High-resolution mapping of SrTm4, a recessive resistance gene to wheat stem rust

Abstract

The diploid wheat recessive stem rust resistance gene SrTm4 was fine-mapped to a 754-kb region on chromosome arm 2A^mL and potential candidate genes were identified. Race Ug99 of Puccinia graminis f. sp. tritici (Pgt), the causal agent of wheat stem (or black) rust is one of the most serious threats to global wheat production. The identification, mapping, and deployment of effective stem rust resistance (Sr) genes are critical to reduce this threat. In this study, we generated SrTm4 monogenic lines and found that this gene confers resistance to North American and Chinese Pgt races. Using a large mapping population (9522 gametes), we mapped SrTm4 within a 0.06 cM interval flanked by marker loci CS4211 and 130K1519, which corresponds to a 1.0-Mb region in the Chinese Spring reference genome v2.1. A physical map of the SrTm4 region was constructed with 11 overlapping BACs from the resistant Triticum monococcum PI 306540. Comparison of the 754-kb physical map with the genomic sequence of Chinese Spring and a discontinuous BAC sequence of DV92 revealed a 593-kb chromosomal inversion in PI 306540. Within the candidate region, we identified an L-type lectin-domain containing receptor kinase (LLK1), which was disrupted by the proximal inversion breakpoint, as a potential candidate gene. Two diagnostic dominant markers were developed to detect the inversion breakpoints. In a survey of T. monococcum accessions, we identified 10 domesticated T. monococcum subsp. monococcum genotypes, mainly from the Balkans, carrying the inversion and showing similar mesothetic resistant infection types against Pgt races. The high-density map and tightly linked molecular markers developed in this study are useful tools to accelerate the deployment of SrTm4-mediated resistance in wheat breeding programs.

Fig. 1

Stem rust reactions to Pgt race TTTTF (isolate 01MN84A-1-2) inoculated on leaves of segregating resistant and susceptible plants from cross G3116 × PI 306540. 1, PI 306540 (SrTm4); 2, G3116; 3–5, resistant F_3:4 plants; 6–8, susceptible F_3:4 plants. R, resistant; S, susceptible

Fig. 2

High-density genetic maps for stem rust resistance locus SrTm4. a Genomic region containing SrTm4 (marked in gray) on the long arm of wheat chromosome 2A. b Genetic map based on 811 F₂ plants and 14 molecular markers; c High-density genetic map based on 4761 F₂ plants and 10 molecular markers; d Physical map of Chinese Spring. Coordinates are based on CS RefSeq v2.1

Fig. 3

Transcript levels of high-confidence genes annotated in the candidate gene region. Differentially expressed genes (DEGs) between homozygous susceptible lines (G3116, S-A13 and S-E14) and homozygous resistant lines (PI 306540, R-F14 and R-K18) were identified using RNA-seq data. The heatmap was generated using the pheatmap package (Kolde and Kolde 2018)

Fig. 4

Chromosomal inversion in the candidate region. (a) Comparison of the SrTm4 physical map in PI 306540 with the genomic sequence of Chinese Spring (RefSeqv2.1). The figure was generated using the Python drawing library matplotlib (Hunter 2007). The inverted region is highlighted by the red square. (b) Syntenic relationships between Chinese Spring and PI 306540. The figure was created using the NGenomeSyn program (https://github.com/hewm2008/NGenomeSyn). Blue arrows represent the inverted regions. (c) Dominant markers used to characterize the inversion breakpoints. Two dominant markers HNPI30F1R1 and HNPI30F4R4 (Table S2) were developed on the breakpoint junctions. These primers amplify PCR products of 1007-bp and 1859-bp when the 593-kb chromosomal inversion is present and no product when its absent. The amplification products are marked with a red arrow. + , PCR product present; -, no PCR product (colour figure online)

Fig. 5

Infection types of T. monococcum accessions PI 277131-2, PI 306547, PI 428158, PI 435001, PI 306540, and G3116 in response to Puccinia graminis f. sp. tritici race TTTTF (isolate 01MN84A-1-2). Plants were grown in a growth chamber at 18 °C day/15 °C night with 16 h light/8 h darkness. R, resistant; S, susceptible

Fig. 6

A collection of 79 T. monococcum accessions was used to test the presence/absence of the chromosomal inversion. Dominant markers HNPI30F1R1, HNPI30F4R4, and DV92F1R1 (Table S2) were used to genotype these T. monococcum accessions. Green circles, accessions without the inversion; Red triangles, genotypes with the inversion (colour figure online)