Molecular mapping of the py-1 gene for resistance to corky root rot (Pyrenochaeta lycopersici) in tomato

 We report the molecular mapping of the py-1 gene for resistance to corky root rot [Pyrenochaeta lycopersici (Schneider and Gerlach)] in tomato using RAPD and RFLP marker analysis. DNA from near-isogenic lines (NILs) of tomato differing in corky root rot resistance was screened with 575 random oligonucleotide primers to detect polymorphic DNAs linked to py-1. Three primers (OPW-04, OPC-02, OPG-19) revealed polymorphisms between the NILs. Twelve resistant and eight susceptible DNA pools derived from segregating F₃ families were used to confirm that the RAPD markers were linked to the py-1 gene. Two of the linked amplified fragments, corresponding to OPW-04 and OPC-02, were subsequently cloned and mapped on the tomato molecular linkage map as RFLPs. These clones were located between TG40 and CT31 on the short arm of chromosome 3. Further analysis with selected RFLP markers showed that 7% (8.8 cM) of chromosome 3 of the resistant line ‘Moboglan’ was introgressed from the L. peruvianum donor parent. Three RFLP markers (TG40, TG324, and TG479) from the introgressed part of chromosome 3 were converted to cleaved amplified polymorphism (CAP) markers for use in a polymerase chain reaction (PCR) assay. These PCR markers will allow rapid large-scale screening of tomato populations for corky root rot resistance. Received: 2 January 1998 / Accepted: 12 January 1998     

Linkage analysis of genes for resistance to downy mildew (Bremia lactucae) in lettuce (Lactuca sativa)

Summary The genetics of specific resistance was studied in F₂ populations which segregated for either one or two resistance genes. The resistance factors 1, 11 and 14 which had not previously been characterized genetically segregated as single dominant genes (Dm). Resistance was determined by three linkage groups; R 1/14, 2, 3, and 6 in the first, R 5/8, and 10 in the second and R 4, 7 and 11 in the third. Cultivars of lettuce commonly used in the differential series to detect virulence to R3 and R10, were demonstrated to carry two tightly linked resistance genes. Implications of this linkage arrangement to the manipulation and characterization of these resistance genes are discussed.     

The genetics of race specific resistance in lettuce (Lactuca sativa) to downy mildew (Bremia lactucae)

Data are presented on the segregation of resistance to five British races and two Dutch races of Bremia lactucae in the F₂ progenies of crosses involving seven resistant and several susceptible lettuce cultivars. These data and also those previously published by other workers are considered in relation to the systematic model proposed by Crute & Johnson (1976) to explain the genetics of race specific resistance to B. lactucae in lettuce. It is shown that, with minor modifications, the model accommodates almost all of the previously published data and correctly predicts the new data, except for one set which cannot at present be interpreted. It is concluded that genetic evidence exists for the presence, among various cultivars of lettuce, of at least four and possibly five different dominant resistance genes of major effect designated Dm₂, Dm₃, Dm₄, Dm₆ and Dm₈; and of a pair of dominant genes with complementary effect designated Dm7/1 and Dm7/2. The resistance conferred by these genes is specified in relation to five British races, five Dutch, three Israeli and one United States race of the fungus. Resistance genotypes are proposed for cultivars Avoncrisp, Avondefiance, Calmar, Great Lakes 659, Kares, Meikoningen, Mildura, Proeftuins Blackpool, Solito, Valverde, Ventura and the USDA line PI 164937.     

Vesicular-arbuscular mycorrhizal infection in lettuce (Lactuca sativa) in relation to calcium supply

Summary Infection of lettuce roots (Lactuca sativa L.) by the vesicular-arbuscular mycorrhizal fungiGlomus caledonium andGlomus mosseae was dependent on the amount of calcium (supplied as CaCl₂·2H₂O or CaSO₄·2H₂O) in the nutrient solution; those plants growing at low calcium concentrations being poorly infected.     

Transactivation of Ds elements in plants of lettuce (Lactuca sativa)

The maize transposable element, Activator (Ac), is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. In this paper, we describe somatic and germinal transactivation of Ds by chimeric transposase genes in whole plants. Constructs containing either the Ds element or the Ac transposase open reading frame (ORF) were introduced into lettue. The Ds element was located between either the 35S or the Nos promoter and a chimeric spectinomycin resistance gene (which included a transit peptide), preventing expression of spectinomycin resistance. The genomic coding region of the Ac transposase was expressed from the 35S promoter. Crosses were made between 104 independent R₁ plants containing Ds and three independent R₁ plants expressing transposase. The excision of Ds in F₁ progenies was monitored using a phenotypic assay on spectinomycin-containing medium. Green sectors in one-third of the F₁ families indicated transactivation of Ds by the transposase at different developmental stages and at different frequencies in lettuce plants. Excision was confirmed using PCR and by Southern analysis. The lack of green sectors in the majority of F₁ families suggests that the majority of T-DNA insertion sites are not conducive to excision. In subsequent experiments, the F₁ plants containing both Ds and the transposase were grown to maturity and the F₂ seeds screened on medium containing spectinomycin. Somatic excision was again observed in several F₂ progeny; however, evidence for germinal excision was observed in only one F₂ family.     

Development of EST-SSR markers for the study of population structure in lettuce (Lactuca sativa L.)

A set of 61 simple sequence repeat (SSR) markers was developed from the 19,523 Lactuca sativa and Lactuca serriola unigenes. Approximately 4.5% of the unigenes contained a perfect SSR at least 20 bp long, corresponding to roughly 1 perfect SSR per 14.7 kb. Marker polymorphism was tested on a set comprising 96 accessions representing all major horticultural types and 3 wild species (L. serriola, Lactuca saligna, and Lactuca virosa). Both the average marker heterozygosity (UHe = 0.32) and the number of different alleles per locus (Na = 3.56) were significantly reduced in expressed sequence tag (EST)-SSRs as compared with anonymous SSRs (UHe = 0.59, Na = 5.53). Marker transfer rate to the wild species corresponded to the decreasing sexual compatibility with L. sativa and was higher for EST-SSRs (100% L. serriola, 87% L. saligna, and 75% L. virosa) than for anonymous SSRs (93%, 66%, and 42%, respectively). Assessment of population structure among 90 L. sativa cultivars with SSRs was in good agreement with classification into the horticultural types. The average marker heterozygosity was smallest in iceberg (0.097), Latin (0.140), and romaine-type (0.151) cultivars while highest in leaf (green leaf 0.208 and red leaf 0.240) lettuces. The level of marker heterozygosity is in accord with morphological variability observed in different horticultural types.     

Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa)

Two sets of overlapping experiments were conducted to examine recombination and spontaneous mutation events within clusters of resistance genes in lettuce. Multiple generations were screened for recombinants using PCR-based markers flanking Dm3. The Dm3 region is not highly recombinagenic, exhibiting a recombination frequency 18-fold lower than the genome average. Recombinants were identified only rarely within the cluster of Dm3 homologs and no crossovers within genes were detected. Three populations were screened for spontaneous mutations in downy mildew resistance. Sixteen Dm mutants were identified corresponding to spontaneous mutation rates of 10(-3) to 10(-4) per generation for Dm1, Dm3, and Dm7. All mutants carried single locus, recessive mutations at the corresponding Dm locus. Eleven of the 12 Dm3 mutations were associated with large chromosome deletions. When recombination could be analyzed, deletion events were associated with exchange of flanking markers, consistent with unequal crossing over; however, although the number of Dm3 paralogs was changed, no novel chimeric genes were detected. One mutant was the result of a gene conversion event between Dm3 and a closely related homolog, generating a novel chimeric gene. In two families, spontaneous deletions were correlated with elevated levels of recombination. Therefore, the short-term evolution of the major cluster of resistance genes in lettuce involves several genetic mechanisms including unequal crossing over and gene conversion.     

Further work on the genetics of race specific resistance in lettuce (Lactuca sativa) to downy mildew (Bremia lactucae)

Data are presented on the segregation of resistance to four races of Bremia lactucae in the F2 progenies of crosses involving 15 resistant and various susceptible lettuce cultivars. Most of these data and those recently published by other workers fit the systematic model for the genetics of race specific resistance to B. lactucae proposed by Crute & Johnson(1976). Seven different dominant resistance genes of major effect were found. There was also evidence of a pair of dominant genes with complementary effect, one of which may sometimes be effective on its own. Two of the genes may be linked and another may have two different alleles for resistance at the same locus. The resistance conferred by these genes is specified in relation to two British, two Dutch and four French races of the fungus. Resistance genotypes are proposed for 16 cultivars.     

Induction of phenylalanine ammonia-lyase (PAL) in germinating lettuce seeds (Lactuca sativa)

Dry lettuce seeds (achenes of Lactuca sativa L. cv. Grand Rapids) contain no detectable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity. Enzyme activity could be detected in these seeds within 4 h of imbibition under white light. The specific activity of PAL increased rapidly during the next 12–16 h of imbibition. Far-red light completely suppressed germination as well as the development of PAL. Gibberellic acid (GA₃, 0.1 m M ), although effective in causing almost 100% germination in dark, did not induce proportionate increases in PAL. Seed germination as well as PAL activity were substantially inhibited by cis -4-cyclohexene-l, 2-dicarboximide (CHDC, 1.0 m M ) both in light and dark. Both GA₃ and benzyladenine (BA, 0.1 m M ) retarded radicle elongation in light. Concomitantly, a decrease in PAL activity was observed. Benzyladenine was able to reverse the effects of CHDC on germination but PAL activity was still highly reduced, probably due to the inhibitory effects of BA on elongation of the radicle. More than 95% of the extractable PAL was found to be present in the radicle. When seeds incubated in white light for 10 h were transferred to FR, further increases in PAL activity as well as the growth of the radicle were severely inhibited. It is suggested that the induction of PAL in light-sensitive lettuce seeds is coincidental with the germination of seeds, and the amount of PAL per germinated seed is related to the extent of elongation of the embryonic axes.     

Leaf traits as indicators of limiting growing conditions for lettuce (Lactuca sativa)

Lettuce growth under unstressed conditions was compared to growth under four limiting conditions, i.e. no phosphorus fertilization (0_P), no nitrogen fertilization (0_N), low light (LR) and water stress (WR) over two different growing periods. We investigated the adaptive changes in terms of the morphological and physiological leaf traits, identifying stress‐specific and ‘stable’ indicators suitable for use in breeding programmes. The plants subjected to the WR treatments had lower leaf expansion and specific leaf area (SLA), as well as lower soil–plant analysis development (SPAD) values, stomatal conductance (POR), water index (WI) and leaf temperature (TIR) compared with plants in the unstressed CONTROL. Low light increased the leaf area (LA), SLA and leaf mass ratio (LMR). The 0_N treatment induced a general reduction in the normalised difference vegetation index (NDVI) values, as well as strong changes in LMR and SLA. In general, 0_P induced less pronounced effects than the other treatments. Principal component analysis indicated that the stable and suitable selection indicators of adaptive changes for low nitrogen and low light conditions were LA, SLA, leaf area per unit total plant mass (LAR), LMR, SPAD and POR, while SPAD, POR, TIR and WI were suitable indicators for drought.     

Identification of a phenylalanine ammonia-Iyase inactivating factor in harvested head lettuce (Lactuca sativa)

Exposing head lettuce ( Lactuca sativa L., crisphead or Iceberg type) leaf tissue to hormonal levels of ethylene (10 μl l⁻¹) at 5°C promotes the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and an increase in its activity. It also promotes the appearance of the postharvest physiological disorder called russet spotting (RS). Discontinuing ethylene exposure after 4 days resulted in a rapid decline in PAL activity which was delayed by treating excised midrib leaf tissue with actinornycin D or cycloheximide at 5°C. Only cycloheximide delayed the loss of PAL activity in tissue that was transferred from 5 to 15°C. Activity of PAL from Rhodolorula glutinis was. slowly lost during incubation in buffer alone, but there was a logarithmic decline in its activity over time when it was incubated with aliquots of the resuspended 10000 g pellet from homogenized, lettuce tissue affected with RS. The in vitro loss in PAL activity was 9–fold higher in extracts from lettuce showing RS symptoms than from control lettuce, boiled samples or the buffer control. The PAL-inactivating factor isolated from lettuce affected with RS had a pH optimum around 8.0. It appears that the rapid loss in PAL activity after the discontinuation of exposure to ethylene is dependent on the de novo synthesis of a PAL-inactivating factor.     

Association analysis of bacterial leaf spot resistance and SNP markers derived from expressed sequence tags (ESTs) in lettuce (Lactuca sativa L.)

Bacterial leaf spot of lettuce, caused by Xanthomonas campestris pv. vitians, is a devastating disease of lettuce worldwide. Since there are no chemicals available for effective control of the disease, host-plant resistance is highly desirable to protect lettuce production. A total of 179 lettuce genotypes consisting of 29 leaf, 15 crisphead, one stem, 21 romaine, and 113 butterhead types were evaluated for response to X. c. vitians. One source of high resistance and five sources of moderate resistance were identified with four being butterhead lettuce and two leaf lettuce. The population genetic structure based on 350 expressed-sequence-tag-derived single nucleotide polymorphism generated two clades: Clade I and Clade II. The butterhead type was genetically distinct from romaine and crisphead types, while the leaf type was found to frequently exchange genes with other types through the history of breeding. Association mapping identified one single nucleotide polymorphism (QGB19C20.yg-1-OP5) associated with disease severity in Q general linear model and Q + K mixed linear model. Two SNP markers (Contig15389-1-OP1 and Contig6039-19-OP1) were associated with the resistance in the leaf lettuce PI 358000-1 which had no disease symptoms. The marker QGB19C20.yg-1-OP5 is in linkage group 2, while both of Contig15389-1-OP1 and Contig6039-19-OP1 are in linkage group 4. The resistant lines and the associated SNPs should be useful to develop resistant cultivars to battle against the devastating disease in lettuce.     

An assessment of the role of ethylene in mediating lettuce (Lactuca sativa) root growth at high temperatures

Growth of temperate lettuce (Lactuca sativa) plants aeroponically in tropical greenhouses under ambient root-zone temperatures (A-RZTs) exposes roots to temperatures of up to 40 degrees C during the middle of the day, and severely limits root and shoot growth. The role of ethylene in inhibiting growth was investigated with just-germinated (24-h-old) seedlings in vitro, and 10-d-old plants grown aeroponically. Compared with seedlings maintained at 20 degrees C, root elongation in vitro was inhibited by 39% and root diameter increased by 25% under a temperature regime (38 degrees C/24 degrees C for 7 h/17 h) that simulated A-RZT in the greenhouse. The effects on root elongation were partially alleviated by supplying the ethylene biosynthesis inhibitors aminooxyacetic acid (100-500 microM) or aminoisobutyric acid (5-100 microM) to the seedlings. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to seedlings grown at 20 degrees C mimicked the high temperature effects on root elongation (1 microM) and root diameter (1 mM). Compared with plants grown at a constant 20 degrees C root-zone temperature, A-RZT plants showed decreased stomatal conductance, leaf relative water content, photosynthetic CO(2) assimilation, shoot and root biomass, total root length, the number of root tips, and root surface area, but increased average root diameter. Addition of 10 microM ACC to the nutrient solution of plants grown at a constant 20 degrees C root-zone temperature mimicked the effects of A-RZT on these parameters but did not influence relative water content. Addition of 30 microM aminoisobutyric acid or 100 microM aminooxyacetic acid to the nutrient solution of A-RZT plants increased stomatal conductance and relative water content and decreased average root diameter, but had no effect on other root parameters or root and shoot biomass or photosynthetic CO(2) assimilation. Although ethylene is important in regulating root morphology and elongation at A-RZT, the failure of ethylene biosynthesis inhibitors to influence shoot carbon gain limits their use in ameliorating the growth inhibition induced by A-RZT.     

Identification of the novel recessive gene pi55(t) conferring resistance to Magnaporthe oryzae

The elite rice cultivar Yuejingsimiao 2 (YJ2) is characterized by a high level of grain quality and yield, and resistance against Magnaporthe oryzae. YJ2 showed 100% resistance to four fungal populations collected from Guangdong, Sichuan, Liaoning, and Heilongjiang Provinces, which is a higher frequency than that shown by the well-known resistance (R) gene donor cultivars such as Sanhuangzhan 2 and 28zhan. Segregation analysis for resistance with F₂ and F₄ populations indicated the resistance of YJ2 was controlled by multiple genes that are dominant or recessive. The putative R genes of YJ2 were roughly tagged by SSR markers, located on chromosomes 2, 6, 8, and 12, in a bulked-segregant analysis using genome-wide selected SSR markers with F₄ lines that segregated into 3 resistant (R):1 susceptible (S) or 1R:3S. The recessive R gene on chromosome 8 was further mapped to an interval ≈1.9 cM/152 kb in length by linkage analysis with genomic position-ready markers in the mapping population derived from an F₄ line that segregated into 1R:3S. Given that no major R gene was mapped to this interval, the novel R gene was designated as pi55(t). Out of 26 candidate genes predicted in the region based on the reference genomic sequence of the cultivar Nipponbare, two genes that encode a leucine-rich repeat-containing protein and heavy-metal-associated domain-containing protein, respectively, were suggested as the most likely candidates for pi55(t).     

Randomization of cortical microtubules in root epidermal cells induces root hair initiation in lettuce (Lactuca sativa L.) seedlings

Root hair formation is induced when lettuce seedlings are transferred from liquid medium at pH 6.0 to fresh medium at pH 4.0. If seedlings are transferred to pH 6.0, no root hairs are formed. We investigated the role of microtubules in this low pH-induced root hair initiation in lettuce. At the hair-forming zone in root epidermal cells, microtubules were perpendicular to the longitudinal axis of the cell just after pre-culture. This arrangement became disordered as early as 5 min after transfer to pH 4.0, and became random by 30 min later. At pH 4.0, the randomization extended to the entire hair-forming zone of seedlings; at pH 6.0, however, randomization did not occur and transverse microtubules were maintained. When seedlings at pH 6.0 were treated with microtubule-depolymerizing drugs, root hairs were formed. In contrast, when a microtubule-stabilizing drug, taxol, was added to the medium, no root hairs formed, even at pH 4.0. These results suggest that the transverse cortical microtubules inhibit root hair formation, and that their destruction is necessary for initiation. Furthermore, the microfilament-disrupting drugs cytochalasin B and latrunculin B inhibited root hair initiation, suggesting that actin filaments are necessary for root hair initiation.     

Intracellular localization of sucrose-Phosphate Phosphate in photosynthetic cells of lettuce (Lactuca sativa)

The intracellular localization of sucrose-phosphate phosphatase (SPP) in photosynthetic cells of lettuce ( Lactuca sativa ) was investigated by using protoplasts isolated directly from leaf tissue. No SPP activity was detected in either vacuolar, chloroplast or mitochondrial preparations. The recovered activity of SPP was found in the cytosolic fractions at proportions parallel with those of the cytoplasmic marker alcohol dehydrogenase. Maximal activity was measured between pH 6. 0 and 7. 0. The data demonstrate a cytosolic location for SPP. Neither sucrose nor any other saccharide tested at 100 m M was a strong inhibitor of the enzyme, which was inactive in the presence of 35 m M ethylenediammetetraacetic acid.     

lyc1, a transgenic mutant of Lactuca sativa (lettuce) tightly linked to chloroplast biogenesis.

A T-DNA tagging mutant of lettuce was studied. The transgenic lettuce mutant, designated light yellowish cotyledon1 (lyc1), exhibited a chloroplast mutant phenotype in which the chloroplast development was arrested at the cotyledon stage and the cotyledon became a light yellowish due to the reduced level of chlorophyll content. A microscopic observation of the mutant showed that the chloroplasts failed to position along the cell wall, but were scattered in the cytoplasm. The number of chloroplasts in the mutant was reduced to 66% compared to that in the wild-type plant. These results suggest that the lyc1 mutation is due to a defect in the biogenesis of chloroplasts. Also, the homozygous lyc1 mutant became lethal in a T2 and F1 progeny analysis, indicating that the mutation is recessive.     

FRET hybridization probes for the rapid detection of disease resistance alleles in plants: detection of corky root resistance in lettuce

We describe the development of a non-electrophoresis PCR-based assay for allele discrimination at a disease resistance locus. The assay is based on the emission of light by fluorescence resonance energy transfer (FRET) upon annealing of two hybridization probes. The analysis of melting curve profiles of the probes and templates allowed the detection of single nucleotide polymorphisms. The assay was applied to the detection of alleles at the cor locus in lettuce (Lactuca sativa) that confers recessive resistance to corky root disease. Probes and primers for the assay were designed after the characterization of a single nucleotide polymorphism between alleles of PCR products amplified using a linked marker. That polymorphism was validated in a collection of lettuce varieties representing different genetic backgrounds. The FRET hybridization probes approach provided fast and accurate genotyping of breeding material directly in a one-tube reaction. The absence of electrophoresis makes this approach suitable for applications that require automation and high-throughput genotyping analyses such as marker-assisted selection programs.     

Evidence for a race-specific resistance factor in some lettuce (Lactuca sativa L.) cultivars previously considered to be universally susceptible to Bremia lactucae regel

Summary Previously undetected race-specific resistance to Bremia lactucae (downy mildew) was located in many lettuce cultivars hitherto considered to be universally susceptible to this disease. This resistance factor(s) may also be widely distributed in other cultivars known to carry combinations of already recognised factors R1 to R11. Specific virulence to match this resistance is almost invariably present in pathogen collections. This situation may be either a relic of the evolutionary history of the B. lactucae — L. sativa asssociation or may reflect a rare mutation in B. lactucae for avirulence on all but a few specialised L. sativa genotypes.