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.

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.].

The Control of the Crossover Localization in Allium.

Meiotic crossovers/chiasmata are not randomly distributed and strictly controlled. The mechanisms behind crossover (CO) patterning remain largely unknown. In Allium cepa, as in the vast majority of plants and animals, COs predominantly occur in the distal 2/3 of the chromosome arm, while in Allium fistulosum they are strictly localized in the proximal region. We investigated the factors that may contribute to the pattern of COs in A. cepa, A. fistulosum and their F1 diploid (2n = 2x = 8C + 8F) and F1 triploid (2n = 3x = 16F + 8C) hybrids. The genome structure of F1 hybrids was confirmed using genomic in situ hybridization (GISH). The analysis of bivalents in the pollen mother cells (PMCs) of the F1 triploid hybrid showed a significant shift in the localization of COs to the distal and interstitial regions. In F1 diploid hybrid, the COs localization was predominantly the same as that of the A. cepa parent. We found no differences in the assembly and disassembly of ASY1 and ZYP1 in PMCs between A. cepa and A. fistulosum, while F1 diploid hybrid showed a delay in chromosome pairing and a partial absence of synapsis in paired chromosomes. Immunolabeling of MLH1 (class I COs) and MUS81 (class II COs) proteins showed a significant difference in the class I/II CO ratio between A. fistulosum (50%:50%) and A. cepa (73%:27%). The MLH1:MUS81 ratio at the homeologous synapsis of F1 diploid hybrid (70%:30%) was the most similar to that of the A. cepa parent. F1 triploid hybrid at the A. fistulosum homologous synapsis showed a significant increase in MLH1:MUS81 ratio (60%:40%) compared to the A. fistulosum parent. The results suggest possible genetic control of CO localization. Other factors affecting the distribution of COs are discussed.

Hidden Pitfalls of Using Onion Pollen in Molecular Research.

There is little information on the use of pollen in molecular research, despite the increased interest in genome editing by pollen-mediated transformation. This paper presents an essential toolbox of technical procedures and observations for molecular studies on onion (Allium cepa L.) pollen. PCR is a useful tool as an express method to evaluate editing results before pollination. A direct PCR protocol for pollen suspension has been adapted without needing DNA pre-extraction. We showed that the outer layer of lipids known as pollenkitt is a limiting factor for successful PCR on pollen. A simple pre-washing step of pollen suspension was able to eliminate the pollenkitt and enormously affect the PCR results. Additionally, our pollenkitt study helped us develop a simple and effective pollination method using wetted onion pollen grains. Classical manual pollination usually is conducted by intact pollen without wetting. Most existing methods of the editing system delivery into pollen are carried out in a wet medium with consequent drying before pollination, which adversely affects the viability of pollen. The optimal medium for wet pollination was 12% sucrose water solution. Our method of using wetted pollen grains for pollination might be very beneficial for pollen genetic manipulation.

Two-Step Identification of N-, S-, R- and T-Cytoplasm Types in Onion Breeding Lines Using High-Resolution Melting (HRM)-Based Markers.

High-resolution melting (HRM) analysis is a powerful detection method for fast, high-throughput post-PCR analysis. A two-step HRM marker system was developed for identification of the N-, S-, R- and T-cytoplasms of onion. In the first step for the identification of N-, S- and R-cytoplasms, one forward primer was designed to the identical sequences of both cox1 and orf725 genes, and two reverse primers specific to the polymorphic sequences of cox1 and orf725 genes were used. For the second step, breeding lines with N-cytoplasm were evaluated with primers developed from the orfA501 sequence to distinguish between N- and T-cytoplasms. An amplicon with primers to the mitocondrial atp9 gene was used as an internal control. The two-step HRM marker system was tested using 246 onion plants. HRM analysis showed that the most common source of CMS, often used by Russian breeders, was S-cytoplasm; the rarest type of CMS was R-cytoplasm; and the proportion of T-cytoplasm among the analyzed breeding lines was 20.5%. The identification of the cytoplasm of a single plant by phenotype takes from 4 to 8 years. The HRM-based system enables quick and easy distinguishing of the four types of onion cytoplasm.

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.

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.

Integrating Genetic and Chromosome Maps of Allium cepa: From Markers Visualization to Genome Assembly Verification.

The ability to directly look into genome sequences has opened great opportunities in plant breeding. Yet, the assembly of full-length chromosomes remains one of the most difficult problems in modern genomics. Genetic maps are commonly used in de novo genome assembly and are constructed on the basis of a statistical analysis of the number of recombinations. This may affect the accuracy of the ordering and orientation of scaffolds within the chromosome, especially in the region of recombination suppression. Moreover, it is impossible to assign contigs lacking DNA markers. Here, we report the use of Tyr-FISH to determine the position of the short DNA sequence of markers and non-mapped unique copy sequence on the physical chromosomes of a large-genome onion (Allium cepa L.). In order to minimize potential background masking of the target signal, we improved our earlier developed pipeline for probe design. A total of 23 markers were located on physical chromosomes 2 and 6. The order of markers was corrected by the integration of genetic, pseudochromosome maps and cytogenetic maps. Additionally, the position of the mlh1 gene, which was not on the genetic map, was defined on physical chromosome 2. Tyr-FISH mapping showed that the order of 23.1% (chromosome 2) and 27.3% (chromosome 6) of the tested genes differed between physical chromosomes and pseudochromosomes. The results can be used for the improvement of pseudochromosome 2 and 6 assembly. The present study aims to demonstrate the value of the in situ visualization of DNA sequences in chromosome-scaffold genome assembly.

Transposons Hidden in Arabidopsis thaliana Genome Assembly Gaps and Mobilization of Non-Autonomous LTR Retrotransposons Unravelled by Nanotei Pipeline.

Long-read data is a great tool to discover new active transposable elements (TEs). However, no ready-to-use tools were available to gather this information from low coverage ONT datasets. Here, we developed a novel pipeline, nanotei, that allows detection of TE-contained structural variants, including individual TE transpositions. We exploited this pipeline to identify TE insertion in the Arabidopsis thaliana genome. Using nanotei, we identified tens of TE copies, including ones for the well-characterized ONSEN retrotransposon family that were hidden in genome assembly gaps. The results demonstrate that some TEs are inaccessible for analysis with the current A. thaliana (TAIR10.1) genome assembly. We further explored the mobilome of the ddm1 mutant with elevated TE activity. Nanotei captured all TEs previously known to be active in ddm1 and also identified transposition of non-autonomous TEs. Of them, one non-autonomous TE derived from (AT5TE33540) belongs to TR-GAG retrotransposons with a single open reading frame (ORF) encoding the GAG protein. These results provide the first direct evidence that TR-GAGs and other non-autonomous LTR retrotransposons can transpose in the plant genome, albeit in the absence of most of the encoded proteins. In summary, nanotei is a useful tool to detect active TEs and their insertions in plant genomes using low-coverage data from Nanopore genome sequencing.

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.

SORL1 mutation in a Greek family with Parkinson's disease and dementia.

Whole exome sequencing and linkage analysis were performed in a three generational pedigree of Greek origin with a broad phenotypic spectrum spanning from Parkinson's disease and Parkinson's disease dementia to dementia of mixed type (Alzheimer disease and vascular dementia). We identified a novel heterozygous c.G1135T (p.G379W) variant in SORL1 which segregated with the disease in the family. Mutation screening in sporadic Greek PD cases identified one additional individual with the mutation, sharing the same 12.8Mb haplotype. Our findings provide support for SORL1 mutations resulting in a broad range of additional phenotypes and warrants further studies in neurodegenerative diseases beyond AD.

A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps.

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

Comparative Tyramide-FISH mapping of the genes controlling flavor and bulb color in Allium species revealed an altered gene order.

Evolutionarily related species often share a common order of genes along homeologous chromosomes. Here we report the collinearity disruption of genes located on homeologous chromosome 4 in Allium species. Ultra-sensitive fluorescence in situ hybridization with tyramide signal amplification (tyr-FISH) allowed the visualization of the alliinase multigene family, chalcon synthase gene and EST markers on Allium cepa and Allium fistulosum chromosomes. In A. cepa, bulb alliinase, root alliinase (ALL1) and chalcon synthase (CHS-B) genes were located in the long arm but EST markers (API18 and ACM082) were located in the short arm. In A. fistulosum, all the visualized genes and markers were located in the short arm. Moreover, root alliinase genes (ALL1 and AOB249) showed contrast patterns in number of loci. We suppose that the altered order of the genes/markers is the result of a large pericentric inversion. To get insight into the evolution of the chromosome rearrangement, we mapped the bulb alliinase gene in phylogenetically close and distant species. In the taxonomic clade including A. fistulosum, A. altaicum, A. oschaninii and A. pskemense and in phylogenetically distant species A. roylei and A. nutans, the bulb alliinase gene was located on the short arm of chromosome 4 while, in A. cepa and A. schoenoprasum, the bulb alliinase gene was located on the long arm of chromosome 4. These results have encouraging implications for the further tracing of inverted regions in meiosis of interspecific hybrids and studding chromosome evolution. Also, this finding may have a practical benefit as closely related species are actively used for improving onion crop stock.