Browsing by Subject "Genomics"

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Breeding for resistance to Fusarium ear diseases in maize and small-grain cereals using genomic tools
(2021) Gaikpa, David Sewordor; Miedaner, Thomas
The world’s human and livestock population is increasing and there is the need to increase quality food production to achieve the global sustainable development goal 3, zero hunger by year 2030 (United Nations, 2015). However, biotic stresses such as Fusarium ear infections pose serious threat to cereal crop production. Breeding for host plant resistance against toxigenic Fusarium spp. is a sustainable way to produce more and safer cereal crops such as maize and small-grain winter cereals. Many efforts have been made to improve maize and small-grain cereals for ear rot (ER) and Fusarium head blight (FHB) resistances, using conventional and genomic techniques. Among small-grain cereals, rye had the shortest maturity period followed by the descendant, hexaploid triticale while both wheat species had the longest maturity period. In addition, rye and triticale were more robust to Fusarium infection and deoxynivalenol accumulation, making them safer grain sources for human and animal consumption. However, a few resistant cultivars have been produced by prolonged conventional breeding efforts in durum wheat and bread wheat. High genetic variation was present within each crop species and can be exploited for resistance breeding. In this thesis, the genetic architecture of FHB resistance in rye was investigated for the first time, by means of genome-wide association study (GWAS) and genomic prediction (GP). GWAS detected 15 QTLs for Fusarium culmorum head blight severity, of which two had major effects. Both weighted and unweighted GP approaches yielded higher prediction abilities than marker-assisted selection (MAS) for FHB severity, heading stage and plant height. Genomics-assisted breeding can shorten the duration of breeding rye for FHB resistance. In the past decade, genetic mapping and omics were used to identify a multitude of QTLs and candidate genes for ear rot resistances and mycotoxin accumulation in maize. The polygenic nature of resistance traits, high genotype x environment interaction, and large-scale phenotyping remain major bottlenecks to increasing genetic gains for ear rots resistance in maize. Phenotypic and molecular analyses of DH lines originating from two European flint landraces (“Kemater Landmais Gelb”, KE, and “Petkuser Ferdinand Rot”, PE) revealed high variation for Gibberella ear rot (GER) severity and three agronomic traits viz. number of days to female flowering, plant height and proportion of kernels per cob. By employing multi-SNP GWAS method, we found four medium-effect QTLs and many small-effect (10) QTLs for GER severity in combined DH libraries (when PCs used as fixed effects), none co-localized with the QTLs detected for the three agronomic traits analyzed. However, one major QTL was detected within KE DH library for GER severity. Two prioritized SNPs detected for GER resistance were associated with 25 protein-coding genes placed in various functional categories, which further enhances scientific knowledge on the molecular mechanisms of GER resistance. Genome-based approaches seems promising for tapping GER resistance alleles from European maize landraces for applied breeding. After several cycles of backcrossing and sufficient selection for agronomic adaptation traits, the resistant lines identified in this thesis can be incorporated into existing maize breeding programs to improve immunity against F. graminearum ear infection. Breeding progress can be faster using KE landrace than PE. A successful validation of QTLs identified in this thesis can pave way for MAS in rye and marker-assisted backcrossing in maize. Effective implementation of genomic selection requires proper design of the training and validation sets, which should include part of the current breeding population.
Phenotypic and genomics-assisted breeding of soybean for Central Europe : from environmental adaptation to tofu traits
(2022) Kurasch, Alena; Würschum, Tobias
Soybean (Glycine max Merr.) is one of the major crops in the world providing an important source of protein and oil for food and feed; however it is still a minor crop in Central Europe. Soybean cultivation can play an important role in a more sustainable agricultural system by increasing local and regional protein production in Europe. The demand for locally produced soybean products is still growing in Europe. The key for a successful establishment of soybean cultivation in Europe is adaptation of soybean varieties to the Central European growing conditions. For the latitudinal adaptation to long-day conditions in Central to Northern Europe, an adapted early flowering and maturity time is of crucial importance for a profitable cultivation. The key traits flowering and maturity are quantitatively inherited and mainly affected by photoperiod responsiveness and temperature sensitivity. The most important loci for an early flowering and maturity are E1-E4 and the various allelic combinations condition soybean flowering and maturity time and therefore strongly contribute to the wide adaptability (Jiang et al., 2014; Tsubokura et al., 2014; M. Xu et al., 2013). Besides the main usage as protein source for animal feeding, soybean is also a very valuable source for human consumption. Tofu is enjoying ever greater popularity in Europe, as it is one of the best sources of plant protein with additional health benefits, rich in essential amino acids, beneficial lipids, vitamins, and minerals, as well as other bioactive compounds, such as isoflavones, soyasaponin, and others, (Lima et al., 2017; Zhang et al., 2018). Thus, plant breeding has to provide not only well-adapted varieties with good agronomic and quality properties, but also provide varieties well-suited to the further processing into soymilk and tofu. Therefore, a good knowledge about the breeding target, how to assess it and how it is inherited is crucial. The conducted studies covered a broad range of aspects relevant to improve a soybean breeding program. By combining environmental analysis, E-gene analysis, genomic approaches (QTL mapping and genomic prediction), and tofu phenotyping, breeder decisions become more accurate and targeted in the way of selection thereby increasing the genetic gain. In addition, combining the results of the different aspects helps to optimize the resources of a breeding program. Increasing the knowledge about the different aspects from environment to tofu QTL enables a breeder to be more precise and focused. But the more targeted and specific, the more complex a breeding program gets, which requires adequate tools to handle all the different information in a meaningful and efficient way to enable a quick and precise breeding decision.
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.
Speciation and domestication genomics of Amaranthus spp.
(2017) Stetter, Markus; Schmid, Karl J.
The genus Amaranthus consists of 50 to 70 species, including several cultivated and weedy species. The seeds of the three grain amaranth species, A. caudatus, A. cruentus and A. hypochondriacus have a high nutritional value and are gluten free. In this work, three main aspects of amaranth genetics are studied, because previous work was limited to few species and few genetic markers: First, the evolutionary relationship between species in the genus; second, the domestication syndrome of South American grain amaranth; and third, crossing methods and controlled growth conditions for amaranth breeding. The genus has been taxonomically split into three subgenera, A. Amaranthus, A. Albersia and A. Acnida. Together with their two relatives A. hybridus and A. quitensis, the three grain amaranths form the Hybridus complex within the A. Amaranthus subgenus. We used genotyping by sequencing (GBS) of 94 genebank accessions, representing 35 species to infer the phylogeny of Amaranthus. SNPs were called using de novo and reference genome based methods and genome sizes of the species were measured using flow cytometry. The analysis of genome size evolution within the genus revealed that with the exception of two lineages polyploidization played a minor role in the history of the genus. A distancebased neighbor joining tree of individual accessions and a species tree based on the multispecies coalescent were constructed. Both phylogenies supported the previous taxonomic classification into three subgenera, but split the A. Acnida subgenus into two distant groups. Analyzing the Hybridus complex gave insights into the domestication history of grain amaranth. The complex was well separated from the other species in the A. Amaranthus subgenus and included the three grain amaranth species and their wild relatives. Individuals within the Hybridus complex did not cluster by species, but rather by their geographic origin from South and Central America. Geographically separated lineages of A. hybridus appeared to be the common ancestor of the three cultivated grain amaranths, while A. quitensis was involved in the domestication of A. caudatus. The domestication of grain amaranth remains unclear and seems to be complex, because the domestication syndrome that differentiates crops from their wild ancestors is only weakly pronounced. Therefore, the domestication syndrome in South American grain amaranth (A. caudatus) was studied by characterizing genetic and phenotypic diversity of A. caudatus and the two potential wild relatives, A. hybridus and A. quitensis. To investigate the evolutionary relationship of A. caudatus and its potential wild ancestors, 119 amaranth accession from the Andean region were characterized using 9,485 GBS derived SNPs. Additionally, two domestication related phenotypes, seed color and seed size, were analyzed. None of the accessions of wild amaranths had white seeds, while this was the predominant seed color in A. caudatus. The seed size did not significantly differ between species, but a genetically distinct group of A. caudatus from Bolivia had significantly larger seeds than the other groups. The genetic analysis revealed a strong differentiation of A. caudatus from its wild relatives. The two relatives did not cluster according to their species assignment, but rather by their geographic origins from Peru and Ecuador. Surprisingly, A. caudatus had a higher genetic diversity than its two close relatives and shared a high proportion of polymorphisms with them, consistent with the absence of strong bottlenecks or high levels of gene flow between them. Efficient crosses are an essential tool for plant research and breeding. Three different crossing methods (open pollination, hot water emasculation and hand emasculation) were evaluated for their efficiency and validated with low cost genetic markers. We identified controlled growth conditions for amaranth that allow short generation times of only six weeks instead of six months. All three crossing methods successfully produced offspring, but with different success rates. Open pollination had the lowest (10%) and hand emasculation the highest success rate (74%). Hot water emasculation showed an intermediate success rate (26%), but high maximum of 94%. Additionally, this method is easy to perform and suitable for large-scale hybrid production. Eleven PCR-based SNP markers were found to be sufficient for intra- and interspecific hybrid identification. Despite the very small flowers, amaranth crosses can be carried out efficiently and verified with inexpensive SNP markers with short generation times under suitable conditions. The phylogeny and population genetic analysis suggest that amaranth domestication was incomplete. Gene flow from A. quitensis into A. caudatus might have prevented the fixation of domestication related alleles. The genus phylogeny and the over 200 genotyped accessions in this work provide the largest genomic resource for amaranth so far.
Strategies for selecting high-yielding and broadly adapted maize hybrids for the target environment in Eastern and Southern Africa
(2012) Windhausen, Sandra Vanessa; Melchinger, Albrecht E.
Maize is a major food crop in Africa and primarily grown by small-holder farmers under rain-fed conditions with low fertilizer input. Projections of decreasing precipitation and increasing fertilizer prices accentuate the need to provide farmers with maize varieties tolerant to random abiotic stress, especially drought and N deficiency. Genetic improvement for the target environment in Eastern and Southern Africa can be achieved by: (i) direct selection of grain yield in random abiotic stress environments, (ii) indirect selection for a secondary trait or grain yield in optimal, low-N and/or managed stress environments, or (iii) index selection using information from all test environments. At present, the maize hybrid testing programs of the International Maize and Wheat Improvement Center (CIMMYT) select primarily for grain yield under managed stress and optimal environments and subdivide the target environment according to geographic and climatic differences. It is not known to what extend the current strategy contributes to selection gains. The same holds true for genomic prediction, a strategy that is not yet implemented into the CIMMYT maize breeding program but that may accelerate breeding progress and reduce cycle length by predicting genotype performance based on molecular markers. Regarding the different strategies mentioned for selecting high-yielding and broadly adapted maize hybrids, the breeder needs to decide which of them are most promising to increase genetic gains. Consequently, the objectives of my thesis were to (1) evaluate the potential of leaf and canopy spectral reflectance as novel secondary traits to predict grain yield across different environments, (2) estimate to what extent indirect selection in managed drought and low-N stress environments is predictive of grain yield in random abiotic stress environments, (3) investigate whether subdividing the target environment into climate, altitude, geographic, yield level or country subregions is likely to increase rates of genetic gain, and (4) evaluate the prospects of genomic prediction in the presence of population structure. The measurement of spectral reflectance (495 ? 1853 nm) of both leaves and canopy at anthesis and milk grain stage explained less than 40% of the genetic variation in grain yield after validation. Consequently, selection based on predicted grain yield is only suitable for pre-screening, while final yield evaluation will still be necessary. Nevertheless, the prospect of developing inexpensive and easy to handle devices that can provide, at anthesis, precise estimates of final grain yield warrants further research. Based on a retrospective analysis across 9 years, more than 600 trials and 448 maize hybrids, it was shown that maize hybrids were broadly adapted to climate, altitude, geographic and country subregions in Eastern and Southern Africa. Consequently, I recommend that the maize breeding programs of CIMMYT in the region should be consolidated. Within the consolidated breeding programs, genotypes should be selected for performance in low- and high yielding environments as the genotype-by-yield level interaction variance was high relative to the genetic variance and genetic correlations between low- and high-yielding environments were moderate. Genetic gains were maximized by index selection, considering the yield-level effect as fixed and appropriately weighting information from all trials. To allow better allocation of resources, locations with high occurrence of random abiotic stress need to be identified. Heritability in trials conducted at these locations may be increased by the use of row- and column designs and/or spatial adjustment. Furthermore, resources invested into managed drought trials should be maintained during early breeding stages but shifted to the conduct of low-N trials at later breeding stages. Investments in a larger number of low-N trials may increase selection gain, because performance under low-N and random abiotic stress was highly correlated and genotypes can be easily selected under different levels of soil N. Prospects are promising to accelerate breeding cycles by the use of genomic prediction. Based on two large data sets on the performance of eight breeding populations, it was shown that prediction accuracy resulted primarily from differences in mean performance of these populations. Genomic prediction may be implemented into the CIMMYT maize breeding program to predict the performance of lines from a diversity panel, segregating lines from the same or related crosses, and progenies from closed populations within a recurrent selection program. The breeding scenarios in which genomic prediction is most promising still need to be defined. Generally, the construction of larger training sets with strong relationship to the validation set and a detailed analysis of the population structure within the training and validation sets are required. In conclusion, combining index and genomic selection is the most promising strategy for providing high-yielding and broadly adapted maize genotypes for the target environments in Eastern and Southern Africa.