CRISPR/Cas9-mediated мultiplexed multi-allelic mutagenesis of genes located on A, B and R subgenomes of hexaploid triticale.

KEY MESSAGE: The first-time generation of hexaploid triticale plants harbouring variable panels of novel mutations in gene families involved in starch biosynthesis has been achieved by the subgenome-independent multiplexed CRISPR/Cas9-mediated editing.

Comparative Characterization of Pseudoroegneria libanotica and Pseudoroegneria tauri Based on Their Repeatome Peculiarities.

Pseudoroegneria species play an important role among Triticeae grasses, as they are the putative donors of the St genome in many polyploid species. Satellite repeats are widely used as a reliable tool for tracking evolutionary changes because they are distributed throughout the genomes of plants. The aim of our work is to perform a comparative characterization of the repeatomes of the closely related species Ps. libanotica and Ps. tauri, and Ps. spicata was also included in the analysis. The overall repeatome structures of Ps. libanotica, Ps. tauri, and Ps. spicata were similar, with some individual peculiarities observed in the abundance of the SIRE (Ty1/Copia) retrotransposons, Mutator and Harbinger transposons, and satellites. Nine new satellite repeats that have been identified from the whole-genome sequences of Ps. spicata and Ps. tauri, as well as the CL244 repeat that was previously found in Aegilops crassa, were localized to the chromosomes of Ps. libanotica and Ps. tauri. Four satellite repeats (CL69, CL101, CL119, CL244) demonstrated terminal and/or distal localization, while six repeats (CL82, CL89, CL168, CL185, CL192, CL207) were pericentromeric. Based on the obtained results, it can be assumed that Ps. libanotica and Ps. tauri are closely related species, although they have individual peculiarities in their repeatome structures and patterns of satellite repeat localization on chromosomes. The evolutionary fate of the identified satellite repeats and their related sequences, as well as their distribution on the chromosomes of Triticeae species, are discussed. The newly developed St genome chromosome markers developed in the present research can be useful in population studies of Ps. libanotica and Ps. tauri; auto- and allopolyploids that contain the St genome, such as Thinopyrum, Elymus, Kengyilia, and Roegneria; and wide hybrids between wheat and related wild species.

Erratum: Aegilops crassa Boiss. repeatome characterized using low-coverage NGS as a source of new FISH markers: application in phylogenetic studies of the Triticeae.

[This corrects the article DOI: 10.3389/fpls.2022.980764.].

Allelic Variation of Glu-A1 and Glu-B1 Genes in Winter Durum Wheat and Its Effect on Quality Parameters.

Winter durum wheat is a relatively young crop that is highly adaptable due to its winter type of growth habit. The priority of breeding and genetic improvement of winter durum wheat is to improve grain quality and pasta quality, largely determined by the glutenin storage proteins. In the present study, a collection of 76 accessions of winter durum wheat from P.P. Lukyanenko National Grain Centre was studied. The allelic state of high-molecular-weight glutenin genes, Glu-A1 and Glu-B1, using PCR markers and SDS-PAGE was identified and grain and pasta quality traits were assessed in a two-year field experiment. The positive effect of the Glu-A1a allele and a negative effect of Glu-A1c on the gluten index were shown. It was found that Glu-B1al and Glu-B1f have a positive effect on the quality and quantity of protein and gluten, while the Glu-A1c + Glu-B1al genotypes were closest to the high-quality category in protein-associated quality traits.

Permanent Spreading of 1RS.1AL and 1RS.1BL Translocations in Modern Wheat Breeding.

Wheat-rye translocations 1RS.1BL and 1RS.1AL are used in bread wheat breeding worldwide because a short arm of rye chromosome 1 (1RS) when introgressed into the wheat genome confers resistance to diseases, pests and better performance under drought-stress conditions. However, in durum wheat genotypes, these translocations occur only in experimental lines, although their advantages could enhance the potential of this crop. P.P. Lukyanenko National Grain Centre (NGC) has successfully developed commercially competitive cultivars of bread and durum wheat demanded by many agricultural producers in the South of Russia for decades. Here, 94 accessions of bread and 343 accessions of durum wheat, representing lines and cultivars from collection, competitive variety trials and breeding nursery developed at NGC were screened for 1RS using PCR markers and genomic in situ hybridization. The 1RS.1BL and 1RS.1AL translocations were detected in 38 and 6 bread wheat accessions, respectively. None of the durum wheat accessions showed translocation, despite the fact that some of them had 1RS.1BL donors in their pedigree. The absence of translocations in the studied durum wheat germplasm can be caused by the negative selection of 1RS carriers at different stages of the breeding process due to low quality and difficulties in transferring rye chromatin through wheat gametes.

The launch of satellite: DNA repeats as a cytogenetic tool in discovering the chromosomal universe of wild Triticeae.

Fluorescence in situ hybridization is a powerful tool that enables plant researchers to perform systematic, evolutionary, and population studies of wheat wild relatives as well as to characterize alien introgression into the wheat genome. This retrospective review reflects on progress made in the development of methods for creating new chromosomal markers since the launch of this cytogenetic satellite instrument to the present day. DNA probes based on satellite repeats have been widely used for chromosome analysis, especially for "classical" wheat probes (pSc119.2 and Afa family) and "universal" repeats (45S rDNA, 5S rDNA, and microsatellites). The rapid development of new-generation sequencing and bioinformatical tools, and the application of oligo- and multioligonucleotides has resulted in an explosion in the discovery of new genome- and chromosome-specific chromosome markers. Owing to modern technologies, new chromosomal markers are appearing at an unprecedented velocity. The present review describes the specifics of localization when employing commonly used vs. newly developed probes for chromosomes in J, E, V, St, Y, and P genomes and their diploid and polyploid carriers Agropyron, Dasypyrum, Thinopyrum, Pseudoroegneria, Elymus, Roegneria, and Kengyilia. Particular attention is paid to the specificity of probes, which determines their applicability for the detection of alien introgression to enhance the genetic diversity of wheat through wide hybridization. The information from the reviewed articles is summarized into the TRepeT database, which may be useful for studying the cytogenetics of Triticeae. The review describes the trends in the development of technology used in establishing chromosomal markers that can be used for prediction and foresight in the field of molecular biology and in methods of cytogenetic analysis.

Aegilops crassa Boiss. repeatome characterized using low-coverage NGS as a source of new FISH markers: Application in phylogenetic studies of the Triticeae.

Aegilops crassa Boiss. is polyploid grass species that grows in the eastern part of the Fertile Crescent, Afghanistan, and Middle Asia. It consists of tetraploid (4x) and hexaploid (6x) cytotypes (2n = 4x = 28, D1D (Abdolmalaki et al., 2019) XcrXcr and 2n = 6x = 42, D1D (Abdolmalaki et al., 2019) XcrXcrD2D (Adams and Wendel, 2005), respectively) that are similar morphologically. Although many Aegilops species were used in wheat breeding, the genetic potential of Ae. crassa has not yet been exploited due to its uncertain origin and significant genome modifications. Tetraploid Ae. crassa is thought to be the oldest polyploid Aegilops species, the subgenomes of which still retain some features of its ancient diploid progenitors. The D1 and D2 subgenomes of Ae. crassa were contributed by Aegilops tauschii (2n = 2x = 14, DD), while the Xcr subgenome donor is still unknown. Owing to its ancient origin, Ae. crassa can serve as model for studying genome evolution. Despite this, Ae. crassa is poorly studied genetically and no genome sequences were available for this species. We performed low-coverage genome sequencing of 4x and 6x cytotypes of Ae. crassa, and four Ae. tauschii accessions belonging to different subspecies; diploid wheatgrass Thinopyrum bessarabicum (Jb genome), which is phylogenetically close to D (sub)genome species, was taken as an outgroup. Subsequent data analysis using the pipeline RepeatExplorer2 allowed us to characterize the repeatomes of these species and identify several satellite sequences. Some of these sequences are novel, while others are found to be homologous to already known satellite sequences of Triticeae species. The copy number of satellite repeats in genomes of different species and their subgenome (D1 or Xcr) affinity in Ae. crassa were assessed by means of comparative bioinformatic analysis combined with quantitative PCR (qPCR). Fluorescence in situ hybridization (FISH) was performed to map newly identified satellite repeats on chromosomes of common wheat, Triticum aestivum, 4x and 6x Ae. crassa, Ae. tauschii, and Th. bessarabicum. The new FISH markers can be used in phylogenetic analyses of the Triticeae for chromosome identification and the assessment of their subgenome affinities and for evaluation of genome/chromosome constitution of wide hybrids or polyploid species.

Compensatory Effect of the ScGrf3-2R Gene in Semi-Dwarf Spring Triticale (x Triticosecale Wittmack).

The dwarfness in many triticale cultivars is provided by the dominant Ddw1 (Dominant dwarf 1) allele found in rye. However, along with conferring semi-dwarf phenotype to improve resistance to lodging, this gene also reduces grain size and weight and delays heading and flowering. Grf (Growth-regulating factors) genes are plant-specific transcription factors that regulate plant growth, including stem growth, in terms of length and thickness, and leaf and fruit size. In this work, we partially sequenced the rye gene ScGrf3 on chromosome 2R homologous to the wheat Grf3 gene, and found multiple polymorphisms in intron 3 and exon 4 complying with two alternative alleles (haplotypes ScGrf3-2Ra and ScGrf3-2Rb). For the identification of these, we developed a codominant PCR marker. Using a new marker, we studied the effect of ScGrf3-2R alleles in combination with the Ddw1 dwarf gene on economically valuable traits in F4 and F5 recombinant lines of spring triticale from the hybrid combination Valentin 90 x Dublet, grown in the Non-Chernozem zone for 2 years. Allele ScGrf3-2Ra was associated with greater thousand-grain weight, higher spike productivity, and earlier heading and flowering, which makes ScGrf3-2R a perspective compensator for negative effects of Ddw1 on these traits and increases prospects for its involvement in breeding semi-dwarf cultivars of triticale.

qPCR as a Selective Tool for Cytogenetics.

qPCR is widely used in quantitative studies of plant genomes and transcriptomes. In this article, this method is considered as an auxiliary step in the preparation and selection of markers for FISH analysis. Several cases from the authors' research on populations of the same species were reviewed, and a comparison of the closely related species, as well as the adaptation of the markers, based on satellite tandem repeats (TRs) using quantitative qPCR data was conducted. In the selected cases, TRs with contrast abundance were identified in the cases of the Dasypyrum, Thinopyrum and Aegilops species, and the transfer of TRs between the wheat and related species was demonstrated. TRs with intraspecific copy number variation were revealed in Thinopyrum ponticum and wheat-wheatgrass partial amphidiploids, and the TR showing predominant hybridization to the sea buckthorn Y chromosome was identified. Additionally, problems such as the absence of a reference gene for qPCR, and low-efficiency and self-complementary primers, were illustrated. In the cases considered here, the qPCR results clearly show high correlation with the subsequent results of the FISH analysis, which confirms the value of this method for cytogenetic studies.

TaGRF3-2A Improves Some Agronomically Valuable Traits in Semi-Dwarf Spring Triticale.

The breeding improvement of triticale is tightly associated with the introgression of dwarfing genes, in particular, gibberellin (GA)-insensitive Ddw1 from rye. Despite the increase in harvest index and resistance to lodging, this gene adversely affects grain weight and size. Growth regulation factor (GRF) genes are plant-specific transcription factors that play an important role in plant growth, including GA-induced stem elongation. This study presents the results of a two-year field experiment to assess the effect of alleles of the TaGRF3-2A gene in interaction with DDW1 on economically valuable traits of spring triticale plants grown in the Non-Chernozem zone. Our results show that, depending on the allelic state, the TaGRF3-2A gene in semi-dwarf spring triticale plants influences the thousand grain weight and the grain weight of the main spike in spring triticale, which makes it possible to use it to compensate for the negative effects of the dwarfing allele Ddw1. The identified allelic variants of the TaGRF3-2A gene can be included in marker-assisted breeding for triticale to improve traits.

Effects of Rht17 in combination with Vrn-B1 and Ppd-D1 alleles on agronomic traits in wheat in black earth and non-black earth regions.

BACKGROUND: Plant height is an important wheat trait that is regulated by multiple genes, among which Rht is of the utmost value. In wheat, Rht-B1p (=Rht17) is a mutant allele of the Rht gene that encodes for a DELLA-protein and results in the development of gibberellin-insensitive plants with a dwarfing phenotype. The pleiotropic effects of dwarfing genes on yield are highly dependent on both the genetic background and the environmental conditions. In Russia, the Central Non-Black Earth Region and Krasnodar Krai are two economically important regions that require differing management for sustainable wheat production for food, feed and industry. The purpose of our study was to compare the pleiotropic effects of Rht-B1p on the main valuable agronomic traits in the F3:4 families of the spring bread wheat Chris Mutant/Novosibirskaya 67 in the genetic background of Vrn-B1a/vrn-B1 (spring/winter phenotype) and Ppd-D1a/Ppd-D1b (insensitivity/sensitivity to photoperiod) alleles in a field experiment in Moscow and Krasnodar Krai. RESULTS: Plant height was reduced on average by 21 cm (28%) and 25 cm (30%), respectively; Ppd-D1a slightly strengthened the dwarfing effect in Moscow and mitigated it in Krasnodar Krai. Grain weight of the main spike was reduced by Rht-B1p in Moscow and to lesser extent in Krasnodar; Ppd-D1a and Vrn-B1a tended to partially compensate for this loss in Krasnodar Krai. Thousand grain weight was reduced on average by 5.3 g (16%) and 2.9 g (10%) in Moscow and Krasnodar Krai, respectively, but was partially compensated for by Ppd-D1a in Krasnodar Krai. Harvest index was increased due to Rht-B1p by 6 and 10% in Moscow and Krasnodar Krai, respectively. Rht-B1p resulted in a delay of heading by 1-2 days in Moscow. Ppd-D1a accelerated heading by 1 day and 6 days in Moscow and in Krasnodar Krai, respectively. CONCLUSIONS: Rht-B1p could be introduced into wheat breeding along with dwarfing genes such as Rht-B1b and Rht-D1b. Special attention should be paid to its combination with Ppd-D1a and Vrn-B1a as regulators of developmental rates, compensators of adverse effects of Rht-B1p on productivity and enhancers of positive effect of Rht-B1p on harvest index.

Development of Specific Thinopyrum Cytogenetic Markers for Wheat-Wheatgrass Hybrids Using Sequencing and qPCR Data.

The cytogenetic study of wide hybrids of wheat has both practical and fundamental values. Partial wheat-wheatgrass hybrids (WWGHs) are interesting as a breeding bridge to confer valuable genes to wheat genome, as well as a model object that contains related genomes of Triticeae. The development of cytogenetic markers is a process that requires long and laborious fluorescence in situ hybridization (FISH) testing of various probes before a suitable probe is found. In this study, we aimed to find an approach that allows to facilitate this process. Based on the data sequencing of Thinopyrum ponticum, we selected six tandem repeat (TR) clusters using RepeatExplorer2 pipeline and designed primers for each of them. We estimated the found TRs' abundance in the genomes of Triticum aestivum, Thinopyrum ponticum, Thinopyrum intermedium and four different WWGH accessions using real-time qPCR, and localized them on the chromosomes of the studied WWGHs using fluorescence in situ hybridization. As a result, we obtained three tandem repeat cytogenetic markers that specifically labeled wheatgrass chromosomes in the presence of bread wheat chromosomes. Moreover, we designed and tested primers for these repeats, and demonstrated that they can be used as qPCR markers for quick and cheap monitoring of the presence of certain chromosomes of wheatgrass in breeding programs.

Allele mining of TaGRF-2D gene 5'-UTR in Triticum aestivum and Aegilops tauschii genotypes.

The low diversity of the D-subgenome of bread wheat requires the involvement of new alleles for breeding. In grasses, the allelic state of Growth Regulating Factor (GRF) gene is correlated with nitrogen uptake. In this study, we characterized the sequence of TaGRF-2D and assessed its diversity in bread wheat and goatgrass Aegilops tauschii (genome DD). In silico analysis was performed for reference sequence searching, primer pairs design and sequence assembly. The gene sequence was obtained using Illumina and Sanger sequencing. The complete sequences of TaGRF-2D were obtained for 18 varieties of wheat. The polymorphism in the presence/absence of two GCAGCC repeats in 5' UTR was revealed and the GRF-2D-SSR marker was developed. Our results showed that the alleles 5' UTR-250 and 5' UTR-238 were present in wheat varieties, 5' UTR-250 was presented in the majority of wheat varieties. In Ae. tauschii ssp. strangulata (likely donor of the D-subgenome of polyploid wheat), most accessions carried the 5' UTR-250 allele, whilst most Ae. tauschii ssp. tauschii have 5' UTR-244. The developed GRF-2D-SSR marker can be used to study the genetic diversity of wheat and Ae. tauschii.

Molecular Characterization of the Dwarf53 Gene Homolog in Dasypyrum villosum.

The Dwarf53 (D53) gene, first studied in rice, encodes a protein that acts as a repressor of the physiological response of plants to strigolactones-substances that regulate the activity of axillary buds, stem growth, branching of roots and other physiological processes. In this work, we isolated and sequenced the homolog of the D53 gene in several accessions of the wild grass Dasypyrum villosum of different geographical origins, resulting in the discovery of large allelic variety. A molecular marker was also created that allows us to differentiate the D. villosum D53 gene from common wheat genes. Using this marker and monosomic addition, substitution and translocation wheat lines carrying the known D. villosum chromosomes, the D53 gene was localized on the long arm of the 5V chromosome.

Copy Number Variation of Transposable Elements in Thinopyrum intermedium and Its Diploid Relative Species.

Diploid and polyploid wild species of Triticeae have complex relationships, and the understanding of their evolution and speciation could help to increase the usability of them in wheat breeding as a source of genetic diversity. The diploid species Pseudoroegneria spicata (St), Thinopyrum bessarabicum (Jb), Dasypyrum villosum (V) derived from a hypothetical common ancestor are considered to be possible subgenome donors in hexaploid species Th. intermedium (JrJvsSt, where indices r, v, and s stand for the partial relation to the genomes of Secale, Dasypyrum, and Pseudoroegneria, respectively). We quantified 10 families of transposable elements (TEs) in P. spicata, Th. bessarabicum, D. villosum (per one genome), and Th. intermedium (per one average subgenome) using the quantitative real time PCR assay and compared their abundance within the studied genomes as well as between them. Sabrina was the most abundant among all studied elements in P. spicata, D. villosum, and Th. intermedium, and among Ty3/Gypsy elements in all studied species. Among Ty1/Copia elements, Angela-A and WIS-A showed the highest and close abundance with the exception of D. villosum, and comprised the majority of all studied elements in Th. bessarabicum. Sabrina, BAGY2, and Angela-A showed similar abundance among diploids and in Th. intermedium hexaploid; Latidu and Barbara demonstrated sharp differences between diploid genomes. The relationships between genomes of Triticeae species based on the studied TE abundance and the role of TEs in speciation and polyploidization in the light of the current phylogenetic models is discussed.

Development of new cytogenetic markers for Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang.

Thinopyrum ponticum (Podpera, 1902) Z.-W. Liu & R.-C.Wang, 1993 is an important polyploid wild perennial Triticeae species that is widely used as a source of valuable genes for wheat but its genomic constitution has long been debated. For its chromosome identification, only a limited set of FISH probes has been used. The development of new cytogenetic markers for Th. ponticum chromosomes is of great importance both for cytogenetic characterization of wheat-wheatgrass hybrids and for fundamental comparative studies of phylogenetic relationships between species. Here, we report on the development of five cytogenetic markers for Th. ponticum based on repetitive satellite DNA of which sequences were selected from the whole genome sequence of Aegilops tauschii Cosson, 1849. Using real-time quantitative PCR we estimated the abundance of the found repeats: P720 and P427 had the highest abundance and P132, P332 and P170 had lower quantity in Th. ponticum genome. Using fluorescence in situ hybridization (FISH) we localized five repeats to different regions of the chromosomes of Th. ponticum. Using reprobing multicolor FISH we colocalized the probes between each other. The distribution of these found repeats in the Triticeae genomes and its usability as cytogenetic markers for chromosomes of Th. ponticum are discussed.

Effect of Dwarfing Gene Ddw1 on Height and Agronomic Traits in Spring Triticale in Greenhouse and Field Experiments in a Non-Black Earth Region of Russia.

Triticale is a relatively new crop which still possesses serious drawbacks that can be significantly improved by breeding. The dwarfing genes proved to be very useful in the development of new lodging resistant and productive cultivars of winter triticale. The aim of our research was to assess the effect of the Ddw1 dwarfing gene from rye on the agronomic valuable traits in spring triticale. The Ddw1 effect was studied in the greenhouse experiment in segregating the F2:3 population and in the field of F3:4 and F4:5 families derived from crossing winter triticale 'Hongor' (Ddw1Ddw1) and spring triticale 'Dublet' (ddw1ddw1). As a result, in all three generations, a strong decrease in plant height was demonstrated that was accompanied by a decrease in grain weight per spike and 1000-grain weight. In field experiments, a decrease in spike length and increase in spike density and delay in flowering and heading were observed. As a result of decrease in culm vegetative weight due to Ddw1, the harvest index measured in F4:5 increased. The spike fertility and number of grains were not affected by Ddw1. The comparison of Ddw1 in rye, winter, and spring triticale, and the possible role of Ddw1 in improving spring triticale are discussed.

Pipeline for the Rapid Development of Cytogenetic Markers Using Genomic Data of Related Species.

Repetitive DNA including tandem repeats (TRs) is a significant part of most eukaryotic genomes. TRs include rapidly evolving satellite DNA (satDNA) that can be shared by closely related species, their abundance may be associated with evolutionary divergence, and they have been widely used for chromosome karyotyping using fluorescence in situ hybridization (FISH). The recent progress in the development of whole-genome sequencing and bioinformatics tools enables rapid and cost-effective searches for TRs including satDNA that can be converted into molecular cytogenetic markers. In the case of closely related taxa, the genome sequence of one species (donor) can be used as a base for the development of chromosome markers for related species or genomes (target). Here, we present a pipeline for rapid and high-throughput screening for new satDNA TRs in whole-genome sequencing of the donor genome and the development of chromosome markers based on them that can be applied in the target genome. One of the main peculiarities of the developed pipeline is that preliminary estimation of TR abundance using qPCR and ranking found TRs according to their copy number in the target genome; it facilitates the selection of the most prospective (most abundant) TRs that can be converted into cytogenetic markers. Another feature of our pipeline is the probe preparation for FISH using PCR with primers designed on the aligned TR unit sequences and the genomic DNA of a target species as a template that enables amplification of a whole pool of monomers inherent in the chromosomes of the target species. We demonstrate the efficiency of the developed pipeline by the example of FISH probes developed for A, B, and R subgenome chromosomes of hexaploid triticale (BBAARR) based on a bioinformatics analysis of the D genome of Aegilops tauschii (DD) whole-genome sequence. Our pipeline can be used to develop chromosome markers in closely related species for comparative cytogenetics in evolutionary and breeding studies.

Variation in Copy Number of Ty3/Gypsy Centromeric Retrotransposons in the Genomes of Thinopyrum intermedium and Its Diploid Progenitors.

Speciation and allopolyploidization in cereals may be accompanied by dramatic changes in abundance of centromeric repeated transposable elements. Here we demonstrate that the reverse transcriptase part of Ty3/gypsy centromeric retrotransposon (RT-CR) is highly conservative in the segmental hexaploid Thinopyrum intermedium (JrJvsSt) and its possible diploid progenitors Th. bessarabicum (Jb), Pseudoroegneria spicata (St) and Dasypyrum villosum (V) but the abundance of the repeats varied to a large extent. Fluorescence in situ hybridization (FISH) showed hybridization signals in centromeric region of all chromosomes in the studied species, although the intensity of the signals drastically differed. In Th. intermedium, the strongest signal of RT-CR probe was detected on the chromosomes of Jv, intermediate on Jr and faint on Js and St subgenome suggesting different abundance of RT-CR on the individual chromosomes rather than the sequence specificity of RT-CRs of the subgenomes. RT-CR quantification using real-time PCR revealed that its content per genome in Th. bessarabicum is ~ 2 times and P. spicata is ~ 1,5 times higher than in genome of D. villosum. The possible burst of Ty3/gypsy centromeric retrotransposon in Th. intermedium during allopolyploidization and its role in proper mitotic and meiotic chromosome behavior in a nascent allopolyploid is discussed.