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Molecular markers and phenotyping have been widely used to evaluate wheat germplasm diversity. However, the feasibility of using chromosome fluorescence in situ hybridization (FISH) to evaluate wheat genetic diversity has not been well investigated. In this study, seventy-six representative Chinese wheat lines in main wheat production area were selected and investigated with multicolour FISH using Oligo-pTa535, Oligo-pSc119.2 and Oligo-(GAA)8 probes. The results indicated that wheat chromosomes can be clearly recognized by FISH. For wheat A, B and D genomes, the number of FISH types ranged from 2 to 7, 2 to 6 and 1 to 5, respectively. The average number of FISH types in the A and B genomes was higher than that in the genome D. The rye-derived 1RS chromosome in wheat background could also be clearly detected by these probes. The frequency of 1RS in Chinese wheat lines investigated was 48.7%, and most (94.6%) of them belonged to 1BL.1RS. The genetic relationships among the seventy-six Chinese wheat lines subjected to FISH were divided into three clusters, e.g., CL1, CL2 and CL3. Those wheat lines derived from Shandong and Henan Provinces were mainly located in clusters CL1 and CL3, respectively, which may suggest that the FISH type is associated with the adaptation of wheat. These results also indicated that multicolour FISH using a combination of three different oligo-probes generates sufficiently diverse hybridization patterns among wheat lines to evaluate the genetic diversity of wheat.

A physical map of Thinopyrum intermedium chromosome 7J was constructed using translocation mapping, and a new seedling purple coleoptile gene was mapped to the bin of FL 0.35–0.63 of 7JS. Thinopyrum intermedium (2n = 6x = 42, JJJsJsStSt), a wild relative of common wheat, harbors numerous beneficial genes for wheat improvement. Previous studies showed that wheat–Th. intermedium partial amphiploid TAF46 and its derived addition line L1 had a purple coleoptile, which was derived from Th. intermedium chromosome 7J. To identify and physically map the purple coleoptile gene, 12 wheat–Th. intermedium 7J translocation lines were analyzed by sequential multicolor fluorescence in situ hybridization (mc-FISH), PCR-based landmark unique gene (PLUG) and intron targeting (IT) markers. A physical map of the 7J chromosome was constructed, consisting of eight chromosomal bins with 89 markers. Seedling evaluation of the coleoptile colors of all tested materials indicated that the purple coleoptile gene was located to the bin with a fraction length (FL) of 0.35–0.63 on chromosome 7JS. Furthermore, based on the syntenic relationships between Th. intermedium and wheat chromosomes, we developed a new chromosome 7J-specific EST-PCR marker from the chromosomal region corresponding to the purple coleoptile gene through the Triticeae multi-omics database. The approach of designing chromosome-specific markers has facilitated fine mapping of the Thinopyrum-specific purple coleoptile gene, and these translocation lines will be valuable for studying the function of the purple coleoptile gene in anthocyanin biosynthesis.

Background There are no current national estimates of the candidaemia burden in China, and epidemiological candidaemia data from the underdeveloped region of China are lacking. Methods A 7-year retrospective study was carried out to analyse the prevalence, species distribution, antifungal susceptibility, risk factors and inpatient mortality of candidaemia among paediatric and adult patients in a regional tertiary teaching hospital in China. Results During the seven-year study period, a total of 201 inpatients with candidaemia were identified. The median age of the patients was 65 years (range, 1 day to 92 years), and 114 of the patients (56.7%) were male. The mean annual incidence of candidaemia was 0.26 cases per 1000 admissions (0.42 cases per 1000 paediatric admissions vs 0.24 cases per 1000 adult admissions, P Candida albicans was the most common fungal species (81/201, 40.3%) in all patients, Candida glabrata was the most common fungal species (18/35, 51.4%) in paediatric patients. Most isolates were susceptible to flucytosine (99.0%) and amphotericin B (99.0%), and the activity of antifungal agents against Candida species was no significant difference in satisfaction between paediatric and adult patients (P > 0.05). The all-cause mortality rate was 20.4% (paediatric patients: 11.4% vs adult patients:22.3%, P > 0.05). Fewer univariate predictors of poor outcomes were identified for paediatric patients than for adult patients (4 vs 11 predictors). Respiratory dysfunction and septic shock were independent predictors of 30-day mortality for all patients. Conclusions The epidemiological data of candidaemia in paediatric and adult patients are only different in the distributions of Candida species and the mean annual incidence of candidaemia. Flucytosine and amphotericin B can be used as first-choice agents when no antifungal susceptibility test results are available.

Background Introgression of chromatin from Secale species into common wheat has for decades been a successful strategy for controlling the wheat diseases. The wild Secale species, Secale africanum Stapf., is a valuable source for resistance to foliar disease of wheat. A wheat-S. africanum chromosome 6Rafr substitution line displayed resistance to both powdery mildew and stripe rust at the adult-plant stage. Results Wheat-S. africanum chromosome 6Rafr deletion and translocation lines were produced and identified by sequential non-denaturing fluorescence in situ hybridization (ND-FISH) using multiple Oligo-based probes. Different ND-FISH patterns were observed between S. cereale 6R and S. africanum 6Rafr. With reference to the physical map of the draft genome sequence of rye inbred line Lo7, a comprehensive PCR marker analysis indicated that insertions and deletions had occurred by random exchange between chromosomes 6R and 6Rafr. A survey of the wheat- S. africanum 6Rafr lines for disease resistance indicated that a powdery mildew resistance gene(s) was present on the long arm of 6Rafr at FL0.85–1.00, and that a stripe rust resistance gene(s) was located in the terminal region of 6RafrS at FL0.95–1.00. The wheat-S. africanum 6Rafr introgression lines also displayed superior agronomic traits, indicating that the chromosome 6Rafr may have little linkage drag in the wheat background. Conclusions The combination of molecular and cytogenetic methods allowed to precisely identify the chromosome rearrangements in wheat- S. africanum 6Rafr substitution, deletion and translocation lines, and compare the structural difference between chromosomes 6R and 6Rafr. The wheat- S. africanum 6Rafr lines containing gene(s) for powdery mildew and stripe rust resistance could be used as novel germplasm for wheat breeding by chromosome engineering.

A general distribution of tandem repeats (TRs) in the wheat genome was predicted and a new web page combined with fluorescence in situ hybridization experiments, and the newly developed Oligo probes will improve the resolution for wheat chromosome identification. Comprehensive sequence analysis of tandem repeats (TR) in the wheat reference genome permits discovery and application of TRs for chromosome identification. Genome-wide localization of TRs was identified in the reference sequences of Chinese Spring using Tandem Repeat Finder (TRF). A database of repeats unit size, array number, and physical coverage length of TRs in the wheat genome was built. The distribution of TRs occupied 3–5% of the wheat chromosomes, with non-random dispersal across the A, B, and D genomes. Three classes of TRs surrounding the predicted genes were compared. An optimized computer-assisted website page B2DSC was constructed for the general distribution and chromosomally enriched zones of TR sequences to be displayed graphically. The physical distribution of predicted TRs in the wheat genome by B2DSC matched well with the corresponding hybridization signals obtained with fluorescence in situ hybridization (FISH). We developed 20 oligonucleotide probes representing 20–60 bp lengths of high copy number of TRs and verified by FISH. An integrated physical map of TR-Oligo probes for wheat chromosome identification was constructed. Our results suggest that the combination of both molecular cytogenetics and genomic research will significantly benefit wheat breeding through chromosome manipulation and engineering.

New molecular markers were developed for targeting Thinopyrum intermedium 1St#2 chromosome, and novel FISH probe representing the terminal repeats was produced for identification of Thinopyrum chromosomes. Thinopyrum intermedium has been used as a valuable resource for improving the disease resistance and yield potential of wheat. A wheat-Th. intermedium ssp. trichophorum chromosome 1St#2 substitution and translocation has displayed superior grain protein and wet gluten content. With the aim to develop a number of chromosome 1St#2 specific molecular and cytogenetic markers, a high throughput, low-cost specific-locus amplified fragment sequencing (SLAF-seq) technology was used to compare the sequences between a wheat-Thinopyrum 1St#2 (1D) substitution and the related species Pseudoroegneria spicata (St genome, 2n = 14). A total of 5142 polymorphic fragments were analyzed and 359 different SLAF markers for 1St#2 were predicted. Thirty-seven specific molecular markers were validated by PCR from 50 randomly selected SLAFs. Meanwhile, the distribution of transposable elements (TEs) at the family level between wheat and St genomes was compared using the SLAFs. A new oligo-nucleotide probe named Oligo-pSt122 from high SLAF reads was produced for fluorescence in situ hybridization (FISH), and was observed to hybridize to the terminal region of 1St#L and also onto the terminal heterochromatic region of Th. intermedium genomes. The genome-wide markers and repetitive based probe Oligo-pSt122 will be valuable for identifying Thinopyrum chromosome segments in wheat backgrounds.

Species containing E genome of Thinopyrum offered potential to increase the genetic variability and desirable characters for wheat improvement. However, E genome specific marker was rare. The objective of the present report was to develop and identify sequenced characterized amplified region (SCAR) markers that can be used in detecting E chromosome in wheat background for breeding purpose. Total 280 random amplified polymorphic DNA (RAPD) primers were amplified for seeking of E genome specific fragments by using the genomic DNA of Thinopyrum elongatum and wheat controls as templates. As a result, six RAPD fragments specific for E genome were found and cloned, and then were converted to SCAR markers. The usability of these markers was validated using a number of E- genome-containing species and wheat as controls. These markers were subsequently located on E chromosomes using specific PCR and fluorescence in situ hybridization (FISH). SCAR markers developed in this research could be used in molecular marker assisted selection of wheat breeding with Thinopyrum chromatin introgressions.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, was one of the most disaster foliar diseases for wheat-growing areas of the world. Thinopyrum intermedium has provided novel resistance genes to multi-fungal disease, and new wheat-Th. intermedium derivatives for stripe rust resistance still need to develop for wheat breeding. Wheat line X484-3 was selected from a cross between wheat line MY11 and wheat-Th. intermedium ssp. trichophorum partial amphiploid TE-1508, and was characterized by genomic in situ hybridization (GISH) and functional molecular markers. Chromosome counting revealed that the X484-3 was 2n = 44 and GISH analysis using Pseudoroegneria spicata genomic DNAas a probe demonstrated that X484-3 contained a pair of St-chromosomes from Th. intermedium donor parents. The functional molecular markers confirmed that introduced St-chromosomes belonging to linkage group 7, indicating that line X484-3 was a 7St addition line. The resistance observation displayed that the introduced Th. intermedium ssp. trichophorum derived chromosomes 7St were responsible for the stripe rust resistances at adult plant. The identified wheat-Th. intermedium chromosome 7St addition line X484-3 can be used as a donor in wheat breeding for stripe rust resistance.

The wild species, Secale africanum Stapf., serves as a valuable germplasm resource for increasing the diversity of cultivated rye (Secale cereale L.) and providing novel genes for wheat improvement. The objective of this study was to identify new wheat-S. africanum chromosome 2Rafr derivatives by molecular markers and in situ hybridization, and to test the effects of centric translocations of S. africanum chromosome 2Rafr on disease resistance and agronomic performance of wheat. The T2RafrS.2DL and T2DS.2RafrL translocations, and other S. africanum chromosome introgressions such as a 2Rafr substitution as well as monotelo-2RafrS and 2RafrL addition lines, were identified by the genomic in situ hybridization and sequential fluorescence in situ hybridization (FISH). Twenty-three molecular markers were localized on chromosome 2Rafr arms which will facilitate future identification of their introgressions into wheat. A comparison of FISH patterns and the molecular marker distribution between the S. africanum chromosome 2Rafr and cultivated rye chromosome 2R indicates differentiation between the wild and cultivated Secale genomes. Tests of disease resistance and agronomic performance of wheat-S. africanum chromosome 2Rafr derivatives indicated that chromosome 2RafrL carried gene(s) for dwarfing and stripe rust resistance, while the 2RafrS translocation line was the most favorable for agronomic performance when compared with the substitution and 2RafrL translocation lines. These results encourage the continued use of novel wheat-S. africanum 2Rafr derivative lines in wheat improvement.

Thinopyrum intermedium has been hybridized extensively with wheat (Triticum aestivum L.) and several genes for disease resistance have been introgressed to cultivated wheat. However, there are very few reports about the Th. intermedium-derived seed storage protein genes which have been transferred into a wheat background by chromosome manipulation. Our aim is to identify several wheat–Th. intermedium ssp. trichophorum derivatives, and document these lines by genomic in situ hybridization (GISH), molecular markers and seed storage protein analysis. We found that a novel Th. intermedium 1St#2 chromosome-specific high-molecular-weight glutenin subunit (HMW-GS) was transferred to the wheat–Thinopyrum derivative lines. The genomic sequence of the Thinopyrum-derived HMW-GS was characterized and designated Glu-1St#2x, since it resembled x-type glutenins in both the N-terminal domain and C-terminal domain. It is much shorter than that of reported HMW-GS genes. The Glu-1St#2x sequence was successfully expressed in Escherichia coli and resulted in the identical weight to the native protein. The GISH and newly developed chromosome Thinopyrum-specific DNA markers enabled physically location of Glu-1St#2x to the region FL0.60–1.00 on Th. intermedium 1St#2L chromosome arm. Phylogenetic analysis revealed that the Glu-1St#2x evolved earlier than other x-type HMW-GS homoeologues in modern wheat genomes. The effect of Glu-1St#2x on protein content, sodium dodecyl sulphate sedimentation value and improvement of solvent retention capacity in wheat background suggested that Th. intermedium chromosome 1St#2 may have potential for improvement of wheat end-product quality.

A synthetic autopolyploid was developed from diploid Aegilops tauschii, D genome progenitor of common wheat (Triticum aestivum). The tetraploid Ae. tauschii displayed a markedly larger organ size than the diploid donor. Fluorescence in situ hybridization (FISH) and DNA marker analysis revealed that there is no clear variation at either the chromosomal or DNA level between the diploid and tetraploid plants. We analyzed the variation in cytosine methylation patterns between the diploid and tetraploid plants by methylation-sensitive amplified polymorphism (MSAP) and detected 228 and 232 methylated sites in diploid and tetraploid plants, respectively. Statistical comparison indicated that the tetraploid Ae. tauschii genotype displayed no significant difference in polymorphic methylation level compared to the diploid ones. Twenty-two different genomic fragments displaying different methylation behavior during the ploidy conversions were isolated and sequenced. It demonstrated that alterations in the level of methylation have the most profound effects on coding genes. We demonstrated that there are some genes expressions modified by DNA methylation may be correlated with phenotypic alteration after autotetraploidization.

Background The uncultivated species Dasypyrum breviaristatum carries novel diseases resistance and agronomically important genes of potential use for wheat improvement. The development of new wheat-D. breviaristatum derivatives lines with disease resistance provides an opportunity for the identification and localization of resistance genes on specific Dasypyrum chromosomes. The comparison of wheat-D. breviaristatum derivatives to the wheat-D. villosum derivatives enables to reveal the genomic divergence between D. breviaristatum and D. villosum. Results The mitotic metaphase of the wheat- D. breviaristatum partial amphiploid TDH-2 and durum wheat -D. villosum amphiploid TDV-1 were studied using multicolor fluorescent in situ hybridization (FISH). We found that the distribution of FISH signals of telomeric, subtelomeric and centromeric regions on the D. breviaristatum chromosomes was different from those of D. villosum chromosomes by the probes of Oligo-pSc119.2, Oligo-pTa535, Oligo-(GAA)7 and Oligo-pHv62-1. A wheat line D2139, selected from a cross between wheat lines MY11 and TDH-2, was characterized by FISH and PCR-based molecular markers. FISH analysis demonstrated that D2139 contained 44 chromosomes including a pair of D. breviaristatum chromosomes which had originated from the partial amphiploid TDH-2. Molecular markers confirmed that the introduced D. breviaristatum chromosomes belonged to homoeologous group 7, indicating that D2139 was a 7Vb disomic addition line. The D2139 displayed high resistance to wheat stripe rust races at adult stage plant, which may be inherited from, D. breviaristatum chromosome 7Vb. Conclusion The study present here revealed that the large divergence between D. breviaristatum and D. villosum with respected to the organization of different repetitive sequences. The identified wheat- D. breviaristatum chromosome addition line D2139 will be used to produce agronomically desirable germplasm for wheat breeding.

Thinopyrum elongatum serves as an excellent gene pool for wheat improvement. Genes for resistance to many biotic and abiotic stresses have been transferred from Th. elongatum to wheat through chromosome manipulation. For breeding programs, molecular markers enable screening of a large number of genotypes for alien chromosome introgressions. The main objective of the present study was to develop and characterize EST (expressed sequence tags) and PLUG (PCR-based Landmark Unique Gene) markers that can distinguish Th. elongatum chromatin from the wheat genomes. A total of 258 mapped EST primer pairs and 46 PLUG primer pairs were tested on DNA from wheat Chinese Spring (CS) and CS-Th. elongatum addition lines. The results showed that 43 primer pairs could be effectively mapped to specific Th. elongatum chromosomes. Twenty-two of the 43 markers displayed similar homoeologous chromosome locations to hexaploid wheat. Nine markers mapped to different linkage groups between wheat and Th. elongatum, while 12 makers mapped on two or three different Th. elongatum chromosomes. A comparison of molecular marker locations indicated that Th. elongatum genome was closely related to the D genome of wheat, and chromosome rearrangements and duplication had occurred in Th. elongatum and the wheat genomes. The markers will be useful in comparative gene mapping, chromosome evolutionary analysis, and gene introgression for wheat improvement using Th. elongatum accessions as gene donors.

A species-specific DNA sequence (marker) that can detect the presence of Secale cereale chromatin in common wheat background was developed by using wheat microsatellite primer Xgwm614. One rye-specific DNA amplified fragment of 416bp (pSa614416) was obtained from Secale africanum and a wheat - S. africanum amphiploid. The primer Xgwm614 also gave rise to specific bands in all Chinese Spring-Imperial rye addition lines 1R to 7R. Sequence analysis revealed that pSa614416 was strongly homologous to a miniature inverted transposable element (MITE) stowaway-like element. Results of fluorescence in situ hybridization showed that the signal of pSa614416 was distributed along all S. cereale. cv Jingzhou chromosomes, but the signal strengths were unbalanced on the seven rye genome chromosomes. This repetitive element may be useful as a molecular marker for the introgression of rye germplasm into the wheat genome.

In the present study, we report on a new triticale (×Triticosecale Wittm.) ZH-1, derived from the progeny of common wheat (Triticum aestivum L.) cv. MY15 × Chinese Weining rye (Secale cereale L.). ZH-1, exhibiting shorter plant height and higher tillering ability compared to MY15, is immune to both powdery mildew and stripe rust and has stable fertility. Genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and C-banding revealed that the chromosome composition of triticale ZH-1 was 14 A-genome (1A-7A), 12 B-genome (1B-2B, 4B-7B), 12 R-genome (1R, 3R-7R), chromosomes 6D and T2DS.2DL-?R. Moreover, the PCR results of PLUG and EST-SSR markers also strongly supported the above stated chromosome composition of triticale ZH-1. In addition, the physical mapping of chromosome T2DS.2DL-?R showed a minute chromosomal fragment derived from rye was attached at the distal end of 2DL. The new triticale ZH-1 could be a valuable source for wheat improvement, especially for resistance to disease.

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.

A new wheat-Thinopyrum substitution line AS1677, developed from a cross between wheat line ML-13 and wheat-Thinopyrum intermedium ssp. trichophorum partial amphiploid TE-3, was characterized by fluorescence in situ hybridization (FISH), sequential Giemsa-C banding, genomic in situ hybridization (GISH), seed storage protein electrophoresis, molecular marker analysis and disease resistance screening. Sequential Giemsa-C banding and GISH using Pseudoroegneria spicata genomic DNA as probe indicated that a pair of St-chromosomes with strong terminal bands were introduced into AS1677. FISH using pTa71 as a probe gave strong hybridization signals at the nuclear organization region and in the distal region of the short arms of the St chromosome. Moreover, FISH using the repetitive sequence pAs1 revealed that a pair of wheat 1D chromosomes was absent in accession AS1677. Seed storage proteins separated by acid polyacrylamide gel electrophoresis (APAGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that AS1677 lacked the gliadin and glutenin bands encoded by Gli-D1 and Glu-D1, further confirming the absence of chromosome 1D. The introduced St chromosome pair belonging to homoeologous group 1 was identified by newly produced genome specific markers. AS1677 is a new 1St (1D) substitution line. When inoculated with stripe rust and powdery mildew isolates, AS1677 expressed stripe rust resistance possibly derived from its Thinopyrum parent. AS1677 can be used as a donor source for introducing novel disease resistance genes to wheat in breeding programs aided by molecular and cytogenetic markers.

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.

Genomic in situ hybridization (GISH) with Secale cereale cv. ‘Jingzhou rye’ DNA as a probe to chromosomes of hexaploid triticale line Fenzhi-1 revealed that not only were all chromosomes of rye strongly hybridized along the entire chromosome length, but there were also stronger signals in terminal or subtelomeric regions. This pattern of hybridization signals is referred to as GISH banding. After GISH banding, sequential fluorescene in situ hybridizaion (FISH) with tandem repeated sequence pSc200 and pSc250 as probes showed that the chromosomal distribution of pSc200 is highly coincident with the GISH banding pattern, suggesting that GISH banding revealed chromosomal distribution of pSc200 in rye. In addition, FISH using pSc200 and pSc250 as probes to chromosomes of 11 species of the genus Secale and two artificial amphiploids (Triticum aestivum-S. strictum subsp. africanum amphiploid and Aegilops tauschii-S. silvestre amphiploid) showed that (1) the chromosomal distribution of pSc200 and pSc250 differed greatly in Secale species, and the trend towards an increase in pSc200 and pSc250 binding sites from wild species to cultivated rye suggested that pSc200 and pSc250 sequences gradually accumulated during Secale evolution; (2) the chromosomal distribution of pSc200 and pSc250 presented polymorphism on homologous chromosomes, suggesting that the same species has two heterogeneous homologous chromosomes; (3) the intensity and number of hybridization signals varied differently on chromosomes between pSc200 and pSc250, suggesting that each repetitive family evolved independently.

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.

During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH (genome in situ hybridization) and AFLP (amplified fragment length polymorphism) on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism (MSAP) analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines (CN12, 20.15%; CN17, 20.91%; CN18, 22.42%), but the ratios of hemimethylated sites were significantly lowered (CN12, 21.41%; CN17, 23.43%; CN18, 22.42%), whereas 16.37% were fully-methylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns (33.74%), 9 demethylation patterns (22.76%) and 7 uncertain patterns (4.07%). In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.

Rye (Secale cereale) plays an important role in wheat improvement. Here we report a new triticale, named Fenzhi-1, derived from the wide cross MY11 (Triticum aestivum) × Jingzhou (Secale cereale) after the in vitro rye pollen has been irradiated by He-Ne laser. Morphologically, Fenzhi-1 is characterized by branched-spikes. Genetically, Fenzhi-1 displays stable fertility and immunity to wheat powdery mildew and stripe rust. In situ hybridization (FISH) and seed storage protein electrophoresis revealed that Fenzhi-1 is a new primary hexaploid triticale (AABBRR). The present study not only provides a new method to synthesize an artificial species, but also shows that Fenzhi-1 could be a valuable source for wheat improvement.

A genome specific DNA sequence that detects Secale africanum chromatin incorporated into wheat was developed in this study. Random amplified polymorphic DNA (RAPD) analysis was used to search for genome specific DNA sequences of S. africanum in lines, R111, “mianyang11” (MY11) and wheat-rye 1RS/1BL translocations R25 and R57. A high copy rye-specific DNA segment pSaD15940 of the S. africanum genome was obtained. The sequence of pSaD15 did not show any significant homology to other reported sequences in databases and it is therefore a new repetitive sequence of Secale. PCR primers were designed for pSaD15940, which amplify a clear 887 bp fragment in S. africanum but not in any wheat. The primers also amplified an 887 bp fragment in other accessions of rye, Chinese Spring-Imperial rye chromosome additions and a diverse range of material carrying different rye chromosomes or chromosomal segments. In situ hybridization showed that probe pSaD15940 was specifically hybridized throughout all rye chromosomes arms except for the terminal regions. The advantage of the rye-specific probe developed herein compared to those of previous reports is that it has been shown to be widely applicable to other Secale species. The probe will be useful as a molecular marker for the introgression of S. africanum and other rye chromosome segments into the wheat genome.

Two wheat–Thinopyrum substitution lines X479 and X482 selected from the progenies of wheat “Mianyang26 (MY26)” × wheat–Thinopyrum intermedium ssp. trichophorum partial amphiploid were characterized by seed storage protein electrophoresis, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), and PCR-based molecular markers. Seed storage protein analysis showed that X479 expressed some of Th. intermedium ssp. trichophorum-specific gliadin and glutenin bands. Chromosome counting and GISH probed by Pseudoroegneria spicata genomic DNA indicated that two pairs of Thinopyrum-derived chromosomes (St genome and St–JS translocated chromosomes) substituted for two pairs of wheat chromosomes in both X479 and X482. FISH using pAs1 and pHvG38 as probes showed that chromosomes 1B and 4B, and 4D and 6D were absent in X479 and X482, respectively. Using the newly isolated JS chromosome-specific repetitive sequence pDb12H as a probe, the FISH signals revealed that the translocation of St–JS chromosomes in X479 and X482 occurred in repetitive sequence regions of the short arm. The molecular markers based on wheat–rice colinearity confirmed that the chromosome constitutions of X479 and X482 were 1St (1B) + 4St–4JS (4B) and 4St–JS (4D) + 6St (6D), respectively. The substitution lines were both fully fertile which suggests that the Th. intermedium chromosomes in X479 and X482 substitute well for the corresponding wheat chromosomes. The rust resistance and novel agronomic traits revealed that the substitution lines will be potentially useful for genetic improvement of wheat.

High molecular weight glutenin subunit (HMW-GS) 1Bx23, an x-type subset encoded by Glu-B1p, which is only distributed in Triticum turgidum, was successfully transferred from hexaploid triticale to common wheat line SY95-71. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that subunit 1Bx23 has a faster mobility than subunit 1Bx7 and 1Bx20, but slower than 1Bx17. Primers designed from the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of SY95-71. Total nucleotide sequences of 3426 bp including an open reading frame of 2385 bp and upstream sequence of 1038 bp were obtained. Compared with the reported gene sequences of Glu-B1-1 alleles, including 1Bx7, 1Bx14, 1Bx20 and 1Bx17, the promoter region of the 1Bx23 was displayed close to 1Bx7 and 1Bx17. The deduced amino acid sequence of coding region of 1Bx23 exhibited 34, 30, 20 and 22 amino acid substitutions from that of 1Bx14, 1Bx20, 1Bx7 and 1Bx17, respectively. A phylogenetic tree based on the nucleotide sequence alignment of the Glu-1Bx alleles shows that the 1Bx23 are apparently clustered with 1Bx7 and 1Bx17, and more ancient than 1Bx14 and 1Bx20, suggesting that the evolution speeds are different among Glu-1Bx genes. Additionally, the potential use of wheat line SY95-71 to further screen the quality contribution of unique subunit 1Bx23 is also discussed.

Dasypyrum breviaristatum and nine related species in Triticeae were analyzed using the random amplified polymorphic DNA (RAPD) technique, in order to understand the genetic relationship and to develop species specific markers. The genome relationship dendrogram shows that D. breviaristatum and D. villosum could not be grouped together, indicating that D. breviaristatum was unlikely to be directly derived from D. villosum, while D. breviaristatum was closest to Thinopyrum intermedium, which implied that they might have similar breeding behaviors when introducing their chromatins into wheat. A D. breviaristatum genome specific RAPD product of 1182bp, was cloned and designated as pDb12H. Sequence analysis revealed that pDb12H was strongly homologuos to a long terminal repeat (LTR) Sabrina retrotransposon newly reported in Hordeum. The pDb12H was converted into a PCR based marker, which allows effectively monitoring the D. breviaristatum chromatin introgression into wheat. Fluorescence in situ hybridization (FISH) suggested that pDb12H was specifically hybridized throughout all D. breviaristatum chromosomes arms except for the terminal and centromeric regions, which can be used to characterize wheat –D. breviaristatum chromosome translocation. The genomes repetitive element will also be useful to study gene interactions between the wheat and alien genomes after the polyploidization.

A wheat-Dasypyrum breviaristatum partial amphiploid and its derivatives were analyzed by molecular cytological observation and tested for disease resistance in order to evaluate the potential use of the D. breviaristatum for wheat improvement. A fertility-improved partial amphiploid, TDH-2, was produced from the selfing population of Triticum aestivum cv. Chinese spring (CS)-D. breviaristatum amphiploid. Based on the results obtained from genomic in situ hybridization (GISH) and seed protein electrophoresis, we found the presence of fourteen D. breviaristatum chromosomes and the absence of D genome in TDH-2, indicating that the genomic composition of TDH-2 was AABBVbVb. GISH analysis on BC1F4 progenies of TDH-2×wheat demonstrated that alien D. breviaristatum chromosomes or segments were frequently transmitted. A survey of diseases resistance revealed that powdery mildew resistance from D. breviaristatum was totally expressed, however, the expression of stripe rust resistance from D. breviaristatum was dependent on the wheat background. The comparison of polymerase chain reaction (PCR), which was carried out using molecular marker SCAR1400 linked to Pm21 D. villosum-derived powdery mildew resistance gene, suggested that D. breviaristatum possessed new resistance gene(s) different from that in D. villosum. The present study showed that the partial amphiploid TDH-2 and its derivatives could serve as novel sources for transfer of disease resistance genes to wheat.

In order to solve the problem of mining biomedical knowledge, a biomedical semantic-based knowledge discovery method (Bio-SKDM) is proposed. Using the semantic types and semantic relations of the biomedical concepts, Bio-SKDM can identify the relevant concepts collected from Medline and generate the novel hypothesis between these concepts. The experiment result shows that compared with ARROWSMITH and LITLINKER, Bio-SKDM generates less but more relevant novel hypotheses and requires less human intervention in the discovery procedure.

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.