A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps.

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

Genome-enabled discovery of anthraquinone biosynthesis in Senna tora.

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we reveal the genome sequence of S. tora at the chromosome level with 526 Mb (96%) assembled into 13 chromosomes. Comparison among related plant species shows that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora. Combining genomics, transcriptomics, metabolomics, and biochemistry, we identify a CHS-L gene contributing to the biosynthesis of anthraquinones. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants.

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.

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.

Exploration of acute genotoxic effects and antigenotoxic potential of gambogic acid using Allium cepa assay.

The plant derived xanthanoid gambogic acid (GA) is well known for its anticancer activity. To date, biological actions of GA on plant system have not been reported. In the present study, we evaluated the potential acute genotoxic activity of GA, and its antigenotoxic potential against H2O2 induced genetic damage using Allium cepa root chromosomal aberration assay under hydroponic conditions. There was a significant decrease in the percentage of mitotic index/prophase index with the increase in clastogenicity percentage in a dose and time-dependent manner when Allium cepa bulbs were exposed to GA at 0.1 mM and 1 mM concentration for 1 h, 2 h, and 4 h. Total genomic DNA integrity analyzed by agarose gel electrophoresis and cell viability revealed pronounced DNA degradation and loss of viability when treated with 1  mM GA for 4 h. In situ histochemical localization by Schiff's staining and 3, 3-diaminobenzidine confirmed increased levels of lipid peroxide and H2O2 in GA treated roots respectively. Scanning electron microscopy and FT-IR suggested surface damage and biomolecular intervention of GA in root cells. In addition, possible antigenotoxic effect of GA at lower concentration was explored by employing standard assays using H2O2. We observed a higher percentage of nuclear lesions upon treatment with 3% H2O2 (97.21 ± 0.76) that reduced significantly after modulatory treatment with 0.01 mM GA (70.44 ± 4.42). The results suggest that GA is a Janus-faced compound as it demonstrates a genotoxic activity at higher doses and genoprotective action at lower precise doses.

A comparative study of the dynamic accumulation of polyphenol components and the changes in their antioxidant activities in diploid and tetraploid Lonicera japonica.

Polyploidization is an effective method to achieve a higher yield of secondary metabolism active ingredients in medicinal plants. Polyphenols are the main active substances that contribute to the antioxidant activity of Lonicera japonica. For studying on the effect of chromosome doubling and harvest time on the dynamic accumulation of the main active substances and antioxidant capabilities of L. japonica, the polyphenol composition contents (7 phenolic acids and 3 flavonoids) and the antioxidant capacity in buds and flowers of diploid and tetraploid L. japonica at six different growth stages were determined by HPLC-DAD and three common antioxidant assays (FRAP, OH RSC and DPPH ARP), and the correlation between the dynamic accumulation of the polyphenol components and antioxidant capacity was also analyzed in current research. The results indicated that the content of the most determined phenolic acids and flavonoids and the antioxidant capacity in most of the growth stages from tetraploid plants were significantly higher than those in the diploid plants. Furthermore, the changes in the antioxidant activity presented a significant positive correlation with the variations in the chlorogenic acid, rutin, hyperoside, luteoloside in the two ploidy levels of L. japonica plants. The higher yields of chlorogenic acid (158.97, 164.00, 199.85 mg), luteoloside (5.44, 4.03, 6.31 mg), hyperoside (1.15, 1.06, 1.30 mg) and total flavonoids (9.87, 8.67, 11.10 mg) from 100 buds or flowers in tetraploid plants occurred during the S3-S5 stages, and these stages also exhibited higher antioxidant activities. Therefore, the stages of S3-S5 are recommended as the best time for harvesting high-yield, high-quality tetraploid Flos Lonicerae Japonicae.

Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination.

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ∼6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice.

Distinct chromatin environment associated with phosphorylated H3S10 histone during pollen mitosis I in orchids.

Pollen developmental pathway in plants involving synchronized transferal of cellular divisions from meiosis (microsporogenesis) to mitosis (pollen mitosis I/II) eventually offers a unique "meiosis-mitosis shift" at pollen mitosis I. Since the cell type (haploid microspore) and fate of pollen mitosis I differ from typical mitosis (in meristem cells), it is immensely important to analyze the chromosomal distribution of phosphorylated H3S10 histone during atypical pollen mitosis I to comprehend the role of histone phosphorylation in pollen development. We investigated the chromosomal phosphorylation of H3S10 histone during pollen mitosis I in orchids using immunostaining technique. The chromosomal distribution of H3S10ph during pollen mitosis I revealed differential pattern than that of typical mitosis in plants, however, eventually following the similar trends of mitosis in animals where H3S10 phosphorylation begins in the pericentromeric regions first, later extending to the whole chromosomes, and finally declining at anaphase/early cytokinesis (differentiation of vegetative and generative cells). The study suggests that the chromosomal distribution of H3S10ph during cell division is not universal and can be altered between different cell types encoded for diverse cellular processes. During pollen development, phosphorylation of histone might play a critical role in chromosome condensation events throughout pollen mitosis I in plants.

Global DNA methylation variations after short-term heat shock treatment in cultured microspores of Brassica napus cv. Topas.

Heat stress can induce the cultured microspores into embryogenesis. In this study, whole genome bisulphite sequencing was employed to study global DNA methylation variations after short-term heat shock (STHS) treatments in cultured microspores of Brassica napus cv. Topas. Our results indicated that treatment on cultured Topas microspores at 32 °C for 6 h triggered DNA hypomethylation, particularly in the CG and CHG contexts. And the total number of T32 (Topas 32 °C for 6 h) vs. T0 (Topas 0 h) differentially methylated region-related genes (DRGs) was approximately two-fold higher than that of T18 (Topas 18 °C for 6 h) vs. T0 DRGs, which suggested that 32 °C might be a more intense external stimulus than 18 °C resulting in more changes in the DNA methylation status of cultured microspores. Additionally, 32 °C treatment for 6 h led to increased CHG differential methylations of transposons (DMTs), which were mainly constituted by overlaps between the hypomethylated differentially methylated regions (hypo-DMRs) and transposon elements (TEs). Further analysis demonstrated that the DRGs and their paralogs exhibited differential methylated/demethylated patterns. To summarize, the present study is the first methylome analysis of cultured microspores in response to STHS and may provide valuable information on the roles of DNA methylation in heat response.

Integration of multi-omics data for prediction of phenotypic traits using random forest.

BACKGROUND: In order to find genetic and metabolic pathways related to phenotypic traits of interest, we analyzed gene expression data, metabolite data obtained with GC-MS and LC-MS, proteomics data and a selected set of tuber quality phenotypic data from a diploid segregating mapping population of potato. In this study we present an approach to integrate these ~ omics data sets for the purpose of predicting phenotypic traits. This gives us networks of relatively small sets of interrelated ~ omics variables that can predict, with higher accuracy, a quality trait of interest. RESULTS: We used Random Forest regression for integrating multiple ~ omics data for prediction of four quality traits of potato: tuber flesh colour, DSC onset, tuber shape and enzymatic discoloration. For tuber flesh colour beta-carotene hydroxylase and zeaxanthin epoxidase were ranked first and forty-fourth respectively both of which have previously been associated with flesh colour in potato tubers. Combining all the significant genes, LC-peaks, GC-peaks and proteins, the variation explained was 75 %, only slightly more than what gene expression or LC-MS data explain by themselves which indicates that there are correlations among the variables across data sets. For tuber shape regressed on the gene expression, LC-MS, GC-MS and proteomics data sets separately, only gene expression data was found to explain significant variation. For DSC onset, we found 12 significant gene expression, 5 metabolite levels (GC) and 2 proteins that are associated with the trait. Using those 19 significant variables, the variation explained was 45 %. Expression QTL (eQTL) analyses showed many associations with genomic regions in chromosome 2 with also the highest explained variation compared to other chromosomes. Transcriptomics and metabolomics analysis on enzymatic discoloration after 5 min resulted in 420 significant genes and 8 significant LC metabolites, among which two were putatively identified as caffeoylquinic acid methyl ester and tyrosine. CONCLUSIONS: In this study, we made a strategy for selecting and integrating multiple ~ omics data using random forest method and selected representative individual peaks for networks based on eQTL, mQTL or pQTL information. Network analysis was done to interpret how a particular trait is associated with gene expression, metabolite and protein data.

The wheat calcium-dependent protein kinase TaCPK7-D positively regulates host resistance to sharp eyespot disease.

Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, limits wheat production worldwide. Here, TaCPK7-D, encoding a subgroup III member of the calcium-dependent protein kinase (CPK) family, was identified from the sharp eyespot-resistant wheat line CI12633 through comparative transcriptomic analysis. Subsequently, the defence role of TaCPK7-D against R. cerealis infection was studied by the generation and characterization of TaCPK7-D-silenced and TaCPK7-D-overexpressing wheat plants. Rhizoctonia cerealis inoculation induced a higher transcriptional level of TaCPK7-D in the resistant wheat line CI12633 than in the susceptible cultivar Wenmai 6. The expression of TaCPK7-D was significantly induced after exogenous application of 1-aminocyclopropane-1-carboxylic acid (an ethylene biosynthesis precursor). The green fluorescent protein signal distribution assays indicated that TaCPK7-D localizes to the plasma membrane in both onion epidermal cells and wheat protoplasts. Following R. cerealis inoculation, TaCPK7-D-silenced wheat CI12633 plants displayed more severe sharp eyespot symptoms than control CI12633 plants. Four defence-associated genes (ß-1,3-glucanase, chitinase 1, defensin and TaPIE1) and an ethylene biosynthesis key gene, ACO2, were significantly suppressed in the TaCPK7-D-silenced wheat plants compared with control plants. Conversely, TaCPK7-D-overexpressing wheat lines showed increased resistance to sharp eyespot compared with untransformed recipient wheat Yangmai 16. Furthermore, the transcriptional levels of these four defence-related genes and ACO2 gene were significantly elevated in TaCPK7-D-overexpressing plants compared with untransformed recipient wheat plants. These results suggest that TaCPK7-D positively regulates the wheat resistance response to R. cerealis infection through the modulation of the expression of these defence-associated genes, and that TaCPK7-D is a candidate to improve sharp eyespot resistance in wheat.

[In vitro autotetraploid induction and analysis on DNA methylation diversity of Platycodon grandiflorum].

In order to investigate the epigenetic variations between diploid and autotetraploid of Platycodon grandiflorus. The diploid buds of P. grandiflorus were soaked in the mixture of different concentration colchicines and 0.002 g•mL ⁻¹ dimethyl sulphoxide (DMSO)．The identification of autotetraploid plants were based on morphological characteristics, chromosome number and flow cytometry. And then the level and pattern of DNA methylation explored by using the technology of methylation sensitive amplified polymorphism (MSAP)．The result demonstrated that the buds soaked in 0.2% colchicines and 0.002 g•mL ⁻¹ DMSO solution for 12 h was ideal conditions to induce autotetraploid of P. grandiflorus, with induction rate of 32.0%.The diploid and tetraploid plants existed distinctly differences in morphological indexes.Totally,1 586 bands were amplified by 20 pairs of selective primers, of which 764 and 822 bands were detected in diploid and autotetraploid respectively. The total methylation ratio,full methylation ratio and hemimethylated ratio were 91.25%,61.25% and 30.65% in diploid of P. grandiflorus,respectively.However,the total methylation ratio,full methylation ratio and hemimethylated ratio of autotetraploid of P. grandiflorus were 86.13%,54.38% and 31.75%, respectively. Compared with diploid, the genomic DNA total methylate ratio and full methylation ratio of autotetration plants decreased by 6.02% and 7.14%.But the hemimethylated ratio of autotetraploid was higher than that of diploid, which more than 1.6%. All this results indicated that DNA methylation patterns have adjusted during the polyploidy process．.

Pre-apoptotic activity of aqueous extracts of Cynanchum sarcomedium Meve & Liede on cells of Allium cepa and human erythrocytes.

Cynanchum sarcomedium Meve & Liede is a member of Apocynaceae, seen in dry and rocky areas. The present study highlights the cytotoxic potential of C. sarcomedium mediated by apoptosis on cells of Allium cepa and human red blood cells (RBCs). Cytogenetic changes in A. cepa and in situ visualization of cell death were revealed through acetocarmine and Evans blue staining techniques. Quantitative estimation of cell death was carried out at 600 nm in a spectrophotometer. Membrane characteristics of RBC in response to the treatment were evaluated by May-Grünwald-Giemsa staining and scanning electron microscopy (SEM). Cell membrane damage is a major factor for assessing apoptosis which is observed in the present study (90.91 %). Cell shrinkage, cytoplasmic fragmentation, condensed chromatin and presence of apoptotic bodies were the common cytological changes in A. cepa associated with apoptosis. Blebs in RBC evidenced by SEM revealed the membrane damage potential of the plant. Results obtained hereby suggest that the plant is an effective source to be used in toxicological studies and anti-cancer therapy.

The role of OsMSH4 in male and female gamete development in rice meiosis.

Meiosis is essential for gametogenesis in sexual reproduction in rice (Oryza sativa L.). We identified a MutS-homolog (MSH) family gene OsMSH4 in a trisomic plant. Cytological analysis showed that developments of both pollen and embryo sacs in an Osmsh4 mutant were blocked due to defective chromosome pairing. Compared with the wild type, the Osmsh4 mutant displayed a significant ~21.9% reduction in chiasma frequency, which followed a Poisson distribution, suggesting that class I crossover formation in the mutant was impaired. Temporal and spatial expression pattern analyses showed that OsMSH4 was preferentially expressed in meiocytes during their meiosis, indicating a critical role in gametogenesis. Subcellular localization showed that OsMSH4-green fluorescent protein was predominantly located in the nucleus. OsMSH4 could interact with another MSH member (OsMSH5) through the N-terminus and C-terminus, respectively. Direct physical interaction between OsMSH5, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c was identified by yeast two-hybrid assays and further validated by pull-down assays. Our results supported the conclusion that the OsMSH4/5 heterodimer plays a key role in regulation of crossover formation during rice meiosis by interaction with the RPA complex.

Analysis of the age of Panax ginseng based on telomere length and telomerase activity.

Ginseng, which is the root of Panax ginseng (Araliaceae), has been used in Oriental medicine as a stimulant and dietary supplement for more than 7,000 years. Older ginseng plants are substantially more medically potent, but ginseng age can be simulated using unscrupulous cultivation practices. Telomeres progressively shorten with each cell division until they reach a critical length, at which point cells enter replicative senescence. However, in some cells, telomerase maintains telomere length. In this study, to determine whether telomere length reflects ginseng age and which tissue is best for such an analysis, we examined telomerase activity in the main roots, leaves, stems, secondary roots and seeds of ginseng plants of known age. Telomere length in the main root (approximately 1 cm below the rhizome) was found to be the best indicator of age. Telomeric terminal restriction fragment (TRF) lengths, which are indicators of telomere length, were determined for the main roots of plants of different ages through Southern hybridization analysis. Telomere length was shown to be positively correlated with plant age, and a simple mathematical model was formulated to describe the relationship between telomere length and age for P. ginseng.

Meiotic studies in some species of tribe Cichorieae (Asteraceae) from Western Himalayas.

The present paper deals with meiotic studies in 15 species belonging to 6 genera of the tribe Cichorieae from various localities of Western Himalayas. The chromosome number has been reported for the first time in Hieracium crocatum (2n = 10) and Lactuca lessertiana (2n = 2x = 16). Further, intraspecific variability has been reported for the first time in H. umbellatum (2n = 2x = 10 and 2n = 6x = 54), Tragopogon dubius (2n = 2x = 14 and 2n = 4x = 28), and T. gracilis (2n = 2x = 14). The chromosome report of 2n = 2x = 10 in Youngia tenuifolia is made for the first time in India. Maximum numbers of the populations show laggards, chromosome stickiness, and cytomixis from early prophase to telophase-II, leading to the formation of aneuploid cells or meiocytes with double chromosome number. Such meiotic abnormalities produce unreduced pollen grains and the reduced pollen viability.

Induction of microtubule damage in Allium cepa meristematic cells by pharmaceutical formulations of thiabendazole and griseofulvin.

Microtubules (MT) are formed by the assembly of α- and ß-tubulins and MT-associated proteins. We characterized the effects of pharmaceutical formulations containing the microtubule disruptors thiabendazole (TBZ) and griseofulvin (GF) on the mitotic machinery of plant (A. cepa) meristematic cells. GF concentrations between 10 and 250 µg/ml were tested. GF induced mitotic index inhibition and genotoxic effects, including chromosome fragments, bridges, lagged chromosomes, C-metaphases, tripolar cell division, disorganized anaphases and nuclear abnormalities in interphase cells. Efects on the mitotic machinery were studied by direct immunofluorescence with ß-tubulin labeling and by DNA counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Exposure of meristematic root cells to TBZ or GF, 100 µg/ml, caused microtubular damage which led to abnormal MT arrays. Our results suggest that GF induces abnormalities in spindle symmetry/polarity, while TBZ causes chromosome missegregation, polyploidy, and lack of cytokinesis.

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes.

Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48A(NPL4) complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction.

The conserved chimeric transcript UPGRADE2 is associated with unreduced pollen formation and is exclusively found in apomictic Boechera species.

In apomictic Boechera spp., meiotic diplospory leads to the circumvention of meiosis and the suppression of recombination to produce unreduced male and female gametes (i.e. apomeiosis). Here, we have established an early flower developmental staging system and have performed microarray-based comparative gene expression analyses of the pollen mother cell stage in seven diploid sexual and seven diploid apomictic genotypes to identify candidate factors for unreduced pollen formation. We identified a transcript unique to apomictic Boechera spp. called UPGRADE2 (BspUPG2), which is highly up-regulated in their pollen mother cells. BspUPG2 is highly conserved among apomictic Boechera spp. genotypes but has no homolog in sexual Boechera spp. or in any other taxa. BspUPG2 undergoes posttranscriptional processing but lacks a prominent open reading frame. Together with the potential of stably forming microRNA-like secondary structures, we hypothesize that BspUPG2 functions as a long regulatory noncoding messenger RNA-like RNA. BspUPG2 has apparently arisen through a three-step process initiated by ancestral gene duplication of the original BspUPG1 locus, followed by sequential insertions of segmentally duplicated gene fragments, with final exonization of its sequence structure. Its genesis reflects the hybridization history that characterizes the genus Boechera.

Construction of reference chromosome-scale pseudomolecules for potato: integrating the potato genome with genetic and physical maps.

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker-based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal "pseudomolecules".