Globally, soil salinity has been on the rise owing to various factors that are both human and environmental. The abiotic stress caused by soil salinity has become one of the most damaging abiotic stresses faced by crop plants, resulting in significant yield losses. Salt stress induces physiological and morphological modifications in plants as a result of significant changes in gene expression patterns and signal transduction cascades. In this comprehensive review, with a major focus on recent advances in the field of plant molecular biology, we discuss several approaches to enhance salinity tolerance in plants comprising various classical and advanced genetic and genetic engineering approaches, genomics and genome editing technologies, and plant growth-promoting rhizobacteria (PGPR)-based approaches. Furthermore, based on recent advances in the field of epigenetics, we propose novel approaches to create and exploit heritable genome-wide epigenetic variation in crop plants to enhance salinity tolerance. Specifically, we describe the concepts and the underlying principles of epigenetic recombinant inbred lines (epiRILs) and other epigenetic variants and methods to generate them. The proposed epigenetic approaches also have the potential to create additional genetic variation by modulating meiotic crossover frequency.

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In eukaryotes, hundreds of ribosomal RNA (rRNA) genes are clustered at chromosomal loci called nucleolus organizer regions (NORs). Arabidopsis thaliana has two NORs, one on chromosome 2 (NOR2) and the other on chromosome 4 (NOR4). Each NOR consists of ~ 400 rRNA gene copies. We recently showed that rRNA gene subtypes that map to the NOR2 are silenced during development, whereas those that map to NOR4 are active. In several DNA methylation mutants of Arabidopsis, we show release of the NOR2 silencing to varying degrees. Significantly, the highest release of NOR2 silencing correlates with a maximum loss of cytosine methylation in the CHH context followed by CG, but independent of RNA-directed DNA methylation (RdDM). Next, we show that NOR2 is relatively hypermethylated and NOR4 is hypomethylated using methylation analysis of genomic DNA carried out with different types of methylation-sensitive restriction enzymes. We demonstrate similar differential methylation status between NOR2 and NOR4 in genotypes carrying NORs from multiple ecotypes. Lastly, using Tn5 transposon-mediated transposition assay, we show that NOR2 chromatin is in more condensed state than NOR4.

Bioinformatic alignment of the assembled genomes of Col-0 and Sha ecotypes of Arabidopsis thalianarevealed ~ 10,000 genome-wide structural variants involving simple insertions or deletions and repeat contractions or expansions. Using some of these structural variants, we developed new, rapid, and low-cost PCR-based molecular markers that are genetically linked to the nucleolus organizer regions (NORs). A. thaliana has two NORs, one each on chromosome 2 (NOR2) and chromosome 4 (NOR4). Both NORs are ~ 4 Mb each, and hundreds of 45S ribosomal RNA (rRNA) genes are tandemly arrayed at these loci. Using previously characterized recombinant inbred lines (RILs) derived from Col-0 x Sha crosses, we validated the utility of the newly developed NOR-linked markers in genetically mapping rRNA genes and the associated telomeres to either NOR2 or NOR4. Lastly, we sequenced Sha genome using Oxford Nanopore Technology (ONT) and used the data to obtain sequences of NOR-telomere junctions, and with the help of RILs, we mapped them as new genetic markers to their respective NORs (NOR2-TEL2N and NOR4-TEL4N). The structural variants obtained from this study would serve as valuable data for genomewide association studies (GWAS), developing synteny, and to rapidly design more genome-wide genetic (molecular) markers for new gene/trait mapping purposes. Key MessageGenome-wide structural variants we identi ed and new NOR-linked markers we developed would be useful for future genome-wide association studies (GWAS), developing synteny, and for new gene/trait mapping purposes.