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

Abstract

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.

The draft chromosome-level genome assembly of tetraploid ground cherry (Prunus fruticosa Pall.) from long reads

“…Genome sequencing advances breeding processes enormously by providing insights into evolution and comparative studies with related species, determining the positions of putative genes, which may control different traits, and allowing for the possibility for marker-assisted selection. Hence several genomes of other Prunus species [23–30] as well as other members of the Rosaceae family [31–33] have been sequenced in recent years. The sizes of Prunus genomes so far sequenced range between 250-300 Mbp with high synteny of the eight basic chromosomes [3].…”

Robust Response to Plum pox virus Infection via Plant Biotechnology

“…A high diversity of research was developed to better understand the relationship between the virus sequence transgene introduced in the HoneySweet plum and the high level of resistance phenotype of the clone [5][6][7]. The recent publication of the whole genome sequencing of plum and the insertion events of HoneySweet [8] demonstrated that a multiple viral transgene copy has 2 of 14 been introduced into the plum genome. These new findings based on these results clarified remarkably the relationship between the number of transgene copy and virus resistance.…”

“…These new findings based on these results clarified remarkably the relationship between the number of transgene copy and virus resistance. One of the two insertion events in HoneySweet is designated "insertion event 2" [8] which consists of two inverted repeats of the PPV CP gene split by a hairpin and is potentially the key to the resistance. The 132 bp of the 3 untranslated sequence and unpaired in the duplication of the PPV sequence, reshapes the hairpin and together they represent the inverse repeat of CP sequence split by a hairpin (IRSH) gene construct responsible for the PPV resistance [8,9].…”

“…One of the two insertion events in HoneySweet is designated "insertion event 2" [8] which consists of two inverted repeats of the PPV CP gene split by a hairpin and is potentially the key to the resistance. The 132 bp of the 3 untranslated sequence and unpaired in the duplication of the PPV sequence, reshapes the hairpin and together they represent the inverse repeat of CP sequence split by a hairpin (IRSH) gene construct responsible for the PPV resistance [8,9]. Scorza et al have reengineered it in the BlueByrd (BO70146) [10] plum and have also successfully confirmed through the majority of clones obtained that they were resistant to PPV infection [9] in a two-year greenhouse experiment.…”

Evidence for high intergenic sequence variation in heterozygous Italian ryegrass (Lolium multiflorumLam.) genome revealed by a high-quality draft diploid genome assembly

“…Despite de novo genome assembly methods are being developed since more than two decades [14], only recently the community started focusing on highly heterozygous genomes [15][16][17][18][19][20][21]. Typically, high amounts of sequence variation between alleles prevent contig extension and scaffolding.…”