Zheng-Long Ren

Isolation of a new repetitive DNA sequence from Secale africanum enables targeting of Secale chromatin in wheat background

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 887xa0bp fragment in S. africanum but not in any wheat. The primers also amplified an 887xa0bp 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. Inxa0situ 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.

Diversified chromosomal distribution of tandemly repeated sequences revealed evolutionary trends in Secale (Poaceae).

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.

Identification of α-gliadin genes in Dasypyrum in relation to evolution and breeding

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.xa0villosum. (L.) Candargy and D.xa0breviaristatum (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.xa0breviaristatum 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.xa0breviaristatum displayed higher variation than those from D.xa0villosum, 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 6xa0VS. The SCAR markers can be used to target the introgression of Dasypyrum α-gliadin genes in wheat–Dasypyrum derivatives.

Molecular cytogenetic characterization of wheat– Secale africanum amphiploids and derived introgression lines with stripe rust resistance

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.

Discrimination of Repetitive Sequences Polymorphism in Secale cereale by Genomic In Situ Hybridization-Banding

Genomic in situ hybridization banding (GISH-banding), a technique slightly modified from conventional GISH, was used to probe the Chinese native rye (Secale cereale L.) DNA, and enabled us to visualize the individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat (Triticum aestivum L.) background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S. cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattern of Secale vavilovii was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromatin is discussed.

Characterization of a new T2DS.2DL-?R translocation triticale ZH-1 with multiple resistances to diseases

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. MY15xa0×xa0Chinese 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.

Molecular Characterization of a HMW Glutenin Subunit Allele Providing Evidence for Silencing of x-type Gene on Glu-B1

Understanding the molecular structure of high-molecular-weight glutenin subunit (HMW-GS) may provide useful evidence for the study on the improvement of quality of cultivated wheat and the evolution of Glu-1 alleles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the subunits encoded by Glu-B1 were null, named 1Bxm, in a Triticum turgidum var. dicoccoides line PI94640. Primers based on the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of line PI94640. The PCR products were sequenced, and the total nucleotide sequence of 3,442 bp including upstream sequence of 1,070 bp was obtained. Compared with the reported gene sequences of Glu-1Bx alleles, the promoter region of the Glu-1Bxm showed close resemblance to 1Bx7. The Glu-1Bxm coding region differs from the other Glu-1Bx alleles for a deduced mature protein with only 212 residues, and a stop codon (TAA) at 637 bp downstream from the start codon was present, which was probably responsible for the silencing of x-type subunit genes at the Glu-B1 locus. Phylogenetic tree based on the nucleotide sequence alignment of HMW glutenin subunit genes showed that 1Bxm was the most ancient type of Glu-B1 alleles, suggesting that the evolution rates are different among Glu-1Bx genes. Further study on the contribution of the unique silenced Glu-B1 alleles to quality improvement was also discussed.

Molecular characterization of a novel HMW-GS 1Dx5′ associated with good bread making quality (Triticum aestivum L.) and the study of its unique inheritance

A novel 1Dx type high molecular weight glutenin subunit (HMW-GS) associated with good bread-making quality was identified in the bread wheat line W958. This glutenin subunit was designated as 1Dx5′ here for the same electrophoretic mobility as the traditional one 1Dx5 in the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. In this work, the 5′ flanking region, N-terminal as well as partial central repetitive domain of this allele was cloned and sequenced. Comparison of the amino acid sequence of 1Dx5′ with that of 1Dx5 and 1Dx2 showed the main difference being the substitution of one cysteine residue located in the central repetitive domain of 1Dx5 with serine residue in 1Dx5′ or an additional proline being inserted at the N-terminal of 1Dx5′ compared with 1Dx2. Allelic specific-polymerase chain reaction (AS-PCR) molecular markers discriminating the alleles of Glu-D1 locus were designed and applied to two F2 segregation populations. Meanwhile, the dough properties of the F2 population were measured and analyzed, showing that the 1Dx5′ subunit is as good as 1Dx5 and superior than 1Dx2 in terms of dough property. The result illustrated that the inter-chain hydrogen bonds formed between the subunit repetitive domains rather than the cysteine may play the main role in bread making quality. Notably, the novel 1Dx5′ have a great portion in the F2 segregation population, suggesting the phenomenon of segregation distortion which could facilitate the introduction of 1Dx5’ into the wheat breeding program.

Eukaryotic translation initiation factor 5A of wheat: identification, phylogeny and expression.

In the present study, we report on the characterization of a full-length cDNA clone (TaeIF5A-1) and as well as two genomic sequences (TaeIF5A-2 and TaeIF5A-3) encoding eIF5A in wheat (Triticum aestivum). In addition, 9 partial DNA sequences of eIF5A gene were also isolated from different species of triticeae tribe. Phylogenetic analysis of eIF5A coding genes in general revealed that TaeIF5A-1 represents ancestral types of the gene family in plant. Chromosome location analysis shows that TaeIF5A-2 was located on the chromosome 2BL. The mRNA level of TaeIF5A-1 has spatial and temporal difference, indicating it mainly plays important role in leaf development.