IntroductionEfficient genetic transformation is essential for the delivery of the CRISPR/Cas9 genome editing system and thus represents an important technology for breeding-oriented research in barley (Hordeum vulgare L.). However, transformation and plant regeneration from tissue culture remain challenging in non-model barley genotypes. Previous studies demonstrated that expression of a chimeric fusion between two interacting transcription factors, GROWTH-REGULATING FACTOR 4 (GRF4) and GRF-INTERACTING FACTOR 1 (GIF1), enhances regeneration capacity in wheat and other species.MethodsIn this study, we evaluated the effect of the wheat-derived GRF4-GIF1 morphogenic regulators on biolistic transformation and genome editing efficiency in three commercial barley cultivars: Tselinniy 5, Aley, and G-23035.ResultsThe JD633 construct carrying GRF4-GIF1 enabled recovery of stable transformants in all three genotypes, with efficiencies ranging from 2.5% to 5%, whereas the control construct lacking morphogenic regulators resulted in no transgenic events in any of the tested varieties. Among transformed T0 plantlets, genome editing efficiency reached 64.3%, with predominantly biallelic mutations that were stably inherited in the T1 generation. Molecular screening revealed the presence of plasmid-free edited plants in the T0 generation, likely arising from transient Cas9 expression, and provided evidence of tissue chimerism.DiscussionThese results demonstrate that the GRF-GIF system facilitates genome editing, providing a practical framework for accelerating precision breeding in barley.

The hexaploid wheat (Triticum aestivum L., genome BBAADD, 2n = 6x = 42) is one of the most important food crops. Enhancement and preservation its biodiversity is a key issue in the agrobiology of the twenty-first century. Related cultivated and wild species are the most practical source of this enhancement. The problem of genetic introgression is most successfully addressed by producing artificial amphidiploids and their subsequent use as "bridges" in breeding programs. Artificial amphidiploids are also a convenient model for solving genealogical issues of wheat. The karyotype formation and the genome stabilization of allopolyploid species are some of the most interesting points in evolutionary biology, because it is not enough to obtain between-species hybrids and to double their haploid genomes-it is required to restore their fertility. Intergeneric crosses undertaken within the tribe Triticeae Dumort led to quite a lot of fertile artificial amphidiploids possessing a wide variety of genome combinations, including the three elementary genomes of cultivated wheat. Among them are new forms (species) previously unknown in the nature. Following this study, we have added a new artificial hulled hexaploid amphidiploid, genome formula BBAASS, to the taxonomy of the genus Triticum under the name Triticum aminovii N.P. Gontsch. The karyotype of T. aminovii is provided, and its genome formula BBAASS has been validated by fluorescent in situ hybridization. The holotype of the new species is held in the I.M. Krasnoborov Herbarium (NS) of the Central Siberian Botanical Garden of the Siberian Branch of the Russian Academy of Sciences, voucher number NS0000947.

The use of the gene pool of wild relatives for expanding the genetic diversity of common wheat is an important task of breeding programs. However, the practical application of common wheat lines with alien genetic material is constrained by the lack of information on chromosomal rearrangements and the negative impact of the transferred material on agronomically important traits. This research is aimed at studying 14 introgression lines with the T2DL.2DS-2SS translocation and the 5S(5D) substitution from Aegilops speltoides obtained from crossing common wheat varieties (Aurora, Krasnodarskaya 99, Nika Kubani) with the genome-substituted form Avrodes (BBAASS). Hybrid lines with different combinations of T2DL.2DS-2SS and T1BL.1RS translocations and 5S(5D) substitution were characterized by resistance to leaf and yellow rusts, productivity components and technological qualities of grain. The assessment of the varieties’ resistance to rust diseases showed that Krasnodarskaya 99, Nika Kubani and the Aurora variety, which is a carrier of the T1BL.1RS translocation, are highly susceptible to diseases, while the presence of the T2DL.2DS-2SS translocation and the 5S(5D) substitution, both together and separately, provides resistance to fungal pathogens. The analysis of the lines using markers designed for known resistance genes of Ae. speltoides did not reveal the presence of the Lr28, Lr35 and Lr51 genes in the lines. The results suggest that the genetic material of Ae. speltoides transferred to chromosomes 2D and 5D contains new resistance genes. To determine the effect of the T2DL.2DS-2SS translocation and the 5S(5D) substitution on the productivity and technological qualities of grain, the lines were assessed by weight of 1000 grains, grain weight and number of ears per 1 m2, by protein and gluten content, gluten quality and general baking evaluation. A positive effect was determined upon the weight of 1000 grains, protein and gluten content. There were no significant differences in other characteristics. The T2DL.2DS-2SS trans-location and the 5S(5D) substitution did not have a negative effect on the productivity and technological quality of grain, and are of interest for breeding practice.

The genome-substituted synthetic form Avrodes (AABBSS) was used for transferring resistance to yellow rust (Puccinia striiformis f. sp. tritici Eriks.) from Aegilops speltoides Tausch, (2n = 14) to bread wheat. The study involved 24 introgressive lines of bread wheat obtained using the Avrodes form. Yellow rust resistant lines P07-L.02, P07-L.1, P07-L.17, P07-L.43, P07-L.19, AS12-88, AS12-06, AS12-07, AS12- 51, Asp81-21, Asp63-21, Asp053-21, Asp04-21, Asp022-19, Asp023-19 and Asp029-20 were selected and can be used as new donors of disease resistance. The use of differential chromosome staining (C-banding) and fluorescence in situ hybridization (FISH) identified the genetic material of Ae. speltoides transmitted in the form of 5S(5D) chromosome substitution and translocations of T5BS.5BL-5SL, T2DL.2DS-2SS, T5D, as well as translocation of T1BL.1RS from Secale cereale L. The work revealed that the lines with single translocations of T1BL.1RS and T5BS.5BL-5SL were susceptible to yellow rust, while the lines in which the T2DL.2DS-2SS translocation and 5S(5D) substitutions were identified, as well as the lines with translocations of T1BL.1RS, T2DL.2DS-2SS and T5D showed resistance to the disease. Presumably, the selected introgression lines, obtained by means of crosses with Avrodes, may carry new genes or loci for yellow rust resistance.

Anticipatory wheat breeding for pathogen resistance is key to preventing economically significant crop losses caused by diseases. Recently, the harmfulness of a dangerous wheat disease, stem rust, caused by Puccinia graminis f. sp. tritici, was increased in the main grain-producing regions of the Russian Federation. At the same time, importation of the Ug99 race (TTKSK) is still a possibility. In this regard, the transfer of effective resistance genes from related species to the bread wheat breeding material followed by the chromosomal localization of the introgressions and a marker analysis to identify known resistance genes is of great importance. In this work, a comprehensive analysis of ten spring bread wheat introgressive lines of the Federal Center of Agricultural Research of the South-East Region (L657, L664, L758, L935, L960, L968, L971, L995/1, L997 and L1110) was carried out. These lines were obtained with the participation of Triticum dicoccum, T. timopheevii, T. kiharae, Aegilops speltoides, Agropyron elongatum and Secale cereale. In this study, the lines were evaluated for resistance to the Ug99 race (TTKSK) in the Njoro, Kenya. Evaluation of introgression lines in the field for resistance to the Ug99 race (TTKSK) showed that four lines were immune, two were resistant, three were moderately resistant, and one had an intermediate type of response to infection. By cytogenetic analysis of these lines using fluorescent (FISH) and genomic (GISH) in situ hybridization, introgressions from Ae. speltoides (line L664), T. timopheevii (lines L758, L971, L995/1, L997 and L1110), Thinopyrum ponticum = Ag. elongatum (2n = 70) (L664, L758, L960, L971, L997 and L1110), as well as introgressions from T. dicoccum (L657 and L664), T. kiharae (L960) and S. cereale (L935 and L968) were detected. Molecular markers recommended for marker-oriented breeding were used to identify known resistance genes (Sr2, Sr25, Sr32, Sr1A.1R, Sr36, Sr38, Sr39 and Sr47). The Sr36 and Sr25 genes were observed in lines L997 and L1110, while line L664 had the Sr39+Sr47+Sr25 gene combination. In lines L935 and L968 with 3R(3D) substitution from S. cereale, gene resistance was presumably identified as SrSatu. Thus, highly resistant to both local populations of P. graminis and the Ug99 race, bread wheat lines are promising donors for the production of new varieties resistant to stem rust.

Triticum timopheevii Zhuk. attracts the attention of bread wheat breeders with its high immunity to the leaf rust pathogen. However, introgressions from this species in Triticum aestivum L. are little used in practical breeding. In the presented study, the agronomic value of T. aestivum/T. timopheevii line L624 was studied in comparison with the parent cultivars Saratovskaya 68, Dobrynya and the standard cultivar Favorit during 2017–2022. Introgressions from T. timopheevii in L624 were detected by the FISH method with probes pSc119.2, pAs1 and Spelt1, as well as micro satellite markers Xgwm312, Xgpw4480 and Xksum73. Translocations of 2AS.2AL-2A t L and on 2DL were detected as well. Line L624 is highly resistant to Puccinia triticina both under the background of natural epiphytotics and under laboratory conditions. PCR analysis with the DNA marker of the LrTt1 gene (Xgwm312) revealed that it is not identical to the Lr gene(s) in L624. According to a five-year study, the grain yield of L624 was, on average, higher than that of Favorit and Dobrynya, but lower than that of Saratovskaya 68. Line L624 had a lower weight of 1000 grains than the recipients, and was at the same level with the standard cultivar Favorit. Introgressions from T. timopheevii in L624 increased the grain protein content by comparison with Saratovskaya 68 and Favorit, but it was at the same level as in Dobrynya. As for parameters of flour and bread, L624 was not inferior to the recipient cultivars, but by volume and porosity of bread, it surpassed Saratovskaya 68. Moreover, L624 surpassed Favorit by the elasticity of the dough, the ratio of the elas ticity of the dough to the extensibility and the strength of the flour. Thus, the results obtained suggest that introgressions in chromosomes 2A and 2D in L624 do not impair baking properties.

This chapter is dedicated to using fluorescence in situ hybridization (FISH) for the genotyping of Triticeae tribe species and hybrids. The basic method of FISH on metaphase chromosomes is presented with a discussion on its modifications, and deoxyribonucleic acid (DNA) probes that can be useful for genotyping are proposed.

CRISPR/Cas technology makes it possible to induce mutations at defined positions. In breeding-oriented research, this opens up exciting opportunities for the targeted improvement of many agricultural crops. Wheat and barley are among the most important cereals in the world. However, the transformation poses a particular challenge for cereals and is strongly genotype dependent. This is because agrobacteria, which is mostly used for delivering the CRISPR/Cas system, have a limited compatibility with these non-host plants. Transformation of wheat is additionally difficult due to the large genome size and polyploidy. Besides obtaining improved genotypes, the object of the current study was to optimize the method of genomic editing based on the CRISPR/Cas system using particle bombardment for non-model varieties of barley and wheat. In barley, we targeted theNudgene that controls hulled/naked phenotype of the grain. Since the regeneration rate remains an issue for the cultivated cultivars, we used the JD633 vector that carries theGRF4-GIF1chimera to increase the efficiency of regeneration. We obtained five T0plants, carrying mutations. In wheat, targetingPpd-1genes that control photoperiod-dependent floral induction results in Cas9-induced mutations in 52 of 210 T0plants. The developed collection of wheat plants with different new alleles ofPpd-D1andPpd-B1genes is being studied for the expression under short day conditions and the effect on the vegetation period. Thus, we have obtained plants of the cultivated varieties of barley and wheat with edited agronomically important genes, using the improved protocols of biolistic transformation. This work was done within the framework of State Assignment Kurchatov Genomic Center of ICG SB RAS (No. 075-15-2019-1662).

Enrichment of grains with anthocyanins is considered a feasible approach to improving the nutritional properties of bread wheat. Here, two black-grained substitution lines with either 4B or 4D chromosomes substituted by wheatgrass (Thinopyrum ponticum) 4Th were created via marker-assisted combining the anthocyanin biosynthesis regulatory genes Pp-1, Pp3 (Purple pericarp), and Ba1 (Blue aleurone) in the genetics background of cv. Saratovskaya 29. The black grains manifested the simultaneous accumulation of anthocyanins in aleurone, and the pericarp layers resulted in the highest total anthocyanin content (TAC), which amounted to approximately the sum of TACs of the purple and blue grains. The lines with substitution 4Th(4B) had long trichomes on the leaves, whereas the lines with substitution 4Th(4D) did not differ from parental cv. Saratovskaya 29. Despite the chromosome substitution having a positive effect on the main spike length, the other yield-related traits (grain weight per main spike, the spike number per plant, and the 1000-grain weight) were decreased in the lines with chromosome substitution. The developed lines together with the parental ones constitute a valuable model for comparative studies and genetic stock for breeding commercial wheat cultivars featuring high levels of anthocyanins in grains.

Background. The basic approach to the production of new common wheat genotypes involving introgressive hybridization entails a long-term process. Doubled haploid production could accelerate it. However, this method is not widely used in breeding programs due to its main limitation: the genotype dependence. Due to genetic differences between wheat and related species, it was assumed that alien genetic materials are different in their capacity to affect androgenesis. The effect of alien translocations on androgenesis has been shown earlier. The aim of this study was to develop a set of DH wheat lines containing a wheat-alien translocation in the genome and study the effect of alien translocations on androgenesis of anther culture in such lines.Materials and methods. The plant material included: the spring wheat cultivar ‘Novosibirskaya 16’, line Velut 991 carrying wheat-alien translocations 1RS.1BL from rye and 5BS.5BL-5SL from Aegilops speltoides Tausch, and four hybrid F3 generation lines (10-7, 14-8, 15-8, 15-12) from their crossing, differing in the content of alien translocations.Results. It was shown that parameters of androgenesis such as the number of embryo-like structures per 100 anthers, the number of albino regenerants per 100 anthers, and the number of green regenerants per 100 anthers varied depending on the line. The best -responding lines Velut 991, 10-7 and 14-8 are characterized by the presence of a 1RS.1BL wheat-rye translocation chromosome. Regeneration frequency of green plants was recorded to be 8,6%, 3,6% and 10,1% respectively. The values of the parameters for lines 15-12 (carrying 5BS.5BL-5SL translocation) and 15-8 (without translocations) did not differ significantly.Conclusion. Therefore, it can be concluded that the presence of the introgressive fragment of chromosome 5S did not affect the efficiency of androgenesis and the short shoulder of chromosome 1R carries genes that stimulated androgenesis in anther culture.

Vaviloid spike branching, also called sham ramification, is a typical trait of Triticum vavilovii Jakubz. and is characterized by a lengthening of the spikelet axis. In this article, we present the results of a study of three triticale–wheat hybrid lines with differences in terms of the manifestation of the vaviloid spike branching. Lines were obtained by crossing triticale with hexaploid wheat, T. aestivum var. velutinum. The parental triticale is a hybrid of synthetic wheat (T. durum × Ae. tauschii var. meyrei) with rye, S. cereale ssp. segetale. Line 857 has a karyotype corresponding to hexaploid wheat and has a spike morphology closest to normal, whereas Lines 808/1 and 844/4 are characterized by the greatest manifestation of vaviloid spike branching. In Lines 808/1 and 844/4, we found the substitution 2RL(2DL). The karyotypes of the latter lines differ in that a pair of telocentric chromosomes 2DS is detected in Line 808/1, and these telocentrics are fused into one unpaired chromosome in Line 844/4. Using molecular genetic analysis, we found a deletion of the wheat domestication gene Q located on 5AL in the three studied hybrid lines. The deletion is local since an analysis of the adjacent gene B1 showed the presence of this gene. We assume that the manifestation of vaviloid spike branching in two lines (808/1 and 844/4) is associated with a disturbance in the joint action of genes Q and AP2L2-2D, which is another important gene that determines spike morphology and is located on 2DL.

This review is devoted to the introgressive hybridization of common wheat, the process of integrating the genes of other cultivated or wild species from the secondary and tertiary gene pools into its genome in order to increase the genetic diversity of this most important agricultural crop. The results achieved in the introgressive hybridization of common wheat over the past 20 years are summarized, the distribution of alien translocations by subgenomes and chromosomes of common wheat is analyzed, and the possibilities for further development of this direction of research and selection based on the use of alien genetic material are discussed.

Synthetic recombination form RS7 (BBAAUS), in which the first two genomes, A and B, originate from common wheat, and the third recombinant genome consists of Aegilops speltoides (S) and Ae. umbellulata (U) chromosomes, was obtained from crossing synthetic forms Avrodes (BBAASS) and Avrolata (BBAAUU). Resistant to leaf rust, yellow rust and powdery mildew, introgression lines have been obtained from backcrosses with the susceptible varieties of common wheat Krasnodarskaya 99, Fisht and Rostislav. PCR analysis showed the presence of amplification fragments with marker SCS421 specific for the Lr28 gene in the line 4991n17. The cytological study (С-banding and FISH) of 14 lines has revealed chromosomal modifications in 12 of them. In most cases, the lines carry translocations from Ae. speltoides, which were identified in chromosomes 1D, 2D, 3D, 2B, 4B, 5B and 7B. Also, lines with the substituted chromosomes 1S (1B), 4D (4S), 5D (5S) and 7D (7S) were identified. Lines that have genetic material from Ae. speltoides and Ae umbellulata at once were revealed. In the line 3379n14, translocations in the short arm of chromosome 7D from Ae. umbellulata and chromosomes 5BL, 1DL, 2DL from Ae. speltoides were revealed. The line 4626p16 presumably has a translocation on the long arm of chromosome 2D from Ae. umbellulata and the T7SS.7SL-7DL translocation from Ae. speltoides. The T1DS.1DL-1SL and T3DS.3DL-3SL translocations from Ae. speltoides, and T2DS.2DL-2UL and T7DL.7DS-7US from Ae. umbellulata have been obtained for the first time. These lines may carry previously unidentified disease resistance genes and, in particular, leaf rust resistance genes from Ae. speltoides and Ae. umbellulata.

Triticale-wheat hybrid lines from collection of the Genetic Resources Institute of ANAS (Azerbaijan) significantly differ in the ear morphology (manifestation of the vaviloid type of spike branching), in some quantitative traits, and in karyotypes.Karyotypic differences are mainly associated with chromosomes of the second homeologous group.The chromosomes 2D are replaced by a pair of rye chromosomes, presumably 2R in all the studied lines.Some lines have telocentric chromosomes, presumably corresponding to the short arm of a wheat chromosome 2D.We also observed rye chromosomes variability in different lines: loss of one arm or the deletion of subtelomeric heterochromatin accompanied by the loss of the pSc119.2signal.

Cytogenetic methods such as chromosome banding and in situ hybridization remain relevant in the post-genomic era, especially for allopolyploid species where genome duplication in some cases makes it difficult to assess the reorganization of chromosomes during evolution. In this review, we give a brief description of cytogenetic methods for the analysis of homoeological chromosomes in cereals. Emphasis is placed on the development of methods for the study of polyploid wheat and its progenitors and on tandem repeats and retrotransposons as markers to evaluate chromosome reorganization throughout evolution and breeding. The most effective cytological probes used for the identification of chromosomes in wheat and Triticeae species by fluorescence and genomic in situ hybridization are described. Particular attention is paid to ribosomal genes used as markers in phylogenetic studies and for chromosome identification. Utility of these cytogenetic methods in the evaluation of breeding lines is demonstrated. A strategy for cytological analysis of wheat hybrids according to the degree of relationships between the species involved in crosses is also discussed.

The synthesis of new allopolyploid cereal genotypes is an important task aimed at involving new genetic resources in breeding programs. Diploid species of the genera Triticum and Aegilops – bread wheat relatives – are an important source of agronomically valuable traits. A tetraploid synthetic with genomic formula DDAuAu was obtained by N.A. Navruzbekov through crossing Aegilops tauschii Coss. and Triticum urartu Thum. ex Gandil. The purpose of this work was to study the chromosomal composition and biological and commercially important traits of the tetraploid. Cytogenetic analysis using fluorescent in situ hybridization showed the presence of all chromosomes of the D genome in the chromosomal complement of the synthetic. By means of stepwise vernalization, the winter habit was established for the tetraploid synthetic with the optimum vernalization requirement of 45 days. Under greenhouse conditions, two groups of genotypes were found whose flowering dates differed by 6.5 days, which may indicate an allelism at the Vrn-3 locus. The coloring of various organs of the tetraploid plant, such as coleoptile, stem, anthers, and glumes of the spike, was revealed. The coloration of the aleurone layer of the grain may indicate that the donor species T. urartu is a carrier of the Ba gene that controls its blue color. A new morphotype of leaf pubescence was found. In terms of productivity, the tetraploid is comparable to bread wheat. Grains are characterized by a supersoft structure and high wet gluten content, from 39–45 to 65 %, in the field and greenhouse conditions, respectively. Thus, the tetraploid can be used to create new wheat genotypes as a source of untapped genetic diversity, as well as a new genetic model for studying the patterns of evolution of polyploid plants.