1. INTRODUCTION
The most serious disease of potatoes (Solanum tuberosum L.) is late blight (LB) disease, which is caused by Phytophthora infestans. It can entirely devastate a potato field [1]. LB is managed by foliar spraying using fungicides, which causes pollution of the environment. Furthermore, the appearance of novel P. infestans populations has led to novel strategies for the pathogen control, because the novel races will display increased virulence and resistance to fungicides. Therefore, traditional breeding programs for potatoes focused on using potato cultivars resistant to LB [2]. Conventional breeding programs take about 12 years, starting from the crossing to obtaining a novel potato variety. Besides, the selection process (involving many agronomic and quality characteristics) is laborious and needs time, so breeders must search for novel sources of LB resistance by DNA molecular markers linked to the resistance loci. This will save time, and will also help to better understand the host and pathogen interactions [3,4]. Up to now, quantitative trait loci (QTL) for LB disease resistance have been identified on S. tuberosum L. chromosome, which have clusters of resistance R loci, so R genes are candidates for the QTL effect [5,6]. The “candidate gene study” allows the characterization of molecular markers which can be used in potato breeding programs as marker-aided selection (MAS) [7]. Many investigations were carried out on potato genotypes resistant to P. infestans. Furthermore, multiple R loci were genotyped and identified in the potato [8]. To improve traditional methods in potato breeding programs, DNA markers closely linked to LB resistance loci may be simple to use in diploid or tetraploid potatoes. Tagging of resistance genes with DNA marker technology displays a possibility for MAS to screen disease resistance trait in the seedling stage and choose the best plants to be characterized for backcrossing. Moreover, recessive genes can be screened, which is difficult to do using traditional breeding methods [9].
The majority of the DNA markers applied formerly in mapping were either cleaved amplified polymorphic sequence (CAPS) or sequence-characterized amplified regions (SCARs) are simple to apply [10]. SCAR marker depends on variations in the primer sites, resulting in the presence or absence of an amplicon, while CAPS assays are based on polymorphism in the restriction site after polymerase chain reaction (PCR) amplification. Genetic markers representing resistance allele fragments are most suitable for testing big genetic populations during breeding. The disadvantage of DNA markers is that they cannot differentiate between functionally active genes and inactive structural homologs [11]. Furthermore, with the recent progress in molecular biology of S. tuberosum L., the ease of use of DNA marker technology linked to resistance alleles in potato germplasm screening was reported. To make MAS an integrated part of traditional breeding, it will be important for breeders to identify the possibility molecular markers. Wherefore, to promote breeders, updates on advancements in DNA marker development for P. infestans resistance loci/QTLs. Genetic markers are expected to play an important role in MAS due to low costs, ease of use, linkage to target gene, and the proportion of phenotypic variance illustrated by molecular markers [10,12].
Genetic resistance to LB disease is acquisition its importance [13] due to several agents, involving the increasing demands to generate potato yields without applying fungicides [14], and alterations to legislation related to the application of chemicals [15]. Breeding of potato varieties/cultivars resistant to LB disease depends on the 11 dominant R loci (R1 to R11), which are produced by species Solanum demissum and DNA markers linked to those dominant alleles were identified through MAS [16,17].
The purpose of the study was to use DNA markers to discriminate between resistant and susceptible potato genotypes to LB. Moreover, we will correlate phenotype data obtained from the detached leaf assay with genotypic results to confirm the PCR-based screening results.
2. MATERIALS AND METHODS
2.1. Plant Materials
Eleven potato varieties Annabelle, Bellini, Cara, Deta, Diamond, Herms, Jelly, Lady Rosetta, Metro, Mondial, and Spunta were collected from the brown rot project, Dokki, Giza, Egypt), as well as six wild potato species Solanum acule CGN 17674, Solanum chacoense CGN 17903, S. demissum CGN 17788, S. demissum CGN 17797, Solanum stoloniferum CGN 17605, and S. tuberosum CGN 17609 (supported by Centre for Genetic Resources, Netherland (http://www.wur.nl).
2.2. Source of P. infestans Isolate
The P. infestans isolate was obtained from the Plant Pathology Department, Agriculture Faculty, Ain Shams University [18].
2.3. LB Leaflet Tests
Five newly expanded leaflets of each potato genotype (grown in a greenhouse) were detached and used for the experiment (equivalent to five replications per genotype). Prior to inoculation, leaflets were washed and placed abaxial surface–up on filter paper in five Petri dishes (one leaflet/Petri plate). One drop (50 μl) of inoculum (3 × 103 sporangia ml–1) of P. infestans was inoculated onto each leaflet using a micropipette. The Petri dishes were sealed with Parafilm to prevent desiccation. Then, inoculated leaflets were incubated at 16 ± 0.5°C with 12 h light cycle for the appearance of symptoms. On the 7th day after inoculation, the percent disease severity [%DS] was recorded after the disease prevailing using 1-9 Henfling scale [19].
2.4. Primer Design and Selection
In this research, nine resistance genes to LB named R1, R3a, R3b, R9a, R8 & R9a, Rpi-phu1, Rpi-ber, Rpi-blb1, and Rpi-blb3 were used depending on prior publications. A total of 15 gene-specific primer pairs were screened, depending on the DNA nucleotide sequences of the nine candidate genes; nine primers were based on publications, and six primers were designed in our laboratory using the Primer3 online tool (http://biotools.umassmed.edu/bioapps/primer3_www.cgi). The primer pairs used to amplify the nine resistance R loci are shown in Table 1 [21-28].
Table 1: Sequence of primers used in this study.
| Specific primers/marker type | Resistance gene name | Chromosome No. | Annealing temperature (AT) °C | Primer name | Productsize (bp) | Forward (3?–5?) | Reverse (5?–3?) | Solanum species | Ref. and accessions used for primers in the study |
|---|---|---|---|---|---|---|---|---|---|
| Specific primers | R3a | XI | 55 | R3a1 | 194 | Tgctcggtcttcagattgtg | Ttgctggttgcttgtttctg | Solanum tuberosum | Accession (AY849382) (present study) |
| R3a | XI | 55 | R3a2 | 247 | Gccagcactaggacaactcc | Gggtaccgtctccaaactga | Solanum tuberosum | Accession (AY849382) (present study) | |
| R3b | XI | 55 | R3b1 | 226 | Ggagttgccgaatgacttgt | Tagcggcgtcttcaagaaat | Solanum demissum | Accession (JF900492) (present study) | |
| R3b | XI | 55 | R3b2 | 244 | Gccgaaaagagtggcattta | Aaaggaagcccttcttccag | Solanum demissum | Accession (JF900492) (present study) | |
| R8 | IX | 55 | R8a | 220 | Gtgggatctcctcaaggtca | Tccttcattgcggaactacc | Solanum demissum | Accession (KU530153) (present study) | |
| R8 | IX | 55 | R8b | 237 | Acccttttccaggaacatca | Ctcacaatgaatgccgaatg | Solanum demissum | Accession (KU530153) (present study) | |
| SCARs | R1 | V | 58 | R1-517 | 517 | Gaatggaaagtgagcaatggcaagt | Gtagtacctatcttatttctgcaagaat | Solanum demissum | [20] |
| R1 | V | 60 | 76-2s | 1400 | Cactcgtgacatatcctcacta | Caaccctggcatgccacg | Solanum tuberosum | [21] | |
| Rpi-phu1 | IX | 55 | phu6 | 298 | Agagaccctggatatatttcatagctct | Cgctctaggcacagggctcaatgctgat | Solanum phureja | [22] | |
| Rpi-ber | X | 50 | Rpi-ber1-Q133 | 395 | Atcatctcctcaaagaatcaag | Atctccccattgacaaccaa | Solanum berthaultii | [23] | |
| Rpi-blb1 | VIII | 58 | BLB1 | 821 | Aacctgtatggcagtggcatg | Gtcagaaaagggcactcgtg | Solanum stoloniferum | [24] | |
| Rpi-blb3 | IV | 50 | RGH2 | 320 | Ggsaagaccactcttgcaag | Tggtyataatyactctgctgc | Solanum bulbocastanum | [25] | |
| R9a | IX | 54 | EDN61 | 450 | Gcatcatgtctgcacctatg | Ctttgatgtggatggatggtg | Solanum edinense | [26] | |
| CAPS | R8&R9a | IX | 58 | 184-81F | 680 | Ccaccgtatgctccgccgtc | Gttccacttagccttgtcttgctca | Solanum demissum | [27] |
| R3a | XI | 54 | TG105 | 650 | Tcacatgagctgggagaaat | Aaaggcctgttgctgagag | Solanum tuberosum | [28] |
CAPS: Cleaved amplified polymorphic sequence, SCARs: Sequence-characterized amplified regions
2.5. DNA Extraction and PCR Amplification
Total genomic DNA was extracted from fresh potato leaves of 17 potato genotypes, using the DNeasy plant mini-prep kit (Qiagen, CA). PCR amplification was performed in a thermal cycler (Biometra, biomedizinische Analytik GmbH) in a total volume of 25 μl containing 50 ng DNA, 10 μM of each primer, 200 mM dNTPs, 1.5 mM MgCl2, and 0.5 U Taq DNA polymerase (GoTaq® DNA Polymerase, Promega, USA). PCR was performed under the following conditions: 94°C at 4 min and then 35 cycles of 94°C at 1 min, 50–60°C at 1 min, and 72°C at 1 min, and a final extension step at 72°C for 5 min.
All the PCR products were electrophoresed on 1% agarose gel electrophoresis in 1× TBE buffer. The genomic DNA was stained with RedSafe Nucleic Acid Staining Solution (1/20,000) (iNtRON Biotechnology, Inc. Kr) and was visualized and photographed with Gel-Documentation system (Bio-Rad Laboratories, Inc., Cali, USA). The size of each fragment was estimated with reference to a size marker of the 100 bp DNA ladder (BioRoN, Germany).
2.6. Statistical Analysis
Correlation analysis was performed using Microsoft Excel 2010 with an evaluation of Pearson’s correlation coefficients.
3. RESULTS
3.1. Infection Assay in a Detached Leaf of Potato
To identify potato genotypes resistant and susceptible to P. infestans, 17 potato genotypes were tested against LB by detached leaf assay [Table 2 and Figure 1]. Some of the inoculated potato leaves appeared to have a few lesions. They were recorded as highly resistant, such as S. chacoense CGN 17903 and Jelly, when the leaves showed necrosis without sporulation, they were identified as resistant, for example, S. acule CGN 17674, while the leaves did not show the same reaction or sporulation were not clear. They were classified as either moderately resistant, that is, Cara, Diamond, Lady Rosetta, Metro, Mondial, Spunta, Deta, Herms, S. demissum CGN 17788, S. demissum CGN 17797, S. stoloniferum CGN 17605, and S. tuberosum CGN 17609, or moderately susceptible, such as Annabelle and Bellini [Table 2].
Table 2: SCAR and CAPS markers of P. infestans resistance R genes in 17 potato genotypes.
| No. | Potato genotype | Phytophthora infestans | Phytophthora infestans resistance genes and DNA markers | The total number of R gene markers | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| R1 | R1 | Rpi-phu1 | Rpi-ber | Rpi-blb1 | Rpi-blb3 | R9a | R8 & R9a | R3a | |||||||
| Scale value | Pheno-type | SCAR* | CAPS** | ||||||||||||
| R1-517 | R1-76-2s | phu6 | Rpi-ber1-Q133 | BLB1 | RG H2 | Edn61 | 184-81 | TG105 | |||||||
| 1 | Jelly | 1 | HRa | + | - | + | + | + | + | + | - | HoSe | + | HeRg | 7 |
| 2 | Lady Rosetta | 3 | MRb | + | - | + | + | - | + | - | + | HeR | - | HoS | 4 |
| 3 | Mondial | 3 | MR | + | - | + | - | + | + | + | - | HoS | + | HeR | 6 |
| 4 | Diamond | 4 | MR | + | - | + | + | - | + | - | - | HoS | + | HeR | 5 |
| 5 | Metro | 3 | MR | + | - | + | - | + | + | + | - | HoS | - | HoS | 5 |
| 6 | Spunta | 4 | MR | + | + | + | - | - | + | + | - | HoS | + | HeR | 6 |
| 7 | Cara | 3 | MR | + | - | + | + | - | + | - | - | HoS | + | HeR | 5 |
| 8 | Deta | 3 | MR | - | - | + | - | - | + | - | - | HoS | + | HeR | 3 |
| 9 | Herms | 3 | MR | + | + | + | - | - | + | - | - | HoS | + | HeR | 5 |
| 10 | Bellini | 5 | MSc | + | - | + | - | - | + | + | - | HoS | + | HeR | 5 |
| 11 | Annabelle | 5 | MS | - | - | + | + | + | + | - | - | HoS | - | HoS | 4 |
| 12 | Solanum tuberosum CGN 17609 | 3 | MR | - | - | - | + | + | + | - | - | HoS | + | HeR | 4 |
| 13 | Solanum acule CGN 17674 | 2 | Rd | - | - | + | + | - | + | - | + | HoRf | - | HoS | 4 |
| 14 | Solanum chacoense CGN 17903 | 1 | HR | + | - | + | + | - | + | - | + | HoR | - | HoS | 5 |
| 15 | Solanum stoloniferum CGN 17605 | 3 | MR | + | + | + | + | + | + | + | + | HoR | - | HoS | 8 |
| 16 | Solanum demissum CGN 17788 | 3 | MR | + | + | + | + | - | + | + | + | HoR | - | HoS | 7 |
| 17 | Solanum demissum CGN 17797 | 3 | MR | - | - | + | + | - | + | + | + | HoR | - | HoS | 5 |
| A total number of bands | 12 | 4 | 16 | 11 | 6 | 17 | 8 | 6 | 9 | ||||||
SCAR
*: Sequence characterized amplified region, CAPS
**: Cleaved amplified polymorphic sequence, HR
a: Highly resistant, MRb: Moderately resistant, MSc: Moderately susceptible, Rd: Resistant, HoSe: Homozygous susceptible, HoRf: Homozygous resistant, HeRg: Heterozygous resistant, +: Presence of band, -: Absence of band
 | Figure 1: Assessment of detached leaf assay seven days after inoculation with Phytophthora infestans sporangia. Jelly cultivar a resistant phenotype that possessed genes (R1, Rpi-phu1, Rpi-ber, Rpi-blb1, Rpi-blb3, R9a, and R3a); Lady Rosetta showing moderate resistant, which possessed loci (R1, Rpi-phu1, Rpi-ber, Rpi-blb3, and R8 and R9a); Bellini showing moderately susceptible, which possess (R1, Rpi-phu1, Rpi-blb3, R9a, and R3a), and cultivar Annabelle appeared moderately susceptible, which have genes (Rpi-phu1, Rpi-ber, Rpi-blb1, and Rpi-blb3). C: Control, I: Infected. [Click here to view] |
3.2. Detection of Resistance Genes R3a, R3b, and R8 in Potato Genotypes
Six specific primer pairs were designed to detect three R genes, R3a, R3b, and R8 in 17 potato genotypes as are shown in Table 1. PCR products of R3a, R3b, and R8 resistance genes scored one specific band of (194 and 247), (226 and 244), and (220 and 237), respectively, in all the tested potato genotypes [Figure 2]. These primers have not recorded any polymorphic variations between resistant and susceptible potato genotypes. Therefore, seven SCAR and two CAPS markers were used to discriminate between resistant and susceptible potato genotypes.
 | Figure 2: Polymerase chain reaction products of R3a, R3b, and R8 genes using six designed primers in 17 potato genotypes. Marker: 100 bp DNA ladder. [Click here to view] |
3.3. Identification of R Genes by PCR Markers
Seven SCAR and two CAPS markers were screened to determine whether the eight candidate resistance R genes, R1, Rpi-phu1, Rpi-ber, Rpi-blb1, Rpi-blb3, R9a, R8 and R9a, and R3a were present or absent in the resistant and susceptible potato genotypes to LB disease [Table 2]. PCR amplicons for the R1 gene using primer SCAR R1-517 amplified a fragment of 517 bp in 12 out of the 17 potato genotypes [Figure 3 and Table 2]. Furthermore, PCR results for the R1 gene using SCAR 76-2s scored one band of 1400 bp in 4 out of 17 genotypes [Figure 3 and Table 2]. On the other hand, the Rpi-phu1 gene amplified using the primer pairs SCAR phu6 gave one fragment of 298 bp in 16 out of 17 potato genotypes [Figure 3 and Table 2]. Furthermore, the Rpi-ber gene amplified by SCAR Rpi-ber1-Q133 recorded a single amplified fragment of 395 bp in a total of 11 from 17 genotypes [Figure 3 and Table 2]. Moreover, the Rpi-blb1 gene amplified by primer combination SCAR BLB1 gave one amplicon with the expected size of 821 bp in 6 of 17 potato genotypes [Figure 3 and Table 2]. Besides, PCR results of R9a using the primer pair EDN61 yielded an amplicon of 450 bp in 8 of the 17 genotypes of S. tuberosum. The presence or absence of the Rpi-blb3 gene was analyzed using the primer set SCAR RGH2, which gave one amplified fragment of 320 bp in all tested potato genotypes. Therefore, this primer has not displayed any polymorphisms between the resistant and susceptible potato genotypes [Figure 3 and Table 2].
 | Figure 3: Polymerase chain reaction products of R1, Rpi-phu1, Rpi-ber, Rpi-blb1, and Rpi-blb3 genes using different sequence-characterized amplified region primers amplified from 17 potato genotypes. Marker: 100 bp DNA ladder. [Click here to view] |
Amplification of the R8 and R9a gene combination, using primer CAPS 184-81 gave one band of 680 bp in all studied potato genotypes [Figure 4 and Table 2]. An amplicon of 680 bp was subjected to digestion by the restriction enzyme RsaI, to determine which potato genotypes are susceptible and resistant-homozygous or heterozygous for R8 and R9a resistance genes. Restriction fragment, 480 bp to homozygous resistance were detected in wild species S. acule CGN 17674, S. chacoense CGN 17903, S. stoloniferum CGN 17605, S. demissum CGN 17788, and S. demissum CGN 17797 [Figure 4 and Table 2]. Three bands of 280, 400, and 480 bp to heterozygous resistance were found in the variety Lady Rosetta. Other potato genotypes produced restriction fragments at 280 and 400 bp, indicating susceptibility to P. infestans.
 | Figure 4: Detection of resistance genes R8 and R9a combination and R3a using cleaved amplified polymorphic sequence (CAPS) primer 184-81 and CAPS primer TG105, respectively. (a) CAPS marker using primer pair 184-81 before and after cut with RsaI, (b) cleaved amplified polymorphic sequence marker using primer set TG105 before and after cut with HinfI. [Click here to view] |
On the other hand, resistance gene R3a was amplified using primer CAPSTG105, which gave one band of 650 bp in all tested potato genotypes [Figure 4 and Table 2]. This band was digested using the Hinf1 restriction enzyme, which gave more polymorphic fragments. Eight genotypes scored two amplicons at 150 and 500 bp only to indicate homozygous susceptible to the LB, such as Lady Rosetta, Metro, Annabelle, S. acule CGN 17674, S. chacoense CGN 17903, S. stoloniferum CGN 17605, S. demissum CGN 17788, and S. demissum CGN 17797 [Figure 4 and Table 2]. However, the remaining genotypes produced three bands of 150, 350, and 500 bp for heterozygous resistance [Figure 4 and Table 2], while the amplicon of 350 bp, which detects homozygous resistance. There are no genotypes for this trait [Figure 4 and Table 2].
3.4. The Relation between the Number of Markers and LB Resistance
The results of PCR amplification for all eight genes in potato genotypes resistant and susceptible, using the nine markers, are summarized in Table 2 and Figure 5. The results Spearman’s correlation coefficient showed no correlation between the number of R gene markers and levels of resistance (r = –0.186 ns). For example, the number of R gene markers in the highly resistant varieties ranged from 5 to 7 markers, for example, Jelly and S. chacoense CGN 17903, followed by resistant varieties like S. acule CGN 17674 (4), and moderately resistant (3-8) such as, Diamond, Lady Rosetta, Metro, Mondial, Spunta, Cara, Deta, Herms, S. stoloniferum CGN 17605, S. tuberosum CGN 17609, S. demissum CGN 17788, and S. demissum CGN 17797. Finally, the moderately susceptible potato varieties have from 4 to 5 markers, for example, Annabelle and Bellini [Table 2 and Figure 5]. Depending on the PCR results, 17 potato genotypes were classified into eight groups. The first group: composed of 17 potato genotypes have Rpi-blb3. The second group: contained 16 genotypes including only the RPi-phu1 gene. The third group: consisted of 12 genotypes involved R1 (using marker SCAR R1-517). The fourth group: composed of 11 genotypes have Rpi-ber. The fifth group: included nine genotypes have R3a locus. The sixth groups: contained eight genotypes have R9a. The seventh group: involved six potato genotypes have BLB1 and R8 and R9 genes. The eighth group: included four genotypes have R1 gene (using SCAR 76-2s) [Table 2].
 | Figure 5: Illustrate the total number of late blight resistance (R) gene markers in 17 potato genotypes. [Click here to view] |
4. DISCUSSION
A gene-based marker is considered a powerful tool for the detection of the presence or absence of resistance (R) genes to LB disease in potatoes [29]. In this investigation, we evaluated 17 potato genotypes for resistance against P. infestans using a detached leaf assay. Results of this study showed that S. chacoense CGN 17903 and Jelly genotypes were highly resistant to LB (scale value; 1), while S. acule CGN 17674 was resistant (scale value; 2). Cara, Diamond, Lady Rosetta, Metro, Mondial, Spunta, Deta, Herms, S. stoloniferum CGN 17605, S. tuberosum CGN 17609, S. demissum CGN 17788, and S. demissum CGN 17797 were moderately resistant (scale value; 3-4), and Annabelle and Bellini were moderately susceptible (scale value; 5). In the previous study, we assessed the previous potato genotypes for LB resistance under greenhouse conditions. The results showed that the data from the laboratory trial were run in parallel with the greenhouse results, except two potato varieties, Deta and Herms, which were moderately resistant under the laboratory conditions, were moderately susceptible under the greenhouse conditions [18]. Rogozina et al. [30] evaluated 50 hybrids and cultivars of potatoes for resistance for LB disease in the field and laboratory for 7 years. These genotypes gave different responses to P. infestans infection, which varied from highly resistant to susceptible hybrids. Furthermore, the results of the laboratory evaluation agreed with the field experiments for several years. On the contrary, there are some potato genotypes, which were moderately resistant to P. infestans in the field experiments, were recorded as moderately susceptible under laboratory conditions. Fry [31] observed the presence of differences between results of the laboratory and field trials (1-3 points) for most potato genotypes which can be illustrated by more favorable conditions for microorganism development in laboratory experiments. Furthermore, other plant organs may be share in the resistance [32].
In this study, 17 potato genotypes were tested for the presence or absence of eight resistance (R) genes to LB, namely R1, Rpi-phu1, Rpi-ber, Rpi-blb1, Rpi-blb3, R9a, R8 and R9a, and R3a, using seven SCAR and two CAPS markers. PCR amplicons for the R1 gene amplified using primers SCAR R1-517 and SCAR 76-2s gave clear polymorphisms between the resistant and susceptible genotypes. The results of PCR of the Rpi-phu1, Rpi-ber, Rpi-blb1, Rpi-blb3, and R9a genes, using SCAR phu6, SCAR Rpi-ber1-Q133, SCAR BLB1, SCAR RGH2, and SCAR Edn61, respectively, also showed polymorphisms between the resistant and susceptible genotypes. For the Rpi-blb3 gene, the PCR products obtained from the primer set SCAR RGH2 have not displayed any polymorphisms between the resistant and susceptible genotypes. For (R8 and R9a) and R3a genes, the results of CAPS markers differentiated between homozygous resistant or susceptible potato genotypes from heterozygous resistant ones. Furthermore, the digestion of fragments by a restriction enzyme revealed clear polymorphisms between susceptible and resistant potato genotypes. Therefore, these polymorphic PCR amplicons could be used as DNA markers in potato breeding programs to differentiate resistant and susceptible genotypes. These results were confirmed by Tiwari et al. [10] found that CAPS markers give clear polymorphisms between potato cultivars resistant and susceptible to LB after digestion by a restriction site. Cao et al. [33] mentioned that SCAR markers produced from randomly amplified polymorphic DNA and amplified fragment length polymorphism markers are considered important tools in the characterization of R genes. These markers depend on the marker sequence data, which enhances the reliability and reproducibility of PCR results [34]. Bisognin et al. and Pattanayak et al. [3,35] who mentioned that potato breeding programs can be improved with the aid of DNA markers linked to resistance loci against LB.
In the current work, it has been observed that there is no correlation between the number of markers and resistance levels to LB. In spite of, the potato variety Deta has three resistance gene markers; it was moderately resistant to LB disease. On the contrary, potato variety Bellini contained five resistance gene markers; it was moderately susceptible to LB. These results agree with Sedlák et al. [36], who mentioned that there is no correlation between the presence of a marker and the degree of resistance in the plant. Mu et al. [37] observed the presence of a negative correlation between a number of stripe rust resistance alleles and disease scores in wheat. Rogozina et al. [30] indicated that some potato hybrids having only one or two markers of Rpi loci; they were resistant or moderately resistant to LB disease. According to Kim et al.; Rogozina et al.; and Zhu et al. [17,30,38] who discovered that the presence of multiple R resistance loci is required for a genotype to give high levels of resistance against P. infestans. Bouwman and Fadina et al. [11,39] who indicated that conventional hybridization methods using 2-3 resistance genes (stacking genes) give resistance to a broad spectrum of P. infestans races, compared with genetic engineering methods.
In this finding, it has been shown that the Rpi-blb3 gene was the most common in 17 potato genotypes, followed by the RPi-phu1 gene, while R1 (using marker SCAR 76-2s) was the least popular in potato genotypes. On the other hand, it has been observed that some domesticated potato cultivars contain R genes. These cultivars were introgressed from wild species. Similar findings were reported by Beketova et al.; Goss et al. [40,41] who reported that the R locus was introgressed into different potato cultivars from wild species, involving R1 to R11, R3a, R3b, and R9a from S. demissum [27,42,43], Rpi-blb1, Rpi-blb2, and Rpi-blb3 from Solanum bulbocastanum [25,43,44], and RPi-phu1 from Solanum phureja [22].
5. CONCLUSION
Gene-targeted markers were used successfully to characterize potato genotypes with resistance loci to LB disease. The selection of potato genotypes resistant against LB is the most effective strategy for the control of the pathogen. In this study, the collection of 17 potato genotypes was estimated in the laboratory using the detached leaf assay. Besides, these genotypes were assayed by the seven SCAR and two CAPS markers for R genes. According to the study’s findings, there is no correlation between LB resistance levels and the number of R gene markers. For instance, moderately resistant S. stoloniferum CGN 17605 has the highest number of markers (8), followed by highly resistant cultivar Jelly (7). On the contrary, the lowest number of resistance gene markers was scored in the moderately resistant potato genotype Deta (3). Therefore, these genotypes have more than one resistance gene that could be taken into consideration in the potato breeding programs for resistance against LB disease.
6. AUTHORS’ CONTRIBUTIONS
Dr. HAM performed SCAR and CAPS markers, and analysis data, Prof. Dr. SAM wrote the manuscript, and Prof. Dr. OEE edited the manuscript.
7. FUNDING
This work was funded by National Research Centre (Project ref. 12020111), Dokki, Giza, Egypt.
8. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
9. ETHICS APPROVAL
This study does not involve experiments on animals or human subjects.
10. DATA AVAILABILITY
All data generated or analyzed during this paper already exist in this paper.
11. PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
12. AUTHORS’ INFORMATION
Genetics and Cytology Department, Biotechnology Research Institute, National Research Centre (NRC), Dokki, 12622, Egypt.
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