Genome Variability in Artificial Allopolyploid Hybrids of Avena sativa L. and Avena macrostachya Balansa ex Coss. et Durieu Based on Marker Sequences of Satellite DNA and the ITS1-5.8S rDNA Region.

Artificial hybrids between cultivated Avena species and wild Avena macrostachya that possess genes for resistance to biotic and abiotic stresses can be important for oat breeding. For the first time, a comprehensive study of genomes of artificial fertile hybrids Avena sativa × Avena macrostachya and their parental species was carried out based on the chromosome FISH mapping of satellite DNA sequences (satDNAs) and also analysis of intragenomic polymorphism in the 18S-ITS1-5.8S rDNA region, using NGS data. Chromosome distribution patterns of marker satDNAs allowed us to identify all chromosomes in the studied karyotypes, determine their subgenomic affiliation, and detect several chromosome rearrangements. Based on the obtained cytogenomic data, we revealed differences between two A. macrostachya subgenomes and demonstrated that only one of them was inherited in the studied octoploid hybrids. Ribotype analyses showed that the second major ribotype of A. macrostachya was species-specific and was not represented in rDNA pools of the octoploids, which could be related to the allopolyploid origin of this species. Our results indicate that the use of marker satDNAs in cytogenomic studies can provide important data on genomic relationships within Avena allopolyploid species and hybrids, and also expand the potential for interspecific crosses for breeding.

Hybridization in the Subtribe Alopecurinae Dumort. (Poaceae) According to Molecular Phylogenetic Analysis: Different Ploidy Level Tells Different Origin of the Groups.

We performed next-generation sequencing of the 18S rDNA-ITS1-5.8S rDNA region along with traditional Sanger sequencing of rbcL, matK, ndhF, and ITS1-5.8S rDNA-ITS2 to clarify the hybridization pattern in the subtribe Alopecurinae and in the genus Alopecurus in particular. Our data support the hybrid origin of Alopecurus × brachystylus from hybridization between A. geniculatus (sect. Alopecurium) and A. pratensis (sect. Alopecurus). Moreover, in the rDNA of hybrid A. × brachystylus, only A. aequalis-like ribotypes from tetraploid A. geniculatus participated. Surprisingly, we found the traces of introgression of A. arundinaceus-like ribotypes not only in hybrid A. × marssonii (A. geniculatus × A. arundinaceus) but in A. aequalis s. str. as well. A high-polyploid group from the section Alopecurus, A. aggr. alpinus has undoubted hybrid origin: e. g., A. brachystachyus has rDNA from the sect. Alopecurium. Alopecurus alpinus, with its allies, is clearly distinct from other members of the sect. Alopecurus (especially by maternal line) and thus we can re-establish a previous opinion about the separate group to which A. alpinus belongs. Species from the section Colobachne (presumably Alpine grasses from Ancient Mediterranean region) probably hybridized with the A. alpinus group. Even A. myosuroides (sect. Pseudophalaris) that could be referred to the separate genus has ribotypes common with the species of the section Alopecurium (A. aequalis, A. geniculatus) in one of the accessions. Additionally, we found that the possible polyphyletic origin of the genus Limnas. Limnas stelleri is very close to Alopecurus magellanicus according to NGS data, while L. malyschevii is more or less distinct from other studied species of the genus Alopecurus.

Characterization of a HIR-Fab-IDS, Novel Iduronate 2-Sulfatase Fusion Protein for the Treatment of Neuropathic Mucopolysaccharidosis Type II (Hunter Syndrome).

BACKGROUND: Mucopolysaccharidosis type II is a severe lysosomal storage disease caused by deficient activity of the enzyme iduronate-2-sulfatase. The only medicinal product approved by the US Food and Drug Administration for enzyme replacement therapy, recombinant iduronate-2-sulfatase (idursulfase, Elaprase®), is a large molecule that is not able to cross the blood-brain barrier and neutralize progressive damage of the central nervous system caused by the accumulation of glycosaminoglycans. Novel chimeric protein HIR-Fab-IDS is an anti-human insulin receptor Fab fragment fused to recombinant modified iduronate-2-sulfatase. This modification provides a highly selective interaction with the human insulin receptor, which leads to the HIR-Fab-IDS crossing the blood-brain barrier owing to internalization of the hybrid molecule by transcytosis into endothelial cells adjacent to the nervous system by the principle of a 'molecular Trojan horse'. OBJECTIVES: In this work, the physicochemical and biological characterization of a blood-brain barrier-penetrating fusion protein, HIR-Fab-IDS, is carried out. HIR-Fab-IDS consists of an anti-human insulin receptor Fab fragment fused to recombinant iduronate-2-sulfatase. METHODS: Comprehensive analytical characterization utilizing modern techniques (including surface plasmon resonance and mass spectrometry) was performed using preclinical and clinical batches of HIR-Fab-IDS. Critical quality parameters that determine the therapeutic effect of iduronate-2-sulfatase, as well as IDS enzymatic activity and in vitro cell uptake activity were evaluated in comparison with the marketed IDS product Elaprase® (IDS RP). In vivo efficiency of HIR-Fab-IDS in reversing mucopolysaccharidosis type II pathology in IDS-deficient mice was also investigated. The affinity of the chimeric molecule for the INSR was also determined by both an enzyme-linked immunosorbent assay and surface plasmon resonance. We also compared the distribution of 125I-radiolabeled HIR-Fab-IDS and IDS RP in the tissues and brain of cynomolgus monkeys after intravenous administration. RESULTS: The HIR-Fab-IDS primary structure investigation showed no significant post-translational modifications that could affect IDS activity, except for the formylglycine content, which was significantly higher for HIR-Fab-IDS compared with that for IDS RP (~ 76.5 vs ~ 67.7%). Because of this fact, the specific enzyme activity of HIR-Fab-IDS was slightly higher than that of IDS RP (~ 2.73 × 106 U/µmol vs ~ 2.16 × 106 U/µmol). However, differences were found in the glycosylation patterns of the compared IDS products, causing a minor reduced in vitro cellular uptake of HIR-Fab-IDS by mucopolysaccharidosis type II fibroblasts compared with IDS RP (half-maximal effective concentration ~ 26.0 vs ~ 23.0 nM). The efficacy of HIR-Fab-IDS in IDS-deficient mice has demonstrated a statistically significant reduction in the level of glycosaminoglycans in the urine and tissues of the main organs to the level of healthy animals. The HIR-Fab-IDS has revealed high in vitro affinity for human and monkey insulin receptors, and the radioactively labeled product has been shown to penetrate to all parts of the brain and peripheral tissues after intravenous administration to cynomolgus monkeys. CONCLUSIONS: These findings indicate that HIR-Fab-IDS, a novel iduronate-2-sulfatase fusion protein, is a promising candidate for the treatment of central nervous system manifestations in neurological mucopolysaccharidosis type II.

Diversity of Arbuscular Mycorrhizal Fungi in Distinct Ecosystems of the North Caucasus, a Temperate Biodiversity Hotspot.

BACKGROUND: Investigations that are focused on arbuscular mycorrhizal fungus (AMF) biodiversity is still limited. The analysis of the AMF taxa in the North Caucasus, a temperate biodiversity hotspot, used to be limited to the genus level. This study aimed to define the AMF biodiversity at the species level in the North Caucasus biotopes. METHODS: The molecular genetic identification of fungi was carried out with ITS1 and ITS2 regions as barcodes via sequencing using Illumina MiSeq, the analysis of phylogenetic trees for individual genera, and searches for operational taxonomic units (OTUs) with identification at the species level. Sequences from MaarjAM and NCBI GenBank were used as references. RESULTS: We analyzed >10 million reads in soil samples for three biotopes to estimate fungal biodiversity. Briefly, 50 AMF species belonging to 20 genera were registered. The total number of the AM fungus OTUs for the "Subalpine Meadow" biotope was 171/131, that for "Forest" was 117/60, and that for "River Valley" was 296/221 based on ITS1/ITS2 data. The total number of the AM fungus species (except for virtual taxa) for the "Subalpine Meadow" biotope was 24/19, that for "Forest" was 22/13, and that for "River Valley" was 28/24 based on ITS1/ITS2 data. Greater AMF diversity, as well as number of OTUs and species, in comparison with that of forest biotopes, characterized valley biotopes (disturbed ecosystems; grasslands). The correlation coefficient between "Percentage of annual plants" and "Glomeromycota total reads" r = 0.76 and 0.81 for ITS1 and ITS2, respectively, and the correlation coefficient between "Percentage of annual plants" and "OTUs number (for total species)" was r = 0.67 and 0.77 for ITS1 and ITS2, respectively. CONCLUSION: High AMF biodiversity for the river valley can be associated with a higher percentage of annual plants in these biotopes and the active development of restorative successional processes.

Origin of the Rare Hybrid Genus ×Trisetokoeleria Tzvelev (Poaceae) According to Molecular Phylogenetic Data.

In our article, we analyzed new data on the origin of the hybrid genus ×Trisetokoeleria. According to the morphological criteria ×T. jurtzevii is a hybrid between Koeleria asiatica s. l. and Trisetum spicatum, ×T. taimyrica, and originated from Koeleria asiatica s. l. and Trisetum subalpestre, ×T. gorodkowii, a hybrid between Koeleria asiatica and Trisetum ruprechtianum. Later ×T. taimyrica was transferred to Koeleria. Parental taxa are prone to active hybridization themselves, thus, new methods of next-generation sequencing (NGS) were needed to clarify the relationships of these genera. For NGS we used the fragment 18S rDNA (part)-ITS1-5.8S rDNA (totally 441 accessions). We analyzed ITS1-5.8S rDNA-ITS2 region, trnL-trnF and trnK-rps16 from eight samples of the five species, using the Sanger method: ×Trisetokoeleria jurtzevii, ×T. taimyrica, Koeleria asiatica, Sibirotrisetum sibiricum (=Trisetum sibiricum), and Trisetum spicatum. We also studied the pollen fertility of ×Trisetokoeleria and its possible progenitors. Our data partly contradicted previous assumptions, based on morphological grounds, and showed us a picture of developed introgression within and between Koeleria and Trisetum. ×T. jurtzevii, a totally sterile hybrid formed rather recently. We can suppose that ×T. jurtzevii is a hybrid between K. asiatica and some Trisetum s. str. Species, but not T. spicatum. ×T. gorodkowii, a hybrid in the stage of primary stabilization; it has one unique ribotype related to T. spicatum s. l. The second parental species is unrelated to Trisetum ruprechtianum. ×T. taimyrica and is a stabilized hybrid species; it shares major ribotypes with the T. spicatum/T. wrangelense group and has a minor fraction of rDNA related to genus Deyeuxia s. l.

New Insights into the Genomic Structure of Avena L.: Comparison of the Divergence of A-Genome and One C-Genome Oat Species.

We used next-generation sequencing analysis of the 3'-part of 18S rDNA, ITS1, and a 5'-part of the 5.8S rDNA region to understand genetic variation among seven diploid A-genome Avena species. We used 4−49 accessions per species that represented the As genome (A. atlantica, A. hirtula, and wiestii), Ac genome (A. canariensis), Ad genome (A. damascena), Al genome (A. longiglumis), and Ap genome (A. prostrata). We also took into our analysis one C-genome species, A. clauda, which previously was found to be related to A-genome species. The sequences of 169 accessions revealed 156 haplotypes of which seven haplotypes were shared by two to five species. We found 16 ribotypes that consisted of a unique sequence with a characteristic pattern of single nucleotide polymorphisms and deletions. The number of ribotypes per species varied from one in A. longiglumis to four in A. wiestii. Although most ribotypes were species-specific, we found two ribotypes shared by three species (one for A. damascena, A. hirtula, and A. wiestii, and the second for A. longiglumis, A. atlantica, and A. wiestii), and a third ribotype shared between A. atlantica and A. wiestii. A characteristic feature of the A. clauda ribotype, a diploid C-genome species, is that two different families of ribotypes have been found in this species. Some of these ribotypes are characteristic of Cc-genome species, whereas others are closely related to As-genome ribotypes. This means that A. clauda can be a hybrid between As- and C-genome oats.

Correction: Rodionov, A.V., et al. Intragenomic Polymorphism of the ITS 1 Region of 35S rRNA Gene in the Group of Grasses with Two-Chromosome Species: Different Genome Composition in Closely Related Zingeria Species. Plants 2020, 9, 1647.

The authors wish to make the following corrections to their paper [...].

Intragenomic Polymorphism of the ITS 1 Region of 35S rRNA Gene in the Group of Grasses with Two-Chromosome Species: Different Genome Composition in Closely Related Zingeria Species.

Zingeria (Poaceae) is a small genus that includes Z. biebersteiniana, a diploid species with the lowest chromosome number known in plants (2n = 4) as well as hexaploid Z. kochii and tetraploid Z. pisidica, and/or Z. trichopoda species. The relationship between these species and the other low-chromosomes species Colpodium versicolor are unclear. To explore the intragenomic polymorphism and genome composition of these species we examined the sequences of the internal transcribed spacer 1 of the 35S rRNA gene via NGS approach. Our study revealed six groups of ribotypes in Zingeria species. Their distribution confirmed the allopolyploid nature of Z. kochii, whose probable ancestors were Colpodium versicolor and Z. pisidica. Z. pisidica has 98% of rDNA characteristic only for this species, and about 0.3% of rDNA related to that of Z. biebersteiniana. We assume that hexaploid Z. kochii is either an old allopolyploid or a homodiploid that has lost most of the rRNA genes obtained from Z. biebersteiniana. In Z. trichopoda about 81% of rDNA is related to rDNA of Z. biebersteiniana and 19% of rDNA is derived from Poa diaphora sensu lato. The composition of the ribotypes of the two plants determined by a taxonomy specialist as Z. pisidica and Z. trichopoda is very different. Two singleton species are proposed on this base with ribotypes as discriminative characters. So, in all four studied Zingeria species, even if the morphological difference among the studied species was modest, the genomic constitution was significantly different, which suggests that these are allopolyploids that obtained genomes from different ancestors.

Molecular cytogenetics of valuable Arctic and sub-Arctic pasture grass species from the Aveneae/Poeae tribe complex (Poaceae).

BACKGROUND: Grasslands in the Arctic tundra undergo irreversible degradation due to climatic changes and also over-exploitation and depletion of scarce resources. Comprehensive investigations of cytogenomic structures of valuable Arctic and sub-Arctic grassland species is essential for clarifying their genetic peculiarities and phylogenetic relationships, and also successful developing new forage grass cultivars with high levels of adaptation, stable productivity and longevity. We performed molecular cytogenetic characterization of insufficiently studied pasture grass species (Poaceae) from related genera representing two neighboring clades: 1) Deschampsia and Holcus; 2) Alopecurus, Arctagrostis and Beckmannia, which are the primary fodder resources in the Arctic tundra. RESULTS: We constructed the integrated schematic maps of distribution of these species in the northern, central and eastern parts of Eurasia based on the currently available data as only scattered data on their occurrence is currently available. The species karyotypes were examined with the use of DAPI-banding, multicolour FISH with 35S rDNA, 5S rDNA and the (GTT)9 microsatellite motif and also sequential rapid multocolour GISH with genomic DNAs of Deschampsia sukatschewii, Deschampsia flexuosa and Holcus lanatus belonging to one of the studied clades. Cytogenomic structures of the species were specified; peculiarities and common features of their genomes were revealed. Different chromosomal rearrangements were detected in Beckmannia syzigachne, Deschampsia cespitosa and D. flexuosa; B chromosomes with distinct DAPI-bands were observed in karyotypes of D. cespitosa and H. lanatus. CONCLUSIONS: The peculiarities of distribution patterns of the examined chromosomal markers and also presence of common homologous DNA repeats in karyotypes of the studies species allowed us to verify their relationships. The obtained unique data on distribution areas and cytogenomic structures of the valuable Arctic and sub-Arctic pasture species are important for further genetic and biotechnological studies and also plant breeding progress.

Interstitial (TTAGGG)(n) sequences are not hot spots of recombination in the chicken lampbrush macrochromosomes 1-3.

To study the role of telomere (TTAGGG)(n) sequences in promoting of crossing over in chicken female meiosis, we have localized telomere repeats by FISH and studied the distribution of chiasmata in the giant diplotene bivalents, the chicken lampbrush macrochromosomes 1--3. We show that all interstitial clusters of the (TTAGGG)(n) repeat in these chromosomes do not coincide with hot spots of genetic recombination (crossing over) in the chicken female. Moreover, terminal TTAGGG-positive chromomeres also are not chiasma hot spots. We conclude that, at least in chicken macrochromosomes in female meiosis, a role for canonical telomere sequences in promoting of crossing over is not confirmed.