Spontaneous chromosome instability and tissue culture-induced karyotypic alteration in wheat-Thinopyrum intermedium alien addition lines.

MAIN CONCLUSION: Wheat lines harboring wild-relative chromosomes can be karyotypically unstable during long-term maintenance. Tissue culture exacerbates chromosomal instability but appears inefficient to induce somatic homoeologous exchange between alien and wheat chromosomes. We assessed if long-term refrigerator storage with regular renewal via self-fertilization, a widely used practice for crop germplasm maintenance, would ensure genetic fidelity of alien addition lines, and explored the possibility of inducing somatic homoeologues exchange by tissue culture. We cytogenetically characterized sampled stock seeds of originally confirmed 12 distinct wheat-Thinopyrum intermedium alien addition lines (dubbed TAI lines), and subjected immature embryos of the TAI lines to tissue culture. We find eight of the 12 TAI lines were karyotypically departed from their original identity as bona fide disomic alien addition lines due to extensive loss of whole-chromosomes of both Th. intermedium and wheat origins during the ca. 3-decade storage. Rampant numerical chromosome variations (NCVs) involving both alien and wheat chromosomes were detected in regenerated plants of all 12 studied TAI lines, but at variable rates among the wheat sub-genomes and chromosomes. Compared with NCVs, structural chromosome variations (SCVs) occurred at substantially lower rates, and no SCV involving the added alien chromosomes was observed. The NCVs manifested only moderate effects on phenotypes of the regenerated plants under field conditions.

Overexpression of a Cinnamyl Alcohol Dehydrogenase-Coding Gene, GsCAD1, from Wild Soybean Enhances Resistance to Soybean Mosaic Virus.

Soybean mosaic virus (SMV) is the most prevalent soybean viral disease in the world. As a critical enzyme in the secondary metabolism of plants, especially in lignin synthesis, cinnamyl alcohol dehydrogenase (CAD) is widely involved in plant growth and development, and in defense against pathogen infestation. Here, we performed RNAseq-based transcriptome analyses of a highly SMV-resistant accession (BYO-15) of wild soybean (Glycine soja) and a SMV-susceptible soybean cultivar (Williams 82), also sequenced together with a resistant plant and a susceptible plant of their hybrid descendants at the F3 generation at 7 and 14 days post-inoculation with SMV. We found that the expression of GsCAD1 (from G. soja) was significantly up-regulated in the wild soybean and the resistant F3 plant, while the GmCAD1 from the cultivated soybean (G. max) did not show a significant and persistent induction in the soybean cultivar and the susceptible F3 plant, suggesting that GsCAD1 might play an important role in SMV resistance. We cloned GsCAD1 and overexpressed it in the SMV-susceptible cultivar Williams 82, and we found that two independent GsCAD1-overexpression (OE) lines showed significantly enhanced SMV resistance compared with the non-transformed wild-type (WT) control. Intriguingly, the lignin contents in both OE lines were higher than the WT control. Further liquid chromatography (HPLC) analysis showed that the contents of salicylic acid (SA) were significantly more improved in the OE lines than that of the wild-type (WT), coinciding with the up-regulated expression of an SA marker gene. Finally, we observed that GsCAD1-overexpression affected the accumulation of SMV in leaves. Collectively, our results suggest that GsCAD1 enhances resistance to SMV in soybeans, most likely by affecting the contents of lignin and SA.

Mutation of GmAITR Genes by CRISPR/Cas9 Genome Editing Results in Enhanced Salinity Stress Tolerance in Soybean.

Breeding of stress-tolerant plants is able to improve crop yield under stress conditions, whereas CRISPR/Cas9 genome editing has been shown to be an efficient way for molecular breeding to improve agronomic traits including stress tolerance in crops. However, genes can be targeted for genome editing to enhance crop abiotic stress tolerance remained largely unidentified. We have previously identified abscisic acid (ABA)-induced transcription repressors (AITRs) as a novel family of transcription factors that are involved in the regulation of ABA signaling, and we found that knockout of the entire family of AITR genes in Arabidopsis enhanced drought and salinity tolerance without fitness costs. Considering that AITRs are conserved in angiosperms, AITRs in crops may be targeted for genome editing to improve abiotic stress tolerance. We report here that mutation of GmAITR genes by CRISPR/Cas9 genome editing leads to enhanced salinity tolerance in soybean. By using quantitative RT-PCR analysis, we found that the expression levels of GmAITRs were increased in response to ABA and salt treatments. Transfection assays in soybean protoplasts show that GmAITRs are nucleus proteins, and have transcriptional repression activities. By using CRISPR/Cas9 to target the six GmAITRs simultaneously, we successfully generated Cas9-free gmaitr36 double and gmaitr23456 quintuple mutants. We found that ABA sensitivity in these mutants was increased. Consistent with this, ABA responses of some ABA signaling key regulator genes in the gmaitr mutants were altered. In both seed germination and seedling growth assays, the gmaitr mutants showed enhanced salt tolerance. Most importantly, enhanced salinity tolerance in the mutant plants was also observed in the field experiments. These results suggest that mutation of GmAITR genes by CRISPR/Cas9 is an efficient way to improve salinity tolerance in soybean.

Analysis of expression characteristics of soybean leaf and root tissue-specific promoters in Arabidopsis and soybean.

The characterization of tissue-specific promoters is critical for studying the functions of genes in a given tissue/organ. To study tissue-specific promoters in soybean, we screened tissue-specific expressed genes using published soybean RNA-Seq-based transcriptome data coupled with RT-PCR analysis. We cloned the promoters of three genes, GmADR1, GmBTP1, and GmGER1, and constructed their corresponding ß-Glucuronidase (GUS) promoter-GUS reporter vectors. We generated transgenic Arabidopsis plants and examined the expression patterns of these promoters by GUS staining and RT-PCR analysis. We also transformed the promoter-GUS reporter vectors into soybean to obtain hairy roots, and examined promoter expression by GUS staining. We found a root-specific expression pattern of GmADR1 and GmBTP1 in both Arabidopsis and soybean, and the promoter of GmGER1 showed a leaf-specific pattern in transgenic Arabidopsis plants. To test the potential utility of these promoters in soybean improvement by transgenic means, we used the GmADR1 promoter to drive expression of a salt resistance gene in soybean, GmCaM4, by generating transgenic soybean plants. We found that the transgenic plants had significantly enhanced salt tolerance compared to non-transformed wild-type, suggesting that introducing endogenous promoters by transgenic means can drive the expression of functional genes in specific tissues and organs in soybean.

Over-expression of GmKR3, a TIR-NBS-LRR type R gene, confers resistance to multiple viruses in soybean.

KEY MESSAGE: That overexpression of GmKR3 enhances innate virus resistance by stimulating. Soybean mosaic virus (SMV) is found in many soybean production areas, and SMV infection is one of the prevalent viral diseases that can cause significant yield losses in soybean. In plants, resistance (R) genes are involved in pathogen reorganization and innate immune response activation. Most R proteins have nucleotide-binding site and leucine-rich repeat (NBS-LRR) domains, and some of the NBS-LRR type R proteins in dicots have Toll/Interleukin-1 Receptor (TIR) motifs. We report here the analysis of the over-expression of GmKR3, a soybean TIR-NBS-LRR type R gene on virus resistance in soybean. When over-expressed in soybean, GmKR3 enhanced the plant's resistance to several strains of SMV, the closely related potyviruses bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), and the secovirus bean pod mottle virus (BPMV). Importantly, over-expression of GmKR3 did not affect plant growth and development, including yield and qualities of the seeds. HPLC analysis showed that abscisic acid (ABA) content increased in the 35S:GmKR3 transgenic plants, and in both wild-type and 35S:GmKR3 transgenic plants in response to virus inoculation. Consistent with this observation, we found that the expression of two ABA catabolism genes was down-regulated in 35S:GmKR3 transgenic plants. We also found that the expression of Gm04.3, an ABA responsive gene encoding BURP domain-containing protein, was up-regulated in 35S:GmKR3 transgenic plants. Taken together, our results suggest that overexpression of GmKR3 enhanced virus resistance in soybean, which was achieved at least in part via ABA signaling.

The chromatin remodeler ZmCHB101 impacts expression of osmotic stress-responsive genes in maize.

KEY MESSAGE: The maize chromatin remodeler ZmCHB101 plays an essential role in the osmotic stress response. ZmCHB101 controls nucleosome densities around transcription start sites of essential stress-responsive genes. Drought and osmotic stresses are recurring conditions that severely constrain crop production. Evidence accumulated in the model plant Arabidopsis thaliana suggests that core components of SWI/SNF chromatin remodeling complexes play essential roles in abiotic stress responses. However, how maize SWI/SNF chromatin remodeling complexes function in osmotic and drought stress responses remains unknown. Here we show that ZmCHB101, a homolog of A. thaliana SWI3D in maize, plays essential roles in osmotic and dehydration stress responses. ZmCHB101-RNA interference (RNAi) transgenic plants displayed osmotic, salt and drought stress-sensitive phenotypes. Genome-wide RNA-sequencing analysis revealed that ZmCHB101 impacts the transcriptional expression landscape of osmotic stress-responsive genes. Intriguingly, ZmCHB101 controls nucleosome densities around transcription start sites of essential stress-responsive genes. Furthermore, we identified that ZmCHB101 associates with RNA polymerase II (RNAPII) in vivo and is a prerequisite for the proper occupancy of RNAPII on the proximal regions of transcription start sites of stress-response genes. Taken together, our findings suggest that ZmCHB101 affects gene expression by remodeling chromatin states and controls RNAPII occupancies in maize under osmotic stress.

Zinc oxide nanoparticle exposure triggers different gene expression patterns in maize shoots and roots.

The potential impacts of environmentally accumulated zinc oxide nanoparticles (nZnOs) on plant growth have not been well studied. A transcriptome profile analysis of maize exposed to nZnOs showed that the genes in the shoots and roots responded differently. Although the number of differentially expressed genes (DEGs) in the roots was greater than that in the shoots, the number of up- or down-regulated genes in both the shoots and roots was similar. The enrichment of gene ontology (GO) terms was also significantly different in the shoots and roots. The "nitrogen compound metabolism" and "cellular component" terms were specifically and highly up-regulated in the nZnO-exposed roots, whereas the categories "cellular metabolic process", "primary metabolic process" and "secondary metabolic process" were down-regulated in the exposed roots only. Our results revealed the DEG response patterns in maize shoots and roots after nZnO exposure.

Over-expression of GmSN1 enhances virus resistance in Arabidopsis and soybean.

KEY MESSAGE: GmSN1 enhances virus resistance in plants most likely by affecting the expression of signal transduction and immune response genes. Soybean mosaic virus (SMV) infection causes severe symptom and leads to massive yield loss in soybean (Glycine max). By comparative analyzing gene expression in the SMV-resistant soybean cultivar Rsmv1 and the susceptible cultivar Ssmv1 at a transcriptome level, we found that a subgroup of Gibberellic Acid Stimulated Transcript (GAST) genes were down-regulated in SMV inoculated Ssmv1 plants, but not Rsmv1 plants. Sequence alignment and phylogenetic analysis indicated that one of the GAST genes, GmSN1, was closely related to Snakin-1, a well-characterized potato microbial disease resistance gene. When over-expressed in Arabidopsis and soybean, respectively, under the control of the 35S promoter, GmSN1 enhanced turnip mosaic virus resistance in the transgenic Arabidopsis plants, and SMV resistance in the transgenic soybean plants, respectively. Transcriptome analysis results showed that the up-regulated genes in the 35S:GmSN1 transgenic Arabidopsis plants were largely enriched in functional terms including "signal transduction" and "immune response". Real-time PCR assay indicated that the expression of GmAKT2, a potassium channel gene known to enhance SMV resistance when over-expressed in soybean, was elevated in the 35S:GmSN1 transgenic soybean plants. Taken together, our results suggest that GmSN1 enhances virus resistance in plants most likely by affecting the expression of signal transduction and immune response genes.

The Core Subunit of A Chromatin-Remodeling Complex, ZmCHB101, Plays Essential Roles in Maize Growth and Development.

ATP-dependent chromatin remodeling complexes play essential roles in the regulation of diverse biological processes by formulating a DNA template that is accessible to the general transcription apparatus. Although the function of chromatin remodelers in plant development has been studied in A. thaliana, how it affects growth and development of major crops (e.g., maize) remains uninvestigated. Combining genetic, genomic and bioinformatic analyses, we show here that the maize core subunit of chromatin remodeling complex, ZmCHB101, plays essential roles in growth and development of maize at both vegetative and reproductive stages. Independent ZmCHB101 RNA interference plant lines displayed abaxially curling leaf phenotype due to increase of bulliform cell numbers, and showed impaired development of tassel and cob. RNA-seq-based transcriptome profiling revealed that ZmCHB101 dictated transcriptional reprogramming of a significant set of genes involved in plant development, photosynthesis, metabolic regulation, stress response and gene expressional regulation. Intriguingly, we found that ZmCHB101 was required for maintaining normal nucleosome density and 45 S rDNA compaction. Our findings suggest that the SWI3 protein, ZmCHB101, plays pivotal roles in maize normal growth and development via regulation of chromatin structure.

Immediate Genetic and Epigenetic Changes in F1 Hybrids Parented by Species with Divergent Genomes in the Rice Genus (Oryza).

BACKGROUND: Inter-specific hybridization occurs frequently in higher plants, and represents a driving force of evolution and speciation. Inter-specific hybridization often induces genetic and epigenetic instabilities in the resultant homoploid hybrids or allopolyploids, a phenomenon known as genome shock. Although genetic and epigenetic consequences of hybridizations between rice subspecies (e.g., japonica and indica) and closely related species sharing the same AA genome have been extensively investigated, those of inter-specific hybridizations between more remote species with different genomes in the rice genus, Oryza, remain largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the immediate chromosomal and molecular genetic/epigenetic instability of three triploid F1 hybrids produced by inter-specific crossing between species with divergent genomes of Oryza by genomic in situ hybridization (GISH) and molecular marker analysis. Transcriptional and transpositional activity of several transposable elements (TEs) and methylation stability of their flanking regions were also assessed. We made the following principle findings: (i) all three triploid hybrids are stable in both chromosome number and gross structure; (ii) stochastic changes in both DNA sequence and methylation occurred in individual plants of all three triploid hybrids, but in general methylation changes occurred at lower frequencies than genetic changes; (iii) alteration in DNA methylation occurred to a greater extent in genomic loci flanking potentially active TEs than in randomly sampled loci; (iv) transcriptional activation of several TEs commonly occurred in all three hybrids but transpositional events were detected in a genetic context-dependent manner. CONCLUSIONS/SIGNIFICANCE: Artificially constructed inter-specific hybrids of remotely related species with divergent genomes in genus Oryza are chromosomally stable but show immediate and highly stochastic genetic and epigenetic instabilities at the molecular level. These novel hybrids might provide a rich resource of genetic and epigenetic diversities for potential utilization in rice genetic improvements.

[Analgesic effect of DICLORAL on radiation-induced oral mucositis].

PURPOSE: To assess the effects of DICLORAL's alleviation of pain caused by radiation-induced oral mucositis. METHODS: With compound borax solution as a comparative drugs' the solution of DICLORAL was adopted for patients with radiation-induced oral mucositis due to radiotherapy for nasal NK/T cell lymphoma. All 59 patients were randomly divided into 2 groups: experimental group (using DICLORAL) and control group (using Compound Borax Solution). During the treatment, we observed drugs' ability of alleviate oral pain or pain on swallowing, when it worked and how long it could sustain in controlling pain. Statistical analysis was performed using SPSS 16.0 software package. RESULTS: Drugs' ability of alleviate oral pain or pain on swallowing and the onset time was not significantly different between the 2 groups (P>0.05). Effective duration in experimental group was longer than that in control group (P<0.05). CONCLUSIONS: DICORAL can alleviate and cease pain caused by radiation-induced oral mucositis effectively.

Multigene engineering of starch biosynthesis in maize endosperm increases the total starch content and the proportion of amylose.

Maize (Zea mays spp. mays) is a staple crop for more than 900 million people. The seeds or kernels provide a rich source of calories because ~70% of the weight is carbohydrate, mostly in the form of starch. The content and composition of starch are complex traits controlled by many genes, offering multiple potential targets for intervention. We used a multigene engineering approach combining the overexpression of Bt2, Sh2, Sh1 and GbssIIa (to enhance the activity of sucrose synthase, AGPase and granule-bound starch synthase) with the suppression of SbeI and SbeIIb by RNA interference (to reduce the activity of starch branching enzyme). Maize plants expressing all six genes plus the selectable marker showed a 2.8-7.7% increase in the endosperm starch content and a 37.8-43.7% increase in the proportion of amylose, which was significant compared to untransformed control plants. We also observed improvements in other agronomic traits, such as a 20.1-34.7% increase in 100-grain weight, a 13.9-19.0% increase in ear weight, and larger kernels with a better appearance, presumably reflecting the modified starch structure within the kernels. Our results confirm that multigene engineering applied to the starch biosynthesis pathway can not only modulate the quality and quantity of starch but can also improve starch-dependent agronomic traits.

Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast.

DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5'-CCGG-3' containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92-9.12%, and 2.88-6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA methyltransferase gene with conserved activity in both prokaryotic and eukaryotic non-host species.

Structural genes of wheat and barley 5-methylcytosine DNA glycosylases and their potential applications for human health.

Wheat supplies about 20% of the total food calories consumed worldwide and is a national staple in many countries. Besides being a key source of plant proteins, it is also a major cause of many diet-induced health issues, especially celiac disease. The only effective treatment for this disease is a total gluten-free diet. The present report describes an effort to develop a natural dietary therapy for this disorder by transcriptional suppression of wheat DEMETER (DME) homeologs using RNA interference. DME encodes a 5-methylcytosine DNA glycosylase responsible for transcriptional derepression of gliadins and low-molecular-weight glutenins (LMWgs) by active demethylation of their promoters in the wheat endosperm. Previous research has demonstrated these proteins to be the major source of immunogenic epitopes. In this research, barley and wheat DME genes were cloned and localized on the syntenous chromosomes. Nucleotide diversity among DME homeologs was studied and used for their virtual transcript profiling. Functional conservation of DME enzyme was confirmed by comparing the motif and domain structure within and across the plant kingdom. Presence and absence of CpG islands in prolamin gene sequences was studied as a hallmark of hypo- and hypermethylation, respectively. Finally the epigenetic influence of DME silencing on accumulation of LMWgs and gliadins was studied using 20 transformants expressing hairpin RNA in their endosperm. These transformants showed up to 85.6% suppression in DME transcript abundance and up to 76.4% reduction in the amount of immunogenic prolamins, demonstrating the possibility of developing wheat varieties compatible for the celiac patients.

Global transgenerational gene expression dynamics in two newly synthesized allohexaploid wheat (Triticum aestivum) lines.

BACKGROUND: Alteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. Common wheat (Triticum aestivum) was formed via allohexaploidization about 10,000 years ago, and became the most important crop plant. To gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat. RESULTS: Multi-color GISH (genomic in situ hybridization) was used to identify individual plants from two nascent allohexaploid wheat lines between Triticum turgidum (2n=4x=28; genome BBAA) and Aegilops tauschii (2n=2x=14; genome DD), which had a stable chromosomal constitution analogous to that of common wheat (2n=6x=42; genome BBAADD). Genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from T. turgidum and Ae. tauschii, using the Affymetrix Gene Chip Wheat Genome-Array. Comparison with the parental plants coupled with inclusion of empirical mid-parent values (MPVs) revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. Genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. Strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse Gene Ontology (GO) terms. Nonetheless, those genes showing non-additive expression exhibited a significant enrichment for vesicle-function. CONCLUSIONS: Our results show that two patterns of global alteration in gene expression are conditioned by allohexaploidization in wheat, that is, parental dominance expression and non-additive expression. Both altered patterns of gene expression but not the identity of the genes involved are likely to play functional roles in stabilization and establishment of the newly formed allohexaploid plants, and hence, relevant to speciation and evolution of T. aestivum.

Rapid genomic changes in polyploid wheat and related species: implications for genome evolution and genetic improvement.

A polyploid organism by possessing more than two sets of chromosomes from one species (autopolyploidy) or two or more species (allopolyploidy) is known to have evolutionary advantages. However, by what means a polyploid accommodates increased genetic dosage or divergent genomes (allopolyploidy) in one cell nucleus and cytoplasm constitutes an enormous challenge. Recent years have witnessed efforts and progress in exploring the possible mechanisms by which these seemingly intangible hurdles of polyploidy may be ameliorated or eventually overcome. In particular, the documentation of rapid and extensive non-Mendelian genetic and epigenetic changes that often accompany nascent polyploidy is revealing: the resulting non-additive and novel gene expression at global, regional and local levels, and timely restoration of meiotic chromosomal behavior towards bivalent pairing and disomic inheritance may ensure rapid establishment and stabilization as well as its long-term evolutionary success. Further elucidation on these novel mechanisms underpinning polyploidy will promote our understanding on fundamental issues in evolutionary biology and in our manipulation capacities in future genetic improvement of important crops that are currently polyploids in genomic constitution. This review is intended to provide an updated discussion on these interesting and important issues within the scope of a specific yet one of the most important plant groups-polyploid wheat and its related species.

Tissue culture-induced transpositional activity of mPing is correlated with cytosine methylation in rice.

BACKGROUND: mPing is an endogenous MITE in the rice genome, which is quiescent under normal conditions but can be induced towards mobilization under various stresses. The cellular mechanism responsible for modulating the activity of mPing remains unknown. Cytosine methylation is a major epigenetic modification in most eukaryotes, and the primary function of which is to serve as a genome defense system including taming activity of transposable elements (TEs). Given that tissue-culture is capable of inducing both methylation alteration and mPing transposition in certain rice genotypes, it provides a tractable system to investigate the possible relationship between the two phenomena. RESULTS: mPing transposition and cytosine methylation alteration were measured in callus and regenerated plants in three rice (ssp. indica) genotypes, V14, V27 and R09. All three genotypes showed transposition of mPing, though at various frequencies. Cytosine methylation alteration occurred both at the mPing-flanks and at random loci sampled globally in callus and regenerated plants of all three genotypes. However, a sharp difference in the changing patterns was noted between the mPing-flanks and random genomic loci, with a particular type of methylation modification, i.e., CNG hypermethylation, occurred predominantly at the mPing-flanks. Pearson's test on pairwise correlations indicated that mPing activity is positively correlated with specific patterns of methylation alteration at random genomic loci, while the element's immobility is positively correlated with methylation levels of the mPing's 5'-flanks. Bisulfite sequencing of two mPing-containing loci showed that whereas for the immobile locus loss of CG methylation in the 5'-flank was accompanied by an increase in CHG methylation, together with an overall increase in methylation of all three types (CG, CHG and CHH) in the mPing-body region, for the active locus erasure of CG methylation in the 5'-flank was not followed by such a change. CONCLUSION: Our results documented that tissue culture-induced mPing activity in rice ssp. indica is correlated with alteration in cytosine methylation patterns at both random genomic loci and the elements' flanks, while the stability of mPing positively correlates with enhanced methylation levels of both the flanks and probably the elements per se. Thus, our results implicate a possible role of cytosine methylation in maintaining mPing stability under normal conditions, and in releasing the element's activity as a consequence of epigenetic perturbation in a locus-specific manner under certain stress conditions.

Construction of the seed-coat cDNA microarray and screening of differentially expressed genes in barley.

Some barley mutants can synthesize neither anthocyanins nor proanthocyanidins in the seed coat, which is related to several genes in locus Ant13, but the exact model of action remains unknown. We used the cDNA microarray technology with barley transcription-deficient mutant (ant13-152) that does not synthesize proanthocyanidins as the tester, and its wild type genotype (Triumph) as the driver, to study this question. Six-thousand and forty-eight clones from the wild type Morex testa+pericarp cDNA library were amplified using PCR, and the DNA fragments were spotted on commercial amino-modified glass slide as microarray. The mRNAs from the developing seed coat (8-15 days) of both the mutant and the wild-type barley plants were isolated, and labeled respectively with Cy3-dUTP and Cy5-dUTP when reversely transcribed to cDNAs. The labeled cDNAs were used as probes, mixed at the same molar concentration, and hybridized with the DNA fragments on the slide. Seventy clones exhibiting marked differential expression (ratio>4) were identified from the microarray. All the 25 cDNA clones that showed an over-expression in wild type in comparison to the mutant ant13-152 were sequenced. It was found that most of these overexpressing clones were transcription/translation and hordein-associated genes. These results have laid a solid material basis for further elucidation of the metabolic pathway in proanthocyanidin synthesis in barley and likely other plants.