Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.     

Allozyme genetics in permanent translocation heterozygotes of the Oenothera biennis complex

Allozyme inheritance and transmission genetics of 11 enzyme systems were determined in the permanent translocation heterozygotes Oenothera biennis, Oe. strigosa, and Oe. parviflora. Electrophoretic variation was examined first among 164 strains of structural heterozygotes. Allelic configurations were then judged from inheritance patterns in reciprocal F1 hybrids between each of 22 ring-forming strains and tester strains of the related bivalent-formers, Oe. hookeri and Oe. grandiflora. Allozymes are inherited as codominant markers, and, as dictated by the genetic system, within a strain individual allelic variants are generally transmitted through only one germ line. Of the 20 loci resolved, only eight are polymorphic in any species, and, within species, generally only two alleles are present at each polymorphic locus. Despite the relatively meager allelic array, each of the 22 strains whose chromosome complexes were characterized is genotypically unique. Generally, within taxa, alpha (egg) and beta (sperm) complexes differ in allele frequency at several polymorphic loci. Such variability is correlated with differences in the phylogenetic origins of complexes and not with differences in segmental arrangement within a group of related complexes.     

Inactivation of pollen and other effects of genome-plastome incompatibility inOenothera

A series of strains of the homozygous speciesOenothera grandiflora (characterized by the genome BB and plastome III) were combined with plastome IV fromO. parviflora (BC-IV) by means of appropriate crosses. An incompatibility between genome B and plastome IV is expressed in the haplo- and diplophase: (1) B-IV pollen, though normally developed, is largely inactive. The extent of the inactivation varies between different strains and shows a seasonal fluctuation as determined by seed set in outcrossing and selfing experiments. (2) In most of the strains lethality of BB-IV embryos is the rule, leading to empty seeds. This can be ameliorated by including another plastome in the zygotes and developing embryos on account of the biparental plastid transmission inOenothera. It can best be demonstrated in crosses with a seed parent having normal green plastids of plastome IV and mutated chlorophyll deficient plastids from a different plastome in the pollen parent, leading to variegated progeny as well as a remainder of empty seeds. (3) In about one-half of the strains the BB-IV plants exhibit a temporary bleaching of thevirescens type. The incompatibily between genome B and plastome IV does not support the earlier assumption that plastome IV is the ancestor of plastomes II, III, and V. Instead, a precursor plastome is postulated from which plastomes II, III, and IV are descended. While plastome I can be derived from II, only plastome V can be descended from plastome IV.Deceased August 28, 1998.     

Cytogenetic investigations onOenothera wolfii (Onagraceae)

Oenothera wolfii, endemic in coastal Northern California and Southern Oregon, has been analysed cytogenetically. It is a permanent structural heterozygote composed of two nearly identical genomic complexes which are closely related to the neighboring maritime ecotype of the homozygousOe. elata subsp.hookeri. — Oe wolfii is believed to have evolved recently from its homozygous ancestor by the accumulation of reciprocal translocations and the acquisition of balanced lethals. Forms such asOe. wolfii represent an important connecting link in our understanding of the evolution of the complexheterozygous species ofOenothera. Dedicated to Prof. Dr.Josef Straub for His 75th birthday.     

Genetically Programmed Chloroplast Dedifferentiation as a Consequence of Plastome-Genome Incompatibility in Oenothera

Comparision of chloroplast from plants with one of four plastome types (I, II, III, IV) in the nuclear background of Oenothera elata strain Johansen addressed the effects of plastome-genome incompatibility with respect to leaf pigmentation, plastid ultrastructure, chlorophyll a/chlorophyll b ratio, and photosynthetic electron transport. Previous observations of plastomes I, II, and IV in this nuclear background have revealed no indications of incompatibility, but the studies reported here demonstrate that chloroplasts of plastome IV have subtle alterations in their photosynthetic abilities, in particular, deficiencies in photosystem II. The well-characterized "hybrid bleaching" of plants with the AA genotype and plastome III involves leaves that become bleached in the center while remaining green at the tips, edges, and veins. Electron transport assays performed on fractionated bleached and green tissue from the same plants show photosynthetic defects in both the green and bleached regions, although defects in the latter are more severe. Ultrastructural studies show that chloroplasts in the bleached areas enlarge, thylakoid membranes become swollen and vesiculated, and production of new thylakoids is blocked, with chloroplasts appearing to undergo a programmed senescence. A time course revealed that the senescence is actually a reversible dedifferentiation. Alterations in the composition of medium to which AA/III seedlings were transferred showed that the presence of auxin can prevent the development of the typical incompatibility response, with leaf tissue remaining green rather than bleaching. It is proposed that differences in concentrations of plant growth regulators may be responsible for the persistence of normal chloroplasts near the vascular tissue and leaf blade edges and that seasonal fluctuations in auxin levels could explain the periodic bleaching that occurs in older plants.     

A spontaneous point mutation in the Rubisco large subunit gene impairing holoenzyme assembly renders the IVβ plastome mutant of Oenothera extremely light‐ and chilling sensitive

A spontaneously occurring chloroplast genome (plastome) mutant of Oenothera , IVβ, was identified as a single point mutation in the Rubisco large subunit gene (G337 → C), leading to an V113L exchange, which topologically occurs at the interface of two adjacent large subunits (LSU). The minor sterical hindrance of dimer formation by this amino acid exchange strongly impairs holoenzyme assembly, leading to an accumulation of a processing precursor of the holoenzyme, the B‐complex, consisting of one LSU and 14 units of chaperonine 60 (cpn60). It is associated with very low holoenzyme concentrations in the mutant tissue, but does not affect the kinetic properties of the enzyme once assembled. When grown under moderate or low light, leaf tissue containing the plastome mutant showed decreased Chl contents and Chl a / b ratios, increased relative carotenoid contents and violaxanthin deepoxidation activity, but very low CO₂ fixation and O₂ evolution rates and was very sensitive to photoinhibition. The light dependence of chlorophyll fluorescence quenching components at low temperature resembled an extremely chilling sensitive Oenothera genotype as compared to the wild‐type. The IVβ mutant thus behaves similarly to the Rubisco SSU antisense plants analysed by Stitt and co‐workers (summarised by Stitt and Schulze 1994) and gives an example of the possible influence of plastome mutations on the sensitivity of the photosynthetic apparatus to excess light by modifying the capacity of the Calvin cycle.     

Plastome mutator-induced alterations arise in Oenothera chloroplast DNA through template slippage.

The plastome mutator of Oenothera hookeri strain Johansen causes deletions and duplications at target sites defined by direct repeats in the plastid genome. Previous studies characterized the mutations long after they had occurred and could not discriminate between the possibilities that the plastome mutator acted through unequal homologous recombination or template slippage. From the known hotspots, the rRNA spacer in the large inverted repeat was chosen for this study because it contains both direct and indirect repeats. Identical deletions were recovered from independently derived plants; the altered regions were always flanked by direct repeats. The regions in which the deletions occurred have the potential to form secondary structures that would stabilize the intervening sequence. Of the two affected regions, the one with the stronger potential secondary structure was altered more frequently. Because no duplication products or inversions were recovered, it is proposed that the plastome mutator acts through template slippage rather than through a recombination mechanism.     

Cytogenetic investigations onOenothera nutans (Onagraceae)

Oenothera nutans, common to the Appalachian Mts between 650 and 1 700 m altitude, was investigated cytogenetically and taxonomically. The species is permanently structurally heterozygous. It consists of two genomes of the B-type which are more or less indistinguishable phenotypically. Nearly all of the strains investigated possess a self-incompatibility factor in one of the two complexes. Both complexes show a close relationship to the predominantly homozygousO. grandiflora, a native of the southern lowlands.O. nutans andO. grandiflora possess the same plastid type, plastome III. Probably,O. nutans evolved by an accumulation of reciprocal translocations within an originally structurally homozygous population, which must be regarded ancestral to the present forms ofO. grandiflora.     

Identification of plastid genotypes in Oenothera subsect. Munzia by restriction fragment length polymorphisms (RFLPs)

 Five discrete plastid genotypes (plastomes), designated I–V and typified by Oenothera Hookeri, biennis, Lamarckiana, parviflora and argillicola respectively, have been previously characterized within the European subsect. Euoenothera. The evolutionarily more-derived plastome types (I, II and V) are generally less tolerant of new hybridization events than the ancestral types (III and IV), and were first identified based on their incompatibility reactions with standard hybrid nuclei. Restriction maps for all five plastomes are available for the enzymes PvuII, SalI, KpnI and PstI (Gordon et al. 1982). The present study employs PvuII and KpnI restriction digests to compare 28 of the 45 species of subsect. Munzia with Euoenothera plastomes I–V. The results of plastome RFLP fingerprinting show uniform divergence of the South American taxa from their European congeners; all share the previously documented 45-kb inversion in the large single-copy region reported by Hachtel et al. (1991). However, at least six new plastome types have evolved within subsect. Munzia, giving rise to small-fragment size differences of 0.1–0.7 kb. In two of these cases (Oe. featherstonei and Oe. longiflora) unique fragments occurred. For Oe. featherstonei the unique KpnI fragment resulted from a novel 2.2 kb insertion, whereas in Oe. longiflora an additional PvuII restriction site has been created. Received: 2 June 1998 / Accepted: 14 July 1998     

Development of polarity in the ovules of the F2-progeny of theOenothera hybridalbicans · hhookeri

In the F₂-progeny of hybrids from crosses betweenOenothera biennis orsuaveolens andOe. hookeri with theRenner-complexesalbicans and^hhookeri, the development of callose pattern in meiocytes and megaspore tetrads is the same as in the F₁ and the parentOe. hookeri. During the development of the megaspore tetrads and the embryo sacs primary and secondary heteropolarity as well as homopolarity is observed. Estimates for the initial frequency of homo- and heteropolar tetrads at the end of the degeneration of megaspores in the tetrads immediately before the start of embryo sac development could be calculated. The F₂-plants can be arranged in three groups, distinguished by the frequency of the two polarity types. One of these groups behaves similar to the parentOe. hookeri, the two others have more homopolar tetrads. The segregation can be interpreted as recombination of genes, which influence the development of the polarity in the ovules. This is possible by crossing-over of genes between the twoRenner-complexes of the hybrid.     

Chromosome studies in Andean taxa of Alstroemeria (Alstroemeriaceae)

Meiotic or mitotic chromosomes of seven Alstroemeria taxa, native in Argentina and Chile and with Andean distribution were studied: A. andina ssp. venustula, A. hookeri ssp. cummingiana , A. hookeri ssp. recumbens , A. pallida , A. patagonica , A. pseudospathulata and A. pygmaea . All were diploid with 2 n = 16. Karyotypes of A. andina ssp. venustula and A. pygmaea were analysed, revealing similarity to previously analysed species. Thus, to all existing arguments for not retaining Schickendantzia as a separate genus, we can add another one which merges A. pygmaea with other Alstroemeria species, and does not support its taxonomic uniqueness. In general, the meiotic behaviour was normal, with regular formation of eight bivalents except in A. hookeri ssp. cummingiana , in one plant of which meiotic irregularities at various stages were observed. At the tetrad stage a large percentage of the cells presented micronuclei. The presence of 0–2 supernumerary chromosomes in A. hookeri ssp. recumbens is recorded. The karyotype asymmetry presented by most Alstroemeria species is discussed. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 138 , 451–459.     

The chicken or the egg? Plastome evolution and an independent loss of the inverted repeat in papilionoid legumes

The plastid genome (plastome), while surprisingly constant in gene order and content across most photosynthetic angiosperms, exhibits variability in several unrelated lineages. During the diversification history of the legume family Fabaceae, plastomes have undergone many rearrangements, including inversions, expansion, contraction and loss of the typical inverted repeat (IR), gene loss and repeat accumulation in both shared and independent events. While legume plastomes have been the subject of study for some time, most work has focused on agricultural species in the IR-lacking clade (IRLC) and the plant model Medicago truncatula. The subfamily Papilionoideae, which contains virtually all of the agricultural legume species, also comprises most of the plastome variation detected thus far in the family. In this study three non-papilioniods were included among 34 newly sequenced legume plastomes, along with 33 publicly available sequences, to assess plastome structural evolution in the subfamily. In an effort to examine plastome variation across the subfamily, approximately 20% of the sampling represents the IRLC with the remainder selected to represent the early-branching papilionoid clades. A number of IR-related and repeat-mediated changes were identified and examined in a phylogenetic context. Recombination between direct repeats associated with ycf2 resulted in intraindividual plastome heteroplasmy. Although loss of the IR has not been reported in legumes outside of the IRLC, one genistoid taxon was found to completely lack the typical plastome IR. The role of the IR and non-IR repeats in the progression of plastome change is discussed.     

An evaluation of putative sympatric speciation within Limnanthes (Limnanthaceae)

Limnanthes floccosa ssp. floccosa and L. floccosa ssp. grandiflora are two of five subspecies within Limnanthes floccosa endemic to vernal pools in southern Oregon and northern California. Three seasons of monitoring natural populations have quantified that L. floccosa ssp. grandiflora is always found growing sympatrically with L. floccosa ssp. floccosa and that their flowering times overlap considerably. Despite their subspecific rank within the same species crossing experiments have confirmed that their F1 hybrids are sterile. An analysis of twelve microsatellite markers, with unique alleles in each taxon, also shows exceedingly low levels of gene flow between populations of the two subspecies. Due to the lack of previous phylogenetic resolution among L. floccosa subspecies, we used Illumina next generation sequencing to identify single nucleotide polymorphisms from genomic DNA libraries of L. floccosa ssp. floccosa and L. floccosa ssp. grandiflora. These data were used to identify single nucleotide polymorphisms in the chloroplast, mitochondrial, and nuclear genomes. From these variable loci, a total of 2772 bp was obtained using Sanger sequencing of ten individuals representing all subspecies of L. floccosa and an outgroup. The resulting phylogenetic reconstruction was fully resolved. Our results indicate that although L. floccosa ssp. floccosa and L. floccosa ssp. grandiflora are closely related, they are not sister taxa and therefore likely did not diverge as a result of a sympatric speciation event.     

Molecular marker systems for Oenothera genetics

The genus Oenothera has an outstanding scientific tradition. It has been a model for studying aspects of chromosome evolution and speciation, including the impact of plastid nuclear co-evolution. A large collection of strains analyzed during a century of experimental work and unique genetic possibilities allow the exchange of genetically definable plastids, individual or multiple chromosomes, and/or entire haploid genomes (Renner complexes) between species. However, molecular genetic approaches for the genus are largely lacking. In this study, we describe the development of efficient PCR-based marker systems for both the nuclear genome and the plastome. They allow distinguishing individual chromosomes, Renner complexes, plastomes, and subplastomes. We demonstrate their application by monitoring interspecific exchanges of genomes, chromosome pairs, and/or plastids during crossing programs, e.g., to produce plastome-genome incompatible hybrids. Using an appropriate partial permanent translocation heterozygous hybrid, linkage group 7 of the molecular map could be assigned to chromosome 9.8 of the classical Oenothera map. Finally, we provide the first direct molecular evidence that homologous recombination and free segregation of chromosomes in permanent translocation heterozygous strains is suppressed.     

Membrane composition and physiological activity of plastids from an oenothera plastome mutator-induced chloroplast mutant

Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly.     

Plastid genome structure and loss of photosynthetic ability in the parasitic genus Cuscuta

The genus Cuscuta (dodder) is composed of parasitic plants, some species of which appear to be losing the ability to photosynthesize. A molecular phylogeny was constructed using 15 species of Cuscuta in order to assess whether changes in photosynthetic ability and alterations in structure of the plastid genome relate to phylogenetic position within the genus. The molecular phylogeny provides evidence for four major clades within Cuscuta. Although DNA blot analysis showed that Cuscuta species have smaller plastid genomes than tobacco, and that plastome size varied significantly even within one Cuscuta clade, dot blot analysis indicated that the dodders possess homologous sequence to 101 genes from the tobacco plastome. Evidence is provided for significant rates of DNA transfer from plastid to nucleus in Cuscuta. Size and structure of Cuscuta plastid genomes, as well as photosynthetic ability, appear to vary independently of position within the phylogeny, thus supporting the hypothesis that within Cuscuta photosynthetic ability and organization of the plastid genome are changing in an unco-ordinated manner.     

Chloroplast DNA differences between two species of Oenothera subsection Munzia: location in the chloroplast genome and relevance to possible interactions between nuclear and plastid genomes

Summary The chloroplast DNAs (cpDNAs) of Oenothera berteriana and Oe. odorata (subsection Munzia) were examined by restriction endonuclease analysis with Sal I, Pvu II, Kpn I, Pst I, Hind III, and Bam HI. The fragment patterns show that these cpDNAs have all 133 restriction sites in common as well as a lot of individual bands. Nevertheless the cpDNAs of the two species can be distinguished by distinct differences in size between a small number of fragments. The 42 cleavage sites produced by Sal I, Pvu II and Kpn I were mapped on the circular cpDNAs. This was achieved by an approach which combined experimental and mathematical procedures. The overall serial order of the fragments was found to be the same for both cpDNAs. The size differences of individual fragments in the Sal I, Pvu II and Kpn I patterns between Oe. berteriana and Oe. odorata cpDNA are located within five regions scattered along the plastid chromosome. Two of these regions have been localized in the larger and one in the smaller of the two single-copy parts of the cpDNA molecule. The remaining two overlap the borders between the large single-copy and each of the duplicated parts of the molecule. The positions of distinct restriction sites are altered among the two Oenothera plastome DNAs by 0.02–0.4 MDa (30–600 base pairs). These alterations probably result from insertions/deletions.Abbreviations cpDNA chloroplast, plastid DNA - Oe. Oenothera - MDa Megadalton - rRNA, rDNA ribosomal RNA, DNA Dedicated to Professor Berthold Schwemmle, Tübingen, on the occasion of his 60th birthday     

Complete sequencing and comparative analyses of the pepper (Capsicum annuum L.) plastome revealed high frequency of tandem repeats and large insertion/deletions on pepper plastome

Plants in the family Solanaceae are used as model systems in comparative and evolutionary genomics. The complete chloroplast genomes of seven solanaceous species have been sequenced, including tobacco, potato and tomato, but not peppers. We analyzed the complete chloroplast genome sequence of the hot pepper, Capsicum annuum. The pepper chloroplast genome was 156,781 bp in length, including a pair of inverted repeats (IR) of 25,783 bp. The content and the order of 133 genes in the pepper chloroplast genome were identical to those of other solanaceous plastomes. To characterize pepper plastome sequence, we performed comparative analysis using complete plastome sequences of pepper and seven solanaceous plastomes. Frequency and contents of large indels and tandem repeat sequences and distribution pattern of genome-wide sequence variations were investigated. In addition, a phylogenetic analysis using concatenated alignments of coding sequences was performed to determine evolutionary position of pepper in Solanaceae. Our results revealed two distinct features of pepper plastome compared to other solanaceous plastomes. Firstly, large indels, including insertions on accD and rpl20 gene sequences, were predominantly detected in the pepper plastome compared to other solanaceous plastomes. Secondly, tandem repeat sequences were particularly frequent in the pepper plastome. Taken together, our study represents unique features of evolution of pepper plastome among solanaceous plastomes.