Physical mapping of the albostrians locus

The albostrians gene was delimited to a 0.06 cM genetic interval by high-resolution genetic mapping. As half of the physical map of barley was contained in contigs larger than 904 Kbp (N50 contig length = 904 Kbp) (International Barley Genome Sequencing Consortium, 2012; Ariyadasa et al., 2014) it should be feasible to find the markers flanking the albostrians locus present on a single physical BAC contig.

Therefore, the flanking markers Zip_2661 and CAPS_2551 as well as their respective co-segregating markers were used to identify a physical map contig that would theoretically contain the gene albostrians.

3.2.1 Linking albostrians flanking markers to the physical map of barley by sequence comparison and PCR-based screening

Two approaches were used to anchor the closest albostrians flanking markers to the physical map of barley (International Barley Genome Sequencing Consortium, 2012).

This was either based on in silico analysis through sequence comparison (BLAST = Basic Local Alignment Search Tool) or by experimental PCR-screening of a barley

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BAC library HVVMRXALLeA derived from the North American six-rowed malting variety ‘Morex’ (Schulte et al., 2011). All except one marker could be anchored in silico (Table 3-4). Marker Contig_1596897 was anchored experimentally by BAC library screening. Two markers Zip_2672 and Contig_1575446 were anchored both by sequence comparison and PCR-based screening.

The two proximally flanking markers Contig_37952_1 and Zip_2661 as well as the co-segregating marker Zip_2662 were allocated to FingerPrinted BAC contig 7112 (FPcontig_7112). FPcontig_7615 was identified by the distally flanking marker 3_0168. All the remaining co-segregating, but the gene albostrians distally flanking markers (Zip_2665, Zip_2667_4, Contig_1596897, Zip_2672, Contig_1575446, Contig_49785_5, and CAPS_2551) were allocated to FPcontig_7506 (Table 3-4).

Thus instead of a single large BAC contig the identified genetic interval was represented by three individual physical BAC contigs. Based on information provided by the genetically anchored physical map (Mascher et al., 2013a) two additional FP contigs (FPcontig_4483 and FPcontig_44845) were predicted to be located between contigs carrying the flanking markers. In order to identify a possible candidate gene on any of these five BAC contigs the minimally overlapping and non-redundant path of BAC clones was selected for shotgun sequencing (Appendix Table 5).

Table 3-4: Allocating albostrians flanking markers to the physical map of barley.

Marker ID Morex_WGS_Contig Corresponding BAC FPcontig Anchoring Method

Contig_37952_1 Contig_37952 HVVMRXALLeA0035F11 7112 In silico

Zip_2661 Contig_65209 HVVMRXALLhB0102L04 7112 In silico

Zip_2662 Contig_104939 HVVMRX83KhA0187H24 7112 In silico

Zip_2665 Contig_263534 HVVMRXALLmA0412B01 7506 In silico

3_0168 Contig_137310 HVVMRXALLrA0254F14 7615 In silico

Zip_2667_4 Contig_65509 HVVMRXALLmA0412B01 7506 In silico

Contig_1596897 Contig_1596897 HVVMRXALLeA0275D08 7506 BAC screening Zip_2672 Contig_1575446 HVVMRXALLeA0135I17 7506 In silico/BAC screening Contig_1575446 Contig_1575446 HVVMRXALLeA0135I17 7506 In silico/BAC screening

Contig_49785_5 Contig_49785 HVVMRXALLmA0055G09 7506 In silico

CAPS_2551 Contig_49785 HVVMRXALLMA0055G09 7506 In silico

3.2.2 Sequence analysis of BAC contigs identified by markers that are flanking or co-segregating with the albostrians locus

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A total number of 60 minimal tiling path (MTP) BAC clones was selected from 5 FPcontigs (Appendix Table 5) for high-throughput short read shotgun sequencing.

The obtained sequencing reads were assembled with CLC assembly cell 3.2.2 (http://www.clcbio.com/) after quality trimming and vector / adaptor removal. All-against-all BLAST searches of individual BAC assemblies revealed that FPcontig_7112 and FPcontig_7615 were overlapping by BAC clones HVVMRXALLrA0395M21 and HVVMRXALLmA0230A06. No overlaps could be identified for the remaining three physical contigs (FPcontig_4483, FPcontig_7506 and FPcontig_44845). The proximal flanking marker Zip_2661 and co-segregating marker Zip_2662 were located on the same BAC clone HVVMRXALLrA0395M21 (FP Contig_7112) (Figure 3-3B). The distally flanking marker 3_0168 was located on a contiguous sequence scaffold of FPcontig_7615 thus markers flanking the locus albostrians on both sides were allocated to a single sequence scaffold represented by the MTP sequence of the physical contigs FPcontig_7112 and FPcontig_7615. A new marker Contig_92279 could be developed based on the sequence information of BAC HVVMRXALLrA0395M21, which contained the co-segregating marker Zip_2662 as well as the proximal flanking marker Zip_2661. Mapping of marker Contig_92279 revealed its distal allocation in regard of the gene albostrians, thus a physical region of about 46 Kbp was characterized genetically and physically to comprise the albostrians locus (Figure 3-3C). The ratio of physical to genetic distance of this region equalled about 0.77 Mb / cM, indicating that the albostrians gene was located in a

‘hot spot’ of recombination (Kunzel et al., 2000). In order to identify (a) possible candidate gene(s), gene models were predicted for non-repetitive sequences of the target interval through sequence comparison to annotated gene models defined on the Morex reference WGS assembly (International Barley Genome Sequencing Consortium, 2012). Only one single gene MLOC_670 (GenBank accession ID AK366098) could be identified within the target interval (Figure 3-3C). Re-sequencing of the gene MLOC_670 revealed a 4 bp deletion in mutant M4205 compared to wild type. This mutation was predicted to induce a frame-shift in the reading frame and as a consequence a premature stop codon in the second exon of the gene (Figure 3-3D) which ultimately might result in loss of function of the gene in the mutant.

Therefore, the identified gene was a strong candidate for representing the albostrians gene.

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Figure 3-3: Summary of genetic and physical mapping of the albostrians gene. (A) High-resolution genetic mapping of the albostrians gene. One co-segregating marker (Zip_2662) is identified and the recombination between contiguous markers is indicated by the numbers below. (B) Physical anchoring of markers to the sequenced MTP BACs. The two flanking markers Zip_2661 and Contig_92279 as well as the co-segregating marker Zip_2662 are located on the same BAC clone HVVMRXALLrA0395M21 (FPcontig_7112) as indicated in green color. (C) Gene prediction based on repeat masked BAC assemblies of the target interval through alignment to barley gene models published by IBSC (2012). A single gene, MLOC_670 (GenBank accession ID: AK366098) as indicated by the arrow, is identified within the target interval between two flanking markers Zip_2661 and Contig_92279, flanking a physical distance of around 46 Kbp. (D) Structure of the gene MLOC_670: The mutant allele harbors a 4 bp deletion (red bar) near the end of the first exon as compared to the wild type allele found in genotype Morex.

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3.3 Structural and functional annotation of the albostrians candidate gene