Contrasting patterns of evolution of 45S and 5S rDNA families uncover new aspects in the genome constitution of the agronomically important grass Thinopyrum intermedium (Triticeae).

We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

Mahelka V ，Kopecky D ，Baum BR 《-》

Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species.

Nicotiana tabacum (tobacco) is an allotetraploid derived from ancestors of the modern diploids, N. sylvestris and N. tomentosiformis. We identified and characterized two distinct families of 5S ribosomal DNA (rDNA) in N. tabacum; one family had an average 431 bp unit length and the other a 646 bp unit length. In the diploid species, N. sylvestris and N. tomentosiformis, the 5S rDNA unit lengths are 431 bp and 644 bp respectively. The non-coding spacer sequence of the short unit in tobacco had high sequence homology to the spacer of N. sylvestris5S rDNA, while the longer spacer of tobacco had high homology with the 5S spacer of N. tomentosiformis. This suggests that the two 5S families in tobacco have their origin in the diploid ancestors. The longer spacer sequence had a GC rich sub-region (called the T-genome sub-region) that was absent in the short spacer. Pulsed field gel analysis and fluorescent in situ hybridization to tobacco metaphase chromosomes showed that the two families of 5S rDNA units are spatially separate at two chromosomal loci, on chromosomes S8 (short family) and T8 (long family). The repeat copy number at each chromosomal locus showed heterogeneity between different tobacco cultivars, with a tendency for a decrease in the copy number of one family to be compensated by an increase in the copy number of the second family. Sequence analysis reveals there is as much diversity in 5S family units within the diploid species as there is within the T and S-genome 5S family units respectively, suggesting 5S diversification within each family had occurred before tobacco speciation. There is no evidence of interlocus homogenization of the two 5S families in tobacco. This is therefore substantially different to 18-26S rDNA where interlocus gene conversion has substantially influenced most sequences of S and T genome origin; possible reasons are discussed.

Fulnecek J ，Lim KY ，Leitch AR ，Kovarík A ，Matyásek R ... -  《HEREDITY》

Physical localization of the 18S-5.8S-26S rDNA and sequence analysis of ITS regions in Thinopyrum ponticum (Poaceae: Triticeae): implications for concerted evolution.

Fluorescence in situ hybridization was used in Thinopyrum ponticum, a decaploid species, and its related diploid species, to investigate the distribution of the 18S-5.8S-26S rDNA. The distribution of rDNA was similar in all three diploid species (Th. bessarabicum, Th. elongatum and Pseudoroegneria stipifolia). Two pairs of loci were observed in each somatic cell at metaphase and interphase. One pair was located near the terminal end and the other in the interstitial regions of the short arms of one pair of chromosomes. However, all of the major loci in Th. ponticum were located on the terminal end of the short arms of chromosomes, and one chromosome had only one major locus. The maximum number of major loci detected on metaphase spreads was 20, which was the sum of that of its progenitors. The interstitial loci that exist in the possible diploid genome donor species were probably 'lost' during the evolutionary process of the decaploid species. A number of minor loci were also detected on whole regions of two pairs of homologous chromosomes. These results suggested that the position of rDNA loci in the Triticeae might be changeable rather than fixed. Positional changes of 18S-5.8S-26S rDNA loci between Th. ponticum and its candidate genome donors indicate that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donors. The possible evolutionary significance of the distribution of the rDNA is also discussed. Internal transcribed spacer (ITS) regions of nuclear DNA in Th. ponticum were investigated by PCR amplification and sequencing. The sequence data from five positive clones selected at random, together with restriction site analysis, indicated that the ITS repeated units are nearly homogeneous in this autoallodecapolypoid species. Combined with in situ hybridization results, the data led to the conclusion that the ITS region has experienced interlocus as well as intralocus concerted evolution. Phylogenetic analyses showed that the sequences from Th. ponticum have concerted to the E genome repeat type.

Li D ，Zhang X 《-》

Polymorphic nuclear gene sequences indicate a novel genome donor in the polyploid genus Thinopyrum.

For decades, the wheatgrass genus Thinopyrum has been of interest to plant breeders as a source of genes that confer competitive traits. This genus has been a considerable challenge to plant systematists because of the impacts of polyploidization on the evolution of this group. This study was aimed to augment existing cytogenetic data with a sequence-based investigation of the genomes of these species. Sequences of the internal transcribed spacer 1 (ITS1), introns 9 through 11 of the granule-bound starch synthase (GBSSI) gene and intron III of the beta-amylase gene (Bmy1) were isolated from the genomes of polyploid Thinopyrum species by PCR, cloning and sequencing and the evolutionary distances between these species and putative diploid ancestors were estimated with Kimura's two-parameter method. Phylogenetic analysis of these sequences largely agrees with what has been established through cytogenetic means for the Th. caespitosum (Koch) Liu & Wang and Ps geniculata (Trin.) Á. Löve, and suggests a contribution of the St genome of Ps. spicata (Pursh) Á. Löve to the tetraploids Th. scirpeum (Presl) Dewey and Th. junceiforme (Á. Löve & D. Löve) Á. Löve. A unique Bmy1 allele, divergent from other Triticeae but shared between Th. caespitosum, Th. intermedium (Host) Barkworth & Dewey, Th. junceum (L.) Á. Löve and Th. ponticum Barkworth & Dewey, implies a connection between these species. Distinct oligonucleotide polymorphisms and distance calculations based on the three loci implicate Crithopsis delileana (Schult.) Roshev. and Taeniatherum caput-medusae (L.) Nevski in the evolution of the hexaploid Th. intermedium and the decaploid Th. ponticum and also suggest a potential connection of these polyploids with Elytrigia repens (L.) Desv. ex Nevski. None of these species have been previously associated with the Thinopyrum genus. Allele-specific PCR was employed to detect the putative Crithopsis allele of ITS1 in a number of accessions. Real-time PCR indicates that two of six genomes of the hexaploid Th. intermedium have the Crithopsis-type ITS1 allele and that all ITS1 loci in the decaploid Th. ponticum are of this type. These results are supportive of the hypothesis that concerted evolution has homogenized the rDNA of Th. ponticum to the allele derived from the Crithopsis or Taeniatherum ancestor. Discovery of these novel alleles, with close homology to Ta. caput-medusae, may represent a fundamental change in the view of the evolution of Th. intermedium and Th. ponticum.

Arterburn M ，Kleinhofs A ，Murray T ，Jones S ... -  《HEREDITAS》

On the genome constitution and evolution of intermediate wheatgrass (Thinopyrum intermedium: Poaceae, Triticeae).

The wheat tribe Triticeae (Poaceae) is a diverse group of grasses representing a textbook example of reticulate evolution. Apart from globally important grain crops, there are also wild grasses which are of great practical value. Allohexaploid intermediate wheatgrass, Thinopyrum intermedium (2n = 6x = 42), possesses many desirable agronomic traits that make it an invaluable source of genetic material useful in wheat improvement. Although the identification of its genomic components has been the object of considerable investigation, the complete genomic constitution and its potential variability are still being unravelled. To identify the genomic constitution of this allohexaploid, four accessions of intermediate wheatgrass from its native area were analysed by sequencing of chloroplast trnL-F and partial nuclear GBSSI, and genomic in situ hybridization. The results confirmed the allopolyploid origin of Thinopyrum intermedium and revealed new aspects in its genomic composition. Genomic heterogeneity suggests a more complex origin of the species than would be expected if it originated through allohexaploidy alone. While Pseudoroegneria is the most probable maternal parent of the accessions analysed, nuclear GBSSI sequences suggested the contribution of distinct lineages corresponding to the following present-day genera: Pseudoroegneria, Dasypyrum, Taeniatherum, Aegilops and Thinopyrum. Two subgenomes of the hexaploid have most probably been contributed by Pseudoroegneria and Dasypyrum, but the identity of the third subgenome remains unresolved satisfactorily. Possibly it is of hybridogenous origin, with contributions from Thinopyrum and Aegilops. Surprising diversity of GBSSI copies corresponding to a Dasypyrum-like progenitor indicates either multiple contributions from different sources close to Dasypyrum and maintenance of divergent copies or the presence of divergent paralogs, or a combination of both. Taeniatherum-like GBSSI copies are most probably pseudogenic, and the mode of their acquisition by Th. intermedium remains unclear. Hybridization has played a key role in the evolution of the Triticeae. Transfer of genetic material via extensive interspecific hybridization and/or introgression could have enriched the species' gene pools significantly. We have shown that the genomic heterogeneity of intermediate wheatgrass is higher than has been previously assumed, which is of particular concern to wheat breeders, who frequently use it as a source of desirable traits in wheat improvement.

Mahelka V ，Kopecký D ，Paštová L 《BMC EVOLUTIONARY BIOLOGY》