Ilona Krämer

Diagnostic value of molecular markers for Lr genes and characterization of leaf rust resistance of German winter wheat cultivars with regard to the stability of vertical resistance

Breeding for resistance is an efficient strategy to manage wheat leaf rust caused by Puccinia triticina f. sp. tritici. However, a prerequisite for the directed use of Lr genes in breeding and the detection of new races virulent to these Lr genes is a detailed knowledge on Lr genes present in wheat cultivars. Therefore, respective molecular markers for 18 Lr genes were tested for specificity and used to determine Lr genes in 115 wheat cultivars. Results obtained were compared to available pedigree data. Using respective molecular markers, genes Lr1, Lr10, Lr26, Lr34 and Lr37 were detected, but data were not always in accordance with pedigree data. However, leaf rust scoring data of field trials confirmed the reliability of DNA markers. These reliable marker data facilitated the analyses of the development of virulent leaf rust races from 2002 to 2009 based on released cultivars. A sudden change from low infection rates to susceptibility was observed for Lr1, Lr3, Lr10, Lr13, Lr14, Lr16, Lr26 and Lr37 since 2006. Cultivars carrying several leaf rust resistance genes showed no significant shift to susceptibility except one cultivar which revealed an increasing infection rate at a low level. In summary, it turned out that pedigree data are often not reliable and a detection of Lr genes by diagnostic markers is fundamental to combine Lr genes in cultivars for a durable resistance against leaf rust, and to conduct reliable surveys based on released cultivars, instead of ‘Thatcher’ NILs.

Validity of selected DNA markers for breeding leaf rust resistant wheat

The set of 44 near-isogenic lines of spring wheat cv. Thatcher and wheat genotypes known to carry specific leaf rust resistance genes were included in the present study for the preliminary validation of twelve STS and SCAR markers linked to leaf rust resistance genes Lr9, Lr19, Lr20, Lr21, Lr24, Lr25, Lr26, Lr28, Lr29, Lr37. Seven Lr genes were specifically tagged by STS and SCAR markers. The presence of genes Lr9, Lr19, Lr20, Lr24, Lr28, Lr29, Lr37 in the tested plant materials was confirmed by a unique amplification of markers SCS5 550, SCS265 512 and SCS253 736, STS638, SCS73 719, SCS421 570, IPY10 and Lr29F24/R24, cslVrgal3, PS10R/L, respectively. Evaluation of the repeatability and the reliability of selected markers (pTAG621 for Lr1, STS683 for Lr20, D14L for Lr21, Lr25F20/R19 for Lr25, Lr29F24/R24, IPY10 for Lr29, cslVrgal3 for Lr37 and PS10R/L for Lr47) across four European laboratories and PCR conditions demonstrated the usability of STS638, Lr29F24/R24, IPY10, cslVrgal3 and PS10R/L markers in marker-assisted selection. STS markers pTAG621 for gene Lr1, D14L for gene Lr21, Lr25F20/R19 for gene Lr25 were found to be unsuitable for effective screening of large segregating populations in breeding programs.

Intraspecific genetic diversity of Pyrenophora tritici-repentis (Died.) Drechs. (Drechslera tritici-repentis[Died.] Shoem.) detected by random amplified polymorphic DNA assays

Abstract Random amplified polymorphic DNAs (RAPDs) were used to study the genetic variation of Pyrenophora tritici-repentis isolates causing wheat tan spot. Two independent experiments were conducted in 2002 – 2003. In 2002, 40 isolates collected in Russia (Krasnodar region, Bashkiria), Germany, and the Czech Republic were studied and 35 unique RAPD genotypes were identified. Most of the genetic variation (72%) was observed within populations and 28% between them. In 2003, 69 new isolates from Russia (Dagestan, North Osetia, Bashkiria), Germany, and the Czech Republic were studied and 47 unique RAPD genotypes were identified. As in 2002, most of the genetic variation (75%) was observed within populations and 25% between them. Total gene diversity in each group ranged from 0.67 – 1.00 for 2002 and was 1.00 for 2003. The average gene diversity was estimated between 0.13 and 0.20 in 2002 and between 0.07 and 0.18 in 2003. A dendrogramme based on genetic distances between isolates illustrates that the variation is distributed on a small scale (0.3 – 4.0%). Estimated FST values and clustering of isolates on dendrogrammes suggest that groups of isolates from Bashkiria and groups of isolates from Dagestan and North Osetia are separated from others and may be considered as different geographical populations. No clear differentiation between isolates from other sites was revealed.

High-resolution mapping of the barley Ryd3 locus controlling tolerance to BYDV

Barley yellow dwarf disease (BYD) is transmitted by aphids and is caused by different strains of Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV). Economically it is one of the most important diseases of cereals worldwide. Besides chemical control of the vector, growing of tolerant/resistant cultivars is an effective way of protecting crops against BYD. The Ryd3 gene in barley (Hordeum vulgare L.) confers tolerance to BYDV-PAV and BYDV-MAV and the locus was previously mapped on the short arm of barley chromosome 6H near the centromere. We applied a strategy for high-resolution mapping and marker saturation at the Ryd3 locus by exploiting recent genomic tools available in barley. In a population of 3,210 F2 plants, 14 tightly linked markers were identified, including 10 that co-segregated with Ryd3. The centromeric region where Ryd3 is located suffers suppressed recombination or reduced recombination rate, suggesting potential problems in achieving (1) map-based cloning of Ryd3 and (2) marker selection of the resistance in breeding programmes without the introduction of undesirable traits via linkage drag.

Genetic fine mapping of a novel leaf rust resistance gene and a Barley Yellow Dwarf Virus Tolerance (BYDV) Introgressed from Hordeum Bulbosum by the Use of the 9K iSelect Chip

Leaf rust and barley yellow dwarf, caused by Puccinia hordei Otth and barley yellow dwarf virus (BYDV)/cereal yellow dwarf virus (CYDV), are important diseases of barley (Hordeum vulgare L.) worldwide. Screening of spring barley landraces from Serbia led to the identification of the accession ‘MBR1012’ carrying resistance to the most widespread virulent leaf rust pathotypes in Europe, while a barley line carrying an introgression derived from H. bulbosum on chromosome 2HL was found to be highly tolerant to BYDV-PAV. In a population of 91 doubled haploid lines, derived from the cross MBR1012 (R) × Scarlett (S), the resistance gene against leaf rust was mapped in the telomeric region of chromosome 1HS by using simple sequence repeats (SSR). In parallel, the population was genotyped on the newly developed Illumina iSelect custom 9K BeadChipQ1, resulting in the identification of closer linked markers. To exploit BYDV tolerance, DH lines derived from the cross (Emir x H. bulbosum) x Emir have been analysed in three steps. In a first step, out of 221 DH lines, 27 plants carrying a recombination event in the H. bulbosum fragment were selected based on nine markers specific for chromosome 2HL. In a next step, selected recombinant plants were analysed on a custom-made Illumina BeadXpress Array (384 SNPs) and on the 9k iSelect BeadChip. Finally, artificially inoculated DH lines carrying introgressions of different sizes will be screened for BYDV virus tolerance by artificial inoculation in order to map this tolerance. Results obtained revealed the presence of novel resistance/tolerance genes and in parallel provide the tools for their efficient deployment in barley breeding

A saturated SNP linkage map for the orange wheat blossom midge resistance gene Sm1

Key messageSNP markers were developed for the OWBM resistance geneSm1that will be useful for MAS. The wheatSm1region is collinear with an inverted syntenic interval inB. distachyon.AbstractOrange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin) is an important insect pest of wheat (Triticum aestivum) in many growing regions. Sm1 is the only described OWBM resistance gene and is the foundation of managing OWBM through host genetics. Sm1 was previously mapped to wheat chromosome arm 2BS relative to simple sequence repeat (SSR) markers and the dominant, sequence characterized amplified region (SCAR) marker WM1. The objectives of this research were to saturate the Sm1 region with markers, develop improved markers for marker-assisted selection (MAS), and examine the synteny between wheat, Brachypodium distachyon, and rice (Oryza sativa) in the Sm1 region. The present study mapped Sm1 in four populations relative to single nucleotide polymorphisms (SNPs), SSRs, Diversity Array Technology (DArT) markers, single strand conformation polymorphisms (SSCPs), and the SCAR WM1. Numerous high quality SNP assays were designed that mapped near Sm1. BLAST delineated the syntenic intervals in B. distachyon and rice using gene-based SNPs as query sequences. The Sm1 region in wheat was inverted relative to B. distachyon and rice, which suggests a chromosomal rearrangement within the Triticeae lineage. Seven SNPs were tested on a collection of wheat lines known to carry Sm1 and not to carry Sm1. Sm1-flanking SNPs were identified that were useful for predicting the presence or absence of Sm1 based upon haplotype. These SNPs will be a major improvement for MAS of Sm1 in wheat breeding programs.

Bewegungsmuster von Wanderratten (Rattus norvegicus) imResistenzgebiet von Deutschland für die Entwicklung einer effektiven Bekämpfungsstrategie

Recent studies on endophytic Kosakonia radicincitans DSM 16656T demonstrated a wide plant growth-promoting activity. The application of this Gram-negative bacterium as a biofertilizer is limited due to the lack of adequate formulation that protects the cells during drying and storage and supports plant colonization. Here we set out to elucidate the compatible solutes accumulation influence of K. radicincitans triggered by osmotic stress on its capacity as an endophyte in radish plants. We found that physiological modifications by osmotic stress treatments and accumulation of compatible solutes during cultivation, improve the capability of K. radicincitans formulated in dry beads to colonize and to promote radish growth. Thus, pre-conditioning of cells with NaCl 4% and by adding compatible solutes such as hydroxyectoine during cultivation induced a positive effect on relative gene expression response, enhancing significantly the ability to colonize plant tissue up to 10 fold. Additionally, when this osmolyte was added, either dry matter of tuber or leaves increased by 3 % and 13.59 %, respectively in comparison to beads without the bacterium. These first results indicate that a systematic approach to cultivation and formulation may increase the endophytic capacity of this bacterium.

Evaluation of genetic resources in the genus Asparagus for resistance to Asparagus virus 1 (AV-1)

Forty-four Asparagus officinalis cultivars, gene bank accessions and breeding lines as well as thirty-four accessions of wild relatives of Asparagus were evaluated for resistance to Asparagus virus 1. Three different test strategies were developed for the assessment of individual plants: (1) natural infection under field conditions, or two vector-mediated infection assays using the green peach aphid Myzus persicae (2) in an insect-proof gauze cage or (3) in a climate chamber. The AV-1 infections were verified by DAS-ELISA and RT-PCR approaches. All tested 660 individual plants of A. officinalis germplasm were susceptible to AV-1 infection. In contrast, in 276 plants of 29 Asparagus wild accessions no virus infection could be detected. These resistant accessions comprised of nineteen diploid, tetraploid and hexaploid species of both the Eurasian clade and the African clade of the asparagus germplasm. Data of the AV-1 resistance evaluation are discussed in relation to the genetic distance of the resistance carrier and potential application in breeding.

High resolution mapping of resistance genes against barley mosaic disease and Barley yellow dwarf virus

Ryegrass (Lolium spec.) is the most important cool-season forage crop in temperate regions. Though, the seed production is considerably affected by several fungal and bacterial obligate biotrophic pathogens. The overall purpose of this study is directed to developing ryegrass cultivars with multiple pathogen resistance and agronomic adaption to Germany’s agricultural conditions. This aim shall be achieved by combining genes for resistances to stem rust, crown rust and bacterial wilt. The pyramidisation shall be accomplished by the use of specific molecular markers which will be derived by bulked segregant analysis combined with next generation sequencing based massive analysis of cDNA ends (MACE) transcriptome profiling. RNA was isolated from bulks of infected and noninfected leaf segments from susceptible and resistant genotypes of various fullsibling mapping populations (n ≥ 200) and their respective parental lines for every investigated pathogen. After MACE was performed, bioinformatic analysis detects SNPs and transcripts that were exclusively expressed in the resistant bulk. Thus, 30 molecular markers were genetically mapped to a 50.8 cM spanning region surrounding the stem rust resistance locus LpPg1. The development of this high efficient molecular selection tool marks MACE as a fast and reliable method that detects polymorphisms for genetic mapping of candidate genes and obtains to be the method of choice for investigating the molecular and genetic base of resistances to stem rust, crown rust and bacterial wilt.The current production systems in arable farming have reached their limits. Sizes of machinery are continuously increasing. Compaction and limits on the road are the consequences. Productionrelated restrictions like nitrogen pressure and development of resistances against plant protection products are further problems. Last but not least, the sociopolitical acceptance of crop production is questioned in public opinion. Due to these circumstances the question arises if the system of crop farming which has been adapted to the machinery available on the market is the right strategy for the future. Why not going the other way round and decide what a plant production system has to look like to be at an optimum and then decide what kind of machinery is needed to cultivate? Following this idea the plants must be in the focus.

Lokale Ausbreitungsmuster zu Tyr139Cys-Polymorphismen von Wanderratten im westfälischen Resistenzgebiet

Die Zusammensetzung der Ackerunkrautflora entspricht einer Anpassung an die standortspezifischen Umweltbedingungen, dem Produktionsverfahren hinsichtlich insbesondere der Fruchtfolge und Bodenbearbeitungstechnik, sowie dem jeweiligen Herbizidmanagement. Da die Art und Intensität der Unkrautflora einen erheblichen Einfluss auf die Ertragsfähigkeit und Wirtschaftlichkeit im Ackerbau ausübt, sollte die Produktionstechnik eine nachhaltige Zunahme des Unkrautbesatzes vermeiden. Dies gilt insbesondere für die Art und Intensität der chemischen Unkrautregulierung. In einem Langzeitversuch bei Zurnhausen (11.769° öL., 48.426° nB., WGS84), Landkreis Freising wird seit 2006 der Einfluss einer unterschiedlichen Pflanzenschutzmittelintensität in einer Fruchtfolge mit Winterweizen, Wintergerste und Silomais untersucht. Die vollständige, stationäre Fruchtfolge wird zudem unter dem Regime einer tief-wendenden und nicht-wendenden Bodenbearbeitung durchgeführt. Der Exaktversuch wird auf Großparzellen mit je 60 m2 in vierfacher Wiederholung nach den Vorgaben der einschlägigen EPPO-Richtlinien durchgeführt. Die Herbizidbehandlung erfolgt in den Intensitätsstufen 100 %, 75 % und 50 % Aufwandmenge einer ortsüblichen Herbizidauswahl bzw. -kombination. Die Entwicklung der Unkrautflora wird durch Auszählungen vor dem jeweiligen Herbizideinsatz erfaßt. Der Einfluss der Herbizidbehandlungsintensität auf das Unkrautspektrum und die Unkrautbesatzdichte wird in Abhängigkeit von der Art der Grundbodenbearbeitung detailiert dargestellt und hinsichtlich den Anpassungspotenzial der Herbizidaufwandmenge diskutiert. Hierzu werden auch die erhobenen Ertragsdaten und die Produktionswirtschaftlichkeit herangezogen.