Whole-Genome Sequencing and Analysis of Tumour-Forming Radish (Raphanus sativus L.) Line

Abstract

Spontaneous tumour formation in higher plants can occur in the absence of pathogen invasion, depending on the plant genotype. Spontaneous tumour formation on the taproots is consistently observed in certain inbred lines of radish (Raphanus sativus var. radicula Pers.). In this paper, using Oxford Nanopore and Illumina technologies, we have sequenced the genomes of two closely related radish inbred lines that differ in their ability to spontaneously form tumours. We identified a large number of single nucleotide variants (amino acid substitutions, insertions or deletions, SNVs) that are likely to be associated with the spontaneous tumour formation. Among the genes involved in the trait, we have identified those that regulate the cell cycle, meristem activity, gene expression, and metabolism and signalling of phytohormones. After identifying the SNVs, we performed Sanger sequencing of amplicons corresponding to SNV-containing regions to validate our results. We then checked for the presence of SNVs in other tumour lines of the radish genetic collection and found the ERF118 gene, which had the SNVs in the majority of tumour lines. Furthermore, we performed the identification of the CLAVATA3/ESR (CLE) and WUSCHEL (WOX) genes and, as a result, identified two unique radish CLE genes which probably encode proteins with multiple CLE domains. The results obtained provide a basis for investigating the mechanisms of plant tumour formation and also for future genetic and genomic studies of radish.

Keywords: CLE; Raphanus sativus; WOX; genomic sequence; inbred lines; single nucleotide variants; spontaneous tumours.

Figure 1

Spontaneous tumour formation in inbred radish lines: (a). taproots of related lines 19 (left) and 18 (right) contrasting in the tumour formation trait; (b). a family tree of the radish genetic collection showing the origin of the inbred lines; tumour lines 10, 11, 12, 13, 14, 16, 19, 20, 21, 32, 34 are marked in red; the squares indicate the intended progeny of each radish line. Different boxes represent lines of diverse cultivars. The sector that includes lines originating from the Saxa cultivar is highlighted in green.

Figure 2

Comparative characteristics of the genomes of radish lines 18 and 19 sequenced in this work and the radish reference genome (GCA_019705955.1). The analysis was carried out using the BUSCO programme.

Figure 3

Chromosomal location of radish genes with (a) InDels or (b) SNPs identified in tumour line 19 compared to non-tumour line 18 performed using the MapChart 2.32 software (https://www.wur.nl/en/show/mapchart.htm (accessed on 30 April 2024)).

Figure 4

Schematic representation of the insertion (marked with an asterisk) detected in the RsERF018 gene. (a) The scheme of an ERF18 gene. The insertion is located on the border of the 5’-UTR and the start codon. (b) 5’-UTR insertion of the RsERF018 gene in radish lines and its possible consequences. The amino acid content of the protein synthesised during translation of the normal sequence is marked in black, and the protein synthesised during translation in the case of the CAG insertion is marked in white. Radish tumour lines are highlighted in red.

Figure 5

Chromosomal location of radish (a) CLE and (b) WOX family genes performed using the MapChart 2.32 software (https://www.wur.nl/en/show/mapchart.htm (accessed on 30 April 2024)).

Figure 6

Radish CLE gene family (RsCLEs). (a) Phylogenetic tree of RsCLE genes constructed using the Neighbour-joining algorithm. The colour indicates RsCLEm1 and RsCLEm2 genes, which encode proteins with multiple CLE domains. (b) CLE domain consensus sequences of all RsCLE peptides identified in radish.

Figure 7

The proteins with multiple CLE domains probably encoded by RsCLEm genes. (a) A representation of the domain organisation of RsCLEm proteins, including the positions of signal peptide (SP) and CLE domains. Signal motifs were predicted with the SignalP-6.0 tool (https://services.healthtech.dtu.dk/service.php?SignalP (accessed on 30 April 2024)). Identical sequences of CLE domains are marked with the same colour. (b) CLE domain consensus sequences of Raphanus sativus and Brassica napus. (c) Sequence alignment of the putative 12-amino-acid CLE domain sequences encoded by the CLEm genes of Raphanus sativus and Brassica napus. (d) Phylogenetic analysis of the BnCLEm and RsCLEm peptides.

Figure 8

Pipeline of the experiment for the analysis of tumour and non-tumour lines of Raphanus sativus. Coloured blocks indicate different stages of this work. SNVs marked with a red circle are the main SNVs investigated in this paper.