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.

Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci.

The sweet potato whitefly, Bemisia tabaci, harbors Portiera aleyrodidarum, an obligatory symbiotic bacterium, as well as several secondary symbionts including Rickettsia, Hamiltonella, Wolbachia, Arsenophonus, Cardinium and Fritschea, the function of which is unknown. Bemisia tabaci is a species complex composed of numerous biotypes, which may differ from each other both genetically and biologically. Only the B and Q biotypes have been reported from Israel. Secondary symbiont infection frequencies of Israeli laboratory and field populations of B. tabaci from various host plants were determined by PCR, in order to test for correlation between bacterial composition to biotype and host plant. Hamiltonella was detected only in populations of the B biotype, while Wolbachia and Arsenophonus were found only in the Q biotype (33% and 87% infection, respectively). Rickettsia was abundant in both biotypes. Cardinium and Fritschea were not found in any of the populations. No differences in secondary symbionts were found among host plants within the B biotype; but within the Q biotype, all whiteflies collected from sage harboured both Rickettsia and Arsenophonus, an infection frequency which was significantly higher than those found in association with all other host plants. The association found between whitefly biotypes and secondary symbionts suggests a possible contribution of these bacteria to host characteristics such as insecticide resistance, host range, virus transmission and speciation.

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.

Simple-sequence repeat markers used in merging linkage maps of melon (Cucumis melo L.).

A set of 118 simple sequence repeat (SSR) markers has been developed in melon from two different sources: genomic libraries (gSSR) and expressed sequence-tag (EST) databases (EST-SSR). Forty-nine percent of the markers showed polymorphism between the 'Piel de Sapo' (PS) and PI161375 melon genotypes used as parents for the mapping populations. Similar polymorphism levels were found in gSSR (51.2%) and EST-SSR (45.5%). Two populations, F2 and a set of double haploid lines (DHLs), developed from the same parent genotypes were used for map construction. Twenty-three SSRs and 79 restriction fragment length polymorphisms (RFLPs), evenly distributed through the melon genome, were used to anchor the maps of both populations. Ten cucumber SSRs, 41 gSSRs, 16 EST-SSR, three single nucleotide polymorphism (SNP) markers, and the Nsv locus were added in the DHL population. The maps developed in the F2 and DHL populations were co-linear, with similar lengths, except in linkage groups G1, G9, and G10. There was segregation distortion in a higher proportion of markers in the DHL population compared with the F2, probably caused by selection during the construction of DHLs through in vitro culture. After map merging, a composite genetic map was obtained including 327 transferable markers: 226 RFLPs, 97 SSRs, three SNPs, and the Nsv locus. The map length is 1,021 cM, distributed in 12 linkage groups, and map density is 3.11 cM/marker. SSR markers alone cover nearly 80% of the map length. This map is proposed as a basis for a framework melon map to be merged with other maps and as an anchor point for map comparison between species of the Cucurbitaceae family.

Comparative mapping of ZYMV resistances in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.).

Zucchini yellow mosaic virus (ZYMV) routinely causes significant losses in cucumber ( Cucumis sativus L.) and melon ( Cucumis melo L.). ZYMV resistances from the cucumber population 'TMG1' and the melon plant introduction (PI) 414723 show different modes of inheritance and their genetic relationships are unknown. We used molecular markers tightly linked to ZYMV resistances from cucumber and melon for comparative mapping. A 5-kb genomic region (YCZ-5) cosegregating with the zym locus of cucumber was cloned and sequenced to reveal single nucleotide polymorphisms and indels distinguishing alleles from ZYMV-resistant (TMG1) and susceptible (Straight 8) cucumbers. A low-copy region of the YCZ-5 clone was hybridized to bacterial artificial chromosome (BAC) clones of melon and a 180-kb contig assembled. One end of this melon contig was mapped in cucumber and cosegregated with ZYMV resistance, demonstrating that physically linked regions in melon show genetic linkage in cucumber. However the YCZ-5 region segregated independently of ZYMV resistance loci in two melon families. These results establish that these sources of ZYMV resistances from cucumber TMG1 and melon PI414723 are likely non-syntenic.

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.

Spectral imaging of multi-color chromogenic dyes in pathological specimens.

We have investigated the use of spectral imaging for multi-color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.

Use of a murine xenograft model for canine transmissible venereal tumor.

OBJECTIVE: To develop a murine model for canine transmissible venereal tumor (CTVT). ANIMALS: Thirty-three 6-week-old NOD/LtSz-scid (NOD/SCID) mice and seven 6-week-old C57BL/6J mice. PROCEDURE: Samples of CTVT were excised from a 3-year-old dog and inoculated SC into ten 6-week-old NOD/SCID mice to induce growth of xenograft transmissible venereal tumor (XTVT). To establish mouse-to-mouse transmission, samples of XTVT were removed and inoculated SC into 4 groups of 6-week-old NOD/SCID mice and into a control group. Samples of CTVT were also inoculated into immunocompetent C57BL/6J mice for a mouse antibody production (MAP) test. The canine and xenografted tumors were evaluated cytologically and histologically, and polymerase chain reaction was performed for detection of the rearranged LINE/c-MYC junction. RESULTS: 8 of 10 NOD/SCID mice that were inoculated with CTVT developed tumors 3 to 10 weeks after inoculation. In the second-generation xenograft, all mice developed tumors by postinoculation day 47; 1 X 10(6) of XTVT cells were enough to create a xenograft. Metastases developed in 4 of 20 mice. Xenografted and metastatic tumors retained cytologic, histologic, and molecular characteristics of CTVT. Results of the MAP test were negative for all pathogens. CONCLUSION: We established an NOD/SCID murine model for XTVT and metastasis of CTVT. This model should facilitate study of tumor transplantation, progression, and metastasis and should decrease or eliminate the need for maintaining allogenic transfer in dogs.

Acetyl-coa: alcohol acetyltransferase activity and aroma formation in ripening melon fruits.

Melon varieties (Cucumis melo L.) differ in a range of physical and chemical attributes. Sweetness and aroma are two of the most important factors in fruit quality and consumer preference. Volatile acetates are major components of the headspace of ripening cv. Arava fruits, a commercially important climacteric melon. In contrast, volatile aldehydes and alcohols are most abundant in cv. Rochet fruits, a nonclimacteric melon. The formation of volatile acetates is catalyzed by alcohol acetyltransferases (AAT), which utilize acetyl-CoA to acetylate several alcohols. Cell-free extract derived from Arava ripe melons exhibited substantial levels of AAT activity with a variety of alcohol substrates, whereas similar extracts derived from Rochet ripe melons had negligible activity. The levels of AAT activity in unripe Arava melons were also low but steadily increased during ripening. In contrast, similar extracts from Rochet fruits displayed low AAT activity during all stages of maturation. In addition, the benzyl- and 2-phenylethyl-dependent AAT activity levels seem well correlated with the total soluble solid content in Arava fruits.

Simple sequence repeats in Cucumis mapping and map merging.

Thirty-four polymorphic simple-sequence repeats (SSRs) were evaluated for length polymorphism in melon (Cucumis melo L.) and cucumber (Cucumis sativus L.). SSR markers were located on three melon maps (18 on the map of 'Vedrantais' and PI 161375, 23 on the map of 'Piel de Sapo' and PI 161375, and 16 on the map of PI 414723 and 'Dulce'). In addition, 14 of the markers were located on the cucumber map of GY14 and PI 183967. SSRs proved to be randomly distributed throughout the melon and cucumber genomes. Mapping of the SSRs in the different maps led to the cross-identification of seven linkage groups in all melon maps. In addition, nine SSRs were common to both melon and cucumber maps. The potential of SSR markers as anchor points for melon-map merging and for comparative mapping with cucumber was demonstrated.

Signal to noise analysis of multiple color fluorescence imaging microscopy.

BACKGROUND: Various approaches that were recently developed demonstrate the ability to simultaneously detect all human (or other species) chromosomes by using combinatorial labeling and fluorescence in situ hybridization (FISH). With the growing interest in this field, it is important to develop tools for optimizing and estimating the accuracy of different experimental methods. METHODS: We have analyzed the principles of multiple color fluorescence imaging microscopy. First, formalism based on the physical principles of fluorescence microscopy and noise analysis is introduced. Next, a signal to noise (S/N) analysis is performed and summarized in a simple accuracy criterion. The analysis assumes shot noise to be the dominant source of noise. RESULTS: The accuracy criterion was used to calculate the S/N of multicolor FISH (M-FISH), spectral karyotyping, ratio imaging, and a method based on using a set of broad band filters. Spectral karyotyping is tested on various types of samples and shows accurate classifications. We have also tested classification accuracy as a function of total measurement time. CONCLUSIONS: The accuracy criterion that we have developed can be used for optimizing and analyzing different multiple color fluorescence microscopy methods. The assumption that shot noise is dominant in these measurements is supported by our measurements.

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.

Length polymorphism and homologies of microsatellites in several Cucurbitaceae species.

The objectives of this research were to assess (1) the degree of Simple Sequence Repeats (SSR) DNA length polymorphism in melon (Cucumis melo L.) and other species within the Cucurbitaceae family and (2) the possibility of utilizing SSRs flanking primers from single species to other genera or species of Cucurbitaceae. Five melon (CT/GA) n SSRs were isolated from a genomic library. Two cucumber (Cucumis sativus L.) SSRs were detected through a search of DNA sequence databases, one contained a (CT)8 repeat, the other a (AT)13 repeat. The seven SSRs were used to test a diverse sample of Cucurbitaceae, including 8 melon, 11 cucumber, 5 squash, 1 pumpkin, and 3 watermelon genotypes. Five of the seven SSRs detected length polymorphism among the 8 melon genotypes. PCR amplification revealed between three and five length variants (alleles) for each SSR locus, with gene diversity values ranging from 0.53 to 0.75. Codominant segregation of the alleles among F2 progeny was demonstrated for each of the five SSR loci. Four of the seven SSRs detected polymorphism among the 11 cucumber genotypes, with gene diversity values ranging between 0.18 and 0.64. Primers specific to SSRs of C. melo and C. sativus also amplified DNA extracted from genotypes belonging to other genera of the Cucurbitaceae family.

Analysis of rearranged immunoglobulin genes indicating a process of clonal evolution in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is known to be a stable monoclonal neoplasm. In contrast to early studies demonstrating no more than two hybridizing immunoglobulin heavy chain bands corresponding to the two expected alleles, we have demonstrated an unexpected multiband pattern when the HindIII-digested DNA samples from 38 CLL patients were analysed by Southern blot hybridization using JH and C mu gene probes. In order to characterize the genetic basis for the multiband pattern, we molecularly cloned the immunoglobulin heavy chain genes of one of the patients whose leukaemic DNA sample demonstrated three hybridizing JH bands and a loss of the germline band. The cloned rearranged immunoglobulin genes could be divided, based on the restriction mapping and the hybridization with the various probes, into two basic patterns representing two alleles. In one of the cloned rearranged immunoglobulin genes a secondary rearrangement occurred that resulted in the addition of 300 base-pair long sequence into the switch region, and the creation of a HindIII restriction site. The results of the study suggest that clonal evolution occurs in some CLL, and that many of these neoplasms are indeed oligoclonal due to the accumulation of secondary genetic changes.

Identity of rearranged LINE/c-MYC junction sequences specific for the canine transmissible venereal tumor.

The canine transmissible venereal tumor is a naturally occurring neoplastic disease that affects the external genitalia of both sexes and is transmitted during coitus. Cytogenetic and immunologic studies demonstrated that tumors from different parts of the world are very similar, suggesting that they are transferred from one animal to another by the transplantation of viable cells. We found that the c-MYC oncogene was rearranged in this tumor by the insertion of a transposable genetic element sequence (known as LINE, long interspersed element) 5' to the first exon. The amplification of a DNA segment located in the junction of the LINE genome and c-MYC upstream sequences enabled the testing of the similarity of transmissible venereal tumor samples collected independently in different parts of the world. Oligonucleotide primers flanking the LINE/c-MYC junction were used to amplify a 340-base-pair segment and nested primers amplified a 280-base-pair segment. A fifth oligonucleotide used as a probe contained the actual junction sequence. All of the tumors analyzed revealed the existence of the specific bands, which were absent in normal canine DNA samples. The amplified segments obtained from all of the tumors analyzed were identical in size and nucleotide sequence, suggesting transmission of the original rearranged cell itself, as opposed to independent events of LINE insertion in a "hot spot."

Kaposi's sarcoma among AIDS patients: transmissible venereal tumour by cell engraftment?

The epidemiologic findings of Kaposi's sarcoma (KS) among patients with the acquired immunodeficiency syndrome (AIDS) suggest that human immunodeficiency virus (HIV) infection is insufficient for the development of KS. It was speculated that another sexually transmitted infection is responsible for the markedly increased incidence of KS among patients who acquired HIV infection through sexual intercourse. However, no such contributing infectious agent was consistently identified. The canine transmissible venereal tumour (TVT) is a malignant tumour that can be transplanted by viable cells across major histocompatibility complex (MHC) barriers. Recent findings suggest that all canine TVTs originated from the same tumour and were transferred from one animal to the other during sexual intercourse. It is suggested that, in analogy with the canine TVT model, the characteristics of KS epidemic among AIDS patients may be explained by transmission and engraftment of viable malignant cells during intercourse.

Immunoglobulin heavy chain gene rearrangements in chronic lymphocytic leukaemia: correlation with clinical stage.

A search for a correlation between the clinical stage of chronic lymphocytic leukaemia (CLL) and the pattern of immunoglobulin heavy chain gene rearrangements was undertaken. DNA samples from the leukaemic cells of 38 CLL patients were analysed by Southern blot hybridization. Using probes for the immunoglobulin heavy chain J (JH) and C mu regions a marked heterogeneity of the hybridization patterns was observed in both regions. The number of JH hybridization bands varied from one to four and more than two were found in 58% of the patients. In 42% of the patients no germline JH genes were found. One to three additional C mu bands were observed in 34%, but the germline was preserved in all samples. There was no correlation between the clinical stage and the number of hybridizing JH bands; however, a significant correlation was found between the loss of JH germline band or a C mu multiband pattern and advanced stage of the disease. The genetic events in the immunoglobulin genes observed in advanced CLL patients are assumed to result from clonal evolution and tumour progression.

A search for bcl1, bcl2, and c-myc oncogene rearrangements in chronic lymphocytic leukemia.

Three cellular or putative oncogenes: c-myc, bcl1, and bcl2 were previously found to be rearranged in some B cell malignancies due to chromosomal translocations. Data concerning the role of such genetic rearrangements in B-CLL are very scanty and limited to few cases in which bcl1 rearrangements were found. We studied DNA samples from 38 cases of B-CLL by Southern blot technique in order to find out the existence and frequency of such events. No bcl1 or bcl2 rearrangements were found in any of the studied cases; thus, involvement of these genes in CLL must be rare. In one patient who had an aggressive and resistant disease, c-myc rearrangement was found.