Molecular and Cytogenetic Characterization of a Powdery Mildew-Resistant Wheat-Aegilops mutica Partial Amphiploid and Addition Line.

Aegilops mutica Boiss., a diploid species (2n = 2x = 14, TT), has been rarely studied before. In this research, a hexaploid wheat (cv. Chinese Spring)-Ae. mutica partial amphiploid and a wheat-Ae. mutica addition line were characterized by chromosome karyotyping, FISH using oligonucleotides Oligo-pTa535-1, Oligo-pSc119.2-1, and (GAA)8 as probes, and EST-based molecular markers. The results showed that the partial amphiploid strain consisted of 20 pairs of wheat chromosomes and 7 pairs of Ae. mutica chromosomes, with both wheat 7B chromosomes missing. EST-based molecular marker data suggested that the wheat-Ae. mutica addition line carries the 7T chromosome. Resistance tests indicated that both the partial amphiploid and the 7T addition line were highly resistant to powdery mildew, whereas the wheat control line Chinese Spring was highly susceptible, indicating the presence of a potentially new powdery mildew resistance gene on the Ae. mutica 7T chromosome. The karyotype, FISH patterns, and molecular markers can now be used to identify Ae. mutica chromatin in a wheat background, and the 7T addition could be used as a new powdery mildew resistance source for wheat breeding.

A novel wheat-Dasypyrum breviaristatum substitution line with stripe rust resistance.

The introduction of genetic variation from wild and cultivated Triticeae species has been a long-standing approach for wheat improvement. Dasypyrum breviaristatum species harbor novel and agronomically important genes for resistance against multi-fungal diseases. The development of new wheat-D. breviaristatum introgression lines offers chances for the identification of stripe rust resistance gene(s). A wheat line, D11-5, was selected from a cross between wheat line MY11 and wheat-D. breviaristatum partial amphiploid TDH-2. It was characterized by FISH and PCR-based molecular markers. Chromosome counting revealed that the D11-5 line shows a hexaploid set of 2n = 6x = 42 chromosomes. FISH analysis using the Dasypyrum repetitive sequence pDb12H as a probe demonstrated that D11-5 contained a pair of D. breviaristatum chromosomes, while FISH with wheat D-genomic repetitive sequences revealed that the chromosome 2D was absent in D11-5. The functional molecular markers confirmed that the introduced D. breviaristatum chromosomes belong to the homoeologous group 2, indicating that D11-5 was a 2V(b) (2D) disomic substitution line. Field resistance showed that the introduced D. breviaristatum chromosomes 2V(b) were responsible for the stripe rust resistance at the adult plant stage. FISH, C-banding, and PCR-based molecular marker analysis indicated that the chromosome 2V(b) of D. breviaristatum was completely different from the chromosome 2V of D. villosum. The identified wheat-D. breviaristatum chromosome substitution line D11-5 may be applied to produce agronomically desirable stripe rust resistance germplasm.

Optimization of large-scale culture conditions for the production of cordycepin with Cordyceps militaris by liquid static culture.

Cordycepin is one of the most important bioactive compounds produced by species of Cordyceps sensu lato, but it is hard to produce large amounts of this substance in industrial production. In this work, single factor design, Plackett-Burman design, and central composite design were employed to establish the key factors and identify optimal culture conditions which improved cordycepin production. Using these culture conditions, a maximum production of cordycepin was 2008.48 mg/L for 700 mL working volume in the 1000 mL glass jars and total content of cordycepin reached 1405.94 mg/bottle. This method provides an effective way for increasing the cordycepin production at a large scale. The strategies used in this study could have a wide application in other fermentation processes.

Characterization and phylogenetic analysis of α-gliadin gene sequences reveals significant genomic divergence in Triticeae species.

Although the unique properties of wheat α-gliadin gene family are well characterized, little is known about the evolution and genomic divergence of α-gliadin gene family within the Triticeae. We isolated a total of 203 α-gliadin gene sequences from 11 representative diploid and polyploid Triticeae species, and found 108 sequences putatively functional. Our results indicate that α-gliadin genes may have possibly originated from wild Secale species, where the sequences contain the shortest repetitive domains and display minimum variation. A miniature inverted-repeat transposable element insertion is reported for the first time in α-gliadin gene sequence of Thinopyrum intermedium in this study, indicating that the transposable element might have contributed to the diversification of α-gliadin genes family among Triticeae genomes. The phylogenetic analyses revealed that the α-gliadin gene sequences of Dasypyrum, Australopyrum, Lophopyrum, Eremopyrum and Pseudoroengeria species have amplified several times. A search for four typical toxic epitopes for celiac disease within the Triticeae α-gliadin gene sequences showed that the α-gliadins of wild Secale, Australopyrum and Agropyron genomes lack all four epitopes, while other Triticeae species have accumulated these epitopes, suggesting that the evolution of these toxic epitopes sequences occurred during the course of speciation, domestication or polyploidization of Triticeae.

Characterization of wheat: Secale africanum introgression lines reveals evolutionary aspects of chromosome 1R in rye.

Wild Secale species, Secale africanum Stapf., serve as a valuable source for increasing the diversity of cultivated rye (Secale cereale L.) and provide novel genes for wheat improvement. New wheat - S. africanum chromosome 1R(afr) addition, 1R(afr)(1D) substitution, 1BL.1R(afr)S and 1DS.1R(afr)L translocation, and 1R(afr)L monotelocentric addition lines were identified by chromosome banding and in situ hybridization. Disease resistance screening revealed that chromosome 1R(afr)S carries resistance gene(s) to new stripe rust races. Twenty-nine molecular markers were localized on S. africanum chromosome 1R(afr) by the wheat - S. africanum introgression lines. Twenty markers can also identically amplify other reported wheat - S. cereale chromosome 1R derivative lines, indicating that there is high conservation between the wild and cultivated Secale chromosome 1R. Nine markers displayed polymorphic amplification between S. africanum and S. cereale chromosome 1R(afr) derivatives. The comparison of the nucleotide sequences of these polymorphic markers suggested that gene duplication and sequence divergence may have occurred among Secale species during its evolution and domestication.

Diversity and evolution of four dispersed repetitive DNA sequences in the genus Secale.

We present the first characterization of 360 sequences in six species of the genus Secale of both cultivated and wild accessions. These include four distinct kinds of dispersed repetitive DNA sequences named pSc20H, pSc119.1, pSaO5(411), and pSaD15(940) belonging to the Revolver family. During the evolution of the genus Secale from wild to cultivated accessions, the pSaO5(411)-like sequences became shorter mainly because of the deletion of a trinucleotide tandem repeating unit, the pSc20H-like sequences displayed apparent homogenization in cultivated rye, and the second intron of Revolver became longer. In addition, the pSc20H-, pSc119.1-, and pSaO5(411)-like sequences cloned from wild rye and cultivated rye could be divided into two large clades. No single case of the four kinds of repetitive elements has been inherited by each Secale accession from a lone ancestor. It is reasonable to consider the vertical transmission of the four repetitive elements during the evolution of the genus Secale. The pSc20H- and pSaO5(411)-like sequences showed evolutionary elimination at specific chromosomal locations from wild species to cultivated species. These cases imply that different repetitive DNA sequences have played different roles in the chromosome development and genomic evolution of rye. The present study adds important information to the investigations dealing with characterization of dispersed repetitive elements in wild and cultivated rye.

Molecular characterization of a wheat -Thinopyrum ponticum partial amphiploid and its derived substitution line for resistance to stripe rust.

Stripe rust (caused by Puccinia striiformis) occurs annually in most wheat-growing areas of the world. Thinopyrum ponticum has provided novel rust resistance genes to protect wheat from this fungal disease. Wheat - Th. ponticum partial amphiploid line 7430 and a substitution line X005 developed from crosses between wheat and 7430 were resistant to stripe rust isolates from China. Genomic in situ hybridization (GISH) analysis using Pseudoroegneria spicata genomic DNA as a probe demonstrated that the partial amphiploid line 7430 contained ten J(s) and six J genome chromosomes, and line X005 had a pair of J(s)-chromosomes. Giemsa-C banding further revealed that both lines 7430 and X005 were absent of wheat chromosomes 6B. The EST based PCR confirmed that the introduced J(s) chromosomes belonging to linkage group 6, indicating that line X005 was a 6J(s)/6B substitution line. Both resistance observation and sequence characterized amplified region (SCAR) markers displayed that the introduced chromosomes 6J(s) were responsible for the stripe rust resistances. Therefore, lines 7430 and X005 can be used as a donor in wheat breeding for stripe rust resistance.

[Development and application of a genome specific marker for Secale africanum].

Genome in situ hybridization (GISH) analysis of wheat-Secale africanum amphiploid revealed that the S. africanum 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.

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.

[Analysis of St-chromosome-containing triticeae polyploids using specific molecular markers].

In this study, random amplified polymorphic DNA (RAPD) analysis was performed on Pseudoroegneria spicata, Aegilops ventricosa, Secale cereale cv. Jingzhou Rye, Dasypyrum villosum and other 11 triticeae materials using 200 random 10-based primers. A 542 bp specific RAPD fragment (Accession No. DQ992032), named OPH11542, was obtained from Ps. Spicata. Based on the sequence of OPH11542, a pair of sequence characterized amplified region PCR (SCAR-PCR) primers was designed and used to amplify the materials of triticeae. The result demonstrated that 3 specific DNA segments of 542 bp, 742 bp (DQ992033)and 743 bp (EF014218) respectively, were obtained from St chromosome, however, these 3 segments were not appear in materials not contain St chromosome. Sequence similarity analysis revealed that these 3 segments were new repeated DNA sequences. SCAR-PCR was also performed on 15 materials containing St chromosome, and the result showed that OPH11742 or OPH11743 was always found in materials containing StY chromosome, whereas OPH11542 in materials containing StH chromosome. These results indicated that, chromosome recombination or modification often occurred in St chromosome in the course of combination of St and other chromosome to form a polypolid, and OPH11542, OPH11742 and OPH11743 could be used as molecular markers for the detection of St chromosome.

[Cloning and expression of Gymnadenia conopsea GcSec61beta gene encoding endosplasmic reticulum membrane translocation channel protein].

Protein translocation channel in endosplasmic reticulum (ER) of eukaryotes is composed of several subunits of Sec61 complex, which is essential for protein secretion. In the present study, we cloned a full-length cDNA fragment of 621 bp coding 107 amino acids from a psychrophile and endangered plant Gymnadenia conopsea, which grows in high land. Sequence analysis revealed that the gene was highly homologous to the member Sec61beta of ER protein transporter channel, which was thus designated as GcSec61beta. Phylogenetic tree shows that the GcSec61beta was closely related to the corresponding genes from Arabidopsis thaliana and Oryza sativa. Results of semi-quantitative RT-PCR showed that the expression of GcSec61beta was high both in leaves and the bud, and also induced by low temperature treatment. The sequence of the GcSec61beta was introduced into pET28a vector and transformed to E. coli strain BL21. The growth of E. coli was slowed down but the cold resistance was increased by the expression of GcSec61beta, which provides a new function of GcSec61beta protein.

[Sequence variation of chloroplast gene infA-rpl36 region occurred in some Triticeae species].

Based on the sequenced wheat chloroplast genome (cpDNA), a pair of primers in infA-rp136 region was designed and used to amplify DNA from 12 diploid or polyploid Triticeae species. The 12 PCR products were cloned and sequenced. The resulting sequences ranged from 584 to 601 bp. DNA sequence analysis revealed that variation was higher in their intergenic regions than in their coding regions. Among these 12 species, the DNA sequence of coding region of infA gene showed homology as high as 97 percent, indicating that the infA gene is highly conserved among species. However, substantial deletions and insertions were found in 5 out of 12 deduced amino acid sequences, confirming that infA is one of the most evolutionally active cpDNA genes. Whereas the low variation was observed in rp136 gene, implying that the different genes has different evolutionary speed. The constructed phylogenetic trees demonstrate that the polyploidy species Thinopyrum intermedium might have different origin of cytoplasm, and their cytoplasm origins are as complex as their nuclear genome origins.

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.

Studies on genome relationship and species-specific PCR marker for Dasypyrum breviaristatum in Triticeae.

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.

Molecular characterization of high molecular weight glutenin subunit allele 1Bx23 in common wheat introduced from hexaploid triticale.

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.

Molecular cytogenetic characterization and disease resistance observation of wheat-Dasypyrum breviaristatum partial amphiploid and its derivatives.

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 AABBV(b)V(b). GISH analysis on BC(1)F(4) progenies of TDH-2xwheat 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 SCAR(1400) 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.