Development and identification of a novel wheat-Thinopyrum ponticum disomic substitution line DS5Ag(5D) with new genes conferring resistance to powdery mildew and leaf rust

Abstract

Background: Powdery mildew (caused by Blumeria graminis f. sp. tritici (Bgt)) and leaf rust (caused by Puccinia triticina (Pt)) are prevalent diseases in wheat (Triticum aestivum L.) production. Thinopyrum ponticum (2n = 10x = 70, E^eE^eE^bE^bE^xE^xStStStSt) contains genes that confer high levels of resistance to these diseases.

Results: An elite wheat-Th. ponticum disomic substitution line, DS5Ag(5D), was developed in the Bainong Aikang 58 (AK58) background. The line was assessed using genomic in situ hybridization (GISH), oligo-nucleotide probe multiplex (ONPM) fluorescence in situ hybridization (FISH), and molecular markers. Twenty eight chromosome-specific molecular markers were identified for the alien chromosome, and 22 of them were co-dominant. Additionally, SNP markers from the wheat 660 K SNP chip were utilized to confirm chromosome identification and they provide molecular tools for tagging the chromosome in concern. The substitution line demonstrated high levels of resistance to powdery mildew throughout its growth period and to leaf rust at the adult stage. Based on the resistance evaluation of five F₅ populations between the substitution lines and wheat genotypes with different levels of sensitivity to the two diseases. Results showed that the resistance genes located on 5Ag confered stable resistance against both diseases across different backgrounds. Resistance spectrum analysis combined with diagnostic marker detection of known resistance genes of Th. ponticum revealed that 5Ag contained two novel genes, Pm5Ag and Lr5Ag, which conferred resistance to powdery mildew and leaf rust, respectively.

Conclusions: In this study, a novel wheat-Th. ponticum disomic substitution line DS5Ag(5D) was successfully developed. The Th. ponticum chromosome 5Ag contain new resistance genes for powdery mildew and leaf rust. Chromosomic-specific molecular markers were generated and they can be used to track the 5Ag chromosome fragments. Consequently, this study provides new elite germplasm resources and molecular markers to facilitate the breeding of wheat varieties that is resistant to powdery mildew and leaf rust.

Keywords: Thinopyrum ponticum; Disomic substitution line; Fluorescence in situ hybridization; Leaf rust; Powdery mildew.

Fig. 1

FISH/GISH karyotype pattern of PY5814 and its parents. A, E FISH/GISH karyotypes of PY5814 (A) and AK58 (E) RTC with ONPM#4–1. B, D GISH analysis of PY5814 RTC (B) and PMC (D), the green signals are for the Th. ponticum gDNA probe labeled by fluorescein-12-dUTP (green). C, F FISH karyotypes of PY5814 (C) and Th. ponticum (F) with ONPM#7.Common wheat chromosomes were blue (DAPI); arrows and asterisks refer to 5Ag chromosome and T1RS•1BL translocation, respectively; bar = 10 μm

Fig. 2

Molecular marker analysis of PY5814. M: Marker DL2000; 1: Th. ponticum; 2: PY5814; 3: AK58; 4: KY818; 5: LKAZ8; 6: CSN5BT5D; 7: CSN5DT5B; Solid and hollow arrows indicate the specific fragment of chromosome 5Ag and 5D, respectively

Fig. 3

Wheat 660 K SNP array analysis of PY5814 and its parents. A Lowest homozygous, highest heterozygous and miss rate on the 5D chromosome. B The difference of SNPs on 5D chromosome between PY5814 and its two parents was the most obvious

Fig. 4

Resistance evaluation of PY5814 and its parents. A-C Reactions to powdery mildew at seedling stage (A) and adult stage (B) and leaf rust at adult stage (C). 1: Th. ponticum; 2: DS5Ag(5D); 3: AK58; 4: KY18; 5: LKAZ8; 6: CS; 7: MX169

Fig. 5

FISH/GISH analysis was performed on the F₅ population using ONPM#4–1 probes. A–F FISH/GISH analysis of wheat-Th. ponticum DS5Ag(5D) resistant lines, which did not contain 1RS•1BL translocation lines, of PY5814/CS (A, D), PY5814/WM6 (B, E), and PY5814/Q03073(C, F) F₅ populations. G–I GISH analysis of F₅ susceptible lines of PY5814/CS (G), PY5814/WM6 (H), and PY5814/Q03073 (I). Arrows refer to 5Ag chromosome, bar = 10 μm

Fig. 6

Agronomic performance of PY5814 and its recurrent wheat parent AK58. A Plants of PY5814 (right) and AK58 (left). B Seeds of PY5814 (right) and AK58 (left)