Genome wide analysis of the heavy-metal-associated (HMA) gene family in tomato and expression profiles under different stresses.

The heavy-metal-associated (HMA) family plays a major role in the transportation of metals. Despite having the genome sequence of the tomato (Solanum lycopersicum), the HMA gene family has not been studied yet. In this study, we identified 48 HMA genes and categorized them into Cu/Ag P1B-ATPase and Zn/Co/Cd/Pb P1BATPase sub-families according to their phylogenic relationship with Arabidopsis and rice. The SlHMA genes were distributed throughout the 12 chromosomes. Analysis of gene structure, chromosomal position, and synteny, revealed that segmental duplications bestowed their evolution. The high numbers of stress-related cis-elements were found to be present in the putative promoter regions indicate the involvement of SlHMAs in stress modulation pathways. RNA-seq data revealed that SlHMAs had divergent expression in different tissues and developmental stages, where members of Cu/Ag P1B-ATPase subfamily were strongly expressed in the roots. RT-qPCR analysis of nine selected SlHMAs showed that most of the genes were up-regulated in response to heavy metals and moderately regulated in response to different abiotic stresses such as salt, drought, and cold.

Genome-wide identification and expression analysis of the bZIP transcription factor family genes in response to abiotic stress in Nicotiana tabacum L.

BACKGROUND: The basic leucine zipper (bZIP) transcription factor (TF) is one of the largest families of transcription factors (TFs). It is widely distributed and highly conserved in animals, plants, and microorganisms. Previous studies have shown that the bZIP TF family is involved in plant growth, development, and stress responses. The bZIP family has been studied in many plants; however, there is little research on the bZIP gene family in tobacco. RESULTS: In this study, 77 bZIPs were identified in tobacco and named NtbZIP01 through to NtbZIP77. These 77 genes were then divided into eleven subfamilies according to their homology with Arabidopsis thaliana. NtbZIPs were unevenly distributed across twenty-two tobacco chromosomes, and we found sixteen pairs of segmental duplication. We further studied the collinearity between these genes and related genes of six other species. Quantitative real-time polymerase chain reaction analysis identified that expression patterns of bZIPs differed, including in different organs and under various abiotic stresses. NtbZIP49 might be important in the development of flowers and fruits; NtbZIP18 might be an important regulator in abiotic stress. CONCLUSIONS: In this study, the structures and functions of the bZIP family in tobacco were systematically explored. Many bZIPs may play vital roles in the regulation of organ development, growth, and responses to abiotic stresses. This research has great significance for the functional characterisation of the tobacco bZIP family and our understanding of the bZIP family in higher plants.

OsMLH1 interacts with OsMLH3 to regulate synapsis and interference-sensitive crossover formation during meiosis in rice.

Meiotic recombination is essential for reciprocal exchange of genetic information between homologous chromosomes and their subsequent proper segregation in sexually reproducing organisms. MLH1 and MLH3 belong to meiosis-specific members of the MutL-homolog family, which are required for normal level of crossovers (COs) in some eukaryotes. However, their functions in plants need to be further elucidated. Here, we report the identification of OsMLH1 and reveal its functions during meiosis in rice. Using CRISPR-Cas9 approach, two independent mutants, Osmlh1-1 and Osmlh1-2, are generated and exhibited significantly reduced male fertility. In Osmlh1-1, the clearance of PAIR2 is delayed and partial ZEP1 proteins are not loaded into the chromosomes, which might be due to the deficient in resolution of interlocks at late zygotene. Thus, OsMLH1 is required for the assembly of synapsis complex. In Osmlh1-1, CO number is dropped by ~53% and the distribution of residual COs is consistent with predicted Poisson distribution, indicating that OsMLH1 is essential for the formation of interference-sensitive COs (class I COs). OsMLH1 interacts with OsMLH3 through their C-terminal domains. Mutation in OsMLH3 also affects the pollen fertility. Thus, our experiments reveal that the conserved heterodimer MutLγ (OsMLH1-OsMLH3) is essential for the formation of class I COs in rice.

Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba.

The survival of cells depends on their ability to replicate correctly genetic material. Cells exposed to replication stress can experience a number of problems that may lead to deregulated proliferation, the development of cancer, and/or programmed cell death. In this article, we have induced prolonged replication arrest via hydroxyurea (HU) treatment and also premature chromosome condensation (PCC) by co-treatment with HU and caffeine (CF) in the root meristem cells of Vicia faba. We have analyzed the changes in the activities of retinoblastoma-like protein (RbS807/811ph). Results obtained from the immunocytochemical detection of RbS807/811ph allowed us to distinguish five unique activity profiles of pRb. We have also performed detailed 3D modeling using Blender 2.9.1., based on the original data and some final conclusions. 3D models helped us to visualize better the events occurring within the nuclei and acted as a high-resolution aid for presenting the results. We have found that, despite the decrease in pRb activity, its activity profiles were mostly intact and clearly recognizable, with some local alterations that may correspond to the increased demand in transcriptional activity. Our findings suggest that Vicia faba's ability to withstand harsh environments may come from its well-developed and highly effective response to replication stress.

Altered chromatin conformation and transcriptional regulation in watermelon following genome doubling.

Polyploidy has played a crucial role in plant evolution, development and function. Synthetic autopolyploid represents an ideal system to investigate the effects of polyploidization on transcriptional regulation. In this study, we deciphered the impact of genome duplication at phenotypic and molecular levels in watermelon. Overall, 88% of the genes in tetraploid watermelon followed a >1:1 dosage effect, and accordingly, differentially expressed genes were largely upregulated. In addition, a great number of hypomethylated regions (1688) were identified in an isogenic tetraploid watermelon. These differentially methylated regions were localized in promoters and intergenic regions and near transcriptional start sites of the identified upregulated genes, which enhances the importance of methylation in gene regulation. These changes were reflected in sophisticated higher-order chromatin structures. The genome doubling caused switching of 108 A and 626 B compartments that harbored genes associated with growth, development and stress responses.

Validating a segment on chromosome 7 of japonica for establishing low-cadmium accumulating indica rice variety.

Cadmium (Cd) contamination of rice is a serious food safety issue that has recently been gaining significant public attention. Therefore, reduction of Cd accumulation in rice grains is an important objective of rice breeding. The use of favourable alleles of Cd accumulating genes using marker-assisted selection (MAS) is theoretically feasible. In this study, we validated a segment covering OsHMA3-OsNramp5-OsNramp1 on chromosome 7 of japonica for establishing low-cadmium accumulating indica rice variety. The OsHMA3-OsNramp5-OsNramp1jap haplotype significantly decreased grain Cd concentration in middle-season indica genetic background. The improved 9311 carrying the OsHMA3-OsNramp5-OsNramp1jap haplotype with recurrent parent genome recovery of up to 91.6% resulted in approximately 31.8% decrease in Cd accumulation in the grain and with no penalty on yield. There is a genetic linkage-drag between OsHMA3-OsNramp5-OsNramp1 jap and the gene conditioning heading to days (HTD) in the early-season indica genetic background. Because the OsHMA3-OsNramp5-OsNramp1-Ghd7jap haplotype significantly increases grain Cd concentration and prolongs growth duration, the linkage-drag between OsHMA3-OsNramp5-OsNramp1 and Ghd7 should be broken down by large segregating populations or gene editing. A novel allele of OsHMA3 was identified from a wide-compatibility japonica cultivar, the expression differences of OsNramp1 and OsNramp5 in roots might contribute the Cd accumulating variation between japonica and indica variety.

Genome-wide identification and expression profiling of glutathione S-transferase family under multiple abiotic and biotic stresses in Medicago truncatula L.

Glutathione transferases (GSTs) constitute an ancient, ubiquitous, multi-functional antioxidant enzyme superfamily that has great importance on cellular detoxification against abiotic and biotic stresses as well as plant development and growth. The present study aimed to a comprehensive genome-wide identification and functional characterization of GST family in one of the economically important legume plants-Medicago truncatula. Here, we have identified a total of ninety-two putative MtGST genes that code for 120 proteins. All these members were classified into twelve classes based on their phylogenetic relationship and the presence of structural conserved domain/motif. Among them, 7 MtGST gene pairs were identified to have segmental duplication. Expression profiling of MtGST transcripts revealed their high level of organ/tissue-specific expression in most of the developmental stages and anatomical tissues. The transcripts of MtGSTU5, MtGSTU8, MtGSTU17, MtGSTU46, and MtGSTU47 showed significant up-regulation in response to various abiotic and biotic stresses. Moreover, transcripts of MtGSTU8, MtGSTU14, MtGSTU28, MtGSTU30, MtGSTU34, MtGSTU46 and MtGSTF8 were found to be highly upregulated in response to drought treatment for 24h and 48h. Among the highly stress-responsive MtGST members, MtGSTU17 showed strong affinity towards its conventional substrates reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) with the lowest binding energy of-5.7 kcal/mol and -6.5 kcal/mol, respectively. Furthermore, the substrate-binding site residues of MtGSTU17 were found to be highly conserved. These findings will facilitate the further functional and evolutionary characterization of GST genes in Medicago.

Disruption of actin filaments delays accumulation of cell plate membranes after chromosome separation.

Phragmoplasts, which comprise microtubules, actin filaments, and membrane vesicles, are responsible for cell plate formation and expansion during plant cytokinesis. Our previous research using the actin polymerization inhibitor latrunculin B (LatB) to investigate the role of actin filaments suggested the existence of two types of microtubules: 1) initial microtubules sensitive to LatB but unassociated with NACK1 kinesin and 2) later LatB-insensitive, NACK1-associated microtubules. The organization of initial phragmoplast microtubules might have been disrupted by the LatB treatment; this hypothesis remained unverified, however, as the exact timing of cell plate membrane accumulation could not be determined. In the present study, we further investigated the timing of cell plate formation during LatB treatment. We monitored chromosome separation during anaphase as well as accumulation of FM4-64-stained cell plate membranes in dividing transgenic tobacco BY-2 cells expressing RFP-tagged histone H2B. We observed that LatB treatment prolonged the time between the slowdown of daughter chromosome migration and the accumulation of cell plate membranes. This result suggests that disruption of actin filaments resulted in delayed cell plate formation possibly by perturbation of initial phragmoplast microtubules or cell plate assembly.

Global Profiling of lncRNAs Expression Responsive to Allopolyploidization in Cucumis.

Long non-coding RNAs (lncRNAs) play critical regulatory roles in various biological processes. However, the presence of lncRNAs and how they function in plant polyploidy are still largely unknown. Hence, we examined the profile of lncRNAs in a nascent allotetraploid Cucumis hytivus (S14), its diploid parents, and the F1 hybrid, to reveal the function of lncRNAs in plant-interspecific hybridization and whole genome duplication. Results showed that 2206 lncRNAs evenly transcribed from all 19 chromosomes were identified in C. hytivus, 44.6% of which were from intergenic regions. Based on the expression trend in allopolyploidization, we found that a high proportion of lncRNAs (94.6%) showed up-regulated expression to varying degrees following hybridization. However, few lncRNAs (33, 2.1%) were non-additively expressed after genome duplication, suggesting the significant effect of hybridization on lncRNAs, rather than genome duplication. Furthermore, 253 cis-regulated target genes were predicted for these differentially expressed lncRNAs in S14, which mainly participated in chloroplast biological regulation (e.g., chlorophyll synthesis and light harvesting system). Overall, this study provides new insight into the function of lncRNAs during the processes of hybridization and polyploidization in plant evolution.

State changes of the HORMA protein ASY1 are mediated by an interplay between its closure motif and PCH2.

HORMA domain-containing proteins (HORMADs) play an essential role in meiosis in many organisms. The meiotic HORMADs, including yeast Hop1, mouse HORMAD1 and HORMAD2, and Arabidopsis ASY1, assemble along chromosomes at early prophase and the closure motif at their C-termini has been hypothesized to be instrumental for this step by promoting HORMAD oligomerization. In late prophase, ASY1 and its homologs are progressively removed from synapsed chromosomes promoting chromosome synapsis and recombination. The conserved AAA+ ATPase PCH2/TRIP13 has been intensively studied for its role in removing HORMADs from synapsed chromosomes. In contrast, not much is known about how HORMADs are loaded onto chromosomes. Here, we reveal that the PCH2-mediated dissociation of the HORMA domain of ASY1 from its closure motif is important for the nuclear targeting and subsequent chromosomal loading of ASY1. This indicates that the promotion of ASY1 to an 'unlocked' state is a prerequisite for its nuclear localization and chromosomal assembly. Likewise, we find that the closure motif is also necessary for the removal of ASY1 by PCH2 later in prophase. Our work results in a unified new model for PCH2 and HORMADs function in meiosis and suggests a mechanism to contribute to unidirectionality in meiosis.

The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis.

Meiosis is key to sexual reproduction and genetic diversity. Here, we show that the Arabidopsis cyclin-dependent kinase Cdk1/Cdk2 homolog CDKA;1 is an important regulator of meiosis needed for several aspects of meiosis such as chromosome synapsis. We identify the chromosome axis protein ASYNAPTIC 1 (ASY1), the Arabidopsis homolog of Hop1 (homolog pairing 1), essential for synaptonemal complex formation, as a target of CDKA;1. The phosphorylation of ASY1 is required for its recruitment to the chromosome axis via ASYNAPTIC 3 (ASY3), the Arabidopsis reductional division 1 (Red1) homolog, counteracting the disassembly activity of the AAA+ ATPase PACHYTENE CHECKPOINT 2 (PCH2). Furthermore, we have identified the closure motif in ASY1, typical for HORMA domain proteins, and provide evidence that the phosphorylation of ASY1 regulates the putative self-polymerization of ASY1 along the chromosome axis. Hence, the phosphorylation of ASY1 by CDKA;1 appears to be a two-pronged mechanism to initiate chromosome axis formation in meiosis.

The condensin subunits SMC2 and SMC4 interact for correct condensation and segregation of mitotic maize chromosomes.

Structural Maintenance of Chromosomes 2 (SMC2) and Structural Maintenance of Chromosomes 4 (SMC4) are the core components of the condensin complexes, which are required for chromosome assembly and faithful segregation during cell division. Because of the crucial functions of both proteins in cell division, much work has been done in various vertebrates, but little information is known about their roles in plants. Here, we identified ZmSMC2 and ZmSMC4 in maize (Zea mays) and confirmed that ZmSMC2 associates with ZmSMC4 via their hinge domains. Immunostaining revealed that both proteins showed dynamic localization during mitosis. ZmSMC2 and ZmSMC4 are essential for proper chromosome segregation and for H3 phosphorylation at Serine 10 (H3S10ph) at pericentromeres during mitotic division. The loss of function of ZmSMC2 and ZmSMC4 enlarges mitotic chromosome volume and impairs sister chromatid separation to the opposite poles. Taken together, these findings confirm and extend the coordinated role of ZmSMC2 and ZmSMC4 in maintenance of normal chromosome architecture and accurate segregation during mitosis.

Involvement of abscisic acid-responsive element-binding factors in cassava (Manihot esculenta) dehydration stress response.

Cassava (Manihot esculenta) is a major staple food, animal feed and energy crop in the tropics and subtropics. It is one of the most drought-tolerant crops, however, the mechanisms of cassava drought tolerance remain unclear. Abscisic acid (ABA)-responsive element (ABRE)-binding factors (ABFs) are transcription factors that regulate expression of target genes involved in plant tolerance to drought, high salinity, and osmotic stress by binding ABRE cis-elements in the promoter regions of these genes. However, there is little information about ABF genes in cassava. A comprehensive analysis of Manihot esculenta ABFs (MeABFs) described the phylogeny, genome location, cis-acting elements, expression profiles, and regulatory relationship between these factors and Manihot esculenta betaine aldehyde dehydrogenase genes (MeBADHs). Here we conducted genome-wide searches and subsequent molecular cloning to identify seven MeABFs that are distributed unevenly across six chromosomes in cassava. These MeABFs can be clustered into three groups according to their phylogenetic relationships to their Arabidopsis (Arabidopsis thaliana) counterparts. Analysis of the 5'-upstream region of MeABFs revealed putative cis-acting elements related to hormone signaling, stress, light, and circadian clock. MeABF expression profiles displayed clear differences among leaf, stem, root, and tuberous root tissues under non-stress and drought, osmotic, or salt stress conditions. Drought stress in cassava leaves and roots, osmotic stress in tuberous roots, and salt stress in stems induced expression of the highest number of MeABFs showing significantly elevated expression. The glycine betaine (GB) content of cassava leaves also was elevated after drought, osmotic, or salt stress treatments. BADH1 is involved in GB synthesis. We show that MeBADH1 promoter sequences contained ABREs and that MeBADH1 expression correlated with MeABF expression profiles in cassava leaves after the three stress treatments. Taken together, these results suggest that in response to various dehydration stresses, MeABFs in cassava may activate transcriptional expression of MeBADH1 by binding the MeBADH1 promoter that in turn promotes GB biosynthesis and accumulation via an increase in MeBADH1 gene expression levels and MeBADH1 enzymatic activity. These responses protect cells against dehydration stresses by preserving an osmotic balance that enhances cassava tolerance to dehydration stresses.

Patronus is the elusive plant securin, preventing chromosome separation by antagonizing separase.

Chromosome distribution at anaphase of mitosis and meiosis is triggered by separase, an evolutionarily conserved protease. Separase must be tightly regulated to prevent the untimely release of chromatid cohesion and disastrous chromosome distribution defects. Securin is the key inhibitor of separase in animals and fungi, but has not been identified in other eukaryotic lineages. Here, we identified PATRONUS1 and PATRONUS2 (PANS1 and PANS2) as the Arabidopsis homologs of securin. Disruption of PANS1 is known to lead to the premature separation of chromosomes at meiosis, and the simultaneous disruption of PANS1 and PANS2 is lethal. Here, we show that PANS1 targeting by the anaphase-promoting complex is required to trigger chromosome separation, mirroring the regulation of securin. We showed that PANS1 acts independently from Shugosins. In a genetic screen for pans1 suppressors, we identified SEPARASE mutants, showing that PANS1 and SEPARASE have antagonistic functions in vivo. Finally, we showed that the PANS1 and PANS2 proteins interact directly with SEPARASE. Altogether, our results show that PANS1 and PANS2 act as a plant securin. Remote sequence similarity was identified between the plant patronus family and animal securins, suggesting that they indeed derive from a common ancestor. Identification of patronus as the elusive plant securin illustrates the extreme sequence divergence of this central regulator of mitosis and meiosis.

Reducing MSH4 copy number prevents meiotic crossovers between non-homologous chromosomes in Brassica napus.

In allopolyploids, correct chromosome segregation requires suppression of non-homologous crossovers while levels of homologous crossovers are ensured. To date, no mechanism able to specifically inhibit non-homologous crossovers has been described in allopolyploids other than in bread wheat. Here, we show that reducing the number of functional copies of MSH4, an essential gene for the main crossover pathway, prevents non-homologous crossovers in allotetraploid Brassica napus. We show that non-homologous crossovers originate almost exclusively from the MSH4-dependent recombination pathway and that their numbers decrease when MSH4 returns to single copy in B. napus; by contrast, homologous crossovers remain unaffected by MSH4 duplicate loss. We also demonstrate that MSH4 systematically returns to single copy following numerous independent polyploidy events, a pattern that is probably not by chance. These results suggest that stabilization of allopolyploid meiosis can be enhanced by loss of a key meiotic recombination gene.

Genome-wide association study of seed protein, oil and amino acid contents in soybean from maturity groups I to IV.

KEY MESSAGE: Genomic regions associated with seed protein, oil and amino acid contents were identified by genome-wide association analyses. Geographic distributions of haplotypes indicate scope of improvement of these traits. Soybean [Glycine max (L.) Merr.] protein and oil are used worldwide in feed, food and industrial materials. Increasing seed protein and oil contents is important; however, protein content is generally negatively correlated with oil content. We conducted a genome-wide association study using phenotypic data collected from five environments for 621 accessions in maturity groups I-IV and 34,014 markers to identify quantitative trait loci (QTL) for seed content of protein, oil and several essential amino acids. Three and five genomic regions were associated with seed protein and oil contents, respectively. One, three, one and four genomic regions were associated with cysteine, methionine, lysine and threonine content (g kg-1 crude protein), respectively. As previously shown, QTL on chromosomes 15 and 20 were associated with seed protein and oil contents, with both exhibiting opposite effects on the two traits, and the chromosome 20 QTL having the most significant effect. A multi-trait mixed model identified trait-specific QTL. A QTL on chromosome 5 increased oil with no effect on protein content, and a QTL on chromosome 10 increased protein content with little effect on oil content. The chromosome 10 QTL co-localized with maturity gene E2/GmGIa. Identification of trait-specific QTL indicates feasibility to reduce the negative correlation between protein and oil contents. Haplotype blocks were defined at the QTL identified on chromosomes 5, 10, 15 and 20. Frequencies of positive effect haplotypes varied across maturity groups and geographic regions, providing guidance on which alleles have potential to contribute to soybean improvement for specific regions.

Genome-wide analysis of oligopeptide transporters and detailed characterization of yellow stripe transporter genes in hexaploid wheat.

Oligopeptide transporters (OPT) are integral cell membrane proteins that play a critical role in the transport of small peptides, secondary amino acids, glutathione conjugates, and mineral uptake. In the present study, 67 putative wheat yellow stripe-like transporter (YSL) proteins belonging to the subfamily of OPT transporters were identified. Phylogeny analysis resulted in the distribution of wheat YSLs into four discrete clades. The highest number of YSLs was present on the A genome and the chromosome 2 of hexaploid wheat. The identified wheat YSL genes showed differential expression in different tissues and during grain development suggesting the importance of this subfamily. Gene expression pattern of TaYSLs during iron starvation experiments suggested an early high transcript accumulation of TaYS1A, TaYS1B, TaYSL3, TaYSL5, and TaYSL6 in roots. In contrast, delayed expression was observed in shoots for TaYS1A, TaYS1B, TaYSL5, TaYSL12, and TaYSL19 as compared to control. Further, their expression under biotic and abiotic response emphasized their alternative functions during the plant growth and development. In conclusion, this work is the first comprehensive study of wheat YSL transporters and would be an important resource for prioritizing genes towards wheat biofortification.

A Low Resolution Epistasis Mapping Approach To Identify Chromosome Arm Interactions in Allohexaploid Wheat.

Epistasis is an important contributor to genetic variance. In inbred populations, pairwise epistasis is present as additive by additive interactions. Testing for epistasis presents a multiple testing problem as the pairwise search space for modest numbers of markers is large. Single markers do not necessarily track functional units of interacting chromatin as well as haplotype based methods do. To harness the power of multiple markers while minimizing the number of tests conducted, we present a low resolution test for epistatic interactions across whole chromosome arms. Epistasis covariance matrices were constructed from the additive covariances of individual chromosome arms. These covariances were subsequently used to estimate an epistatic variance parameter while correcting for background additive and epistatic effects. We find significant epistasis for 2% of the interactions tested for four agronomic traits in a winter wheat breeding population. Interactions across homeologous chromosome arms were identified, but were less abundant than other chromosome arm pair interactions. The homeologous chromosome arm pair 4BL/4DL showed a strong negative relationship between additive and interaction effects that may be indicative of functional redundancy. Several chromosome arms appeared to act as hubs in an interaction network, suggesting that they may contain important regulatory factors. The differential patterns of epistasis across different traits demonstrate that detection of epistatic interactions is robust when correcting for background additive and epistatic effects in the population. The low resolution epistasis mapping method presented here identifies important epistatic interactions with a limited number of statistical tests at the cost of low precision.

Chromosome Doubling-Enhanced Biomass and Dihydrotanshinone I Production in Salvia miltiorrhiza, A Traditional Chinese Medicinal Plant.

The root of Chinese sage (Salvia miltiorrhiza Bunge) was regarded as top-grade Chinese medicine two thousand years ago, according to Shen Nong Materia Medica. The aim of this study is to develop an easy and reliable means for obtaining tetraploids (4x plants) via thidiazuron-induced direct organogenesis in the presence of colchicine. The resulting 4x plants showed significantly enhanced agronomic traits, including the size of stomata, leaflet, pollen, and seed as well as shoot length, root diameter, number of leaves, and fresh weight of plant. In addition, an obvious reduction of length to width ratio was found in the 4x plants, including stomata, leaflets, pollens, seeds, and roots. The 4x ploidy state of the plants was stable as was proved by evaluation of selection indicators as well as consistent ploidy level at 10th generation plantlets and also on 4x seedlings obtained via self-pollination. The major bioactive compounds, salvianolic acid B, tanshinone I, tanshinone IIA, dihydrotanshinone I and cryptotanshinone, as well as total tanshinones were determined by high performance liquid chromatography (HPLC). The concentrations of dihydrotanshinone I and total tanshinones in the root extract of the 4x plants were significantly higher when compared with the 2x plants. This present study developed a simple and efficient system for inducing and subculture of tetrapolids which have stable ploidy level, enhanced growth characteristics as well as the content of dihydrotanshinone I in the root of S. miltiorrhiza.

Centromeric DNA characterization in the model grass Brachypodium distachyon provides insights on the evolution of the genus.

Brachypodium distachyon is a well-established model monocot plant, and its small and compact genome has been used as an accurate reference for the much larger and often polyploid genomes of cereals such as Avena sativa (oats), Hordeum vulgare (barley) and Triticum aestivum (wheat). Centromeres are indispensable functional units of chromosomes and they play a core role in genome polyploidization events during evolution. As the Brachypodium genus contains about 20 species that differ significantly in terms of their basic chromosome numbers, genome size, ploidy levels and life strategies, studying their centromeres may provide important insight into the structure and evolution of the genome in this interesting and important genus. In this study, we isolated the centromeric DNA of the B. distachyon reference line Bd21 and characterized its composition via the chromatin immunoprecipitation of the nucleosomes that contain the centromere-specific histone CENH3. We revealed that the centromeres of Bd21 have the features of typical multicellular eukaryotic centromeres. Strikingly, these centromeres contain relatively few centromeric satellite DNAs; in particular, the centromere of chromosome 5 (Bd5) consists of only ~40 kb. Moreover, the centromeric retrotransposons in B. distachyon (CRBds) are evolutionarily young. These transposable elements are located both within and adjacent to the CENH3 binding domains, and have similar compositions. Moreover, based on the presence of CRBds in the centromeres, the species in this study can be grouped into two distinct lineages. This may provide new evidence regarding the phylogenetic relationships within the Brachypodium genus.