[Prime-Editing Methods and pegRNA Design Programs].

It has been 10 years since CRISPR/Cas technology was applied to edit the genomes of various organisms. Its ability to produce a double-strand break in a DNA region specified by the researcher started a revolution in bioengineering. Later, the Base Editing (BE) method was developed. BE is performed via the formation of single-strand breaks by the mutant form of Cas nuclease (nickase), fused with deaminases and other enzymes. It can be used to promote A ↔ G and C ↔ T transitions, and a C → G transversion. Just over 3 years ago, a new Prime Editing (PE) variant of CRISPR/Cas was invented. Unlike BE, in PE the nickase is fused with reverse transcriptase, capable of building a new DNA chain using the pegRNA template. The pegRNA consists of an elongated guide RNA with an extra sequence at the 3'-end. Prime editing makes it possible to insert the desired mutations into this extra sequence and to carry out any substitutions and indels of bases without the use of special donor DNA. To date, a number of PE variants have been proposed; they are briefly considered in this review with an emphasis on prime editing of plant genomes. Some attention is also paid to pegRNA design programs, as well as evaluation of the efficiency of the editing. Such a variety of PE techniques is due to the opportunities of high-precision introduction of desired changes with a rather low frequency of off-target mutations in the genomes of various organisms. The relatively low efficiency of prime editing inspires researchers to offer new approaches. There is hope that further development of the technology will improve PE enough to take its rightful place among the genome targeting methods that are suitable for any organisms, and will have a positive impact on the agricultural sector, industrial biotechnologies, and medicine.

[Design of Guide RNA for CRISPR/Cas Plant Genome Editing].

CRISPR/Cas technology of genome editing is a powerful tool for making targeted changes in the DNA of various organisms, including plants. The choice of the precise nucleotide sequence (protospacer) in the gene to be edited is important in the design of guide RNA, which can be carried out by specialized software. We review and compare all the known on-line and off-line resources for guide RNA design, with special attention paid to tools capable of searching for off-target edits sites in plant genomes. The use of Cas12a may be preferable to Cas9. Techniques allowing C→T and G→A base editing without DNA cleavage are discussed along with the basic requirements for the design of effective and highly specific guide RNAs. Ways for improving guide RNA design software are presented. We also discuss the lesser risks of off-target editing in plant genomes as opposed to animal genomes. Examples of edited plant genomes including those that do not lead to the creation of transgenic plants are reviewed.

[Inheritance of reversions to male fertility in male-sterile sorghum hybrids with 9E cytoplasm male sterility induced by environmental conditions].

Heritable phenotypic alterations occurring during plant ontogenesis under the influence of environmental factors are among the most intriguing genetic phenomena. It was found that male-sterile sorghum hybrids in the 9E cytoplasm from the F1 and F2 generations, which were obtained by crossing CMS lines with different fertile lines grown in field conditions, were transferred to greenhouse produce fertile tillers. Lines created by the self-pollination of revertant tillers exhibit complete male fertility upon cultivation under various environments (in the field, Tdry plot,(y) Tirrigated plot(y)). In a number of test-crosses of revertants to CMS lines in the 9E cytoplasm, restoration of male fertility in F1 hybrids was found, indicating that revertants possess functional fertility-restoring genes. A high positive correlation was found between the fertility level of the test-cross hybrids and the hydrothermal coefficient (the ratio of the sum of precipitation to the sum of temperatures) during the booting stage and pollen maturation (r = 0.75...0.91; P<0.01), suggesting that a high level of plant water availability is needed for the expression of fertility-restoring genes of revertants. These data show that the fertility-restoring genes for the 9E cytoplasm are dominant in conditions of high water availability and recessive in drought conditions; reversions to male fertility are due to up-regulation of fertility-restoring genes by a high level of water availability. Comparative MSAP-analysis of DNA of male-sterile and male-fertile test-cross hybrids using HpaII/MspI restrictases and primers to polygalacturonase gene ADPG2, which is required for cell separation during reproductive development, and gene MYB46, the transcription factor regulating secondary wall biosynthesis, revealed differences in the number and the length of amplified fragments. Changes in the methylation of these genes in conditions of drought stress are apparently the reason for male sterility of sorghum hybrids in the 9E cytoplasm. These data demonstrate that methylation of nuclear genes in sterility-inducing cytoplasm may be one of mechanisms causing the CMS phenomenon.

[Genetic variation in a sorghum line with multiple genetic instability induced with ethidium bromide in an in vitro culture].

Ethidium bromide treatment (15 mg/l, 26 degrees C, 18 h) of a sorghum Zheltozernoe 10 callus culture yielded line Zh10-brl displaying multiple genetic instability. The line was characterized by a broad variety of mutations, which were identified in consecutive generations obtained from one initial regenerant via self-pollination. The mutation caused male sterility (male sterility, generation R1), a low plant height (dwarfness, R2), a reduced awn length (awnless, R3), yellow leaves in seedlings (xantha, R6), leaf variegation (leaf variegation, R6), leaf bleaching (virescence, R6), etc. In some cases, segregation in families suggested a monogenic recessive inheritance for the induced mutations. Male sterility was due to a range of defects that affected microsporogenesis and microgametogenesis and were probably caused by mutations of several genes. Leaf variegation was due to the appearance of green areas in originally albino seedling leaves; the reversion occurred only in somatic tissues without affecting male and female gametes. In male-sterile and variegated mutants, sequence-specific amplified polymorphism (SSAP) analysis with primers to the Isaak transposon revealed new DNA fragments, which were absent from the original line. The results supported the hypothesis that the mutations isolated in line Zh10-brl result from transposon mobilization induced by ethidium bromide and/or in vitro culture conditions.

[Mobile genetic elements in plant sex evolution].

The most significant theories of the appearance and maintenance of sex are presented. However, in the overwhelming majority of existing theories, the problem of sex, which is the central problem of evolutionary biology, is considered primarily through the prism of reproductive features of living organisms, whereas the issue of molecular driving forces of sexual reproduction id restricted to the possible role of mobile genetic elements (MGEs) in the appearance of sexual reproduction. The structural and functional significance of MGEs in the genomic organization of plants is illustrated. It is shown that MGEs could act as important molecular drivers of sex evolution in plants. The involvement of MGEs in the formation of sex chromosomes and possible participation in seeds-without-sex reproduction (apomixis) is discussed. Thus, the hypothesis on the active MGE participation in sex evolution is in good agreement with the modern views on pathways and directions of sex evolution in plants.

[Effect of ubiquinone 50 and viral infection on phytohemagglutinin activity in development of induced resistance in tobacco plants].

The effects of ubiquinone 50 (synthetic coenzyme Q10) and viral infection on phytohemagglutinin activity were studied in the tobacco (Nicotiana tabacum L.) cultivar Samsun NN, which carries the necrosis-formation locus and exhibits hypersensitivity to tobacco mosaic virus (TMV) and ubiquinone (Q10). The treatment with Q10 and inoculation with VTM were accompanied by an increase in phytohemagglutinin activity, indicating that lectins may be involved in the development of defense responses in plants.

[Hormonal status of tobacco variety Samsun NN exposed to synthetic coenzyme Q10 (ubiquinone 50) and TMV infection].

Hormonal system status has been analyzed in leaf disks of hypersensitive tobacco Nicotiana tabacum L. variety Samsun NN during the development of resistance to tobacco mosaic virus (TMV) induced by synthetic coenzyme Q10 (ubiquinone 50). The absolute and relative content of abscisic acid (ABA), indoleacetic acid (IAA), and cytokinins (CKs) was determined after the exposure of leaves to Q10 solution and the subsequent TMV infection. In plants not treated with Q10, CK content increased about 2.5 times 1 day after TMV infection, while a significant increase in the ABA level and a decrease in the IAA level were observed only after 2 days. In the dynamics, Q10 treatment had a protective antiviral effect, significantly decreased the ABA level, and increased the IAA level in sensitized plants compared to nonsensitized ones.

[RAPD-PCR analysis of the variability of spring common wheat cultivar genomes and their androclinal double haploid form]. / RAPD-PTsR-analiz izmenchivosti genoma sortov iarovoi iarovoi miagkoi pshenitsy i ikh androklinnykh digaploidnykh form.

Based on RAPD-PCR analysis with 15 primers including those homologous to particular loci, the level of genetic polymorphism in the collection of spring common wheat genotypes and their androclinal double haploid (ADH) lines was determined. Intraspecific polymorphism of the original wheat forms was 20%. By the absence of polymorphism in the molecular patterns of ADH lines and original forms, it was shown that, under the conditions used, no genetic changes in the genomic DNA of spring common wheat in anther culture occurred. Thus, the technology of direct in vitro androgenesis does not lead to genome rearrangements and may be used for rapid production of pure lines of such a complex allopolyploid as spring common wheat.