Year 1 (2007-8)
The first phase of the EXBARDIV project involved the selection and expansion of plant samples for our germplasm collections, namely the Hordeum vulgare Cultivar Collection (HVCC), the Landrace Collection (LRC), the Hordeum spontaneum Collection (HSC) and Advanced Backcross Collections (ABC). Following extensive discussions, 451 candidate lines were selected for the HVCC. Seed for these, obtained from germplasm collections, breeders and our own resources, were grown over the summer of 2007, before the project formally commenced (see Figure 1) in duplicate locations to avoid accidental losses. For each line, seed was collected from a single plant to ensure genetic purity. These seed samples were grown in bulk plots in North Italy over the Winter-Spring to increase stocks (Figure 2) and a small sample of each line was also grown in the greenhouse in Scotland (Figure 3) to provide reference seed stocks and DNAs for subsequent use in the project and for posterity. Some lines were lost due to poor fertility and we expect the HVCC to be finalised at roughly 400 lines, following the harvest in Italy in early July 2008. DNAs for these lines were extracted in June 2008 and these were analysed using 1536 gene-targetted Single Nucleotide Polymorphism (SNP) molecular markers over the summer of 2008 (Figure 5).

For the LRC we were fortunate to have access to a well-studied population of 480 lines carefully collected from multiple recorded locations in Syria and Jordan. We received 100 pure seed per sample from our generous external collaborator (Dr Stefania Grando, ICARDA), avoiding the need for the first growth step. These seed have been treated in exactly the same way as the HVCC (as described above) thereafter (Figures 2 and 3).

Figure 3 The HVCC, LRC and HSC growing in the greenhouse in Invergowrie, Scotland, Spring 2008

Figure 4 Testing ABC lines for their ability to grow under nitrogen starvation conditions

Year 2 (2008-9)

The HSC collection has been given a further round of single-seed purification and amplification by selfed-growth in the greenhouse during Spring-Summer 2009 by Partner Rasmussen (Denmark)
(wild barley has significant residual heterozygosity resulting from out-crossing in the wild).

All HVCC and LRC single seed descent-purified seed were subjected to field seed increase by Cattivelli - Italy in bulk plots in Fiorenzuola Italy during Winter 2007–Spring 2008. During this stage, basic descriptive information (row type, heading date, thousand kernel weight) were collected. After harvesting (July 2008), the HVCC was finalised to 122 winter and 285 spring barley cultivars. For the LRC, pure seed of the well-studied population of 480 lines collected in Syria and Jordan received from ICARDA (see above) were directly multiplied. All 480 LRC lines progressed successfully into the finalised LRC.

HVCC and LRC samples (roughly 50g seed per sample) were distributed by partner Cattivelli (Italy) to partners Flavell and Russell (UK), Pillen (Germany) and Graner (Germany) for detailed phenotyping at 4 European locations (Italy was also used). In Winter 2008 – Summer 2009 the two collections of germplasm (HVCC and LRC) are under evaluation in multi-environment field trials at these four locations (Figure 6). Phenology, yield and yield related traits will be recorded during Summer 2009 on replicated plots (~ 4 m2 for each genotype), and used for association analyses.

In separate experiments, the complete HVCC Spring barley and LRC (Partners Schulman, Graner) and LRC alone (Partners Flavell, Russell) collections are being scored for resistance to Net Blotch (Pyrenophora teres teres; Schulman; glasshouse), powdery mildew (Blumeria graminis; Graner; detached leaf assay) and Rynchosporium (Flavell and Russell; field nursery plot) respectively. For the latter the plants are at an early stage of growth and no data are yet available. For powdery mildew 899 HVCC and LRC genotypes have been screened so far. Two virulent isolates of Blumeria graminins tritici were used for screening. All genotypes were grown in the greenhouse in 96 well trays under optimal conditions. Two week old seedlings were used for the detached leaf assay. The second leaf of each individual was divided and the two halves were exposed separately to the two fungal isolates. Two individuals of each genotype were used. The genotypes that were resistant to both isolates were screened again and in total 23% of the winter barley cultivars, 58% of the spring cultivars and 14% of the barley landraces were resistant to both isolates. Data generated for the powdery mildew assay along with the genotypic data from ILLUMINA Golden Gate Assays will be used in the near future for association studies. Associated regions/candidate genes will be re-sequenced.

In addition, 285 barley spring cultivars have been evaluated for their tolerance to frost by Partner Cattivelli (Italy) in the glasshouse. For each accession, 8 first-leaf stage plants have been cold acclimated for 4 weeks (3°C, 8 h light and 2°C, 16 h dark), then exposed to two different freezing conditions (-12°C and -10°C) for 10 h. To evaluate the effect of freezing on the functionality of the Photosystem II (PSII) reaction centers, the maximum quantum yield of the PSII photochemistry has been measured by the ratio of variable (Fv) to maximal (Fm) fluorescence in a dark-adapted state (Fv/Fm), using a Pulse Amplitude-Modulated fluorometer, after a 24 h recovery time. Lastly, phenotypic measurements are ongoing for the HSC plants by Partner 6. 3 plants of each HSC line are being grown in a randomized grid in the glasshouse, which will be scored and tested for relevant phenotype characters during Summer 2009.

After promising associations are identified by marker-trait comparative analysis the next step will be to re-sequence alleles for candidate genes showing high levels of association with the phenotyped traits. To this end, Partners Flavell and Schulman are working on parallel new methods to achieve this eficiently. Partner Flavell has developed a novel PCR approach for tagging genomic DNA before amplification of individual amplicon segments from pooled samples. Partner Schulman has been establishing a “fishing” protocol for isolation of regions of interest by hybridization and their subsequent amplification in emulsion PCR. P4 has also been involved in annotation of the Brachypodium distachyon genome, which will aid in intentification of candidate genes in mapping intervals. These method will be tested against each other and against array capture approaches in 2009. The optimal approach will then be used for the EXBARDIV Project

Association analysis using a H. spontaneum introgression library of cultivated barley:
Thirty-nine introgression lines (S42ILs) were developed previously by marker-assisted selection of BC3S4 lines from the original cross of the spring barley cultivar ‘Scarlett’ and the H. spontaneum accession ISR42-8 (Schmalenbach et al. 2008). The ILs contain single relatively small regions of the wild barley genome within the genetic background of the cultivated barley genome. The idea is to search for useful new exotic alleles in the S42IL library to enrich the cultivated germplasm. This task involves Partner Pillen (Germany) exclusively.

Currently, the introgression lines are growing under glass house conditions with two levels of nitrogen fertilization. The recurrent parent ‘Scarlett’ is used as a control. We expect to identify N efficiency responsible quantitative trait loci (QTLs) by the mixed model ANOVA statistical approach. Exotic QTL alleles, improving N efficiency under high or low N fertilization, will be located by Dunnett tests.

Development of a barley meta population by advanced backcrossing
Fifty different H. spontaneum lines from the Fertile Crescent were crossed with the European cultivar ‘Barke’. The F1s were backcrossed to produce BC1s. ‘Barke’ was always used as the recurrent parent. Currently, additional BC1 and BC2 backcrosses plus selfings are being conducted in order to produce 10-50 sub-populations which will be used in consecutive projects for wild barley allele mining studies. This task involves Partner Pillen (Germany) exclusively.