Towards the Reference Sequence of Chromosome 5B of Bread Wheat Triticum aestivum

Salina E.A.¹*, Sergeeva E.M.¹, Nesterov M.A.¹, Kiseleva A.A., ¹ Frenkel Z.², Korol A.² Muterko A.F. ¹, Mardanov A.V.³, Ravin N.V³, Jacobs JJMR⁴, International Wheat Genomic Sequencing Consortium

¹Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia

²University of Haifa, Haifa, Israel

³Centre of Bioengineering, Moscow, Russia

⁴Bayer CropScience N.V. Technologiepark 38, Gent, Belgium

The IWGSC strategy for sequencing the bread wheat genome is based on first obtaining physical maps of the individual chromosomes. Our aim is to develop and use the physical map for construction of the reference sequence of wheat chromosome 5B which bears a number of agronomically important genes such as Vrn, Skr, Ne1, Ph1 and genes conferring resistance to fungal diseases.

The 5B chromosome-specific BAC-libraries were obtained with approximately 15-fold coverage for the short arm (290 Mbp) and the long arm (580 Mbp).

BAC clones of 5BS were fingerprinted using the SNaPshot^TM fluorescent restriction fragment labeling method and the 5BL library was fingerprinted as part of a collaborative project between Bayer Crop Science and the IWGSC using Whole Genome Profiling (WGP^TM) technology. Each set of fingerprint data was  assembled with the LTC program.

A minimal tiling path (MTP) consisting of 3161 overlapping BAC clones covered 258 Mbp (~89%) of chromosome 5BS and 6187 MTP BAC clones covered 567 Mbp (~98%) of 5BL. The 5BS BAC clones were assembled into 275contigs, of which 90 with a size greater than 300 kb were designated as supercontigs. More than 40% of the supercontigs were anchored by using seventy-one markers mapped to chromosome 5BS using a F2 population generated from a cross of CS and CS-5B dic.

39 BAC-supercontigs were linked to  chromosome 5B using the GenomeZipper approach.

Further SNP-based anchoring of BAC clones was undertaken by genotyping the population of 116 Recombinant Inbred Chromosome Lines (RICLs) produced from a cross of CS and CS-5Bdic using the Illumina Infinium 15k Wheat SNP array (TraitGenetics GmbH). 418 markers polymorphic between the parental lines were integrated into the 5B map.

The first 9 supercontigs (384 ВАС clones) including gene-rich regions have been sequenced using the Roche/454 platform. The sequences of BAC shotgun libraries produced from pools of 10 BACs were supplemented by sequences obtained from pooled BAC 8-kb mate pair libraries. Assembly of reference sequences of these 5BS regions is in progress.

The 5BS physical mapping was supported by the RSF (Project No. 14-14-00161) and 5BS sequencing was supported by FTP of the RF (agreement no. 14.604.21.0106).