Institut für Pflanzenzüchtung, Saatgutforschung und Populationsgenetik

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Markergestützte Vererbungsanalyse der Pollenfertilitätsrestauration bei Winterroggen (Secale cereale L.)
(2001) Wolf, Markus; Geiger, Hartwig H.
In rye (Secale cereale L.) as in a number of other crossfertilized crops, hybrid breeding allows a targeted use of heterosis for improving yield and yield stability. The creation of hybrids in outbred rye requires an efficient system of pollination control because of the monoclinous inflorescence. In commercial seed production, mostly the Pampa (P-) Cytoplasma is used, which induces cytoplasmatic male sterility (CMS). Restoration of pollen fertility is achieved by means of pollinator lines carrying nuclear dominant restorer genes. To obtain information about the number, localization, and efficacy of the restorer genes, molecular marker based inheritance analysis in rye was performed. The results were compared to already localized restorer sources in rye and other species. Ninety-two marker loci were mapped by RFLP technique, which covered a genome-length of 845 cM. QTL (quantitative trait loci) analysis revealed a major gene on the short arm of chromosome 1R. Two RFLP markers, psr596 and bcd1124, were linked with a distance of about 0.3 cM to this major gene. Moreover, two modifying minor genes on chromosome arms 3RL and 5RL were detected. One of these minor genes showed epistatic interaction with the above mentioned major gene.
Entwicklung, Charakterisierung und Kartierung von Mikrosatellitenmarkern bei der Zuckerrübe (Beta vulgaris L.)
(2001) Dörnte, Jost; Geiger, Hartwig H.
Simple sequence repeats (SSRs) or microsatellites were isolated from a sugarbeet (Beta vulgaris L.) genomic phage library. The size-fractionated library was screened for the occurrence of the motifes (GA)n, (GT)n, (TGA)n, (AGA)n and (CCG)n. The motifes (GA)n and (GT)n were found to occur most frequently in the sugarbeet genome (every 225 kb). In contrast, the trimer motifes were half as frequent (every 527 kb). A total of 217 microsatellite sequences were found in the sequenced clones. Most of the repeats were imperfect and/or compound. Sequence comparison revealed that 23% of the clones wich containing the (GT)n motif are variants of a previously described satellite DNA (SCHMIDT et al. 1991). Of 102 primer pairs tested on sugarbeet DNA, 71 gave a single product in the expected size. On 23 sugarbeet samples 64 of the 71 SSR-markers reveald length polymorphisms. The number of detected alleles per marker ranged from 2 to 13 (average 4,9) and the PIC-values ranged from 0,17 to 0,86 (average 0,58). A cluster analysis of the 23 samples confirms the pedigree data. The developed SSR markers were compared with RFLP and AFLP markers. Therefore nine sugarbeet lines, each with five single plants per line, were analysed. The SSR analyse shows the lowest similarity between the nine lines. The similarity inside the lines revealed no differences between the marker assays. Thirtythree SSR markers were genetically mapped into the RFLP framework maps of 2 F2-populations. The markers are randomly distributed over eight linkage groups of sugar beet.
Optimizing the development of seed-parent lines in hybrid rye breeding
(2001) Tomerius, Alexandra-Maria; Geiger, Hartwig H.
In hybrid rye breeding, seed-parent and pollinator lines are developed from two divergent gene pools. Line development comprises selection for line performance per se followed by selection for combining ability to the opposite gene pool. Cytoplasmic-genic male sterility (CMS) is employed as hybridizing mechanism. This study deals with model calculations aiming to optimize and compare alternative schemes of seed-parent line development in hybrid rye breeding on the basis of their expected selection gain per year in an index comprising the most important breeding objectives. Prediction of selection gains rests on current estimates of quantitative-genetic and economic parameters. The schemes are optimized for the number of candidates, testers to assess testcross performance, test locations, and replicates at the individual selection stages. Optimization is carried out assuming a fixed annual budget. Five schemes are investigated which differ in the basic genetic material assumed, in the type of test units and the number of selection stages for line and testcross selection, and in the length. The standard scheme employs second cycle material. First, S2-lines are evaluated per se. Selection for combining ability is then carried out at two stages employing testcross progenies of the CMS analogues of the candidate lines in backcross generations BC1 resp. BC2. The first alternative scheme employs an additional stage of BC1L-testcross selection. Another scheme is suited for developing seed-parent lines from broader-based population material. In addition to these 'conventional' methods, a scheme using doubled haploid lines is investigated as well as a scheme in which testcross progenies are produced by means of a gametocide instead of CMS. The optimum dimensioning and relative efficiency of the schemes is investigated for various genetical and economical situations.
Genetische und physiologische Einflußfaktoren sowie deren Wechselwirkungen auf die Trichothecenbildung bei Roggen, Triticale und Weizen nach Inokulation mit Fusarium culmorum (W. G. Sm.) Sacc.
(2002) Reinbrecht, Carsten; Geiger, Hartwig H.
Fusarium culmorum causes specific hazards of cereal quality by the producion of trichothecenes. Prophylaxis by plant breeding can be highly effective. The aim of this study was to investigate the accumulation of trichothecenes in cereals with regard to host and fungal genotype, to physiological factors and the resulting interactions. To determine the effects of 6 environments (E), 2 inoculation dates (ID), 2 fungal isolates (I), and 5 conidia concentrations (C) and their interactions, field trials with up to 12 rye, 6 triticale, and 8 wheat genotypes (G) were conducted in 1995-1997. Kinetics of trichothecene in the heads were described with 6 harvest dates (H) in 2 host genotypes each. In a growth chamber, 2 levels of temperature (T) and 2 of relative humidity (R) were investigated by using 2 host genotypes each. Average deoxynivalenol (DON) accumulation of rye, triticale, and wheat was 41, 46, and 82 mg kg-1, respectively. Genotypes differed significantly in rye and wheat. In all cereal species, GxE interactions were important. In wheat, DON content was highly correlated to all resistance traits, whereas in rye only a tight correlation existed to the relative specific grain weight. In triticale and wheat, inoculation at full anthesis resulted in higher DON contents than inoculation at heading. In rye, no effect of inoculation date was found. In contrast, GxID interaction was significant in rye. The nivalenol (NIV) producing isolate led to lower trichothecene contents than the DON producing isolate. This effect was found to be significant only in rye and triticale. Significant GxI interactions occurred in wheat only. Even one week after inoculation, considerable DON concentrations could be obtained in harvested heads, especially in wheat. Maximum DON contents were observed between 3 and 6 weeks after inoculation (in wheat: partially above 300 mg kg-1). NIV contents were always lower than DON contents. Until full ripening, DON contents slightly decreased, whereas NIV contents increased continuously. HxE and HxI interactions were most important. Trichothecene content in chaff and spindles was 2-4 fold higher than in the respective kernels at 6 and 8 weeks after inoculation. With higher conidia concentrations, increasing contents of DON+3-Acetyl-DON were measured. GxC interactions were highly significant. Highest heritabilities were found in the upper concentration levels. When the relative humidity was high, trichothecene concentrations of kernels were superior. With the temperature, an inverse effect was obtained. It seems that GxT interaction contributed most to GxE in rye and wheat, in triticale also the GxR interaction. In conclusion, assisting resistance traits may replace an expensive quantification of trichothecenes in early generations. In advanced generations, tests should be conducted in several environments with high conidia concentrations, and a toxin analysis should be carried out directly.
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.
Untersuchungen zur Bedeutung der Stickstoffeffizienz für die Ertragssicherheit bei Mais
(2002) Thiemt, Elisabeth-M.; Geiger, Hartwig H.
Increased fertilization with nitrogen (N) in maize production areas often leads to pollution. Maize varieties with improved N-use efficiency under low soil N conditions can therefore contribute to sustainable agriculture. The objectives of this study were to investigate, whether i) hybrids with special adaptation to low soil nitrogen condition show higher yield stability than those which were selected in high nitrogen environments , ii) N-efficient hybrids are more tolerant to drought conditions, iii) combination of parent lines with differences in N-efficiency leads to increased heterosis , and iiii) hybrids show differences concerning components of N-efficiency, in particular N-uptake and N-utilization efficiency. A set of hybrids was generated with parent lines showing superior testcross performance at low or high N-levels, designated L-lines and H-lines, respectively. Field trials were conducted in 14 environments: each trial was grown under high (NH) and low (NL) nitrogen level. Under NL-conditions LxL-hybrids outyielded HxH-hybrids significantly, while at NH the HxH-hybrids showed higher grain yield than LxL hybrids. N-efficient hybrids did not show increased drought tolerance. LxL-hybrids tended to have higher yield stability than HxH-hybrids. Significant increase of heterosis for the traits dry matter yield and dry matter content was not found, neither at NL nor at NH-level. Under NL-conditions N-uptake was reduced, but N-utilization efficiency increased.
Resistance gene analogues as a tool for basic and applied resistance genetics exemplified by sugarcane mosaic virus resistance in maize (Zea mays L.)
(2003) Quint, Marcel; Melchinger, Albrecht E.
With the recent cloning of a number of plant disease resistance genes (R genes) it became apparent that R genes share certain homologies in conserved amino acid domains. PCR amplification of genomic DNA using degenerate primers on the basis of these conserved amino acid domains identified sequences with homologies to plant disease R genes - resistance gene analogues (RGAs). RGAs exist in large numbers in plant genomes and provide new possibilities for the investigation of resistance genetics in general and also for the analysis of certain plant disease resistances. The overall objective of this thesis was to evaluate the use of RGAs for plant breeding for the example of sugarcane mosaic virus (SCMV) resistance in maize. SCMV is one of the most important virus diseases of maize and causes serious yield losses in susceptible cultivars. Owing to the non-persistent manner of transmission, control of aphid vectors by chemical means is not effective and therefore, cultivation of resistant maize varieties is the most efficient method of virus control. Previous studies on the inheritance of oligogenic SCMV resistance located two major quantitative trait loci (QTLs) - Scmv1 and Scmv2 - on chromosomes 6S and 3L, respectively. The objectives of this study were to (1) give an overview on the current status of breeding for virus resistance in maize, (2) identify and genetically map candidate genes for Scmv1 and Scmv2, (3) use potential sequence homologies of linked RGAs for targeted increase of the number of candidate genes in the target regions, (4) convert closely linked amplified fragment length polymorphism (AFLP) markers into codominant, simple PCR-based markers as a tool for marker-assisted selection (MAS) and map-based cloning, (5) evaluate RGAs for the development of molecular markers, MAS, and map-based cloning, and (6) investigate the consequences of duplicate markers for the construction of linkage maps and their implications for MAS and map-based cloning. Three previously published RGAs, pic13, pic21, and pic19 were cloned from six maize inbred lines, converted to cleaved amplified polymorphic sequence (CAPS) markers, and mapped in relation to SCMV R genes (Scmv1, Scmv2) in maize. Pairwise sequence alignments among the six inbreds revealed a frequency of one single nucleotide polymorphism (SNP) per 33 bp for the three RGAs, indicating a high degree of polymorphism and a high probability of success in converting RGAs into codominant CAPS markers compared to other sequences. Therefore, RGAs meet important requirements for the development of molecular markers, i.e., a high degree of polymorphism and availability in great numbers throughout the genome. In contrast to this, the degree of polymorphism for AFLPs closely linked to Scmv1 an Scmv2 was significantly lower in the same six inbred lines compared to RGAs. Only two of eight AFLP markers could be converted into one CAPS and one indel (insertion/deletion) marker. By genetic mapping, pic21 was shown to be different from Scmv2, whereas pic19 and pic13 could be mapped as single-copy markers to the target regions and are candidates for Scmv1 and Scmv2, respectively, due to genetic mapping and consistent restriction patterns of ancestral lines. Subsequently, pic19 was used as candidate for Scmv1 to screen a maize BAC library to identify homologous sequences in the maize genome and to investigate their genomic organisation. Fifteen positive BAC clones were identified and classified into five physically independent contigs consisting of overlapping clones. Genetic mapping clustered three contigs into the same genomic region as Scmv1 on chromosome 6S. The two remaining contigs mapped to the same region as a QTL for SCMV resistance on chromosome 1. Thus, RGAs mapping to a target region can be successfully used to identify further linked candidate sequences. The pic19 homologous sequences of these clones revealed a sequence similarity of 94-98% at the nucleotide level. The high sequence similarity and the multi-locus character of the previously single-copy mapped RGA pic19 show potential problems for the use of RGAs as molecular markers. The existence of ghost markers analogous to ghost QTL was suggested to be a result of simultaneous mapping of several homologous gene family members which cannot be distinguished at the level of PCR. The idea of ghost loci derived by potentially duplicated sequences such as expressed sequence tags (ESTs), AFLPs, or simple sequence repeats (SSRs) was the subject of a theoretical and computer simulation study. Simultaneous amplification of homologous sequences results in an excess of heterozygotes causing distorted segregation ratios. We were able to theoretically prove the existence of such ghost markers resulting in changes of the correct marker orders. If these fictive ghost markers are part of a genetic map which is the subject of MAS or map-based cloning this may have fatal effects like locating a target gene into an incorrect marker interval. This incorrect locus order caused by duplicate marker loci can negatively affect the assignment of target genes to chromosome regions in a map-based cloning experiment, hinder indirect selection for a favourable allele at a QTL, and decrease the efficiency of reducing the chromosome segment attached to the target gene in marker-assisted backcrossing. In conclusion, this thesis demonstrates the use of RGAs for plant breeding and resistance genetics in general. RGAs provide a good source for the development of simple PCR-based markers. Furthermore, RGAs are an excellent tool for MAS, the identification of candidate genes and effective increase of such candidates in target regions using sequence homologies between RGAs. The duplicate nature of RGAs revealed potential problems for genetic mapping of potentially duplicated sequences which are widespread in eukaryote genomes and existent for several types of molecular markers. For resistance genetics in general, investigation of RGAs is important for the understanding of R gene organisation and evolutionary genetics of plant disease resistance.
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.
Untersuchungen zur Vererbung von Qualitätseigenschaften bei Silomais (Zea mays L.)
(2004) Krützfeldt, Birte A. E.; Geiger, Hartwig H.
In central Europe silage maize (Zea mays L.) is a major source of cattle feed. The quality or the feeding value of a silage maize variety mainly depends on its digestibility and energy content. The establishing of the near-infrared-reflectance-spectroscopy- (NIRS) technique allows the analysis of more than one quality determining trait simultaneously in an easy and short way. In this study one objective was the influence of stover quality on whole plant quality. In hybrid breeding indirect selection on the basis of inbred line performance has a great advantage because the number of testcrosses can be reduced. Therefore it was tested, if the stover quality of the testcrosses could be predicted on the line per se value. Besides the correlation between agronomic and quality traits was analysed. In the years 1999 and 2000 the evaluation of the stover of the lines and testcrosses and the whole plant of the testcrosses was conducted at four climatically diverse sites in Germany. Three data sets with flint-lines and dent-lines, each proved with one tester-line, were evaluated for the correlation between inbred line and testcross performance. The test for combining ability was performed with three smaller data sets also consisting of flint-lines and dent-lines with two tester-lines per data set. The coefficients of heritability were high for the agronomic and quality traits in the data sets of the inbred lines. In the data sets of the testcrosses the variation attributed to the genotypic variance was smaller, genotype × location-interactions were of lower importance. In the data sets, each with two tester-lines it was obvious that for quality traits of stover and whole plant the interaction between line and tester was mostly not significant. The genotypic correlation between inbred line and testcross performance was highly significant for almost all quality traits of the stover, but the correlation coefficients were mostly only moderate. Only the expected success of an indirect selection on line per se- value for cell-wall digestibility of the stover exceeded that of the direct selection on testcross performance in all data sets. However, a selection of extremes on line per se value should be possible for stover digestibility. The genotypic correlations between comparable traits in stover and whole plant were mainly low. The cell-wall digestibility was the only trait which was independent of dry matter content. For evaluation of the further quality traits attention has to be paid to the maturity stage, to prevent a maturity-based bias of the results. In the testcrosses stover digestibility increased and whole plant digestibility was reduced with an increase in whole plant dry matter yield. But the genotypic correlations were only moderate and a simultaneous selection to improve quality and yield seems to be possible.
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.
Identification of essentially derived varieties in maize (Zea mays L.) using molecular markers, morphological traits, and heterosis
(2004) Heckenberger, Martin; Melchinger, Albrecht E.
The ‘breeder’s exemption’ as fixed in the UPOV convention on plant variety protec-tion allows the use of protected germplasm for the development of new plant varieties. The aim of this concept is the creation of new genetic variation to guarantee a continuous breeding progress. However, the use of molecular markers in backcrossing programs and genetic engineering has created the technical basis to develop new plant varieties without original breeding efforts. Therefore, the concept of ‘essential derivation’ was implemented into the 1991 Act of the UPOV convention to distinguish between varieties that resulted from intensive and creative selection programs and cultivars that were developed without major genetic changes from these former varieties. Accordingly, a variety is deemed to be essentially derived from an initial variety (IV), if it (i) was predominantly derived from the IV, (ii) is clearly distinguishable from the IV, and (iii) genetically conforms to the IV in the expression of it’s essential characteristics. The goal of this thesis was to evaluate and compare different approaches to assess conformity in the expression of the essential characteristics between IV and essentially derived varieties (EDVs) and to derive a theoretical and experimental basis for the devel-opment of thresholds to distinguish between independently derived varieties and EDVs in maize (Zea mays L.). The main focus was set on the evaluation of genetic distances based on ‘simple sequence repeats’ (SSRs) and ‘amplified fragment length poly¬morphisms’ (AFLPs) as well as the factors contributing to the GD between parental inbreds and their progeny lines. Furthermore, the ability of heterosis and morphological distances for identification of EDVs was examined. In detail, the objectives were to (1) analyze the factors influencing genetic distances (GD) based on SSRs and AFLPs between related maize inbred lines, (2) investigate the power of SSR- and AFLP-based GD estimates, morphological distances and heterosis for discriminating between progenies derived from F2, BC1, and BC2 populations, (3) exemplify theoretical and simulated results with experimental data, and (4) draw conclusions with regard to EDV thresholds suggested in the literature. A total of 220 flint, dent, and US maize inbred lines was genotyped with 100 SSRs equally distributed across the maize genome. The 220 lines comprised 163 triplets. A triplet consisted of one progeny and both parental lines, where the former was developed from an F2-, BC1-, or BC2 population. A subset of 58 lines (38 triplets) was genotyped addition-ally with 20 AFLP primer combinations. Furthermore, morphological traits and heterosis were observed for these 38 triplets in a field experiment over two years and three locations. The distributions of GD values for parental lines and their F2- and BC1-derived progeny overlapped for simulated as well as for experimen-tal data. Assuming that the derivation of a line from an F2 population was an accepted breed-ing procedure and the derivation from a BC1 population would not be accepted, we ob-served Type II errors (β) ranging from 0.23 to 0.37 depending on the germplasm pool for a given Type I error (α) of 0.05. For a threshold between BC1 and BC2, β ranged from 0.40 to 0.60 with an increasing tendency for higher BC levels. For fixed GD thresholds of T=0.25, 0.20, 0.15, and 0.10 suggested in the literature, substantial differences for α and β were found between different germplasm pools. Therefore, thresholds need to be gene pool specific and different thresholds for potential EDVs from intra-pool crosses than for progenies from inter-pool crosses must be applied. Discrimination of F2-, BC1-, and BC2-derived progeny lines on the basis of heterosis and morphological distances revealed β values ranging from 0.50 to 0.95 depending on the trait or combination of traits. Therefore, heterosis and morphological distances were fairly inappropriate tools for identification of EDVs due to the larger overlaps of F2-, BC1-, and BC2-distributions compared to GDs based on molecular markers. In general, SSRs and AFLPs were the most adequate tools to uncover close pedigree relationships between maize inbred lines and to discriminate among lines derived with ac-cepted or non-accepted breeding procedures. Therefore, the results presented in this study provide an example for identification of EDVs and can be transferred to other diploid crops by adjusting the corresponding thresholds.
Resistance of Maize (Zea mays L.) Against the European Corn Borer (Ostrinia nubilalis Hb.) and its Association with Mycotoxins Produced by Fusarium spp.
(2004) Magg, Thomas; Melchinger, Albrecht E.
The European corn borer (ECB, Ostrinia nubilalis Hübner) is a major pest of maize (Zea mays L.) in Europe and continues to spread to northern maize growing regions. The ECB severely affects commercial maize production by decreasing yield stability. In addition, damaged plants often show an increased susceptibility to secondary infections caused by Fusarium spp.. Information about the potential of Bt hybrids (event 176, MON810) to reduce yield losses and mycotoxin contamination under Central European growing conditions is still lacking. However, such monogenic resistances with a strong negative effect on the ECB will break down rapidly. Improving the natural host plant resistance of maize could provide an economical and ecological tool for an integrated pest management system. The overall goal of this study was to evaluate alternative breeding strategies for improving resistance of maize against ECB damage and Fusarium spp.. The objectives were to (1) initiate a selection experiment in the early maturing European flint pool and evaluate a breeding program for ECB resistance in the European dent pool, (2) compare the efficiency of host plant resistance vs. Bt resistance in maize, (3) determine Fusarium-caused mycotoxin contamination of maize genotypes with improved host plant resistance to ECB, and (4) study the association between important agronomic traits, ECB resistance traits, and mycotoxin concentration in early European maize germplasm. The goal of the Hohenheim ECB breeding program, initiated in 1992, was to select lines with improved per se and testcross performance for multiple agronomic traits and ECB resistance. In the standard breeding scheme, line development started from a segregating S1 population. Genotypes were evaluated for their line per se ECB resistance in generations S1, S3, and S5. Lines from the S2, S4, and S5 generations were testcrossed and evaluated for their agronomic performance. Selection was based on ECB resistance and TC performance for grain yield and maturity. In order to compare transgenic Bt maize hybrids carrying event 176 or MON810 with their isogenic counterparts and commercial hybrids or experimental hybrids, field trials in multiple environments were conducted in 1998 to 2000. Furthermore, a laboratory bioassay with neonate ECB larvae was performed to assess mortality and subsequently the level of Bt antibiosis present in the used hybrids of 1998. Resistance traits such as damage rating of stalks, number of damaged plants, and number of larvae per plant were assessed exclusively in manually ECB infested plots. Grain yield, grain dry matter content and plant height were determined in the insecticide protected and the ECB infested main plots. In addition, grain samples from each subplot were drawn at random and analyzed separately for Fusarium mycotoxins such as type B trichothecenes (DON, NIV), Zearalenon (ZEN), Fumonisins (FUM), and Moniliformin (MON). The inbred lines displayed a significant genotypic variance for all ECB resistance traits evaluated. However, in the further course of selection and topcross testing, most dent and flint lines, especially those displaying improved resistance to ECB larvae feeding, were discarded because of their poor agronomic performance. Negative correlations between grain yield, early maturity and the damage rating of stalks were identified. However, three dent lines (P028, P029, P030) with moderate resistance to ECB were developed. In all experiments, Bt hybrids were superior to other hybrids in the control of ECB larvae. Non-Bt hybrids displayed a significant genotypic variance for all evaluated resistance traits; grain yield reductions ranged from 8.6 to 21.8% under manual infestation of ECB. All evaluated resistance traits were highly significantly correlated with each other and showed significant negative correlations to grain yield reduction. Bt hybrids did not differ from their isogenic counterparts for most agronomic traits. Highly significant location and genotype × location interactions were identified for all mycotoxins evaluated, except MON. MON concentration doubled under manual infestation of ECB compared to insecticide protected conditions and a similar trend was found for FUM. Bt hybrids displayed significantly lower MON concentrations than non-Bt hybrids and significantly lower DON concentrations than their isogenic counterparts under ECB infestation. Highly significant correlations between ECB resistance traits and MON were found. However, a significant genotypic variance was observed for DON, 15-A-DON, FUM, and MON concentrations, suggesting variation for resistance against Fusarium spp. in current elite hybrids. By combining different sources of monogenic Bt resistance and quantitatively inherited resistances to ECB, it may be possible to develop hybrids with multiple resistance by pyramiding the underlying genes in one genotype. Therefore, further research is required to identify new sources of ECB resistance and new breeding strategies should be developed. Furthermore, there is indication that an improved resistance against Fusarium spp. possesses a greater potential for reducing mycotoxin contamination of maize kernels than a high level of ECB resistance. Since resistance to ECB and resistance to Fusarium spp. are inherited fairly independently, simultaneous improvement of both resistances seems to be necessary for improving the stability and quality of future maize hybrids.
QTL mapping of resistance to Sclerotinia sclerotiorum (Lib.) De Bary in sunflower (Helianthus annuus L.)
(2005) Micic, Zeljko; Melchinger, Albrecht E.
Sclerotinia sclerotiorum (Lib.) de Bary is one of the most important pathogens of sunflower. Three different disease symptoms can be caused by S. sclerotiorum: Sclerotinia wilt, midstalk- and head rot. An improvement of the resistance against S. sclerotiorum would contribute to yield security and thus increase the profitability of sunflower cultivation. We investigated resistance to midstalk rot with respect to the prospects of marker-assisted selection (MAS). The bjectives were to (1) identify quantitative trait loci (QTL) involved in resistance against Sclerotinia sclerotiorum, (2) map their position in the genome, (3) characterize their gene effects, and (4) estimate their consistency across generations of the cross NDBLOSsel x CM625. Two sunflower lines with high resistance level to S. sclerotiorum and different genetic origins (NDBLOSsel and TUB-5-3234) were used as parents. They were crossed with a highly susceptible line CM625 to develop two mapping populations. A modified leaf test was used for the evaluation of midstalk-rot resistance. Three resistance traits and two morphological traits were measured. Disease resistance of 354 F3 families of the population NDBLOSsel x CM625 was screened in field trials with two different sowing times in 1999. A total 317 recombinant inbred lines (RIL) derived from F3 families were tested in 2002/2003. The 434 F3 families of cross CM625 x TUB-5-3234 were screened in 2000/2001. The field trials were conducted by using generalized lattice designs with three replications and five infected plants per replication. Highly significant genetic variation between F3 families and RIL was observed for the resistance traits in all field trials. Heritabilities ( ) were highest for stem lesion and lowest for leaf lesion for all three experiments. The resistance traits were moderately correlated with each other. For the construction of the genetic map of population NDBLOSsel x CM625, 352 F2 individuals were analyzed with 117 SSR marker loci. On the basis of results from the QTL mapping in F3 families, 41 markers were selected and genotyped in 248 RIL. A "selective genotyping" (SG) approach was used for population CM625 x TUB-5-3234. Based on the results measured in F3 families for stem lesion, the SSR genotype at 72 marker loci was determined for the 60 most resistant and 60 most susceptible F2 individuals. For QTL mapping and estimation, the method of the "composite interval of mapping" was used. For stem lesion in the population NDBLOSsel x CM625, eight QTL were detected explaining 33.7% of the genetic variance ( ). The QTL on LG8 explained 36.7% of the phenotypic variance (R2adj). All other QTL for this trait explained between 3.3 and 6.0% of R2adj. Nine QTL were detected for leaf lesion. The proportion of the phenotypic variance explained by individual QTL ranged from 3.4 to 11.3%. All detected QTL for leaf lesion explained 25.3% of the genetic variance in cross validation. For speed of fungal growth, 6 QTL were detected, which explained from 4.6 to 10.2% R2adj. Cross validation explained 24.4% of. Most QTL showed additive gene action. QTL occurring consistently across generations can be recommended for MAS and therefore, the QTL results between RIL and F3 families of population NDBLOSsel x CM625 were compared. One common QTL was identified for leaf lesion, two for stem lesion and three for speed of fungal growth. In population CM625 x TUB-5-3234, four QTL for stem lesion, three QTL for leaf lesion and three QTL for speed of fungal growth were identified. Owing to the SG approach we conjecture that not all QTL were found. The comparison of QTL results between two F3 populations showed two common QTL for stem lesion on LG4 and LG8. The QTL on LG4 originated from the susceptible parent CM625. The QTL on LG8 probably corresponds to the QTL with the largest effect determined in the population NDBLOSsel x CM625. Regarding MAS, our results indicate that two QTL detected for stem lesion and speed of fungal growth in population NDBLOSsel x CM625 are promising. They were consistent across environments, and showed no adverse correlation to leaf morphology in trials with the RIL. In mapping population CM625  TUB-5-3234, it remained unclear whether TUB-5-3234 can contribute new alleles with sufficiently large effects for resistance that were not identified in line NDBLOSsel and would be useful in MAS. The genomic region on LG10 should be analyzed in more detail with respect to its importance for resistance in multiple plant parts (head and stalk) and to verify its association with leaf morphology. Resistance breeding of sunflower against S. sclerotiorum is difficult due to the complex inheritance of the trait. This study showed that both the resistance source NDBLOSsel and the identified markers are promising in improving resistance by MAS. For a broader resistance against S. sclerotiorum, it is necessary to detect new resistance genes from different sources to pyramide them in elite lines.
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.
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.
Resistance breeding in maize (Zea mays L.) against the European corn borer (Ostrinia nubilalis Hübner) and the use of DNA-markers for marker-assisted selection
(2005) Papst, Christine; Melchinger, Albrecht E.
The European corn borer (Ostrinia nubilalis Hb., ECB) is an important pest in maize production. Feeding of ECB larvae causes grain yield losses of up to 30% and promotes ear and stalk rots caused by Fusarium spp.. Maize cultivars carrying the Bt gene are highly resistant to ECB larvae feeding. However, the use of transgenic cultivars is controversially discussed. In contrast, the natural host plant resistance (HPR) is regarded as more durable. The main objective of this study was to identify quantitative trait loci (QTL) for HPR against ECB and to draw conclusions about their usefulness in marker-assisted selection (MAS). The specific research questions were: (1) Where are QTL for ECB resistance and related agronomic traits located in the maize genome and what are their genetic effects? (2) How consistent are QTL detected across unrelated populations? (3) How consistent are QTL detected for line per se and testcross performance? (4) Which physiological mechanisms underlie the resistance against ECB larvae feeding? (5) What is the association between ECB resistance and mycotoxin concentrations in grain maize? Two unrelated dent populations (A and B) were developed. For Experiment 1 the F2:3 families were evaluated for line per se performance for ECB resistance. All F2:3 families of Population B were testcrossed with a susceptible tester line and also evaluated for ECB resistance (Experiment 2). Two sets of F2:3 families from Population B, each comprising the most resistant and the most susceptible lines, were selected (Experiment 3). In Experiment 4, 10 maize cultivars consisting of four pairs of transgenic hybrids and their isogenic counterparts were used to determine the association between mycotoxin concentration and ECB resistance. All entries in Experiment 4 were analyzed for mycotoxin concentration of deoxynivalenol (DON), fumonisin (FUM), fusarenon-X (FUS), moniliformin (MON) and nivalenol (NIV) in grain samples. In all four experiments, resistance to ECB larvae feeding was evaluated using manual infestation with ECB larvae. Furthermore agronomic and quality traits were recorded. In Experiment 1, two QTL for resistance were detected in Population A, both explaining about 25% of the genotypic variance. No common QTL for resistance traits was found across Populations A and B. Possible explanations for the low consistency of QTL across populations are a low power of QTL detection caused by small population sizes, sampling, and environmental effects. Furthermore, population-specific QTL regions cannot be ruled out. In Experiment 2, six QTL for resistance explaining 27% of the genotypic variance were found for testcross performance. Three common QTL for resistance were detected for line per se and testcross performance. Phenotypic as well as genotypic correlations between line per se and testcross performance were low for resistance, indicating a moderate consistency across the different types of progeny. The low consistency across both types of progeny is presumably attributable to the low power of QTL detection in TC progenies caused by a decreased genotypic variance and masking effects of the tester allele. Despite the low consistency of QTL across populations and progenies in the present study, a comparison with other reports from the literature revealed that most of the QTL occurred in clusters. Given the low percentage of genotypic variance explained by QTL-marker associations, we conclude that MAS will not be efficient for resistance breeding against ECB with the current molecular marker techniques. In Experiment 3, significant correlations were observed between resistance and quality traits, such as digestibility and stalk strength. These findings confirm the importance of increased cell-wall fortification for resistance against ECB larvae feeding, and support the hypothesis that candidate genes for resistance are involved in lignin biosynthesis. The analyses of mycotoxin concentrations in Experiment 4 showed that DON, FUM, and MON were the most prevalent mycotoxins in maize kernels. Differences between protected and infested plots were only significant for DON and FUM. Transgenic Bt hybrids showed lower mycotoxin concentrations in kernels than the other hybrids. However, only low correlations were found between ECB resistance and mycotoxin concentrations across all 10 hybrids. Therefore, selection for ECB resistance does not necessarily reduce mycotoxin concentration, suggesting that each complex of characters must be improved simultaneously by breeding. Even if MAS for resistance against the ECB does not seem promising at the moment, the information about QTL regions may be a first step for further research on possible candidate genes, e.g., brown midrib genes located in the common QTL regions with effects on the lignin biosynthesis. Genotypes with an improved digestibility, without impairing ECB resistance by reduced cell-wall strength, would be most promising.
Experimental and simulation studies on introgressing genomic segments from exotic into elite germplasm of rye (Secale cereale L.) by marker-assisted backcrossing
(2005) Susic, Zoran; Geiger, Hartwig H.
The introgression of exotic germplasm is a promising approach to increase the genetic diversity of elite rye breeding materials. Even though exotic germplasm may contain genomic segments that can improve oligo- and polygenically inherited traits, it has not been intensively utilized in modern rye breeding due to its agronomically inferior phenotypes and low performance level. Introgression of exotic germplasm requires techniques that would minimize negative side effects attributable to genetic interactions between recipient and donor. This appears achievable by the introgression library approach involving the systematic transfer of donor chromosome (DC) segments from an agriculturally unadapted source (donor) into an elite line (recipient, recurrent parent). A set of introgression lines (ILs) is thus developed, in which introgression is restricted to one or a few short DC segments. Ideally, the introgressed DC segments are evenly distributed over the whole recipient genome and the total genome of the exotic donor is comprised in the established set of ILs. The systematic development of an introgression library in rye has not been described yet. The main objectives of this study were to i) establish two rye introgression libraries by marker-assisted backcrossing, comprising of ILs each harbouring one to three DC segments and jointly covering most of the donor genome (DG), and ii) apply computer simulations to develop a highly effective and cost-efficient marker-assisted introgression strategy for the creation of introgression libraries in rye. A cross between a homozygous elite rye inbred line L2053-N (recurrent parent) and a heterozygous Iranian primitive rye population Altevogt 14160 (donor) was used as base material to generate the two libraries (F and G). Repeated backcrossing (BC) and subsequent selfing (S) until generation BC2S3 were chosen as the introgression method. The AFLP and SSR markers were employed to select individuals carrying desired DC segments, starting from generation BC1 to generation BC2S2. The chromosomal localization of DC segments, the number of DC segments per IL, and the proportion of recurrent parent genome were used as criteria to select parent individuals. This procedure resulted in the first two rye introgression libraries worldwide, comprising 40 BC2S3 ILs per library and covering 72% of the total DG in library F and 63% in library G (jointly approximately 80%). Most of the established ILs harboured one to three homozygous DC segments (on average 2.2 in both libraries), with a mean length of 18.3 cM in library F and 14.3 cM in library G. Computer simulations were conducted using the software PLABSIM version 2 to evaluate and optimize strategies for developing an introgression library in rye. Simulations were based on map-length estimates obtained from genotyping the BC1 generation of population F (7 chromosome pairs, genome size 665 cM). Six strategies differing in the number of BC and S generations were analysed, by setting the restrictions of sufficient DG coverage and RPG recovery. The medium-long BC3S1 strategy proved to be the most recommendable. It allows to achieve close to 100% DG coverage with moderate progeny sizes (19 individual per IL) in the individual generations and an acceptable total number of marker data points (52700), thus providing a good compromise between the cost and speed of an introgression procedure. Longer strategies are somewhat more cost-efficient but too time-demanding. The reverse is true for shorter strategies. An optimal allocation of resources is achieved by starting an introgression strategy with a small BC1 population (between 60 and 200 individuals) and stepwise increasing the progeny size per IL from about 15 to about 25-35 individuals in the succeeding generations. Targeting longer DC segments and using genetic maps with lower marker density allow a remarkable reduction in resources. This approach, however, possesses shortcomings when implementation in breeding is considered. The longer DC segments more likely carry i) unfavourable loci as well, ii) more than one gene controlling the trait in question, or iii) many additional loci affecting other traits. The major disadvantage of genetic maps with large marker distances is the unknown information about possible double cross-overs within marker intervals. All above-mentioned disadvantages may cause problems in the process of identification and isolation of genes controlling the trait of interest. Thus, a lower initial effort for the establishment of an introgression library will later on require additional efforts for using the ILs in breeding and genomics. Since the results of the simulation study became available after the marker-assisted establishment of the two rye introgression libraries had been finished, the dimensioning of the experimental study deviated from the optimum dimensioning determined in the simulation study: i) The BC2S2 introgression strategy was used in the empirical approach, whereas the BC3S1 strategy proved to be most recommendable in the simulation study. ii) The BC1 population sizes of libraries F and G (68 and 69, respectively) were far below the optimum value (200) determined in the simulation study for the chosen BC2S2 strategy. iii) The mean progeny sizes per IL from generation BC2 onwards varied between 7 and 21, whereas the optimum progeny size would have been two to three times higher. iv) The total number of analysed individuals (690 in library F, 684 in library G) was considerably lower than the optimum determined in the simulation study (3440). As a consequence, the coverage of the donor genome in the two libraries was incomplete and most ILs harboured more than a single DC segment. The potential application of the results of the simulation study would have increased the value of the developed ILs (higher DG coverage, lower number of DC segments per IL) considerably, despite limited resources. The effects of the introgressed DC segments on agronomically important qualitative and quantitative traits still need to be examined in multi-environmental field experiments. Introgression lines with beneficial DC segments may directly be used in practical hybrid rye breeding programs. Moreover, such ILs may be further backcrossed to create near isogenic lines (NILs) each carrying a single marker-characterized short DC segment. These NILs are an ideal starting point for high-resolution mapping and for the isolation and functional characterisation of candidate genes. The two rye introgression libraries and the results of the simulation study mark important milestones for the targeted exploitation of exotic rye germplasm and provide a promising opportunity to proceed towards functional genomics in rye.
Response to modified recurrent full-sib selection in two European F2 maize populations analyzed with quantitative genetic methods
(2006) Flachenecker, Christian; Melchinger, Albrecht E.
Many plant breeding strategies lead to a reduction in the genetic variance of the source population. However, a sufficient genetic variance is essential for the long-term selection response. Hence, the aim of recurrent selection (RS) is a continuous increase in the frequencies of favorable alleles while maintaining genetic variability in the population. Several intrapopulation RS methods have been proposed in maize: e.g., mass selection, half-sib selection, full-sib (FS) selection, S1 selection. Among them, recurrent FS selection is characterized by a short cycle length, complete parental control, and a high selection response. The goal of this thesis was to investigate the changes in the population structure over several cycles of a modified recurrent FS selection program in two European F2 maize populations. In detail, the objectives were to (i) monitor trends across selection cycles in the estimates of population mean, inbreeding coefficients, and variance components, (ii) determine selection response for per se and testcross performance, (iii) compare predicted with realized selection response, (iv) extend the population diallel analysis under full consideration of inbreeding depression due to random genetic drift, (v) separate genetic effects due to selection from those due to random genetic drift, and (vi) investigate the usefulness of best linear unbiased prediction (BLUP) estimates of parents for predicting progeny performance under the recurrent FS selection scheme applied. Four early maturing European flint inbreds were used as parents to produce two F2 populations (A×B and C×D). Both populations were three times intermated by chain crossing to reduce the gametic phase disequilibrium. Starting from the F2Syn3 population obtained in this manner, a modified recurrent FS selection program was conducted over four cycles in population A×B and over seven cycles in population C×D. In each cycle, 144 FS families were tested in field trials and, in parallel, six plants from each FS family were selfed. The selfed ears of the 36 families with the highest selection index (SI = 2 × dry matter content + grain yield) were selected and intermated according to a pseudo-factorial mating scheme. In this mating scheme, the gametic contribution of the best selected FS families is doubled compared with the gametic contribution of the remaining selected FS families. Afterwards, all cycles of both populations were tested in two population diallel analyses in six environments. Based on the known pedigree records, the inbreeding coefficient of each FS family and the coancestry coefficients among them were calculated. Variance components and BLUP values were obtained using phenotypic means and coancestry coefficients. For grain yield, the selection response per cycle, which could be expected after correcting for the effects of random genetic drift, was higher than reported in the literature (14.1% and 8.3% in populations A×B and C×D, respectively). We ascribe the comparatively high selection response mainly to the pseudo-factorial mating scheme. This mating scheme is expected to increase the selection response compared with commonly applied random mating schemes, without a major reduction in the effective population size (Ne). In this study, the expected Ne was 32, suggesting a minor influence of random genetic drift compared with that of selection. This assumption was verified by an extended population diallel analysis, showing that random genetic drift reduced the selection response only by about 1-2% in both populations. In contrast to an estimation of variance components with moment estimators, the REML procedure has no special requirements on the mating scheme and accounts for any relationship among families in a breeding population. As expected from the high Ne applied in our study, we observed only a moderate decrease in additive variance for grain yield and grain moisture in both populations. Nevertheless, the variance components were still associated with high standard errors, which prevented the revealing of trends across cycles. A larger number of test locations and larger population size would reduce the standard errors of variance components at the cost of oversized and expensive field trials. Methods for predicting the performance of progenies are important to optimize RS programs. Due to simplifying assumptions, a prediction with phenotypic means is often inaccurate. An alternative method is BLUP, which was suggested for predicting the performance of untested single-cross hybrids but has not been applied in RS programs. In our study, the prediction of progeny performance based on BLUP was only marginally better than prediction based on the phenotypic mean. However, higher degree of relationship between the entries and lower heritabilities would increase the advantage of BLUP compared with phenotypic means.
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.
Linkage disequilibrium and association mapping in elite germplasm of European maize
(2006) Stich, Benjamin; Melchinger, Albrecht E.
Linkage mapping has become a routine tool for the identification of quantitative trait loci (QTL) in plants. An alternative, promising approach is association mapping, which has been successfully applied in human genetics to detect QTL coding for diseases. The objectives of this research were to examine the feasibility of association mapping in elite maize breeding populations and develop for this purpose appropriate biometric methods. The feasibility of association mapping depends on the extent of linkage disequilibrium (LD) as well as on the forces generating and conserving LD in the population under consideration. The objectives of our studies were to (i) examine the extent and genomic distribution of LD between pairs of simple sequence repeat (SSR) marker loci, (ii) compare these results with those obtained with amplified fragment length polymorphism (AFLP) markers, and (iii) investigate the forces generating and conserving LD in plant breeding populations. Our studies were based on experimental data of European elite maize inbreds as well as on computer simulations modeling the breeding history of the European flint heterotic group. The experimental results on European elite maize germplasm suggested that the extent of LD between SSR markers as well as AFLP markers are encouraging for the detection of marker-phenotype associations in genomewide scans. In populations with a short history of recombination, SSRs are advantageous over AFLPs in that they have a higher power to detect LD. In contrast, in populations with a long history of recombination, for which no LD is expected between pairs of SSR markers, AFLP markers should be favored over SSRs because then their higher marker density that is generated with a fixed budget can be used. Furthermore, the results of our experimental and simulation studies indicated that not only physical linkage is a cause of LD in plant breeding populations, but also relatedness, population stratification, genetic drift, and selection. So far, in plant genetics the logistic regression ratio test (LRRT) has been applied as a population-based association mapping approach. However, this test does only correct for LD caused by population stratification. The objectives of the presented study were to (i) adapt the quantitative pedigree disequilibrium test to typical pedigrees of inbred lines produced in plant breeding programs and (ii) compare the newly developed quantitative inbred pedigree disequilibrium test (QIPDT) and the commonly employed LRRT with respect to the power and type I error rate of QTL detection. This study was based on computer simulations modeling the breeding history of the European maize heterotic groups. In QIPDT the power of QTL detection was higher with 75 extended pedigrees than in LRRT with 75 independent inbreds. Furthermore, while the type I error rate of LRRT surpassed the nominal ® level, the QIPDT adhered to it. These results suggested that the QIPDT is superior to the LRRT for genome-wide association mapping if data collected routinely in plant breeding programs are available. Epistatic interactions among QTL contribute substantially to the genetic variation in complex traits. The main objectives of our study were to (i) investigate by computer simulations the power and proportion of false positives for detecting three-way interactions among QTL involved in a metabolic pathway in populations of recombinant inbred lines (RILs) derived from a nested design and (ii) compare these estimates to those obtained for detecting three-way interactions among QTL in RIL populations derived from diallel and different partial diallel mating designs. The computer simulations of this study were based on single nucleotide polymorphism haplotype data of 26 diverse maize inbreds. The power and proportion of false positives to detect three-way interactions with 5000 RILs derived from a nested design was relatively high for both the 4 QTL and the 12 QTL scenario. Higher power to detect three-way interactions was observed for RILs derived from optimally allocated distancebased designs than for RILs derived from a nested or diallel design. Our results suggested that association mapping methods adapted to the special features of plant breeding populations have the potential to overcome the limitations of classical linkage mapping methods.

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