Abstract
Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.). Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i) identification of genomic and coding sequences (CDS) of candidate genes, (ii) intron- and exon-structure reconstruction, (iii) identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv); testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT) lines, a set of 65 primer pairs (54.6%), corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.
Highlights
IntroductionWheat (Triticum aestivum L.) is the cereal with the largest acreage worldwide [1]
Genomic resources in wheatWheat (Triticum aestivum L.) is the cereal with the largest acreage worldwide [1]
Alignment of candidate gene sequences with corresponding genomic sequences retrieved from the International Wheat Genome Sequencing Consortium, the Bristol Wheat Genomics and NCBI allowed the identification of exon-intron splicing positions, and the identification of coding and non coding regions
Summary
Wheat (Triticum aestivum L.) is the cereal with the largest acreage worldwide [1]. It belongs to the family Poaceae and has a complex allohexaploid genome of about 17 Giga-base pairs (Gbp). 300.000–500.000 years ago the first hybridisation between the wild diploid wheat (Triticum urartu, 2n = 2x = 14, genome AuAu) and an ancestor closest related to goat grass (Aegilops speltoides, 2n = 2x = 14, genome SS) took place [4, 5] leading to the generation of wild emmer wheat (Triticum dicoccoides, 2n = 4x = 28, genome AuAuBB) [6]. By a spontaneous hybridisation of cultivated emmer with another goat grass (Aegilops tauschii 2n = 2x = 14, genome DD) in combination with a natural mutation, bread wheat (Triticum aestivum, 2n = 6x = 42, genome AABBDD) was created [7]. Due to the hexaploid genome and a very high homology of the three sub-genomes in wheat, the genome sequence information has an inestimable value for molecular breeding, comparative genomics and association studies
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
