Defining the 'HoneySweet' insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica)

Abstract

'HoneySweet' plum (Prunus domestica) is resistant to Plum pox potyvirus, through an RNAi-triggered mechanism. Determining the precise nature of the transgene insertion event has been complicated due to the hexaploid genome of plum. DNA blots previously indicated an unintended hairpin arrangement of the Plum pox potyvirus coat protein gene as well as a multicopy insertion event. To confirm the transgene arrangement of the insertion event, 'HoneySweet' DNA was subjected to whole genome sequencing using Illumina short-read technology. Results indicated two different insertion events, one containing seven partial copies flanked by putative plum DNA sequence and a second with the predicted inverted repeat of the coat protein gene driven by a double 35S promoter on each side, flanked by plum DNA. To determine the locations of the two transgene insertions, a phased plum genome assembly was developed from the commercial plum 'Improved French'. A subset of the scaffolds (2447) that were >10 kb in length and representing, >95% of the genome were annotated and used for alignment against the 'HoneySweet' transgene reads. Four of eight matching scaffolds spanned both insertion sites ranging from 157,704 to 654,883 bp apart, however we were unable to identify which scaffold(s) represented the actual location of the insertion sites due to potential sequence differences between the two plum cultivars. Regardless, there was no evidence of any gene(s) being interrupted as a result of the insertions. Furthermore, RNA-seq data verified that the insertions created no new transcriptional units and no dramatic expression changes of neighboring genes.

Fig. 1. Diagram of the initial transformation T-DNA and the rearrangements resulting at the insertion event 1 and insertion event 2.

Each of the transgene regions is color coded with the NPTII gene, promoter, and terminator in yellow, the PPV-CP in red, the UIDA in blue, the MUA-10 in green, and plum in dark blue. The regions in pink represent the fragments of the initial T-DNA specified by the base numbers

Fig. 2. Diagram of the plum scaffolds that contain sites that match the insertion events.

Under each scaffold number is the size of the scaffold in bases. The insertion sites are marked with a triangle and the flanking genes with a bar. The key identifies each by color

Fig. 3. Synteny between peach and the eight scaffolds that have matching sequence to the flanking sequences for insertion 1 and 2.

Like color blocks are orthologous genes. The names of all the genes for peach are listed on the side. Light gray blocks for the plum represent unique genes, deep purple blocks represent the repetitive DMR6-like genes and the forest green represent arabinogalactan-9 family genes. Blank space are gene gaps in the sequences. The asterisk for Scaffold 1650 is to indicate that there is an inversion in the middle of the synteny chart. The red stars indicate the location of insertion 1. The yellow stars are indications of where insertion 2 is