Isolation and characterisation of the Rvi5 (Vm) apple scab resistance gene in Malus ×domestica

Apple scab, caused by the fungal pathogen Venturia inaequalis (Cke.) Wint.is the major disease affecting apples grown in temperate climates around the world. The disease can cause a significant economic impact as diseased fruit are not marketable. Complete crop loss is possible if appropriate control measures are not taken. Control of the disease is achieved through good cultural practices, such as leaf litter reduction to prevent primary inoculum production, but prevention is primarily achieved through frequent applications of fungicides. Whilst the application of chemical fungicides is effective at controlling the spread of the disease, they are not environmentally friendly, and V. inaequalis has successfully developed resistance against some fungicides. Thus, there is a need for more sustainable methods to control the disease, and as a result, research into identifying and exploiting natural resistance to scab from Malus germplasm is well established. Breeding for apple scab resistance primarily involves the introgression of resistance genes from the varieties of cultivated and wild apple species, and to date, a total of 17 scab resistance loci have been characterised. The apple scab resistance gene Rvi6 (Vf) derived from the Asiatic wild apple accession Malus floribunda 821 is the most studied and mostly utilized scab resistance gene in resistance breeding programs. However, identification of V. inaequalis isolates that can overcome natural Rvi6-based resistance demonstrates the importance of the use of various sources of resistance in apple varietal development and highlights the need to develop durable resistance through pyramiding of resistant genes from various sources. The apple scab resistance gene Rvi5, mapped on the distal end of LG 17 believed to be inherited from Malus micromalus 245-38 and Malus atrosanguinea 804, described as a pit type gene induces hypersensitive response for the pathogen of apple scab after 3-5 days of inoculation. V.inaequalis race 5 has overcome the gene, which is first discovered in England. However, recent surveys conducted under the frame of VINQUEST initiative, suggested that this virulence is not widely widespread. The well-studied apple scab resistance gene Rvi15 also induces the hypersensitive response for the pathogen, but Rvi15 elicits slow response compared to the Rvi5, which requires 15 days to elicit the resistance symptoms after the inoculation. The time differences required to elicit the symptoms in Rvi15 and Rvi5 may be due to the different genes responsible for the resistance reaction. The limited spread of V.inaequalis race 5 and the differences of time required to elicit the resistance reaction with respect to Rvi15, makes this gene interesting to study aiming resistance gene pyramiding. The Rvi5 gene was found to segregate for a single dominant locus and a co-segregating molecular marker linked with the resistance has been reported. For the purpose of isolation and characterisation of the Rvi5 (Vm) apple scab resistance gene, we first fine mapped the region surrounding the Rvi5 locus using 1243 progeny plants of ‘Golden Delicious’ × ‘Murray’ and ‘Galaxy’ × ‘Murray’, which permitted to delimits the locus controlling the resistance to a 1 cM in ‘Murray’ genome and developed three co-segregating markers for the resistance gene (Vm_ SCAR1, Vm_SNP5, FMACH_Vm3). The locus controlling the resistance was flanked by newly designed two molecular markers, SSR FMACH_Vm2 and SSR FMACH_Vm4. The Bacterial Artificial chromosome (BAC) library of ‘Murray’ was first screened for the resistance allele of SSR FMACH_Vm2 and continued by chromosome walking until found a positive clone for the SSR FMACH_Vm4 to isolate the Rvi5 resistance locus. Three bacterial artificial chromosome (BAC) clones spanning the resistance locus were identified, completely sequenced and assembled, which allowed identifying the putative Rvi5 locus in a region of 154kb in size. The open reading frame prediction and functional annotation of the identified region revealed the presence of one putative gene homologous to TMV resistance protein of Malus × domestica, characterized by a Toll and mammalian interleukin-1 receptor protein nucleotide-binding site leucine-rich repeat structure. The identified candidate gene has been transformed to the scab susceptible cultivar ‘Gala’ with the help of gateway directional cloning technology and Agrobacterium mediated transformation. Plant DNA extracted from obtained transgenic plants was used to confirm the insertion of the gene of interest. Five confirmed transgenic ‘Gala’ plants with inserted Rvi5 candidate gene were obtained as first transgenic plants inserted with Rvi5 gene. Before conducting the scab inoculation experiments, transgenic plants are needed in order to undertake multiplication and regeneration to obtain sufficient plant materials for inoculation experiments. Once a sufficient amount of plant material has been obtained from transgenic plants, the shoots will be micro-grafted onto ‘Golden Delicious’ rootstocks. When the micro-grafted shoots reach the ten leaf stage, scab inoculation experiments will be continued with monoconidial V. inaequalis isolates, with differential interaction with Rvi

Fine mapping and isolation of Rvi5 (Vm) scab resistance locus in apple (Malus x domestica Borkh.)

TheRvi5(Vm) apple scab resistance gene has been previously mapped on the distal end of Linkage group 17 by analysing 95 plants of a cross population between ‘Golden Delicious’ and ‘Murray’. For the purpose of fine mapping and isolation of putative Rvi5 locus, two new populations were developed from crosses between ‘Golden Delicious’ × ‘Murray’ and ‘Galaxy’ × ‘Murray’. A total of 1243 plants that were obtained from above crosses were screened by using Simple Sequence Repeat (SSR) markers. SSR markers Hi07h02 and CH05d08 previously reported as co-segregating and not co-segregating with Rvi5(Vm) gene were used as starting and end point of ‘Murray’ BAC library screening. Nine new SSR markers were designed for the region in between SSR Hi07h02 and CH05d08 using the ‘Golden Delicious’ genome sequence. Among the designed new SSR markers only four were polymorphic for the parents and Polymorphic markers (SSR FMACH_Vm1 to SSR FMACH_Vm4) were used to identify the positive BAC clones. With this strategy five BAC clones were identified, BAC extremities were sequenced using universal M13 forward and reverse primers to check the complete coverage of region and orientations of BAC clones. Using bio-informatics tools it has been confirmed the complete coverage of region of interest by spanning about 545kbps region with respect to ‘Golden Delicious’ genome sequence, then the BACs were sequenced by developing paired end libraries and assembled. By analyzing recombinant plants, we were able to recognize the Rvi5 (Vm) locus in between SSR FMACH_Vm2 and FMACH_Vm4 in a region of 228 kbps in size. In addition, our study revealed that Rvi5 locus is more towards the SSR CH05d08 and newly developed marker, SSR FMACH_Vm3 is co-segregating with the locu

Isolation of RVI5 (VM) Locus from Malus × domestica 'Murray'

Scab resistance gene Rvi5 (Vm) is derived from Malus micromalus or Malus atrosanguinea 804 was introgressed into the apple variety ‘Murray’. This gene has the ability to induce a clear hypersensitive response after three days from Venturia inaqualis infection. Being ‘Murray’, a cultivar with an acceptable fruit quality compared to the old ancestors, this genotype could be used in breeding to pursue a durable resistance against Venturia inaequalis. This goal could be accomplished by two different approaches: resistance gene pyramiding by classical breeding or by a cisgenic approach, once the resistance gene will be identified. The isolation of the locus that harbors the resistance is an essential step toward the identification of the candidate genes. A pooling strategy enabled the identification of three overlapping Bacterial Artificial Chromosome (BAC) clones from a Murray library that are covering the Rvi5 region. Then, thanks to the availability of the Malus × domestica ‘Golden Delicious’ genome sequence, we were able to position the Rvi5 locus on the ‘Golden’ genome sequence in a region estimated of about 228 kbps in size. These three clones will be sequenced and assembled to obtain the complete sequence of Rvi5 locus. The assembled sequences will then be used for candidate gene identificatio