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

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.

近年来测序成本的持续下降，推动了诸多大型基因组物种的测序工作，其中包括六倍体普通小麦（*Triticum aestivum* L.）。尽管普通小麦的草图基因组序列已被解析，但由于其三套亚基因组间存在高度同源性，开发适用于标记辅助选择（marker assisted selection）流程的位点特异性引物仍是主要挑战之一。本研究描述了一种高效的位点特异性引物开发方法，其包含四个步骤：(i) 候选基因的基因组序列与编码序列（CDS）鉴定；(ii) 内含子与外显子结构重构；(iii) 小麦A、B、D亚基因组序列的识别，并基于三套亚基因组间的序列差异开发引物；(iv) 引物的功能、扩增片段大小与定位准确性验证。该方法被应用于参与小麦抗冻调控的单拷贝、低拷贝与高拷贝基因。针对从普通小麦、一粒小麦（*Triticum monococcum*）与大麦（*Hordeum vulgare*）中获取序列的27个基因，本研究共开发了119对引物。经缺体-四体（Nulli-tetrasomic, NT）系验证后，其中65对引物（占比54.6%）可实现特异性扩增，对应19个候选基因。随后从上述特异性引物中选取35个扩增片段，通过桑格（Sanger）扩增子重测序进行验证。除1个片段外，其余所有片段均能匹配至原始参考序列。相较于目前普通小麦研究中所采用的引物开发技术，本研究提出的方法展现出更高的特异性，且可推广至其他已获取草图基因组序列的多倍体物种。