A Novel Approach to Obtain Resistance Genes. Resistance is termed durable if it continues to be effective over multiple years of widespread use, but it sometimes appears limited value because invaders may evolve quickly to overcome this resistance. However, it is very time-consuming (even only the analysis step may take several months) use recent mapping based technology to discovery new R genes. Hence, it is importance to find a new method to cut the time of identifying advanced R genes There are three approaches available to isolate or generate new candidate resistance genes as shown below:
1. Map-based cloning or positional cloning : A technology make use of phenotypic databases to isolate novel R genes. Generally speaking, it is the basic method to rescue candidate gene. First of all, the marker linked to the target gene firmly is identified. Then, a typical yeast artificial chromosome or bacterial artificial chromosome clone is found to hybridize the marker probe. Third, new markers are created and used to segregate the target gene. Once find the location of the gene, genetic complementation is performed to get the candidate and then functional analysis would be performed afterwards.
2. Effector genomics: Combination of effectors and assays with the phenotypic data of R genes accelerates engineering of functional R genes (Vleeshouwers et al., 2008). As detailed before, LRR motifs are involved in protein-protein interactions, so RXLR effectors of P. Infestans can be used to identify R genes. The expression of RXLR effectors result in a variety of effector responses in which R-AVR interactions may be found.
3. Allele mining strategy: Alleles using or the combinations of alleles can make significant changes in expression. And the variations in coding sequences or exons, even in non-coding regions (introns, promoter included) have dramatic effect on protein structure and function. This is the most efficient method to identify strong resistance genes.
Nowadays, it is widely recognized that resistance genes to late blight potentially overcome by P. Infestans. Combination of multi-genes, up to four, have been tried. Although they revealed much stronger resistance than single gene, Phytophthora infestans can bypass the stucking genes. Hence, researchers continuously working on the discovery of novel R genes. Under this circumstances, a previously uncharacterized strong R gene to late blight, Rpi-vnt1.1, was identified by Pel et al. ( 2009) from Solanum venturii.
This gene, discoveried by the classical map-based cloning (positional cloning) approach (Pel et al., 2009), was found belongs to the CC-NBS-LRR class (CC domain is responsible for dewnstream signaling involved in protein-protein interactions). Rpi-vnt1.1 is highly similar to Tm-2. Tm-2 is a class of R genes in tomato confer resistance to tomato mosaic virus (ToMV). It includes three alleles, resistance genes Tm-2~2, Tm-2 and susceptible gene Tm-2 (F. Ciccarese, 1980). The expressing of the two resistance genes brought necrotic spot and system necrosis symptoms in resistance response. (Cloning§) Rpi-vnt1.1 shows fine resistance in either transgenic tomato or potato to P. Infestans.
Rpi-vnt1.1 was found in the Centre for Genetics Resources (CGN) in Netherlands. After cross of the five selected resistant varieties, Rpi-vnt1.1 and its two other alleles, Rpi-vnt1.2, Rpi-vnt1.3, were identified. Bulked segregant analysis (BSA) (Michelmore et al., 1991) was used to generate candidate gene markers.
Bulked segregant analysis was first developed by Michelmore et al. (1991) to identify specific markers rapidly. Two bulked DNA samples are made from extreme phenotypes from a single cross. Each bulk contains individuals that are identical for a particular trait or genomic region but arbitrary at all unlinked regions (Monika Singh, 2006). The two bulks are screened for differences using restriction fragement length polymorphism probs or random amplified polymorphic DNA primers. Onlly differing in the selected region, the two bulks are able to harbour that trait to divide the alleles into two groups. Bacterial artificial chromosome clone was used to hybridize the marker probe and constructure a contig to make a allele-specific marker (named vntNBSHae in the paper). After amplified using PCR, Rpi-vnt1.1 and its alleles were analyzed by southern blotting and high-resolution mapping. The identity between Rpi-vnt1.1 and Rpi-vnt1.2 with Tm-22 is 72.1% and 71.1% respectively at the amino acid level. At the nucleic acid level this amount turned to 80.9% and 79.7% respectively. While the similarity in the conserved CC and NB-ARC domains are both higher than 80% indicate Rpi-vnt1.1 and Tm-22 are identical. Despite all of these, the similar of three genes in LRR domain differ greatly. At last, the complementation analysis declared strong resistance of Rpi-vnt1.1. All the leaves detached from 29 plants inoculated with a range of Phytophthora infestans isolates exhibited capability of survive the infection. Despite of some showed signs of a hypersensitive response.
As detailed before, modernday agriculture need mass of novel R genes to deal with late blight. However, functional analysis of target resistance gene homologs is quite time-consuming. In general, it takes months to cultivate stable transformation of a susceptible genotype makes this method so inefficient. According to Pel et al. (2009), they found a complementation assay developed in Nicotiana benthamiana which is susceptible to Phytophthora infestans is capable of performing this analysis. The functional analysis using this assay would be revolutionary (Becktell et al., 2006). In addition, before the analysis step, the authors abandon the conventional method, position cloning, to identify novel genes, employing allele mining instead which also is a time- and energy- saving approach.
This approach is capable of generating many candidate based on the size of the gene family. Certain alleles and their combinations potentially make dramatic changes in trait expression when moved to a suitable genetic background by overcoming the genetic bottlenecks which restricted their introgression to cultivars. Normally, promoters are chosen as the targets for allele mining because of its importance in sequencing and transcription. Silico analysis performed after promoter mining aimed to identify transcription factors under various regulation. However, if the target located in the promoter region, it can hardly be called a true allele mining. Because the amplicon would lack specific and meaningful functions, and may share the conserved motifs with stretch sequences. Thus, to obtain all the information amplicon needed, it is requested the primers to be located between the terminators and the promoters.
There are two allele mining approaches available currently which are Eco-Tilling and sequencing based allele mining (Kumar et al., 2010). The method using Targeting Induced Local Lesions in Genomes (TILLING) to identify natural variation in individuals called Eco-TILLING. TILLING is a technique that can identify point mutations in a specific target gene, nevertheless, the lesions are not induced manually. Indeed, they are naturally existing alleles.
