Narrowing down the region of the Vf locus for scab resistance in apple using AFLP-derived SCARs.

A narrow-down strategy to restrict the Vf region, which controls resistance to the fungal disease apple scab in apple, to a genetic distance of 0.4 cM is presented. Using 11 AFLP-derived SCARs and three RAPD-derived SCARs, all linked to the Vf gene, we subjected 1,412 scab-resistant individuals from 16 mapping populations to genotype analysis. Eleven recombinant individuals were identified within a genetic distance of 0.9 cM around the Vf gene. Using these 11 recombinants, we achieved fine-resolution of several AFLP-derived SCAR markers surrounding the Vf gene, resulting in the following genetic linkage map: ACS-6 and ACS are located left of the Vf gene at genetic distances of 0.2 cM and 0.1 cM, respectively; ACS-7 and ACS-9 are inseparable from the Vf gene; ACS-8, ACS-10, and ACS-4 are located to the right of the Vf gene at genetic distances of 0.1 cM, 0.4 cM, and 0.5 cM, respectively; the remaining five SCARs-ACS-11, ACS-5, ACS-2, ACS-1, and AL07-are inseparable and are located right of the Vf gene at a genetic distance of 0.7 cM. By integrating this linkage data with our previous physical map, we generated a revised map of the narrowed-down region of Vf.

Transcriptional regulation of Arabidopsis thaliana phytochelatin synthase (AtPCS1) by cadmium during early stages of plant development.

Transcriptional regulation of Arabidopsis thaliana (L.) Heynh. phytochelatin synthase (AtPCS1) by cadmium (Cd) was analyzed at various stages of plant development using transgenic Arabidopsis and wild-type plants. Histochemical analysis of beta-glucuronidase (GUS) activity in transgenic lines carrying a uidA gene driven by a 2.0-kb AtPCS1 promoter revealed higher GUS activities in 5-day-old seedlings subjected to 50 microM Cd treatment for 5 days, beginning at seed germination, than in non-treated plants. This high level of GUS activity gradually decreased as plants continued their growth until no differences were observed between Cd-treated and non-treated transgenic plants. The observed GUS activity due to Cd treatment during the early stage of plant development corresponded with induction of AtPCS1 mRNA as confirmed by RNA blot analysis of wild-type Arabidopsis. The steady-state level of AtPCS1 mRNA increased by 2-fold in 5-day-old Cd-treated wild-type Arabidopsis compared to non-treated seedlings. Moreover, AtPCS1 protein levels increased following Cd treatment as observed in western blot analysis of transgenic Arabidopsis lines carrying a C-terminal FLAG-tagged AtPCS1 genomic DNA driven by a 2.0-kb AtPCS1 promoter. The transcriptional regulation of AtPCS1 by Cd during the early stage of seedling development seemed to be correlated with a higher Cd sensitivity in cad2, an Arabidopsis mutant deficient in phytochelatin synthesis, during early stages of plant development. This was supported by our finding that Cd sensitivity in cad2 was reduced as plants continued their growth, and was comparable to that of wild-type plants.

Characterization and expression of beta-1,3-glucanase genes in peach.

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.

Development of sequence-characterized amplified regions (SCARs) from amplified fragment length polymorphism (AFLP) markers tightly linked to the Vf gene in apple.

Amplified fragment length polymorphism (AFLP) markers have become widely used in saturating the region of a gene of interest for the ultimate goal of map-based cloning of the gene or for marker-assisted selection. However, conversion of AFLP markers into restriction fragment length polymorphism (RFLP) or polymerase chain reaction (PCR)-based markers will greatly expand their usefulness in genetic applications. Previously, we have identified 15 AFLP markers tightly linked to the Vf gene conferring scab resistance in apple. In this study, we have successfully converted 11 of these AFLPs into sequence-characterized amplified region (SCAR) markers. Of the remaining four nonconverted AFLP markers, one, ET2MC8-1, has been found to be very short (83 base pairs) and is an A/T rich (90%) marker; a second, EA2MG11-1, has shown identical sequences between Malus floribunda 821 (the original source of the Vf gene) and scab-susceptible apple cultivars; while the other two, EA12MG16-1 and ET8MG1-1, have not been cloned. Using the 11 converted SCAR markers along with 5 previously identified SCAR markers, a high-resolution linkage map around the Vf gene has been constructed, and found to be consistent with its corresponding AFLP map. Three converted SCAR markers (ACS-3, -7, and -9) are inseparable from the Vf gene; whereas one (ACS-6) is located left of, and the remaining seven (ACS-1, -2, -4, -5, -8, -10, and -11) are located right of the Vf gene at genetic distances of 0.4 and 0.2 cM, respectively. A reliable and robust procedure for development of SCAR markers from AFLP markers is presented.

A bacterial artificial chromosome (BAC) library of Malus floribunda 821 and contig construction for positional cloning of the apple scab resistance gene Vf.

The apple scab resistance gene Vf, originating from the wild species Malus floribunda 821, has been incorporated into a wide variety of apple cultivars through a classical breeding program. With the aim of isolating the Vf gene, a bacterial artificial chromosome (BAC) library consisting of 31 584 clones has been constructed from M. floribunda 821. From the analysis of 88 randomly selected BAC clones, the average insert size is estimated at 125 kb. If it is assumed that the genome size of M. floribunda 821 is 769 Mb/haploid, the library represents about 5x haploid genome equivalents. This provides a 99% probability of finding any specific sequence from this library. PCR-based screening of the library has been carried out using eight random genomic sequence-characterized amplified regions (SCARs), chloroplast- and mitochondria-specific SCARs, and 13 high-density Vf-linked SCAR markers. An average of five positive BAC clones per random SCAR has been obtained, whereas less than 1% of BAC clones are derived from the chloroplast or mitochondrial genomes. Most BAC clones identified with Vf-linked SCAR markers are physically linked. Three BAC contigs along the Vf region have been obtained by assembling physically linked BAC clones based on their fingerprints. The overlapping relatedness of BAC clones has been further confirmed by cytogenetic mapping using fiber fluorescence in situ hybridization (fiber-FISH). The M. floribunda 821 BAC library provides a valuable genetic resource not only for map-based cloning of the Vf gene, but also for finding many other important genes for improving the cultivated apple.

Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response.

Respiratory syncytial virus (RSV) is one of the most important pathogens of infancy and early childhood. Here a fruit-based edible subunit vaccine against RSV was developed by expressing the RSV fusion (F) protein gene in transgenic tomato plants. The F-gene was expressed in ripening tomato fruit under the control of the fruit-specific E8 promoter. Oral immunization of mice with ripe transgenic tomato fruits led to the induction of both serum and mucosal RSV-F specific antibodies. The ratio of immunoglobulin subclasses produced in response to immunization suggested that a type 1 T-helper cell immune response was preferentially induced. Serum antibodies showed an increased titer when the immunized mice were exposed to inactivated RSV antigen.

Screening apples for OPD20/600 using sequence-specific primers.

Apple scab, caused by Venturia inaequalis (Cke.) Wint., is the most serious disease of apple trees in many areas of the world. Resistance to V. inaequalis, derived from the small-fruited species Malus floribunda 821, is determined by a major dominant gene, Vf. Using random decamer primers, we identified a RAPD marker, OPD20/600, which is linked to the Vf gene. OPD20/600 was then cloned and sequenced. Sequence-specific primers based on the marker were used to further screen M. floribunda 821, 7 scab-susceptible apple cultivars, 10 scab-resistant apple cultivars, and 28 scab-resistant Coop selections. The sequence-specific primers allowed identification of polymorphisms of OPD20/600 based on the presence or absence of a single band. The advantages of sequence-specific primers over decamer primers for developing genetic markers are discussed.

The complete nucleotide sequence of apple mosaic virus RNA-3.

The complete nucleotide sequence of apple mosaic ilarvirus (ApMV) RNA-3 has been determined from cloned viral cDNAs. The 5' terminus of RNA-3 was determined by direct RNA sequencing, while the 3' end was determined by polyadenylation of genomic RNA and sub-cloning using oligo dT. ApMV RNA-3 is 2056 bases in length and encodes at least two open reading frames. It is similar in size and genome organization to the RNA-3 of other members of the Bromoviridae, which includes ilarviruses. The CP gene is in the 3' half of the molecule, and another large open reading frame is upstream of the CP gene and can potentially encode a protein of 32,400 daltons. This peptide is the same size and shows limited sequence homology to an open reading frame located at the 5' end of RNA 3 in tobacco streak and prune dwarf ilarviruses and alfalfa mosaic virus, which is postulated to be the viral movement protein. The nucleic acid sequence was not homologous to tobacco streak virus, prune dwarf virus, alfalfa mosaic virus or other members of the Bromoviridae. The 5'-non-coding region of ApMV RNA-3 contains a 15 base palindromic sequence which encloses a sequence resembling the ICR-2 regions of eukaryotic tRNA gene promoters.

The nucleotide sequence of apple mosaic virus coat protein gene has no similarity with other Bromoviridae coat protein genes.

A double-stranded cDNA was synthesized from in vitro polyadenylated apple mosaic virus (ApMV) RNA 3 using oligo(dT) and sequence-specific primers, and was cloned into plasmid vectors. A set of overlapping cDNA clones was used to determine the nucleotide sequence of RNA 4. ApMV RNA 4 was found to contain an open reading frame (ORF) of 666 nucleotides, which was flanked by 5' and 3' non-translated sequences of 55 and 264 nucleotides, respectively. The ORF encoding the coat protein was identified by comparing the predicted amino acid sequence with that obtained from direct protein microsequencing of the native viral coat protein. The ORF encodes a protein with an M(r) of 25,056. The nucleotide sequence of the ApMV coat protein gene showed no similarity to those of alfalfa mosaic virus, tobacco streak virus (TSV), brome mosaic virus or cucumber mosaic virus. The predicted amino acid sequence of the amino-terminal region of the ApMV coat protein is basic, rich in cysteine residues and may contain a zinc finger motif similar to that found in TSV.

Adventitious shoot regeneration from leaf tissue of three pear (Pyrus sp.) cultivars in vitro.

To develop an adventitious regeneration system for pear cultivars, several experiments were conducted with 2 cultivars of Pyrus communis L. ('Seckel' and 'Louise Bonne') and one cultivar of P. bretschneideri Rehd. ('Crystal Pear'). Half-leaves, taken from shoots proliferating on Lepoivre medium, were plated in petri-dishes on medium supplemented with various combinations of cytokinins and auxins. Cultures of the above cultivars had been established from mature trees. Among the growth regulators tested, thidiazuron (TDZ), combined with naphthalene-acetic acid (NAA), was the most efficient for stimulation of adventitious shoots. The optimum level of TDZ was about 3 uM; shoot regeneration was observed over a wide range of TDZ and NAA concentrations (0.5 to 5 uM and 2.5 to 13 um, respectively). Among different macronutrient compositions, 1/2 and 1/4 Murashige and Skoog were the most effective. Sucrose concentrations (10 to 50 g L-1) had a linear significant effect on shoot regeneration of 'Crystal Pear'.

Callus formation from Malus x domestica cv. 'Jonathan' protoplasts.

Protoplasts could be successfully isolated and cultured from callus and suspension cultures of Malus xdomestica cv. 'Jonathan'. Protoplast-derived colonies were recovered when the osmoticum (glucose) was gradually reduced in semi-solid 8p medium or by the use of feeder plates. Formation of embryo-like structures was induced from the protoplast-derived callus on media supplemented with IAA and BA. These structures formed roots but plants failed to develop. Protoplasts could be isolated from leaves, but not from stems or petioles. The leaf protoplasts failed to divide.