Oligonucleotide Fluorescence In Situ Hybridization: An Efficient Chromosome Painting Method in Plants.

Fluorescence in situ hybridization (FISH) is an indispensable technique for studying chromosomes in plants. However, traditional FISH methods, such as BAC, rDNA, tandem repeats, and distributed repetitive sequence probe-based FISH, have certain limitations, including difficulties in probe synthesis, low sensitivity, cross-hybridization, and limited resolution. In contrast, oligo-based FISH represents a more efficient method for chromosomal studies in plants. Oligo probes are computationally designed and synthesized for any plant species with a sequenced genome and are suitable for single and repetitive DNA sequences, entire chromosomes, or chromosomal segments. Furthermore, oligo probes used in the FISH experiment provide high specificity, resolution, and multiplexing. Moreover, oligo probes made from one species are applicable for studying other genetically and taxonomically related species whose genome has not been sequenced yet, facilitating molecular cytogenetic studies of non-model plants. However, there are some limitations of oligo probes that should be considered, such as requiring prior knowledge of the probe design process and FISH signal issues with shorter probes of background noises during oligo-FISH experiments. This review comprehensively discusses de novo oligo probe synthesis with more focus on single-copy DNA sequences, preparation, improvement, and factors that affect oligo-FISH efficiency. Furthermore, this review highlights recent applications of oligo-FISH in a wide range of plant chromosomal studies.

Production and cytology of Brassica autoallohexaploids with two and four copies of two subgenomes.

KEY MESSAGE: Different digenomic Brassica autoallohexaploids were produced from the crosses of three allotetraploids and ancestral diploids and characterized for the cytological behavior of two subgenomes with two and four copies. Interspecific hybridization and allopolyploidization present an important pathway for plant evolution and breeding. In this study, different types of digenomic autoallohexaploids with two or four copies of two subgenomes (AAAACC, AACCCC, AAAABB, BBBBCC, BBCCCC) were synthesized by the crosses between three Brassica allotetraploids and their diploid progenitors and the chromosome doubling, and their meiotic behaviors were analyzed by fluorescence in situ hybridization (FISH). These autoallohexaploids showed some variations in pollen fertility and seed-sets and produced both euploid and aneuploid progenies with some chromosomes lost. Two subgenomes in these autoallohexaploids showed some aberrant pairings and segregations, and the degrees of meiotic regularity were negatively associated with the genome affinities. The chromosomes of the subgenome with four copies formed few quadrivalents with the average number < 2, and mainly paired as bivalents, and majority of the chromosomes from the subgenome with two copies gave the expected bivalents. The different extents of the equal and unequal segregations corresponded to the chromosome pairings. The development and cytological investigation of these autoallohexaploids provide not only the new germplasm for genetic research and breeding but also the new clues for the genome behavior and interplay between these subgenomes with different copies.

Dosage-Dependent Gynoecium Development and Gene Expression in Brassica napus-Orychophragmus violaceus Addition Lines.

Distant hybridization usually leads to female sterility of the hybrid but the mechanism behind this is poorly understood. Complete pistil abortion but normal male fertility was shown by one Brassica napus-Orychophragmus violaceus monosomic alien addition line (MA, AACC + 1 IO, 2n = 39) produced previously. To study the effect of a single O. violaceus chromosome addition on pistil development in different genetic backgrounds, hybrids between the MA and B. carinata (BBCC), B. juncea (AABB), and two synthetic hexaploids (AABBCC) were firstly produced in this study which show complete female sterility. A microspore culture was further performed to produce the haploid monosomic alien addition line (HMA, AC + 1 IO, 2n = 20) and disomic addition line (DA, AACC + 2 IO, 2n = 40) together with haploid (H, AC, 2n = 19) and double haploid (DH, AACC, 2n = 38) plants of B. napus from MA to investigate the dosage effect of the alien O. violaceus chromosome on pistil development and gene expression. Compared to MA, the development of the pistils of DA and HMA was completely or partially recovered, in which the pistils could swell and elongate to a normal shape after open pollination, although no seeds were produced. Comparative RNA-seq analyses revealed that the numbers of the differentially expressed genes (DEGs) were significantly different, dosage-dependent, and consistent with the phenotypic difference in pairwise comparisons of HMA vs. H, DA vs. DH, MA vs. DH, MA vs. DA, and MA vs. HMA. The gene ontology (GO) enrichment analysis of DEGs showed that a number of genes involved in the development of the gynoecium, embryo sac, ovule, and integuments. Particularly, several common DEGs for pistil development shared in HMA vs. H and DA vs. DH showed functions in genotoxic stress response, auxin transport, and signaling and adaxial/abaxial axis specification. The results provided updated information for the molecular mechanisms behind the gynoecium development of B. napus responding to the dosage of alien O. violaceus chromosomes.