Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.).

Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of <5, compared to the sweet cultivars that are generally characterized by mature fruit pH values of >6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit acidity within the species.

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes.

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and 'Dulce' (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.

Genes expressed during the development and ripening of watermelon fruit.

A normalized cDNA library was constructed using watermelon flesh mRNA from three distinct developmental time-points and was subtracted by hybridization with leaf cDNA. Random cDNA clones of the watermelon flesh subtraction library were sequenced from the 5' end in order to identify potentially informative genes associated with fruit setting, development, and ripening. One-thousand and forty-six 5'-end sequences (expressed sequence tags; ESTs) were assembled into 832 non-redundant sequences, designated as "EST-unigenes". Of these 832 "EST-unigenes", 254 ( approximately 30%) have no significant homology to sequences published so far for other plant species. Additionally, 168 "EST-unigenes" ( approximately 20%) correspond to genes with unknown function, whereas 410 "EST-unigenes" ( approximately 50%) correspond to genes with known function in other plant species. These "EST-unigenes" are mainly associated with metabolism, membrane transport, cytoskeleton synthesis and structure, cell wall formation and cell division, signal transduction, nucleic acid binding and transcription factors, defense and stress response, and secondary metabolism. This study provides the scientific community with novel genetic information for watermelon as well as an expanded pool of genes associated with fruit development in watermelon. These genes will be useful targets in future genetic and functional genomic studies of watermelon and its development.

Assessment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using DNA markers.

Cucurbita pepo (pumpkin, squash, gourd), an economically important species of the Cucurbitaceae, is extremely variable in fruit characteristics. The objective of the present study was to clarify genetic relationships across a broad spectrum of the C. pepo gene pool, with emphasis on domesticates, using AFLP, ISSR and SSR markers. Forty-five accessions were compared for presence or absence of 448 AFLP, 147 ISSR, and 20 SSR bands, their genetic distances (GDs) were estimated and UPGMA cluster analysis was conducted. The results obtained from these three marker systems were highly correlated (P << 0.001). Clustering was in accordance with the division of C. pepo into three subspecies, fraterna, texana and pepo, with the first two less distant to one another than to the last one. Within the clusters, sub-clustering occurred in accordance with fruit shape and size. The subsp. texana cluster consisted of six sub-clusters, one each for the representatives of its five cultivar-groups (Acorn, Crookneck, Scallop, Straightneck and Ovifera Gourd) and wild gourds. Within the subsp. pepo cluster, the representatives of two cultivar-groups (Zucchini and Orange Gourd) formed distinct sub-clusters and the representatives of two other groups (Cocozelle and Vegetable Marrow) tended to sub-cluster separately from one another but formed an assemblage with the representatives of the remaining group (Pumpkin). Within-group GDs were less than corresponding between-group GDs in nearly all comparisons. The smallest-fruited accession, 'Miniature Ball', appears to occupy a genetically central position within C. pepo.

A reference map of Cucumis melo based on two recombinant inbred line populations.

A composite genetic melon map was generated based on two recombinant inbred line (RI) populations. By analyzing the segregation of 346 AFLPs, 113 IMAs and phenotypic characters on a RI population of 163 individuals derived from the cross Védrantais x PI 161375, a first map was constructed. About 20% of the molecular markers were skewed, and the residual heterozygosity was estimated at 4.43% which was not significantly different from the theoretical value of 4.2%. The genome distribution of molecular markers among the 12 linkage groups was not different from a random distribution with the exception of linkage group XII which was found significantly less populated. The genome distributions of IMAs and AFLPs were complementary. AFLPs were found mainly in the middle of each linkage group and sometimes clustered, whereas IMAs were found mainly at the end. A total of 318 molecular markers, mainly AFLP and IMA markers, were mapped on 63 RIs of the second population, Védrantais x PI 414723. Comparison of the maps enables one to conclude that AFLPs and IMAs of like molecular size, amplified with the same primer combination, correspond to the same genetic locus. Both maps were joined through 116 common markers comprising 106 comigrating AFLPs/IMAs, plus five SSRs and five phenotypic markers. The integrated melon map contained 668 loci issuing from the segregation of 1,093 molecular markers in the two RI populations. The composite map spanned 1,654 cM on 12 linkage groups which is the haploid number of chromosomes in melon. Thirty two known-function probes, i.e. known-function genes (9) and morphological traits (23), were included in this map. In addition, the composite map was anchored to previously published maps through SSRs, RFLPs and phenotypic characters.

The early light-induced protein is also produced during leaf senescence of Nicotiana tabacum.

To better understand the genetic controls of leaf senescence, a tobacco (Nicotiana tabacum L. cv. SR1) mRNA that is up-regulated during senescence was isolated by the cDNA-amplified restriction fragment polymorphism method and the cDNA was cloned. The mRNA coded for the early light-induced protein (ELIP), a member of the chlorophyll a/b-binding protein family that has been implicated in assembly or repair of the photosynthetic machinery during early chloroplast development and abiotic stress. A protein antigenically recognized by antibodies to ELIP appeared during senescence with kinetics similar to those of its mRNA. The mRNA, designated ELIP-TOB, was detected earlier when senescence was enhanced by leaf detachment and treatment with 1-amino-cyclopropane-1-carboxylic acid, and was detected later when senescence was retarded by benzyladenine. However, no ELIP-TOB mRNA was seen in the dark even though senescence was accelerated under these conditions. Furthermore, water stress and anaerobiosis stimulated the appearance of ELIP-TOB mRNA before losses of chlorophyll could be detected. We discuss the conditions that may lead to the up-regulation of ELIP-TOB during senescence and speculate as to the role of the gene product in this terminal phase of leaf development.

Use of random amplified polymorphic DNA (RAPD) markers in the study of the parasitic weed Orobanche.

Despite the tremendous economic impact of broomrapes (Orobanche spp.) on agriculture in many countries little is known of the pattern of genetic variation within this group of parasitic weeds. The present paper describes the use of RAPD markers for the study of five Orobanche species in agricultural fields in Israel. Pronounced genetic differentiation was found between the species, and RAPD markers were raised for the identification of each of them. Southern-hybridization patterns of RAPD products of the various species were used to confirm the interpretation. The same markers were valid both for broomrapes collected in agricultural fields and for those collected in natural habitats. The validity of the markers found for O. cumana and O. crenata was confirmed on plants of the same species that were collected in Spain. Parsimony analysis of 86 RAPD characters produced a tree that clearly distinguishes between the five studied Orobanche species, separates the two Orobanche species belonging to sect. Trionychon from those belonging to sect. Osproleon, and supports the separation of O. cumana from O. cernua and of O. aegyptiaca from O. ramosa.

Electrophysiologic aspects of moricizine HCl.

Moricizine HCl, a phenothiazine derivative, is a new antiarrhythmic drug that has quinidine-like effects on the myocardium. Moricizine HCl, 1 X 10(-7) g/ml, significantly decreases Vmax of the transmembrane action potential of canine Purkinje fibers and reduces the duration of the action potential at 50% and 90% of repolarization, whereas resting potential is unchanged. The drug decreases the fast inward sodium current (lNa), as measured by the double sucrose gap technique in frog atrium trabeculae; the processes of activation, inactivation and reactivation of current did not change even when a high concentration of drug (5 X 10(-6) g/ml) was used. At the same dose, the slow inward calcium current (lCa) increased, but the total outward current did not change. Using the patch-clamp technique, it was shown that moricizine HCl, 1 X 10(-4) g/ml, did not alter single-channel conductance, but essentially decreased the mean open tonic and open-state probability of potassium channels either at positive or negative holding potentials. lNa measured in a single myocyte preparation decreased faster when drug was administered in the external bath compared with intracellular injection. Moricizine HCl action on lNa of the single cell and on Vmax of the action potential is frequency dependent. When lNa was recorded directly, there was a cumulative effect of drug. Similar to other quinidine-like agents, moricizine HCl enhanced arrhythmogenicity within the first few minutes after coronary artery occlusion, but prevented arrhythmias 24 hours after acute myocardial infarction.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate-blood cardioplegia improves ATP preservation in human myocardium.

Two groups of patients subjected to radical correction of Fallot's tetrad and defects of interventricular septum were investigated to ascertain whether the addition of glutamic acid to blood cardioplegic perfusate could improve preservation of myocardial ATP during cardiac arrest. In the control group (17 patients) the myocardial protection was performed by repeated infusions of cold blood potassium cardioplegic solution; in the 2nd group (24 patients) cardioplegic perfusate containing glutamic acid (20 mmol/l) was used. Left ventricular biopsies were taken during the first minute after cross-clamping of the aorta and before the release of the aortic clamp to determine ATP, glutamate and lactate. The cross-clamping time averaged 32 min in both groups. In the patients of the control group the losses of ATP correlated with the decrease in glutamate during the clamping period. A maintenance of a higher myocardial glutamate content by glutamate-containing cardioplegic perfusate prevented ATP fall or increased its level in patients of the 2nd group. There was no significant difference in lactate levels between the two groups by the end of the cardiac arrest. We conclude that enrichment of blood cardioplegic solution by glutamic acid, which may act as a substrate for anaerobic energy production, provides more effective myocardial protection during ischemic heart arrest.

Ischemic heart arrest: nitrogenous metabolism in energy-depleted human myocardium.

Seventeen patients undergoing radical correction of Fallot's tetrad or defects of interventricular septum were investigated. Needle biopsies from the left ventricular apex region were obtained at the 1st min after cross-clamping of the aorta and at the end of cardiac arrest to determine adenosine triphosphate (ATP), glutamate, aspartate, alanine and ammonia. The losses of ATP during clamping period were related to decrease in glutamate. The fall in ATP by more than 20% of the initial level was accompanied by a significant decrease in aspartate, an accumulation of alanine and ammonia in cardiac tissue but did not affect glutamine content. The data obtained prove the participation of specific nitrogenous compounds of human heart, and especially glutamate, in response to energy depletion during ischemia.

The effect of burn injury on the heart in the whole body and on the extracorporeally perfused isolated heart.

Cardiac performance during the initial stage of burn shock in dogs was studied in the whole body and compared with the function of the isolated hearts, perfused from donor animals similarly burned. Continuous recording of the functional indices using electro-magnetic flowmetry and ECG with on-line processing of the data enabled us to establish the following facts. Progressive decrease of cardiac output secondary to the depression of myocardial contractility against the background of stable inflow takes place as early as 2-5 minutes after massive burning in the whole body. No significant changes of the functional indices of the isolated hearts, perfused from the perfusion donors similarly burned, were observed in the second group of experiments. The underlying mechanisms of the above phenomena are discussed.

Modified perfusion and loading technique of the isolated canine heart.

The technique of donor perfusion of the isolated canine heart is described. It is carried out with the aid of a specially developed system providing both retrograde resuscitational coronary perfusion of the heart which sustained ischemia and perfusion with functional volume-systolic loadings of the left ventricle of the working heart under nearly physiologic conditions. This technique was used in the first group of experiments (control) to study the performance of the heart which did not sustain ischemia, and in two groups of experiments with cardioplegia. In the experiments of group II the corrected autologous blood which served as perfusate had the following composition: Hb 8.3 g%; K+ 2.4 mEq/l; pH 7.7; osmolarity 307 mosm/l; pO2 250-450 mm Hg; temperature 13-15 degree C. The hearts were preserved for 2 h following single-dose perfusion . In the experiments of group III the corrected autologous blood had the following composition: Hb 8 g%; K+ 20-25 mEq/l; pH 7.7; osmolarity 320 mosm/l; pO2 300-500 mm Hg; temperature 13-15 degrees C. Coronary perfusion was carried out every 20 min during the 2-hour preservation period. The studies of coronary hemodynamics and cardiac function using the developed system showed practically identical results in groups I and III. In group II deep depression of myocardial function due to ischemic injury was found. The presented data on a comparative assessment of cardiac function in three groups of experiments demonstrate the high value of the developed technique of perfusion and functional loading of the myocardium.

Homologous blood syndrome in dogs. Effect of erythrocyte and platelet antibodies.

Isovolemic perfusion of 50% of the recipient circulating blood volume was carried out in intact mongrel dogs using whole donor blood as well as plasma or platelet suspensions in saline. Perfusion against the background of circulating red blood cells (RBC) or platelets antibodies in the donor or in the recipient was followed by severe and sometimes fatal homologous blood syndrome in a majority of the dogs. Weak and short-term reactions were seen in a few dogs if red blood cells and platelet antibodies were absent.