Interstitial Telomeric-like Repeats (ITR) in Seed Plants as Assessed by Molecular Cytogenetic Techniques: A Review

The discovery of telomeric repeats in interstitial regions of plant chromosomes (ITRs) through molecular cytogenetic techniques was achieved several decades ago. However, the information is scattered and has not been critically evaluated from an evolutionary perspective. Based on the analysis of currently available data, it is shown that ITRs are widespread in major evolutionary lineages sampled. However, their presence has been detected in only 45.6% of the analysed families, 26.7% of the sampled genera, and in 23.8% of the studied species. The number of ITR sites greatly varies among congeneric species and higher taxonomic units, and range from one to 72 signals. ITR signals mostly occurs as homozygous loci in most species, however, odd numbers of ITR sites reflecting a hemizygous state have been reported in both gymnosperm and angiosperm groups. Overall, the presence of ITRs appears to be poor predictors of phylogenetic and taxonomic relatedness at most hierarchical levels. The presence of ITRs and the number of sites are not significantly associated to the number of chromosomes. The longitudinal distribution of ITR sites along the chromosome arms indicates that more than half of the ITR presences are between proximal and terminal locations (49.5%), followed by proximal (29.0%) and centromeric (21.5%) arm regions. Intraspecific variation concerning ITR site number, chromosomal locations, and the differential presence on homologous chromosome pairs has been reported in unrelated groups, even at the population level. This hypervariability and dynamism may have likely been overlooked in many lineages due to the very low sample sizes often used in cytogenetic studies.

Prospects of telomere-to-telomere assembly in barley: analysis of sequence gaps in the MorexV3 reference genome

The first gapless, telomere-to-telomere (T2T) sequence assemblies of plant chromosomes were reported recently. However, sequence assemblies of most plant genomes remain fragmented. Only recent breakthroughs in accurate long-read sequencing have made it possible to achieve highly contiguous sequence assemblies with a few tens of contigs per chromosome, i.e. a number small enough to allow for a systematic inquiry into the causes of the remaining sequence gaps and the approaches and resources needed to close them. Here, we analyze sequence gaps in the current reference genome sequence of barley cv. Morex (MorexV3). Optical map and sequence raw data, complemented by ChIP-seq data for centromeric histone variant CENH3, were used to estimate the abundance of centromeric, ribosomal DNA and subtelomeric repeats in the barley genome. These estimates were compared with copy numbers in the MorexV3 pseudomolecule sequence. We found that almost all centromeric sequences and 45S ribosomal DNA repeat arrays were absent from the MorexV3 pseudomolecules and that the majority of sequence gaps can be attributed to assembly breakdown in long stretches of satellite repeats. However, missing sequences cannot fully account for the difference between assembly size and flow cytometric genome size estimates. We discuss the prospects of gap closure with ultra-long sequence reads.

Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris

India holds a significant rank in production and consumption of the age old protein rich crop Lentil with only one cultivated species and a large number of phenotypically similar cultivars. The need for a reliable and cost effective method of genetic characterization to unravel differences within the Lentil cultivars was felt. The present paper adopted EMA based chromosome preparation followed by staining with two contrasting fluorochromes dyes CMA and DAPI that bind directly to GC and AT rich heterochromatic segments on chromosomes. Analysis of fluorochrome banding pattern furnished a comparative account of genetic diversity within the cultivars that could not be achieved by traditional karyotyping. The marker pair of nucleolar chromosomes (4th and 3rd, majorly) occupied a pivotal position to intensify differences between cultivars in terms of banding patterns around secondary constrictions, suggestive of yet unknown variation in heterochromatin composition. Our study has strengthened genetic background and relationships of Lentil cultivars. We observed certain types of unusual fluorochrome bands that put forward the exclusivity of Indian germplasm and have questioned the mainstream heterochromatin elements of plant chromosomes captured by CMA-DAPI stains. The comprehensive fluorescent karyotypes of 30 L. culinaris cultivars prepared for the first time, serve as an archetype for the benefit of future breeding programmes in any Indian crop.

Plant DNA Repair and Agrobacterium T−DNA Integration

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T−DNA integration into the plant genome.

Genome mapping and gene expression of NDP-sugar pathways in the giant duckweed Spirodela polyrhiza and its relevance for bioenergy

Duckweeds are fast-growing aquatic plants suitable for bioenergy due to fermentable-rich biomass with low lignin. The duckweed sub-families Lemnoideae and Wolffioideae are also distinguished by the distribution of two pectin classes (apiogalacturonan and xylogalacturonan), which seem to be related to their growing capacity and the starch content. The plant cell wall is built from pathways of nucleotide sugars syntheses that culminate in cell wall synthesis and deposition. Therefore, understanding these pathways through mapping the genes involved and their expression would be important to develop tools to improve bioenergy production. Here we used the available information of NDP-sugar metabolism to search for orthologous genes involved in the synthesis of cell wall polysaccharides in Spirodela polyrhiza . We detected 190 genes and mapped them onto the plant chromosomes . The genes were roughly arranged in groups according to their category: "Starch and sucrose metabolism," "Pectins," "Hemicelluloses," and "Cellulose." We followed the expression of thirty-eight of the orthologues’ transcripts – the higher expression being starch ( SBE ), pectin ( GAUT1, MUR, USP , and GER ), and mannan ( CSLA ) syntheses - corroborating the chemical composition of S. polyrhiza cell wall . We further investigated the carbohydrate metabolism pathways and discussed the implications of altering the NDP pathways for bioenergy and biorefinery. We conclude that S. polyrhiza displays suitable features for future genetic transformations leading to the adaptation of its cell wall for biofuels. However, such strategies will have to consider the trade-offs between fermentation and ethanol production benefits and the potential adverse effects of genetic transformation on plant growth and development.

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.

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 refin-ing of genetic maps and validation genome assembly at the chromosomal level. Despite tremen-dous progress in genome sequencing the plant genome assembly at chromosome level still remain a challenge. Recently developed optical and Hi-C mapping aim to assist in genome assembly. For high-confidence in the genome assembly at chromosome level more independent approaches will be required. The present study aimed to refined an ultrasensitive Tyr-FISH technique and to de-velop a reliable and easy method for in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH technique to find out the reasons for the failures of using the method. It has been shown that successful visualization of marker/gene depends significantly on method of chromosome slide preparation, probe design and labelling, high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.7Kb with a frequency of 51.6%. Based on our results, we developed a reliable and simple protocol for dual-color Tyr-FISH visualization of short unique DNA sequences on plant chromosomes. New protocol allows more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

CRISPR/Cas-mediated chromosome engineering: opening up a new avenue for plant breeding

The advent of powerful site-specific nucleases, particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, which enables precise genome manipulation, has revolutionized plant breeding. Until recently, the main focus of researchers has been to simply knock-in or knock-out single genes, or to induce single base changes, but constant improvements of this technology have enabled more ambitious applications that aim to improve plant productivity or other desirable traits. One long-standing aim has been the induction of targeted chromosomal rearrangements (crossovers, inversions, or translocations). The feasibility of this technique has the potential to transform plant breeding, because natural rearrangements, like inversions, for example, typically present obstacles to the breeding process. In this way, genetic linkages between traits could be altered to combine or separate favorable and deleterious genes, respectively. In this review, we discuss recent breakthroughs in the field of chromosome engineering in plants and their potential applications in the field of plant breeding. In the future, these approaches might be applicable in shaping plant chromosomes in a directed manner, based on plant breeding needs.