Conservation of an Agrobacterium cT-DNA insert in Camellia section Thea reveals the ancient origin of tea plants from a genetically modified ancestor

Zhang Q., Zhao L., Folk R.A., Zhao J., Zamora N.A., Yang S., Soltis D.E., Soltis P.S., Gao L., Peng H., Yu X.

Abstract Background and Aims Theaceae, with three tribes, nine genera and more than 200 species, are of great economic and ecological importance. Recent phylogenetic analyses based on plastomic data resolved the relationships among the three tribes and the intergeneric relationships within two of those tribes. However, generic-level relationships within the largest tribe, Theeae, were not fully resolved. The role of putative whole-genome duplication (WGD) events in the family and possible hybridization events among genera within Theeae also remain to be tested further. Methods Transcriptomes or low-depth whole-genome sequencing of 57 species of Theaceae, as well as additional plastome sequence data, were generated. Using a dataset of low-copy nuclear genes, we reconstructed phylogenetic relationships using concatenated, species tree and phylogenetic network approaches. We further conducted molecular dating analyses and inferred possible WGD events by examining the distribution of the number of synonymous substitutions per synonymous site (Ks) for paralogues in each species. For plastid protein-coding sequences , phylogenies were reconstructed for comparison with the results obtained from analysis of the nuclear dataset. Results Based on the 610 low-copy nuclear genes (858 606 bp in length) investigated, Stewartieae was resolved as sister to the other two tribes. Within Theeae, the Apterosperma–Laplacea clade grouped with Pyrenaria, leaving Camellia and Polyspora as sister. The estimated ages within Theaceae were largely consistent with previous studies based mainly on plastome data. Two reticulation events within Camellia and one between the common ancestor of Gordonia and Schima were found. All members of the tea family shared two WGD events, an older At-γ and a recent Ad-β; both events were also shared with the outgroups (Diapensiaceae, Pentaphylacaceae, Styracaceae and Symplocaceae). Conclusions Our analyses using low-copy nuclear genes improved understanding of phylogenetic relationships at the tribal and generic levels previously proposed based on plastome data, but the phylogenetic position of the Apterosperma–Laplacea clade needs more attention. There is no evidence for extensive intergeneric hybridization within Theeae or for a Theaceae-specific WGD event. Land bridges (e.g. the Bering land bridge) during the Late Oligocene may have permitted the intercontinental plant movements that facilitated the putative ancient introgression between the common ancestor of Gordonia and Schima.

Matveeva T.V.

Agrobacterium mediated transformation in nature is the cause of the development of diseases: crown galls and hairy roots. These neoplasms are transgenic tissues on a non-transgenic plant. However, in nature, full-fledged GMOs arise, containing agrobacterial transgenes in every cell and transmitting them in a series of sexual generations. These plants are called naturally transgenic plants or natural GMOs. Over the past 3 years, the list of natural GMO species has been significantly expanded. Due to this, it became possible to make certain generalizations and more substantively discuss the possible evolutionary role of this phenomenon. The presented mini-review is devoted to the generalization of data on the possible functions of genes of agrobacterial origin in plant genomes.

Zhang X., Chen S., Shi L., Gong D., Zhang S., Zhao Q., Zhan D., Vasseur L., Wang Y., Yu J., Liao Z., Xu X., Qi R., Wang W., Ma Y., et. al.

Tea is an important global beverage crop and is largely clonally propagated. Despite previous studies on the species, its genetic and evolutionary history deserves further research. Here, we present a haplotype-resolved assembly of an Oolong tea cultivar, Tieguanyin. Analysis of allele-specific expression suggests a potential mechanism in response to mutation load during long-term clonal propagation. Population genomic analysis using 190 Camellia accessions uncovered independent evolutionary histories and parallel domestication in two widely cultivated varieties, var. sinensis and var. assamica. It also revealed extensive intra- and interspecific introgressions contributing to genetic diversity in modern cultivars. Strong signatures of selection were associated with biosynthetic and metabolic pathways that contribute to flavor characteristics as well as genes likely involved in the Green Revolution in the tea industry. Our results offer genetic and molecular insights into the evolutionary history of Camellia sinensis and provide genomic resources to further facilitate gene editing to enhance desirable traits in tea crops. Haplotype-resolved genome assembly of an Oolong tea cultivar Tieguanyin and population genomic analyses of 190 Camellia accessions provide insights into the evolutionary history of the tea plant Camellia sinensis.

Hoi Q.V., Thin D.B., Thinh B.B.

Many studies have been conducted to classify the genus Camellia, yet they have not been entirely consistent. Therefore, doing research on some of the taxonomic systems of the genus Camellia will serve as the basis for the classification and arrangement of recently discovered species into a consistent system. A series of studies based on morphology as well as molecular biology techniques, the classification of pollen spores, and the analysis of leaf anatomy were examined. The systems based on morphological characteristics were inherited and widely recognized, thereby becoming an appropriate method to classify the genus Camellia with representatives in Vietnam.

Tamura K., Stecher G., Kumar S.

Abstract The Molecular Evolutionary Genetics Analysis (MEGA) software has matured to contain a large collection of methods and tools of computational molecular evolution. Here, we describe new additions that make MEGA a more comprehensive tool for building timetrees of species, pathogens, and gene families using rapid relaxed-clock methods. Methods for estimating divergence times and confidence intervals are implemented to use probability densities for calibration constraints for node-dating and sequence sampling dates for tip-dating analyses. They are supported by new options for tagging sequences with spatiotemporal sampling information, an expanded interactive Node Calibrations Editor, and an extended Tree Explorer to display timetrees. Also added is a Bayesian method for estimating neutral evolutionary probabilities of alleles in a species using multispecies sequence alignments and a machine learning method to test for the autocorrelation of evolutionary rates in phylogenies. The computer memory requirements for the maximum likelihood analysis are reduced significantly through reprogramming, and the graphical user interface has been made more responsive and interactive for very big data sets. These enhancements will improve the user experience, quality of results, and the pace of biological discovery. Natively compiled graphical user interface and command-line versions of MEGA11 are available for Microsoft Windows, Linux, and macOS from www.megasoftware.net.

Letunic I., Bork P.

Abstract The Interactive Tree Of Life (https://itol.embl.de) is an online tool for the display, manipulation and annotation of phylogenetic and other trees. It is freely available and open to everyone. iTOL version 5 introduces a completely new tree display engine, together with numerous new features. For example, a new dataset type has been added (MEME motifs), while annotation options have been expanded for several existing ones. Node metadata display options have been extended and now also support non-numerical categorical values, as well as multiple values per node. Direct manual annotation is now available, providing a set of basic drawing and labeling tools, allowing users to draw shapes, labels and other features by hand directly onto the trees. Support for tree and dataset scales has been extended, providing fine control over line and label styles. Unrooted tree displays can now use the equal-daylight algorithm, proving a much greater display clarity. The user account system has been streamlined and expanded with new navigation options and currently handles >1 million trees from >70 000 individual users.

Otten L.

Analysis of 350 Agrobacterium wgs sequences reveals complex evolutionary history of T-DNA regions Virulent Agrobacterium strains transfer one or more plasmid DNA fragments to plant cells during a well-characterized transformation process. The transferred DNA sequences (T-DNA regions) are delimited by 25 nucleotide long conserved border sequences. Until recently, relatively few T-DNA regions were known. However, due to increased whole genome sequencing efforts, about 400 Agrobacterium sequences have now become available, 350 of which contain T-DNA regions. Detailed analysis identified 92 different T-DNA regions and several new T-DNA genes. T-DNA regions can be divided into three groups. I. Typical Agrobacterium rhizogenes T-DNA regions with rol genes. II. A large group of T-DNA regions with iaa and ipt genes, which can be further subdivided into seven subgroups. III. A small group of unusual T-DNA regions. The evolutionary relation between the T-DNA regions could not be completely elucidated, because of the lack of evolutionary intermediates. Several clusters of highly related structures suggest that evolution of T-DNA regions proceeds by slow, progressive evolution of gene sequences, accompanied by rapid changes in overall structure, due to recombination between T-DNA regions of different origins, and insertion of bacterial insertion sequences (IS). Divergence values for T-DNA genes suggest that they were recruited at different times in evolution. An attempt was made to link T-DNA region evolution to plasmid evolution. The present study provides a solid basis for further studies on T-DNA region diversity and evolution.

Matveeva T.

Agrobacterium-mediated gene transfer leads to crown gall or hairy roots disease, due to expression of transferred T-DNA genes. Spontaneous plant regeneration from the transformed tissues can produce natural transformants carrying cellular T-DNA (cT-DNA) sequences of agrobacterial origin. In 2019, based on genomic sequencing data, cT-DNA horizontally transferred from Agrobacterium were found in two dozen species of angiosperms. This made it possible to evaluate the spread of this phenomenon, as well as make some generalizations regarding the diversity of horizontally transferred genes. The presented research is a continuation of work in this field. It resulted in the description of new naturally occurring transgenic species Aeschynomene evenia C. Wright, Eperua falcata Aubl., Eucalyptus cloeziana F.Muell., Boswellia sacra Flueck., Kewa caespitosa (Friedrich) Christenh., Pharnaceum exiguum Adamson, Silene noctiflora L., Nyssa sinensis Oliv., Vaccinium corymbosum L., Populus alba L. × Populus glandulosa Moench. The previously identified patterns regarding the frequency of the occurrence of natural transformants and the general properties of the cT-DNAs were confirmed in this study.

Zhang W., Luo C., Scossa F., Zhang Q., Usadel B., Fernie A.R., Mei H., Wen W.

For diploid organisms that are highly heterozygous, a phased haploid genome can greatly aid in functional genomic, population genetic and breeding studies. Based on the genome sequencing of 135 single sperm cells of the elite tea cultivar 'Fudingdabai', we herein phased the genome of Camellia sinensis, one of the most popular beverage crops worldwide. High-resolution genetic and recombination maps of Fudingdabai were constructed, which revealed that crossover (CO) positions were frequently located in the 5' and 3' ends of annotated genes, while CO distributions across the genome were random. The low CO frequency in tea can be explained by strong CO interference, and CO simulation revealed the proportion of interference insensitive CO ranged from 5.2% to 11.7%. We furthermore developed a method to infer the relatedness between tea accessions and detected complex kinship and genetic signatures of 106 tea accessions. Among them, 59 accessions were closely related with Fudingdabai and 31 of them were first-degree relatives. We additionally identified genes displaying allele specific expression patterns between the two haplotypes of Fudingdabai and genes displaying significantly differential expression levels between Fudingdabai and other haplotypes. These results lay the foundation for further investigation of genetic and epigenetic factors underpinning the regulation of gene expression and provide insights into the evolution of tea plants as well as a valuable genetic resource for future breeding efforts.

Zhang M., Tang Y., Xu Y., Yonezawa T., Shao Y., Wang Y., Song Z., Yang J., Zhang W.

Abstract Background and Aims The ribosomal DNA (rDNA) gene family, encoding ribosomal RNA (rRNA), has long been regarded as an archetypal example illustrating the model of concerted evolution. However, controversy is arising, as rDNA in many eukaryotic species has been proved to be polymorphic. Here, a metagenomic strategy was applied to detect the intragenomic polymorphism as well as the evolutionary patterns of 26S rDNA across the genus Camellia. Methods Degenerate primer pairs were designed to amplify the 26S rDNA fragments from different Camellia species. The amplicons were then paired-end sequenced on the Illumina MiSeq platform. Key Results An extremely high level of rDNA polymorphism existed universally in Camellia. However, functional rDNA was still the major component of the family, and was relatively conserved among different Camellia species. Sequence variations mainly came from rRNA pseudogenes and favoured regions that are rich in GC. Specifically, some rRNA pseudogenes have existed in the genome for a long time, and have even experienced several expansion events, which has greatly enriched the abundance of rDNA polymorphism. Conclusions Camellia represents a group in which rDNA is subjected to a mixture of concerted and birth-and-death evolution. Some rRNA pseudogenes may still have potential functions. Conversely, when released from selection constraint, they can evolve in the direction of decreasing GC content and structural stability through a methylation-induced process, and finally be eliminated from the genome.

Wang X., Feng H., Chang Y., Ma C., Wang L., Hao X., Li A., Cheng H., Wang L., Cui P., Jin J., Wang X., Wei K., Ai C., Zhao S., et. al.

Tea is an economically important plant characterized by a large genome, high heterozygosity, and high species diversity. In this study, we assemble a 3.26-Gb high-quality chromosome-scale genome for the ‘Longjing 43’ cultivar of Camellia sinensis var. sinensis. Genomic resequencing of 139 tea accessions from around the world is used to investigate the evolution and phylogenetic relationships of tea accessions. We find that hybridization has increased the heterozygosity and wide-ranging gene flow among tea populations with the spread of tea cultivation. Population genetic and transcriptomic analyses reveal that during domestication, selection for disease resistance and flavor in C. sinensis var. sinensis populations has been stronger than that in C. sinensis var. assamica populations. This study provides resources for marker-assisted breeding of tea and sets the foundation for further research on tea genetics and evolution. Tea is an important beverage crop with a large and heterozygous genome. Here, the authors assemble the genome of the cultivar Longjing 43 and conduct a population genetics study to reveal divergent selection for disease resistance and flavor between the two variety groups.

Zhang W., Zhang Y., Qiu H., Guo Y., Wan H., Zhang X., Scossa F., Alseekh S., Zhang Q., Wang P., Xu L., Schmidt M.H., Jia X., Li D., Zhu A., et. al.

Wild teas are valuable genetic resources for studying domestication and breeding. Here we report the assembly of a high-quality chromosome-scale reference genome for an ancient tea tree. The further RNA sequencing of 217 diverse tea accessions clarifies the pedigree of tea cultivars and reveals key contributors in the breeding of Chinese tea. Candidate genes associated with flavonoid biosynthesis are identified by genome-wide association study. Specifically, diverse allelic function of CsANR, CsF3’5’H and CsMYB5 is verified by transient overexpression and enzymatic assays, providing comprehensive insights into the biosynthesis of catechins, the most important bioactive compounds in tea plants. The inconspicuous differentiation between ancient trees and cultivars at both genetic and metabolic levels implies that tea may not have undergone long-term artificial directional selection in terms of flavor-related metabolites. These genomic resources provide evolutionary insight into tea plants and lay the foundation for better understanding the biosynthesis of beneficial natural compounds. Wild teas are considered as valuable resource for studying domestication and breeding. Here, Zhang et al. report genome of wild tea DASZ and transcriptome of 217 accessions, which clarify pedigree of Chinese tea cultivars and show tea may not have undergone long-term artificial directional selection on flavor-related metabolites.

Xia E., Tong W., Hou Y., An Y., Chen L., Wu Q., Liu Y., Yu J., Li F., Li R., Li P., Zhao H., Ge R., Huang J., Mallano A.I., et. al.

Abstract Tea plant is an important economic crop, which is used to produce the world's oldest and most widely consumed tea beverages. Here, we present a high-quality reference genome assembly of the tea plant (Camellia sinensis var. sinensis) consisting of 15 pseudo-chromosomes. LTR retrotransposons (LTR-RTs) account for 70.38% of the genome, and we present evidence that LTR-RTs play critical roles in genome size expansion and the transcriptional diversification of tea plant genes through preferential insertion in promoter regions and introns. Genes, particularly those coding for terpene biosynthesis proteins, associated with tea aroma and stress resistance were significantly amplified through recent tandem duplications and exist as gene clusters in tea plant genome. Phylogenetic analysis of the sequences of 81 tea plant accessions with diverse origins revealed three well-differentiated tea plant populations, supporting the proposition for the southwest origin of the Chinese cultivated tea plant and its later spread to western Asia through introduction. Domestication and modern breeding left significant signatures on hundreds of genes in the tea plant genome, particularly those associated with tea quality and stress resistance. The genomic sequences of the reported reference and resequenced tea plant accessions provide valuable resources for future functional genomics study and molecular breeding of improved cultivars of tea plants.

Otten L.

Agrobacterium can transfer genetic information to plants, transforming the plants naturally. An Agrobacterium plasmid fragment is transferred to the plant via bacterial infection and stably integrated into the plant’s nuclear DNA. This plasmid fragment (termed transferred DNA or T-DNA) contains several genes that are inserted into the plant’s chromosomes. Some natural plant species contain Agrobacterium T-DNA-like sequences, which have been shown to result from natural transformation. These sequences are called cellular T-DNAs or cT-DNAs. Multiple Nicotiana species have been shown to contain cT-DNA sequences and to express cT-DNA genes, qualifying these species as natural transformants. The composition and organization of the T-DNA sequences vary considerably. Sequencing the genome of the Tomentosae and Noctiflorae sections of the genus Nicotiana has identified seven cT-DNA sequences that are similar to sequences in A. rhizogenes, A. tumefaciens, and A. vitis. As some cT-DNA genes show strong growth effects when expressed in other species, they may influence the growth of the natural transformants as well. The precise mechanisms by which these genes alter growth patterns and their regulation by promoters and by plant transcription factors remain to be elucidated.

Zhang Y., Wang D., Wang Y., Dong H., Yuan Y., Yang W., Lai D., Zhang M., Jiang L., Li Z.

Veremeichik G.N., Solomatina T.O., Khopta A.A., Brodovskaya E.V., Gorpenchenko T.Y., Grigorchuk V.P., Bulgakov D.V., Bulgakov V.P.

Long-term cultured calli may experience a biosynthetic shift due to the IAA-dependent expression of the rolA gene, which also affects ROS metabolism. The “hairy root” syndrome is caused by the root-inducing Ri-plasmid of Rhizobium rhizogenes, also known as Agrobacterium rhizogenes. The Ri-plasmid contains genes known as rol genes or root oncogenic loci, which promote root development. The important implications of the rolA gene from the T-DNA include reduced plant size, resistance to infections, and the activation of specialised metabolism. Nevertheless, rolA does not belong to the plast gene group because its function is still uncertain. Recent investigations have shown two important effects of the rolA gene. First, the production of secondary metabolites has changed in long-term cultivated rolA-transgenic calli of Rubia cordifolia L. Second, the expression of both the rolA and rolB genes has a strong auxin-dependent antagonistic effect on reactive oxygen species (ROS) homeostasis. In this work, we attempted to elucidate two rolA gene phenomena: what caused the secondary metabolism of long-term cultured calli to change? How does the individual expression of the rolA gene affect ROS homeostasis? We analysed SNPs in the 5′ untranslated region and coding region of the rolA gene. These mutations do not affect the known essential amino acids of the RolA proteins. Notably, in the promoter of the rolA gene, an ACTTTA motif for auxin-mediated transcription factors was identified. Using two separate cell cultures, we demonstrated the strong auxin dependence of rolA gene expression. The expression of genes involved in ROS metabolism decreased in response to an auxin-mediated increase in rolA gene expression. Two assumptions can be made. The long-term cultivation of calli may cause changes in the hormonal state of the culture over time, which may modulate the action of the RolA protein. Moreover, auxin-dependent expression of the rolA gene led to a decrease in ROS metabolism. It can be assumed that the antagonistic interaction between rolA and rolB prevents strong rolB-induced auxin sensitivity and oxidative bursts to balance the cell state.

Дейнеко Е.В.

Развитие и совершенствование методов молекулярной и клеточной биологии существенно расширило возможности исследователей по модификации геномов растительных клеток и послужило основой для развития новых технологий получения рекомбинантных белков, используемых в фармацевтике и других отраслях народного хозяйства, а также стимулировало создание новых высокоурожайных сортов важных сельскохозяйственных культур, устойчивых к неблагоприятным абиотическим и биотическим факторам среды. Перенос генов в растительный геном из других гетерологичных систем поставил перед исследователями ряд вопросов, связанных с функционированием трансгенов в новом окружении генома-реципиента, а также с их влиянием на функционирование собственных генов растения. За последние сорок лет с момента получения первого трансгенного растения возможности этих технологий были существенно углублены и расширены за счет разработки методов геномного редактирования, основанных на системе CRISPR/Cas. Это позволило не только изменять функционирование целевых генов путем нокаутов или исправлять нежелательные мутации, но и вносить гены интереса в заданные исследователем районы-мишени растительного генома. В предлагаемом обзоре рассматриваются основные этапы исследований по модификации геномов растений за последние сорок лет, с акцентом не только на практическую значимость созданных агробиотехнологий, но и на важность для фундаментальных исследований функционирования генов и выявления структурных особенностей организации генома растений.

Deineko E.V.

The development and improvement of molecular and cellular biology methods has significantly expanded the capabilities of researchers to modify the genomes of plant cells and served as the basis for the development of new technologies for obtaining recombinant proteins used in pharmaceuticals and other sectors of the national economy and has also stimulated the creation of new high-yielding varieties of important agricultural crops that are resistant to unfavorable abiotic and biotic environmental factors. The transfer of genes into the plant genome from other heterologous systems has raised a number of questions for researchers related to the functioning of transgenes in the new environment of the recipient genome as well as their influence on the functioning of the plant’s own genes. Over the past 40 years since the first transgenic plant was produced, the capabilities of these technologies have been significantly deepened and expanded through the development of genome-editing methods based on the CRISPR/Cas system. This made it possible not only to change the functioning of target genes through knockouts or to correct unwanted mutations but also to introduce genes of interest into target regions of the plant genome specified by the researcher. The proposed review examines the main stages of research on the modification of plant genomes over the past 40 years, with an emphasis not only on the practical significance of the created agrobiotechnologies but also on the importance for fundamental research on the functioning of genes and the identification of structural features of the organization of the plant genome.

Rafiei F., Wiersma J., Scofield S., Zhang C., Alizadeh H., Mohammadi M.

AbstractThe year 2020 was a landmark year for wheat. The wheat HB4 event harboring a drought-resistant gene from sunflowers, received regulatory approval and was grown commercially in Argentina, with approval for food and feed in other countries. This, indeed, is many years after the adoption of genetic modifications in other crops. The lack of consumer acceptance and resulting trade barriers halted the commercialization of the earliest events and had a chilling effect on, especially, private Research & Development (R&D) investments. As regulations for modern breeding technologies such as genome-edited cultivars are being discussed and/or adopted across the globe, we would like to propose a framework to ensure that wheat is not left behind a second time as the potential benefits far outweigh the perceived risks. In this paper, after a review of the technical challenges wheat faces with the generation of trans- and cis-genic wheat varieties, we discuss some of the factors that could help demystify the risk/reward equation and thereby the consumer’s reluctance or acceptance of these techniques for future wheat improvement. The advent of next-generation sequencing is shedding light on natural gene transfer between species and the number of perturbations other accepted techniques like mutagenesis create. The transition from classic breeding techniques and embracing transgenic, cisgenic, and genome editing approaches feels inevitable for wheat improvement if we are to develop climate-resilient wheat varieties to feed a growing world population.

Bogomaz O.D., Bemova V.D., Mirgorodskii N.A., Matveeva T.V.

Naturally transgenic plants are plants that have undergone Agrobacterium-mediated transformation under natural conditions without human involvement. Among Arachis hypogaea L., A. duranensis Krapov. & W.C. Greg, A. ipaensis Krapov. & W.C. Greg, A. monticola Krapov. & Rigoni, and A. stenosperma Krapov. & W.C. Greg are known to contain sequences derived from the T-DNA of “Agrobacterium”. In the present study, using molecular genetics and bioinformatic methods, we characterized natural transgenes in 18 new species from six sections of the genus Arachis. We found that small fragments of genes for enzymes of the agropine synthesis pathway were preserved only in some of the studied samples and were lost in the majority of the species during evolution. At the same time, genes, similar to cucumopine synthases (cus-like), remained intact in almost all of the investigated species. In cultivated peanuts, they are expressed in a tissue-specific manner. We demonstrated the intraspecific variability of the structure and expression of the cus-like gene in cultivated peanuts. The described diversity of gene sequences horizontally transferred from Agrobacterium to plants helps to shed light on the phylogeny of species of the genus Arachis and track possible hybridization events. Data on the ability of certain species to hybridize are useful for planning breeding schemes aimed at transferring valuable traits from wild species into cultivated peanuts.

Val Giddings L.

L. Val Giddings Information Technology and Innovation Foundation, Washington, D.C. 20001, USA Correspondence: vgiddings{at}itif.org

Zhidkin R.R., Zhurbenko P.M., Matveeva T.V.

Genetic colonization by agrobacteria is possible due to agrobacterial transformation, which implies interspecies transfer of genetic material (T-DNA). A transgenic tissue is formed on the whole non-transgenic plant during that process. However, it turned out that in nature there are plants containing T-DNA fragments in their genomes and they can inherit these T-DNAs sexually. Such T-DNA was called cellular, and such plants were called natural transgenic. Examples of such organisms are plants of the genus Vaccinium. In the genomes of two species of this genus we found cT-DNA, represented by a rolB/C-like gene [1]. Previously, analyzing the natural transgenes in another genus (Camellia L.) [2], we showed the importance of reconstructing the allelic states of transgenes for phylogenetic studies. In this study, we performed analysis of the spreading of the rolB/C-like gene for its use as a molecular marker within Vaccinium. We used molecular-genetic and bioinformatics methods for sequencing, assembly, and analysis of the rolB/C-like gene. We discovered 26 new Vaccinium species and Agapetes serpens (Wight) Sleumer as containing the rolB/C-like gene. Most of studied samples are characterized by the presence of full-size genes. This made it possible to develop approaches for alleles phasing of the rolB/C-like gene and reconstruct a Vaccinium phylogenetic relationship. We subjected the studied species to phylogenetic analysis based on sequences of the rolB/C-like gene. The resulting phylogenetic tree of the genus Vaccinium divided the species into sections in accordance with the classical genus system based on morphological characters. At the same time, our tree did not confirm the taxa determined on the basis of ITS.

Chen K., Zhurbenko P.M., Danilov L.G., Matveeva T.V., Otten L.

Horizontal gene transfer (HGT) plays an important role in plant evolution and plant development. Agrobacterium-mediated gene transfer leads to the formation of crown galls or hairy roots, due to expression of transferred T-DNA genes. Spontaneous regeneration of transformed cells can produce natural transformants carrying cellular T-DNA (cT-DNA) sequences of bacterial origin. HGT from Agrobacterium to dicots is remarkably widespread. The production of naturally genome modified plants could play a role in plant evolution and environment. Among these natural GMOs (nGMOs) there are the tea plants. Camellia sinensis var. sinensis cv. Shuchazao contains a single 5.5 kb cT-DNA fragment organized as imperfect inverted repeat with three inactive genes. 142 Camellia accessions, belonging to 10 of 11 species of the section Thea, were studied for the presence of cT-DNA alleles. All of them contain the cT-DNA insert, indicating that they are resulted from the single transformed event. Allele phasing showed that 82 accessions were heterozygous for T-DNA alleles, 60 others were homozygous. A phylogenetic analysis of all found alleles showed existence of two separate groups of them, further divided into subgroups. The alleles of the different Camellia species were distributed mosaically over groups, and different species showed very similar T-DNA alleles. This indicates that the taxonomy of Thea requires revision. The nucleotide divergence of the imperfect cT-DNA repeats indicates that the age of cT-DNA insertion is about 15 mya, which is earlier then emergence of section Thea [1]. We present a working model for the origin and evolution of nGMO plants derived from allogamous transformants.

Chen K., Liu H., Blevins T., Hao J., Otten L.

The genus Camellia underwent extensive natural transformation by Agrobacterium. Over a period of 15 million years, at least 12 different inserts accumulated in 72 investigated Camellia species. Like a wide variety of other wild and cultivated plants, Camellia species carry cellular T-DNA sequences (cT-DNAs) in their nuclear genomes, resulting from natural Agrobacterium-mediated transformation. Short and long DNA sequencing reads of 435 accessions belonging to 72 Camellia species (representing 12 out of 14 sections) were investigated for the occurrence of cT-DNA insertions. In all, 12 different cT-DNAs were recovered, either completely or partially, called CaTA to CaTL. Divergence analysis of internal cT-DNA repeats revealed that the insertion events span a period from 0.075 to 15 Mio years ago, and yielded an average transformation frequency of one event per 1.25 Mio years. The two oldest inserts, CaTA and CaTD, have been modified by spontaneous deletions and inversions, and by insertion of various plant sequences. In those cases where enough accessions were available (C. japonica, C. oleifera, C. chekiangoleosa, C. sasanqua and C. pitardii), the younger cT-DNA inserts showed a patchy distribution among different accessions of each species, indicating that they are not genetically fixed. It could be shown that Camellia breeding has led to intersectional transfer of cT-DNAs. Altogether, the cT-DNAs cover 374 kb, and carry 47 open reading frames (ORFs). Two Camellia cT-DNA genes, CaTH-orf358 and CaTK-orf8, represent new types of T-DNA genes. With its large number of cT-DNA sequences, the genus Camellia constitutes an interesting model for the study of natural Agrobacterium transformants.

Zhidkin R., Zhurbenko P., Bogomaz O., Gorodilova E., Katsapov I., Antropov D., Matveeva T.

A variety of plant species found in nature contain agrobacterial T-DNAs in their genomes which they transmit in a series of sexual generations. Such T-DNAs are called cellular T-DNAs (cT-DNAs). cT-DNAs have been discovered in dozens of plant genera, and are suggested to be used in phylogenetic studies, since they are well-defined and unrelated to other plant sequences. Their integration into a particular chromosomal site indicates a founder event and a clear start of a new clade. cT-DNA inserts do not disseminate in the genome after insertion. They can be large and old enough to generate a range of variants, thereby allowing the construction of detailed trees. Unusual cT-DNAs (containing the rolB/C-like gene) were found in our previous study in the genome data of two Vaccinium L. species. Here, we present a deeper study of these sequences in Vaccinium L. Molecular-genetic and bioinformatics methods were applied for sequencing, assembly, and analysis of the rolB/C-like gene. The rolB/C-like gene was discovered in 26 new Vaccinium species and Agapetes serpens (Wight) Sleumer. Most samples were found to contain full-size genes. It allowed us to develop approaches for the phasing of cT-DNA alleles and reconstruct a Vaccinium phylogenetic relationship. Intra- and interspecific polymorphism found in cT-DNA makes it possible to use it for phylogenetic and phylogeographic studies of the Vaccinium genus.

Veremeichik G.N., Bulgakov D.V., Solomatina T.O., Makhazen D.S.

The biological function of the agrobacterial oncogene rolA is very poorly understood compared to other components of the mechanism of horizontal gene transfer during agrobacterial colonization of plants. Research groups around the world have worked on this problem, and available information is reviewed in this review, but other rol oncogenes have been studied much more thoroughly. Having one unexplored element makes it impossible to form a complete picture. However, the limited data suggest that the rolA oncogene and its regulatory apparatus have great potential in plant biotechnology and genetic engineering. Here, we collect and discuss available experimental data about the function and structure of rolA. There is still no clear understanding of the mechanism of RolA and its structure and localization. We believe this is because of the nucleotide structure of a frameshift in the most well-studied rolA gene of the agropine type pRi. In fact, interest in the genes of agrobacteria as natural tools for the phenotypic or biochemical engineering of plants increased. We believe that a detailed understanding of the molecular mechanisms will be forthcoming. • Among pRi T-DNA oncogenes, rolA is the least understood in spite of many studies. • Frameshift may be the reason for the failure to elucidate the role of agropine rolA. • Understanding of rolA is promising for the phenotypic and biochemical engineering of plants.