Is there hybridization between diploid and tetraploid Euphrasia in a secondary contact zone?

Premise

Strong postzygotic reproductive isolating barriers are usually expected to limit the extent of natural hybridization between species with contrasting ploidy. However, genomic sequencing has revealed previously overlooked examples of natural cross-ploidy hybridization in some flowering plant genera, suggesting that the phenomenon may be more common than once thought. We investigated potential cross-ploidy hybridization in British eyebrights (Euphrasia, Orobanchaceae), a group from which 13 putative cross-ploidy hybrid combinations have been reported based on morphology.

Methods

We analyzed a contact zone between diploid Euphrasia rostkoviana and tetraploid E. arctica in Wales. We sequenced part of the internal transcribed spacer (ITS) of nuclear ribosomal DNA and used genotyping by sequencing (GBS) to look for evidence of cross-ploidy hybridization and introgression.

Results

Common variant sites in the ITS region were fixed between diploids and tetraploids, indicating a strong barrier to hybridization. Clustering analyses of 356 single-nucleotide polymorphisms (SNPs) generated using GBS clearly separated samples by ploidy and revealed strong genetic structure (F_ST = 0.44). However, the F_ST distribution across all SNPs was bimodal, indicating potential differential selection on loci between diploids and tetraploids. Demographic inference suggested potential gene flow, limited to around one or fewer migrants per generation.

Conclusions

Our results suggest that recent cross-ploidy hybridization is rare or absent in a site of secondary contact in Euphrasia. While a strong ploidy barrier prevents hybridization over ecological timescales, such hybrids may form in stable populations over evolutionary timescales, potentially allowing cross-ploidy introgression to take place.     

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.     

PHYLOGENY OF THE ULVOPHYCEAE (CHLOROPHYTA): CLADISTIC ANALYSIS OF NUCLEAR-ENCODED rRNA SEQUENCE DATA1

Cladistic analysis of nuclear-encoded rRNA sequence data provided us with the basis for some new hypotheses of relationships within the green algal class Ulvophyceae. The orders Ulotrichales and Ulvales are separated from the clade formed by the remaining orders of siphonous and siphonocladous Ulvophyceae (Caulerpales, Siphonocladales /Cladophorales [S/C] complex, and the Dasycladales), by the Chlorophyceae and Pleurastrophyceae. Our results suggest that the Ulvophyceae is not a monophyletic group. Examination of inter- and intra-ordinal relationships within the siphonous and siphonocladous ulvophycean algae revealed that Cladophora, Chaetomorpha, Anadyomene, Microdictyon, Cladophoropsis and Dictyosphaeria form a clade. Thus the hypothesis, based on ultrastructural features, that the Siphonocladales and Cladophorales are closely related is supported. Also, the Caulerpales is a monophyletic group with two lineages; Caulerpa, Halimeda, and Udotea comprise one, and Bryopsis and Codium comprise the other. The Dasycladales (Cymopolia and Batophora) also forms a clade, but this clade is not inferred to be the sister group to the S/C complex as has been proposed. Instead, it is either the sister taxon to the Caulerpales or basal to the Caulerpales and S/C clade The Trentepohliales is also included at the base of the siphonous and siphonocladous ulvophycean clade. The Pleurastrophyceae, which, like the Ulvophyceae, posses a counter-clockwise arrangement of flagellar basal bodies, are more closely related to the Chlorophyceae than to the Ulvophyceae based on rRNA sequences. Thus, the arrangement of basal bodies does not diagnose a monophyletic group. Previously reported hypotheses of phylogenetic relationships of ulvophycean algae were tested. In each case, additional evolutionary steps were required to obtain the proposed relationships. Relationships of ulvophycean algae to other classes of green algae are discussed.     

Cloning single-stranded DNA

DNA sequence and expression analyses have greatly benefited from using M13 and pUC derived cloning vectors and their polycloning sites. A chronology of the original concepts and experiments is reviewed.     

SEQUENCES OF THE RRN18 GENES OF CHLAMYDOMONAS HUMICOLA AND C. DYSOSMOS ARE IDENTICAL,IN AGREEMENT WITH THEIR COMBINATION IN THE SPECIES C. APPLANATA (CHLOROPHYTA)1

The nuclear Rrn18 gene coding for small-subunit ribosomal RNA was amplified from Chlamydomonas humicola and C. dysosmos. The sequences were identical, in agreement with the combination of these two species under the name C. applanata on morphological and physiological grounds by Ettl and Schlösser (1992).     

Isolation of restriction enzyme EcoVIII, an isoschizomer of HindIII, produced by Escherichia coli E1585-68

A restriction endonuclease designated EcoVIII, an isoschizomer of HindIII, was isolated from Escherichia coli E1585-68 and purified by dextran-polyethylene glycol (DPG) phase partition, ammonium sulfate precipitation, phospho- and DEAE-cellulose chromatography, and hydroxylapatite chromatography. The purified EcoVIII was stable during the purification procedure and its high specific activity required 10 mM Mg²⁺. Unlike HindIII, Eco VIII exhibited a high specific activity even at low pH (pH 6.3) and showed the highest activity at 48° C. Transformation of purified plasmid DNA from E. coli E1585-68 into K-12 indicated that the EcoVIII gene was carried on a multicopy 4.4-kb miniplasmid. EcoVIII seems to be preferable to HindIII for its production and use because of easier handling of producer cells and a wider range of activity.     

A direct approach to the measurement of genetic variation in fish populations: applications of the polymerase chain reaction to studies of Atlantic cod, Gadus morhua L.

We used the polymerase chain reaction (PCR) and direct DNA sequencing to study genetic variation within and among populations of Atlantic cod, Gadus morhua , in the western North Atlantic. In a 307 bp region of the mitochondrial cytochrome b gene, 24 variable nucleotide positions define 24 genotypes, which differ by from one to six nucleotide substitutions. Greenland cod ( G. ogac ) differs from the most similar G. morhua genotype by an additional 12 nucleotide substitutions. Silent transitions dominate both intra- and interspecific comparisons, however four nucleotide substitutions within morhua result in amino acid replacements. Direct sequencing of DNA reveals substantially more of the genetic variation that exists within and between species than do previous indirect methods based on restriction fragment length polymorphisms, and thus has far greater potential to quantify such differences as may exist among fish stocks. Preliminary experiments also indicate that automation of DNA sequencing provides an efficient, rapid, and accurate means for detection of genetic variation in natural populations offish.     

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.     

Molecular cloning of an alanine aminotransferase from NAD-malic enzyme type C4 plant Panicum miliaceum

We have determined the nucleotide sequence of a cDNA encoding AlaAT-2, which is believed to function in the C4-pathway of Panicum miliaceum. An open reading frame (1446 bp) encodes a protein of 482 amino acid residues. The deduced amino acid sequence of AlaAT-2 shows 44.2 and 44.8% homology with the amino acid sequences of AlaATs from rat and human livers, respectively. Northern blot analysis showed that the gene encoding AlaAT-2 in mesophyll and bundle sheath cells was the same and transcribed similarly in the cells. The level of translatable mRNA for AlaAT-2 increased dramatically during greening.