Overexpression of AtMYB2 Promotes Tolerance to Salt Stress and Accumulations of Tanshinones and Phenolic Acid in Salvia miltiorrhiza.

Salvia miltiorrhiza is a prized traditional Chinese medicinal plant species. Its red storage roots are primarily used for the treatment of cardiovascular and cerebrovascular diseases. In this study, a transcription factor gene AtMYB2 was cloned and introduced into Salvia miltiorrhiza for ectopic expression. Overexpression of AtMYB2 enhanced salt stress resistance in S. miltiorrhiza, leading to a more resilient phenotype in transgenic plants exposed to high-salinity conditions. Physiological experiments have revealed that overexpression of AtMYB2 can decrease the accumulation of reactive oxygen species (ROS) during salt stress, boost the activity of antioxidant enzymes, and mitigate oxidative damage to cell membranes. In addition, overexpression of AtMYB2 promotes the synthesis of tanshinones and phenolic acids by upregulating the expression of biosynthetic pathway genes, resulting in increased levels of these secondary metabolites. In summary, our findings demonstrate that AtMYB2 not only enhances plant tolerance to salt stress, but also increases the accumulation of secondary metabolites in S. miltiorrhiza. Our study lays a solid foundation for uncovering the molecular mechanisms governed by AtMYB2 and holds significant implications for the molecular breeding of high-quality S. miltiorrhiza varieties.

Simultaneous Promotion of Salt Tolerance and Phenolic Acid Biosynthesis in Salvia miltiorrhiza via Overexpression of Arabidopsis MYB12.

Transcription factors play crucial roles in regulating plant abiotic stress responses and physiological metabolic processes, which can be used for plant molecular breeding. In this study, an R2R3-MYB transcription factor gene, AtMYB12, was isolated from Arabidopsis thaliana and introduced into Salvia miltiorrhiza under the regulation of the CaMV35S promoter. The ectopic expression of AtMYB12 resulted in improved salt tolerance in S. miltiorrhiza; transgenic plants showed a more resistant phenotype under high-salinity conditions. Physiological experiments showed that transgenic plants exhibited higher chlorophyll contents, and decreased electrolyte leakage and O2- and H2O2 accumulation when subjected to salt stress. Moreover, the activity of reactive oxygen species (ROS)-scavenging enzymes was enhanced in S. miltiorrhiza via the overexpression of AtMYB12, and transgenic plants showed higher superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities compared with those of the wild type (WT) under salt stress, coupled with lower malondialdehyde (MDA) levels. In addition, the amount of salvianolic acid B was significantly elevated in all AtMYB12 transgenic hair roots and transgenic plants, and qRT-PCR analysis revealed that most genes in the phenolic acid biosynthetic pathway were up-regulated. In conclusion, these results demonstrated that AtMYB12 can significantly improve the resistance of plants to salt stress and promote the biosynthesis of phenolic acids by regulating genes involved in the biosynthetic pathway.

BolTLP1, a Thaumatin-like Protein Gene, Confers Tolerance to Salt and Drought Stresses in Broccoli (Brassica oleracea L. var. Italica).

Plant thaumatin-like proteins (TLPs) play pleiotropic roles in defending against biotic and abiotic stresses. However, the functions of TLPs in broccoli, which is one of the major vegetables among the B. oleracea varieties, remain largely unknown. In the present study, bolTLP1 was identified in broccoli, and displayed remarkably inducible expression patterns by abiotic stress. The ectopic overexpression of bolTLP1 conferred increased tolerance to high salt and drought conditions in Arabidopsis. Similarly, bolTLP1-overexpressing broccoli transgenic lines significantly improved tolerance to salt and drought stresses. These results demonstrated that bolTLP1 positively regulates drought and salt tolerance. Transcriptome data displayed that bolTLP1 may function by regulating phytohormone (ABA, ethylene and auxin)-mediated signaling pathways, hydrolase and oxidoreductase activity, sulfur compound synthesis, and the differential expression of histone variants. Further studies confirmed that RESPONSE TO DESICCATION 2 (RD2), RESPONSIVE TO DEHYDRATION 22 (RD22), VASCULAR PLANT ONE-ZINC FINGER 2 (VOZ2), SM-LIKE 1B (LSM1B) and MALATE DEHYDROGENASE (MDH) physically interacted with bolTLP1, which implied that bolTLP1 could directly interact with these proteins to confer abiotic stress tolerance in broccoli. These findings provide new insights into the function and regulation of bolTLP1, and suggest potential applications for bolTLP1 in breeding broccoli and other crops with increased tolerance to salt and drought stresses.

Parallel Speciation of Wild Rice Associated with Habitat Shifts.

The occurrence of parallel speciation strongly implies the action of natural selection. However, it is unclear how general a phenomena parallel speciation is since it was only shown in a small number of animal species. In particular, the adaptive process and mechanisms underlying the process of parallel speciation remain elusive. Here, we used an integrative approach incorporating population genomics, common garden, and crossing experiments to investigate parallel speciation of the wild rice species Oryza nivara from O. rufipogon. We demonstrated that O. nivara originated multiple times from different O. rufipogon populations and revealed that different O. nivara populations have evolved similar phenotypes under divergent selection, a reflection of recurrent local adaptation of ancient O. rufipogon populations to dry habitats. Almost completed premating isolation was detected between O. nivara and O. rufipogon in the absence of any postmating barriers between and within these species. These results suggest that flowering time is a "magic" trait that contributes to both local adaptation and reproductive isolation in the origin of wild rice species. Our study thus demonstrates a convincing case of parallel ecological speciation as a consequence of adaptation to new environments.

Obtaining World Coordinate Information of UAV in GNSS Denied Environments.

GNSS information is vulnerable to external interference and causes failure when unmanned aerial vehicles (UAVs) are in a fully autonomous flight in complex environments such as high-rise parks and dense forests. This paper presents a pan-tilt-based visual servoing (PBVS) method for obtaining world coordinate information. The system is equipped with an inertial measurement unit (IMU), an air pressure sensor, a magnetometer, and a pan-tilt-zoom (PTZ) camera. In this paper, we explain the physical model and the application method of the PBVS system, which can be briefly summarized as follows. We track the operation target with a UAV carrying a camera and output the information about the UAV's position and the angle between the PTZ and the anchor point. In this way, we can obtain the current absolute position information of the UAV with its absolute altitude collected by the height sensing unit and absolute geographic coordinate information and altitude information of the tracked target. We set up an actual UAV experimental environment. To meet the calculation requirements, some sensor data will be sent to the cloud through the network. Through the field tests, it can be concluded that the systematic deviation of the overall solution is less than the error of GNSS sensor equipment, and it can provide navigation coordinate information for the UAV in complex environments. Compared with traditional visual navigation systems, our scheme has the advantage of obtaining absolute, continuous, accurate, and efficient navigation information at a short distance (within 15 m from the target). This system can be used in scenarios that require autonomous cruise, such as self-powered inspections of UAVs, patrols in parks, etc.

Knockout of the OsNAC006 Transcription Factor Causes Drought and Heat Sensitivity in Rice.

Rice (Oryza sativa) responds to various abiotic stresses during growth. Plant-specific NAM, ATAF1/2, and CUC2 (NAC) transcription factors (TFs) play an important role in controlling numerous vital growth and developmental processes. To date, 170 NAC TFs have been reported in rice, but their roles remain largely unknown. Herein, we discovered that the TF OsNAC006 is constitutively expressed in rice, and regulated by H2O2, cold, heat, abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin (GA), NaCl, and polyethylene glycol (PEG) 6000 treatments. Furthermore, knockout of OsNAC006 using the CRISPR-Cas9 system resulted in drought and heat sensitivity. RNA sequencing (RNA-seq) transcriptome analysis revealed that OsNAC006 regulates the expression of genes mainly involved in response to stimuli, oxidoreductase activity, cofactor binding, and membrane-related pathways. Our findings elucidate the important role of OsNAC006 in drought responses, and provide valuable information for genetic manipulation to enhance stress tolerance in future plant breeding programs.

Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic).

BACKGROUND: Curds are the main edible organs, which exhibit remarkable yield heterosis in F1 hybrid broccoli. However, the molecular basis underlying heterosis in broccoli remains elusive. RESULTS: In the present study, transcriptome profiles revealed that the hybridization made most genes show additive expression patterns in hybrid broccoli. The differentially expressed genes including the non-additively expressed genes detected in the hybrid broccoli and its parents were mainly involved in light, hormone and hydrogen peroxide-mediated signaling pathways, responses to stresses, and regulation of floral development, which suggested that these biological processes should play crucial roles in the yield heterosis of broccoli. Among them, light and hydrogen peroxide-mediated signaling pathways represent two novel classes of regulatory processes that could function in yield or biomass heterosis of plants. Totally, 53 candidate genes closely involved in curd yield heterosis were identified. Methylome data indicated that the DNA methylation ratio of the hybrids was higher than that of their parents. However, the DNA methylation levels of most sites also displayed additive expression patterns. These sites with differential methylation levels were predominant in the intergenic regions. In most cases, the changes of DNA methylation levels in gene regions did not significantly affect their expression levels. CONCLUSIONS: The differentially expressed genes, the regulatory processes and the possible roles of DNA methylation modification in the formation of curd yield heterotic trait were discovered. These findings provided comprehensive insights into the curd yield heterosis in broccoli, and were significant for breeding high-yield broccoli varieties.

Identification of Glutathione Peroxidase (GPX) Gene Family in Rhodiola crenulata and Gene Expression Analysis under Stress Conditions.

Glutathione peroxidases (GPXs) are important enzymes in the glutathione-ascorbate cycle for catalyzing the reduction of H2O2 or organic hydroperoxides to water. GPXs play an essential role in plant growth and development by participating in photosynthesis, respiration, and stress tolerance. Rhodiola crenulata is a popular traditional Chinese medicinal plant which displays an extreme energy of tolerance to harsh alpine climate. The GPXs gene family might provide R. crenulata for extensively tolerance to environment stimulus. In this study, five GPX genes were isolated from R. crenulata. The protein amino acid sequences were analyzed by bioinformation softwares with the results that RcGPXs gene sequences contained three conserve cysteine residues, and the subcellular location predication were in the chloroplast, endoplasmic reticulum, or cytoplasm. Five RcGPXs members presented spatial and temporal specific expression with higher levels in young and green organs. And the expression patterns of RcGPXs in response to stresses or plant hormones were investigated by quantitative real-time PCR. In addition, the putative interaction proteins of RcGPXs were obtained by yeast two-hybrid with the results that RcGPXs could physically interact with specific proteins of multiple pathways like transcription factor, calmodulin, thioredoxin, and abscisic acid signal pathway. These results showed the regulation mechanism of RcGPXs were complicated and they were necessary for R. crenulata to adapt to the treacherous weather in highland.

Comparative Transcriptome Analyses Reveal Potential Mechanisms of Enhanced Drought Tolerance in Transgenic Salvia Miltiorrhiza Plants Expressing AtDREB1A from Arabidopsis.

In our previous study, drought-resistant transgenic plants of Salvia miltiorrhiza were produced via overexpression of the transcription factor AtDREB1A. To unravel the molecular mechanisms underpinning elevated drought tolerance in transgenic plants, in the present study we compared the global transcriptional profiles of wild-type (WT) and AtDREB1A-expressing transgenic plants using RNA-sequencing (RNA-seq). Using cluster analysis, we identified 3904 differentially expressed genes (DEGs). Compared with WT plants, 423 unigenes were up-regulated in pRD29A::AtDREB1A-31 before drought treatment, while 936 were down-regulated and 1580 and 1313 unigenes were up- and down-regulated after six days of drought. COG analysis revealed that the 'signal transduction mechanisms' category was highly enriched among these DEGs both before and after drought stress. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, DEGs associated with "ribosome", "plant hormone signal transduction", photosynthesis", "plant-pathogen interaction", "glycolysis/gluconeogenesis" and "carbon fixation" are hypothesized to perform major functions in drought resistance in AtDREB1A-expressing transgenic plants. Furthermore, the number of DEGs associated with different transcription factors increased significantly after drought stress, especially the AP2/ERF, bZIP and MYB protein families. Taken together, this study substantially expands the transcriptomic information for S. miltiorrhiza and provides valuable clues for elucidating the mechanism of AtDREB1A-mediated drought tolerance in transgenic plants.

Transcriptome Analyses from Mutant Salvia miltiorrhiza Reveals Important Roles for SmGASA4 during Plant Development.

Salvia miltiorrhiza (S. miltiorrhiza) is an important Chinese herb that is derived from the perennial plant of Lamiaceae, which has been used to treat neurasthenic insomnia and cardiovascular disease. We produced a mutant S. miltiorrhiza (MT), from breeding experiments, that possessed a large taproot, reduced lateral roots, and defective flowering. We performed transcriptome profiling of wild type (WT) and MT S. miltiorrhiza using second-generation Illumina sequencing to identify differentially expressed genes (DEGs) that could account for these phenotypical differences. Of the DEGs identified, we investigated the role of SmGASA4, the expression of which was down-regulated in MT plants. SmGASA4 was introduced into Arobidopsis and S. militiorrhiza under the control of a CaMV35S promoter to verify its influence on abiotic stress and S. miltiorrhiza secondary metabolism biosynthesis. SmGASA4 was found to promote flower and root development in Arobidopsis. SmGASA4 was also found to be positively regulated by Gibberellin (GA) and significantly enhanced plant resistance to salt, drought, and paclobutrazol (PBZ) stress. SmGASA4 also led to the up-regulation of the genes involved in salvianolic acid biosynthesis, but inhibited the expression of the genes involved in tanshinone biosynthesis. Taken together, our results reveal SmGASA4 as a promising candidate gene to promote S. miltiorrhiza development.

Ectopic Expression of DREB Transcription Factor, AtDREB1A, Confers Tolerance to Drought in Transgenic Salvia miltiorrhiza.

Drought decreases crop productivity more than any other type of environmental stress. Transcription factors (TFs) play crucial roles in regulating plant abiotic stress responses. The Arabidopsis thaliana gene DREB1A/CBF3, encoding a stress-inducible TF, was introduced into Salvia miltiorrhiza Ectopic expression of AtDREB1A resulted in increased drought tolerance, and transgenic lines had higher relative water content and Chl content, and exhibited an increased photosynthetic rate when subjected to drought stress. AtDREB1A transgenic plants generally displayed lower malondialdehyde (MDA), but higher superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under drought stress. In particular, plants with ectopic AtDREB1A expression under the control of the stress-induced RD29A promoter exhibited more tolerance to drought compared with p35S::AtDREB1A transgenic plants, without growth inhibition or phenotypic aberrations. Differential gene expression profiling of wild-type and pRD29A::AtDREB1A transgenic plants following drought stress revealed that the expression levels of various genes associated with the stress response, photosynthesis, signaling, carbohydrate metabolism and protein protection were substantially higher in transgenic plants. In addition, the amount of salvianolic acids and tanshinones was significantly elevated in AtDREB1A transgenic S. miltiorrhiza roots, and most of the genes in the related biosynthetic pathways were up-regulated. Together, these results demonstrated that inducing the expression of a TF can effectively regulate multiple genes in the stress response pathways and significantly improve the resistance of plants to abiotic stresses. Our results also suggest that genetic manipulation of a TF can improve production of valuable secondary metabolites by regulating genes in associated pathways.

Two-face, two-turn α-helix mimetics based on a cross-acridine scaffold: analogues of the Bim BH3 domain.

The design of a cross-acridine scaffold mimicking the i, i+3, i+5, and i+7 residues distributed over a two-face, two-turn α-helix is described. Docking studies and 2D (1)H, (15)N HSQC NMR spectroscopy provide compelling evidence that compound 3 d accurately reproduces the arrangement of four hotspots in the Bim BH3 peptide to permit binding to the Mcl-1 and Bcl-2 proteins (Ki 0.079 and 0.056 µM, respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.

Genome-wide identification and characterization of miRNAs in the hypocotyl and cotyledon of cauliflower (Brassica oleracea L. var. botrytis) seedlings.

MicroRNAs (miRNAs) are a class of small endogenous, non-coding RNAs that have key regulatory functions in plant growth, development, and other biological processes. Hypocotyl and cotyledon are the two major tissues of cauliflower (Brassica oleracea L. var. botrytis) seedlings. Tissue culture experiments have indicated that the regenerative abilities of these two tissues are significantly different. However, the characterization of miRNAs and their roles in regulating organ development in cauliflower remain unexplored. In the present study, two small RNA libraries were sequenced by Solexa sequencing technology. 99 known miRNAs belonging to 28 miRNA families were identified, in which 6 miRNA families were detected only in Brassicaceae. A total of 162 new miRNA sequences with single nucleotide substitutions corresponding to the known miRNAs, and 32 potentially novel miRNAs were also first discovered. Comparative analysis indicated that 42 of 99 known miRNAs and 17 of 32 novel miRNAs exhibited significantly differential expression between hypocotyl and cotyledon, and the differential expression of several miRNAs was further validated by stem-loop RT-PCR. In addition, 235 targets for 89 known miRNAs and 198 targets for 24 novel miRNAs were predicted, and their functions were further discussed. The expression patterns of several representative targets were also confirmed by qRT-PCR analysis. The results identified that the transcriptional expression patterns of miRNAs were negatively correlated with their targets. These findings gave new insights into the characteristics of miRNAs in cauliflower, and provided important clues to elucidate the roles of miRNAs in the tissue differentiation and development of cauliflower.

LaAP2L1, a heterosis-associated AP2/EREBP transcription factor of Larix, increases organ size and final biomass by affecting cell proliferation in Arabidopsis.

In Larix and in some crops, heterosis is prevalent and has been widely used in breeding to produce excellent varieties. However, the molecular basis of heterosis in Larix remains ambiguous. LaAP2L1, a member of the AP2/EREBP transcription factor family, has been suggested to be involved in heterosis in Larix hybrids. Here, the function and regulation of LaAP2L1 were further explored. Overexpression of LaAP2L1 led to markedly enlarged organs and heterosis-like traits in Arabidopsis. Fresh weight of leaves was almost twice as great as in vector controls. Likewise, seed yield of 35S::LaAP2L1 individual plants was >200% greater than that of control plants. The enlarged organs and heterosis-like traits displayed by 35S::LaAP2L1 plants were mainly due to enhanced cell proliferation and prolonged growth duration. At the molecular level, LaAP2L1 upregulated the expression of ANT, EBP1, and CycD3;1 and inhibited the expression of ARGOS in 35S::LaAP2L1 plants, suggesting an important molecular role of LaAP2L1 in regulating plant organ development. These findings provide new insights into the formation of heterosis in woody plants and suggest that LaAP2L1 has potential applications in breeding high-yielding crops and energy plants. In addition, 50 AP2/EREBP transcription factors, including LaAP2L1, in Larix were identified by transcriptome sequencing, and phylogenetic analysis was conducted. This provided information that will be important in further revealing the functions of these transcription factors.

Optimal Frequency of Hepatitis C Virus (HCV) RNA Testing for Detection of Acute HCV Infection Among At-risk People With Human Immunodeficiency Virus: A Multicenter Study.

Using 3-stage pooled-plasma hepatitis C virus (HCV) RNA testing performed quarterly among at-risk people with human immunodeficiency virus (PWH), we found that if testing had been performed every 6 or 12 months, 58.6%-91.7% of PWH who recently acquired HCV would be delayed for diagnosis and might contribute to onward HCV transmission with longer durations.

Gene expression profiles of two intraspecific Larix lines and their reciprocal hybrids.

Heterosis has been widely explored in Larix breeding for more than a century, but the molecular mechanisms underlying this phenomenon remain elusive. In the present study, the genome-wide transcript profiles from two Larix genotypes and their reciprocal hybrids were analyzed using Arabidopsis 70-mer oligonucleotide microarrays. Despite sharing the same two parental lines, one of the hybrids showed obvious heterosis, while the other did not. In total, 1,171 genes were differentially expressed between the heterotic hybrid and its parents, of which 133 genes were nonadditive expression. The number of differentially expressed genes between the non-heterotic hybrid and the parents was 939, but only 54 of these genes were nonadditive expression. Further, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses indicated that most of these differentially expressed genes in the heterotic hybrid were associated with several important biological functions such as physiological processes, responses to stimulus, and starch and sucrose metabolism. The reliability of the microarray data was further validated by the Real-time quantitative RT-PCR. A high Pearson linear correlation coefficient value was detected (r = 0.759, P < 0.01). In conclusion, the gene expression profile in the Larix heterotic hybrid was significantly different from that obtained from the non-heterotic hybrid, and more nonadditive differentially expressed genes were detected in the heterotic hybrid, implying that nonadditive effects may be closely associated with the formation of heterosis in the intraspecific Larix hybridization.

Knocking Out the Transcription Factor OsNAC092 Promoted Rice Drought Tolerance.

Environmental drought stress threatens rice production. Previous studies have reported that related NAC (NAM, ATAF1/2, and CUC) transcription factors play an important role in drought stress. Herein, we identified and characterized OsNAC092, encoding an NAC transcription factor that is highly expressed and induced during drought tolerance. OsNAC092 knockout lines created using the clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) system exhibited increased drought resistance in rice. RNA sequencing showed that the knockout of OsNAC092 caused a global expression change, and differential gene expression is chiefly associated with "response to light stimulus," "MAPK signaling pathway," "plant hormone signal transduction," "response to oxidative stress," "photosynthesis," and "water deprivation." In addition, the antioxidants and enzyme activities of the redox response were significantly increased. OsNAC092 mutant rice exhibited a higher ability to scavenge more ROS and maintained a high GSH/GSSG ratio and redox level under drought stress, which could protect cells from oxidant stress, revealing the importance of OsNAC092 in the rice's response to abiotic stress. Functional analysis of OsNAC092 will be useful to explore many rice resistance genes in molecular breeding to aid in the development of modern agriculture.

DNA Methylation in Genomes of Several Annual Herbaceous and Woody Perennial Plants of Varying Ploidy as Detected by MSAP.

Polyploidization is known to accompany altered DNA methylation in higher plants, which plays an important role in gene expression regulation and maintaining genome stability. While the characteristics of DNA methylation in different polyploid plants are still to be elucidated; here, status of genomic DNA methylation in a series of diploid, triploid, and tetraploid annual herbaceous plants (watermelon and Salvia) and woody perennials (pear, Poplar, and loquat) were explored by methylation-specific amplified polymorphism analysis. The results indicated that levels of DNA methylation in triploid watermelon and Salvia were lower than their diploid parents. In triploid Poplar and pear, higher levels of DNA methylation were detected, and no significant difference was observed between triploid and tetraploid in all tested materials. Further data analysis suggested that about half of the total detected sites underwent changes of DNA methylation patterns in triploid watermelons and Salvia, as well as an obvious trend towards demethylation. However, the changes of DNA methylation patterns in three triploid woody perennials were only 17.54-33.40%. This implied that the characteristics of DNA methylation are significantly different during the polyploidization of different plant species. Furthermore, the results suggested that the level of DNA methylation was nonlinearly related to the ploidy level, and triploid plants displayed more interesting DNA methylation status. The characteristics and possible functions of DNA methylation in different ploidy series are further discussed.

Enhancement of tanshinone production in Salvia miltiorrhiza hairy root cultures by metabolic engineering.

BACKGROUND: Tanshinones are diterpenoid compounds that are used to treat cardiovascular diseases. As current extraction methods for tanshinones are inefficient, there is a pressing need to improve the production of these bioactive compounds to meet increasing demand. RESULTS: Overexpression of SmMDS (2-c-methyl-d-erythritol 2,4-cyclodiphosphate synthase, a tanshinone biosynthesis gene) in transgenic Salvia miltiorrhiza hairy roots significantly increased the tanshinone yield compared to the control, and total tanshinone content in SmMDS-overexpressing lines increased after elicitor treatment. Total tanshinones increased to 2.5, 2.3, and 3.2 mg/g DW (dry weight) following treatment with Ag+, YE (yeast extract), and MJ (methyl jasmonate), respectively, compared with the non-induced transgenic line (1.7 mg/g DW). Also, qRT-PCR analysis showed that the expression levels of two pathway genes was positively correlated with increased accumulation of tanshinone. CONCLUSIONS: Our study provides an effective strategy for increasing the content of tanshinones and other natural compounds using a combination of genetic engineering and elicitor treatment.

Characters of DNA constitution in the rye B chromosome.

We have used chromosome microdissection and microcloning to construct a DNA library of the entire B chromosome (B) of rye. New rye B-specific sequences have been screened from this pool, blasted with other sequences and analyzed to elucidate the characters of DNA constitution and the possible pathway of the origin of the rye B chromosome. We report the discovery of a new sequence that is specific to the rye B centromere.