Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi.

Jeanette Orbegozo,Dennis Solorzano,Maria Lupe Roman,Jan Kreuze,Wilmer J Cuellar,Ida Bartolini,Marc Ghislain

doi:10.1007/s11248-016-9976-y

Abstract

An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-016-9976-y) contains supplementary material, which is available to authorized users.

Highlights

Potato leaf roll virus (PLRV, Genus: Polerovirus, Family: Luteoviridae) is one of the most widespread and important virus disease in potato cultivation (Barker and Dale 2006)
A fragment of 395 bp from the coat protein encoding sequence from PLRV 3626–4034 position of GenBank accession D13953.1 was used to construct an intron spliced hpRNA. This sequence was cloned in sense and antisense orientation, separated by the IV2 intron (217 bp; Vancanneyt et al 1990) under the control of the Cauliflower Mosaic virus 35S promoter and CaMV35S poly-adenylation signal sequence using standard molecular techniques (Sambrook and Russell 2001). This gene cassette was inserted into the HindIII site of the nptII heat inducible self-excision vector pCIP33 (Cuellar et al 2006) to produce the binary vector pCIP35 bearing a T-DNA with the hpPLRV, the nptII selectable marker gene, and the Cre-loxP excisable selectable marker system whose which complete size was 6896 bp (Fig. 1a)
The present work aimed at obtaining PLRV resistant potato plants without an antibiotic resistance gene using RNA interference (RNAi) (Waterhouse et al 1998) and a selfexcisable Cre-loxP nptII system respectively (Cuellar et al 2006)

Summary

Introduction

Potato leaf roll virus (PLRV, Genus: Polerovirus, Family: Luteoviridae) is one of the most widespread and important virus disease in potato cultivation (Barker and Dale 2006). PLRV damages of the potato crop are controlled by the use of certified seed (Radcliffe and Ragsdale 2002) and by insecticide applications (Mowry 2005) These techniques are not always accessible, may be costly, in particular to small-farmers, and produce environmental and human health concerns (Paoletti and Pimentel 2000; Syller 1996; Thomas et al 1997). The levels of resistance are classified into two modes of operation: (1) resistance against infection by viruliferous aphids; (2) and resistance to accumulation of the virus (Barker and Harrison 1986) Both resistances until recently were polygenic and their use in breeding programs is timeconsuming with an uncertain outcome (Barker and Dale 2006; Davidson 1973; Taliansky et al 2003). A valuable source of resistance in Solanum tuberosum Andigena Group was recently found with a major QTL on chromosome V conferring resistance to PLRV and is introgressed into advanced breeding lines by marker-assisted selection (Mihovilovich et al 2014; Velasquez et al 2007)

Methods

Results

Conclusion