Identification of Chromosomes and Chromosome Rearrangements in Crop Brassicas and Raphanus sativus: A Cytogenetic Toolkit Using Synthesized Massive Oligonucleotide Libraries

Abstract

Crop brassicas include three diploid [Brassica rapa (AA; 2n = 2x = 16), B. nigra (BB; 2n = 2x = 18), and B. oleracea (CC; 2n = 2x = 20)] and three derived allotetraploid species. It is difficult to distinguish Brassica chromosomes as they are small and morphologically similar. We aimed to develop a genome-sequence based cytogenetic toolkit for reproducible identification of Brassica chromosomes and their structural variations. A bioinformatic pipeline was used to extract repeat-free sequences from the whole genome assembly of B. rapa. Identified sequences were subsequently used to develop four c. 47-mer oligonucleotide libraries comprising 27,100, 11,084, 9,291, and 16,312 oligonucleotides. We selected these oligonucleotides after removing repeats from 18 identified sites (500–1,000 kb) with 1,997–5,420 oligonucleotides localized at each site in B. rapa. For one set of probes, a new method for amplification or immortalization of the library is described. oligonucleotide probes produced specific and reproducible in situ hybridization patterns for all chromosomes belonging to A, B, C, and R (Raphanus sativus) genomes. The probes were able to identify structural changes between the genomes, including translocations, fusions, and deletions. Furthermore, the probes were able to identify a structural translocation between a pak choi and turnip cultivar of B. rapa. Overall, the comparative chromosomal mapping helps understand the role of chromosome structural changes during genome evolution and speciation in the family Brassicaceae. The probes can also be used to identify chromosomes in aneuploids such as addition lines used for gene mapping, and to track transfer of chromosomes in hybridization and breeding programs.

Oat chromosome and genome evolution defined by widespread terminal intergenomic translocations in polyploids

“…High coverage shows the reciprocal translocations which are also revealed with the chromosomal probes. Currently it is not possible to survey large numbers of lines, and too complex to reconstruct with high coverage of the recombinant chromosomes of multiple species ( Figure 9 ; chromosomal studies using in situ hybridization as Agrawal et al., 2020 or Huang et al., 2018 to identify translocations within species). It will be interesting to identify with base-pair resolution the chromosomal breakpoints, and further characterize the non-reciprocal nature of the translocations.…”

“…With interspecific crosses for exploitation of biodiversity in the Avena genus by introgression from wild species and recombination between chromosomes, chromosomal translocations between the species may make selection of genetically balanced lines more difficult. Agrawal et al (2020) have developed pools of 20,000 oligonucleotides for use as probes in Brassica. By allowing identification of chromosome arms in chromosome preparations, these probes identified a reciprocal translocation present in one cultivar of Brassica rapa, cv.…”

Oat chromosome and genome evolution defined by widespread terminal intergenomic translocations in polyploids

“…The fundamental research has implications for breeding oats if F1 hybrid combinations include undetected translocations leading to poor performance and restricting recombination; and in interspecific crosses, to exploit biodiversity through introgression from wild species, and perhaps enabling additional introgressions in wheat breeding. Agrawal et al (2020) have developed pools of 20,000 oligonucleotides for use as probes in Brassica . By allowing identification of chromosome arms in chromosome preparations, these probes identified a reciprocal translocation present in one cultivar of Brassica rapa, cv.…”

“…Currently it is not possible to survey large numbers of lines, and too complex to reconstruct with high coverage of the recombinant chromosomes of multiple species (Fig. 9; chromosomal studies using in situ hybridization as Agrawal et al, 2020 or Huang et al, 2018 to identify translocations within species). It will be interesting to identify with base-pair resolution the chromosomal breakpoints, and further characterize the non-reciprocal nature of the translocations.…”

Chromosome‐specific painting unveils chromosomal fusions and distinct allopolyploid species in the Saccharum complex

“…Oligos specific to a chromosome region or to an entire chromosome can be developed to track chromosomes or chromosomal rearrangements in both mitosis (Han et al ., 2015; Braz et al ., 2018, 2020a) and meiosis (Han et al ., 2015; He et al ., 2018; Braz et al ., 2021). Oligo‐based chromosome barcode or painting probes have been developed in an increasing number of plant species (Han et al ., 2015; Qu et al ., 2017; Braz et al ., 2018; Albert et al ., 2019; Martins et al ., 2019; Simonikova et al ., 2019; Agrawal et al ., 2020; He et al ., 2020; Meng et al ., 2020; de Oliveira Bustamante et al ., 2021; Hoang et al ., 2021).…”