Biogeographical patterns and speciation of the genus Pinguicula (Lentibulariaceae) inferred by phylogenetic analyses.

Earlier phylogenetic studies in the genus Pinguicua (Lentibulariaceae) suggested that the species within a geographical region was rather monophyletic, although the sampling was limited or was restricted to specific regions. Those results conflicted with the floral morphology-based classification, which has been widely accepted to date. In the current study, one nuclear ribosomal DNA (internal transcribed spacer; ITS) and two regions of chloroplast DNA (matK and rpl32-trnL), from up to ca. 80% of the taxa in the genus Pinguicula, covering all three subgenera, were sequenced to demonstrate the inconsistency and explore a possible evolutionary history of the genus. Some incongruence was observed between nuclear and chloroplast topologies and the results from each of the three DNA analyses conflicted with the morphology-based subgeneric divisions. Both the ITS tree and network, however, corresponded with the biogeographical patterns of the genus supported by life-forms (winter rosette or hibernaculum formation) and basic chromosome numbers (haploidy). The dormant strategy evolved in a specific geographical region is a phylogenetic constraint and a synapomorphic characteristic within a lineage. Therefore, the results denied the idea that the Mexican group, morphologically divided into the three subgenera, independently acquired winter rosette formations. Topological incongruence among the trees or reticulations, indicated by parallel edges in phylogenetic networks, implied that some taxa originated by introgressive hybridisation. Although there are exceptions, species within the same geographical region arose from a common ancestor. Therefore, the classification by the floral characteristics is rather unreliable. The results obtained from this study suggest that evolution within the genus Pinguicula has involved; 1) ancient expansions to geographical regions with gene flow and subsequent vicariance with genetic drift, 2) acquirement of a common dormant strategy within a specific lineage to adapt a local climate (i.e., synapomorphic characteristic), 3) recent speciation in a short time span linked to introgressive hybridisation or multiplying the ploidy level (i.e., divergence), and 4) parallel evolution in floral traits among lineages found in different geographical regions (i.e., convergence). As such, the floral morphology masks and obscures the phylogenetic relationships among species in the genus.

Agrobacterium-mediated Tnt1 mutagenesis of moss protonemal filaments and generation of stable mutants with impaired gametophyte.

The gametophyte of moss exhibits a simple body plan, yet its growth is regulated by complex developmental phenomena similar to angiosperms. Because moss can be easily maintained under laboratory conditions, amenable for gene targeting and the availability of genome sequence, P. patens has become an attractive model system for studying evolutionary traits. Until date, there has been no Agrobacterium-mediated Tnt1 mutagenesis protocol for haploid protonemal filaments of moss. Hence, we attempted to use the intact tobacco Tnt1 retrotransposon as a mutagen for P. patens. Bioinformatic analysis of initiator methionyl-tRNA (Met-tRNAi), a critical host factor for Tnt1 transposition process, suggested that it can be explored as a mutagen for bryophytes. Using protonemal filaments and Agrobacterium-mediated transformation, 75 Tnt1 mutants have been generated and cryopreserved. SSAP analysis and TAIL-PCR revealed that Tnt1 is functional in P. patens and has a high-preference for gene and GC-rich regions. In addition, LTR::GUS lines exhibited a basal but tissue-specific inducible expression pattern. Forward genetic screen resulted in 5 novel phenotypes related to hormonal and gravity response, phyllid, and gamete development. SSAP analysis suggests that the Tnt1 insertion pattern is stable under normal growth conditions and the high-frequency phenotypic deviations are possibly due to the combination of haploid explant (protonema) and the choice of mutagen (Tnt1). We demonstrate that Agrobacterium-mediated Tnt1 insertional mutagenesis could generate stable P. patens mutant populations for future forward genetic studies.

Quantitative and qualitative assessment of pollen DNA metabarcoding using constructed species mixtures.

Pollen DNA metabarcoding-marker-based genetic identification of potentially mixed-species pollen samples-has applications across a variety of fields. While basic species-level pollen identification using standard DNA barcode markers is established, the extent to which metabarcoding (a) correctly assigns species identities to mixes (qualitative matching) and (b) generates sequence reads proportionally to their relative abundance in a sample (quantitative matching) is unclear, as these have not been assessed relative to known standards. We tested the quantitative and qualitative robustness of metabarcoding in constructed pollen mixtures varying in species richness (1-9 species), taxonomic relatedness (within genera to across class) and rarity (5%-100% of grains), using Illumina MiSeq with the markers rbcL and ITS2. Qualitatively, species composition determinations were largely correct, but false positives and negatives occurred. False negatives were typically driven by lack of a barcode gap or rarity in a sample. Species richness and taxonomic relatedness, however, did not strongly impact correct determinations. False positives were likely driven by contamination, chimeric sequences and/or misidentification by the bioinformatics pipeline. Quantitatively, the proportion of reads for each species was only weakly correlated with its relative abundance, in contrast to suggestions from some other studies. Quantitative mismatches are not correctable by consistent scaling factors, but instead are context-dependent on the other species present in a sample. Together, our results show that metabarcoding is largely robust for determining pollen presence/absence but that sequence reads should not be used to infer relative abundance of pollen grains.

Topological assessment of metabolic networks reveals evolutionary information.

Evolutionary information was inferred from the topology of metabolic networks corresponding to 17 plant species belonging to major plant lineages Chlorophytes, Bryophytes, Lycophytes and Angiosperms. The plant metabolic networks were built using the substrate-product network modeling based on the metabolic reactions available on the PlantCyc database (version 9.5), from which their local topological properties such as degree, in-degree, out-degree, clustering coefficient, hub-score, authority-score, local efficiency, betweenness and eigencentrality were measured. The topological measurements corresponding to each metabolite within the networks were considered as a set of metabolic characters to compound a feature vector representing each plant. Our results revealed that some local topological characters are able to discern among plant kinships, since similar phylogenies were found when comparing dendrograms obtained by topological metrics to the one obtained by DNA sequences of chloroplast genes. Furthermore, we also found that even a smaller number of metabolic characters is able to separate among major clades with high bootstrap support (BS > 95), while for some suborders a bigger content has been required.

Study of Some Zanthoxylum Species by Chemical and DNA Analysis Approaches.

The authentication and traceability of spices is a major concern for industrials and consumers. We focused on species from Zanthoxylum genera which are used for many different applications by local populations and also for trading as spices (dried pericarps or whole fruits). In this case, literature gives contradictory data about botanical names, and commercial labelling is often confusing. We studied commercial fruits pericarps extracts obtained by supercritical CO2 and analyzed them by GC/MS. The very complex volatile and semi volatile fractions composition of each extract is described. The barcoding method including molecular biology and phylogenetic analyses was also developed in order to check the commercial botanical identification of the raw material. This is a robust method to identify species in berries samples. We used one genetic marker to identify two Rutaceae clusters, including several species of Zanthoxylum genus. These results indicate that Fagara and Zanthoxylum groups could be considered as two different genera. Combination of chemical analysis and DNA analysis provides an original approach to increase chemical and botanical Zanthoxylum genus knowledge.

Species identification approach for both raw materials and end products of herbal supplements from Tinospora species.

BACKGROUND: Nowadays herbal products used in traditional medicine are sold in processed forms and thus morphological authentication is almost impossible. With herbal industry rapidly growing size, consumer safety becomes an important issue that requires special attention. Identification of herbal species in the products is therefore needed. METHODS: Sequences from the selected regions (matK, rbcL, trnL and ITS1) were retrieved and analysed. Then the most suitable barcode was assessed for discrimination of T. crispa from closely related species by HRM analysis and used in authentication of commercial products. RESULTS: The ITS1 barcode was found to be the suitable primer as melting data from the HRM assay proved to be capable of distinguishing T. crispa from its related species. The developed protocol was then employed to authenticate medicinal products in powdered form. HRM analysis of all tested samples here revealed that five out of eight products contained not only the indicated species T. crispa but also other Tinospora, that have a high level of morphological similarity. CONCLUSION: Misrepresentation, poor packaging and inappropriate labeling of the tested medicinal herbal products are thought to be the reason of the results here. Using Bar-HRM with the ITS marker lead to success in authenticating the tested herbal products.

Character-based DNA barcoding for authentication and conservation of IUCN Red listed threatened species of genus Decalepis (Apocynaceae).

The steno-endemic species of genus Decalepis are highly threatened by destructive wild harvesting. The medicinally important fleshy tuberous roots of Decalepis hamiltonii are traded as substitute, to meet the international market demand of Hemidesmus indicus. In addition, the tuberous roots of all three species of Decalepis possess similar exudates and texture, which challenges the ability of conventional techniques alone to perform accurate species authentication. This study was undertaken to generate DNA barcodes that could be utilized in monitoring and curtailing the illegal trade of these endangered species. The DNA barcode reference library was developed in BOLD database platform for candidate barcodes rbcL, matK, psbA-trnH, ITS and ITS2. The average intra-specific variations (0-0.27%) were less than the distance to nearest neighbour (0.4-11.67%) with matK and ITS. Anchoring the coding region rbcL in multigene tiered approach, the combination rbcL + matK + ITS yielded 100% species resolution, using the least number of loci combinations either with PAUP or BLOG methods to support a character-based approach. Species-specific SNP position (230 bp) in the matK region that is characteristic of D. hamiltonii could be used to design specific assays, enhancing its applicability for direct use in CITES enforcement for distinguishing it from H. indicus.

DNA barcoding analysis and phylogenetic relationships of tree species in tropical cloud forests.

DNA barcoding is a useful tool for species identification and phylogenetic construction. But present studies have far reached a consistent result on the universality of DNA barcoding. We tested the universality of tree species DNA barcodes including rbcL, matK, trnH-psbA and ITS, and examined their abilities of species identification and phylogenetic construction in three tropical cloud forests. Results showed that the success rates of PCR amplification of rbcL, matK, trnH-psbA and ITS were 75.26% ± 3.65%, 57.24% ± 4.42%, 79.28% ± 7.08%, 50.31% ± 6.64%, and the rates of DNA sequencing were 63.84% ± 4.32%, 50.82% ± 4.36%, 72.87% ± 11.37%, 45.15% ± 8.91% respectively, suggesting that both rbcL and trnH-psbA are universal for tree species in the tropical cloud forests. The success rates of species identification of the four fragments were higher than 41.00% (rbcL: 41.50% ± 2.81%, matK: 42.88% ± 2.59%, trnH-psbA: 46.16% ± 5.11% and ITS: 47.20% ± 5.76%), demonstrating that these fragments have potentiality in species identification. When the phylogenetic relationships were built with random fragment combinations, optimal evolutionary tree with high supporting values were established using the combinations of rbcL + matK + trnH-psbA in tropical cloud forests.

Establishment of the most comprehensive ITS2 barcode database to date of the traditional medicinal plant Rhodiola (Crassulacaee).

The roots and rhizomes of Rhodiola crenulata and R. rosea have been used worldwide as adaptogens for hundreds of years. However, rapid growth in demand has resulted in merchants using other species of Rhodiola as adulterants. Here, we surveyed 518 individuals representing 47 of the 55 species in the genus, including 253 R. crenulata individuals from 16 populations and 98 R. rosea individuals from 11 populations, to evaluate the utility of the internal transcribed spacer 2 (ITS2) barcode for identification of Rhodiola species. We detected six haplotypes in R. crenulata and only one haplotype in R. rosea. An obvious overlap between intra- and inter-specific distance was detected, and the authentication efficacy of ITS2, which was assessed by BLAST1, a nearest distance method, and a tree test, was much lower than in other groups. However, R. crenulata and R. rosea could be exactly identified. Analysis showed that the secondary structure of ITS2 differs in R. crenulata and its closest relatives. Our results demonstrated that both a mini barcode from ITS2 and the structure of ITS2 are effective markers for the identification of R. crenulata and R. rosea. This study represents the most comprehensive database of ITS2 barcodes in Rhodiola to date and will be useful in Rhodiola species identification.

Insight into Central Asian flora from the Cenozoic Tianshan montane origin and radiation of Lagochilus (Lamiaceae).

The Tianshan Mountains play a significant role in the Central Asian flora and vegetation. Lagochilus has a distribution concentration in Tianshan Mountains and Central Asia. To investigate generic spatiotemporal evolution, we sampled most Lagochilus species and sequenced six cpDNA locations (rps16, psbA-trnH, matK, trnL-trnF, psbB-psbH, psbK-psbI). We employed BEAST Bayesian inference for dating, and S-DIVA, DEC, and BBM for ancestral area/biome reconstruction. Our results clearly show that the Tianshan Mountains, especially the western Ili-Kirghizia Tianshan, as well as Sunggar and Kaschgar, was the ancestral area. Ancestral biome was mainly in the montane steppe zone of valley and slope at altitudes of 1700-2700 m, and the montane desert zone of foothill and front-hill at 1000-1700 m. Here two sections Inermes and Lagochilus of the genus displayed "uphill" and "downhill" speciation process during middle and later Miocene. The origin and diversification of the genus were explained as coupled with the rapid uplift of the Tianshan Mountains starting in late Oligocene and early Miocene ca. 23.66~19.33 Ma, as well as with uplift of the Qinghai-Tibetan Plateau (QTP) and Central Asian aridification.

Entire plastid phylogeny of the carrot genus (Daucus, Apiaceae): Concordance with nuclear data and mitochondrial and nuclear DNA insertions to the plastid.

PREMISE OF THE STUDY: We explored the phylogenetic utility of entire plastid DNA sequences in Daucus and compared the results with prior phylogenetic results using plastid and nuclear DNA sequences. METHODS: We used Illumina sequencing to obtain full plastid sequences of 37 accessions of 20 Daucus taxa and outgroups, analyzed the data with phylogenetic methods, and examined evidence for mitochondrial DNA transfer to the plastid (DcMP). KEY RESULTS: Our phylogenetic trees of the entire data set were highly resolved, with 100% bootstrap support for most of the external and many of the internal clades, except for the clade of D. carota and its most closely related species D. syrticus. Subsets of the data, including regions traditionally used as phylogenetically informative regions, provide various degrees of soft congruence with the entire data set. There are areas of hard incongruence, however, with phylogenies using nuclear data. We extended knowledge of a mitochondrial to plastid DNA insertion sequence previously named DcMP and identified the first instance in flowering plants of a sequence of potential nuclear genome origin inserted into the plastid genome. There is a relationship of inverted repeat junction classes and repeat DNA to phylogeny, but no such relationship with nonsynonymous mutations. CONCLUSIONS: Our data have allowed us to (1) produce a well-resolved plastid phylogeny of Daucus, (2) evaluate subsets of the entire plastid data for phylogeny, (3) examine evidence for plastid and nuclear DNA phylogenetic incongruence, and (4) examine mitochondrial and nuclear DNA insertion into the plastid.

Creation of reference DNA barcode library and authentication of medicinal plant raw drugs used in Ayurvedic medicine.

BACKGROUND: Ayurveda is a system of traditional medicine that originated in ancient India, and it is still in practice. Medicinal plants are the backbone of Ayurveda, which heavily relies on the plant-derived therapeutics. While Ayurveda is becoming more popular in several countries throughout the World, lack of authenticated medicinal plant raw drugs is a growing concern. Our aim was to DNA barcode the medicinal plants that are listed in the Ayurvedic Pharmacopoeia of India (API) to create a reference DNA barcode library, and to use the same to authenticate the raw drugs that are sold in markets. METHODS: We have DNA barcoded 347 medicinal plants using rbcL marker, and curated rbcL DNA barcodes for 27 medicinal plants from public databases. These sequences were used to create Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL). This library was used to authenticate 100 medicinal plant raw drugs, which were in the form of powders (82) and seeds (18). RESULTS: Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL) was created with high quality and authentic rbcL barcodes for 374 out of the 395 medicinal plants that are included in the API. The rbcL DNA barcode differentiated 319 species (85 %) with the pairwise divergence ranging between 0.2 and 29.9 %. PCR amplification and DNA sequencing success rate of rbcL marker was 100 % even for the poorly preserved medicinal plant raw drugs that were collected from local markets. DNA barcoding revealed that only 79 % raw drugs were authentic, and the remaining 21 % samples were adulterated. Further, adulteration was found to be much higher with powders (ca. 25 %) when compared to seeds (ca. 5 %). CONCLUSIONS: The present study demonstrated the utility of DNA barcoding in authenticating medicinal plant raw drugs, and found that approximately one fifth of the market samples were adulterated. Powdered raw drugs, which are very difficult to be identified by taxonomists as well as common people, seem to be the easy target for adulteration. Developing a quality control protocol for medicinal plant raw drugs by incorporating DNA barcoding as a component is essential to ensure safety to the consumers.

Identification of Chinese Herbs Using a Sequencing-Free Nanostructured Electrochemical DNA Biosensor.

Due to the nearly identical phenotypes and chemical constituents, it is often very challenging to accurately differentiate diverse species of a Chinese herbal genus. Although technologies including DNA barcoding have been introduced to help address this problem, they are generally time-consuming and require expensive sequencing. Herein, we present a simple sequencing-free electrochemical biosensor, which enables easy differentiation between two closely related Fritillaria species. To improve its differentiation capability using trace amounts of DNA sample available from herbal extracts, a stepwise electrochemical deposition of reduced graphene oxide (RGO) and gold nanoparticles (AuNPs) was adopted to engineer a synergistic nanostructured sensing interface. By using such a nanofeatured electrochemical DNA (E-DNA) biosensor, two Chinese herbal species of Fritillaria (F. thunbergii and F. cirrhosa) were successfully discriminated at the DNA level, because a fragment of 16-mer sequence at the spacer region of the 5S-rRNA only exists in F. thunbergii. This E-DNA sensor was capable of identifying the target sequence in the range from 100 fM to 10 nM, and a detection limit as low as 11.7 fM (S/N = 3) was obtained. Importantly, this sensor was applied to detect the unique fragment of the PCR products amplified from F. thunbergii and F. cirrhosa, respectively. We anticipate that such a direct, sequencing-free sensing mode will ultimately pave the way towards a new generation of herb-identification strategies.

DNA Barcoding and Pharmacovigilance of Herbal Medicines.

Pharmacovigilance of herbal medicines relies on the product label information regarding the ingredients and the adherence to good manufacturing practices along the commercialisation chain. Several studies have shown that substitution of plant species occurs in herbal medicines, and this in turn poses a challenge to herbal pharmacovigilance as adverse reactions might be due to adulterated or added ingredients. Authentication of constituents in herbal medicines using analytical chemistry methods can help detect contaminants and toxins, but are often limited or incapable of detecting the source of the contamination. Recent developments in molecular plant identification using DNA sequence data enable accurate identification of plant species from herbal medicines using defined DNA markers. Identification of multiple constituent species from compound herbal medicines using amplicon metabarcoding enables verification of labelled ingredients and detection of substituted, adulterated and added species. DNA barcoding is proving to be a powerful method to assess species composition in herbal medicines and has the potential to be used as a standard method in herbal pharmacovigilance research of adverse reactions to specific products.

Proxy comparison in ancient peat sediments: pollen, macrofossil and plant DNA.

We compared DNA, pollen and macrofossil data obtained from Weichselian interstadial (age more than 40 kyr) and Holocene (maximum age 8400 cal yr BP) peat sediments from northern Europe and used them to reconstruct contemporary floristic compositions at two sites. The majority of the samples provided plant DNA sequences of good quality with success amplification rates depending on age. DNA and sequencing analysis provided five plant taxa from the older site and nine taxa from the younger site, corresponding to 7% and 15% of the total number of taxa identified by the three proxies together. At both sites, pollen analysis detected the largest (54) and DNA the lowest (10) number of taxa, but five of the DNA taxa were not detected by pollen and macrofossils. The finding of a larger overlap between DNA and pollen than between DNA and macrofossils proxies seems to go against our previous suggestion based on lacustrine sediments that DNA originates principally from plant tissues and less from pollen. At both sites, we also detected Quercus spp. DNA, but few pollen grains were found in the record, and these are normally interpreted as long-distance dispersal. We confirm that in palaeoecological investigations, sedimentary DNA analysis is less comprehensive than classical morphological analysis, but is a complementary and important tool to obtain a more complete picture of past flora.

[Molecular-genetic polymorphism of barley (Hordeum vulgare L.) cultivars detected with AFLP method].

Analysis of molecular-genetic polymorphism of barley varieties was performed using AFLP-method. A system for identification and differentiation of barley varieties based on AFLP-markers was developed. Results of testing for 19 varieties indicate a high differential ability of the developed system. Identification of varieties can be conducted using one of two proposed discriminatory sets of AFLP-markers. Based on the calculated genetic distances a dendrogram of phylogenetic relations between the varieties was constructed. The dendrogram revealed a separated origin of varieties of malting and feed directions.

Insight into the karyotype evolution of brachypodium species using comparative chromosome barcoding.

Paleogenomic studies based on bioinformatic analyses of DNA sequences have enabled unprecedented insight into the evolution of grass genomes. They have revealed that nested chromosome fusions played an important role in the divergence of modern grasses. Nowadays, studies on karyotype evolution based on the sequence analysis can also be effectively complemented by the fine-scale cytomolecular approach. In this work, we studied the karyotype evolution of small genome grasses using BAC-FISH based comparative chromosome barcoding in four Brachypodium species: diploid B. distachyon (2n = 10) and B. sylvaticum (2n = 18), diploid (2n = 18) and allopolyploid (2n = 28) B. pinnatum as well as B. phoenicoides (2n = 28). Using BAC clones derived from the B. distachyon genomic libraries for the chromosomes Bd2 and Bd3, we identified the descending dysploidy events that were common for diploids with x = 9 and B. distachyon as well as two nested chromosome fusions that were specific only for B. distachyon. We suggest that dysploidy events that are shared by different lineages of the genus had already appeared in their common ancestor. We also show that additional structural rearrangements, such as translocations and duplications, contributed to increasing genome diversification in the species analysed. No chromosomes structured exactly like Bd2 and Bd3 were found in B. pinnatum (2n = 28) and B. phoenicoides. The structure of Bd2 and Bd3 homeologues belonging to the two genomes in the allopolyploids resembled the structure of their counterparts in the 2n = 18 diploids. These findings reinforce the hypothesis which excludes B. distachyon as a potential parent for Eurasian perennial Brachypodium allopolyploids. Our cytomolecular data elucidate some mechanisms of the descending dysploidy in monocots and enable reconstructions of the evolutionary events which shaped the extant karyotypes in both the genus Brachypodium and in grasses as a whole.

Karyotypic changes through dysploidy persist longer over evolutionary time than polyploid changes.

Chromosome evolution has been demonstrated to have profound effects on diversification rates and speciation in angiosperms. While polyploidy has predated some major radiations in plants, it has also been related to decreased diversification rates. There has been comparatively little attention to the evolutionary role of gains and losses of single chromosomes, which may or not entail changes in the DNA content (then called aneuploidy or dysploidy, respectively). In this study we investigate the role of chromosome number transitions and of possible associated genome size changes in angiosperm evolution. We model the tempo and mode of chromosome number evolution and its possible correlation with patterns of cladogenesis in 15 angiosperm clades. Inferred polyploid transitions are distributed more frequently towards recent times than single chromosome gains and losses. This is likely because the latter events do not entail changes in DNA content and are probably due to fission or fusion events (dysploidy), as revealed by an analysis of the relationship between genome size and chromosome number. Our results support the general pattern that recently originated polyploids fail to persist, and suggest that dysploidy may have comparatively longer-term persistence than polyploidy. Changes in chromosome number associated with dysploidy were typically observed across the phylogenies based on a chi-square analysis, consistent with these changes being neutral with respect to diversification.

TARE1, a mutated Copia-like LTR retrotransposon followed by recent massive amplification in tomato.

Long terminal repeat retrotransposons (LTR-RTs) are the major DNA components in flowering plants. Most LTR-RTs contain dinucleotides 'TG' and 'CA' at the ends of the two LTRs. Here we report the structure, evolution, and propensity of a tomato atypical retrotransposon element (TARE1) with both LTRs starting as 'TA'. This family is also characterized by high copy numbers (354 copies), short LTR size (194 bp), extremely low ratio of solo LTRs to intact elements (0.05∶1), recent insertion (most within 0.75∼1.75 million years, Mys), and enrichment in pericentromeric region. The majority (83%) of the TARE1 elements are shared between S. lycopersicum and its wild relative S. pimpinellifolium, but none of them are found in potato. In the present study, we used shared LTR-RTs as molecular markers and estimated the divergence time between S. lycopersicum and S. pimpinellifolium to be <0.5 Mys. Phylogenetic analysis showed that the TARE1 elements, together with two closely related families, TARE2 and TGRE1, have formed a sub-lineage belonging to a Copia-like Ale lineage. Although TARE1 and TARE2 shared similar structural characteristics, the timing, scale, and activity of their amplification were found to be substantially different. We further propose a model wherein a single mutation from 'G' to 'A' in 3' LTR followed by amplification is responsible for the origin of TARE1, thus providing evidence that the proliferation of a spontaneous mutation can be mediated by the amplification of LTR-RTs at the level of RNA.

Formation and expression of pseudogenes on the B chromosome of rye.

B chromosomes (Bs) are dispensable components of the genomes of numerous species. In contrast with the prevalent view that Bs do not harbor genes, our recent sequence analysis revealed that Bs of rye (Secale cereale) are rich in gene-derived sequences. We compared these gene-like fragments of the rye B with their ancestral A-located counterparts and confirmed an A chromosomal origin and the pseudogenization of B-located gene-like fragments. About 15% of the pseudogene-like fragments on Bs are transcribed in a tissue-type and genotype-specific manner. In addition, B-located sequences can cause in trans down- or upregulation of A chromosome-encoded genic fragments. Phenotypes and effects associated with the presence of Bs might be explained by the activity of B-located pseudogenes. We propose a model for the evolution of B-located pseudogenes.