Extreme haustorial parasites have long captured the interest of naturalists and scientists with their greatly reduced and highly specialized morphology. Along with the reduction or loss of photosynthesis, the plastid genome often decays as photosynthetic genes are released from selective constraint. This makes it challenging to use traditional plastid genes for parasitic plant phylogenetics, and has driven the search for alternative phylogenetic and molecular evolutionary markers. Thus, evolutionary studies, such as molecular clock-based age estimates, are not yet available for all parasitic lineages. In the present study, we extracted 14 nuclear single copy genes (nSCG) from Illumina transcriptome data from one of the “strangest plants in the world”, Hydnora visseri (Hydnoraceae). A ~15,000 character molecular dataset, based on all three genomic compartments, shows the utility of nSCG for reconstructing phylogenetic relationships in parasitic lineages. A relaxed molecular clock approach with the same multi-locus dataset, revealed an ancient age of ~91 MYA for Hydnoraceae. We then estimated the stem ages of all independently originated parasitic angiosperm lineages using a published dataset, which also revealed a Cretaceous origin for Balanophoraceae, Cynomoriaceae and Apodanthaceae. With the exception of Santalales, older parasite lineages tend to be more specialized with respect to trophic level and have lower species diversity. We thus propose the “temporal specialization hypothesis” (TSH) implementing multiple independent specialization processes over time during parasitic angiosperm evolution.

Extreme haustorial parasites have long captured the interest of naturalists and scientists with their greatly reduced and highly specialized morphology. Along with the reduction or loss of photosynthesis, the plastid genome often decays as photosynthetic genes are released from selective constraint. This makes it challenging to use traditional plastid genes for parasitic plant phylogenetics, and has driven the search for alternative phylogenetic and molecular evolutionary markers. Thus, evolutionary studies, such as molecular clock-based age estimates, are not yet available for all parasitic lineages. In the present study, we extracted 14 nuclear single copy genes (nSCG) from Illumina transcriptome data from one of the “strangest plants in the world”, Hydnora visseri (Hydnoraceae). A ~15,000 character molecular dataset, based on all three genomic compartments, shows the utility of nSCG for reconstructing phylogenetic relationships in parasitic lineages. A relaxed molecular clock approach with the same multi-locus dataset, revealed an ancient age of ~91 MYA for Hydnoraceae. We then estimated the stem ages of all independently originated parasitic angiosperm lineages using a published dataset, which also revealed a Cretaceous origin for Balanophoraceae, Cynomoriaceae and Apodanthaceae. With the exception of Santalales, older parasite lineages tend to be more specialized with respect to trophic level and have lower species diversity. We thus propose the “temporal specialization hypothesis” (TSH) implementing multiple independent specialization processes over time during parasitic angiosperm evolution.

The elucidation of the composition and organization of genomes of higher plants is a fundamental problem of modern molecular biology. The genus Beta containing 14 species assigned to the sections Beta, Corollinae, Nanae and Procumbentes provides a suitable system for the comparative study of the nuclear genomes. Sugar beet Beta vulgaris has a genome size of 758 Mbp DNA with estimated 63 % repetitive sequences and the number of chromosomes n=9. The wild beet Beta procumbens is an important natural pool of resistance against pests and tolerance to unfavorable growth conditions. The subject of this research was the isolation and description of new repetitive DNA families from genomes of this Beta species. This work presents the molecular investigation and cytogenetic characterization by high-resolution multicolor fluorescent in situ hybridization (FISH) of the satellite and dispersed repetitive sequences in wild and cultivated beet species and in their hybrids. New repetitive sequences were isolated from the B. procumbens genome. The AluI restriction satellite repeats pAp11 are 229-246 bp long and form subfamilies. The satellite is amplified in the section Procumbentes, but also found in distantly related section Beta. Thus, pAp11 is probably an ancient component of Beta genomes. It could be the ancestor of the satellite subfamily pEV4 in B. vulgaris based on sequence analysis, Southern hybridization and comparative FISH. pAp11 was found at centromeric and a few intercalary sites in B. procumbens and formed intercalary blocks on B. vulgaris chromosomes where it co-localized with pEV4. These remarkable differences in the chromosomal position of pAp11 between Procumbentes and Beta species indicate that both satellites were likely involved in the expansion or rearrangement of the intercalary heterochromatin of B. vulgaris. Other two sequence families characterized on molecular, genomic and chromosomal levels are the non-homologous repeats pAp4 and pAp22, 1354 and 582 bp long. They have a dispersed organization in the genome and are widely scattered along B. procumbens chromosomes. pAp4 and pAp22 are specific for the section Procumbentes and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. High-resolution FISH on meiotic chromosomes showed that the both sequences mostly co-localize. The PCR analysis of their flanking regions revealed that pAp22 is a part of a Long Terminal Repeat (LTR) of an Athila-like env-class retrotransposon. This is the first indication that the retrovirus-like DNA elements exist in Beta. An ancient family of subtelomeric satellite DNA pAv34 was isolated from all four sections of the genus Beta and from spinach, a related Chenopodiaceae. Five clones were analyzed from each of the five species. The genomic organization and species distribution of the satellites were studied by sequencing and Southern hybridization. The repeating units in all families are 344-362 bp long and share 46.2-98.8 % similarity. Each monomer consists of two subunits SU1 and SU2 of 165-184 bp. The maximum likelihood and neighbor joining analyses of the 25 subtelomeric satellite monomers and their subunits indicated, that the duplication leading to the emergence of the 360 bp satellite should have occurred early in the phylogeny. The two directions of diversification are the clustering of satellites in two groups of subunits SU1 and SU2 and the arrangement of satellite repeats in section-specific groups. The comparative chromosomal localization of the telomeric repeat, pAv34 and rDNA was investigated by multicolor FISH. B. vulgaris chromosome termini showed unique physical organization of telomeric repeat and the subtelomeric satellite, as studied by high-resolution FISH on extended DNA fibers. The estimated length of the telomeric array was 0.55 - 62.65 kb, the length of pAv34 was 5.0-125.25 kb, the spacer between these sequences spanned 1.0-16.60 kb. Eight various classes of repeats were used to characterize the minichromosomes of the sugar beet fragment addition lines PRO1 and PAT2 by comparative multi-color FISH. The study allowed to propose a schematic pattern of repetitive DNA organization on the PRO1 and PAT2 minichromosomes. PRO1 has an acrocentric minichromosome, while PAT2 possesses a metacentric or submetacentric chromosome fragment. The functional integrity of the fragment addition line centromeres was confirmed by an immunostaining localization of the proteins specific to the active kinetochore. The serine 10-phosphorylated histone H3 was detected in pericentromeric regions of the PRO1 chromosomes. The microtubuli attachment sites were visualized as parts of kinetochore complexes.

No description available.

The elucidation of the composition and organization of genomes of higher plants is a fundamental problem of modern molecular biology. The genus Beta containing 14 species assigned to the sections Beta, Corollinae, Nanae and Procumbentes provides a suitable system for the comparative study of the nuclear genomes. Sugar beet Beta vulgaris has a genome size of 758 Mbp DNA with estimated 63 % repetitive sequences and the number of chromosomes n=9. The wild beet Beta procumbens is an important natural pool of resistance against pests and tolerance to unfavorable growth conditions. The subject of this research was the isolation and description of new repetitive DNA families from genomes of this Beta species. This work presents the molecular investigation and cytogenetic characterization by high-resolution multicolor fluorescent in situ hybridization (FISH) of the satellite and dispersed repetitive sequences in wild and cultivated beet species and in their hybrids. New repetitive sequences were isolated from the B. procumbens genome. The AluI restriction satellite repeats pAp11 are 229-246 bp long and form subfamilies. The satellite is amplified in the section Procumbentes, but also found in distantly related section Beta. Thus, pAp11 is probably an ancient component of Beta genomes. It could be the ancestor of the satellite subfamily pEV4 in B. vulgaris based on sequence analysis, Southern hybridization and comparative FISH. pAp11 was found at centromeric and a few intercalary sites in B. procumbens and formed intercalary blocks on B. vulgaris chromosomes where it co-localized with pEV4. These remarkable differences in the chromosomal position of pAp11 between Procumbentes and Beta species indicate that both satellites were likely involved in the expansion or rearrangement of the intercalary heterochromatin of B. vulgaris. Other two sequence families characterized on molecular, genomic and chromosomal levels are the non-homologous repeats pAp4 and pAp22, 1354 and 582 bp long. They have a dispersed organization in the genome and are widely scattered along B. procumbens chromosomes. pAp4 and pAp22 are specific for the section Procumbentes and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. High-resolution FISH on meiotic chromosomes showed that the both sequences mostly co-localize. The PCR analysis of their flanking regions revealed that pAp22 is a part of a Long Terminal Repeat (LTR) of an Athila-like env-class retrotransposon. This is the first indication that the retrovirus-like DNA elements exist in Beta. An ancient family of subtelomeric satellite DNA pAv34 was isolated from all four sections of the genus Beta and from spinach, a related Chenopodiaceae. Five clones were analyzed from each of the five species. The genomic organization and species distribution of the satellites were studied by sequencing and Southern hybridization. The repeating units in all families are 344-362 bp long and share 46.2-98.8 % similarity. Each monomer consists of two subunits SU1 and SU2 of 165-184 bp. The maximum likelihood and neighbor joining analyses of the 25 subtelomeric satellite monomers and their subunits indicated, that the duplication leading to the emergence of the 360 bp satellite should have occurred early in the phylogeny. The two directions of diversification are the clustering of satellites in two groups of subunits SU1 and SU2 and the arrangement of satellite repeats in section-specific groups. The comparative chromosomal localization of the telomeric repeat, pAv34 and rDNA was investigated by multicolor FISH. B. vulgaris chromosome termini showed unique physical organization of telomeric repeat and the subtelomeric satellite, as studied by high-resolution FISH on extended DNA fibers. The estimated length of the telomeric array was 0.55 - 62.65 kb, the length of pAv34 was 5.0-125.25 kb, the spacer between these sequences spanned 1.0-16.60 kb. Eight various classes of repeats were used to characterize the minichromosomes of the sugar beet fragment addition lines PRO1 and PAT2 by comparative multi-color FISH. The study allowed to propose a schematic pattern of repetitive DNA organization on the PRO1 and PAT2 minichromosomes. PRO1 has an acrocentric minichromosome, while PAT2 possesses a metacentric or submetacentric chromosome fragment. The functional integrity of the fragment addition line centromeres was confirmed by an immunostaining localization of the proteins specific to the active kinetochore. The serine 10-phosphorylated histone H3 was detected in pericentromeric regions of the PRO1 chromosomes. The microtubuli attachment sites were visualized as parts of kinetochore complexes.

No description available.