The potato R locus codes for dihydroflavonol 4-reductase

The potato R locus is required for the production of red pelargonidin-based anthocyanin pigments in potato (Solanum tuberosum L.). Red color also requires tissue-specific regulatory genes, such as D (for expression in tuber skin) and F (expression in flowers). A related locus, P, is required for production of blue/purple anthocyanins; P is epistatic to R. We have previously reported that the dihydroflavonol 4-reductase gene (dfr) co-segregates with R. To test directly whether R corresponds to dfr, we placed the allele of dfr associated with red color under the control of the CaMV 35S promoter and introduced it into the potato cultivar Prince Hairy (genotype dddd rrrr P-), which has white tubers and pale blue flowers. Transgenic Prince Hairy tubers remained white, but flower color changed to purple. Three independent transgenic lines, as well as a vector-transformed line, were then crossed with the red-skinned variety Chieftain (genotype D-R-pppp), to establish populations that segregated for D, R, P, and the dfr transgene or empty vector. Markers were used to genotype progeny at D and R. Progeny carrying the empty vector in the genetic background D-rrrr produced white or purple tubers, while progeny with the same genotype and the dfr transgene produced red or purple tubers. HPLC and LC–MS/MS analyses of anthocyanins present in Chieftain and in a red-skinned progeny clone with the dfr transgene in a D-rrrr background revealed no qualitative differences. Thus, dfr can fully complement R, both in terms of tuber color and anthocyanin composition.     

An allele of dihydroflavonol 4-reductase associated with the ability to produce red anthocyanin pigments in potato (Solanum tuberosum L.)

The potato R locus is necessary for the production of red pelargonidin-based anthocyanin pigments in any tissue of the plant, including tuber skin and flower petals. The production of pelargonidins in plants requires the activity of dihydroflavonol 4-reductase (DFR) to catalyze the reduction of dihydrokaempferol into leucopelargonidin. To test the hypothesis that potato R encodes DFR, portions of both dfr alleles were sequenced from a diploid potato clone known to be heterozygous Rr. Sequence comparison revealed a polymorphic BamHI restriction site. The presence or absence of this site was monitored in three diploid populations that segregated for R, as well as in a wide range of tetraploid breeding clones and cultivars, by amplifying a fragment of dfr and digesting the products with BamHI. An identically sized dfr restriction fragment lacking the BamHI site was present in all potato clones that produced red anthocyanin pigments, while the same fragment was absent in many potato clones with white tuber skin and flowers. An independent RFLP test using DraI to detect sequence polymorphism was performed on a subset of the potato clones. This test also revealed dfr-derived bands that were present in all red-colored potatoes and absent in several white clones. The presence of shared restriction fragments in all red-colored potatoes provides strong evidence that R does indeed code for DFR. The data are also consistent with a 48 year-old hypothesis by Dodds and Long, that R was selected just once during the domestication of potato. A cDNA clone corresponding to the red allele of dfr was sequenced and compared to two other alleles. The red allele is predicted to encode a 382 amino acid protein that differs at ten amino acid positions from the gene products of the two alternative alleles. Several of these differences map in a region known to influence DFR substrate specificity in Gerbera.Communicated by J. Dvorak     

Application of high-resolution DNA melting for genotyping and variant scanning of diploid and autotetraploid potato

The ideal marker system for tetraploid potato would be dosage-sensitive and have the ability to distinguish heterozygous genotypes with multiple haplotypes within the genomic region targeted by the marker. The objective of this study was to evaluate the utility of high-resolution DNA melting (HRM) for genotyping and polymorphism detection in diploid and tetraploid potato. Amplicon scanning, unlabelled probe, and short amplicon assays were developed for four candidate genes affecting tuber skin and flesh colour, and starch, and a marker linked to nematode resistance. Genotyping a set of 95 potato clones revealed several examples of clones with three distinct haplotypes. Combined probe and amplicon analysis identified between 29 and 44 unique genotypes for the same assays. Assays developed for four of the five target genes are suitable for marker-assisted selection in potato breeding programs. This study illustrates the use of HRM in potato genetics. Further advances in the technology and associated data analysis should make HRM a useful tool for basic and applied studies of potato.     

Development of a codominant CAPS marker for allelic selection between canary yellow and red watermelon based on SNP in lycopene β-cyclase (<Emphasis Type="Italic">LCYB</Emphasis>) gene

Flesh color of watermelon is an agronomically important trait that is predominantly determined by a network of the carotenoid biosynthetic pathway, which also contributes to the nutritional value of the fruit through the health-promoting function of carotenoids. We have identified a key gene, lycopene β-cyclase (LCYB) that may determine canary yellow and red flesh color of watermelon and developed a zero-distance molecular marker that identifies a critical single nucleotide polymorphism (SNP) that distinguishes different alleles of the LCYB gene. Analysis of the flesh color inheritance in segregating populations indicated that a single gene determines the color difference between canary yellow and red flesh in watermelon. The sequence comparison of full-length cDNA of LCYB, which was isolated using degenerate PCR and RACE, identified three SNPs in the coding region of LCYB between canary yellow and red. These SNPs showed perfect co-segregation with flesh color phenotypes. One of the SNPs introduces an amino acid replacement of evolutionarily conserved Phe226 to Val, which may impair the catalytic function of LCYB. This SNP was used to develop a cleaved amplified polymorphic sequence (CAPS) marker, which perfectly cosegregated with flesh color phenotype. Our results strongly suggest that LCYB may be the genetic determinant for canary yellow or red flesh color and our CAPS marker will allow breeders to economically distinguish between canary yellow and red watermelon fruit color at the seedling stage.     

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.     

Validation of candidate gene markers for marker-assisted selection of potato cultivars with improved tuber quality

Tuber yield, starch content, starch yield and chip color are complex traits that are important for industrial uses and food processing of potato. Chip color depends on the quantity of reducing sugars glucose and fructose in the tubers, which are generated by starch degradation. Reducing sugars accumulate when tubers are stored at low temperatures. Early and efficient selection of cultivars with superior yield, starch yield and chip color is hampered by the fact that reliable phenotypic selection requires multiple year and location trials. Application of DNA-based markers early in the breeding cycle, which are diagnostic for superior alleles of genes that control natural variation of tuber quality, will reduce the number of clones to be evaluated in field trials. Association mapping using genes functional in carbohydrate metabolism as markers has discovered alleles of invertases and starch phosphorylases that are associated with tuber quality traits. Here, we report on new DNA variants at loci encoding ADP-glucose pyrophosphorylase and the invertase Pain-1, which are associated with positive or negative effect with chip color, tuber starch content and starch yield. Marker-assisted selection (MAS) and marker validation were performed in tetraploid breeding populations, using various combinations of 11 allele-specific markers associated with tuber quality traits. To facilitate MAS, user-friendly PCR assays were developed for specific candidate gene alleles. In a multi-parental population of advanced breeding clones, genotypes were selected for having different combinations of five positive and the corresponding negative marker alleles. Genotypes combining five positive marker alleles performed on average better than genotypes with four negative alleles and one positive allele. When tested individually, seven of eight markers showed an effect on at least one quality trait. The direction of effect was as expected. Combinations of two to three marker alleles were identified that significantly improved average chip quality after cold storage and tuber starch content. In F1 progeny of a single-cross combination, MAS with six markers did not give the expected result. Reasons and implications for MAS in potato are discussed.     

Engineering heat tolerance in potato by temperature‐dependent expression of a specific allele of HEAT‐SHOCK COGNATE 70

For many commercial potato cultivars, tuber yield is optimal at average daytime temperatures in the range of 14–22 °C. Further rises in ambient temperature can reduce or completely inhibit potato tuber production, with damaging consequences for both producer and consumer. The aim of this study was to use a genetic screen based on a model tuberization assay to identify quantitative trait loci (QTL) associated with enhanced tuber yield. A candidate gene encoding HSc70 was identified within one of the three QTL intervals associated with elevated yield in a Phureja–Tuberosum hybrid diploid potato population (06H1). A particular HSc70 allelic variant was linked to elevated yield in the 06H1 progeny. Expression of this allelic variant was much higher than other alleles, particularly on exposure to moderately elevated temperature. Transient expression of this allele in Nicotiana benthamiana resulted in significantly enhanced tolerance to elevated temperature. An TA repeat element was present in the promoter of this allele, but not in other HSc70 alleles identified in the population. Expression of the HSc70 allelic variant under its native promoter in the potato cultivar Desiree resulted in enhanced HSc70 expression at elevated temperature. This was reflected in greater tolerance to heat stress as determined by improved yield under moderately elevated temperature in a model nodal cutting tuberization system and in plants grown from stem cuttings. Our results identify HSc70 expression level as a significant factor influencing yield stability under moderately elevated temperature and identify specific allelic variants of HSc70 for the induction of thermotolerance via conventional introgression or molecular breeding approaches.     

Differential stress responses to NaCl salt application in early- and late-maturing diploid potato (Solanum sp.) clones

Cultivated tetraploid potatoes (Solanum tuberosum L.) are moderately salt sensitive but greater stress tolerance exists in diploid wild types. However, little work has been published on salt-tolerance in diploid potato. This study utilized sensitive and tolerant diploid potatoes as well as a commercially cultivated potato to investigate mechanisms of stress tolerance. Stem cuttings from salt-tolerant (T) and sensitive (S) clones of early-maturing (EM) and late-maturing (LM) diploid potato clones were stressed for 5 days at the tuber initiation stage with 150 mmol NaCl in a hydroponic sand culture under greenhouse conditions. The stress responses of the early- and late-maturing potato clones were distinctly different. Under stress, early-maturing clones accumulated Na⁺ in the leaf tissues while late-maturing clones generally excluded Na⁺ from the leaf tissues. Salt tolerant clones of both maturity types were able to tolerate high levels of Na⁺ in the leaf tissues. The lower leaves accumulated more Na⁺ than the upper leaves in both maturity types. The potassium to sodium ratio was significantly greater in the leaves of the late-maturing types, reflecting differences in Na⁺ accumulation rather than alterations in K⁺ levels. Proline levels increased upon salt exposure but were not clearly associated with salinity tolerance. Tolerance was manifested in maintenance of vegetative growth, tuber yield, and reduced leaf necrosis. These responses require efficient uptake of water and source–sink translocation. Maintenance of stomatal conductance under stress was not associated with these responses but tuber yield was related to lower-leaf osmotic potential (OP) in both early- and late-maturity types. Salt tolerant clones of both maturity types also had less negative tuber OP under salt stress than sensitive types. High yielding EMT and LMT clones either minimized tuber yield loss or even increased yield after exposure to salt stress. Mechanistic studies and screening experiments for salt tolerant clones should consider maturity type, leaf position and source–sink relationships enhancing tuber yield.     

Producing commercially attractive,uniform true potato seed progenies: the influence of breeding scheme and parental genotype

The production of attractive, uniform true potato seed (TPS) progenies was investigated. Four breeding schemes were compared: intercrossing tetraploid cultivars (cv x cv); doubled dihaploids x cultivars (ddh x cv); cultivars x diploid unreduced-gamete producers (cv x FDR) and doubled dihaploids x diploid unreduced-gamete producers (ddh x FDR). Fifty three progenies and five clones were grown in a glasshouse in a randomised complete block design with three replicates of 25 plants per progeny and clone. Each plant's tubers were counted and the colour, shape, quality of skin finish, flesh colour, and commencai attractiveness (which includes yield) recorded. The most uniform progenies were also selected by visual comparison with the clones. For mean attractiveness, differences (P < 0.001) between breeding schemes and between progenies within breeding schemes were detected. The cv x cv and cv x FDR progenies were more attractive than clonal controls. There were significant additive and non-additive effects for attractiveness in all breeding schemes except cv x FDR. There were between-progeny differences (P < 0.001) for uniformity for all characters. Progenies uniform for one character could be variable for other traits. Breeding schemes gave different levels of uniformity (P < 0.001) for all characters except shape and flesh colour, but none gave low levels of variation for all traits. Doubled-dihaploid parents increased the variation in progenies. There were uniformity differences (P < 0.001) between progenies within breeding schemes for all characters. Evidence of additive and nonadditive genetic variation for uniformity in all traits was detected. In each breeding scheme, parents with good general combining ability (GCA) for uniformity in several characters were identified. Visually selected uniform progenies had parents with good GCAs for uniformity in a range of traits and high specific combining abilities (SCAs) for several traits. A desynaptic first-division restitution (FDR) clone and a male-sterile doubled-dihaploid clone had the best GCAs for tuber uniformity in TPS progenies. Achieving multitrait uniformity in TPS is problematic but may be aided by the selection of parents with GCAs for uniformity coupled with progeny testing to allow for non-additive effects.     

Selection of individual tubers in potato breeding

Summary Out of 720 field-grown potato first year seedlings plants with best tuber apearance and large and medium size tubers with best shape were respectively selected. The tuber progeny of each group was evaluated in field experiments. It was demonstrated that the selection of individual tubers was very effective in the elimination of clones with irregular tuber shape and deep eyes. The tuber progeny of selected tubers was not inferior to the tuber progeny of selected plants. Possible applications of the results to practical potato breeding are discussed.     

Inheritance and genetic mapping of tuber eye depth in cultivated diploid potatoes

Tuber eye depth of the potato (Solanum tuberosum L.) is an important trait for the processing quality and appearance of potatoes. In the present study, we used a cultivated diploid potato family (12601) of 107 plants to dissect the mode of inheritance and to map the gene(s) controlling the trait. The family segregated for both eye depth (deep vs shallow) and tuber shape (round vs long) traits. The deep eye (Eyd) phenotype was found to be associated with round tubers (Ro) in most progeny clones. Further evaluation of this population with molecular markers including simple sequence repeats, amplified fragment length polymorphism, and sequence-characterized amplified regions revealed that the primary locus for eye depth is located on chromosome 10. This map location was confirmed by evaluating a second diploid family (12586). The results of this study led to the following conclusions: (1) there is a major locus controlling the eye depth trait; (2) deep eye (Eyd) is dominant to shallow (eyd); (3) the Eyd/eyd locus is located on chromosome 10; and (4) the Eyd/eyd locus is closely linked with the major locus for tuber shape (Ro/ro), at a distance of about 4 cM.     

Field evaluation of tetraploid somatic potato hybrids

Tetraploid somatic hybrids, derived from ten different fusion combinations, were grown in the field to study the inheritance of quantitatively-controlled agronomic traits. The data from two seasons showed that the heterosis in yield varied between 70% and 230% compared to the mid-parent value of the dihaploid fusion clones. The relative yield of the hybrids compared to two standard varieties (= 100%) ranged from 47% to 106%. Despite large differences in yield between the single hybrids of the same fusion combination, no significant differences could be detected between the hybrids. The analysis of the yield components revealed that the heterosis was mainly due to a highly increased tuber weight, whereas the tuber number was in general intermediate to that of the parent clones. In three combinations, the starch content of the hybrids was 15–20% higher than the midparent value and in the others it was intermediate. Fusion of clones with long oval tubers and with round or round-oval ones showed the round/round-oval form was dominant. Fusion combinations of tuber flesh colour indicated that in most cases yellow was dominant to white, while red skin colour was dominant over yellow.     

Novel somatic hybrids (Solanum tuberosum L. + Solanum tarnii) and their fertile BC1 progenies express extreme resistance to potato virus Y and late blight

Solanum tarnii, a wild diploid, tuber-bearing Mexican species belonging to the series Pinnatisecta is highly resistant to Potato virus Y (PVY) and Colorado potato beetle and shows a strong hypersensitive reaction to Phytophthora infestans. Therefore, it could be a potential source of resistance to pathogens for potato breeders. S. tarnii (2n = 2x = 24) is reproductively isolated from tetraploid Solanum tuberosum and hence difficult to include in potato breeding programmes. In this study, interspecific somatic hybrids were produced for the first time by protoplast electrofusion of the cells of potato cv. Delikat (Solanum tuberosum L.) and Solanum tarnii. The hybrid nature of the regenerants was confirmed by simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers and by morphological analysis and flow cytometry. Selected somatic hybrids were successfully backcrossed with cv. Delikat. Parental lines, primary somatic hybrids and BC₁ progeny were assessed for resistance to PVY by mechanical inoculation, grafting and exposure to viruliferous aphid vectors in the field, and resistance to late blight (P. infestans) by detached leaflet and whole tuber tests. The somatic hybrids showed no symptoms of viral infection and most of them displayed high levels of resistance to foliage blight. The BC₁ progenies were highly resistant to PVY and a few were resistant to foliage blight. Selected hybrids and BC₁ clones were evaluated in the field for tuber quality and tuber yield. Some BC₁ clones produced yields of good quality tubers. The results confirm that both the resistance to PVY and to late blight of S. tarnii is expressed in somatic hybrids, and PVY resistance is transferred to BC₁ progeny, whereas blight resistance is harder to transfer. Somatic hybridization again proved to be a valuable tool for producing pre-breeding material with increased genetic diversity.     

Development of a molecular marker associated with Verticillium wilt resistance in diploid interspecific potato hybrids

Verticillium wilt (VW) is a widespread and serious potato (Solanum tuberosum) disease caused by the soilborne fungi Verticillium dahliae and V. albo-atrum. Breeding for VW resistance in potato is challenging due to ambiguous symptom expression, a lack of high throughput screening techniques, and variability in colonization by the fungus among and within plants. Genetic studies have identified major genes that confer resistance in diploid Solanum chacoense (V _c ) and interspecific hybrids (V _w and V _t ). However, to date, these genes have not been used to develop molecular markers for the identification of resistant clones. Tomato Ve1 and Ve2 gene sequence information was used to amplify candidate Ve gene orthologs from both resistant and susceptible diploid potato hybrids. A CAPS marker was generated to track VW resistance in a backcross population segregating for resistance. The marker was also tested for its usefulness in other breeding lines. Our results indicate that this marker is effective for selection of the V _w gene in segregating breeding populations.     

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.     

Two allelic or tightly linked genetic factors at the <Emphasis Type="Italic">PLRV.4</Emphasis> locus on potato chromosome XI control resistance to potato leafroll virus accumulation

A novel locus for potato resistance to potato leafroll virus (PLRV) was characterized by inheritance studies and molecular mapping. The diploid parental clone DW 91-1187 was resistant to PLRV accumulation in both inoculated plants and their tuber progeny. The resistance to PLRV accumulation present in DW 91-1187 was not transmitted to any F₁ offspring when crossed with a PLRV susceptible clone. Instead, one half of the F₁ individuals exhibited undetectable amounts of PLRV as determined by ELISA during the primary infection assay, but accumulated PLRV in their tuber progeny plants. The other half was clearly infected both in the inoculated and tuber-born plants. The inheritance of resistance to PLRV accumulation may be explained by a model of two complementary alleles of a single gene (PLRV.4) or by two complementary genes that are closely linked in repulsion phase. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers linked to the PLRV.4 locus were selected. The two complementary factors were closely linked in coupling phase to the alternative alleles UBC864₆₀₀ and UBC864₈₀₀ of DNA marker UBC864. These markers may be used for marker-assisted selection of genotypes having both factors for resistance to PLRV accumulation. The PLRV.4 locus was mapped to a central position on linkage group XI of the potato molecular map, where no resistance locus has been mapped previously.     

The effects of inbreeding on the parental values of potato dihaploids

Dihaploids obtained from a somatically chromosome-doubled dihaploid potato were crossed with Solanum phureja clones. To test the effect of inbreeding, measurements were made of their seed production and the tuber yield, tuber number and mean tuber weight of their offspring. On average, seed production of the second generation dihaploids was higher than that of the original dihaploid progenitor. Progeny tuber yield and its components were little different from those of the original dihaploid's progeny. Tuber flesh quality, as measured lack of blemishes, was better in the offspring of second generation dihaploids.
It is suggested that the negative effects of producing second generation dihaploids are minor compared with producing the first generation dihaploid from a tetraploid, because most deleterious recessives have already been unmasked. The results indicated residual variation within the original dihaploid which could be exploited for plant breeding purposes.
An indicator of inbred status, alternative to the inbreeding coefficient, is suggested which could be applied to both diploids and tetraploids.     

Alfalfa transposable elements and variegation

Alfalfa with unstable anthocyanin pigmentation has been independently discovered on six occasions since 1958. Genetic studies showed that each of the six unstable stocks was due to an allele mutable at the basic anthocyanin locus C2 in alfalfa. The alleles are designated c2-m1 through c2-m6. Variegated phenotypes of m1, m2, and m3 are similar and express reversion from the recessive to the dominant state. This reversion produces streaks and sectors of pigment in flower petals and seeds that are otherwise white. Reversion occurs at various times in development and may result in periclinal chimeras. The c2-m4 allele is unique in that it arose during tissue culture, whereas the other mutables were discovered in plant populations. Interestingly, m4 is very stable in planta and only rarely produces a sectored flower, but is very unstable in vitro as measured by about 23% revertant plants regenerated from tissue cultures. Most m4 reversion occurs relatively early in development and results in completely pigmented in vitro revertants, and in large sectors on in planta revertants. Alleles m5 and m6 are phenotypically and genetically similar. Their flowers are basic purple with white streaks thus representing mutation from dominant purple to recessive white. White progeny of m5 and m6 are very stable both in planta and in vitro; reversion of white to purple was never observed. Thus, the loss of function of the dominant allele results in a stable recessive or a deficiency. The absolute stability of m5 white derivatives favors the deficiency model, because transposable element mutations might show reversion. Finally, several mutations are described that reoccur in the mutable populations. It is speculated that they are recent mutations due to transposition of transposable elements.