Characterization and mapping of R Pi-ber , a novel potato late blight resistance gene from Solanum berthaultii

Phytophthora infestans, the causal agent of late blight, threatens potato production worldwide. An important tool in the management of the disease is the use of resistant varieties. Eleven major resistance genes have been identified and introgressed from Solanum demissum. However, new sources of resistance are continually sought. Here, we report the characterization and refined genetic localization of a resistance gene previously identified as Rber in a backcross progeny of Solanum tuberosum and Solanum berthaultii. In order to further characterize Rber, we developed a set of P. infestans isolates capable of identifying each of the 11 R-genes known to confer resistance to late blight in potato. Our results indicate that Rber is a new resistance gene, different from those recognized in S. demissum, and therefore, it has been named R _Pi-ber according to the current system of nomenclature. In order to add new molecular markers around R _Pi-ber , we used a PCR-based mapping technique, named MASP-map, which located R _Pi-ber in a 3.9 cM interval between markers CT240 and TG63 on potato chromosome X. The location of R _Pi-ber coincides with an area involved in resistance to different pathogens of potato and tomato.     

The late blight resistance locus Rpi-bib3 from Solanum bulbocastanum belongs to a major late blight R gene cluster on chromosome 4 of potato

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases in cultivated potato. Breeding of new potato cultivars with high levels of resistance to P. infestans is considered the most durable strategy for future potato cultivation. In this study, we report the identification of a new late-blight resistance (R) locus from the wild potato species Solanum bulbocastanum. Using several different approaches, a high-resolution genetic map of the new locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome 4. One amplification fragment length polymorphism marker was identified that cosegregated in 1,396 progeny plants of an intraspecific mapping population with Rpi-blb3. For comparative genomics purposes, markers linked to Rpi-blb3 were tested in mapping populations used to map the three other late-blight R loci Rpi-abpt, R2, and R2-like also to chromosome 4. Marker order and allelic conservation suggest that Rpi-blb3, Rpi-abpt, R2, and R2-like reside in the same R gene cluster on chromosome 4 and likely belong to the same gene family. Our findings provide novel insights in the evolution of R gene clusters conferring late-blight resistance in Solanum spp.     

The R1 gene for late blight resistance in early and late maturing potato cultivars

The method of polymerase chain reaction was used to amplify a fragment of the LZ-NBS-LRR receptor kinase gene R1; the gene was transferred into potato (Solanum tuberosum) from its wild-growing relative S. demissum and confers the race-specific recognition of the pathogen Phytophthora infestans. To verify this method as a test for the presence of the late blight resistance gene R1, the amplified genome fragment was cloned from the potato hybrid comprising the germplasm of S. demissum. The primary structure of this fragment, which corresponded to the receptor domain of kinase, did not practically differ from the matching sequence in S. demissum. In addition, the method was verified by scoring the set of plant differentials, wherein the presence of R1 was established with race-specific Phytophthora isolates. By screening 70 potato cultivars, we established a significant relationship between the presence of the gene R1 fragment and the phenotypic characters of late blight resistance and late maturity. This evidence supports the idea that R1 was introgressed from short-day S. demissum into potato plants together with some gene(s) conferring late transition to flowering.     

Conditional QTL underlying resistance to late blight in a diploid potato population

A large number of quantitative trait loci (QTL) for resistance to late blight of potato have been reported with a "conventional" method in which each phenotypic trait reflects the cumulative genetic effects for the duration of the disease process. However, as genes controlling response to disease may have unique contributions with specific temporal features, it is important to consider the phenotype as dynamic. Here, using the net genetic effects evidenced at consecutive time points during disease development, we report the first conditional mapping of QTL underlying late blight resistance in potato under five environments in Peru. Six conditional QTL were mapped, one each on chromosome 2, 7 and 12 and three on chromosome 9. These QTL represent distinct contributions to the phenotypic variation at different stages of disease development. By comparison, when conventional mapping was conducted, only one QTL was detected on chromosome 9. This QTL was the same as one of the conditional QTL. The results imply that conditional QTL reflect genes that function at particular stages during the host-pathogen interaction. The dynamics revealed by conditional QTL mapping could contribute to the understanding of the molecular mechanism of late blight resistance and these QTL could be used to target genes for marker development or manipulation to improve resistance.     

Race nonspecific resistance for potato late blight

The late blight fungus (Phytophthora infestans) rots susceptible species of potato plants. None of the major varieties of potato (Solanum tuberosum) grown in the USA is resistant to US-8, the most prevalent genotype of the fungus. Now, Junqi Song, James Bradeen and colleagues have cloned the RB gene from the wild diploid potato species, Solanum bulbocastanum, using a map-based approach in combination with long-range PCR. Transgenic plants containing the gene, normally fully susceptible, displayed broad-spectrum late blight resistance.     

Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato

Comparative genomics provides a tool to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. In this report, we adopted a local RGA approach using genomic information from the model Solanaceous plant tomato to isolate R3a, a potato gene that confers race-specific resistance to the late blight pathogen Phytophthora infestans. R3a is a member of the R3 complex locus on chromosome 11. Comparative analyses of the R3 complex locus with the corresponding I2 complex locus in tomato suggest that this is an ancient locus involved in plant innate immunity against oomycete and fungal pathogens. However, the R3 complex locus has evolved after divergence from tomato and the locus has experienced a significant expansion in potato without disruption of the flanking colinearity. This expansion has resulted in an increase in the number of R genes and in functional diversification, which has probably been driven by the co-evolutionary history between P. infestans and its host potato. Constitutive expression was observed for the R3a gene, as well as some of its paralogues whose functions remain unknown.     

Chitin and chitosan derivatives as elicitors of potato resistance to late blight

Water-soluble low-molecular-weight (3–10 kDa) chitosan obtained by enzymatic degradation of high-molecular-weight chitosan, as well as its deaminated derivatives, can be used as elicitors of resistance to late blight in potato.     

Vector integration in triple R gene transformants and the clustered inheritance of resistance against potato late blight

Genetic transformation with resistance (R) genes is expected to enhance resistance durability against pathogens, especially for potato, a vegetatively propagated crop with tetrasomic inheritance and a long-term breeding program. In this study, 128 potato transformants were analysed for the presence of vector T-DNA genes, borders and backbone sequences. They were harvested after transformation using a construct containing neomycin phosphotransferase II (nptII) and three R genes against potato late blight (Phytophthora infestans). Our analysis revealed that 45 % of the R gene-containing transformants possessed a low T-DNA copy number, without the integration of vector backbone and borders. The integration of vector backbone sequences was characterized using eight genes, and backbone gene tetA was selected for the early prediction of plants with backbone sequence integration. Three transformants, two plants harbouring one T-DNA copy and one plant harbouring three T-DNA copies, were crossed with susceptible cv. Katahdin. Based on our results, we conclude that all four T-DNA genes were inherited as one cluster and segregated in a Mendelian fashion. The three T-DNA inserts from the transformant harbouring three T-DNA copies were statistically proven to be un-linked and inherited into the offspring plants independently. All of the R genes were functionally expressed in the offspring plants as in their parental transformants. This functional gene stacking has important implications towards achieving more durable resistance against potato late blight.     

Derivatives of chitin and chitosan as elicitors of potato resistance to late blight

Water-soluble low-molecular-weight (3-10 kDa) chitosan obtained by enzymatic degradation of high-molecular-weight chitosan, as well as its deaminated derivatives, can be used as elicitors of late blight resistance in potato.     

Silencing of six susceptibility genes results in potato late blight resistance

Phytophthora infestans, the causal agent of late blight, is a major threat to commercial potato production worldwide. Significant costs are required for crop protection to secure yield. Many dominant genes for resistance (R-genes) to potato late blight have been identified, and some of these R-genes have been applied in potato breeding. However, the P. infestans population rapidly accumulates new virulent strains that render R-genes ineffective. Here we introduce a new class of resistance which is based on the loss-of-function of a susceptibility gene (S-gene) encoding a product exploited by pathogens during infection and colonization. Impaired S-genes primarily result in recessive resistance traits in contrast to recognition-based resistance that is governed by dominant R-genes. In Arabidopsis thaliana, many S-genes have been detected in screens of mutant populations. In the present study, we selected 11 A. thaliana S-genes and silenced orthologous genes in the potato cultivar Desiree, which is highly susceptible to late blight. The silencing of five genes resulted in complete resistance to the P. infestans isolate Pic99189, and the silencing of a sixth S-gene resulted in reduced susceptibility. The application of S-genes to potato breeding for resistance to late blight is further discussed.     

Effect of methyl jasmonate on arachidonic acid-induced resistance of potato to late blight

Methyl ester of jasmonic acid (Me-JA) influences the induced resistance of potato tubers to late blight caused byPhytophthora infestans. Treatment of potato tuber disk surfaces with Me-JA solution or exposure to an atmosphere containing Me-JA vapors (10⁻⁶–10⁻⁵ M) increased the rate of rishitin biosynthesis induced by arachidonic acid orP. infestans. Methyl jasmonate increased the sensitivity of potato tissue to arachidonic acid. As a result, in the presence of Me-JA, the protective properties of arachidonic acid were observed at lower concentrations than in the absence of Me-JA. In addition, Me-JA reduced the adverse effects of lipoxygenase inhibitors (salicylhydroxamic acid and esculetin) on the induced resistance of potato tubers to late blight. Therefore, the synergistic interaction of Me-JA and biogenic elicitors can be regarded as part of a mechanism of potato defense against diseases.     

Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight

 Solanum bulbocastanum, a wild, diploid (2n=2x=24) Mexican species, is highly resistant to Phytophthora infestans, the fungus that causes late blight of potato. However this 1 EBN species is virtually impossible to cross directly with potato. PEG-mediated fusion of leaf cells of S. bulbocastanum PI 245310 and the tetraploid potato line S. tuberosum PI 203900 (2n=4x=48) yielded hexaploid (2n= 6x=72) somatic hybrids that retained the high resistance of the S. bulbocastanum parent. RFLP and RAPD analyses confirmed the hybridity of the materials. Four of the somatic hybrids were crossed with potato cultivars Katahdin or Atlantic. The BC₁ progeny segregated for resistance to the US8 genotype (A-2 mating type) of P. Infestans. Resistant BC₁ lines crossed with susceptible cultivars again yielded populations that segregated for resistance to the fungus. In a 1996 field-plot in Wisconsin, to which no fungicide was applied, two of the BC₁ lines, from two different somatic hybrids, yielded 1.36 and 1.32 kg/plant under a severe late-blight epidemic. In contrast, under these same conditions the cultivar Russet Burbank yielded only 0.86 kg/plant. These results indicate that effective resistance to the late-blight fungus in a sexually incompatible Solanum species can be transferred into potato breeding lines by somatic hybridization and that this resistance can then be further transmitted into potato breeding lines by sexual crossing. Received: 27 October 1997 / Accepted: 11 November 1997     

A genetic analysis of quantitative resistance to late blight in potato: towards marker-assisted selection

Late blight caused by the oomycete Phytophthora infestans is the most important fungal disease in potato cultivation worldwide. Resistance to late blight is controlled by a few major genes (R genes) which can be easily overcome by new races of P. infestans and/or by an unknown number of genes expressing a quantitative type of resistance which may be more durable. Quantitative resistance of foliage to late blight was evaluated in five F₁ hybrid families originating from crosses among seven different diploid potato clones. Tuber resistance was evaluated in four of the families. Two of the families were scored for both foliage maturity and vigour. The five families were genotyped with DNA-based markers and tested for linkage with the traits analysed. QTL (quantitative trait locus) analysis identified at least twelve segments on ten chromosomes of potato having genes that affect reproducibly foliage resistance. Two of those segments also have major R genes for resistance to late blight. The segments are tagged by 21 markers that can be analyzed based on PCR (polymerase chain reaction) with specific oligonucleotide primers. One QTL was detected for tuber resistance and one for foliage vigour. Two QTLs were mapped for foliage maturity. Major QTL effects on foliage and tuber resistance to late blight and on foliage maturity and vigour were all linked with marker GP179 on linkage group V of potato. Plants having alleles at this QTL, which increased foliage resistance, exhibited decreased tuber resistance, later maturity and more vigour.     

马铃薯抗晚疫病基因Rpi-blb2的LRR区域多态性及分子进化分析

马铃薯抗晚疫病基因Rpi-blb2是来源于马铃薯野生种Solanum bulbocastanum中的一个具有广谱抗性的NBS-LRR类抗病基因。文章用PCR的方法从20个高抗晚疫病的马铃薯栽培种和20个高感晚疫病的马铃薯栽培种以及7个马铃薯野生种中克隆了Rpi-blb2基因的LRR区段。采用生物信息学方法对这些序列的相似性、多态性位点、核酸多样性指数等参数进行了分析,发现Rpi-blb2的LRR区域在核酸水平上变异程度很高,而且存在多处热点突变位点;通过对该区域的Ka/Ks值进行估算,发现Rpi-blb2的LRR区域总体上受到纯化选择,功能保守,但是LRR区域的不同部位所受到的选择压力却不尽相同。同时,从核酸水平来看,Rpi-blb2基因的LRR区域在马铃薯栽培种和马铃薯野生种之间没有发现明显的分化。     

Nucleotide polymorphism and molecular evolution of the LRR region in potato late blight resistance gene Rpi-blb2

Rpi-blb2, which is originally derived from Solanum bulbocastanum, is a broad-spectrum potato late blight resistance gene and belongs to the NBS-LRR family. Here, the LRR homologues of Rpi-blb2 were cloned with PCR method from 40 potato cultivars (including 20 resistant potato cultivars and 20 susceptible ones) and 7 wild potato populations. Then, the similarities of the sequences, polymorphic (segregating) sites, and nucleotide diversities were estimated by bioinformatic methods. The results showed that high nucleotide polymorphism and some hot-spot mutations existed in the LRR region of Rpi-blb2. The test of Ka/Ks ratio showed that the function of LRR was conserved because of the purifying selection, although different positions of the Rpi-blb2 LRR region were under different selection pressures. Moreover, the LRR region of Rpi-blb2 had no clear differentiation between the cultivated and wild potatoes.     

Evaluating between-plant interference in field trials for assessing potato genotypes for resistance to late blight

Field trials were done with four cultivars over 3 years to assess the extent to which the amount of late blight on the foliage of a potato plant could be influenced by that on a neighbouring plant of the same or a different cultivar. Drills containing the test plants were interspersed with those of spreader plants (cv. King Edward) which were artificially inoculated with Phytophthora infestans. The intensity of blight on the test plants was recorded on several occasions.
Resistant cultivars tended to be scored as less resistant in mixtures with other cultivars than in pure stands, and susceptible cultivars tended likewise to be scored as more resistant in mixed stands. However, standard analysis of variance indicated no systematic evidence of a significant effect due to neighbour cultivars, nor of interaction between cultivars and neighbour cultivars. In contrast, Kempton's (1982) neighbour model indicated a significant and positive interference coefficient (β) in each trial, which generally decreased over time. Predicted pure stand scores for each cultivar indicated that the adjustment was greatest for the most resistant and most susceptible cultivars. There was no advantage in using two-plant rather than one-plant plots in withstanding neighbour effects.     

QTL for field resistance to late blight in potato are strongly correlated with maturity and vigour

Field resistance to Phytophthora infestans, the causal agent of foliage and tuber blight in cultivated potatoes, earliness (maturity) and vigour, were examined in a diploid segregating potato population grown in replicated trials over three consecutive growing seasons. A genetic linkage map of this population was constructed in parallel using PCR-based SSR, AFLP and CAPS markers. Analysis of the trait scores alongside the marker segregation data allowed the identification of regions of the genome which were significantly correlated with components of the respective characters. The most significant associations for all four traits were with marker alleles on potato linkage group V originating from the male (susceptible) parent. In the case of foliage resistance to late blight, the positions of the majority of the effects, which were located on eleven of the twelve potato linkage groups, have been detected in previous [16] and parallel studies [21]. The absence of Solanum demissum-derived R genes for hypersensitive response to late blight and the co-localisation of QTL for resistance, vigour and earliness suggest that developmental and/or physiological factors play a major role in determining the level of foliage resistance in this population. In contrast with previous findings, a negative correlation was found between foliage and tuber blight resistance.     

Characterization and fine-mapping of a resistance locus for northern leaf blight in maize bin 8.06

As part of a larger effort to capture diverse alleles at a set of loci associated with disease resistance in maize, DK888, a hybrid known to possess resistance to multiple diseases, was used as a donor in constructing near-isogenic lines (NILs). A NIL pair contrasting for resistance to northern leaf blight (NLB), caused by Setosphaeria turcica, was identified and associated with bin 8.06. This region of the maize genome had been associated in previous studies with both qualitative and quantitative resistance to NLB. In addition, bins 8.05–8.06 had been associated with quantitative resistance to several other diseases, as well as resistance gene analogs and defense response gene homologs. To test the hypothesis that the DK888 allele at bin 8.06 (designated qNLB8.06 _DK888 ) conditions the broad-spectrum quantitative resistance characteristic of the donor, the NILs were evaluated with a range of maize pathogens and different races of S. turcica. The results revealed that qNLB8.06 _DK888 confers race-specific resistance exclusively to NLB. Allelism analysis suggested that qNLB8.06 _DK888 is identical, allelic, or closely linked and functionally related to Ht2. The resistance conditioned by qNLB8.06 was incompletely dominant and varied in effectiveness depending upon allele and/or genetic background. High-resolution breakpoint analysis, using ~2,800 individuals in F₉/F₁₀ heterogeneous inbred families and 98 F₁₀/F₁₁ fixed lines carrying various recombinant events, delimited qNLB8.06 _DK888 to a region of ~0.46 Mb, spanning 143.92–144.38 Mb on the B73 physical map. Three compelling candidate genes were identified in this region. Isolation of the gene(s) will contribute to better understanding of this complex locus.