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Wheat pathogens, especially those causing powdery mildew and stripe rust, seriously threaten yield worldwide. Utilizing newly identified disease resistance genes from wheat relatives is an effective strategy to minimize disease damage. In this study, chromosome-specific molecular markers for the 3S

Background The identification of chromosomes among Avena species have been studied by C-banding and in situ hybridization. However, the complicated results from several cytogenetic nomenclatures for identifying oat chromosomes are often contradictory. A universal karyotyping nomenclature system for precise chromosome identification and comparative evolutionary studies would be essential for genus Avena based on the recently released genome sequences of hexaploid and diploid Avena species. Results Tandem repetitive sequences were predicted and physically located on chromosomal regions of the released Avena sativa OT3098 genome assembly v1. Eight new oligonucleotide (oligo) probes for sequential fluorescence in situ hybridization (FISH) were designed and then applied for chromosome karyotyping on mitotic metaphase spreads of A. brevis, A. nuda, A. wiestii, A. ventricosa, A. fatua, and A. sativa species. We established a high-resolution standard karyotype of A. sativa based on the distinct FISH signals of multiple oligo probes. FISH painting with bulked oligos, based on wheat-barley collinear regions, was used to validate the linkage group assignment for individual A. sativa chromosomes. We integrated our new Oligo-FISH based karyotype system with earlier karyotype nomenclatures through sequential C-banding and FISH methods, then subsequently determined the precise breakage points of some chromosome translocations in A. sativa. Conclusions This new universal chromosome identification system will be a powerful tool for describing the genetic diversity, chromosomal rearrangements and evolutionary relationships among Avena species by comparative cytogenetic and genomic approaches.

Additional file 1: Table S1. The predicted total TRs in the chromosomes of oat genome assembly v1 by TRF., Table S2. Distribution and copy number prediction of centromeric repeats Oligo-CCS1 in oat genome assembly v1., Figure S1. The genomic distribution of TRs in assembled oat genome v1 revealed by each chromosome., Figure S2. ND-FISH karyotype of A. sativa revealed by multiple oligo probes of tandem repeats., Figure S3. Sequential ND-FISH analysis by predicted TR Oligo-probes on metaphase chromosomes of oat BaiyanII., Figure S4. The comparative genome between the A-genome of A. sativa to A-genome of A. atlantica (a), C-genome of A. sativa to AE genome of A. eriantha (b) by Circos software with the annotated genes.

Wheat is one of the major food crops in the world. Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an economically important disease that affects wheat worldwide. The discovery of novel resistance genes and the deployment of effectively resistant cultivars are important for the ongoing control of wheat stripe rust and the maintenance of the agricultural productivity of wheat. CH7086, a new stripe rust-resistant wheat introgression line, was selected by crossing susceptible cultivars with the resistant Thinopyrum ponticum-derived partial amphiploid Xiaoyan 7430. The resistance of CH7086 is effective against all current Chinese P. striiformis f. sp. tritici races. CH7086 was crossed with the stripe rust-susceptible cultivars to develop F1, F2, F3, and BC1 populations for genetic analysis. Segregation in the F2 and BC1 populations and F2:3 lines were tested for resistance against the P. striiformis f. sp. tritici race CYR32. This test showed that CH7086 carries a single dominant gene for stripe rust resistance, which was temporarily designated YrCH86. The closest of the eight simple sequence repeat (SSR) and expressed sequence tag-SSR markers flanking the locus were X2AS33, which is 1.9 cM distal, and Xmag3807, which is 3.1 cM proximal. The resistance gene and its polymorphic markers were placed in deletion bin 2AS-0.78-1.00 using the ‘Chinese Spring’ nullisomic-tetrasomic, ditelosomic, and deletion lines. The tests of both allelism and resistance specificity suggested that the resistance gene found in CH7086 was not Yr17, which was the only current formally named Yr gene on chromosome 2AS. Thus, YrCH86 appeared to be a new locus and was permanently designated Yr69.

A new wheat-Thinopyrum translocation line CH13-21 was selected from the progenies derived from a cross between wheat-Th. intermedium partial amphiploid TAI7047 and wheat line Mianyang11. CH13-21 was characterized by using genomic in situ hybridization (GISH), multicolor-GISH (mc-GISH), multicolor-fluorescence in situ hybridization (mc-FISH) and chromosome-specific molecular markers. When inoculated with stripe rust and powdery mildew isolates, CH13-21 displayed novel resistance to powdery mildew and stripe rust which inherited from its Thinopyrum parent. The chromosomal counting analyses indicated that CH13-21 has 42 chromosomes, with normal bivalent pairing at metaphase I of meiosis. GISH probed by Th. intermedium genomic DNA showed that CH13-21 contained a pair of wheat-Th. intermedium translocated chromosomes. Sequential mc-FISH analyses probed by pSc119.2 and pAs1 clearly revealed that chromosome arm 6BS of CH13-21 was replaced by Thinopyrum chromatin in the translocation chromosome. The molecular markers analysis further confirmed that the introduced Th. intermedium chromatin in CH13-21 belonged to the long arm of homoeologous group 6 chromosome. Therefore, CH13-21 was a new T6BS.6Ai#1L compensating Robertsonian translocation line. It concludes that CH13-21 is a new genetic resource for wheat breeding programs providing novel variation for disease resistances.

Wheat yellow rust resistance gene Yr17 was originated from the wheat-Aegilops ventricosa introgression, and still effective on the adult plant in Southern China. The previous studies located the gene Yr17 on the translocation of 2NS-2AS using the molecular and cytological markers. In the present study, we screened new PCR-based markers to map the gene Yr17 region from the investigation of a segregating 120 F2 population. All markers including four EST-PCR markers, a SCAR (sequence characterized amplified region) and a PLUG (PCR based landmark unique gene) marker specific to Yr17 gene were mapped on the chromosome 2AS, and located on the chromosomal deletion bin 2AS5-0.8–1.00 region. Based on the wheat-rice collinearity, we found that the sequences of the Yr17 gene linked markers were comparatively matched at rice chromosome 4 and chromosome 7. However, the identified closely linked genomic sequence of Yr17 gene is most likely collinear with genomic region of rice chromosome 4. The newly produced PCR based markers closely linked to Yr17 gene will be useful for the marker-assisted selection in wheat breeding for rust resistance.

The genus Dasypyrum (or Haynaldia) consists of two species, D. villosum and D. breviaristatum. However, the genomic relationships between these two species remain unclear. The objective of this study was to provide molecular phylogenic and cytological evidence on the evolutionary relationships of the genus Dasypyrum. Sequences of Chloroplast DNA (cpDNA) and α-gliadin genes both support the hypothesis that diploid D. breviaristatum is the progenitor of tetraploid D. breviaristatum, and the diploid D. villosum and D. breviaristatum evolved parallel from an ancestral species. Genomic and fluorescence in situ hybridization using ribosomal DNA and rye repetitive DNA sequence as probes also indicated that tetraploid D. breviaristatum originated from diploid D. breviaristatum.

The genus of Secale has many agronomically important characters. In order to use the best of this species, markers tracking the rye chromatin incorporated into wheat must be developed. In this study, one rye genome-specific random amplified polymorphic DNA (RAPD) marker was isolated from Secale africanum (Ra genome). Two cloned markers, named OPP131165 and OPP13662, were 1165 bp and 662 bp, respectively. Sequence analysis revealed that OPP131165 was highly homologous to a part of a new class of transposon-like gene called the Revolver family, and OPP13662 was partially similar to LTR gypsy-like retrotransposon. Fluorescence in situ hybridization (FISH) showed only OPP131165 localized within the whole arms of rye except their terminal regions and no signal was detected on wheat chromosomes, while OPP13662 had no hybridization signal detected on wheat and rye genomes. Based on these sequences, two pairs of sequence-characterized amplified region (SCAR) primers were designed, and the resulted SCAR markers were able to target both cultivated and wild Secale species. The FISH patterns and the two SCAR markers should be able to identify and track all wheat-rye translocation lines, especially the S. africanum chromatin.

In order to develop a molecular marker for the detection of alien chromatin, an allele-specific primer targeting pyruvate dehydrogenase (PDHA1) gene was used to analyze 12 taxa representing different basic genomes in Triticeae. Ampli....cation products with different sizes were generated among species. The sequence alignments indicated that the PDHA1 genes contained some deletions/insertions of Miniature Inverted-repeat Terminal Elements (MITE) and simple sequence repeats (SSRs), thus suggesting that the Triticeae genomes have been rapidly evolving during speciation. The genome-specific amplicons and chromosomal location of PDHA1 gene on Triticeae genomes can be used to trace the corresponding alien chromatins from Aegilops, Secale and Dasypyrum species in wheat background.

Knowledge of the chromosomal distribution of long terminal repeats (LTR) is important for understanding plant chromosome structure, genomic organization and evolution, as well as providing chromosomal landmarks that are useful for chromosome engineering. The aim of this study is to investigate the genomic distribution of Sabrina -like LTR pDbH12, which was first isolated from Dasypyrum breviaristatum (V b genome), on Triticeae species in relation to the genomic evolution and chromosome identification. Fluorescence in situ hybridization (FISH) analysis showed that pDbH12 is present on Dasypyrum (V genome) and Hordeum (H genome) species with the hybridized signals covering the entire chromosomes. However, clone pDbH12 did not hybridize to the genomes of Secale, Triticum, Lophopyrum, Pseduoroengeria, Aegilops, Agropyron desertorum and Elymus. Thinopyrum intermedium displayed fourteen chromosomes that hybridized with pDbH12. Sequential FISH identified these chromosomes as belonging to the J s genome. Results ...

Two amphiploids, AF-1(Triticum aestivum L. cv. Anyuepaideng–Secale africanum Stapf.) and BF-1 (T. turgidum ssp. carthlicum–S. africanum), were evaluated by chromosomal banding and in situ hybridization. The individual S. africanum chromosomes were identified in the BF-1 background by sequential C-banding and genomic in situ hybridization (GISH), and were distinguishable from those of S. cereale, because they exhibited less terminal heterochromatin. Fluorescence in situ hybridization (FISH) using the tandem repeat pSc250 as a probe indicated that only 6Ra of S. africanum contained a significant hybrid signal, whereas S. cereale displayed strong hybridization at the telomeres or subtelomeres in all seven pairs of chromosomes. Extensive wheat–S. africanum non-Robertsonian translocations were observed in both AF-1 and BF-1 plants, suggesting a frequent occurrence of chromosomal recombination between wheat and S. africanum. Moreover, introgression lines selected from the progeny of wheat/AF-1 crosses were resistant when field tested with widely virulent strains of Puccinia striiformis f. sp. tritici. Three highly resistant lines were selected. GISH and C-banding revealed that resistant line L9-15 carried a pair of 1BL.1RS translocated chromosomes. This new type of S. africanum derived wheat–Secale translocation line with resistance to Yr9-virulent strains will broaden the genetic diversity of 1BL.1RS for wheat breeding.

Genome in situ hybridization (GISH) analysis of wheat-Secale africanum amphiploid revealed that the S. afri-canum genome displayed significant divergence to the Secale cereale genome. It is thus valuable to deploy genes from S. africanum. We performed the PCR analysis on S. africanum, wheat-S. afticanum amphiploid, T. eastivum cv. Anyuepaideng and other genetic stocks by 100 ISSR primers. A specific segment of 561 bp, named pSaUBC815561, was obtained from S. africanum using primer UBC815. This segment was not amplified from the control wheat lines. Primer UBC815 also am-plified fragments from wild species of genus Secale, including S. vavilovii, S. sylvestre, and other cultivated ryes. Based on the sequence of pSaUBC815561, a pair of special primers U815-F and U815-R was designed and was used to amplify the DNA of wheat related species in Triticeae aimed at validating the specificity of pSaUBC815561. PCR analysis indicated that this specific DNA fragment was amplified not only from a set of Chinese Spring wheat-Imperial rye chromosome addition lines but also from certain wheat-rye introgression lines. Therefore, pSaUBC815561 can be used as a specific marker for detection of chromosomes of Secale genome in wheat.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a globally serious disease adversely affecting wheat production. The Bgt-resistant wheat breeding line CH09W89 was derived after backcrossing a Bgt resistant wheat-Thinopyrum intermedium partial amphiploid TAI7045 with susceptible wheat cultivars. At the seedling stage, CH09W89 exhibited immunity or high resistance to Bgt pathotypes E09, E20, E21, E23, E26, Bg1, and Bg2, similar to its donor line TAI7045 and Th. intermedium. No Th. intermedium chromatin was detected based on genomic in situ hybridization of mitotic chromosomes. To determine the mode of inheritance of the Bgt resistance and the chromosomal location of the resistance gene, CH09W89 was crossed with two susceptible wheat cultivars. The results of the genetic analysis showed that the adult resistance to Bgt E09 in CH09W89 was controlled by a single recessive gene, which was tentatively designated as pmCH89. Two polymorphic SSR markers, Xwmc310 and Xwmc125, were linked to the resistance gene with genetic distances 3.1 and 2.7 cM, respectively. Using the Chinese Spring aneuploid and deletion lines, the resistance gene and its linked markers were assigned to chromosome arm 4BL in the bin 0.68–0.78. Due to its unique position on chromosome 4BL, pmCH89 appears to be a new locus for resistance to powdery mildew. These results will be of benefit for improving powdery mildew resistance in wheat breeding programs.

To better understand molecular evolution of the large α-gliadin gene family and provide a potential value for wheat quality improvement, total 32 α-gliadin gene sequences were isolated from the two Dasypyrum species, D. villosum. (L.) Candargy and D. breviaristatum (Lindb. F.) Frederisksen. Twelve of 32 sequences contained the in-frame stop condons were predicted to be pseudogenes, suggesting the high variation of gliadin genes in Dasypyrum genome. There are five D. breviaristatum α-gliadin sequences present additional cysteine residues. Four peptides which have been identified as T cell stimulatory epitopes in celiac disease (CD) patients through binding to HLA-DQ2/8 were searched to all Dasypyrum α-gliadin gene sequences, and we found that the distribution of the epitopes varied between Dasypyrum genomes. Phylogenetic analysis of the Dasypyrum α-gliadin genes indicated that the sequences from D. breviaristatum displayed higher variation than those from D. villosum, and the genomic differentiation occurred between the two Dasypyrum species. Moreover, the promoter region of the Dasypyrum α-gliadin genes consisted of four different lengths, indicative of the retrotransposons involving the evolution of the gliadin gene promoters. Based on the specific sequences of the Dasypyrum α-gliadin promoter region, we produced sequence-characterized amplified region (SCAR) markers, and localized the Dasypyrum α-gliadin genes on chromosome 6 VS. The SCAR markers can be used to target the introgression of Dasypyrum α-gliadin genes in wheat–Dasypyrum derivatives.

Powdery mildew (PM) is a very destructive disease of wheat (Triticum aestivum L.). Wheat-Thinopyrum ponticum introgression line CH7086 was shown to possess powdery mildew resistance possibly originating from Th. ponticum. Genomic in situ hybridization and molecular characterization of the alien introgression failed to identify alien chromatin. To study the genetics of resistance, CH7086 was crossed with susceptible genotypes. Segregation in F2 populations and F2:3 lines tested with Chinese Bgt race E09 under controlled conditions indicated that CH7086 carries a single dominant gene for powdery mildew resistance. Fourteen SSR and EST-PCR markers linked with the locus were identified. The genetic distances between the locus and the two flanking markers were 1.5 and 3.2 cM, respectively. Based on the locations of the markers by nullisomic-tetrasomic and deletion lines of 'Chinese Spring', the resistance gene was located in deletion bin 2BL-0.89-1.00. Conserved orthologous marker analysis indicated that the genomic region flanking the resistance gene has a high level of collinearity to that of rice chromosome 4 and Brachypodium chromosome 5. Both resistance specificities and tests of allelism suggested the resistance gene in CH7086 was different from previously reported powdery mildew resistance genes on 2BL, and the gene was provisionally designated PmCH86. Molecular analysis of PmCH86 compared with other genes for resistance to Bgt in the 2BL-0.89-1.00 region suggested that PmCH86 may be a new PM resistance gene, and it was therefore designated as Pm51. The closely linked flanking markers could be useful in exploiting this putative wheat-Thinopyrum translocation line for rapid transfer of Pm51 to wheat breeding programs.