No abstract

Background: Rye (Secale cereale L., 2n = 2x = 14, RR), a relative of common wheat, is a large gene resource pool for wheat improvement. Accurate and convenient identification of the rye chromatin in wheat background will facilitate the transfer and utilization of elite genes derived from rye in wheat breeding. Results: In the present study, five rye cultivars including Imperial, German White, Jingzhouheimai, Baili and Guyuan were sequenced by specific-locus amplified fragment sequencing (SLAF-seq) to develop large-scale rye-specific markers. Based on SLAF-seq and bioinformatics analyses, a total of 404 universal PCR-based and a whole set of Kompetitive allele-specific PCR (KASP) markers specific for the 14 individual rye chromosome arms were developed and validated. Additionally, two KASP markers specific for 1RS and 2RL were successfully applied in the detection of 1RS translocations in a natural population and 2RL chromosome arms in wheat-rye derived progenies that conferred adult resistance to powdery mildew. Conclusion: The 404 PCR-based markers and 14 KASP markers specific for the 14 individual rye chromosome arms developed in this study can enrich the marker densities for gene mapping and accelerate the utilization of rye-derived genes in wheat improvement. Especially, the KASP markers achieved high-throughput and accurate detection of rye chromatin in wheat background, thus can be efficiently used in marker-assisted selection (MAS). Besides, the strategy of rye-specific PCR-based markers converting into KASP markers was high-efficient and low-cost, which will facilitate the tracing of alien genes, and can also be referred for other wheat relatives.

Wheat-rye T1RS·1BL translocations have been widely used worldwide in wheat production for multiple disease resistance and superior yield traits. However, many T1RS·1BL translocations have successively lost their resistance to pathogens due to the coevolution of pathogen virulence with host resistance. Because of the extensive variation in rye (Secale cereale L.) as a naturally cross-pollinating relative of wheat, it still has promise to widen the variation of 1RS and to fully realize its application value in wheat improvement. In the present study, the wheat-rye breeding line R2207 was characterized by comprehensive analyses using genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization with multiple probes, multicolor GISH, and molecular marker analysis, and then was proven to be a cytogenetically stable wheat-rye T1RS·1BL translocation line. Based on the disease responses to different isolates of powdery mildew and genetic analysis, R2207 appears to possess a novel variation for resistance, which was confirmed to be located on the rye chromosome arm 1RS. Line R2207 also exhibited high levels of resistance to stripe rust at both seedling and adult stages, as well as enhanced agronomic performance, so it has been transferred into a large number of commercial cultivars using an efficient 1RS-specific kompetitive allele specific PCR marker for marker-assisted selection.

Chinese wheat landrace Dahongtou was resistant to 35 of 38 tested Chinese isolates of Blumeria graminis f. sp. tritici at the seedling stage. Genetic analysis of the F2 populations and their derived F2:3 families of crosses of Dahongtou with the susceptible varieties Mingxian 169 and Huixianhong indicated that the resistance of Dahongtou to B. graminis f. sp. tritici isolate E09 was conferred by a single recessive gene, tentatively designated as pmDHT. The gene was mapped to chromosome arm 7BL and flanked by markers Xwmc526/XBE443877 and Xgwm611/Xwmc511 at genetic distances of 0.8 and 0.3 cM, respectively. The chromosomal position of pmDHT was similar to the multi-allelic Pm5 locus on 7BL. Allelism tests with crosses of Dahongtou with Fuzhuang 30 (Pm5e) and Xiaobaidong (mlxbd) indicated that pmDHT was allelic to both Pm5e and mlxbd. However, pmDHT showed a different pattern of resistance to the 38 B. graminis f. sp. tritici isolates compared with wheat lines with Pm5a, Pm5b, Pm5e, mlxbd, and PmHYM and also differed from PmSGA. Thus, pmDHT was identified most likely as a new allele or at least a closely linked gene of the Pm5 locus. This gene can be transferred into susceptible wheat cultivars/lines and pyramided with other resistance genes through marker-assisted selection to improve powdery mildew resistance.

Powdery mildew and leaf rust, caused by Blumeria graminis f. sp. tritici (Bgt) and Puccinia triticina (Pt), respectively, are widespread diseases of wheat worldwide. Utilizing resistant cultivars is considered as the most economical, environmental-friendly, and effective method to control these diseases. In the present study, a collection of 2,978 wheat accessions consisting of 1,394 advanced breeding lines, 1,078 Chinese cultivars, 291 introduced cultivars, 132 lines containing alien chromosomes, and 83 landraces was tested for reactions to powdery mildew and leaf rust. The results indicated that 659 (22.1%) wheat accessions were highly resistant to a widely prevalent Bgt isolate, E09, at the seedling stage, and 390 were consistently resistant to the mixture of Bgt isolates at the adult-plant stage. Meanwhile, 63 (2.1%) accessions were highly resistant to leaf rust at the adult-plant stage, of which 54 were resistant to a predominant and highly virulent Pt race, THTT, at the seedling stage. Notably, 17 accessions were resistant to both powdery mildew and leaf rust. To detect known genes for resistance to powdery mildew and leaf rust, these accessions were tested with gene-specific or tightly linked markers for seven Pm genes (Pm2, Pm4, Pm5, Pm6, Pm8, Pm21, and Pm24) and ten Lr genes (Lr1, Lr9, Lr10, Lr19, Lr20, Lr24, Lr26, Lr34, Lr37, and Lr46). Of the 659 powdery mildew-resistant accessions, 328 might carry single Pm genes and 191 carry combined Pm genes. Pm2 was detected at the highest frequency of 59.6%, followed by Pm8, Pm6, Pm21, Pm4, and Pm5, while Pm24 was not detected. Besides, 139 accessions might contain unknown Pm genes different from those tested in this study. In the 63 accessions resistant leaf rust, four Lr genes (Lr1, Lr10, Lr26, and Lr34) were detected in 41 accessions either singly or in combination, while six genes (Lr9, Lr19, Lr20, Lr24, Lr37, and Lr46) were not detected. Twenty-two accessions might contain unknown Lr genes different from those tested in this study. This study not only provided important information for rationally distributing resistance genes in wheat breeding programs, but also identified resistant germplasm that might have novel genes to enrich the diversity of resistance sources.

Wheat powdery mildew is a severe disease affecting yield and quality. Host resistance was proved to be effective and environment-friendly. Wheat line Subtil is an elite germplasm resource resistant to 28 of 30 tested Bgt isolates. Genetic analysis showed that the powdery mildew resistance in Subtil was conferred by a single dominant gene, temporarily designated PmSub. Using bulked segregant analysis, PmSub was mapped to chromosome arm 5DS, and flanked by the markers Bwm16 and Cfd81/Bwm21 at 5.0 and 0.9 cM, respectively. Allelism tests further confirmed PmSub was allelic with documented Pm2 alleles. Then, homologous sequences of Pm2a related sequence was cloned from Subtil and Chinese Spring. It was completely identical to the reported Pm2a sequence, but significantly different from that of Chinese Spring. A marker SWGI067 was developed based on the sequence divergence of homologous sequence in Subtil and Chinese Spring. SWGI067 was closely linked to PmSub, indicating that the gene PmSub itself was different from the cloned Pm2a related sequence. Meanwhile, Subtil produced significantly different reaction pattern compared with other genotypes with Pm genes at or near Pm2 locus. Therefore, PmSub was most likely a new allele of Pm2. PmSub has opportunities for marker-assisted selecting for high-efficiency wheat improvement.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive disease of wheat throughout the world. Host resistance is considered the most sustainable way to control this disease. Powdery mildew resistance gene Pm2b was mapped to the same genetic interval with Pm2a and PmCH1357 cloned previously, but showed different resistance spectra from them, indicating that they might be caused by different resistance genes or alleles. In this study, Pm2b was delimited to a 1.64 Mb physical interval using a large segregating population containing 4,354 F2:3 families of resistant parent KM2939 and susceptible cultivar Shimai 15. In this interval, TraesCS5D03G0111700 encoding the coiled-coil nucleotide-binding site leucine-rich repeat protein (CC-NBS-LRR) was determined as the candidate gene of Pm2b. Silencing by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) technology and two independent mutants analysis in KM2939 confirmed the candidate gene TraesCS5D03G0111700 was Pm2b. The sequence of Pm2b was consistent with Pm2a/PmCH1357. Subcellular localization showed Pm2b was located on the cell nucleus and plasma membrane. Pm2b had the highest expression level in leaves and was rapidly up-regulated after inoculating with Bgt isolate E09. The yeast two-hybrid (Y2H) and luciferase complementation imaging assays (LCI) showed that PM2b could self-associate through the NB domain. Notably, we identified PM2b interacting with the transcription factor TaWRKY76-D, which depended on the NB domain of PM2b and WRKY domain of TaWRKY76-D. TaWRKY76-D negatively regulated the resistance to powdery mildew in wheat. The specific KASP marker K529 could take the advantage of high-throughput and high-efficiency for detecting Pm2b and be useful in molecular marker assisted-selection breeding. In conclusion, cloning and disease resistance mechanism analysis of Pm2b provided an example to emphasize a need of the molecular isolation of resistance genes, which has implications in marker assisted wheat breeding.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a serious fungal disease that critically threatens the yield and quality of wheat. Utilization of host resistance is the most effective and economical method to control this disease. In our study, a wheat breeding line ShiCG15–009, released from Hebei Province, was highly resistant to powdery mildew at all stages. To dissect its genetic basis, ShiCG15–009 was crossed with the susceptible cultivar Yannong 21 to produce F1, F2 and F2:3 progenies. After genetic analysis, a single dominant gene, tentatively designated PmCG15–009, was proved to confer resistance to Bgt isolate E09. Further molecular markers analysis showed that PmCG15–009 was located on chromosome 2BL and flanked by markers XCINAU130 and XCINAU143 with the genetic distances 0.2 and 0.4 cM, respectively, corresponding to a physic interval of 705.14–723.48 Mb referred to the Chinese Spring reference genome sequence v2.1. PmCG15–009 was most likely a new gene differed from the documented Pm genes on chromosome 2BL since its different origin, genetic diversity, and physical position. To analyze and identify the candidate genes, six genes associated with disease resistance in the candidate interval were confirmed to be associated with PmCG15–009 via qRT-PCR analysis using the parents ShiCG15–009 and Yannong 21 and time-course analysis post-inoculation with Bgt isolate E09. To accelerate the transfer of PmCG15–009 using marker-assisted selection (MAS), 18 closely or co-segregated markers were evaluated and confirmed to be suitable for tracing PmCG15–009, when it was transferred into different wheat cultivars.