An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L.) and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance

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.

Association genetics studies on frost tolerance in wheat (Triticum aestivum L.) reveal new highly conserved amino acid substitutions in CBF-A3, CBF-A15, VRN3 and PPD1 genes

“…Furthermore, the core collection of the Institute of Field and Vegetable Crops (IFVCNS), Novi Sad, Serbia [ 70 ] and parental lines of Western European hybrid breeding programs were included. For the physical mapping of PCR amplicons to chromosomes and chromosome segments, 21 nulli-tetrasomic lines (NT-lines) and 46 deletion-lines were used [ 9 , 71 , 72 ]. The DNA was extracted at the three leaf stage according to Stein et al [ 73 ].…”

“…Primer development and testing was conducted according to Babben et al [ 9 ]. Furthermore, the primer assignment was verified based on BLASTs of mRNA and genomic regions of close relatives against the Chinese Spring reference assembly v1.0 using NCBI Megablast.…”

“…Nowadays, the efforts of the International Wheat Genome Sequencing Consortium (IWGSC), in the development of a physical map and a reference sequence facilitates many downstream applications, i.e. development of high throughput genotyping platforms [ 8 ], efficient development of genome specific primers [ 9 ], exome sequencing in large genebank collections [ 10 ] etc. The wheat reference sequence, survey sequence and physical map are available at many public data bases.…”

Association Mapping of Effective Leaf Rust Resistance Genes in Collection of Winter Wheat

“…DArT markers can be easily adapted as tool for selection after allele mining through re-sequencing of specific fragments. However, the development of gene specific primers in wheat may be hampered by the size of genome (17 Gbp), the high repeat content of about 80%, and by the high fragment homology resulting from the presence of the three genomes (A, B and D) and gene family members (Babben et al 2015).…”

Genetic behavior of Triticum aestivum–Dasypyrum villosum translocation chromosomes T6V#4S·6DL and T6V#2S·6AL carrying powdery mildew resistance

“…In this investigation, a pair of specific primers of NAU/xibao15F and NAU/xibao15R can efficiently trace the four homoloeogous chromosome arms 6AS, 6BS, 6DS, and 6VS in wheat background. Recently, an efficient approach for the development of locus specific molecular markers was reported and applied in the determination of the genes involved in frost tolerance in bread wheat (Babben et al 2015). By using locus specific primers, three TaVIP2 homologous genes were successfully located on wheat chromosomes 1AL, 1BL and 1DL (Zhao et al 2016).…”