Doris Kopahnke

An integrated approach for comparative mapping in rice and barley with special reference to the Rph16 resistance locus

The accumulated sequence information of the almost completed rice genome and the transcriptome of other cereals provide an excellent starting point for comparative genome analysis. We performed targeted synteny-based marker saturation for the Rph16 leaf rust resistance locus in barley by extensively exploiting these newly available resources. Out of a collection of over 320,000 public barley ESTs 309 non-redundant candidate syntenic clones have been identified for this region in a two-step in silico selection procedure. For mapping, 54 barley cDNA-clones were selected due to the even distribution of their homologs on a putatively collinear 3-Mb rice BAC contig. Out of these, 97% (30) of the polymorphic markers could be genetically assigned in collinearity to the target region in barley and a set of 11 markers was integrated into an rph16 high-resolution map. Although, the collinear target region of rice does not contain an obvious candidate gene for rph16 the results demonstrate the potential of the presented procedure to efficiently utilize EST resources for synteny-based marker saturation. The systematic genome-wide exploitation of the increasing sequence data resources will strongly improve our current view of genome conservation and likely facilitate a synteny-based isolation of genes conserved across cereal species.

Resistance to powdery mildew in Spanish barley landraces is controlled by different sets of quantitative trait loci

Twenty-two landrace-derived inbred lines from the Spanish Barley Core Collection (SBCC) were found to display high levels of resistance to a panel of 27 isolates of the fungus Blumeria graminis that exhibit a wide variety of virulences. Among these lines, SBCC145 showed high overall resistance and a distinctive spectrum of resistance compared with the other lines. Against this background, the main goal of the present work was to investigate the genetic basis underlying such resistance using a doubled haploid population derived from a cross between SBCC145 and the elite spring cultivar Beatrix. The population was genotyped with the 1,536-SNP Illumina GoldenGate Oligonucleotide Pool Assay (Barley OPA-1 or BOPA1 for short), whereas phenotypic analysis was performed using two B. graminis isolates. A major quantitative trait locus (QTL) for resistance to both isolates was identified on the long arm of chromosome 6H (6HL) and accounted for ca. 60% of the phenotypic variance. Depending on the B. graminis isolate tested, three other minor QTLs were detected on chromosomes 2H and 7H, which explained less than 5% of the phenotypic variation each. In all cases, the alleles for resistance derived from the Spanish parent SBCC145. The position, the magnitude of the effect observed and the proportion of phenotypic variation accounted for by the QTL on 6HL suggest this is a newly identified locus for broad-based resistance to powdery mildew.

Identification of quantitative trait loci for resistance to powdery mildew in a Spanish barley landrace

The Spanish landrace-derived inbred line SBCC97, together with other lines from the Spanish Barley Core Collection, displays high resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei. The objective of this study was to map quantitative trait loci (QTLs) for resistance to powdery mildew in a recombinant inbred line population derived from a cross between SBCC97 and the susceptible cultivar ‘Plaisant’. Phenotypic analysis was performed using four B. graminis isolates, and genetic maps were constructed with mainly simple sequence repeat (SSR) markers, following a sequential genotyping strategy. Two major QTLs with large effects were identified on chromosome 7H, and they accounted for up to 45% of the total phenotypic variance. The alleles for resistance at each QTL were contributed by the Spanish parent SBCC97. One locus was mapped to the short arm of chromosome 7HS, and was flanked by the resistance gene analogue (RGA) marker S9202 and the SSR GBM1060. This corresponded to the same chromosomal region in which a major race-specific resistance gene from Hordeum vulgare ssp. spontaneum, designated as mlt, had been identified previously. The second QTL was linked tightly to marker EBmac0755, and it shared its chromosomal location with the qualitative resistance gene Mlf, which has only been described previously in the wild ancestor H. spontaneum. This is the first report of these two QTLs occurring together in cultivated barley, and it paves the way for their use in barley breeding programs that are designed to transfer resistance alleles into elite cultivars.

Resistance to powdery mildew in one Spanish barley landrace hardly resembles other previously identified wild barley resistances

Two major quantitative trait loci (QTLs) associated with resistance to powdery mildew (Blumeria graminis f. sp. hordei) were previously identified on chromosome 7H of the Spanish barley line SBCC097. The two QTLs seemed to share the same chromosomal position as the major genes mlt and Mlf, which were formerly described in Hordeum vulgare ssp. spontaneum-derived lines. In the present work, different lines that carry mlt (RS42-6*O), Mlf (RS137-28*E), or a combination of both (SI-4 and SI-6) were compared with SBCC097 to evaluate their relatedness at the phenotypic, cellular, and genetic levels. The resistance of the lines was characterised by inoculating them with a set of 27 isolates of B. graminis, which displayed a wide range of virulence. It was revealed that SBCC097 possessed a distinctive resistance spectrum. Microscopic assessment of the cytological development of the resistance response showed that SBCC097 clearly formed fewer well-established colonies and secondary hyphae than the other lines. This was confirmed by the infection type recorded after visual inspection. Genetic analyses of all five lines, based on markers flanking the QTLs derived from SBCC097, supported the macroscopic and microscopic data and pointed to the presence of a combination of novel genes or alleles in SBCC097, which may be included in the category of “intermediate-acting” genes, governing resistance mainly at the post-penetration stage.

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.

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 nested association mapping population identifies multiple small effect QTL conferring resistance against net blotch (Pyrenophora teres f. teres) in wild barley

The net form of net blotch caused by the necrotrophic fungus Pyrenophora teres f. teres is a major disease of barley, causing high yield losses and reduced malting and feed quality. Exploiting the allelic richness of wild barley proved to be a valuable tool to broaden the genetic base of resistance of modern elite cultivars. In this study, a SNP-based nested association mapping (NAM) study was conducted to map QTL for P. teres resistance in the barley population HEB-25 comprising 1,420 lines derived from BC1S3 generation. By scoring the percentage of infected leaf area followed by calculation of the average ordinate (AO) and scoring of the reaction type (RT) in two-year field trials a large variability of net blotch resistance across and within families of HEB-25 was observed. Genotype response to net blotch infection showed a range of 48.2% for AO (0.9–49.1%) and 6.4 for RT (2.2–8.6). NAM based on 5,715 informative SNPs resulted in the identification of 24 QTL for resistance against net blotch. Out of these, six QTL are considered novel showing no correspondence to previously reported QTL for net blotch resistance. Overall, variation of net blotch resistance in HEB-25 turned out to be controlled by small effect QTL. Results indicate the presence of alleles in HEB-25 differing in their effect on net blotch resistance. Results provide valuable information regarding the genetic architecture of the complex barley-P. teres f. teres interaction as well as for the improvement of net blotch resistance of elite barley cultivars.

Identification of QTL conferring resistance to stripe rust (Puccinia striiformis f. sp. hordei) and leaf rust (Puccinia hordei) in barley using nested association mapping (NAM)

The biotrophic rust fungi Puccinia hordei and Puccinia striiformis are important barley pathogens with the potential to cause high yield losses through an epidemic spread. The identification of QTL conferring resistance to these pathogens is the basis for targeted breeding approaches aiming to improve stripe rust and leaf rust resistance of modern cultivars. Exploiting the allelic richness of wild barley accessions proved to be a valuable tool to broaden the genetic base of resistance of barley cultivars. In this study, SNP-based nested association mapping (NAM) was performed to map stripe rust and leaf rust resistance QTL in the barley NAM population HEB-25, comprising 1,420 lines derived from BC1S3 generation. By scoring the percentage of infected leaf area, followed by calculation of the area under the disease progress curve and the average ordinate during a two-year field trial, a large variability of resistance across and within HEB-25 families was observed. NAM based on 5,715 informative SNPs resulted in the identification of twelve and eleven robust QTL for resistance against stripe rust and leaf rust, respectively. Out of these, eight QTL for stripe rust and two QTL for leaf rust are considered novel showing no overlap with previously reported resistance QTL. Overall, resistance to both pathogens in HEB-25 is most likely due to the accumulation of numerous small effect loci. In addition, the NAM results indicate that the 25 wild donor QTL alleles present in HEB-25 strongly differ in regard to their individual effect on rust resistance. In future, the NAM concept will allow to select and combine individual wild barley alleles from different HEB parents to increase rust resistance in barley. The HEB-25 results will support to unravel the genetic basis of rust resistance in barley, and to improve resistance against stripe rust and leaf rust of modern barley cultivars.

Genomweite Assoziationsstudien zur Resistenz gegenüber Pyrenophora teres f. teres in Gerste (Hordeum vulgare)

Die Daten der Vergleichsbetriebe stellen den Status quo im Pflanzenschutz in Deutschland dar. Der Behandlungsindex in Mohre und Frischkohl betrug im Durchschnitt der Jahre (2007-2014) bei 6,2 bzw. 9,2. Mohren wurden vorrangig mit Herbiziden und Frischkohl mit Insektiziden behandelt. Unter dem Aspekt des Pflanzenschutzes und zur Reduktion der Behandlungsintensitat mit Insektiziden kann Abbildung 1: Behandlungsindex der Herbizide (Herbizid-BI) in Abhangigkeit der Vorfrucht in den Vergleichsbetrieben fur Mohre, 2007-2014, in Deutschland, ungleiche Buchstaben symbolisieren signifikante Unterschiede (p<0,05), nBlattfrucht=58, nGemuse=28, nHalmfruchte=115 208 72. ALVA-Tagung, Seminarhotel Wesenufer, 2017 der Fruhkohlanbau empfohlen werden. Im Kohl- und Mohrenanbau konnen Halmvorfruchte Schadlingsbefall vorbeugen. Die Mittelaufwandmengen wurden vorrangig bei Herbizidanwendungen reduziert In beiden Kulturen wurden Abweichungen vom notwendigen Mas verzeichnet. Die Pflanzenschutzberatung sowie die regelmasige Durchfuhrung von Bonituren konnen Pflanzenschutzmittelanwendungen dem notwendigen Mas naher bringen und die Umsetzung des IPS in der Praxis verbessern. Fur die Ausschopfung nichtchemischer Masnahmen sind Praxisdemonstrationen und der intensive Austausch zwischen Wissenschaft und Praxis notig, genauso wie Forschungsforderung zur Entwicklung neuer und Optimierung bestehender integrierter Verfahren und neuer Wirkstoffe. Vorbildhaft wird der IPS von den Demonstrationsbetrieben u. a. in den Kulturen Mohre und Weiskohl, seit 2014 umgesetzt. Die Betriebe werden individuell und intensiv betraten, um Pflanzenschutzmittelanwendungen auf das notwendige Mas zu beschranken. Hierfur werden Bonituren unter Nutzung von Schadschwellen, Warndiensthinweisen und Monitoringverfahren durchgefuhrt.

Mapping QTL for resistance to net blotch ( Pyrenophora teres f. teres ) in a wild barley nested association mapping (NAM) population

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.