Molecular Cytogenetic Identification of a New Wheat-Rye 6R Chromosome Disomic Addition Line with Powdery Mildew Resistance

Abstract

Rye (Secale cereale L.) possesses many valuable genes that can be used for improving disease resistance, yield and environment adaptation of wheat (Triticum aestivum L.). However, the documented resistance stocks derived from rye is faced severe challenge due to the variation of virulent isolates in the pathogen populations. Therefore, it is necessary to develop desirable germplasm and search for novel resistance gene sources against constantly accumulated variation of the virulent isolates. In the present study, a new wheat-rye line designated as WR49-1 was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. Using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization), mc-GISH (multicolor GISH) and EST (expressed sequence tag)-based marker analysis, WR49-1 was proved to be a new wheat-rye 6R disomic addition line. As expected, WR49-1 showed high levels of resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici, Bgt) pathogens prevalent in China at the adult growth stage and 19 of 23 Bgt isolates tested at the seedling stage. According to its reaction pattern to different Bgt isolates, WR49-1 may possess new resistance gene(s) for powdery mildew, which differed from the documented powdery mildew gene, including Pm20 on chromosome arm 6RL of rye. Additionally, WR49-1 was cytologically stable, had improved agronomic characteristics and therefore could serve as an important bridge for wheat breeding and chromosome engineering.

Fig 1. Genomic in situ hybridization (GISH) and sequential multicolor fluorescence in situ hybridization (mc-FISH) analysis of the wheat-rye chromosomes addition line WR49-1.

a and c using rye genomic DNA as a probe and Chinese Spring DNA as a blocker, GISH detection of WR49-1 clearly show bright green hybridization signals evenly distributed on rye chromosomes, the wheat chromosomes were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (blue). b Sequential mc-FISH on the same metaphase after GISH analysis (a) of WR49-1 by pAs1 (red) and pSc119.2 (green) simultaneously. d mc-FISH on the same metaphase after GISH analysis (c) of addition line WR49-1 by pAs1 (red) and pHvG38 (green) simultaneously. Arrows note one pair of added 6R chromosomes.

Fig 2. GISH analysis of wheat cultivar Xiaoyan 6.

Using rye genomic DNA as a probe and Chinese Spring DNA as a blocker, GISH detection indicated that no rye chromosome or chromosome segment existed in Xiaoyan 6, the wheat chromosomes were counterstained with DAPI (blue).

Fig 3. Multicolor-genomic in situ hybridization (mc-GISH) analysis of the wheat-rye chromosome addition line WR49-1.

Yellow-green, brown or gray and pink or red are A-genome chromosomes, B-genome chromosomes and D-genome chromosomes, respectively, and bright-green is added R-genome chromosomes. Arrows indicate one pair of added rye chromosomes. Two small translocations between B and D, B and A chromosomes were also detected in WR49-1, respectively. Asterisks note two small B-genome chromosome segments are translocated to D-genome chromosomes, and crosses note other two small B-genome chromosome segments are translocated to A-genome chromosomes.

Fig 4. PCR amplification of expressed sequence tag (EST)-based markers.

CGG143 (a) specific for rye chromosome 6R, and SWES78 (b), SWES206 (c) and SWES231 (d) specific for 6RL, respectively, in wheat-rye lines and controls for detection of 6R in WR49-1. The 110, 260, 200 and 260 bp bands indicate the diagnostic DNA fragments specific for 6R or 6RL, respectively. Lanes M marker pUC18/MspI, 1 Chinese Spring, 2 Xiaoyan 6, 3 German White, 4 WR49-1. 5 WR41, 6 WR81 and 7 WR91 lines derived from 'Xiaoyan 6 × German White' without 6R identified by multicolor fluorescence in situ hybridization (mc-FISH). 8 to 14 are 1R-7R addition lines of 'Chinese Spring × Imperial', respectively. 15 and 16 are triticale lines 10R2-193-2 and 10R2-194-2 (AABBRR). 17 Lovrin 10 and 18 Lovrin 13 are T1BL·1RS chromosome translocation lines.

Fig 5. Disease responses of Mingxian 169, Huixianhong, Xiaoyan 6, German White and WR49-1 (from left to right) to isolate E09 of Blumeria graminis f. sp. tritici (Bgt) at the seedling stage.

R indicates resistant host reaction, and S indicates susceptible host reaction.

Fig 6. Morphology performance of wheat-rye chromosome addition line WR49-1 and its parents.

a Plants of parent Xiaoyan 6 and WR49-1 (left and right). b Spikes and seeds of German White, Xiaoyan 6, and WR49-1 (from left to right).