Linked by Ancestral Bonds: Multiple Whole-Genome Duplications and Reticulate Evolution in a Brassicaceae Tribe

Pervasive hybridization and whole-genome duplications (WGDs) influenced genome evolution in several eukaryotic lineages. Although frequent and recurrent hybridizations may result in reticulate phylogenies, the evolutionary events underlying these reticulations, including detailed structure of the ancestral diploid and polyploid genomes, were only rarely reconstructed. Here, we elucidate the complex genomic history of a monophyletic clade from the mustard family (Brassicaceae), showing contentious relationships to the early-diverging clades of this model plant family. Genome evolution in the crucifer tribe Biscutelleae (∼60 species, 5 genera) was dominated by pervasive hybridizations and subsequent genome duplications. Diversification of an ancestral diploid genome into several divergent but crossable genomes was followed by hybridizations between these genomes. Whereas a single genus (Megadenia) remained diploid, the four remaining genera originated by allopolyploidy (Biscutella, Lunaria, Ricotia) or autopolyploidy (Heldreichia). The contentious relationships among the Biscutelleae genera, and between the tribe and other early diverged crucifer lineages, are best explained by close genomic relatedness among the recurrently hybridizing ancestral genomes. By using complementary cytogenomics and phylogenomics approaches, we demonstrate that the origin of a monophyletic plant clade can be more complex than a parsimonious assumption of a single WGD spurring postpolyploid cladogenesis. Instead, recurrent hybridization among the same and/or closely related parental genomes may phylogenetically interlink diploid and polyploid genomes despite the incidence of multiple independent WGDs. Our results provide new insights into evolution of early-diverging Brassicaceae lineages and elucidate challenges in resolving the contentious relationships within and between land plant lineages with pervasive hybridization and WGDs.     

Oilseed rape: learning about ancient and recent polyploid evolution from a recent crop species

Oilseed rape (Brassica napus) is one of our youngest crop species, arising several times under cultivation in the last few thousand years and completely unknown in the wild. Oilseed rape originated from hybridisation events between progenitor diploid species B. rapa and B. oleracea, both important vegetable species. The diploid progenitors are also ancient polyploids, with remnants of two previous polyploidisation events evident in the triplicated genome structure. This history of polyploid evolution and human agricultural selection makes B. napus an excellent model with which to investigate processes of genomic evolution and selection in polyploid crops. The ease of de novo interspecific hybridisation, responsiveness to tissue culture, and the close relationship of oilseed rape to the model plant Arabidopsis thaliana, coupled with the recent availability of reference genome sequences and suites of molecular cytogenetic and high‐throughput genotyping tools, allow detailed dissection of genetic, genomic and phenotypic interactions in this crop. In this review we discuss the past and present uses of B. napus as a model for polyploid speciation and evolution in crop species, along with current and developing analysis tools and resources. We further outline unanswered questions that may now be tractable to investigation.     

Molecules and morphology: comparative developmental genetics of the Brassicaceae

For approximately 20 years Arabidopsis has been a model system to investigate developmental and physiological questions in plant biology, leading to the identification of genes and genetic systems involved in many processes. Extending ideas arising from knowledge of developmental genetic systems in Arabidopsis to other species of the Brassicaceae will require the application of genomics technologies developed in Arabidopsis and the establishment of additional genetic systems and resources in other species. Morphological variation in all plant organs, as well as in growth habit, mating systems, and physiology are represented in the breadth of Brassicaceae species offering ample opportunity to investigate the molecular basis of morphological evolution in this family. In addition, the frequent recent hybridization events in Brassica and Arabidopsis facilitate study of this pervasive force in the evolution of all plants.     

Microsatellites for three distantly related genera in the Brassicaceae

Microsatellites are important genetic markers both in population genetics and for delimitation of closely related species. However, to develop microsatellites for each target organism is expensive and time consuming. In this study, we have therefore developed 65 new microsatellite primers for the species Draba nivalis and tested cross-species and cross-genus transfer success of these primers for two other genera in the Brassicaceae; Cardamine and Smelowskia. Furthermore, 15 previously developed microsatellites were tested for amplification in these three genera. The microsatellite markers that amplify across these genera may be useful for other genera in the Brassicaceae as well.     

Biosystematics and evolutionary studies in Indian Drimia species

Abstract The taxonomic position and genetic relationship within Indian Drimia species is controversial due to their morphological similarities and genomic complexities. The present work gives an insight on the genetic relationship between Indian Drimia species on the basis of their karyotype, pollen morphology, flower opening characteristics, hybridization behavior, and by use of DNA sequence of two molecular markers (internal transcribed spacers [ITS] and maturase K [matK]). The karyotypic studies of Indian Drimia species revealed various polyploid forms making their identification and delimitation more difficult. The five species of Indian Drimia are grouped into two complexes, indica complex and wightii complex on the basis of their pollen morphology, karyotype, and hybridization behavior. These two groups were found to be evolving separately. The cytomorphological studies of wightii complex revealed that it is evolving through polyploid and chromosome repatterning, while indica complex have adapted polyploid as well as hybridization for evolution. Phylogeny obtained from DNA sequences of molecular markers (ITS and matK) confirmed that the indica complex and wightii complex are evolving parallely, by grouping them in two clusters. Thus, a combination of conventional and molecular methods proved to be of great use for delimiting a small but complex group of Indian Drimia species.     

Allozyme diversity and systematics inAnnonaceae—a pilot project

Five species ofAnnona and one species fromArtabotrys, Cananga, Polyalthia, andRollinia were investigated in regard to 11 different allozyme loci. Preliminary studies on small population samples ofAnnona suggest genetic uniformity in three species and variability within and between populations in two other species. The allotetraploid origin ofA. glabra is clearly shown by its hybrid enzyme bands. The genetic distance between fiveAnnona species partly corresponds with their morphological relationships; onlyA. muricata appears more separated than is suggested by morphology. A comparison of the five genera demonstrates close relationship betweenAnnona andRollinia. Two enzyme loci are identical within all taxa investigated and possibly may serve as a genetic marker for the family.     

A Time-Calibrated Road Map of Brassicaceae Species Radiation and Evolutionary History

The Brassicaceae include several major crop plants and numerous important model species in comparative evolutionary research such as Arabidopsis, Brassica, Boechera, Thellungiella, and Arabis species. As any evolutionary hypothesis needs to be placed in a temporal context, reliably dated major splits within the evolution of Brassicaceae are essential. We present a comprehensive time-calibrated framework with important divergence time estimates based on whole-chloroplast sequence data for 29 Brassicaceae species. Diversification of the Brassicaceae crown group started at the Eocene-to-Oligocene transition. Subsequent major evolutionary splits are dated to ∼20 million years ago, coinciding with the Oligocene-to-Miocene transition, with increasing drought and aridity and transient glaciation events. The age of the Arabidopsis thaliana crown group is 6 million years ago, at the Miocene and Pliocene border. The overall species richness of the family is well explained by high levels of neopolyploidy (43% in total), but this trend is neither directly associated with an increase in genome size nor is there a general lineage-specific constraint. Our results highlight polyploidization as an important source for generating new evolutionary lineages adapted to changing environments. We conclude that species radiation, paralleled by high levels of neopolyploidization, follows genome size decrease, stabilization, and genetic diploidization.     

Chromosome-level genome assembly of a triploid poplar Populus alba ‘Berolinensis’

Many recent studies have provided significant insights into polyploid breeding, but limited research has been carried out on trees. The genomic information needed to understand growth and response to abiotic stress in polyploidy trees is largely unknown, but has become critical due to the threats to forests imposed by climate change. Populus alba ‘Berolinensis,’ also known “Yinzhong poplar,” is a triploid poplar from northeast China. This hybrid triploid poplar is widely used as a landscape ornamental and in urban forestry owing to its adaptation to adverse environments and faster growth than its parental diploid. It is an artificially synthesized male allotriploid hybrid, with three haploid genomes of P. alba ‘Berolinensis’ originating from different poplar species, so it is attractive for studying polyploidy genomic mechanisms in heterosis. In this study, we focused on the allelic genomic interactions in P. alba ‘Berolinensis,’ and generated a high-quality chromosome-level genome assembly consisting of 19 allelic chromosomes. Its three haploid chromosome sets are polymorphic with an average of 25.73 nucleotide polymorphism sites per kilobase. We found that some stress-related genes such as RD22 and LEA7 exhibited sequence differences between different haploid genomes. The genome assembly has been deposited in our polyploid genome online analysis website TreeGenomes ( https://www.treegenomes.com ). These polyploid genome-related resources will provide a critical foundation for the molecular breeding of P. alba ‘Berolinensis’ and help us uncover the allopolyploidization effects of heterosis and abiotic stress resistance and traits of polyploidy species in the future.     

Genome structure and apomixis in Phoenicaulis (Brassicaceae; Boechereae)

Apomixis in crucifer (Brassicaceae) species is rare, reported in just four genera (Boechera, Draba, Erysimum, and Parrya), and one intergeneric hybrid (Raphanobrassica). It is well studied only in Boechera, where it is widespread among 100+ recognized species. However, its occurrence in related genera of the tribe Boechereae has not been documented previously. Here we analyzed genome evolution, mode of reproduction, and fertility of the monospecific Boechereae genus Phoenicaulis (P. cheiranthoides), endemic to the northwestern United States. We discovered that the species encompasses diploid (2n = 2x = 14), triploid (2n = 3x = 21), and tetraploid (2n = 4x = 28) populations. Comparative chromosome painting analyses revealed that the three karyotypes are essentially structurally identical, differing only in the presence of a largely heterochromatic chromosome (Het) in the triploid and tetraploid cytotypes. The genome structure of Phoenicaulis appeared identical to that of Boechera species previously analyzed, suggesting genomic stasis despite the morphological and molecular divergence of the two genera. This genome colinearity extended to the presence and structure of the Het chromosomes, which are closely associated with apomictic reproduction in Boechera. Interestingly, all three cytotypes of Phoenicaulis proved to be apomictic, regardless of the presence or absence of a Het chromosome, and sexual populations have yet to be identified.     

γ-Glutamylcysteine Production by Escherichia coli B Cells Dosed with the γ-Glutamylcysteine Synthetase Gene

Molecular studies on the evolution and systematics of fungi have been established primarily based on the neutral theory by analyzing neutral mutations in some defined segments of housekeeping genes as genetic markers. Such an approach is, however, hardly applicable for analyzing ancient evolutionary radiations. In the present study, we looked for DNA sequences characterizing higher taxa, and discovered a unique macroevolutionary genomic marker, megB1, that specifies the phylum Basidiomycota. megB1 is an approximately 500-bp DNA element, which is defined by terminal sequences and five internal segments conserved throughout the phylum. megB1 resides on the rDNA intergenic spacer 1 (IGS1) from 27 species of 10 Basidiomycota genera examined. While megB1 was not found in IGS1 from the other 92 species of the 27 Basidiomycota genera, several genera representing them carry megB1 in some other genomic regions. No known taxonomic criteria fit into the classification on the basis of whether megB1 resides on rDNA. Neighbor-joining analysis of the megB1 sequence, however, properly assigned species to their respective genera. Thus far, megB1 has not been found in any genomic or genetic databases currently available for other phyla. These results suggest that megB1 may have emerged upon the occurrence of Basidiomycota, and that this phylum evolved thereafter leaving this element conserved throughout their further differentiation. megB1 may be a novel genomic marker useful in the analysis of ancient through the latest evolutionary radiation in Basidiomycota.     

The use of wild edible plants in western and central Anatolia (Turkey)

In this study, 121 wild edible plants used as food in Anatolia were surveyed to determine the plant parts used and their detailed preparation methods. The results of this study show that the plants may be boiled, fried in fat, and eaten raw or as rolled vegetables. They may also be consumed as pickles, fruits, sweets and spices, and drunk as cold and hot drinks. Thirty species (8 genera) were identified as belonging to the Lamiaceae family, 15 species (15 genera) belong to the Asteraceae family, 13 species (5 genera) belong to the Rosaceae family, 8 species (7 genera) belong to the Brassicaceae family, 6 species (3 genera) belong to the Orchidaceae family and 5 species (5 genera) belong to the Apiaceae family. The genera represented by the highest number of species in the study are as follows:Sideritis L. is represented by 13 species, Origanum L. by 7 species,Rubus L. by 5 species,Thymus L. by 4 species andRumex L. by 4 species.     

Analysis of metaphase I chromosome association in species of the genus Aegilops

Summary Metaphase-I chromosome associations in every diploid and polyploid species of the genus Aegilops were studied using C-banding in order to analyse the cytogenetic behaviour of the whole complement as well as of specific genomes in different polyploid species. Differences were observed in the frequency of associations per cell among different species of the same ploidic level and even between species sharing the same genomic constitution. Differences were also found between different genomes within the same polyploid species and between the same genome when present in several diploid and polyploid species. Several factors proposed as having an influence on the frequency of metaphase-I associations, such as chromosome morphology, C-heterochromatin content, genetic control and genome interactions, are discussed. Most of the polyploid Aegilops species showed a diploid-like behaviour at metaphase I although multivalents involving homoeologous associations were occasionally observed in Ae. biuncialis, Ae. juvenalis and Ae. crassa(6x); therefore, the Aegilops diploidising genetic system is not equally effective in all polyploid species.     

10种杜鹃亚属植物种间杂交的可育性研究

为探索杜鹃花亚属内异种杂交的可育性及其规律,对杜鹃亚属有鳞大花亚组(subsect. Maddenia)、三花杜鹃亚组(subsect. Triflora)、亮鳞杜鹃亚组(subsect. Heliolepida)及腋花杜鹃亚组(subsect. Scabrifolia)等4亚组10个杜鹃花种类的22个杂交组合(其中18个数据完整组合)的可育性进行了研究。结果表明:(1)所涉及的杜鹃亚属不同亚组间及三花杜鹃亚组内杂交均较困难,在18个数据完整组合中高可育与可育组合比率明显偏低,不育比率高(55.6%)。(2)在10个不可育或败育组合中,不能坐果(Cab型)、不能结实(Sab型)和可结实而种子不能发芽(Sng型)的数量分布为6∶1∶3,其不亲和或败育发生的阶段可能涵盖了从前合子期到后合子期的整个阶段。(3)亲本一方为多倍体组合的可育率(41.6%),尤其是母本为多倍体时,比二倍体组合的可育率(50.0%)低且无高可育组合出现,部分印证了倍性是导致该亚属植物不同种类杂交不亲和、不育与育性下降的重要原因,但不是唯一原因,亚组间杂交的可育率(16.7%)明显低于亚组内(三花杜鹃亚组内,58.3%)。(4)与相应的母本自然授粉结果相比,杂交明显导致多数可育组合绿苗率比率和单位可育种子数量比率的大幅度下降,这是双亲遗传差异及多倍体亲本介入后所导致的杂交衰退hybrid weakness现象。(5)在多倍体作母本的情况下,杂交单向不育或非对称遗传渗透现象明显。     

Towards the era of comparative evolutionary genomics in Brassicaceae

The vast genetic diversity, specific genome organization and sequencing of the Arabidopsis thaliana genome made crucifers an ideal group for comparative genomic studies. Arabidopsis genomic resources have greatly expedited comparative genomics within Brassicaceae and fostered the establishment of new Arabidopsis relative model systems (ARMS). The extent of genome colinearity, modes and evolutionary rates of genome alterations are being analyzed by genetic mapping with ever increasing levels of precision. Comparative cytogenetic studies in Brassicaceae are employing various chromosome landmarks and cytogenetic techniques, including localization of rDNA, variation in centromeric satellite repeats, genomic in situ hybridization (GISH), fluorescence ISH using bacterial artificial chromosomes (BAC FISH), and large-scale comparative chromosome painting. Some genome alterations may represent rare genomic changes (RGCs) and thus have the potential to resolve complex/conflicting phylogenetic relationships inferred from DNA sequencing. Comparative genomics should increasingly be integrated with molecular phylogenetics and population genetics to elucidate the processes responsible for genetic variation in Brassicaceae.     

Karyological Differentiation in Sapindaceae with Special Reference to Serjania and Cardiospermum

New chromosome counts for 9 species and 2 genera of Sapindaceae are presented and compared with a review of all available chromosome numbers of the family. In 4 species diploid numbers differing from previous reports are found. In 4 species of the tribe Paullinieae (S. diversiflora, S. subdentata, C. grandiflorum and C. halicacabum) detailed studies on interphase nucleus structure, condensing behaviour and chromosome banding patterns are presented. The karyological differentiation of Paullinieae is generally characterized by dysploid reduction of chromosome numbers and the increase of chromosome size. Sequential staining of nuclei with CMA/DAPI and Giemsa-C-banding demonstrates diversification of constitutive heterochromatin (= hc) and different types of chromatin organization in Serjania and Cardiospermum. The obvious lack of polyploid series and the karyological evolution within the family is discussed. The outstanding small genome size found in Cardiospermum halicacabum is considered to be due to a secondary loss of DNA in the course of the change to herbaceous growth.     

CrusView: A Java-Based Visualization Platform for Comparative Genomics Analyses in Brassicaceae Species

In plants and animals, chromosomal breakage and fusion events based on conserved syntenic genomic blocks lead to conserved patterns of karyotype evolution among species of the same family. However, karyotype information has not been well utilized in genomic comparison studies. We present CrusView, a Java-based bioinformatic application utilizing Standard Widget Toolkit/Swing graphics libraries and a SQLite database for performing visualized analyses of comparative genomics data in Brassicaceae (crucifer) plants. Compared with similar software and databases, one of the unique features of CrusView is its integration of karyotype information when comparing two genomes. This feature allows users to perform karyotype-based genome assembly and karyotype-assisted genome synteny analyses with preset karyotype patterns of the Brassicaceae genomes. Additionally, CrusView is a local program, which gives its users high flexibility when analyzing unpublished genomes and allows users to upload self-defined genomic information so that they can visually study the associations between genome structural variations and genetic elements, including chromosomal rearrangements, genomic macrosynteny, gene families, high-frequency recombination sites, and tandem and segmental duplications between related species. This tool will greatly facilitate karyotype, chromosome, and genome evolution studies using visualized comparative genomics approaches in Brassicaceae species. CrusView is freely available at http://www.cmbb.arizona.edu/CrusView/.The Brassicaceae (crucifer) plant family contains more than 3,700 species, including the model plant organism Arabidopsis (Arabidopsis thaliana); economically important crop species, such as Brassica rapa and Brassica napus; and close relatives of Arabidopsis used in abiotic stress research, such as Eutrema salsugineum and Schrenkiella parvula. Because Brassicaceae plants have high scientific and economic importance, several whole-genome sequencing projects of the species in this family have been recently launched (http://www.brassica.info). Moreover, Brassicaceae is also a good system for population genomics. The 1001 Arabidopsis Genomes Project (http://www.1001genomes.org/) plans to generate complete genome sequences for 1,001 Arabidopsis strains to study the associations between genetic variation and phenotypic diversity. The Value-directed Evolutionary Genomics Initiative project aims to understand the genome evolution of Brassicaceae species by sequencing several close relatives of Arabidopsis, such as Arabidopsis lyrata and Capsella rubella. Recent advances in high-throughput sequencing technology have greatly expedited these whole-genome sequencing projects of versatile nonmodel organisms. Although increasingly longer reads can now be produced from high-throughput sequencing experiments, de novo assembler tools can only generate contig and/or scaffold sequences from high-throughput sequencing reads. These tools cannot generate complete chromosome sequences without genetic and/or physical maps that typically require years to create. This limitation makes chromosome-scale structural variation (i.e. translocation, inversion, deletion and insertion, and segmental and tandem duplication) and genomic macrosynteny analyses difficult to perform.In both plants and animals, genomes of species within the same family have evolved with conserved karyotype patterns due to the rearrangements of large chromosomal segments. Chromosomal karyotypes can be obtained from comparative chromosomal painting (CCP) experiments by performing in situ hybridization experiments on bacterial artificial chromosome sequences between related species. The genome of each Brassicaceae member is composed of 24 conserved genomic blocks that have been considered as the basic units of chromosomal rearrangement during genome evolution (Lysak et al., 2006). The sizes of these conserved blocks range from several to dozens of megabases. Currently, karyotypes profiled by CCP experiments in approximately 20 Brassicaceae species are available; such karyotypes include those from Arabidopsis (n = 5), Homungia alpine (n = 6), Eutrema spp. (n = 7), A. lyrata (n = 8), B. rapa (n = 10), and Polyctenium fremontii (n = 14). By utilizing the karyotype information in Brassicaceae, we have developed a tool, KGBassembler (for Karyotype-based Genome assembler for Brassicaceae), to finalize the assembly of chromosomes from scaffolds/contigs without relying on a genetic/physical map (Ma et al., 2012).Over the past 2 years, complete whole-genome sequences of several Brassicaceae species have been released, including the aforementioned A. lyrata, S. parvula, B. rapa, and E. salsugineum (Dassanayake et al., 2011; Hu et al., 2011; Wang et al., 2011; Wright and Agren, 2011; Wu et al., 2012; Yang et al., 2013). These genomic resources have opened a new era of comparative genomics in Brassicaceae to better understand the genomic evolution (Cheng et al., 2012). Numerous tools and databases are available for performing comparative genomics analysis in plants. CoGe is a comparative genomics analysis platform that is now a part of the iPlant Collaborative Project (Goff et al., 2011). The CoGe database currently includes nearly 2,000 genome sequences of approximately 1,500 organisms, allowing users to perform online visual analyses of genome synteny and duplication events (Tang and Lyons, 2012). PLAZA and Vista are also Web-based databases that provide comparative analysis services on the genomic data deposited in the databases (Frazer et al., 2004; Van Bel et al., 2012). Other stand-alone bioinformatic applications for comparative genomic analysis, such as Easyfig and genoPlotR, are commonly used to generate synteny plots of given genome segments at a scale ranging from a single gene to one chromosome (Guy et al., 2010; Sullivan et al., 2011).In this work, we present a Java-based bioinformatic application, CrusView, for performing visualized analyses of genome synteny and karyotype evolution in Brassicaceae species. CrusView features a user-friendly graphical user interface (GUI) implemented with Standard Widget Toolkit (SWT)/Swing graphics libraries and a SQLite database used to manage local genomic data. Compared with the most commonly used tools in comparative genomics, one of the unique features of CrusView is that available karyotype data of a Brassicaceae species are incorporated to facilitate karyotype-based chromosome assembly and analyses of chromosomal structural evolution. Compared with Web-based tools, the stand-alone CrusView tool was also designed to give users higher flexibility in analyzing currently unpublished genome data and integrating self-defined genomic information based on the users’ interests, such as gene families, gene duplications, chromosomal break points, Gene Ontology terms, and groups of orthologs/paralogs, with the genomic synteny maps. In addition, CrusView can generate images representing genomic synteny between two compared genomes in PNG/SVG/PDF high-resolution formats that are suitable for publication.