The AABB genome donor for bread wheat, tetraploid wheat (Triticum turgidum L.) could serve as a valuable genetic resource, because this group include genetic variations not presenting in hexaploid wheat species. We then crossed tetraploid F1 hybrids and a diploid wild wheat, Aegilops tauschii Coss., to rapidly construct synthetic hexaploid wheat NAM populations (SHN population) to detect useful tetraploid-derived genes.

During the development of the NAM population, we observed hybrid incompatibility in a triploid generation. Identification of causal genes and molecular genetic mechanisms for this hybrid incompatibility are essential for the efficient utilization of ancestral wheat species as germplasms. We aimed to identify the causal genes through QTL analysis with dpMIG-seq (Nishimura et al. 2024), mapping with DNA markers, and revealing their expression through RNA-seq analysis.

We crossed two tetraploid wheat lines, Langdon (LDN) and NP29. A triploid wheat population was developed by crossing the tetraploid F1 individual with an Aegilops tauschii Coss. line, KU-2098. This triploid population was named SHN29. In this population, we observed 28 plants of incompatible type and 24 plants of normal type. This ratio fit a 1:1 ratio in the chi-square test (p=0.579). In addition, the QTL analysis using dpMIG-seq revealed a significant peak on chromosome 2BL. These results suggest this hybrid incompatibility is regulated by a single gene located on chromosome 2BL.

For narrowing down the QTL region, indel markers were developed in the QTL region based on whole genome resequencing data of LDN and NP29. Using these markers, 2 plants recombined in the QTL region were selected from 28 incompatible plants of SHN29. Next, using the newly developed indel markers, we were able to narrow down the candidate region, where the causal gene were located, within a 5 Mb region of chromosome 2B.

For RNA-seq, another triploid wheat population was developed by the same procedure as SHN29. Twenty-four plants, 12 normal and 12 incompatible, were selected by MIG-seq. Total RNA was extracted from their crown tissue and used for RNA-seq analysis.

As a result, 58 genes were located within the candidate region, and 5 of them were identified as differentially expressed genes (DEGs) with more than a 4-fold change in expression levels. Functional mutations in proteins within the candidate region were examined using SnpEff, and several mutations were found to significantly impact protein function, such as the occurrence of premature stop codon and frameshift mutations.