QTL analysis reveals quantitative resistant loci for Phytophthora infestans and Tecia solanivora in tetraploid potato (Solanum tuberosum L.).

Late blight and Guatemalan potato tuber moth caused by Phytophthora infestans and Tecia solanivora, respectively, are major phytosanitary problems on potato crops in Colombia and Ecuador. Hence, the development of resistant cultivars is an alternative for their control. However, breeding initiatives for durable resistance using molecular tools are limited due to the genome complexity and high heterozygosity in autotetraploid potatoes. To contribute to a better understanding of the genetic basis underlying the resistance to P. infestans and T. solanivora in potato, the aim of this study was to identify QTLs for resistance to P. infestans and T. solanivora using a F1 tetraploid potato segregant population for both traits. Ninety-four individuals comprised this population. Parent genotypes and their progeny were genotyped using SOLCAP 12K potato array. Forty-five percent of the markers were polymorphic. A genetic linkage map was built with a length of 968.4 cM and 1,287 SNPs showing good distribution across the genome. Severity and incidence were evaluated in two crop cycles for two years. QTL analysis revealed six QTLs linked to P. infestans, four of these related to previous QTLs reported, and two novel QTLs (qrAUDPC-3 and qrAUDPC-8). Fifteen QTLs were linked to T. solanivora, being qIPC-6 and qOPA-6.1, and qIPC-10 and qIPC-10.1 stable in two different trials. This study is one of the first to identify QTLs for T. solanivora. As the population employed is a breeding population, results will contribute significantly to breeding programs to select resistant plant material, especially in countries where P. infestans and T. solanivora limit potato production.

Identification of Novel Associations of Candidate Genes with Resistance to Late Blight in Solanum tuberosum Group Phureja.

The genetic basis of quantitative disease resistance has been studied in crops for several decades as an alternative to R gene mediated resistance. The most important disease in the potato crop is late blight, caused by the oomycete Phytophthora infestans. Quantitative disease resistance (QDR), as any other quantitative trait in plants, can be genetically mapped to understand the genetic architecture. Association mapping using DNA-based markers has been implemented in many crops to dissect quantitative traits. We used an association mapping approach with candidate genes to identify the first genes associated with quantitative resistance to late blight in Solanum tuberosum Group Phureja. Twenty-nine candidate genes were selected from a set of genes that were differentially expressed during the resistance response to late blight in tetraploid European potato cultivars. The 29 genes were amplified and sequenced in 104 accessions of S. tuberosum Group Phureja from Latin America. We identified 238 SNPs in the selected genes and tested them for association with resistance to late blight. The phenotypic data were obtained under field conditions by determining the area under disease progress curve (AUDPC) in two seasons and in two locations. Two genes were associated with QDR to late blight, a potato homolog of thylakoid lumen 15 kDa protein (StTL15A) and a stem 28 kDa glycoprotein (StGP28). Key message: A first association mapping experiment was conducted in Solanum tuberosum Group Phureja germplasm, which identified among 29 candidates two genes associated with quantitative resistance to late blight.

Hydroxycinnamic acids in cooked potato tubers from Solanum tuberosum group Phureja.

Hydroxycinnamic acids are phenolic compounds and are considered to have health promotion properties due to their antioxidant activity. Potato tubers of 113 genotypes of Solanum tuberosum group Phureja belonging to the Colombian Central Collection, landraces of potatoes, and commercial cultivars were evaluated for their hydroxycinnamic acids content. The composition of these compounds was analyzed using cooked tubers in two different agro-climatic conditions. The genotypes were analyzed for chlorogenic acid, neo-chlorogenic acid, crypto-chlorogenic acid, and caffeic acid by ultrahigh-performance liquid chromatography (UHPLC). Chlorogenic acid was the major representative and varied between 0.77 to 7.98 g kg-1 DW (dry weight) followed by crypto-chlorogenic acid (from 0.09 to 1.50 g kg-1 DW). Under moorland agro-climatic conditions even though the chlorogenic acid levels increased with respect to flatland agro-climatic conditions, the related isomer neo-chlorogenic acid decreased as compared to flatland conditions. The correlation between chlorogenic acid with the isomers, and with caffeic acid was positive. This study demonstrated that there is a wide variation in hydroxycinnamic acids contents in the germplasm studied, which can be exploited in breeding programs to contribute to human health.

StWRKY8 transcription factor regulates benzylisoquinoline alkaloid pathway in potato conferring resistance to late blight.

The resistance to late blight is either qualitative or quantitative in nature. Quantitative resistance is durable, but challenging due to polygenic inheritance. In the present study, the diploid potato genotypes resistant and susceptible to late blight, were profiled for metabolites. Tissue specific metabolite analysis of benzylisoquinoline alkaloids (BIAs) in response to pathogen infection revealed increased accumulation of morphinone, codeine-6-glucuronide and morphine-3-glucuronides. These BIAs are antimicrobial compounds and possibly involved in cell wall reinforcement, especially through cross-linking cell wall pectins. Quantitative reverse transcription-PCR studies revealed higher expressions of TyDC, NCS, COR-2 and StWRKY8 transcription factor genes, in resistant genotypes than in susceptible genotype, following pathogen inoculation. A luciferase transient expression assay confirmed the binding of the StWRKY8 TF to promoters of downstream genes, elucidating a direct regulatory role on BIAs biosynthetic genes. Sequence analysis of StWRKY8 in potato genotypes revealed polymorphism in the WRKY DNA binding domain in the susceptible genotype, which is important for the regulatory function of this gene. A complementation assay of StWRKY8 in Arabidopsis wrky33 mutant background was associated with decreased fungal biomass. In conclusion, StWRKY8 regulates the biosynthesis of BIAs that are both antimicrobial and reinforce cell walls to contain the pathogen to initial infection.

Targeted and Untargeted Approaches Unravel Novel Candidate Genes and Diagnostic SNPs for Quantitative Resistance of the Potato (Solanum tuberosum L.) to Phytophthora infestans Causing the Late Blight Disease.

The oomycete Phytophthora infestans causes late blight of potato, which can completely destroy the crop. Therefore, for the past 160 years, late blight has been the most important potato disease worldwide. The identification of cultivars with high and durable field resistance to P. infestans is an objective of most potato breeding programs. This type of resistance is polygenic and therefore quantitative. Its evaluation requires multi-year and location trials. Furthermore, quantitative resistance to late blight correlates with late plant maturity, a negative agricultural trait. Knowledge of the molecular genetic basis of quantitative resistance to late blight not compromised by late maturity is very limited. It is however essential for developing diagnostic DNA markers that facilitate the efficient combination of superior resistance alleles in improved cultivars. We used association genetics in a population of 184 tetraploid potato cultivars in order to identify single nucleotide polymorphisms (SNPs) that are associated with maturity corrected resistance (MCR) to late blight. The population was genotyped for almost 9000 SNPs from three different sources. The first source was candidate genes specifically selected for their function in the jasmonate pathway. The second source was novel candidate genes selected based on comparative transcript profiling (RNA-Seq) of groups of genotypes with contrasting levels of quantitative resistance to P. infestans. The third source was the first generation 8.3k SolCAP SNP genotyping array available in potato for genome wide association studies (GWAS). Twenty seven SNPs from all three sources showed robust association with MCR. Some of those were located in genes that are strong candidates for directly controlling quantitative resistance, based on functional annotation. Most important were: a lipoxygenase (jasmonate pathway), a 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate pathway), a P450 protein (terpene biosynthesis), a transcription factor and a homolog of a major gene for resistance to P. infestans from the wild potato species Solanum venturii. The candidate gene approach and GWAS complemented each other as they identified different genes. The results of this study provide new insight in the molecular genetic basis of quantitative resistance in potato and a toolbox of diagnostic SNP markers for breeding applications.

Metabolomics deciphers quantitative resistance mechanisms in diploid potato clones against late blight.

Resistance to late blight in potato is either qualitative or quantitative in nature. The quantitative resistance is durable, but the molecular and biochemical mechanisms underlying quantitative resistance are poorly understood, and are not efficiently utilised in potato breeding. A non-targeted metabolomics, using high resolution hybrid mass spectrometry, was applied to decipher the mechanisms of resistance in the advanced breeding diploid potato genotypes (Solanum tuberosum L. Group Phureja), with valuable sources of genetic diversity. The metabolomics profiles of resistant genotypes (AC04 and AC09) were compared with a susceptible commercial genotype (Criolla Colombia), following Phytophthora infestans or mock-inoculation, to identify the resistance related (RR) metabolites. Metabolites belonging to phenylpropanoids, flavonoid and alkaloid chemical groups were highly induced in resistant genotypes relative to susceptible. Concurrently, the biosynthetic genes, tyrosine decarboxylase (TyDC) and tyramine hydroxycinnamoyl transferase (THT), involved in the biosynthesis of hydroxycinnamic acid amides (HCAAs), and chalcone synthase (CHS) and flavonol synthase (FLS), involved in flavonoid biosynthesis, were also upregulated, as confirmed by quantitative real-time PCR. Probable genes coding for these enzymes were sequenced and nonsynonymous single-nucleotide polymorphisms (nsSNPs) were identified. The resistance to late blight observed in this study was mainly associated with cell wall thickening due to deposition of HCAAs, flavonoids and alkaloids.

Quantitative resistance in potato leaves to late blight associated with induced hydroxycinnamic acid amides.

Late blight is a serious economic threat to potato crop, sometimes leading to complete crop loss. The resistance in potato to late blight can be qualitative or quantitative in nature. Qualitative resistance is not durable. Though quantitative resistance is durable, the breeding is challenging due to polygenic inheritance. Several quantitative trait loci (QTLs) have been identified, but the mechanisms of resistance are largely unknown. A nontargeted metabolomics approach was used to identify resistance-related (RR) metabolites in a resistant genotype (F06025), as compared to a susceptible (Shepody) genotype, mock- or pathogen-inoculated. The RR metabolites, which had high fold change in abundance, mainly belonged to phenylpropanoid, flavonoid, fatty acid, and alkaloid chemical groups. The most important phenylpropanoids identified were hydroxycinnamic acid amides, the polyaromatic domain of suberin that is known to be associated with cell wall reinforcement. These metabolites were mapped on to the potato metabolic pathways, and the candidate enzymes and their coding genes were identified. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay revealed a higher upregulation of 4-coumarate: CoA ligase (4-CL), tyrosine decarboxylase (TyDC), and tyramine hydroxycinnamoyl transferase (THT) in the pathogen-inoculated resistant genotype than in susceptible. These genes were sequenced in both resistant and susceptible genotypes, and nonsynonymous single-nucleotide polymorphisms (nsSNPs) were found. The application of these genes in potato resistance improvement, following validation, is discussed.

Evaluación de marcadores moleculares asociados con resistencia a gota (Phytophthora infestans L) en papas diploides y tetraploides / Evaluation of molecular markers associated with resistance to late blight (Phytophthora infestans L) in diploid and tetraploid potatoes

La papa, cultivo de importancia a nivel mundial es gravemente afectado por gota, enfermedad ocasionada por el oomycete Phytophthora infestans. Actualmente la forma más efectiva para combatir la enfermedad es mediante el desarrollo de cultivares resistentes al patógeno. Para esto, una estrategia es identificar genes que confieran resistencia al patógeno, para lo cual se buscan marcadores asociados con el carácter de resistencia. En este estudio se evaluaron marcadores moleculares tipo SCAR (Sequence Characterized Amplified Region): CosA, GP179, BA47f2 y Prp1 asociados con resistencia a P. infestans y el gen de resistencia R1, en 22 cultivares tetraploides pertenecientes a la subespecie andigena y cinco especies silvestres. Se evaluó el polimorfismo y se determinó si los alelos polimórficos permitían diferenciar genotipos resistentes de susceptibles. Se comparó el tamaño de los fragmentos obtenidos con los fragmentos esperados asociados con resistencia de acuerdo a reportes. El análisis se realizó considerando presencia/ausencia de los fragmentos: CosA210, CosA250, R11400, R11800, BA47f2500, GP179570, Prp1300, Prp1600, y Prp1900. Los resultados indicaron que en los cultivares tetraploides y silvestres, se presentaron polimorfismos en todos los marcadores evaluados, con excepción del marcador GP179. No se encontró correlación entre el rasgo de resistencia y los alelos. Los resultados de este estudio muestran que hay repuesta diferencial a los marcadores entre las subsp. tuberosum y subsp. Andigena.

Potato is an important worldwide crop seriously affected by late blight disease caused by the oomycete Phytophthora infestans. Currently, the most effective way to control the disease is developing resistant cultivars to the pathogen by identifying genes that confer resistance to the pathogen. For this purpose it is important to find molecular markers associated with the trait. In this study, the SCAR (Sequence Characterized Amplified Region) markers: CosA, GP179, BA47f2 y Prp1, associated with late blight and the resistant gen R1 were evaluated in 22 tetraploid cultivars from subspecie andigena and five wild potato species. Polymorphism was evaluated and it was evaluated if polymorphic alleles allow differentiating resistant from susceptible genotypes. The fragment length for each marker was compared to the allele size reported associated to resistance. The analysis considered the presence/absence of the fragments: CosA210, CosA250, R11400, R11800, BA47f2500, GP179570, Prp1300, Prp1600 and Prp1900. The results indicated that both, tetraploid cultivars and wild potatoes, showed polymorphisms with all these markers, except with the GP179 marker. It was not found correlation between resistance and the presence of specific alleles. Evidence found in this study indicates that results obtained with molecular markers differed between subsp. tuberosum and subsp. andigena.