distance between adjacent markers was 1.84 cM suggesting that the marker density is not a limiting factor for QTL analyses. Moreover, we observed medium to high heritabilities for all traits. Consequently, the quality of both genotypic and phenotypic data should allow QTL detection with substantial power. We applied two different MC-QTL mapping models on the data assuming fixed allele effects. The disconnected model estimates QTL effects nested within populations, whereas the connected model takes into account the relationship between the populations. Both models outperformed the single population analyses with regard to QTL detection rate, variance explained by the detected QTL, and the size of the confidence intervals. In all analyses, the disconnected model outperformed the connected model in terms of number of QTL and size of confidence intervals. This superiority seems to be caused by the high background dependencies of QTL effects in connected crosses, which was revealed by a modified diallel analysis of the QTL effects. We investigated the genetic architecture of grain yield, grain moisture, adaptation to maturity zones, and flowering time. Our findings suggest that all traits exhibit a complex genetic architecture with an absence of large QTL effects. Some of the studied traits appear to be influenced by epistasis, interactions between loci. In particular, for flowering time, the two-dimensional scan for epistatic interactions suggested the presence of digenic epistasis. The absence of QTL with large effects suggests that marker-assisted selection is not an appropriate tool to breed for adapted maize hybrids with improved grain yield. Consequently, more suitable approaches for complex traits such as genomic selection should be applied. The joint analyses across populations resulted in higher QTL detection power and resolution compared to single population analyses. Thus, for traits with a less complex genetic architecture, MC-QTL mapping is a powerful tool for the identification of robust diagnostic markers.
Phenotypic and molecular analyses of grain and biomass productivity under irrigated and rainfed conditions in hybrid rye
(2014) Gottwald, Marlen; Miedaner, Thomas
Rye (Secale cereale L.) is a small grain cereal used for bread making, livestock feeding and as renewable energy source. These types of usages are leading to different breeding goals. Rye growing regions are affected by climate change and consequently by drought. Germany is touched by rainless periods in spring and early summer in the last years. Again, in spring 2012 farmers in Brandenburg and Lower Saxony were affected by drought periods. Yield losses in those regions, especially in combination with sandy soils are expected. Therefore much attention is paid for breeding of drought resistant germplasm. Briefly, our objectives of this study were to (1) estimate the biomass and biogas potential of different plant materials, their quantitative genetic parameters and biogas-related traits, (2) analyze two recombinant inbred lines and differences in their yield potential between irrigated and rainfed regime, as well as the relative efficiency for indirect selection for drought resistance in irrigated regime, and (3) investigate the phenotypic performance for ten agronomic and quality traits across multiple environments and estimated the number and effects underlying QTL. For the biomass-/ biogas analyses a wide range of plant material was analysed. Germplasm resources, full-sib families selected for grain and forage use were tested for their per se and testcross performance and experimental hybrids selected for grain use and population cultivars selected for grain and forage use were analyzed. Dry matter yields varying across environments from 106 to 177 dt/ha for per se and testcross performance, respectively. For testcross performance, germplasm resources showed similar values to forage rye. The later the maturity stage, the more dry matter yield on the whole plant level was achieved. Estimates of genotypic variances for biomass yield were significant for all rye materials, whereas the variances per se and for testcrosses were for germplasm resources exorbitant higher than for forage and grain rye. Typical cumulative methane production curves were obtained for the whole plant material from the Hohenheim biogas yield test. Methane yield showed large differences between second and third harvest date for individual plant fractions. Differences between genotypes were not substantial for methane yield although significant in some instances. At EC77/83 hybrids and forage rye reached similar methane yield of about 5000 m3/ha. A high correlation between dry matter yield and methane yield was observed (r=0.95). Concerning high cost and time consuming analysis of biogas tests, for breeders the main breeding goal should be maximum dry matter yield. Direct selection on dry matter yield should indirect improve methane yield. Two biparental populations were used for the analysis of drought tolerance. The analysis was performed in duplicate. Both populations were grown under irrigated and rainfed regimes. Striking less rainfall compared to long-term precipitation occurred between April and July, during critical phases of plant development. Grain yield reduction between irrigated and non-irrigated regime ranged from 2% to 29.6% for population A and 2% to 40% for population B, whereas differences between both regimes were significant (P<0.05) for five and four environments, respectively. Genotypic variances of grain yield were significant in all instances, whereas genotype by irrigation interaction variance between both regimes being significant only in three and four environments for population A and B, respectively. Analysis across those environments revealed significant difference for genotype by irrigation interaction variance and the three-way interaction variance in both populations. Heritability estimates were higher for the irrigated than for the rainfed regime. High interaction variance with environment and no clustering of the two regimes in a multi-dimensional analysis were found. This illustrates the different soil and whether conditions between locations and additionally every location suffered from a different drought stress. The correlation between both regimes was significant but moderate, but genotypic coefficients considerably higher (Pop-A: 0.86, Pop-B: 0.84), which could be substantiated that testcrosses differed not substantially in drought-resistance. Indirect selection for drought in the irrigated regime was predicted to be equally or more efficient than direct selection in the non-irrigated regime. Phenotypic and genotypic analysis was done across ten environments for both biparental populations for the general improvement of agronomic and quality traits in rye. Population A were genotyped with a Rye5K SNP array and for population B DArT genotyping was done with a 3K rye array. Additionally both populations were genotyped with about 150 SSRs. The genetic linkage maps comprised 1,819 and 1,265 markers for population A and B, respectively and were used for the QTL analysis for ten agronomic and quality traits. Phenotyping revealed large genetic variation for ten agronomic and quality traits. Intensive phenotyping at up to ten environments led to moderate to high heritabilities. Across environments explained genotypic variance of the individual QTL ranged from 5 to 55%. For 1000-kernel weight, test weight, falling number, and starch content, several QTL with high effects and a frequency of recovery of about 90% were identified in both population. Rye suffered from drought stress in the last decade. Focusing on general improvement of rye regarding yield and quality, as well as improving rye regarding drought-resistance is important. Future research should be done in fine mapping and validation of the detected QTLs, for exploiting their potential in marker assisted breeding.
Development and assessment of a multi-sensor platform for precision phenotyping of small grain cereals under field conditions
(2014) Busemeyer, Lucas; Würschum, Tobias
The growing world population, changing food habits especially to increased meat consumption in newly industrialized countries, the growing demand for energy and the climate change pose major challenges for tomorrows agriculture. The agricultural output has to be increased by 70% by 2050 to achieve food and energy security for the future and 90% of this increase must be achieved by increasing yields on existing agricultural land. Achieving this increase in yield is one of the biggest challenges for the global agriculture and requires, among other things, an efficient breeding of new, higher-yielding varieties adapted to the predicted climate change. To achieve this goal, new methods need to be established in plant breeding which include efficient genotyping and phenotyping approaches of crops. Enormous progress has been achieved in the field of genotyping which enables to gain a better understanding of the molecular basis of complex traits. However, phenotyping must be considered as equally important as genomic approaches rely on high quality phenotypic data and as efficient phenotyping enables the identification of superior lines in breeding programs. In contrast to the rapid development of genotyping approaches, phenotyping methods in plant breeding have changed only little in recent decades which is also referred to as phenotyping bottleneck. Due to this discrepancy between available phenotypic and genotypic information a significant potential for crop improvement remains unexploited. The aim of this work was the development and evaluation of a precision phenotyping platform for the non-invasive measurement of crops under field conditions. The developed platform is assembled of a tractor with 80 cm ground clearance, a carrier trailer and a sensor module attached to the carrier trailer. The innovative sensors for plant phenotyping, consisting of several 3D Time-of-Flight cameras, laser distance sensors, light curtains and a spectral imaging camera in the near infrared reflectance (NIR) range, and the entire system technology for data acquisition were fully integrated into the sensor module. To operate the system, software with a graphical user interface has been developed that enables recording of sensor raw data with time- and location information which is the basis of a subsequent sensor and data fusion for trait determination. Data analysis software with a graphical user interface was developed under Matlab. This software applies all created sensor models and algorithms on sensor raw data for parameter extraction, enables the flexible integration of new algorithms into the data analysis pipeline, offers the opportunity to generate and calibrate new sensor fusion models and allows for trait determination. The developed platform facilitates the simultaneous measurement of several plant parameters with a throughput of over 2,000 plots per day. Based on data of the years 2011 and 2012, extensive calibrations were developed for the traits plant height, dry matter content and biomass yield employing triticale as a model species. For this purpose, 600 plots were grown each year and recorded twice with the platform followed by subsequent phenotyping with state-of-the-art methods for reference value generation. The experiments of each year were subdivided into three measurements at different time points to incorporate information of three different developmental stages of the plants into the calibrations. To validate the raw data quality and robustness of the data collection and reduction process, the technical repeatability for all developed data analysis algorithms was determined. In addition to these analyses, the accuracy of the generated calibrations was assessed as the correlations between determined and observed phenotypic values. The calibration of plant height based on light curtain data achieved a technical repeatability of 0.99 and a correlation coefficient of 0.97, the calibration of dry matter content based on spectral imaging data a of 0.98 and a of 0.97. The generation and analysis of dry biomass calibrations revealed that a significant improvement of measurement accuracy can be achieved by a fusion of different sensors and data evaluations. The calibration of dry biomass based on data of the light curtains, laser distance sensors, 3D Time-of-Flight cameras and spectral imaging achieved a of 0.99 and a of 0.92. The achieved excellent results illustrate the suitability of the developed platform, the integrated sensors and the data analysis software to non-invasively measure small grain cereals under field conditions. The high utility of the platform for plant breeding as well as for genomic studies was illustrated by the measurement of a large population with a total of 647 doubled haploid triticale lines derived from four families that were grown in four environments. The phenotypic data was determined based on platform measurements and showed a very high heritability for dry biomass yield. The combination of these phenotypic data with a genomic approach enabled the identification of quantitative trait loci (QTL), i.e., chromosomal regions affecting this trait. Furthermore, the repeated measurements revealed that the accumulation of biomass is controlled by temporal genetic regulation. Taken together, the very high robustness of the system, the excellent calibration results and the high heritability of the phenotypic data determined based on platform measurements demonstrate the utility of the precision phenotyping platform for plant breeding and its enormous potential to widen the phenotyping bottleneck.
Quantitative-trait loci (QTL) mapping of important agronomical traits of the grain and biomass production in winter rye (Secale cereale L.)
(2015) Haffke, Stefan; Miedaner, Thomas
Rye is an important crop in Northern and Eastern Europe and mainly used for food and feed and became most recently important for biogas production. Hybrid rye varieties dominate the cultivated area, which is mainly on light and sandy soils, because rye has a relatively high tolerance to biotic and abiotic stress factors. Climate change will also affect Central Europe, causing higher temperatures and less precipitation in spring and summer. Rye will be influenced more by these effects than other cereals because it is mainly grown on marginal environments. Rye has a high potential for being used as a biogas substrate, but detailed information on improving this trait in hybrid rye is missing. Until now, no study that analyzed phenotypic and genotypic agronomic traits for using rye for biogas production exists. Further, there is only one study, which dealt with the influence of periodic drought stress in rye cultivated areas. Beside this, we analyzed yield stability over a wide range of environments in consideration of drought stress in Central Europe. We analyzed an interpool hybrid population (Pop-D) in 2011 and 2012 at seven environments in Germany for the biomass yield and grain yield (Publication I). This study showed low correlations between grain yield and dry matter yield (r = 0.33). Higher correlations were obtained with two plant height measurements (at heading time, r = 0.64; before harvest, r = 0.52) and dry matter yield. The indirect selection via plant height was superior in contrast to the direct selection of dry matter yield by factor 1.24. Genotypic results confirmed phenotypic results as no overlapping QTL for grain yield and dry matter yield were detected (Publication II). However, we identified common gene regions for plant height and dry matter yield due to the high correlation between both. Plant height is a promising trait for indirectly selecting high biomass yielding varieties. The paradigm shift from shorter plants with high grain yield to taller hybrids as a resource for biogas substrate needs additional breeding efforts for lodging resistance. In Publication III we analyzed two intrapool populations (Pop-A and -B) and one interpool population (Pop-C) at 16 – 18 environments (location x year combinations) under irrigated and rainfed conditions in Germany and Poland. Yield stability was high over a wide range of environments, even when drought stress environments were included. This illustrates the adaption of rye to marginal and drought stress environments. The analyzed populations showed no differences within yield stability, but yield differences between inter- (Pop-C) and intra-pool (Pop-A and -B) crosses were visible. Selection for yield stability is possible due to the genetic variance for this trait within all three populations. Therefore, it is important to select genotypes with low genotype x environment interaction. All three populations showed high yield stability on a high yield level and were already well adapted to extreme weather events caused by climate change. It is recommended to use highly diverse environments with irrigated and rainfed conditions to select on yield stability and high yielding varieties under optimum and drought conditions
Phenotypic and genotypic assessment of traits with relevance for hybrid breeding in European winter wheat
(2015) Langer, Simon Martin; Würschum, Tobias
Hybrid breeding in wheat has recently received increased interest, especially in Europe, and large public and private projects investigating hybrid wheat breeding have been launched. Hybrid breeding has been a great success story for allogamous crops and is seen as a promising approach to increase the yield potential in wheat. Wheat covers more of the world’s surface than any other food crop and is the second main staple crop for human consumption. It can be produced under widely varying conditions and is grown all around the globe, yet, yield gain has declined and is lagging behind the needs of the constantly growing human population. Future challenges in wheat breeding such as the establishment of hybrid varieties and the adaptation of breeding germplasm to increasing stresses caused by climatic changes also in Europe require knowledge-based improvements of relevant traits and phenotyping approaches suited for applied high-throughput plant breeding. A major limitation for the establishment and the production of hybrid wheat is the lack of a cost-efficient hybrid seed production system. This requires the generation of parental ideotypes which maximize the cross-fertilization capability. Male parents should have an extended time of flowering, extrude anthers and widely shed large amounts of viable pollen. Females need increased receptivity for male pollen by opening the glumes and extruding stigmatic hair. Furthermore, male plants should be taller than females and a synchronized timing of flowering between the two parents is also of utmost importance. Employing a set of European elite winter wheat lines, we developed and evaluated phenotyping methods for important floral and flowering traits with relevance for improved cross-pollination (Publication I). We observed high heritabilities for important traits such as ‘pollen mass’ (h2=0.72) and ‘anther extrusion’ (h2=0.91). In addition, genotypic variances were significant which warrants further breeding success. Positive correlations were found among important flowering and floral characteristics which enables the improvement of outcrossing by indirect selection. ‘Pollen mass’ for example, was associated with ‘anther extrusion’, ‘anther length’ and ‘plant height’. Our findings suggest the utility of the developed phenotyping approaches for applied plant breeding and the potential of the traits to assist in the design of the male ideotype for increased cross-fertilization. We investigated the genetic architecture of flowering time and plant height (Publication II and III). A panel of 410 European winter wheat varieties was genotyped by a genotyping-by-sequencing approach and in addition, analyzed for the effects of specific candidate genes. The major factor affecting flowering time was the photoperiod regulator Ppd-D1 (58.2% of explained genotypic variance) followed by Ppd-B1 copy number variation (3.2%). For plant height, the two candidate loci Rht-D1 (37.0%) and Rht-B1 (14.0%) had the largest effects on the trait but contrary to reports in the literature did not contribute to flowering time control. In addition, we identified several small effect QTL and epistatic QTL responsible for fine-adjustments of these two traits. Population structure and genetic relatedness in European elite wheat lines was assessed using different types of markers (Publication IV). Results for relatedness differed for the marker types but consistently showed the absence of a major population structure. Regarding the large wheat genome our results revealed that a high number of markers is necessary as there are regions with only low coverage. Concordantly, we were not able to identify the major flowering locus Ppd-D1 without targeted candidate gene analysis. Observations on the findings on population structure could be confirmed in Publication II and III and in addition, the geographical distribution of important flowering time and plant height genes displayed the historical development of wheat breeding in Europe. This information on genetic relatedness among lines can also be employed to assist the establishment of hybrid wheat.
Breeding winter durum wheat for Central Europe : assessment of frost tolerance and quality on a phenotypic and genotypic level
(2015) Sieber, Alisa-Naomi; Würschum, Tobias
Durum wheat (Triticum durum) is a tetraploid wheat that is used for pasta and other semolina products. Quality standards for semolina requested by the pasta industry are very high. Different characteristics should come with the cereal as raw material for an optimal end product. Vitreosity, the glassy and amber quality feature of durum wheat kernels, is an indicator for high semolina yield. The complex protein-starch matrix of glassy kernels breaks the grain into the typical semolina granulate instead of flour during milling. Humid conditions, like late summer rains in Central Europe, have a huge effect on this characteristic, changing this matrix irreversibly. Such processes in the kernel are less understood and challenge plant breeders to find genotypes with improved vitreosity. A set of F5 winter durum wheat lines (Chapter 2) was used to investigate the relationship between protein content and vitreosity as well as the impact of humidity on the stability of the trait. A method to evaluate the mealy part in kernels was improved and enabled to test for the influence of humidity on vitreosity. Furthermore, it was revealed that the vitreosity of a durum wheat kernel depends on the protein content up to a specific threshold as well as on the genotypic potential to form the complex endosperm matrix. The ability to maintain this kernel quality under humid conditions also highly depends on the genetics of a variety. In the Mediterranean region, durum wheat is grown as autumn-sown spring type. The mild winters as well as rain during spring allow the plants to develop well, and the dry summers enable an early harvest in June. Durum wheat production in Central Europe, on the other hand, is confronted with harsh winters and recurring severe frosts. The lack of a sufficient frost tolerance in combination with high quality, forces farmers to use the spring type with a spring sowing. Growing winter durum instead of spring durum wheat, would allow an autumn sowing. Using the winter type in this growing area, could have several advantages like an increased yield and stability due to a prolonged growing time. Further, the constant soil coverage would prevent soil erosion and the growth vigor of winter durum has advantages against weeds. The success of winter durum breeding depends on frost tolerance as a key factor for varieties with excellent winter survival. Discontinuous occurrence of frosts across years and protective snow coverage, however, limit the phenotypic selection for this trait under field conditions. Greenhouses or climate chambers could be used as alternative to test under the necessary conditions, but those fully-controlled tests are time consuming and labor-intensive. The ‘Weihenstephaner Auswinterungsanlage’ are wooden boxes with movable glass lids used as a semi-controlled test. Plants are exposed to all seasonal conditions, including frost stresses, in this test, but they can be protected from snow coverage. While this method is already successfully used to test for frost tolerance in bread wheat, the application in durum wheat has not been evaluated yet. The frost tolerance scorings of winter durum elite lines (F5 and F6) based on the ‘Weihenstephaner Auswinterungsanlage’ were compared to the field evaluation (Chapter 3). It was demonstrated that this semi-controlled test produces reliable and highly heritable (h2 = 0.83-0.86) frost tolerance data. The correlation of those results compared with the field data (r = 0.71) suggests this semi-controlled test as an indirect selection platform. Since it is now possible to test cost-efficient at early stages for frost tolerance, the next challenge was to determine whether the kernel quality or the grain yield suffers from an increased frost tolerance. In a survey with F5 winter durum elite lines, no negative association between frost tolerance and quality or other important agronomic traits could be found in European breeding material (Chapter 4). In order to support classical plant breeding, which relies predominantly on phenotypic data and parental information, molecular markers can be taken into account. Molecular markers can provide an in-depth look into the genetic architecture of traits, enable the determination of the relatedness of genotypes, identify the genetic variation in a population, or can assess the effect of geographic selection preferences. Furthermore, it is possible to assist knowledge-based selection. This improves plant breeding programs on a genetic level. The population structure in spring durum has already been examined with molecular methods in several studies. Winter durum, on the other hand, was only analyzed as a small group as part of spring durum studies or in groups of landraces. A highly diverse and unique panel of 170 winter durum and 14 spring durum lines was analyzed using a genotyping-by-sequencing (GBS) approach. A total of 30,611 markers, well distributed across the chromosomes, were obtained after filtering for marker quality. A principal coordinate analysis and a cluster analysis were applied. Together they revealed the absence of a major population structure (Chapter 5). The lines, however, grouped in a certain way, depending on their origin, associated with decreasing quality and increasing frost tolerance moving from South to Continental Europe. These groups allow breeders to conduct targeted crosses to further improve the frost tolerance in the Central European material. Another possibility is to build heterotic groups for hybrid breeding. The linkage disequilibrium (LD) decay was within 2-5 cM, indicating a high diversity in winter durum. The high marker density together with the extent of LD observed in this analysis allows to perform high-resolution association mapping in the present winter durum panel. The 30,611 markers and additional markers for candidate genes in frost tolerance were used to assess the genetic architecture of frost tolerance in durum wheat (Chapter 6). A major QTL was identified on chromosome 5A, likely being Frost Resistance-A2 (Fr-A2). Additional analysis of copy number variation (CNV) of CBF-A14 at Fr-A2 support this conclusion. CBF-A14 CNV explains about 90% of the proportion of genotypic variance. Two markers found in the QTL region were combined into a haploblock and enabled to capture the genetic variance of this QTL. Furthermore, the frequency of the QTL allele for frost tolerance shows a latitudinal gradient which is likely associated with winter conditions. In summary, the selection tools for vitreosity and frost tolerance provided in this study create a platform for winter durum breeding to select for high quality genotypes with excellent winter survival utilizing phenotypic as well as genotypic information.
Differences in yield performance and yield stability between hybrids and inbred lines of wheat, barley, and triticale
(2015) Mühleisen, Jonathan; Reif, Jochen Christoph
Hybrids of wheat, barley, and triticale are expected to possess higher yield performance and yield stability compared to inbred lines. Assessment of yield performance as well as yield stability requires the evaluation of genotypes in plot-based yield trials across multiple environments. Evaluation of genotypes under stress conditions can be associated with increased field heterogeneity, which may result in imprecise estimates of genotypic values. The assessment of yield stability requires intensive testing in many environments, and it would be interesting to know how many test environments are required to reliably estimate yield stability. The key objectives of the present thesis were to (1) investigate optimal strategies to analyze field trials with high error variance due to spatially varying drought stress, (2) identify the required number of test environments to precisely estimate yield stability of individual barley genotypes, and (3) examine yield performance and yield stability of wheat, barley, and triticale hybrids and lines. Drought stress at two locations of a winter triticale trial caused increased field heterogeneity, resulting in lower heritabilities compared to the four non-stress locations. It was found that heritability could be increased by modeling incomplete block and row effects, by using visual scorings of drought stress intensity as covariates in an analysis of covariance, and by modeling a spatial covariance between adjacent plots. The most suitable model can be identified using the Akaike Information Criterion. In addition, it has to be ensured that the covariate is independent from genotypic effects and that it is linearly related with the response variable. Dynamic yield stability of genotypes was frequently found to depend strongly on the specific set of test environments. When the genotypes were evaluated in different environments, e.g. in the following year, the ranking in yield stability could be different. This would result in a low heritability. Theoretical assumptions and empirical studies showed that heritability can be increased when the number of test environments is increased. Five series of barley registration trials with a reduced number of 16 to 27 genotypes evaluated in 39 to 45 environments were used to investigate the relationship between magnitude of heritability of yield stability and number of test environments. Based on a cross-validation approach, it was found, that at least 40 test environments should be used to obtain a heritability of 0.5. Magnitude of heritability, however, varied strongly within and between series. Therefore, depending on the respective set of environments and genotypes, more or less test environments can be needed. Yield performance of wheat hybrids produced using chemical hybridizing agents (CHA) or cytoplasmic male sterility (CMS) was well investigated in other studies reporting around 10% midparent heterosis for grain yield. In the present thesis, CMS-based barley hybrids were compared with parental inbred lines and unrelated commercial inbred lines in breeding and registration trials. Midparent heterosis was around 10%. The comparison with commercial inbred lines in the registration trials revealed that hybrids could compete with and partially surpass outstanding inbred lines. Triticale hybrids, produced using CMS, were evaluated for grain yield at up to 20 environments with their parents and commercial inbred lines. Midparent heterosis amounted to 3% and no hybrid outyielded the best inbred line. The low yield performance of triticale hybrids is probably associated with CMS-system, since CHA-based triticale hybrids showed a midparent heterosis around 10% in early studies, which is comparable to the midparent heterosis found in wheat and barley. Yield stability of CHA-based wheat as well as CMS-based hybrids of barley and triticale was compared with yield stability of parental and commercial inbred lines on group level. The wheat and barley hybrids showed on average significantly higher dynamic yield stability compared to inbred lines, but the triticale hybrids did not. In the barley registration trials, hybrids had the highest dynamic yield stability on average. The CMS-based triticale hybrids, however, showed on average significantly lower dynamic yield stability as their female parents and the commercial inbred lines across 20 environments. In conclusion, hybrids of wheat and barley possessed an increased yield potential as well as an enhanced dynamic yield stability. In contrast, the CMS-based triticale hybrids showed only marginal yield advantages coupled with low dynamic yield stability. Further research is required to increase economical competitiveness of hybrids in all three crops, to identify and eliminate the reasons for poor performance of CMS-based triticale hybrids and to investigate the suitability of dynamic yield stability measures to identify vigorous and stress tolerant genotypes.
Molecular mapping of resistance and aggressiveness in the cereal/Fusarium head blight pathosystem
(2016) Kalih, Rasha; Miedaner, Thomas
Fusarium head blight (FHB) is one of the most destructive fungal diseases in small-grain cereals worldwide causing significant yield losses and contamination of grain with mycotoxins e.g., deoxynivalenol (DON). This renders the grain unsuitable for human consumption and animal feeding. Exploring the genetic mechanism of FHB resistance is considered the key tool for modern cereal breeding activities. Triticale, the intergeneric hybrid between wheat and rye, is an important cereal crop in Poland and Germany. Resistance breeding using genetic mapping to identify quantitative-trait loci (QTL) associated with FHB resistance represents the best strategy for controlling the disease. In parallel, understanding the mechanism of aggressiveness and DON production of F. graminearum will be a significant contribution to improve FHB management. The objectives of the present work were (1) identification of QTL related to FHB resistance in triticale, together with the analysis of the correlation of FHB severity with other related traits such as plant height and heading stage, (2) correlation between DON production and FHB severity, (3) mapping of dwarfing gene Ddw1 in triticale and studying its effect on FHB resistance, plant height and heading stage, (4) detection of SNPs in candidate genes associated with aggressiveness and DON production of a large Fusarium graminearum population in bread wheat. To study the genetic architecture of FHB resistance in triticale, five doubled-haploid (DH) triticale populations with 120 to 200 progenies were successfully tested under field conditions by inoculation with Fusarium culmorum (FC46) in multiple environments. All genotypes were evaluated for FHB resistance, plant height and heading stage. DArT markers were used to genotype triticale populations. Significant genotypic variances (P<0.001) were observed for FHB severity in all populations combined with high heritability. Twenty-two QTLs for FHB resistance in triticale were reported with two to five QTL per population, thus confirming the quantitative inheritance of FHB resistance in triticale. The most prominent (R2 ≥ 35%) QTLs were located on chromosomes 6A, 3B, 4R, and 5R. QTLs for plant height and heading stage were also detected in our work, some of them were overlapping with QTLs for FHB resistance. Correlation between FHB severity, DON content and Fusarium damaged kernels (FDK) in triticale was studied in the population Lasko x Alamo. Significant genotypic variance was detected for all traits. However, low correlation between FHB severity and DON content (r=0.31) was found. Interestingly, correlation between FHB severity and FDK rating was considerably higher (r=0.57). For FHB severity, two QTLs were detected in this population. A QTL located on chromosome 2A with minor effect for FHB severity was also a common QTL for DON content and FDK rating and explained ≥34% of genotypic variance for these two traits. A second QTL on chromosome 5R was a major QTL but it has no effect on DON content or FDK rating. For analyzing the rye dwarfing gene Ddw1 derived from the father Pigmej, 199 (DH) progenies were genotyped with DArT markers and in addition with conserved ortholog set (COS) markers linked to the Ddw1 locus in rye. QTL analyses detected three, four, and six QTLs for FHB severity, plant height and heading stage, respectively. Two specific markers tightly linked with Ddw1 on rye chromosome 5R explained 48, 77, and 71 % of genotypic variation for FHB severity, plant height, and heading stage, respectively. This is strong evidence, that we indeed detected the rye gene Ddw1 in this triticale population. Another objective was to highlight the association between quantitative variation of aggressiveness and DON production of 152 F. graminearum isolates with single nucleotide polymorphism (SNP) markers in seven candidate genes. One to three significant SNPs (P < 0.01 using cross-validation) were associated to FHB severity in four genes (i.e., Gmpk1, Mgv1, TRI6, and Erf2). For DON content, just one significant SNP was detected in the gene Mgv1 explaining 6.5% of the total genotypic variance. In conclusion, wide genetic variation in FHB resistance in triticale has been observed in five populations. QTL mapping analyses revealed twenty-two QTLs for FHB resistance derived from wheat and rye genomes. QTLs located on the rye genome were reported here for the first time and they are a new source for FHB resistance in triticale. In parallel, analysis of the diversity of four pathogenicity genes in F. graminearum is an important first step in inferring the genetic network of pathogenicity in this fungal pathogen.
Genome-wide prediction of testcross performance and phenotypic stability for important agronomic and quality traits in elite hybrid rye (Secale cereale L.)
(2016) Wang, Yu; Miedaner, Thomas
Genomic selection offers a greater potential for improving complex, quantitative traits in winter rye than marker-assisted selection. Prediction accuracies for grain yield for unrelated test populations have, however, to be improved. Nevertheless, they are already favorable for selecting phenotypic stability of quality traits.
Phenotypic, genetic, and genomic assessment of triticale lines and hybrids
(2017) Losert, Dominik; Würschum, Tobias
Triticale (×Triticosecale Wittmack) is a small grain cereal used for livestock feeding and as renewable energy source. These diverse types of usage lead to different breeding strategies, ideally resulting in continued increase of both, grain and biomass yield. Briefly, the objectives of this thesis were to explore aspects with relevance for line and hybrid breeding in triticale by phenotypic, genetic and genomic assessment of important traits. More specifically, the objectives of this study were to (i) evaluate agronomic traits, assess trait correlations, and investigate the amount of heterosis in triticale hybrids, (ii) examine the potential of line and hybrid cultivars for production of biomass, (iii) assess the phenotypic and genotypic variability in triticale germplasm, (iv) investigate long-term phenotypic trends based on cultivars registered in the past three decades, and (v) identify QTL for agronomical relevant traits. In conclusion, hybrids of triticale possess an increased biomass yield potential compared with their mid-parent values as well as compared with commercial reference cultivars. The findings on triticale germplasm and its breeding history provide important information for breeding programs. Furthermore, based on the obtained results, genomic approaches like marker-assisted or genomic selection appear promising to assist triticale breeding in the future.
Design and assessment of breeding strategies for hybrid wheat in Europe
(2018) Boeven, Philipp Hans Günter; Würschum, Tobias
Wheat is one of the top three global staple crops, possesses the largest global cultivation area, and plays a key role for the world’s future food security. However, its projected yield increase is insufficient to meet the future food and feed demand of an ever-growing world population. Consequently, the rate of breeding progress and productivity of wheat must be increased. Unfortunately, current wheat line breeding has a low return on investment mainly due to high levels of farm saved seeds, which makes wheat less attractive for the plant breeding industry and leads to lower investments and progress compared to other crops where the hybrid technology is established. Hybrid breeding is a worldwide success story in many crops but is not yet established in wheat. Hybrid wheat promises increased yield gain due to the exploitation of heterosis, higher yield stability and stabilized return on investments for breeding companies which warrants further investment and breeding progress in this important stable crop. The self-pollinating nature of wheat is a major bottleneck for hybrid seed production and efficient hybrid wheat breeding requires the redesign of the wheat floral architecture to enhance cross-pollination. Furthermore, the longterm success of hybrid wheat is crucially dependent on the establishment of heterotic groups, on the identification of a high yielding heterotic pattern, and finally, on the realized amount of heterosis and hybrid performance. Therefore, the main objectives of my thesis research were to: (i) analyze the genetic diversity and adaptation in a global winter wheat collection and evaluate how diversity trends could be used to support the development of heterotic groups in wheat; (ii) assess the relationship between heterosis and genetic distance under maximized diversity and evaluate the usefulness of exotic germplasm for hybrid wheat breeding; (iii) dissect the genetic architecture underlying male floral traits in wheat to enable genomics-assisted breeding approaches and investigate the trait seed set which is most crucial for an efficient hybrid seed production. The analyses of genetic diversity in a large worldwide panel of 1,110 winter wheat varieties released during the past decades showed no major population structure but revealed genetically distinct subgroups. Most of the global diversity trends could be explained by breeding history and were associated with geographical origin and long-term domestication. We found that the frequency of the copy number variants at the Photoperiod-B1 (Ppd-B1) and the Vernalization-A1 (Vrn-A1) loci reflect wheat adaptation to the environmental conditions of the different regions of origin. Thus, adaptation issues add an additional layer of complexity and hamper the direct introgression of genetic diversity to support the genetic divergence between heterotic pools. Based on all these analyses, we proposed HyBFrame, a unified framework illustrating how global wheat genetic diversity can be used to support and accelerate reciprocal recurrent selection for the development of genetically distinct heterotic groups in wheat. In a second experiment, we produced 2,046 wheat hybrids by crossing elite with elite lines as well as elite with exotic lines and performed multi-environmental field trials. Interestingly, we found an average midparent heterosis of about 10% in elite crosses as well as in exotic crosses and observed no evidence for a breakdown of heterosis under maximized genetic distance among the hybrid parents. Genetic distance based on genome-wide molecular markers revealed only a very weak association with midparent heterosis for grain yield. Here, we elaborated a functional Rogers’ distance giving weight to heterosis loci and observed a strong positive association between heterosis and this novel distance measure. Hence, considering the genetic architecture of heterosis revealed a more accurate picture of the relationship between heterosis and genetic distance. In addition, the genetic architecture of heterosis in wheat is crucially dependent on the genetic background. We found that a higher number of negative dominance and dominance-by-dominance epistatic effects can reduce the level of absolute heterosis in wide crosses between exotic lines and elite testers. Moreover, hybrid performance in wheat is mainly driven by parental per se performance. Thus, elite lines are favorable for hybrid breeding and should be employed as the starting material for heterotic grouping. Hybrid seed production is the major bottleneck for hybrid wheat breeding and explains the low market share of hybrid wheat varieties. Seed set on the female plants in crossing blocks is the most crucial trait for hybrid seed production in wheat. We tested 31 male lines and evaluated the hybrid seed set on two female tester lines in crossing blocks. Seed set showed a large genotypic variation and a high heritability suggesting that recurrent selection for increased seed set is feasible. The major problem is the synchronized flowering between male and female lines, making the evaluation of seed set in large panels very complex and difficult. Hence, indirect male floral traits with high correlation to the trait seed set would be promising to breed for improved hybrid seed production. We found a strong association between seed set and visual anther extrusion, underscoring that indirect male floral traits have a high potential for preliminary male screenings. We also dissected the genetic architecture underlying promising male floral traits and assessed the potential of genomics-assisted approaches for their improvement. We employed a panel of 209 diverse wheat lines and found a complex genetic architecture underlying all male floral traits. The Reduced height gene Rht-D1 was identified as the only major QTL, for which the commonly used height-reducing allele showed negative effects on male floral traits. This genetic architecture with many moderate- or small-effect QTL limits classical marker-assisted selection. In contrast, genomic prediction yielded moderate to high prediction abilities for anther extrusion. Finally, we proposed a breeding scheme to increase cross-pollination in wheat based on a combination of phenotypic and genomics-assisted selection. Taken together, hybrid breeding in wheat is a very promising approach and the next years will show if all of the current issues can be solved. This thesis research contributed to breeding strategies for hybrid wheat breeding and to the general understanding of heterosis in crops.

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