Michael D. Bennett

Polyploidy in angiosperms

Polyploidy has played a major role in higher plant evolution. Most flowering plants are polyploid, and many are distinct in combining the diploid nuclear genomes from two or more different ancestral species or genera (allopolyploids). Recently, molecular techniques have offered powerful new tools for studying the origin and evolution of polyploids. Genomic in situ hybridization allows unequivocal identification of allopolyploids and visualization of their ancestral genomes. Studies of restriction fragment length polymorphism have shown that maize — hitherto generally viewed as a diploid — is really a tetraploid, and that multiple origins are common, if not the rule, for polyploid plant species. It appears that after polyploid formation, considerable and sometimes very rapid changes in genome structure and gene expression have occurred.

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.

Nuclear DNA content in the genera Zea and Sorghum . Intergeneric, interspecific and intraspecific variation

Microdensitometry measurements showed that 4C DNA content varied significantly both within the genus Zea as a whole and within maize (Zea mays ssp. mays) itself. The DNA contents of diploid teosintes from Mexico and northern Guatemala (Zea mays ssp. mexicana, Zea mays ssp. parviglumis and Zea diploperennis) were within the range recorded for maize (9·84 to 13·49 pg), but the DNA content of a diploid teosinte from southern Guatemala (Zea luxurians) was about 50 per cent higher (18·29 to 18·47 pg). The DNA content of maize was three to four times greater than that of diploid Sorghum bicolor (3·12 to 3·47 pg). In contrast to the situation in maize no significant differences in DNA content were found between accessions of diploid Sorghum bicolor.

Evolution of genome size in the angiosperms.

Genome size varies extensively across the flowering plants, which has stimulated speculation regarding the ancestral genome size of these plants and trends in genome evolution. We investigated the evolution of C-values across the angiosperms using a molecular phylogenetic framework and C-values not previously available for crucial basal angiosperms, including Amborella, Illiciaceae, and Austrobaileya. Reconstructions of genome size across the angiosperms and extant gymnosperms indicate that the ancestral genome size for angiosperms is very small (1C ≤ 1.4 pg), in agreement with an earlier analysis of Leitch et al. (1998). Furthermore, a very small genome size (1C ≤ 1.4 pg) is ancestral not only for the angiosperms in general, but also for most major clades of flowering plants, including the monocots and the eudicots. The ancestral genome of core eudicots may also have been very small given that very low 1C-values appear to be ancestral for major clades of core eudicots, such as Caryophyllales, Saxifragales, and asterids. Very large genomes occur in clades that occupy derived positions within the monocots and Santalales.

Discrimination between closely related Triticeae species using genomic DNA as a probe

SummaryLabelled total genomic DNA was used as a probe in combination with blocking DNA to discriminate between taxonomically closely related species in the genera Hordeum and Secale. Discrimination was possible both by Southern hybridization to size-fractionated restriction enzyme digests of genomic DNA and by in situ hybridization to chromosome preparations. To distinguish between two species (e.g. H. vulgare and H. bulbosum), genomic DNA from one species was used as the labelled probe, while unlabelled DNA from the other species was applied at a much higher concentration as a block. The blocking DNA presumably hybridized to sequences in common between the block and the labelled probe, and between the block and DNA sequences on the membrane or chromosomes in situ. If so, mainly species-specific sequences would remain as sites for probe hybridization. These species-specific sequences are dispersed and represent a substantial proportion of the genome (unlike many cloned, species-specific sequences). Consequently, rapid nonradioactive methods detected probe hybridization sites satisfactorily. The method was able to confirm the parentage of hybrid plants. It has potentially wide application in plant breeding for the detection of alien DNA transfer, and it can be easily adapted to many species.

The use of dna sequencing (ITS and trnL-F), AFLP, and fluorescent in situ hybridization to study allopolyploid Miscanthus (Poaceae).

Two clones of Miscanthus, grown under the names M. ×giganteus and M. sacchariflorus, have been used in biomass trials in Europe, but neither the identity of these clones nor their origin has been established. DNA sequencing, amplified fragment length polymorphism (AFLP), and chromosome studies confirm that M. ×giganteus is an allotriploid (2n = 3x = 57) combining genomes from M. sinensis (2n = 2x = 38) and M. sacchariflorus (2n = 38 or 76). Two alleles of the internal transcribed spacer of 18S-25S nuclear ribosomal DNA (ITS) were discovered in polymerase chain reaction products of M. ×giganteus. Cloning of these revealed that one matched M. sinensis and the other M. sacchariflorus. Plastid trnL intron and trnL-F spacer sequences showed that the maternal lineage of M. ×giganteus was M. sacchariflorus. Fluorescent in situ hybridization, FISH, was used to investigate genome organization in Miscanthus but was unable to differentiate between the different parental genomes present in M. ×giganteus, indicating that two parental genomes are still extremely similar at the repetitive DNA level. This study is an example in which rDNA sequences and AFLP fingerprints permit identification of the parental genomes in a hybrid, but FISH methods, at the repetitive DNA level (including genomic in situ hybridization, GISH), were unable to do so because their sequences remain too similar.

Genome Size Evolution in Plants

Publisher Summary This chapter provides an overview of the current state of knowledge concerning genome size evolution in plants. It includes a review of available data, the patterns of variation both within and among species, and higher taxa. Every cellular organism possesses a genome and genome size evolution is not limited to any one taxon, but rather is of universal biological interest. Along with animals, plants are the best studied group with regard to variation in DNA content, and have played a critical role since the earliest days of genome size study. The field of genome size research in plants may be considered to have reached “the end of the beginning.” Although the Plant DNA C-values Database includes representatives from each of the major land plant groups, the percent coverage at the species level remains very poor (generally

Use of the polymerase chain reaction to detect spacer size heterogeneity in plant 5S-rRNA gene clusters and to locate such clusters in wheat (Triticum aestivum L.)

SummaryWe have used the polymerase chain reaction to analyse variation in the size of individual 5S-ribosomal gene spacer sequences. This reaction can be used to demonstrate inter- and intraspecific variation in spacer size, and combined with DNA sequencing it may thus be a valuable taxonomic tool. Two sets of nested polymerase chain reaction primers were designed to amplify the nontranscribed spacer DNA between repeated 5S-rRNA genes. These “universal” primers were used to generate fragments from the genomic DNA from several unrelated monocotyledonous plants. Ribosomal RNA spacer sequences generated in these experiments could also be used to locate 5S-rRNA gene clusters on specific chromosomes in hexaploid wheat (Triticum aestivum). Three distinct spacer sizes were observed after amplification. These were assigned locations on chromosomes by analysing amplification products of genomic DNA from nullisomic/tetrasomic and ditelosomic wheat stocks. “Large” 508-bp 5S repeats are located on the short arm of chromosome 5B and “short” 416-bp and 425-bp repeat unit variants are located on the short arms of chromosomes 1B and 1D, respectively. No other loci were detected. The spacer fragments were cloned, sequenced, and shown to be homologous to wheat 5S-rRNA spacers previously identified. Spacers of uniform size but with some sequence heterogeneity were shown to be located at each locus.

Ribosomal DNA evolution and phylogeny in Aloe (Asphodelaceae)

All Aloe taxa (∼400 species) share a conserved bimodal karyotype with a basic genome of four large and three small submetacentric/acrocentric chromosomes. We investigated the physical organization of 18S-5.8S-26S and 5S ribosomal DNA (rDNA) using fluorescent in situ hybridization (FISH) to 13 Aloe species. The organization was compared with a phylogenetic tree of 28 species (including the 13 used for FISH) constructed by sequence analysis of the internal transcribed spacer (ITS) of 18S-5.8S-26S rDNA. The phylogeny showed little divergence within Aloe, although distinct, well-supported clades were found. FISH analysis of 5S rDNA distribution showed a similar interstitial location on a large chromosome in all species examined. In contrast, the distribution of 18S-5.8S-26S rDNA was variable, with differences in number, location, and size of loci found between species. Nevertheless, within well-supported clades, all species had the same organizational patterns. Thus, despite the striking stability of karyotype structure and location of 5S rDNA, the distribution of 18S-5.8S-26S rDNA is not so constrained and has clearly changed during Aloe speciation.

Genomic in situ hybridization reveals the allopolyploid nature of Milium montianum (Gramineae)

Molecular techniques that “paint” chromosomes offer exciting new opportunities for testing genome relationships.Milium montianum (2n=22) is a grass whose distinctive bimodal karyotype comprises 8 large (L-) and 14 smaller (S-) chromosomes. The proposal thatM. montianum is an allotetraploid, with diploidMilium vernale (2n=8) as the L-chromosome genome donor, has been impossible to confirm by classical means. To test this hypothesis, biotinylated total genomic DNA of diploidM. vernale (2n=8) was hybridized in situ to root tip chromosomes ofM. montianum. TheM. vernale probe hybridized preferentially to all L-chromosomes, but not to the S-chromosomes. These results (i) confirm the allopolyploid nature ofM. montianum, (ii) strongly support the theory that the L-chromosomes ofM. montianum were donated byM. vernale, or a closely related genotype and (iii) show that subsequently the L-chromosomes have largely retained their genomic integrity in the new allopolyploid backgroud. Clearly, genomic in situ hybridization (GISH) is a potentially powerful tool for studying genome evolution and biosystematics. It will often be useful for investigating the origins of wild and cultivated polyploid plant species, especially where conventional methods have failed, for studying introgression, and for understanding the mechanism(s) of origin of bimodal karyotypes.