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

Neha Agrawal,Surinder S Banga,Js Pat Heslop-Harrison,Mehak Gupta

doi:10.3389/fpls.2020.598039

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.

Highlights

The genus Brassica includes six major vegetable or oil crops: three diploid [B. rapa (AA genome composition, 2n = 20), B. nigra (BB, 2n = 16), and B. oleracea (CC, 2n = 18)] and three allotetraploid species [B. juncea (AABB, 2n = 36), B. napus (AACC, 2n = 38), and B. carinata (BBCC, 2n = 34)]
Simulations had earlier shown that the same oligonucleotide sets could depict 16, 18, and 18 sites in B (B. nigra), C (B. oleracea), and R (R. sativus) genomes respectively
Hybridization of the probes produced the second peak on A01, with a region initializing at 29.5 Mbp from the start; C01 produced this peak at 34.5 Mbp

Summary

Introduction

Despite the erosion of collinearity, high synteny and DNA sequence homologies continue to exist among Brassicaceae genomes (Tang et al, 2008; Cheng et al, 2012), representing regions with more conserved gene order or synteny blocks. Each Brassica genome has three or six regions that are orthologous to Arabidopsis thaliana (Lysak et al, 2005, 2007; Cheng et al, 2014) These harbor highly repeated sequences and complicated centromeric regions relative to A. thaliana (Lagercrantz and Lydiate, 1996; Lagercrantz, 1998; Lan et al, 2000; Chalhoub et al, 2014; Liu et al, 2014; Yang et al, 2016; Zhang et al, 2018). In silico analysis of DNA sequence data has been vital for the understanding of evolutionary mechanisms that framed structure of existing plant genomes (Salse and Feuillet, 2011)

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