TARGETED SEQUENCING AND ANALYSIS OF THINOPYRUM SPP. INTROGRESSIONS ON 7AL ARM OF DURUM WHEAT RECOMBINANTS FOR MARKER DEVELOPMENT AND GENE MINING

Chromosome engineering (CE) is a complex cytogenetic strategy that enables the incorporation of small chromosome segments from wild species into cultivated wheats, thereby making alien genes available for effective breeding. Several durum wheat (DW) recombinants were obtained via CE, carrying segments originating from the wild Thinopyrum genus on their homoeologous 7AL arms. The alien introgressions confer several disease resistance genes to DW, as well as improved yield traits under both control and stress conditions, including drought, heat and salinity. To explore the genomic makeup of targeted Thinopyrum introgressions, we sequenced three individual 7AL-Thinopyrum spp. recombinant chromosomes, which were isolated by chromosome flow sorting following FISHIS labelling. The recombinant chromosomes possess 40%, 28% or 23% of the distal DW 7AL region replaced by either 40% Th. ponticum 7el1 chromatin alone (R23), or by composite segments of Th. ponticum 7el1 + Th. elongatum 7E (spanning 28% or 23% of 7AL in R74-10/R112 and R69-9/R5 recombinants, respectively). The obtained reads were aligned against the genome assemblies of DW Svevo and Th. elongatum, allowing identification of specific introgression breakpoints. Flanking Cleaved Amplified Polymorphic Sequences (CAPS) markers were developed to validate the breakpoint regions. A total of 2,786 putative Thinopyrum spp. orthologues were identified within the three alien segments. Gene ontology (GO) analysis revealed enrichment in several specific GO terms. In the Biological Process category, overrepresented classes included “U6 snRNA 3'−end processing”, “gamma−tubulin complex localization” and “photosystem II stabilization”. In the Molecular Function category, “protein−arginine deiminase activity” and “methionine adenosyltransferase activity” were enriched. In the Cellular Component category, “chloroplast” and “pre-folding process” were notably represented. Finally, nine putative orthologues of known genes involved in plant development (MOC1, PLT2, APO1, PIN2, LRD), molecular stress adaptation (HVP1, LEA, CAT3) and disease resistance (GTS) were sequenced and mapped within the defined alien physical stretches. These results lay the foundation for the functional characterisation of alien genes, genotype-phenotype associations, and the translation of findings to cultivated wheats, aiming at the development of more resilient and productive DW varieties.