Exercise/Homework: ELF3 – Use case for accessing barley genome data Originally developed by: MIPS&Carlsberg Lab April 2013 Manuel Spannagl/Thomas Nussbaumer.

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Exercise/Homework: ELF3 – Use case for accessing barley genome data Originally developed by: MIPS&Carlsberg Lab April 2013 Manuel Spannagl/Thomas Nussbaumer Mats Hansson

Exercise objectives Learn from a „real-world“ use case to query the barley genome resources for: Orthologous ELF3 genes in barley Mapping/Location of barley ELF3 Marker evidence Expression Profile …

Exercise Question Question: Early flowering is an important breeding target. More than 100 genes involved in timing of flowering have been identified in Arabidopsis thaliana. One of them is Early Flowering 3 (ELF3). Find the orthologous ELF3 gene in barley and its cDNA sequence. On which chromosome is it located? Where on the chromosome/WGS contig is it located? Which FPC contigs contain ELF3? Find at least two SNP markers associated with ELF3.

Find the orthologous ELF3 gene in barley and its cDNA/gene sequence There are many ways to identify the orthologous ELF3 gene in barley. One way would be to search “ELF3” in the MIPS PlantsDB Arabidopsis thaliana instance at muenchen.de/plant/athal/searchjsp/index.jsp. muenchen.de/plant/athal/searchjsp/index.jsp In the gene report of AT2G you could click „Gene family“ and retrieve the barley ortholog from there. Another way would be to retrieve the Athal ELF3 sequence and use BLAST to search for the best barley hit at gatersleben.de/barley/viroblast.php. gatersleben.de/barley/viroblast.php

Find the orthologous ELF3 gene in barley and its cDNA/gene sequence As a candidate for a putative orthologous ELF3 barley sequence, we find AK muenchen.de/plant/barley/ga/reportsjsp/geneticElement. jsp?gene=AK (where the gene sequences can be obtained from as well)

Background: Available resources for barley anchored physical map Entry point (genes, expression, anchoring): muenchen.de/plant/barley/download/index.jsp Positioning information: ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/

On which WGS contig is it located? Positioning information for AK The gene was annotated on morex_contig_ (RNA-seq support for almost entire gene-model, flcDNA is longer compared to annotated gene model (MLOC_75281)). The Mapping “gene to WGS contigs” can be extracted here: ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/genes/barley_HighCon f_genes_MIPS_20Aug12_TranscriptsStructure.gtf or from the MIPS PlantsDB gene report of AK

Where on the chromosome is it located & Which FPC contigs contain ELF3? ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/genes_to_physMap_ tab Based on the IBSC publication, morex_contig_67536 is assigned to chrom. 1H at 132 cM. As putative Mb position we estimated 463 Mb considering the 75% anchored physical map. morex_contig_67536 is matching the FPC contig “contig 44855”. Additionally, morex_contig_67536 matches a single clone contig (HVVMRXALLhA0624F14 v3 c11) of the clone/BAC MOKI0166J11. The underlying sequence information for particular sequences (FPC contig, WGS contig, genes) can be obtained from: Anchored FPC contigs ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/FPC_PSEUDO_ANCHORED_280512_AC1. FA ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/FPC_PSEUDO_ANCHORED_280512_AC1. FA Anchored WGS contigs ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/WGS_ANCHORED_280512_AC2.FA ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/WGS_ANCHORED_280512_AC2.FA Anchored barley genes ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/GENES_ANCHORED_280512_AC3.FA ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/GENES_ANCHORED_280512_AC3.FA

FPC contig And associated clones, WGS contigs, in silico marker And experimental marker Approx. 500 Kb Accessible via: bin/gb2/gbrowse/Barley_PhysMap

Find at least two SNP markers associated with ELF3 Markers used to anchor morex_contig_67536 ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/in_silico_marker/mor ex_marker.TXT ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/in_silico_marker/mor ex_marker.TXT

Find at least two SNP markers associated with ELF3 Markers used to anchor FPC contig ftp://ftpmips.helmholtz- muenchen.de/plants/barle y/public_data/anchoring/in _silico_marker/FPC_pseud omolecule_marker.TXT ftp://ftpmips.helmholtz- muenchen.de/plants/barle y/public_data/anchoring/in _silico_marker/FPC_pseud omolecule_marker.TXT

Find at least two SNP markers associated with ELF3 Markers directly associated with AK ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/in_silico_marker/flcD NA_marker.TXT ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/in_silico_marker/flcD NA_marker.TXT Marker sequences in: ftp://ftpmips.helmholtz- muenchen.de/plants/barley/public_data/anchoring/sequences/IN_SILICO _MARKER.FA

Preview: Get the expression profile of a barley gene e.g. MLOC_75281 For AK only a partial gene model was annotated on a WGS contig, but this partial gene model (MLOC_75281) allows us to deduce the expression profile. We e.g. observe a FPKM value of 44 for „nodABC“ conditions. (unpublished)