Simon J. Hiscock

Genomic Clues to the Evolutionary Success of Polyploid Plants

Polyploidy, or the presence of two or more diploid parental genome sets within an organism, is found to an amazing degree in higher plants. In addition, many plant species traditionally considered to be diploid have recently been demonstrated to have undergone rounds of genome duplication in the past and are now referred to as paleopolyploids. Polyploidy and interspecific hybridisation (with which it is often associated) have long been thought to be important mechanisms of rapid species formation. The widespread occurrence of polyploids, which are frequently found in habitats different from that of their diploid progenitors, would seem to indicate that polyploidy is associated with evolutionary success in terms of the ability to colonise new environmental niches. A flurry of recent genomic studies has provided fresh insights into the potential basis of the phenotypic novelty of polyploid species. Here we review current knowledge of genetic, epigenetic, and transcriptional changes associated with polyploidy in plants and assess how these changes might contribute to the evolutionary success of polyploid plants. We conclude by stressing the need for field-based experiments to determine whether genetic changes associated with polyploidy are indeed adaptive.

PCP-A1, a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression.

Self-incompatibility (SI) in Brassica species is controlled by a single polymorphic locus (S) with multiple specificities. Two stigmatically expressed genes that have been cloned from this region encode the S locus glycoprotein (SLG) and S receptor kinase (SRK). Both appear to be essential for the operation of SI. It is believed that rejection of incompatible pollen grains is effected by recognition events between an as yet unidentified S locus–encoded pollen coating–borne protein and the SLG/SRK. We previously identified a small pollen coat protein PCP7 (renamed here PCP-A1, for pollen coat protein, class A, 1) that binds with high affinity to SLGs irrespective of S genotype. Here, we report the cloning of PCP-A1 from Brassica oleracea and demonstrate that it is unlinked to the S locus. In situ localization of PCP-A1 transcripts revealed that they accumulate specifically in pollen at the late binucleate/trinucleate stage of development rather than in the tapetum, which previously was taken to be the principal source of the pollen coat. PCP-A1 is characterized by the presence of a structurally important motif consisting of eight cysteine residues shared by the plant defensins. Based on the presence of this motif and other data, homology modeling has been used to produce a putative structure for PCP-A1. Protein–protein interaction analyses demonstrate that SLG exists in monomeric and dimeric forms, both of which bind PCP-A1. Evidence is also presented for the existence of putative membrane-associated PCP-A1 binding proteins in stigmatic tissue.

Development of anonymous cDNA microarrays to study changes to the Senecio floral transcriptome during hybrid speciation

Interspecific hybridization is an important process through which abrupt speciation can occur. In recent years, genetic changes associated with hybrid speciation have been identified through a variety of techniques, including AFLP/SSR mapping, GISH/FISH and cDNA‐AFLP differential display. However, progress in using microarray technology to analyse whole genome/transcriptome changes associated with hybrid speciation has been limited due to the lack of extensive sequence data for many hybrid species and the difficulties in extrapolating results from commercially available microarrays for model species onto nonmodel hybrid taxa. Increasingly therefore researchers studying nonmodel systems are turning to the development of ‘anonymous’ cDNA microarrays, where the time and cost of producing microarrays is reduced by printing unsequenced cDNA clones, and sequencing only those clones that display interesting expression patterns. Here we describe the creation, testing and preliminary use of anonymous cDNA microarrays to study changes in floral transcriptome associated with allopolyploid speciation in the genus Senecio. We report a comparison of gene expression between the allohexaploid hybrid, Senecio cambrensis, its parental taxa Senecio squalidus (diploid) and Senecio vulgaris (tetraploid), and the intermediate triploid (sterile) hybrid Senecio×baxteri. Anonymous microarray analysis revealed dramatic differences in floral gene expression between these four taxa and demonstrates the power of this technique for studies of the genetic impact of hybridization in nonmodel flowering plants.

Changes to gene expression associated with hybrid speciation in plants: further insights from transcriptomic studies in Senecio

Interspecific hybridization is an important mechanism of speciation in higher plants. In flowering plants, hybrid speciation is usually associated with polyploidy (allopolyploidy), but hybrid speciation without genome duplication (homoploid hybrid speciation) is also possible, although it is more difficult to detect. The combination of divergent genomes within a hybrid can result in profound changes to both genome and transcriptome. Recent transcriptomic studies of wild and resynthesized homoploid and allopolyploid hybrids have revealed widespread changes to gene expression in hybrids relative to expression levels in their parents. Many of these changes to gene expression are ‘non-additive’, i.e. not simply the sum of the combined expression levels of parental genes. Some gene expression changes are far outside the range of gene expression in either parent, and can therefore be viewed as ‘transgressive’. Such profound changes to gene expression may enable new hybrids to survive in novel habitats not accessible to their parent species. Here, we give a brief overview of hybrid speciation in plants, with an emphasis on genomic change, before focusing discussion on findings from recent transcriptomic studies. We then discuss our current work on gene expression change associated with hybrid speciation in the genus Senecio (ragworts and groundsels) focusing on the findings from a reanalysis of gene expression data obtained from recent microarray studies of wild and resynthesized allopolyploid Senecio cambrensis. These data, showing extensive non-additive and transgressive gene expression changes in Senecio hybrids, are discussed in the light of findings from other model systems, and in the context of the potential importance of gene expression change to hybrid speciation in plants.

Evolutionary history of plant microRNAs

microRNAs (miRNAs) are short noncoding regulatory genes that perform important roles in plant development and physiology. With the increasing power of next generation sequencing technologies and the development of bioinformatic tools, there has been a dramatic increase in the number of studies surveying the miRNAomes of plant species, which has led to an explosion in the number of described miRNAs. Unfortunately, very many of these new discoveries have been incompletely annotated and thus fail to discriminate genuine miRNAs from small interfering RNAs (siRNAs), fragments of longer RNAs, and random sequence. We review the published repertoire of plant miRNAs, discriminating those that have been correctly annotated. We use these data to explore prevailing hypotheses on the tempo and mode of miRNA evolution within the plant kingdom.

Cellular and molecular mechanisms of sexual incompatibility in plants and fungi.

Plants and fungi show an astonishing diversity of mechanisms to promote outbreeding, the most widespread of which is sexual incompatibility. Sexual incompatibility involves molecular recognition between mating partners. In fungi and algae, highly polymorphic mating-type loci mediate mating through complementary interactions between molecules encoded or regulated by different mating-type haplotypes, whereas in flowering plants polymorphic self-incompatibility loci regulate mate recognition through oppositional interactions between molecules encoded by the same self-incompatibility haplotypes. This subtle mechanistic difference is a consequence of the different life cycles of fungi, algae, and flowering plants. Recent molecular and biochemical studies have provided fascinating insights into the mechanisms of mate recognition and are beginning to shed light on evolution and population genetics of these extraordinarily polymorphic genetic systems of incompatibility.

Unilateral incompatibility within the Brassicaceae:further evidence for the involvement of the self-incompatibility (S)-locus

Unilateral pollen-pistil incompatibility within the Brassicaceae has been re-examined in a series of interspecific and intergeneric crosses using 13 self-compatible (SC, Sc) species and 12 self-incompatible (SI) species from ten tribes. SC x SC crosses were usually compatible, SI x SC crosses showed unilateral incompatibility, while SI x SI crosses were often incompatible or unilaterally incompatible. Unilateral incompatibility (UI) is shown to be overcome by bud pollination or treating stigmas with cycloheximide — features in common with self-incompatibility. Treating stigmas with pronase prevents pollen tubes from penetrating the stigma in normally compatible intra-and interspecific pollinations. The results presented show that the presence of an incompatibility system is important in predicting the outcome of interspecific and intergeneric crosses and, combined with the physiological similarities between UI and SI, would suggest an involvement of the S-locus in UI.

Identification and localization of an active cutinase in the pollen of Brassica napus L.

Polyclonal antiserum and monoclonal antibodies raised to a purified cutinase from Fusarium solani f. sp. pisi have been used to identify an active cutinase in the pollen of Brassica napus. These antibodies recognized a polypeptide with an estimated molecular weight of 22kDa — a molecular weight indentical to that of the Fusarium cutinase — and localized this polypeptide to the intine of the pollen wall. Enzyme assays on the renatured 22kDa polypeptide after electroelution from a preparative SDS-PAGE gel revealed the polypeptide to be an enzyme capable of catalysing the hydrolysis of tritiated apple cutin and the synthetic substrate p-nitrophenyl butyrate. The molecular weight, immunological properties and substrate specificity of the Brassica cutinase suggest that this enzyme resembles more closely fungal cutinases than it does the cutinase from the pollen of Nasturtium (Tropaeolum majus) — the only angiosperm cutinase so far characterized (Maiti et al., 1979, Arch. Biochem. Biophys. 196, 412–423). These differences between the pollen cutinases from two members of the Dicotyledoneae are unexpected and predict a diversity of this class of pollen enzyme within the angiosperms.

Hybridization and polyploidy as drivers of continuing evolution and speciation in Sorbus

Interspecific hybridization and polyploidy are pivotal processes in plant evolution and speciation. The fate of new hybrid and polyploid taxa is determined by their ability to reproduce either sexually or asexually. Hybrids and allopolyploids with odd chromosome numbers are frequently sterile but some establish themselves through asexual reproduction (vegetative or apomixis). This allows novel genotypes to become established by isolating them from gene flow and leads to complex patterns of variation. The genus Sorbus is a good example of taxonomic complexity arising from the combined effects of hybridization, polyploidy and apomixis. The Avon Gorge in South‐west Britain contains the greatest diversity of Sorbus in Europe, with three endemic species and four putative endemic novel hybrids among its 15 native Sorbus taxa. We used a combination of nuclear microsatellite and chloroplast DNA markers to investigate the evolutionary relationships among these Sorbus taxa within the Avon Gorge. We confirm the genetic identity of putative novel taxa and show that hybridization involving sexual diploid species, primarily S. aria and S. torminalis and polyploid facultative apomictic species from subgenus Aria, has been responsible for generating this biodiversity. Importantly our data show that this creative evolutionary process is ongoing within the Avon Gorge. Conservation strategies for the rare endemic Sorbus taxa should therefore consider all Sorbus taxa within the Gorge and must strive to preserve this evolutionary process rather than simply the individual rare taxa that it produces.

Genetic control of self-incompatibility in Senecio squalidus L. (Asteraceae): a successful colonizing species

Senecio squalidus (Oxford ragwort) is a well-known introduction to the British flora that has proved to be an extremely successful colonist over the last 150 years. Unusually for a colonizing species, S. squalidus is self-incompatible (SI). Being a member of the Asteraceae, SI in S. squalidus is expected to be sporophytic. This paper presents genetic data showing that the SI system of S. squalidus is indeed sporophytic and is controlled by a single multiallelic S locus, alleles of which show the dominance/recessive relationships characteristic of sporophytic SI (SSI). Early indications are that the number of S alleles in populations is low because only four different S alleles were identified in a sample of four plants from two distinct populations; one S allele, S1, a pollen/stigma recessive allele, was present in all four plants. Forced inbreeding, using salt-treatment to overcome SI, was shown to generate ‘pseudo-self-compatible’ individuals with weakened SI and a loss/reduction in stigmatic S-specific discrimination. Relatively high frequencies of unpredictable compatible crossing ‘anomalies’ suggest that a ‘gametophytic element’ may influence the outcome of crosses in certain genetic backgrounds so as to increase levels of compatibility when S alleles are shared. Together, these findings indicate a genetic ‘flexibility’ in the SSI system of S. squalidus that could be crucial to its success as a colonizer.