Molecular and morphological characterization of somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. and <Emphasis Type="Italic">S. etuberosum</Emphasis> Lindl.

Interspecific potato somatic hybrids between Solanum tuberosum L. (di)haploid C-13 and 1 endosperm balance number non-tuberous wild species S. etuberosum Lindl. were produced by protoplasts electrofusion. The objective was to transfer virus resistance from this wild species into the cultivated potatoes. Post-fusion products were cultured in VKM medium followed by regeneration of calli in MS₁₃ K medium at 20°C under a 16-h photoperiod, and regenerants were multiplied on MS medium. Twenty-one somatic hybrids were confirmed by RAPD, SSR and cytoplasm (chloroplast/mitochondria) type analysis possessing species-specific diagnostic bands of corresponding parents. Tetraploid nature of these somatic hybrids was determined through flow cytometry analysis. Somatic hybrids showed intermediate phenotypes (plant, leaves and floral morphology) to their parents in glass-house grown plants. All the somatic hybrids were male-fertile. ELISA assay of somatic hybrids after artificial inoculation of Potato virus Y (PVY) infection reveals high PVY resistance.     

Intertribal somatic hybrids between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Camelina sativa</Emphasis> with high linolenic acid content

Intertribal somatic hybrids of Brassica napus and Camelina sativa were developed by protoplast electrofusion. Hybrid identity of the regenerants was determined using flow cytometric analysis of nuclear DNA content and simple sequence repeat (SSR) marker analysis. Three hybrids exhibited specific bands for B. napus and C. sativa. These hybrids showed intermediate leaf, flower and seed morphology compared with the two parental species. The seeds of these three hybrids had a modified fatty acid profile, indicating higher level of linolenic and eicosanoic acids than those of B. napus. Our results suggest that somatic hybridization offers opportunities for transferring entire genomes between B. napus and C. sativa in improving rapeseed breeding.     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

Early characterization of somatic hybrids from symmetric protoplast electrofusion of <Emphasis Type="Italic">Solanum pinnatisectum</Emphasis> Dun. and <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Variability of 31 somatic hybrids of Solanum pinnatisectum Dun. with Solanum tuberosum L. for leaf morphology, plant vigor, resistance to Phytophthora infestans, ploidy level, and cytoplasm type was evaluated in vitro. The composition of these somatic hybrids was as follows: [S. pinnatisectum Dun. (2n = 2x = 24; cytoplasmic type Wγ) + S. tuberosum L. (2n = 4x = 48; cytoplasmic type Tß)]. Based on leaf morphology and plant growth vigor, plants were divided into three groups, including plants close to tbr parent with unlobed leaves, small plants with scarcely dissected leaves, and vigorous plants with asymmetrically and pinnately lobed leaves. Nine of the somatic hybrids were found to be highly resistant to P. infestans. Somatic hybrids were either tetraploid or hexaploid, with hexaploids being predominant. The cytoplasm of somatic hybrids was either Tßγ or Wßγ, with Tßγ being more common. Overall, in contrast to leaf morphology and growth vigor, level of resistance to P. infestans was not related to either ploidy level or type of cytoplasm. These findings demonstrate that early in vitro selection of promising hybrids can be useful in breeding programs.     

Carbohydrates and resistance to <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in potato plants

Plants generally deal with biotic or abiotic stresses by altering components as for example cell wall constituents and metabolites. Infection by Phytophthora infestans, the causal agent of late blight, constitutes a stress condition for the plants and they react to it with changes arising in their metabolism depending on the resistance level of the plants. The present work compares two potato hybrids differing in their level of horizontal resistance to late blight. Carbohydrate content in stems and leaves of infected and uninfected plants was determined by HPLC. Some carbohydrates accumulated in the stems of the resistant hybrid infected by P. infestans, whereas they remained unchanged in the susceptible hybrid. On the other hand, in the leaves, these carbohydrates accumulated only in the infected susceptible hybrid.     

Production of interspecific somatic hybrids between tuber mustard (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">juncea</Emphasis>) and red cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

In the present investigation, the interspecific somatic hybridization between tuber mustard and red cabbage was established in order to introduce valuable genes from red cabbage (Brassica oleracea) into Brassica juncea. Prior to fusion treatment, protoplasts of red cabbage were inactivated with 2 mM iodoacetamide to inhibit cell division. Micro-calluses were obtained at a frequency of 10.3% after approximately 5 weeks culture following protoplast fusion. Some of the fusion-derived calluses possessed red pigmented cells after being transferred to proliferation medium, and they were presumably considered to be somatic hybrid cell lines. Plantlets were regenerated from 12 cell lines, of which nine plantlets exhibited characteristics intermediate of both parents in terms of plant morphology. With the exception of common protein bands featured by two parents, there were unique banding patterns produced in the hybrids by using SDS-PAGE analysis. By chromosome countings, it was showed that they ranged approximately from 2n=30 to 42 in chromosome numbers. Their hybridity were further confirmed by RAPD analysis revealing that genes of both parents were partially incorporated into the hybrids. Positively, all these hybrids were capable of seed-setting. The pod-setting was 4.2 in somatic hybrid H₇ when backcrossed with tuber mustard.     

Allele mining in <Emphasis Type="Italic">Solanum</Emphasis>: conserved homologues of <Emphasis Type="Italic">Rpi-blb1</Emphasis> are identified in <Emphasis Type="Italic">Solanum stoloniferum</Emphasis>

Allele mining facilitates the discovery of novel resistance (R) genes that can be used in breeding programs and sheds light on the evolution of R genes. Here we focus on two R genes, Rpi-blb1 and Rpi-blb2, originally derived from Solanum bulbocastanum. The Rpi-blb1 gene is part of a cluster of four paralogues and is flanked by RGA1-blb and RGA3-blb. Highly conserved RGA1-blb homologues were discovered in all the tested tuber-bearing (TB) and non-tuber-bearing (NTB) Solanum species, suggesting RGA1-blb was present before the divergence of TB and NTB Solanum species. The frequency of the RGA3-blb gene was much lower. Interestingly, highly conserved Rpi-blb1 homologues were discovered not only in S. bulbocastanum but also in Solanum stoloniferum that is part of the series Longipedicellata. Resistance assays and genetic analyses in several F1 populations derived from the relevant late blight resistant parental genotypes harbouring the conserved Rpi-blb1 homologues, indicated the presence of four dominant R genes, designated as Rpi-sto1, Rpi-plt1, Rpi-pta1 and Rpi-pta2. Furthermore, Rpi-sto1 and Rpi-plt1 resided at the same position on chromosome VIII as Rpi-blb1 in S. bulbocastanum. Segregation data also indicated that an additional unknown late blight resistance gene was present in three populations. In contrast to Rpi-blb1, no homologues of Rpi-blb2 were detected in any material examined. Hypotheses are proposed to explain the presence of conserved Rpi-blb1 homologues in S. stoloniferum. The discovery of conserved homologues of Rpi-blb1 in EBN 2 tetraploid species offers the possibility to more easily transfer the late blight resistance genes to potato varieties by classical breeding.     

Mapping of the <Emphasis Type="Italic">S. demissum</Emphasis> late blight resistance gene <Emphasis Type="Italic">R8</Emphasis> to a new locus on chromosome IX

The use of resistant varieties is an important tool in the management of late blight, which threatens potato production worldwide. Clone MaR8 from the Mastenbroek differential set has strong resistance to Phytophthora infestans, the causal agent of late blight. The F1 progeny of a cross between the susceptible cultivar Concurrent and MaR8 were assessed for late blight resistance in field trials inoculated with an incompatible P. infestans isolate. A 1:1 segregation of resistance and susceptibility was observed, indicating that the resistance gene referred to as R8, is present in simplex in the tetraploid MaR8 clone. NBS profiling and successive marker sequence comparison to the potato and tomato genome draft sequences, suggested that the R8 gene is located on the long arm of chromosome IX and not on the short arm of chromosome XI as was suggested previously. Analysis of SSR, CAPS and SCAR markers confirmed that R8 was on the distal end of the long arm of chromosome IX. R gene cluster directed profiling markers CDP^Sw54 and CDP^Sw55 flanked the R8 gene at the distal end (1 cM). CDP^Tm21-1, CDP^Tm21-2 and CDP^Tm22 flanked the R8 gene on the proximal side (2 cM). An additional co-segregating marker (CDP^Hero3) was found, which will be useful for marker assisted breeding and map based cloning of R8.     

Interspecific somatic hybrids <Emphasis Type="Italic">Solanum bulbocastanum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis> H-8105

Interspecific somatic hybrids between a dihaploid potato clone H-8105 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuberizing species Solanum bulbocastanum were generated and analysed. Only ten regenerants displaying the intermediate morphology with dominating characteristics of the wild parent (simple leaves, anthocyanin pigmentation) were produced in 15 weeks after a single PEG-mediated fusion event. The RAPD patterns confirmed the hybridity of all of them. The hybrids rooted poorly and grew slowly in vitro. The cytological analysis revealed a high degree of aneuploidy in the hybrids with morphological and growth anomalies in vitro, while the morphologically normal hybrids were tetraploids. All the S. bulbocastanum (+) H-8105 hybrids were unstable in culture and three of them were consequently lost during three years of propagation in vitro. The possible reasons for instability of somatic hybrids between the distantly related species are discussed.     

Broad spectrum late blight resistance in potato differential set plants Ma<Emphasis Type="Italic">R8</Emphasis> and Ma<Emphasis Type="Italic">R9</Emphasis> is conferred by multiple stacked <Emphasis Type="Italic">R</Emphasis> genes

Phytophthora infestans is the causal agent of late blight in potato. The Mexican species Solanum demissum is well known as a good resistance source. Among the 11 R gene differentials, which were introgressed from S. demissum, especially R8 and R9 differentials showed broad spectrum resistance both under laboratory and under field conditions. In order to gather more information about the resistance of the R8 and R9 differentials, F1 and BC1 populations were made by crossing Mastenbroek (Ma) R8 and R9 clones to susceptible plants. Parents and offspring plants were examined for their pathogen recognition specificities using agroinfiltration with known Avr genes, detached leaf assays (DLA) with selected isolates, and gene-specific markers. An important observation was the discrepancy between DLA and field trial results for Pi isolate IPO-C in all F1 and BC1 populations, so therefore also field trial results were included in our characterization. It was shown that in MaR8 and MaR9, respectively, at least four (R3a, R3b, R4, and R8) and seven (R1, Rpi-abpt1, R3a, R3b, R4, R8, R9) R genes were present. Analysis of MaR8 and MaR9 offspring plants, that contained different combinations of multiple resistance genes, showed that R gene stacking contributed to the Pi recognition spectrum. Also, using a Pi virulence monitoring system in the field, it was shown that stacking of multiple R genes strongly delayed the onset of late blight symptoms. The contribution of R8 to this delay was remarkable since a plant that contained only the R8 resistance gene still conferred a delay similar to plants with multiple resistance genes, like, e.g., cv Sarpo Mira. Using this “de-stacking” approach, many R gene combinations can be made and tested in order to select broad spectrum R gene stacks that potentially provide enhanced durability for future application in new late blight resistant varieties.     

Exploring the quantitative resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">phaseolicola</Emphasis> in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Pseudomonas syringae pv. phaseolicola is an important disease that causes halo blight in common bean. The genetic mechanisms underlying quantitative halo blight resistance are poorly understood in this species, as most disease studies have focused on qualitative resistance. The present work examines the genetic basis of quantitative resistance to the nine halo blight races in different organs (primary and trifoliate leaf, stem and pod) of an Andean recombinant inbred line (RIL) progeny. Using a multi-environment quantitative trait locus (QTL) mapping approach, 76 and 101 main-effect and epistatic QTLs were identified, respectively. Most of the epistatic interactions detected were due to loci without detectable QTL additive main effects. Main and epistatic QTLs detected were mainly consistent across the environment conditions. The homologous genomic regions corresponding to 26 of the 76 main-effect detected QTLs were positive for the presence of resistance-associated gene cluster encoding nucleotide-binding and leucine-rich repeat (NL) proteins and known defence genes. Main-effect QTLs for resistance to races 3, 4 and 5 in leaf, stem and pod were located on chromosome 2 within a 3.01-Mb region, where a cluster of nine NL genes was detected. The NL gene Phvul.002G323300 is located in this region, which can be considered an important putative candidate gene for the non-organ-specific QTL identified here. The present research provides essential information not only for the better understanding of the plant-pathogen interaction but also for the application of genomic assisted breeding for halo blight resistance in common bean.     

<Emphasis Type="Italic">Sgt1</Emphasis>, but not <Emphasis Type="Italic">Rar1</Emphasis>, is essential for the <Emphasis Type="Italic">RB</Emphasis>-mediated broad-spectrum resistance to potato late blight

Background  

Late blight is the most serious potato disease world-wide. The most effective and environmentally sound way for controlling late blight is to incorporate natural resistance into potato cultivars. Several late blight resistance genes have been cloned recently. However, there is almost no information available about the resistance pathways mediated by any of those genes.     

Fecundity and feeding of <Emphasis Type="Italic">Galerucella calmariensis</Emphasis> and <Emphasis Type="Italic">G. pusilla</Emphasis> on <Emphasis Type="Italic">Lythrum salicaria</Emphasis>

Fecundity and feeding of two introduced sibling biological control species, Galerucella calmariensis and G. pusilla (Coleoptera: Chrysomelidae) on purple loosestrife, Lythrum salicaria L. (Lythraceae) were compared at constant temperatures of 12.5, 15, 20, 25, and 27.5 °C. Larval feeding was also carried out at 30 °C, but at this temperature, larvae developed only to the L2 stage and none pupated. Thus, data for this temperature were not used in the analysis. There were significant species × temperature interactions in fecundity. Of the two species, Galerucella pusilla laid more eggs. Although egg production of both species was lowest at 12.5 °C and increased to 20 °C, at higher temperatures, the two species reacted differently. From 25 to 27.5 °C, egg production decreased for G. pusilla, but G. calmariensis fecundity peaked at 27.5 °C. Significant temperature × species × life-stage interactions were also observed in feeding. For each species, the amount of feeding varied with temperature and stage of development. Galerucella pusilla adults consumed more foliage at 15, 20, and 27.5 °C. However, at 12.5 °C G. calmariensis adults fed more than G. pusilla. G. pusilla larvae consumed an average of 25% less foliage than G. calmariensis. The lower larval consumption of G. pusilla suggests that when food is limited, G. pusilla larvae may have a higher survival rate because of its ability to complete larval development with less food and produce more progeny due to its greater fecundity. When food is not limited neither species would have a competitive advantage and both species could coexist temporally and spatially. However, since G. calmariensis larvae consumed more leaf material, the larval stage of this species would have a greater impact on purple loosestrife than G. pusilla.     

Production and genetic characterization of somatic hybrids between leaf mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) and broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Inter-specific somatic hybrids of leaf mustard (Brassica juncea) and broccoli (Brassica oleracea) were established to introduce cytoplasmic male sterility (CMS) and Verticillium dahliae Kleb. resistance from broccoli to leaf mustard. Protoplasts isolated from hypocotyls and cotyledons of inbred lines of leaf mustard and broccoli were fused using 40% (w/v) polyethelene glycol and then cultured in modified k8p medium supplemented with 0.2 mg/l 2,4-dichlorophenoxi-acetic acid, 0.5 mg/l 6-benzyladenine (BA), 0.1 mg/l naphthaleneacetic acid (NAA), and 0.1 mg/l kinetin (Kin), and 0.4 M mannitol as osmoticum. At the eight- to ten-cell stage, divided cells were transferred to Kao’s basal medium supplemented with 0.3 M sucrose as carbon source and 0.1% agarose, 2 mg/l BA, 2 mg/l zeatin (ZEA), 1 mg/l NAA, and 0.5 mg/l Kin for callus proliferation. After 35 d, when small calli reached 2–3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/l ZEA and 2 mg/l indole-3-acetic acid. The chromosome numbers of putative somatic hybrids varied from 46 to 54. A total of ten plants showed a 0.5-kb, CMS-specific band, while two regenerated plants showed a missing band similar to that of leaf mustard by polymerase chain reaction amplification using Ogura CMS-specific primers. Hybrid state was confirmed by random amplified polymorphic DNA analysis. Regenerated plants had normal petals and stamens, but only two plants produced pollen and set seed.     

Major-effect QTLs for stem and foliage resistance to late blight in the wild potato relatives <Emphasis Type="Italic">Solanum sparsipilum</Emphasis> and <Emphasis Type="Italic">S. spegazzinii</Emphasis> are mapped to chromosome X

To find out new resistance sources to late blight in the wild germplasm for potato breeding, we examined the polygenic resistance of Solanum sparsipilum and S. spegazzinii by a quantitative trait locus (QTL) analysis. We performed stem and foliage tests under controlled conditions in two diploid mapping progenies. Four traits were selected for QTL detection. A total of 30 QTLs were mapped, with a large-effect QTL region on chromosome X detected in both potato relatives. The mapping of literature-derived markers highlighted colinearities with published late blight QTLs or R-genes. Results showed (a) the resistance potential of S. sparsipilum and S. spegazzinii for late blight control, and (b) the efficacy of the stem test as a complement to the foliage test to break down the complex late blight resistance into elementary components. The relationships of late blight resistance QTLs with R-genes and maturity QTLs are discussed.     

Characteristics of the interspecific somatic hybrids <Emphasis Type="Italic">Solanum pinnatisectum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis> H-8105

The morphological, cytological and molecular analyses of the plants regenerated after PEG-induced fusion between mesophyll protoplasts from the dihaploid potato clone H-8105 and the wild tuberous disease-resistant species S. pinnatisectum, were performed. A single fusion experiment yielded 313 calli, although only two calli produced shoots. From the rooted shoots, two stable clones (PT-01-1 and PT-01-2) exhibiting different vigor and habitat, were developed. The plants of PT-01-1 clone grew slowly in vitro, produced tubers after transfer to soil but did not set flowers. In contrast, the plants of the vigorous clone PT-01-2 produced both tubers and flowers after transfer to soil. The flower and tuber morphology of PT-01-1 and PT-01-2 regenerants was intermediate in comparison to the parental species. Cytological analysis revealed that the PT-01-1 clone was pentaploid and the PT-01-2 clone was tetraploid. The molecular (RAPD) analysis confirmed hybridity of both clones. The preliminary tests on late blight resistance of the hybrids showed no differences with a potato parent.     

Detection of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in seeds using a specific TaqMan probe

Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.     

Fine genetic mapping of <Emphasis Type="Italic">xa24</Emphasis>, a recessive gene for resistance against <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating plant bacterial disease worldwide. Different bacterial blight resistance (R) genes confer race-specific resistance to different strains of Xoo. We fine mapped a fully recessive gene, xa24, for bacterial blight resistance to a 71-kb DNA fragment in the long arm of rice chromosome 2 using polymerase chain reaction-based molecular markers. The xa24 gene confers disease resistance at the seedling and adult stages. It mediates resistance to at least the Philippine Xoo races 4, 6 and 10 and Chinese Xoo strains Zhe173, JL691 and KS-1-21. Sequence analysis of the DNA fragment harboring the dominant (susceptible) allele of xa24 suggests that this gene should encode a novel protein that is not homologous to any known R proteins. These results will greatly facilitate the isolation and characterization of xa24. The markers will be convenient tools for marker-assisted selection of xa24 in breeding programs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.