Browsing by Subject "Molekulare Marker"

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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.
Diversity in the tropical multipurpose shrub legumes Cratylia argentea (Desv.) O. Kuntze and Flemingia macrophylla (Willd.) Merrill.
(2006) Andersson, Meike S.; Schultze-Kraft, Rainer
Cratylia argentea (Desv.) O. Kuntze and Flemingia macrophylla (Willd.) Merrill are promising tropical multipurpose shrub legumes. Both are drought-tolerant, well adapted to low-fertility, acid soils, and especially suited for low-input smallholder production systems in the sub-humid and humid tropics. They can be used e.g. as dry season forage supplementation, live soil cover or mulch, erosion barrier hedges, and shade-providing shrubs in young coffee and cocoa plantations. Germplasm collections were assembled from the wild-legume flora in Brazil (C. argentea) and Southeast Asia (F. macrophylla), but research and development are so far based on only a few accessions. Knowledge about the extent of genetic diversity within these collections is very limited. In addition, the potential utilization of F. macrophylla is so far limited by poor forage quality and acceptability of the few evaluated accessions. The objective of the present study, conducted in a research cooperation with the International Centre for Tropical Agriculture (CIAT), Cali, Colombia, was to assess the diversity in the germplasm collections of C. argentea (38 accessions) and F. macrophylla (69 accessions) in terms of morphological and phenological traits, agronomic and forage quality traits, and molecular markers, and to identify superior genotyes. Based on these different characterization approaches, the objective was furthermore to establish core collections for F. macrophylla, and to compare and validate the different strategies, giving particular consideration to their practical implications (time and cost efficiency) for the application to small collections of perennial wild tropical legumes. Cratylia argentea High diversity in terms of phenological and agronomic as well as forage quality traits was detected in the collection, with scope for plant improvement in terms of higher dry season DM production. Accessions CIAT 18674 and 22406 were identified as promising for further evaluation since they were similar to the commercial cultivar "Veraniega" in terms of forage quality, and superior in terms of DM production, particularly in the dry season. Molecular marker analysis with RAPDs showed that the genetic diversity in the collection was relatively low and fairly homogeneously distributed. Accessions CIAT 22373, 22378, 22380, 22381 and 22411 were identified as possible duplicates. Flemingia macrophylla High diversity in terms of morphological and agronomic as well as forage quality traits was detected among the 69 accessions. The identification of four morphotypes in the collection probably has taxonomic implications. Scope for plant improvement was identified with respect to forage quality - one of the species´ main limitations. Accessions CIAT 18437, 21083 and 21090 had similar DM production and higher digestibility than the control accession, and were virtually free of extractable condensed tannins. Problems with low palatability and low seed production of these promising accessions need to be further studied. Genetic diversity in F. macrophylla was higher than in C. argentea, and corresponded closely to the four morphotypes revealed by conventional characterization. Various duplicate accessions were identified, and evidence was provided that the non-Asian accessions are not native to their collection site regions, but rather introduced from Southeast Asia. The results have direct applications for plant improvement of these promising multipurpose legumes. The superior genotypes selected in this study will be used in work with farmers in CIAT-research sites in Central America and distributed to partners. It must be recognized, however, that the diversity assessed is influenced by the climatic and edaphic conditions at the site where the studies were conducted. Therefore, multilocational trials should be considered with a selected subset (including the promising accessions) of C. argentea and F. macrophylla i) to assess the extent of genotype x environment interaction, and ii) to identify genotypes with consistently high performance in a range of distinct environments. Research on the reproduction system of both species is urgently required to determine the potential extent and impact of outcrossing. Beyond the immediate application of these species for farmer utilization, the results of the use and comparison of different approaches to assess diversity and to establish core collections can help to improve germplasm management and characterization of wild tropical legume species in general. Random sampling has been identified as a valuable and resource-efficient strategy for the creation of core collections when no additional information about accessions is available, and in the absence of adequate funds. The validation of the findings of this study with a broader range of perennial tropical wild legumes is necessary to assess their applicability to other species.
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.
Lockerbeerigkeit bei Klonen von Spätburgunder (Pinot noir) : Analyse von molekularen Markern und der Einfluss von Gibberellin auf die Traubenmorphologie
(2014) Hoffmann, Petra; Blaich, Rolf
In viticulture, the architecture of the grape cluster affects the quality of the grapes. Compact grape clusters are more prone to B. cinerea infection, which reduces yield (Vail et al. 1998, Vail et al. 1991). Loose clusters have longer pedicel and rachis structures (Alleweldt 1959) and are less susceptible to B. cinerea. For this reason, the cultivation of clones with the loose cluster trait is of great interest. Loose clusters can result from the application of phytohormones, the spacing of the flower clusters, the thinning of fruit, or a reduced pruning. These treatments reduce berry set and promote pedicle elongation when applied to clones with compact clusters (Alleweldt 1959). Genetically based loose clustered grape phenotypes occur among grapevine cultivars. In this study we are able to differentitate between losse and compact clones using the marker FlExp2 on the basis of sequence data. The loose cluster clones show a 4 bp deletion at 219-222 bp and a C/T transition at 231 bp, unlike the compact cluster clones. In all tested Pinot ssp. clones, the sequence correlated to the phenotype. The marker was tested on other varieties such as Riesling, Gewurztraminer, Chardonnay, Chenin blanc, Cardinal and White Chasselas. The phenotypes were again consistent with the sequence. In the case of loose clustered table grapes, a deletion occurs instead of the transition at 231 bp. Additionally, the variety White Gutedel demonst- rated a C/T transition at 217 bp. These results were confirmed by sequencing 30 clo- nes (loose and compact clusters) in two repetitions in both directions. Both markers shwoed two fragments with a four bp difference. The amplification products are a In- del/SNP mutation for loose cluster and a „CTTT“ mutation for copmact cluster grapes.The CAPS and the SCAR marker identified that the trait of bunch architecture is heterozygous. The sequence of the amplification products was distinct for loose and compact cluster types. The SCAR marker shows two amplification products at 162 bp and 166 bp and the CAPS marker at 283 bp and 287 bp. The heterozygosity didn ́t produce a molecular marker for the MAS. In silico, analysis shows that the identified locus is in the Exon in the Vlexp1 gene. This gene is an expansin gene which is responsible for cell elongation (McQueen- Mason 1992). A part of the role which the hormones play in grape Morphology was analyzed in this study. The inflorescences of the genetically loose clustered clone 1-84 Gm did not show increased gibberellin concentration, indicating that gibberellin does not have a influence on the genetic based loose clone (1-84 Gm). However, the auxin concentra- tion in inflorescences of loose cluster clones increases earlier and remains high lon- ger than in those of the compact cluster clone 18 Gm. After a treatment with gib- berellin, the clone 1-84 Gm exhibited increased concentration of both gibberellin and auxin and formed even looser clusters. Similarly, the same treatment applied to the compact clone 18 Gm resulted in looser clusters and increased concentration of gib- berellin and auxin with a higher concentration of auxin for a longer period of time. It remains unclear precisely how the gibberellin treatment induces looser clusters. It may be that there is an interaction between gibberellin and auxin or that the auxin alone causes the cell extension. It remains an open question whether expansin toge- ther with gibberellin or auxin is responsible for the development of loose clusters, or if it is caused by a gibberellin auxin interaction. The growth pattern of the stalks and inflorescences were identified in order to put these results in context with the results of the hormone and genetic analysis. The stalks and inflorescences of the treated and untreated clones were measured weekly before GA3-application and continued four weeks after application. The growth of the flower clusters ended three weeks af- ter anthesis while the stalks grew continuously. In the loose cluster clone 1-84 Gm, the growth of stalks and flower clusters was significantly larger than in the compact cluster clone 18 Gm. The growth behavior of the peclone 18 Gm when treated with gibberellin was identical to the clone 1-84 Gm without gibberellin treatment. Gib- berellin treatment caused a significant increase in the growth of the stalk and flower clusters. The treated loose cluster clones formed the largest stalks while the untrea- ted compact cluster clone 18 Gm the smallest. Such clone growth behavior results in loose cluster architecture.
Molecular and agronomic assessment of genetic diversity and hybrid breeding in triticale
(2006) Tams, Swenja H.; Melchinger, Albrecht E.
Knowledge of the genetic diversity of a species is of paramount importance for the choice of crossing parents in line and hybrid breeding. Genetic distance (GD) estimates based on molecular markers proved to be well suited for direct exploration of the relationship within a germplasm pool. Triticale hybrid breeding and heterosis have received increasing attention in recent years. Hybrid seed production is highly attractive for autogamous species because of the built-in variety protection of hybrids in comparison to line varieties. The main objective was to appraise the prospect of hybrid breeding in European winter triticale and develop time- and cost-reducing strategies. In particular, the main objectives were to (i) assess and compare genetic diversity estimates in European winter triticale elite germplasm based on molecular markers and pedigree data, (ii) determine hybrid performance and heterosis in multiple environments, and (iii) evaluate prediction methods for hybrid performance and heterosis to support future hybrid breeding programs. Average coancestry coefficient between all pairs of the 128 European elite genotypes was low (f = 0.059) due to scanty information available for the majority of the varieties and breeding lines. Better estimates of genetic distance of triticale genotypes were obtained by molecular marker assessment with 93 simple sequence repeat (SSR) markers and 10 PstI/TaqI primer combinations of amplified fragment length polymorphism (AFLP) markers. While SSR markers have been developed in wheat and rye and are mapped in the genome, the location and distribution of AFLP markers is unknown. Both marker systems resulted in reliable genetic diversity estimates. The moderate correlation between genetic distance estimate (GD) of SSR and AFLP marker analyses (GDSSR; GDAFLP) corresponded with other studies. Cluster analysis and principle coordinate analysis revealed no clear separation of germplasm groups. Supported by a bootstrap analysis, it was concluded that both marker systems provide consistent information for germplasm identification. The lack of grouping is in concordance with the breeding history of triticale as a self-pollinator, the wide adaptation of the inter-generic species and the single end-use purpose. Simultaneously to the marker assessment, 209 F1 hybrids were produced by a chemical hybridizing agent. The hybrids and their parents (57 females and five testers) were evaluated in field trials in six environments in Germany during the season 2001-2002. A combined analysis revealed significant heterosis for all eight traits. The level of mid-parent heterosis was positive for grain yield, 1000-kernel weight, number of kernels per spike, test weight and plant height and negative for number of spikes per m², falling number and protein concentration. Forty-six of the hybrids outyielded modern varieties, which were included as checks, by 10% and more. This aspect is important for the success of hybrids on the market for commercial production. Results regarding hybrid performance, heterosis, GCA/SCA relationship, trait correlation in hybrids and parents and aspects regarding cost-effective high quality F1 seed production appear to be sufficiently positive to encourage further work on hybrid breeding. Approaches to reduce time and costs for the identification of superior parental combinations and the prediction of hybrid performance revealed no reliable method yet. Correlations between SCA and GD of parents based on the different marker systems were low for all traits, which hampers prediction. Grouping of germplasm based on GD estimates or on heterotic response of the hybrids could not be discovered in triticale. As a consequence, a first step for an optimum allocation of resources in commercial hybrid breeding programs is the development of heterotic groups. In the present study, several females have been sub-grouped according to their heterotic response and SCA for grain yield with two tester pairs. Following the early history of hybrid breeding in maize, a multi-stage procedure was suggested for triticale to evaluate and expand the sub-grouping and enhance heterosis among groups.