Metabolic and gene-expression analyses reveal developmental dynamics of cutin deposition in pomegranate fruit grown under different environmental conditions.

The chemical and transcriptional changes in the cuticle of pomegranate (Punica granatum L.) fruit grown under different environmental conditions were studied. We collected fruit from three orchards located in different regions in Israel, each with a distinct microclimate. Fruit were collected at six phenological stages, and cutin monomers in the fruit cuticle were profiled by gas chromatography-mass spectrometry (GC-MS), along with qPCR transcript-expression analyses of selected cutin-related genes. While fruit phenotypes were comparable along development in all three orchards, principal component analyses of cutin monomer profiles suggested clear separation between cuticle samples of young green fruit to those of maturing fruit. Moreover, total cutin contents in green fruit were lower in the orchard characterized by a hot and dry climate compared to orchards with moderate temperatures. The variances detected in total cutin contents between orchards corresponded well with the expression patterns of BODYGUARD, a key biosynthetic gene operating in the cutin biosynthetic pathway. Based on our extraction protocols, we found that the cutin polyester that builds the pomegranate fruit cuticle accumulates some levels of gallic acid-the precursor of punicalagin, a well-known potent antioxidant metabolite in pomegranate fruit. The gallic acid was also one of the predominant metabolites contributing to the variability between developmental stages and orchards, and its accumulation levels were opposite to the expression patterns of the UGT73AL1 gene which glycosylates gallic acid to synthesize punicalagin. To the best of our knowledge, this is the first detailed composition of the cutin polyester that forms the pomegranate fruit cuticle.

Epigenetic Modifications Related to Potato Skin Russeting.

Potato tuber skin is a protective corky tissue consisting of suberized phellem cells. Smooth-skinned varieties are characterized by a clean, shiny appearance compared to the darker hue of russeted potatoes. The rough skin of russeted cultivars is a desired, genetically inherited characteristic; however, unwanted russeting of smooth-skinned cultivars often occurs under suboptimal growth conditions. The involvement of epigenetic modifiers in regulating the smooth skin russeting disorder was tested. We used smooth-skin commercial cultivars with and without the russeting disorder and three lines from a breeding population segregating for russeting. Anatomically, the russet skin showed similar characteristics, whether the cause was environmentally triggered or genetically determined. The old outer layers of the corky phellem remain attached to the newly formed phellem layers instead of being sloughed off. Global DNA methylation analysis indicated a significant reduction in the percentage of 5-methylcytosine in mature vs. immature skin and russet vs. smooth skin. This was true for both the smooth-skin commercial cultivars and the russeted lines. The expression level of selected DNA methyltransferases was reduced in accordance. DNA demethylase expression did not change between the skin types and age. Hence, the reduced DNA methylation in mature and russet skin is more likely to be achieved through passive DNA demethylation and loss of methyltransferase activity.

Potato Periderm Development and Tuber Skin Quality.

The periderm is a corky tissue that replaces the epidermis when the latter is damaged, and is critical for preventing pathogen invasion and water loss. The periderm is formed through the meristematic activity of phellogen cells (cork cambium). The potato skin (phellem cells) composes the outer layers of the tuber periderm and is a model for studying cork development. Early in tuber development and following tuber expansion, the phellogen becomes active and produces the skin. New skin layers are continuously added by division of the phellogen cells until tuber maturation. Some physiological disorders of the potato tuber are related to abnormal development of the skin, including skinning injuries and russeting of smooth-skinned potatoes. Thus, characterizing the potato periderm contributes to modeling cork development in plants and helps to resolve critical agricultural problems. Here, we summarize the data available on potato periderm formation, highlighting tissue characteristics rather than the suberization processes.

Ripening of Pomegranate Skin as Revealed by Developmental Transcriptomics.

The appearance of pomegranate (Punica granatum L.) fruit is highly important for its marketing. The primary concerns are obtaining sufficient red pigment accumulation and minimal cracking of the fruit skin (the outer red layer of the peel). We analyzed the skin transcriptome of pomegranate cv. Wonderful at distinct time points of fruit development to characterize the processes that occur in the skin during fruit ripening and which may reflect on processes in the whole fruit, such as the non-climacteric nature of pomegranate. The data suggested a ripening mechanism in pomegranate skin that differs from that in strawberry-the model plant for non-climacteric fruit where abscisic acid is the growth regulator that drives ripening-involving ethylene, polyamine, and jasmonic acid pathways. The biosynthetic pathways of important metabolites in pomegranate-hydrolyzable tannins and anthocyanins-were co-upregulated at the ripening stage, in line with the visual enhancement of red coloration. Interestingly, cuticle- and cell-wall-related genes that showed differential expression between the developmental stages were mainly upregulated in the skin of early fruit, with lower expression at mid-growth and ripening stages. Nevertheless, lignification may be involved in skin hardening in the mature fruit.

Pomegranate Fruit Growth and Skin Characteristics in Hot and Dry Climate.

Pomegranate (Punica granatum L.) fruit is well known for its health-beneficial metabolites. The pomegranate peel consists of an inner thick spongy white tissue, and an outer smooth skin layer that accumulates anthocyanins in red cultivars when ripe. The skin is made up of epidermis cells covered by a cuticle, the latter being the first target of cracking and russeting. The present study focuses on the effect of Israel's hot and dry climate on pomegranate growth, to elucidate the derived effects on fruit skin characteristics and its putative resistance to the building pressure from fruit expansion. Experiments were conducted for four years, in four orchards located in different regions of the country, each with a different typical microclimate. Fruit-growth parameters were followed using remote-sensing tools, microscopic study, and mineral analysis of the skin, followed by determination of the peel's elastic modulus. Fruit expanded in two phases: a short rapid phase followed by a gradual phase with a sigmoidal growth-rate pattern. Extreme hot and dry climate during the period of maximal growth rate was associated with restricted growth and a high proportion of small-size fruit. Anatomical study indicated that the skin of mature pomegranate fruit is made up of epidermal cells that are relatively flat and spaced apart, and is expected to be less durable against internal pressure. In contrast, skin of early immature fruit has two layers of dense and rounded epidermis, and is expected to be more resistant to cracking. Tensile strength studies confirmed this trend-skin of mature fruit had a lower elastic modulus than young fruit. However, restrained growth due to extreme environmental cues may result in better resistance of the mature pomegranate fruit to cracking, and in better skin quality and appearance, albeit small fruits. On the other hand, temperate climate at the beginning of the growth period, which allows high growth rate and high daily shrinkage, leads to pomegranate skin disorders.

Metabolic changes in pomegranate fruit skin following cold storage promote chilling injury of the peel.

Pomegranate cv. 'Wonderful' fruit are susceptible to chilling injuries of the peel (CIp) when stored at 7 °C in modified-atmosphere bags for more than 3 months. The damage, manifested as superficial browning, is restricted to the fruit skin, i.e., the outer colored layer of the peel. To characterize possible causes of CIp development, fruit were collected at early harvest-when the premature fruit are poorly colored and susceptible to CIp development, and at late harvest-when mature fruit have fully red skin and less susceptibility to CIp. Skin samples were collected on day of harvest and at different time points during storage. Anatomical study of skin with CIp disorder showed a broken cuticle layer with underlying degenerated cells. A high total phenol content, which is associated with high antioxidant capacity, was not sufficient to prevent the development of CIp in the premature fruit. The concentration of punicalagin was the same for premature and mature skin at harvest and during storage, and therefore not associated with CIp development in the premature fruit skin. Furthermore, the expression of antioxidant-related genes CAT2, SOD and GR2 was similar for both premature and mature fruit skin. Poor pigmentation of the premature fruit skin and chilling-induced downregulation of key anthocyanin-biosynthesis genes were associated with CIp development. High total phenol concentration combined with high expression of the gene encoding PPO was also associated with CIp; however, high expression ratio of PAL to PPO was found in mature skin, and may be associated with reduced CIp disorder. The results presented suggest future possibilities for controlling the CIp phenomenon.

Adventitious root formation in crops-Potato as an example.

The root system of potato is made up of adventitious roots (AR) that form at the base of a sprout once it emerges from the mother tuber. By definition, AR originate from dormant preformed meristems, or from cells neighboring vascular tissues in stems or leaves. This may occur as part of the developmental program of the plant (e.g., potato), or when replacing the embryonic primary roots in response to stress conditions, such as flooding, nutrient deprivation, or wounding. AR formation is studied mainly in cereals and model plants, and less is known about its developmental program in root and tuber crops. In this review, we summarize the recent data on AR development in potato and relate this knowledge to what is known from model plants. For example, AR formation following stem cutting in potato follows a pattern of initiation, expression, and emergence phases that are known for other plants and involves auxin, the master regulator of AR induction and development. Molecular regulation of AR formation and the effect of environmental stresses are discussed. Understanding the origin and nature of AR systems in important crops will contribute to increased production and improve global food security.

Effects of Polyhalite Fertilization on Skin Quality of Potato Tuber.

The protective peel of potato tuber consists of periderm tissue, the outmost cell layers of which contain corky cell walls and are termed "skin". The skin protects the tuber from water loss and pathogen invasion, and its visual appearance is a highly important marketing factor. Physiological skin blemishes are of great concern, mainly russeting disorder and skinning injuries. We previously showed that application of calcium (Ca) reduces the rate and severity of skin russeting. Here, polyhalite fertilization was tested as an alternative source of Ca. The polyhalite mineral is a hydrated sulfate of potassium (K), Ca, and magnesium (Mg), and thus contains additional important nutrients that may contribute to skin quality. Furthermore, in view of the direct interaction of soil mineral elements with the tuber skin, we tested application of polyhalite at the end of the growth period, assuming that providing the mineral at the last stages of skin development may enhance its quality. Accordingly, polyhalite was applied at three time points: preplanting, in-season at around 3-4 weeks prior to haulm desiccation, and 2 days post-haulm desiccation. The experiments included several cultivars and locations. Data indicated that late application of polyhalite, after haulm desiccation, results in reduced concentrations of Ca and Mg and increased concentration of K in the tuber peel of fertilized plants compared to controls. Tuber appearance was improved, and the expression of FHT and CYP86A33, indicator genes for skin suberization, was significantly upregulated. Earlier applications of the polyhalite mineral did not alter mineral elements concentrations in the tuber peel compared to control plants. Overall, polyhalite fertilization positively affected tuber skin appearance and skin-related gene expression. However, the effect was moderate, and the mineral did not fully mitigate skin imperfections. The effect of polyhalite may be dependent on local conditions and cultivar type.

The transcriptome of potato tuber phellogen reveals cellular functions of cork cambium and genes involved in periderm formation and maturation.

The periderm is a protective corky tissue that is formed through the cambial activity of phellogen cells, when the outer epidermis is damaged. Timely periderm formation is critical to prevent pathogen invasion and water loss. The outer layers of the potato periderm, the tuber skin, serves as a model to study cork development. Early in tuber development the phellogen becomes active and produces the skin. During tuber maturation it becomes inactive and the skin adheres to the tuber flesh. The characterization of potato phellogen may contribute to the management of costly agricultural problems related to incomplete skin-set and the resulting skinning injuries, and provide us with new knowledge regarding cork development in planta. A transcriptome of potato tuber phellogen isolated by laser capture microdissection indicated similarity to vascular cambium and the cork from trees. Highly expressed genes and transcription factors indicated that phellogen activation involves cytokinesis and gene reprograming for the establishment of a dedifferentiation state; whereas inactivation is characterized by activity of genes that direct organ identity in meristem and cell-wall modifications. The expression of selected genes was analyzed using qPCR in native and wound periderm at distinct developmental stages. This allowed the identification of genes involved in periderm formation and maturation.

Nutritional value of potato (Solanum tuberosum) in hot climates: anthocyanins, carotenoids, and steroidal glycoalkaloids.

MAIN CONCLUSION: Growth in hot climates selectively alters potato tuber secondary metabolism-such as the anthocyanins, carotenoids, and glycoalkaloids-changing its nutritive value and the composition of health-promoting components. Potato breeding for improved nutritional value focuses mainly on increasing the health-promoting carotenoids and anthocyanins, and controlling toxic steroidal glycoalkaloids (SGAs). Metabolite levels are genetically determined, but developmental, tissue-specific, and environmental cues affect their final content. Transcriptomic and metabolomic approaches were applied to monitor carotenoid, anthocyanin, and SGA metabolite levels and their biosynthetic genes' expression under heat stress. The studied cultivars differed in tuber flesh carotenoid concentration and peel anthocyanin concentration. Gene expression studies showed heat-induced downregulation of specific genes for SGA, anthocyanin, and carotenoid biosynthesis. KEGG database mapping of the heat transcriptome indicated reduced gene expression for specific metabolic pathways rather than a global heat response. Targeted metabolomics indicated reduced SGA concentration, but anthocyanin pigments concentration remained unchanged, probably due to their stabilization in the vacuole. Total carotenoid level did not change significantly in potato tuber flesh, but their composition did. Results suggest that growth in hot climates selectively alters tuber secondary metabolism, changing its nutritive value and composition of health-promoting components.

Identification of candidate genes mediating apple fruit-cracking resistance following the application of gibberellic acids 4 + 7 and the cytokinin 6-benzyladenine.

Calyx-end cracking in 'Pink Lady' apple is treated by a solution of gibberellic acids 4 and 7 (GA4+7) and the cytokinin 6-benzyladenine (BA). Although the GA4+7 and BA mixture is applied early in apple fruit development, it mitigates cracking that becomes evident in the mature fruit, implying a long-term treatment effect. The reduced incidence of peel cracking is associated with increased epidermal cell density, which is maintained until fruit maturation. Presently, the expression of genes that have been previously reported to be associated with epidermal cell patterning and cuticle formation, or cracking resistance, was monitored in the peel during fruit development and following GA4+7 and BA treatment. For most of the genes whose expression is naturally upregulated during fruit development, the early GA4+7 and BA treatment maintained or further increased the high expression level in the mature peel. Where the expression of a gene was downregulated during development, no change was detected in the treated mature peel. Gene-networking analysis supported the interaction between gene clusters of cell-wall synthesis, cuticle formation and GA signaling. Overall, the data suggested that the GA4+7 and BA treatment did not modify developmental cues, but promoted or enhanced the innate developmental program.

Lanosterol synthase-like is involved with differential accumulation of steroidal glycoalkaloids in potato.

MAIN CONCLUSION: Phytosterol homeostasis may be maintained in leaves through diversion of intermediates into glycoalkaloid biosynthesis, whereas in tuber flesh, excess intermediates are catalyzed by tuber-specific StLAS - like , resulting in low tuber glycoalkaloids. Lanosterol synthase (LAS) and cycloartenol synthase (CAS) are phylogenetically related enzymes. Cycloartenol is the accepted precursor leading to cholesterol and phytosterols, and in potato, to steroidal glycoalkaloid (SGA) biosynthesis. LAS was also shown to synthesize some plant sterols, albeit at trace amounts, questioning its role in sterol homeostasis. Presently, a potato LAS-related gene (StLAS-like) was identified and its activity verified in a yeast complementation assay. A transgenic approach with targeted gene expression and metabolic profiling of sterols and SGAs was used. Analyses of StLAS-like transcript levels and StLAS-like-promoter::GUS reporter assays indicated specific expression in tuber flesh tissue. Overexpression of Arabidopsis AtLAS in leaves where the endogenic StLAS-like is not expressed, resulted with increased SGA level and reduced phytosterol level, while in the tuber flesh SGA level was reduced. StLAS-like expression only in tuber flesh may explain the differential accumulation of SGAs in commercial cultivars-low in tubers, high in leaves. In leaves, to maintain phytosterol homeostasis, an excess of intermediates may be diverted into SGA biosynthesis, whereas in tuber flesh these intermediates are catalyzed by tuber-specific StLAS-like instead, resulting in low levels of SGA.

Identification of genes related to skin development in potato.

KEY MESSAGE: Newly identified genes that are preferentially expressed in potato skin include genes that are associated with the secondary cell wall and stress-related activities and contribute to the skin's protective function. Microarrays were used to compare the skin and tuber-flesh transcriptomes of potato, to identify genes that contribute to the unique characteristics of the skin as a protective tissue. Functional gene analysis indicated that genes involved in developmental processes such as cell division, cell differentiation, morphogenesis and secondary cell wall formation (lignification and suberization), and stress-related activities, are more highly expressed in the skin than in the tuber flesh. Several genes that were differentially expressed in the skin (as verified by qPCR) and had not been previously identified in potato were selected for further analysis. These included the StKCS20-like, StFAR3, StCYP86A22 and StPOD72-like genes, whose sequences suggest that they may be closely related to known suberin-related genes; the StHAP3 transcription factor that directs meristem-specific expression; and the StCASP1B2-like and StCASP1-like genes, which are two orthologs of a protein family that mediates the formation of Casparian strips in the suberized endodermis of Arabidopsis roots. An examination of microtubers induced from transgenic plants carrying GUS reporter constructs of these genes indicated that these genes were expressed in the skin, with little to no expression in the tuber flesh. Some of the reporter constructs were preferentially expressed in the inner layers of the skin, the root endodermis, the vascular cambium and the epidermis of the stem. Cis-regulatory elements within the respective promoter sequences support this gene-expression pattern.

Potato root system development and factors that determine its architecture.

The potato root system is often characterized as shallow and inefficient, with poor ability to extract water and minerals from the soil. Potato root system architecture (RSA) refers to its 3-dimensional structure as determined by adventitious root (AR) growth and branching through lateral roots (LR). Understanding how the root system develops holds potential to increase plant yield and optimize agricultural land use. Root system development was monitored in greenhouse-grown potato while a root-on-a-plate assay was developed to explore factors that affect AR and LR development. Expression study of LR-related genes was conducted. Transgenic plants carrying DR5:GFP and CycB1:GUS reporter genes were used to monitor auxin signaling and cell division during root primordia formation, respectively. Maximum root development occurred mainly during the 6-week post seed-tuber planting and slowed during the onset of tuberization. AR and LR development was coordinated - a positive correlation was found between the length of AR and LR and between LR length and number. The expression of LR-related genes was higher in LR than in AR. High nitrate levels reduced LR number and length, however ablation of root-cap by high temperature (33°C) or cutting resulted with enhanced formation of LR. Growth conditions affect AR and LR development in potato, determining the final architecture of its root system. The overall results indicate that LR formation in potato follows similar pattern as in model plants, facilitating study and manipulation of its RSA to improve soil exploitation and yield.

Silicon fertilization of potato: expression of putative transporters and tuber skin quality.

MAIN CONCLUSION: A silicon transporter homolog was upregulated by Si fertilization and drought in potato roots and leaves. High Si in tuber skin resulted in anatomical and compositional changes suggesting delayed skin maturation. Silicon (Si) fertilization has beneficial effects on plant resistance to biotic and abiotic stresses. Potatoes, low Si accumulators, are susceptible to yield loss due to suboptimal growth conditions; thus Si fertilization may contribute to crop improvement. The effect of Si fertilization on transcript levels of putative transporters, Si uptake and tuber quality was studied in potatoes grown in a glasshouse and fertilized with sodium silicate, under normal and drought-stress conditions. Anatomical studies and Raman spectroscopic analyses of tuber skin were conducted. A putative transporter, StLsi1, with conserved amino acid domains for Si transport, was isolated. The StLsi1 transcript was detected in roots and leaves and its level increased twofold following Si fertilization, and about fivefold in leaves upon Si × drought interaction. Nevertheless, increased Si accumulation was detected only in tuber peel of Si-fertilized plants--probably due to passive movement of Si from the soil solution--where it modified skin cell morphology and cell-wall composition. Compared to controls, skin cell area was greater, suberin biosynthetic genes were upregulated and skin cell walls were enriched with oxidized aromatic moieties suggesting enhanced lignification and suberization. The accumulating data suggest delayed tuber skin maturation following Si fertilization. Despite StLsi1 upregulation, low accumulation of Si in roots and leaves may result from low transport activity. Study of Si metabolism in potato, a major staple food, would contribute to the improvement of other low Si crops to ensure food security under changing climate.

Benzyladenine and gibberellin treatment of developing "Pink Lady" apples results in mature fruits with a thicker cuticle comprising clusters of epidermal cells.

A mixture of 6-benzyladenine (BA) and gibberellins GA4 plus GA7 applied to "Pink Lady" apple at early phenological stages was previously shown to result in an immediate increase in epidermal cell density and associated reduction in calyx-end cracking disorder in the mature fruit, implying a long-term effect of the BA + GA4+7 mixture. Here, we analyzed the anatomical changes in the mature peel at the calyx end 210 days after full bloom (DAFB), following application of the plant growth regulators (PGRs) at the cell-division phase of fruit development, 21-50 DAFB. Experiments were conducted in northern Israel, and the PGRs were applied as the commercial formulation Superlon™ (Fine Agrochemicals Ltd.), composed of 19 g l(-1) BA and 19 g l(-1) GA4+7. Trees were sprayed with 0.025, 0.1, or 0.2 % (v/v) Superlon™. The most obvious phenomenon was the presence of epidermal cell clusters within the cuticular matrix that were detached from the native epidermal layer located at the bottom of the cuticle and which could not be detected in the untreated control fruits. Treatment with 20 mg l(-1) BA + GA4+7 (0.1 % Superlon™) resulted in a markedly thicker cuticle, a higher percentage of detached epidermal cells within the cuticular membrane and a significant reduction in calyx-end cracking at harvest. The presence of cuticle-embedded epidermal cell clusters may have contributed to strengthening the peel by adding more cell-wall components, thickening the cuticle layer and possibly enhancing crack repair.

Anthocyanin synthesis in native and wound periderms of potato.

Skin color of red potatoes is due to accumulation of anthocyanins in the tuber periderm, a protective tissue that replaces the epidermis at an early stage of tuber development. The periderm consists of external layers of suberized phellem cells making up the skin, and internal layers of parenchyma-like phelloderm cells. Red pigmentation is an important marketing factor for red-skinned potatoes. However, injuries to the tuber surface, which are common in the potato industry, result in the development of a wound periderm that is devoid of the characteristic red coloration. To study the reason for these differences in anthocyanin accumulation, the expression level of anthocyanin biosynthesis genes and regulators was monitored in native and wound periderm using microarray analysis and quantitative polymerase chain reaction. We found significantly higher expression of the anthocyanin pathway in the phelloderm cells compared with the skin and tuber-flesh samples. However, in wound periderm, the anthocyanin pathway was strongly downregulated relative to the native periderm. This was true for two developmental stages of the native periderm--'immature', when the skin is prone to skinning injuries, and 'mature', following skin set--suggesting that anthocyanin synthesis continues postharvest. Wound-induced expression of steroidal glycoalkaloid glycosyltransferases, suberin-related 3-ketoacyl-CoA synthase and actin indicated that downregulation of the anthocyanin-specific pathway does not reflect global repression of the wound-periderm transcriptome. Loss of pigmentation may result from reduced expression of the Myb-bHLH-WD40 anthocyanin regulatory complex--a possible candidate might be the bHLH transcription factor JAF13.

Root architecture and root and tuber crop productivity.

It is becoming increasingly evident that optimization of root architecture for resource capture is vital for enabling the next green revolution. Although cereals provide half of the calories consumed by humans, root and tuber crops are the second major source of carbohydrates globally. Yet, knowledge of root architecture in root and tuber species is limited. In this opinion article, we highlight what is known about the root system in root and tuber crops, and mark new research directions towards a better understanding of the relation between root architecture and yield. We believe that unraveling the role of root architecture in root and tuber crop productivity will improve global food security, especially in regions with marginal soil fertility and low-input agricultural systems.

Allelic variation in genes contributing to glycoalkaloid biosynthesis in a diploid interspecific population of potato.

KEY MESSAGE: Variation for allelic state within genes of both primary and secondary metabolism influences the quantity and quality of steroidal glycoalkaloids produced in potato leaves. Genetic factors associated with the biosynthesis and accumulation of steroidal glycoalkaloids (SGAs) in potato were addressed by a candidate gene approach and whole genome single nucleotide polymorphism (SNP) genotyping. Allelic sequences spanning coding regions of four candidate genes [3-hydroxy-3-methylglutaryl coenzyme A reductase 2 (HMG2); 2,3-squalene epoxidase; solanidine galactosyltransferase; and solanidine glucosyltransferase (SGT2)] were obtained from two potato species differing in SGA composition: Solanum chacoense (chc 80-1) and Solanum tuberosum group Phureja (phu DH). An F2 population was genotyped and foliar SGAs quantified. The concentrations of α-solanine, α-chaconine, leptine I, leptine II and total SGAs varied broadly among F2 individuals. F2 plants with chc 80-1 alleles for HMG2 or SGT2 accumulated significantly greater leptines and total SGAs compared to plants with phu DH alleles. Plants with chc 80-1 alleles at both loci expressed the greatest levels of total SGAs, α-solanine and α-chaconine. A significant positive correlation was found between α-solanine and α-chaconine accumulation as well as between leptine I and leptine II. A whole genome SNP genotyping analysis of an F2 subsample verified the importance of chc 80-1 alleles at HMG2 and SGT2 for SGA synthesis and accumulation and suggested additional candidate genes including some previously associated with SGA production. Loci on five and seven potato pseudochromosomes were associated with synthesis and accumulation of SGAs, respectively. Two loci, on pseudochromosomes 1 and 6, explained phenotypic segregation of α-solanine and α-chaconine synthesis. Knowledge of the genetic factors influencing SGA production in potato may assist breeding for pest resistance.

Sequence diversity in coding regions of candidate genes in the glycoalkaloid biosynthetic pathway of wild potato species.

Natural variation in five candidate genes of the steroidal glycoalkaloid (SGA) metabolic pathway and whole-genome single nucleotide polymorphism (SNP) genotyping were studied in six wild [Solanum chacoense (chc 80-1), S. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, S. stoloniferum] and cultivated S. tuberosum Group Phureja (phu DH) potato species with contrasting levels of SGAs. Amplicons were sequenced for five candidate genes: 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2) and 2.3-squalene epoxidase (SQE) of primary metabolism, and solanidine galactosyltransferase (SGT1), and glucosyltransferase (SGT2) of secondary metabolism. SNPs (n = 337) producing 354 variations were detected within 3.7 kb of sequenced DNA. More polymorphisms were found in introns than exons and in genes of secondary compared to primary metabolism. Although no significant deviation from neutrality was found, dN/dS ratios < 1 and negative values of Tajima's D test suggested purifying selection and genetic hitchhiking in the gene fragments. In addition, patterns of dN/dS ratios across the SGA pathway suggested constraint by natural selection. Comparison of nucleotide diversity estimates and dN/dS ratios showed stronger selective constraints for genes of primary rather than secondary metabolism. SNPs (n = 24) with an exclusive genotype for either phu DH (low SGA) or chc 80-1 (high SGA) were identified for HMG2, SQE, SGT1 and SGT2. The SolCAP 8303 Illumina Potato SNP chip genotyping revealed eight informative SNPs on six pseudochromosomes, with homozygous and heterozygous genotypes that discriminated high, intermediate and low levels of SGA accumulation. These results can be used to evaluate SGA accumulation in segregating or association mapping populations.