111 research outputs found

    Resistance gene analogues as a tool for basic and applied resistance genetics exemplified by sugarcane mosaic virus resistance in maize (Zea mays L.)

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    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.Pflanzliche Krankheitsresistenzgene (R-Gene) weisen Homologien in konservierten Aminosäureregionen auf. Homologien zu R-Genen ausfweisende, sog. Resistenzgenanaloga (RGAs), existieren in großer Anzahl in pflanzlichen Genomen und eröffnen neue Möglichkeiten für die Erforschung der Resistenzgenetik allgemein sowie die Untersuchung spezifischer pflanzlicher Resistenzen. Im Rahmen dieser Dissertation wurde am Beispiel der Zuckerrohrmosaikvirusresistenz (sugarcane mosaic virus, SCMV) bei Mais der Nutzen von RGAs für die Pflanzenzüchtung untersucht und bewertet. SCMV ist eine der wichtigsten Viruserkrankungen bei Mais und verursacht signifikante Ertragsverluste in anfälligen Sorten. Aufgrund der nicht-persistenten Übertragungweise des Virus durch Aphiden ist es nicht möglich, die SCMV-Vektoren auf chemischem Wege effizient zu bekämpfen. Daher ist der Anbau resistenter Sorten die einzig wirksame Bekämpfungsmaßnahme. Vorhergehende Studien zur genetischen Basis der oligogen vererbten SCMV-Resistenz identifizierten zwei Genomregionen (quantitative trait loci, QTLs), Scmv1 und Scmv2, auf den Chromosomen 6S und 3L, die massgeblich an der Ausprägung der Resistenz beteiligt sind. Die Ziele dieser Arbeit waren (1) einen Überblick über den aktuellen Status der Virusresistenzzüchtung bei Mais zu geben, (2) die Identifizierung und genetische Kartierung von Kandidatengenen für die beiden Zielgene Scmv1 und Scmv2, (3) die Nutzung potentieller Sequenzhomologien gekoppelter RGAs zur gezielten Erhöhung der Anzahl von Kandidatengenen in den Zielregionen, (4) die Konvertierung eng gekoppelter AFLP (amplified fragment length polymorphism) Marker in codominante, einfach handhabbare PCR Marker, (5) die Bewertung von RGAs für die Entwicklung molekularer Marker, markergestütze Selektion (marker-assisted selection, MAS) und kartengestützte Klonierung, sowie (6) die Untersuchung der Auswirkungen duplizierter Loci auf die Erstellung genetischer Kopplungskarten, MAS und kartengestützte Klonierung. Die drei zuvor veröffentlichten RGAs pic13, pic21 und pic19 wurden aus sechs Maisinzuchtlinien kloniert und in CAPS (cleaved amplified polymorphic sequence) Marker umgewandelt. Paarweise Sequenzvergleiche ergaben eine Frequenz von einem SNP (single nucleotide polymorphism) alle 33 bp über die drei RGAs. Demnach haben RGAs verglichen mit anderen genomischen Sequenzen einen sehr hohen Polymorphiegrad und lassen sich daher leicht in codominante CAPS Marker umwandeln. RGAs erfüllen wichtige Anforderungen für die Entwicklung molekularer Marker bezüglich des Polymorphiegrades und des zahlreichen Vorkommens in pflanzlichen Genomen. Im Gegensatz dazu war der Polymorphiegrad der eng koppelnden AFLP (amplified fragment length polymorphism) Marker wesentlich kleiner und nur zwei von acht AFLPs konnten in einen CAPS und einen Indel (Insertion/Deletion) Marker umgewandelt werden. Die genetische Kartierung der RGAs zeigte, daß Scmv2 nicht mit pic21 identisch ist, während pic19 und pic13 als single-copy Marker in die Zielregionen kartierten und auch aufgrund der Vorfahrenanalysen als Kandidaten für Scmv1 und Scmv2 in Frage kommen. Daraufhin wurde mit pic19 als Kandidat für Scmv1 in einer Mais BAC-Bibliothek ein Screening durchgeführt, um pic19-homologe Sequenzen im Maisgenom zu identifizieren und deren genomische Organisation zu untersuchen. 15 positive BAC-Klone wurden identifiziert und konnten in fünf physikalisch unabhängige Contigs (Bereiche überlappender Klone), angeordnet werden. Drei Contigs kartierten eng gekoppelt in die Scmv1–Region auf Chromosom 6S, während zwei weitere in dieselbe Region auf Chromosom 1 kartierten wie ein QTL für SCMV-Resistenz. Diese Ergebnisse zeigen beispielhaft, dass RGAs aus Zielregionen unter Nutzung potentieller Sequenzhomologien gekoppelter RGAs erfolgreich genutzt werden können, um benachbarte Kandidatengene zu identifizieren. Paarweise Sequenzvergleiche der pic19-homologen Sequenzen der einzelnen Contigs ergaben eine 94-98%ige Sequenzidentität auf Nukleotidebene. Die hohe Sequenzähnlichkeit und der Multilocuscharakter des ursprünglich single-copy kartierten RGAs pic19 zeigen potentielle Probleme für die Verwendung von RGAs als molekulare Marker auf. Daraufhin wurde als Folge von simultaner Amplifikation homologer Sequenzen, die auf PCR Ebene nicht unterschieden werden können, die Existenz von Ghost-Markern analog zu den bekannten Ghost-QTL vorgeschlagen. Die Idee der auf potentiell duplizierten Sequenzen wie z.B. ESTs (expressed sequence tags), AFLPs oder SSRs (simple sequence repeats) beruhenden Ghost-Marker war Gegenstand einer detaillierten theoretischen Untersuchung und Computersimulationsstudie. Demnach resultiert die simultane Amplifikation homologer Sequenzen in einem Überschuß an Heterozygoten, wodurch es zu schiefen Spaltungsverhältnissen für den betreffenden Marker kommt. Die Existenz von Ghost-Markern und die daraus folgenden Verfälschungen der korrekten Markerfolge konnten theoretisch nachgewiesen werden. Werden diese fiktiven Ghost-Marker über eine genetische Kopplungskarte für MAS oder kartengestützte Klonierung herangezogen, so kann die Kartierung eines Zielgens in ein falsches Markerintervall aufgrund des Ghost-Markers fatale Auswirkungen haben. Die durch duplizierte Loci hervorgerufene inkorrekte Locusfolge kann die Kartierung von Zielgenen negativ beeinflussen und zur Feinkartierung falscher Markerintervalle bei der kartengestützen Klonierung führen. In der MAS behindern inkorrekte Locusfolgen die indirekte Selektion auf vorteilhafte Allele in einer QTL-Region und verringern die Effizienz der Reduzierung des Genomanteils des rekurrenten Elters bei markergestützer Rückkreuzung. Zusammenfassend konnte diese Studie den Nutzen von RGAs sowohl für die Pflanzenzüchtung als auch für die Erforschung der Resistenzgenetik deutlich machen. RGAs stellen aufgrund ihres zahlreichen Vorkommens und des hohen Polymorphiegrades einen hervorragenden Ausgangspunkt für die Entwicklung molekularer Marker dar. Darüber hinaus konnte gezeigt werden, daß RGAs von großem Nutzen für die MAS, die Identifizierung von Kandidatengenen, sowie die effektive Erhöhung der Anzahl der Kandidatengene in einer bestimmten Zielregion durch Nutzung von Sequenzhomologien zwischen RGAs sind. Die potentielle Duplikation von RGAs zeigt mögliche Probleme für die genetische Kartierung von duplizierten Sequenzen auf, die trotz des hohen Anteils von Duplikationen in eukaryotischen Genomen bislang unterschätzt wurden. Diese Problematik trifft nicht nur für RGAs zu, sondern ist über zahlreiche Markertypen hinweg existent. Für die Resistenzgenetik im Allgemeinen liefert die Erforschung von RGAs wichtige Erkenntnisse zum Verständnis der genomischen Organisation und der Evolution von R-Genen und R-Genclustern

    Molecular mechanism of photoperiod sensing

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    ELF3 and GI are two important components of the Arabidopsis circadian clock. They are not only essential for the oscillator function but are also pivotal in mediating light inputs to the oscillator. Lack of either results in a defective oscillator causing severely compromised output pathways, such as photoperiodic flowering and hypocotyl elongation. Although single loss of function mutants of ELF3 and GI have been well-studied, their genetic interaction remains unclear. We generated an elf3 gi double mutant to study their genetic relationship in clock-controlled growth and phase transition phenotypes. We found that ELF3 and GI repress growth during the night and the day, respectively. We also provide evidence that ELF3, for which so far only a growth inhibitory role has been reported, can also act as a growth promoter under certain conditions. Finally, circadian clock assays revealed that ELF3 and GI are essential Zeitnehmers that enable the oscillator to synchronize the endogenous cellular mechanisms to external environmental signals. In their absence, the circadian oscillator fails to synchronize to the light dark cycles even under diurnal conditions. Consequently, clock-mediated photoperiod-responsive growth and development is completely lost in plants lacking both genes, suggesting that ELF3 and GI together convey photoperiod sensing to the central oscillator. Since ELF3 and GI are conserved across flowering plants and represent important breeding and domestication targets, our data highlight the possibility of developing photoperiod-insensitive crops by manipulating the combination of these two key genes

    Photoperiod sensing of the circadian clock is controlled by EARLY FLOWERING 3 and GIGANTEA

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    ELF3 and GI are two important components of the Arabidopsis circadian clock. They are not on-ly essential for the oscillator function but are also pivotal in mediating light inputs to the oscillator. Lack of either results in a defective oscillator causing severely compromised output pathways, such as photoperiodic flowering and hypocotyl elongation. Although single loss of function mu-tants of ELF3 and GI have been well-studied, their genetic interaction remains unclear. We gen-erated an elf3 gi double mutant to study their genetic relationship in clock-controlled growth and phase transition phenotypes. We found that ELF3 and GI repress growth differentially during the night and the day, respectively. Circadian clock assays revealed that ELF3 and GI are essential Zeitnehmers that enable the oscillator to synchronize the endogenous cellular mechanisms to external environmental signals. In their absence, the circadian oscillator fails to synchronize to the light-dark cycles even under diurnal conditions. Consequently, clock-mediated photoperiod-responsive growth and development are completely lost in plants lacking both genes, suggesting that ELF3 and GI together convey photoperiod sensing to the central oscillator. Since ELF3 and GI are conserved across flowering plants and represent important breeding and domestication targets, our data highlight the possibility of developing photoperiod-insensitive crops by adjusting the allelic combination of these two key genes

    Some inequalities for kk-colored partition functions

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    Motivated by a partition inequality of Bessenrodt and Ono, we obtain analogous inequalities for kk-colored partition functions pk(n)p_{-k}(n) for all k2k\geq2. This enables us to extend the kk-colored partition function multiplicatively to a function on kk-colored partitions, and characterize when it has a unique maximum. We conclude with one conjectural inequality that strengthens our results.Comment: 11 pages, 1 tabl

    High temperature perception in leaves promotes vascular regeneration and graft formation in distant tissues

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    Cellular regeneration in response to wounding is fundamental to maintain tissue integrity. Various internal factors including hormones and transcription factors mediate healing, but little is known about the role of external factors. To understand how the environment affects regeneration, we investigated the effects of temperature upon the horticulturally relevant process of plant grafting. We found that elevated temperatures accelerated vascular regeneration in Arabidopsis thaliana and tomato grafts. Leaves were crucial for this effect, as blocking auxin transport or mutating PHYTOCHROME INTERACTING FACTOR 4 (PIF4) or YUCCA2/5/8/9 in the cotyledons abolished the temperature enhancement. However, these perturbations did not affect grafting at ambient temperatures, and temperature enhancement of callus formation and tissue adhesion did not require PIF4, suggesting leaf-derived auxin specifically enhanced vascular regeneration in response to elevated temperatures. We also found that elevated temperatures accelerated the formation of inter-plant vascular connections between the parasitic plant Phtheirospennum japonicum and host Arabidopsis, and this effect required shoot-derived auxin from the parasite. Taken together, our results identify a pathway whereby local temperature perception mediates long distance auxin signaling to modify regeneration, grafting and parasitism.This article has an associated 'The people behind the papers' interview

    myTAI: evolutionary transcriptomics with R.

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    MOTIVATION: Next Generation Sequencing (NGS) technologies generate a large amount of high quality transcriptome datasets enabling the investigation of molecular processes on a genomic and metagenomic scale. These transcriptomics studies aim to quantify and compare the molecular phenotypes of the biological processes at hand. Despite the vast increase of available transcriptome datasets, little is known about the evolutionary conservation of those characterized transcriptomes. RESULTS: The myTAI package implements exploratory analysis functions to infer transcriptome conservation patterns in any transcriptome dataset. Comprehensive documentation of myTAI functions and tutorial vignettes provide step-by-step instructions on how to use the package in an exploratory and computationally reproducible manner. AVAILABILITY AND IMPLEMENTATION: The open source myTAI package is available at https://github.com/HajkD/myTAI and https://cran.r-project.org/web/packages/myTAI/index.html. CONTACT: [email protected]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

    An Inhibitor of Tryptophan-Dependent Biosynthesis of Indole-3- Acetic Acid Alters Seedling Development in Arabidopsis

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    Abstract Although polar transport and the TIR1-dependent signaling pathway of the plant hormone auxin/indole-3-acetic acid (IAA) are well characterized, understanding of the biosynthetic pathway(s) leading to the production of IAA is still limited. Genetic dissection of IAA biosynthetic pathways has been complicated by the metabolic redundancy caused by the apparent existence of several parallel biosynthetic routes leading to IAA production. Valuabl

    Post-embryonic Hourglass Patterns Mark Ontogenetic Transitions in Plant Development

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    The historic developmental hourglass concept depicts the convergence of animal embryos to a common form during the phylotypic period. Recently, it has been shown that a transcriptomic hourglass is associated with this morphological pattern, consistent with the idea of underlying selective constraints due to intense molecular interactions during body plan establishment. Although plants do not exhibit a morphological hourglass during embryogenesis, a transcriptomic hourglass has nevertheless been identified in the model plant Arabidopsis thaliana. Here, we investigated whether plant hourglass patterns are also found postembryonically. We found that the two main phase changes during the life cycle of Arabidopsis, from embryonic to vegetative and from vegetative to reproductive development, are associated with transcriptomic hourglass patterns. In contrast, flower development, a process dominated by organ formation, is not. This suggests that plant hourglass patterns are decoupled from organogenesis and body plan establishment. Instead, they may reflect general transitions through organizational checkpoints

    Clinical, Histologic, and Molecular Characteristics of Anaplastic Lymphoma Kinase-positive Primary Cutaneous Anaplastic Large Cell Lymphoma

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    Unlike systemic anaplastic large cell lymphoma, the vast majority of primary cutaneous anaplastic large cell lymphomas (C-ALCL) do not carry translocations involving the ALK gene and do not express ALK. Expression of ALK protein therefore strongly suggests secondary cutaneous involvement of a systemic anaplastic large cell lymphoma. Recent studies described a small subgroup of ALK-positive C-ALCL, but information on frequency, prognosis, and translocation partners is virtually lacking. A total of 6/309 (2%) C-ALCL patients included in the Dutch registry for cutaneous lymphomas between 1993 and 2019 showed immunohistochemical ALK expression. Clinical and histopathologic characteristics, immunophenotype and disease course were evaluated. Underlying ALK translocations were analyzed with anchored multiplex polymerase chain reaction-based targeted next-generation sequencing. Median age at diagnosis was 39 years (range: 16 to 53 y). All patients presented with a solitary lesion. Treatment with radiotherapy (n=5) or anthracycline-based chemotherapy (n=1) resulted in complete responses in all 6 patients. Three patients developed a relapse, of whom 2 extracutaneous. After a median follow-up of 41 months, 5 patients were alive without disease and 1 patient died of lymphoma. Immunohistochemically, 3 cases (50%) showed combined nuclear and cytoplasmic ALK expression with underlying NPM1-ALK fusions, while 3 cases (50%) showed solely cytoplasmic ALK expression with variant ALK fusion partners (TRAF1, ATIC, TPM3). ALK-positive C-ALCL is extremely uncommon, has a comparable favorable prognosis to ALK-negative C-ALCL, and should be treated in the same way with radiotherapy as first-line treatment
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