Genotyping-by-sequencing uncovers a Thinopyrum 4StS·1JvsS Robertsonian translocation linked to multiple stress tolerances in bread wheat

Abstract

GBS read coverage analysis identified a Robertsonian chromosome from two Thinopyrum subgenomes in wheat, conferring leaf and stripe rust resistance, drought tolerance, and maintaining yield stability. Agropyron glael (GLAEL), a Thinopyrum intermedium × Th. ponticum hybrid, serves as a valuable genetic resource for wheat improvement. Despite its potential, limited knowledge of its chromosome structure and homoeologous relationships with hexaploid wheat (Triticum aestivum) has restricted the full exploitation of GLAEL's genetic diversity in breeding programs. Here, we present the development of a 44-chromosome wheat/GLAEL addition line (GLA7). Multicolor genomic in situ hybridization identified one chromosome arm from the St subgenome of Th. intermedium, while the other arm remained unclassified. Genotyping-by-sequencing (GBS) read coverage analysis revealed a unique Robertsonian translocation between two distinct Thinopyrum subgenomes, identified as 4StS·1J^vsS. The GLA7 line demonstrated strong adult plant resistance to both leaf rust and stripe rust under natural and artificial infection conditions. Automated phenotyping of shoot morphological parameters together with leaf relative water content and yield components showed that the GLA7 line exhibited elevated drought tolerance compared to parental wheat genotypes. Three years of field trials showed that GLA7 exhibits similar agronomic performance and yield components to the wheat parents. This unique addition line holds promise for enhancing wheat's tolerance to multiple stresses through the introduction of new resistance genes, as well as its ability to mitigate the effects of temporary water limitation during flowering, all without negatively impacting wheat performance.

Fig. 1

Development of the wheat/GLAEL introgression line GLA7. The BC₃F₂ and later generations were used for the in situ hybridization investigations. In the BC₃F₆ generation, the plants were phenotypically characterized

Fig. 2

Genomic in situ hybridization (GISH) a and fluorescence in situ hybridization (FISH) b, c patterns of a cell from the wheat/GLAEL GLA7 progeny line. a mcGISH of a 44-chromosome metaphase cell: Two chromosome arms hybridized with Pseudoroegneria spicata (2n = 2x = 14, St) DNA probe (red, yellow arrows); no hybridization with Thinopyrum bessarabicum (2n = 2x = 12, J^b) DNA. DAPI-stained wheat chromosomes are blue. Scale bar = 10 μm. b Repetitive DNA FISH patterns (Afa-family, pSc119.2, oligo-pTa71) showing the identification of all 42 wheat chromosomes and a pair of additional chromosomes (yellow arrows) with Afa-family signals on the long arm. Scale bar = 10 μm. c FISH karyogram displaying the hybridization sites of oligo-pTa71, pSc119.2, and Afa-family probes on all wheat chromosomes and the introgressed Thinopyrum chromosome (Thi). Scale bar = 10 μm

Fig. 3

Results of the GBS read coverage analysis along the in silico T. aestivum-Th. intermedium hybrid chromosomes. a Average number of reads per Mb of the MV9, KAR, GLAEL, and GLA7 genotypes, b Normalized GBS read coverage of the wheat/GLAEL GLA7 addition line along the A, B, D, S, J, V chromosomes of the in silico hybrid. The X-axis represents the genomic position within the chromosome in Mb, while the Y-axis shows the normalized read coverage. Turquoise arrows indicate the introgression of a 4St segment, while purple arrows mark the 1V (J.^vs segment)

Fig. 4

Symptoms of spontaneous stripe rust a and leaf rust b, and induced leaf rust infection c, along with spike and seed morphology of the wheat/GLAEL GLA7 addition line and its parental wheat genotypes d. a Stripe rust infection on a leaf of the susceptible wheat genotypes MV9 and KAR, contrasted with the healthy leaves of the wheat/GLAEL addition line GLA7, observed in the pesticide-free nursery in 2023, Martonvásár, Hungary. b Symptoms of the spontaneous leaf rust infection on the wheat genotype MV9 and KAR, alongside the healthy leaves of the wheat/GLAEL addition line (on the right), observed in the pesticide-free ‘Tükrös’ nursery in 2024). c Artificially induced leaf rust infection on the leaves of the parental wheat genotypes MV9 and KAR, compared to the resistant leaf of the addition line, observed in the ‘Rustgarden’ nursery in 2024. d Spike and seed morphology of the wheat genotypes MV9 and KAR, GLAEL, and the wheat/GLAEL addition line

Fig. 5

Results of the drought tolerance test for MV9 and KAR wheat and the GLA7 addition line. a Control and drought-treated plants were phenotyped automatically for RGB traits using the PlantScreen™ platform both before and after the drought treatment. b Leaf color segmentation for each genotype under control and drought conditions performed by the RGB pixel image analysis. The relative percent of different colors are shown. c–i Mean values of plant area (dm²), relative water content (RWC, %), shoot weight (g), grain weight per plant (g), thousand grain weight (TGW, g), grain length (mm), and grain width (mm) for each genotype. Standard errors of the means are represented as error bars. Two-way ANOVAs with Tukey’s post hoc tests were performed for all tested genotypes for each yield parameter separately under control and drought stress treatments (α = 0.05, n = 8). Different letters above error bars indicate statistically significant differences among treatments as determined by Tukey’s post hoc tests