Cisgenic apple trees; development, characterization, and performance.

Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.

Virulence Characterization of Venturia inaequalis Reference Isolates on the Differential Set of Malus Hosts.

A set of differential hosts has recently been identified for 17 apple scab resistance genes in an updated system for defining gene-for-gene (GfG) relationships in the Venturia inaequalis-Malus pathosystem. However, a set of reference isolates characterized for their complementary avirulence alleles is not yet available. In this paper, we report on improving the set of differential hosts for h(7) and propose the apple genotype LPG3-29 as carrying the single major resistance gene Rvi7. We characterized a reference set of 23 V. inaequalis isolates on 14 differential apple hosts carrying major resistance genes under controlled conditions. We identified isolates that were virulent on at least one of the following defined resistance gene hosts: h(1), h(2), h(3), h(4), h(5), h(6), h(7), h(8), h(9), h(10), and h(13). Sixteen different virulence patterns were observed. In general, the isolates carried one to three virulences, but some of them were more complex, with up to six virulences. This set of well-characterized isolates will be helpful for the identification of additional apple scab resistance genes in apple germplasm and the characterization of new GfG relationships to help improve our understanding of the host-pathogen interactions in the V. inaequalis-Malus pathosystem.

Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples.

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (P(MdRbc)) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar 'Gala' was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. 'Santana'.

Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines.

Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. 'Elstar' and 'Gala' in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable.

Genetic mapping of 14 avirulence genes in an EU-B04 × 1639 progeny of Venturia inaequalis.

Durable resistance to apple scab (Venturia inaequalis (Cke) Wint; anamorph Spilocaea pomi Fries) is one of the major goals of apple (Malus) breeding programs. Since current scab resistance breeding is heavily reliant on genes with gene-for-gene relationships, a good understanding of the genetic basis of host-pathogen interactions needs to be developed for this strategy to be successful. While the genomic organization of apple scab resistance genes has been studied extensively, little is known about the avirulence genes in the pathogen. The progeny of a cross of European V. inaequalis race (1) isolate EU-B04 and race (1,2,8,9) isolate 1639 was used to generate a genetic map based on microsatellite and AFLP markers, and investigated for inheritance of avirulence traits on 20 Malus accessions representing 17 scab resistance genes. The accessions comprised scab differential hosts (0), (1), (2), (8), and (9), and hosts carrying known as well as not previously reported secondary resistance genes, including some identified in crosses that have resistant accessions 'Geneva', 'Dolgo', Malus baccata jackii, M. micromalus, or 'Antonovka' in their pedigree. The latter genes appear to be narrow spectrum genes that showed gene-for-gene relationships as a segregation ratio of Avr:avr=1:1 was observed on 12 accessions, while a ratio of 3:1 was observed on five accessions and a ratio of 7:1 on one host. All progenies were shown to be pathogenic, as all of them were able to infect hosts (0) and (1). A genetic map consisting of 15 major linkage groups (LGs) and spanning 972cM was generated with the aid of 156 markers. The map position of 12 avirulence traits was determined: eight avirulence genes mapped into two separate clusters (1: AvrVdg2, AvrVv1, AvrVu1, AvrVrjrd; and 2: AvrVu2, AvrVh3.2, AvrVs1, AvrVu4), while four avirulence genes (AvrRvi8, AvrVv2, AvrVt57 and AvrVsv) mapped to different LGs. AvrRvi2 and AvrRvi9 also are genetically linked, but showed an interaction with AvrRvi8, the nature of which is unclear. While AvrRvi8 segregated at 1:1 ratio, the other two Avrs segregated at 3:1 ratios. However, all progeny avirulent on hosts (2) and (9) were also avirulent on host (8) and further research is required to determine the avirulence gene relationships. A further two independently segregating loci, AvrRvi1 and AvrRvi6, identified in previous studies, were mapped by inference based on their known linkage to SSR markers. The clustering of avirulence genes in V. inaequalis reflecting the clustering of resistance genes in Malus suggests this pathosystem is a classical example of an "arms race" between host and pathogen. This also seems to apply to the narrow spectrum scab resistance genes, which may imply a larger role in plant defense for these genes than has been assumed to date.

QTL mapping of anthracnose ( Colletotrichum spp.) resistance in a cross between Capsicum annuum and C. chinense.

Anthracnose fruit rot is an economically important disease that affects pepper production in Indonesia. Strong resistance to two causal pathogens, Colletotrichum gloeosporioides and C. capsici, was found in an accession of Capsicum chinense. The inheritance of this resistance was studied in an F(2) population derived from a cross of this accession with an Indonesian hot pepper variety ( Capsicum annuum) using a quantitative trait locus (QTL) mapping approach. In laboratory tests where ripe fruits were artificially inoculated with either C. gloeosporioides or C. capsici, three resistance-related traits were scored: the infection frequency, the true lesion diameter (averaged over all lesions that actually developed), and the overall lesion diameter (averaged over all inoculation points, including those that did not develop lesions). One main QTL was identified with highly significant and large effects on all three traits after inoculation with C. gloeosporioides and on true lesion diameter after inoculation with C. capsici. Three other QTL with smaller effects were found for overall lesion diameter and true lesion diameter after inoculation with C. gloeosporioides, two of which also had an effect on infection frequency. Interestingly, the resistant parent carried a susceptible allele for a QTL for all three traits that was closely linked to the main QTL. The results with C. capsici were based on less observations and therefore less informative. Although the main QTL was shown to have an effect on true lesion diameter after inoculation with C. capsici, no significant QTL were identified for overall lesion diameter or infection frequency.