Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Resistance Research and Stress Tolerance, Erwin-Baur-Str. 27, 06484, Quedlinburg, Saxony-Anhalt, Germany.
Martin Luther University Halle-Wittenberg (MLU), Institute of Agricultural and Nutritional Sciences, Betty-Heimann-Str. 5, 06120, Halle (Saale), Saxony-Anhalt, Germany.
BMC Genomics. 2018 May 29;19(1):409. doi: 10.1186/s12864-018-4795-6.
Understanding the genetic basis of frost tolerance (FT) in wheat (Triticum aestivum L.) is essential for preventing yield losses caused by frost due to cellular damage, dehydration and reduced metabolism. FT is a complex trait regulated by a number of genes and several gene families. Availability of the wheat genomic sequence opens new opportunities for exploring candidate genes diversity for FT. Therefore, the objectives of this study were to identity SNPs and insertion-deletion (indels) in genes known to be involved in frost tolerance and to perform association genetics analysis of respective SNPs and indels on FT.
Here we report on the sequence analysis of 19 candidate genes for FT in wheat assembled using the Chinese Spring IWGSC RefSeq v1.0. Out of these, the tandem duplicated C-repeat binding factors (CBF), i.e. CBF-A3, CBF-A5, CBF-A10, CBF-A13, CBF-A14, CBF-A15, CBF-A18, the vernalisation response gene VRN-A1, VRN-B3, the photoperiod response genes PPD-B1 and PPD-D1 revealed association to FT in 235 wheat cultivars. Within six genes (CBF-A3, CBF-A15, VRN-A1, VRN-B3, PPD-B1 and PPD-D1) amino acid (AA) substitutions in important protein domains were identified. The amino acid substitution effect in VRN-A1 on FT was confirmed and new AA substitutions in CBF-A3, CBF-A15, VRN-B3, PPD-B1 and PPD-D1 located at highly conserved sites were detected. Since these results rely on phenotypic data obtained at five locations in 2 years, detection of significant associations of FT to AA changes in CBF-A3, CBF-A15, VRN-A1, VRN-B3, PPD-B1 and PPD-D1 may be exploited in marker assisted breeding for frost tolerance in winter wheat.
A set of 65 primer pairs for the genes mentioned above from a previous study was BLASTed against the IWGSC RefSeq resulting in the identification of 39 primer combinations covering the full length of 19 genes. This work demonstrates the usefulness of the IWGSC RefSeq in specific primer development for highly conserved gene families in hexaploid wheat and, that a candidate gene association genetics approach based on the sequence data is an efficient tool to identify new alleles of genes important for the response to abiotic stress in wheat.
了解小麦（Triticum aestivum L.）抗寒（FT）的遗传基础对于防止因细胞损伤、脱水和代谢减少而导致的霜冻产量损失至关重要。FT 是一个由多个基因和几个基因家族调控的复杂性状。小麦基因组序列的可用性为探索 FT 相关候选基因多样性提供了新的机会。因此，本研究的目的是鉴定与抗寒相关的已知基因中的 SNP 和插入缺失（indels），并对各自的 SNP 和 indels 进行 FT 的关联遗传学分析。
本研究利用中国春 IWGSC RefSeq v1.0 组装了 19 个与 FT 相关的候选基因，并对其进行了序列分析。其中，串联重复的 C 重复结合因子（CBF），即 CBF-A3、CBF-A5、CBF-A10、CBF-A13、CBF-A14、CBF-A15、CBF-A18、春化反应基因 VRN-A1、VRN-B3、光周期反应基因 PPD-B1 和 PPD-D1 与 235 个小麦品种的 FT 相关。在六个基因（CBF-A3、CBF-A15、VRN-A1、VRN-B3、PPD-B1 和 PPD-D1）中，发现了重要蛋白结构域中的氨基酸（AA）取代。VRN-A1 上的 AA 取代对 FT 的影响得到了证实，并在 CBF-A3、CBF-A15、VRN-B3、PPD-B1 和 PPD-D1 中检测到位于高度保守位点的新 AA 取代。由于这些结果依赖于 2 年内五个地点获得的表型数据，因此可以利用 CBF-A3、CBF-A15、VRN-A1、VRN-B3、PPD-B1 和 PPD-D1 上的 AA 变化与 FT 显著相关的检测结果，在冬小麦抗寒的标记辅助育种中加以利用。
针对上述基因，我们从之前的一项研究中开发了 65 对引物对，并用这些引物对与 IWGSC RefSeq 进行 BLAST 比对，确定了 39 对引物组合，覆盖了 19 个基因的全长。这项工作证明了 IWGSC RefSeq 在六倍体小麦中高度保守基因家族的特定引物开发中的有用性，并且基于序列数据的候选基因关联遗传学方法是鉴定小麦中与非生物胁迫反应相关基因新等位基因的有效工具。
