Identification of RAPD markers linked to the Ns locus in potato

Using the RAPD method and bulked segregant analysis we identified four RAPD markers linked to a dominant gene Ns, responsible for a hypersensitive reaction of potato (Solanum tuberosum L.) to potato virus S (PVS) infection. The markers OPE15⁵⁵⁰, OPJ13⁵⁰⁰, OPG17⁴⁵⁰ and OPH19⁹⁰⁰ were found to be closely linked to the Ns gene in diploid potato clones. They are situated at 2.6, 3.3, 4.6 and 6.6 cM from Ns, respectively. As a source of the gene, clone G-LKS 678147/60, which is known to carry Ns transferred from S. tuberosum ssp. andigena was used. These RAPD markers were not amplified in resistant tetraploid clones containing Ns derived from the clone MPl65 118/3, also having an andigenum origin. This suggests that there may be two separate loci of Ns in the sources identified, or different alleles with the same specificity at a single locus, or that the genetic background of tetraploids tested results in different RAPD amphlification patterns.     

The importance of wild potato species resistant to the potato cyst nematode,Globodera pallida,pathotypes P4A and P5A,in potato breeding. II. The crossability of resistant species

Summary Over 2400 pollinations were made to investigate the crossability relationships between cultivated potatoes, and wild diploid species from Series Tuberosa and Circaeifolia, as well as wild polyploid species in Series Tuberosa and Longipedicellata resistant to potato cyst nematode, Globodera pallida pathotypes P₄A and P₅A. Wild diploids in Series Tuberosa crossed easily with cultivated diploid species, except with Solanum lignicaule where most pollinations failed, and seed set was extremely low (0.2 seeds per pollination or less). It is suggested that this species is 1EBN. S. capsicibaccatum is clearly isolated from Series Tuberosa, but can form hybrids with S. lignicaule, which can act as a bridging species to S. tuberosum haploids. S. gourlayi and S. oplocense can be crossed with both subspecies of S. tuberosum, but S. papita, Series Longipedicellata is reproductively isolated from the tetraploid cultigens. The crossability data are discussed in the light of germplasm utilisation for breeding potato varieties resistant to potato cyst nematode.     

Linkage and quantitative trait locus mapping of foliage late blight resistance in the wild species Solanum vernei

The global cultivation of potato (Solanum tuberosum) is threatened by epidemics caused by new variants of the late blight pathogen, Phytophthora infestans. New sources of durable late blight resistance are urgently needed and these may be found in wild Solanum species. The diploid wild species, S. vernei, has not previously been subjected to mapping of quantitative trait loci (QTLs) for late blight resistance. Two populations designated HGIHJS and HGG, originating from a cross between a clone of S. vernei and two different S. tuberosum clones were evaluated in field trials for late blight infestation. The relative area under the disease progress curve (RAUDPC) was estimated and used for QTL mapping. A linkage map of S. vernei, comprising 11 linkage groups, nine of which could be assigned to chromosomes, was constructed. Results indicated that the resistance in S. vernei was quantitatively inherited. Significant QTLs for late blight resistance were identified on chromosomes VIII (HGG), VI and IX (HGIHJS). In addition, potential QTLs were detected on chromosomes VII (HGIHJS) and IX (HGG). A putative and a significant QTL for tuber yield were found on chromosomes VI and VII in HGG, but no linkage between yield and resistance was indicated. The QTL for late blight resistance, which mapped to chromosome IX, could be useful for late blight resistance breeding as it was located close to the microsatellite marker STM1051 in both populations.     

Induction of novel organelle DNA variation and transfer of resistance to frost and Verticillium wilt in Solanum tuberosum through somatic hybridization with 1EBN S. commersonii

Somatic hybridization can be used to induce genetic variability in plastidial and mitochondrial genomes, and transfer multiple uncloned genes across sexual barriers. Somatic hybrids were produced between a dihaploid clone of the common potato, S. tuberosum subsp. tuberosum, and the wild sexually incongruent diploid species S. commersonii. Fusion products were selected on the basis of callus growth and regeneration in vitro. Genome composition of putative somatic hybrids was determined by flow cytometric analysis of nuclear DNA content, RAPD analysis, and Southern analysis with probes specific to organellar DNA. All regenerated fusion products proved to be hybrids based on RAPD analysis. Seventy per cent of somatic hybrids were (near) tetraploids, 22% (near) hexaploids and 8%(near) octoploids. A high correlation was found between the nuclear DNA content determined by flow cytometry and the number of chloroplasts in stomata guard cell pairs. Somatic hybrids inherited the parental plastids in a random manner. On the contrary, they preferentially inherited the mitochondrial DNA fragments of S. tuberosum. The majority of them had a rearranged mitochondrial genome with fragments from both parents. Hybrids were highly vigorous and morphologically more similar to the cultivated than to the wild parent, produced tubers on long stolons under long photoperiod conditions, showed a high degree of flowering, but did not produce pollen. In addition, somatic hybrids were generally more resistant to frost and Verticillium wilt than the cultivated parent, indicating the introgression of relevant resistance genes from the wild species into the genetic background of S. tuberosum. This revised version was published online in July 2006 with corrections to the Cover Date.     

The breeding potential of wild potato species resistant to the potato tuber moth,Phthorimaea operculella (Zeller)

Summary The potato tuber moth, Phthorimaea operculella (Zeller) is an important pest of potatoes in the field and in stores in warm environments throughout the world. In this study genetic resistance to potato tuber moth was identified in clones of Solanum sparsipilum (coded MBN) originally developed for resistance to bacterial wilt and root-knot nematode. Resistance to this pest in S. sucrense and S. tarijense as well as S. sparsipilum was exploited in wide crosses with diploid and tetraploid cultivated potatoes, and haploids derived from S. tuberosum; hybrid progenies were produced. Crosses between resistant S. pinnatisectum or S. commersonii and cultivated potatoes failed completely, although S. commersonii did hybridise with two bridging species S. lignicaule and S. capsicibaccatum which are slightly compatible with cultivated potatoes. Resistance to potato tuber moth was transferred to all progenies except those in which S. tarijense was the resistant parent. The development of potatoes resistant to potato tuber moth is discussed in the context of population breeding for the lowland tropics.     

Fertile somatic hybrids of Solanum etuberosum (+) dihaploid Solanum tuberosum and their backcrossing progenies: relationships of genome dosage with tuber development and resistance to potato virus Y

The wild non-tuberous species Solanumetuberosum is resistant to biotic andabiotic stresses, but is very difficult tocross with cultivated potato. Therefore,interspecific somatic hybrids between adihaploid clone of potato S.tuberosum (2n=2x=24, AA genome) and thediploid species S. etuberosum(2n=2x=24, EE genome) were produced byprotoplast fusion. Among the 7 fertilefusion hybrids analysed by genomic insitu hybridisation (GISH), three groups ofplants were found with the genomicconstitution of AAEE, AAEEEE and AAAAEE.Four fusion hybrids had exactly theexpected chromosome composition, while eachof the three aneuploid hybrids had lost twochromosomes of S. etuberosum. Twobackcross progenies were developed, andGISH analysis was applied to analysetransmission of the parental chromosomesinto the sexual generations. BC₁hybrids derived from the crosses of thehexaploid somatic hybrids with tetraploidpotato were pentaploid with thetheoretically expected genomic compositionor with slight deviation from thisexpectation. In the three BC₂ hybridsanalysed by GISH seven to 12 chromosomes ofS. etuberosum were detected in thepredominant S. tuberosum background.No recombinant chromosomes in the hybridswere detected. Genome dosage affects tuberformation in hybrids and their progenies,but has less effect on resistance to potatovirus Y (PVY) in fusion hybrids. Severalgenotypes of the fusion hybrids andBC₁ progeny did not show viralinfection even in the graftingexperiments.     

Introduction of an amylose-free (amf) mutant into breeding of cultivated potato,Solanum tuberosum L.

Summary Using an amylose-free (amf)mutant of diploid potato (Solanum tuberosum), diploid and tetraploid clones with different genotypes at the amf-locus were produced. In order to make use of the diploid material in analytic breeding of amf-potatoes, clones were selected that produced a considerable frequencies of 2n-pollen and 2n-eggs. Successful attempts were made to select normal synaptic as well as desynaptic clones producing 2n-gametes. Based on the phenotype of starch in the microspores, tetraploid clones with nulliplex, simplex and duplex genotypes at the amf-locus were selected. Prospects of using this material in conventional as well as in analytic breeding of potato are discussed.     

The Importance of the Bolivian Wild Potato Species in Breeding for Globodera pallida Resistance

Screening for resistance to the potato cyst nematode, Globodera pallida, in potatoes from. Bolivia, was carried out in 1983 and 1984, using a mixture of four nematode populations representing pathotypes Pa₁, Pa₂ and Pa₃ From the 66 accessions of 17 species and subspecies evaluated, highly resistant genotypes were identified in 21 accessions from seven species. All had Pf/Pi values of 2 or less, whereas the susceptible control, Solanum tuberosum cv. ‘Disiree’ had Pf/Pi values of more than 2G in both tests. Two diploid wild species, S. brevicaule and S. leptophyes, showed the best resistant. The geographical distributional of resistant populations and the evolution of resistance in wild potato populations are discussed.     

Flowering behaviour,male sterility and berry setting in tetraploid Solanum tuberosum germplasm

Summary Six hundred and seventy six accessions of cultivated potato (Solanum tuberosum ssp. tuberosum) from 25 countries, were studied for flowering and fruiting behaviour under long days (12–14 h). Flowering intensity ranged from dropping of floral buds just after initiation to profuse blooming. The majority (58.3%) of the accessions bloomed profusely, though 20.4% of the accessions did not bloom at all. Weeks to flowering ranged from 6 to 15 and the majority (66.5%) of the flowering accessions bloomed within 8 to 9 weeks after planting. Duration of flowering ranged from 1 to 10 weeks and the majority (68.1%) of the flowering accessions bloomed for 1 to 4 weeks only. Twentythree per cent of the flowering accessions were completely male sterile. Maximum male fertility was 90% only. No berry setting was observed in 31.8% of the flowering accessions. Only 54.3 per cent of the accessions were found to be fertile in all respects and could be used both as male and female parents. Premature bud abscission was the major cause of sterility. Peru was the best source of profuse-flowering genotypes, Poland was the best source of early flowering genotypes and Mexico was the best source of long duration flowering and good berry setting genotypes. The results suggested that flower bud formation; the growth and development of mature flowers; weeks to flowering and duration of flowering are independent characters controlled by different genes of quantitative nature. Berry setting and duration of flowering were closely associated (r=0.95). Genetic as well as environmental factors interfered with the developmental process leading to flower production and berry setting at different times in different genotypes. The practical implications of these results for true potato seed production are discussed.Publication No. 1298, CPRI, Shimla.     

Inheritance and stability of the virus-resistant gene in the progeny of transgenic sweet potato

Viral diseases of sweet potato are very prevalent and often seriously damaging to the plants. In particular, the severe strain of the sweet potato feathery mottle virus (SPFMV‐S) causes ‘obizyo‐sohi’ disease in Japan. In order to confer viral resistance against SPFMV using current biotechnology, a transgenic sweet potato has been produced, introducing hygromycin‐resistant (hpt) and SPFMV‐S coat protein (CP) genes, which have shown a significant resistance to SPFMV‐S. In the breeding programme, it is important to confirm that the viral resistance conferred in T₀ plants can be inherited by their progeny. In the present study, progeny were obtained from crosses between the transgenic T₀ and a non‐transgenic variety of sweet potato. The results showed that the CP gene was inherited by the next generation and that the stability of viral resistance was also confirmed. Thus, this production system for the virus‐resistant transgenic sweet potato is useful in practical breeding.     

Immunity to Clavibacter michiganensis subsp. sepedonicus: Screening of exotic Solanum species

Summary Accessions from exotic Solanum species, including diploid and tetraploid species, were screened for immunity to Clavibacter michiganensis subsp. sepedonicus, the causal agent of potato ring rot. The diploid species included S. infundibuliforme, S. lesteri, S. megistacrolobum, S. tuberosum Group Phureja, S. polyadenium, S. pinnatisectum, S. raphanifolium, S. sparsipilum, S. sanctae-rosae, S. tuberosum Group Stenotomum, S. toralapanum, and S. verrucosum. The tetraploid species included S. tuberosum Group Andigena, S. acaule, S. fendleri, S. hjertingii, S. oplocense, S. polytrichon, and S. stoloniferum. Apparent immunity was initially found in several diploid species, but was not present during subsequent retesting. Immunity was found in nine accessions of tetraploid S. acaule. These accessions maintained their immunity during testing over an eight-month period. S. acaule appears to be a good source of immunity for introgression studies.     

Breeding behavior for tuber protein in Solanum tuberosum and Tuberosum-phureja hybrids

Summary True protein content among tubers within a plant of Solanum tuberosum cv. Oneida was found to be negatively correlated with tuber size. A similar study of S. phureja genotype 148-17 revealed no correlation. Tuber protein was determined for 250 genotypes in each of four hybrid potato populations in a factorial mating design with four potato cultivars as stylar parents mated to four groups of pollen parents (4x cultivars, 4x high protein selections, 2x S. phureja, 2x high protein S. phureja). The hybrid population derived from the 4x high protein selections was significantly higher in protein content than the other populations. High estimates of general combining ability for tuber protein content were found for both stylar and pollen parents. Poor photoperiodic adaptation to growing conditions in northern latitudes was a possible explanation for the variable protein phenotypes typical of S. phureja and the lack of transmission of the high protein character in phureja to 4x-2x hybrids. Although protein content was negatively correlated with total yield, high protein segregates with good yield potential were identified in all four populations.Scientific Journal Series Article 11, 616 of the Minnesota Agricultural Experiment Station.     

Immunity to Clavibacter michiganensis subsp. sepedonicus: Inheritance of immunity in Solanum acaule

Summary Bacterial ring rot of potato (Solanum tuberosum) has not been successfully controlled through management of certified seed. Therefore, the identification of immunity to this pathogen and its introgression into the cultivated potato is vitally important to the potato industry. Immunity was detected in the disomic tetraploid 2EBN (Endosperm Balance Number) species S. acaule. Immune and nonimmune parents were crossed in a 4 × 4 mating scheme that consisted of four immune × immune crosses, four immune × nonimmune, four nonimmune × immune, and four nonimmune × nonimmune crosses. Analysis was performed on the 16 F₁ populations and 54% of the progeny was found to exhibit an immune response to inoculation with Clavibacter michiganensis subsp. sepedonicus (Spieck & Kotth.) Davis et al. Immunity appears to be conferred by two dominant alleles, one at each locus, and may be associated with minor or modifying genes. Phenotypic expression of nonimmune progeny ranged from susceptible to resistant, probably due to minor or modifying genes. These results indicate that it may not be difficult to transfer immunity into the cultivated potato.     

Influence of suboptimal temperature on biomass production of potato populations of Andean and European origin

Summary Plants grown from seed derived by crossing conventional European S. tuberosum material were compared with plants grown from seed derived by crossing S. tuberosum with various, Andean frost resistant tuberbearing Solanum species. Biomass growth at optimal (20°/10°) and suboptimal (10°/4°) temperatures was studied.Differences in increase of fresh and dry matter were found between populations of Andean and European orgins at 10°/4°. At 20°/10° no such differences were found.At suboptimal temperature, Andean hybrids produced significantly more fresh and dry matter than European crosses in two harvests (64 and 178 days old plants). Statistically, Andean hybrids were found to produce the same amount of dry matter per day at both temperatures, over the complete growth period.Height increase and flower development were strongly depressed in European crosses under the suboptimal temperatures, but much less so in Andean crosses.The Andean material appears to be an under utilized resource in potato breeding for cool climates.     

Breaking the crossability barriers between disomic tetraploid Solanum acaule and tetrasomic tetraploid S. tuberosum

Summary A combination of compatible second pollinations and embryo rescue was applied for systematic production of true tetraploid hybrids from crosses between disomic tetraploid Solanum acaule and tetrasomic tetraploid potato, S. tuberosum. Several genotypes of tetraploid potatoes were pollinated with S. acaule, and the compatible second pollinations were made on the following day, with a genotype of S. phureja, IvP 35 to promote fruit development. Embryo rescue was carried out in 21 families, 14 to 27 days after the first pollination. A total of eight plants were obtained from the embryo rescue and their chromosome numbers were counted in the root tips. Three of the eight plants were identified as tetraploid, and five others as diploid. Morphology, isozyme banding patterns, and pollen stainability, as well as potato spindle tuber viroid (PSTVd) resistance, indicated the hybrid nature of the three plants. This is the first report of successful tetraploid hybrid production between disomic tetraploid S. acaule (4x) and tetrasomic tetraploid potatoes. Seed set from the crosses between one of hybrids and diploid potatoes indicated workable levels of both male and female fertility for introgression of valuable genes from S. acaule into the cultivated potato gene pool. The methodology used may be applied to other disomic tetraploid tuber-bearing Solanum species and with some modifications also to distantly related solanaceous species and genera.     

Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia

Summary Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used as genetic markers and chromosome morphology as a cytological marker. In this combination, development of hybrid callus was late and slow. Only a limited number of non-regenerable hybrid calli have become available. Chromosome loss could clearly be established in these hybrids from loss of markers and from chromosome cytology. Loss of markers occurred independantly.The best conditions to induce loss of chromosomal material in donor cells by irradiation were found by cytological investigations. A very drastic reduction in chromosome transfer by fusion could be effected by irradiation of plant tissue and subsequent preparation of protoplasts after a few days. Following fusion, hybrid callus was recovered with the potato genome drastically reduced. The amount of loss was deduced from the presence of a few fragments in metaphase cells or from interphase nuclei after in situ hybridization with a repetitive potato DNA probe.     

Microprotoplast fusion technique: a new tool for gene transfer between sexually-incongruent plant species

Summary Various aspects of a microprotoplast fusion technique and the strategies followed for intergeneric partial genome transfer (one or a few chromosomes) and alien genes from sexually-incongruent donor species to recipient species are described. The essential requirements of the microprotoplast fusion technique are the induction of micronuclei at high frequencies, as well as the isolation and enrichment of sub-diploid microprotoplasts in donor species, efficient fusion of the donor microprotoplasts with normal recipient protoplasts and stable regeneration of plants from fusion products. The results on the production of microprotoplast hybrid plants between the transformed donor lines of Solanum tuberosum and Nicotiana Plumbaginifolia carrying various genetic markers, and a recipient line of Lycopersicon peruvianum or Nicotiana tabacum, and on the transfer and expression of alien genes (kanamycin resistance, -glucuronidase) are presented. The data obtained on microprotoplast hybrid plants between S. tuberosum and L. peruvianum showed that many of the hybrids contained one potato chromosome carrying nptII and GUS, and 24 or 48 L. peruvianum chromosomes (monosomic additions), and that they were male-and female-fertile. Various applications of chromosome transfer by this technique, especially for economically-important traits (e.g. disease or stress resistance) from sexually-incompatible wild species, for construction of chromosome-specific DNA libraries through microdissection and microcloning of chromosomes, or by flow-sorting of chromosomes for genome analysis, are discussed.     

Mapping loci for chlorosis associated with chlorophyll b deficiency in potato

About 30% of the potato plants from a (Solanum tuberosum × S. berthaultii) × S. tuberosum backcross population had chlorotic, malformed leaves; but a gradation in symptom severity suggested regulation by more than one gene. The study was undertaken to determine whether this was the case, whether any genes previously reported to control chlorosis in potato were involved, and to see how symptoms were related to effects on chlorophyll content. Testing for quantitative trait loci indicated major control by a single recessive gene on chromosome 1, close to one or more loci that have been reported to produce chlorosis in tomato, but distinct from similar genes previously identified in potato. The proposed symbol for the potato gene that confers phenotype with chlorotic and malformed leaves is cml (chlorotic and malformed leaves). The effects of this gene appeared to be accentuated by a second gene, located on chromosome 12. Chlorotic plants showed a 50% decrease in chlorophyll b level in the affected parts of leaves. It is concluded that cml is different from previously reported genes for chlorosis in potato, that at least one other gene modifies the intensity of symptom expression, and that the observed chlorosis is produced through effects on chlorophyll b level.     

The Use of RFLPs (Restriction Fragment Length Polymorphisms) Detects Germplasm Introgressions from Wild Species into Potato (Solanum tuberosum ssp. tuberosum) Breeding Lines

The germplasm of the potato (S. tuberosum ssp. tuberosum) has been modified since the beginning of this century by breeders who introgressed important agronomic traits, for example disease resistance genes, from several wild and cultivated Solanum species of the Americas. In this paper we show that the Rflp analysis of potato-breeding material can be used to detect chromosomal regions descended from more-distantly related Solanum species. The Rflp patterns of individuals of ten Solanum species, ten breeding lines of S. tuberosum ssp. tuberosum and the cultivated variety ‘Bintje’ were analyzed. Rflp data for each of eight single loci of known genomic position were used for the computation of locus-specific phenograms by distance matrix methods. Several of the potato-breeding lines deviated clearly from the clustered species S. tuberosum, S. stenotomum and S. canasense for one or more of the loci considered. These deviations indicated the presence of “exotic” germplasm at a particular locus. The possibility of detecting such germplasm has implications for mapping the agronomic traits for which the wild species were introgressed and which may still be linked to “foreign” chromosome fragments.