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Yoshinori Kanayama, Hitoshi Mori, Hidemasa Imaseki, and Shohei Yamaki

Sorbitol plays a important role in the translocation of photosynthate in apple. Sorbitol-6-phosphate dehydrogenase (S6PDH, is the key enzyme regulating sorbitol biosynthesis. The cloning of functional gene like S6PDH provides the potential to elucidate the mechanism of production and translocation of sugar in the Rosaceae family and to manipulate endogenous sorbitol production in horticultural crops.

Poly(A)+RNA was prepared from apple seedlings and cDNA library constructed in an expression vector was screened by the loquat-S6PDH antibody prepared by Hirai (Natl. Res. Inst. Veg. Ornam. Plants & Tea, Japan). The cloned cDNA contained an open reading frame of 930 base pairs encoding a sequence of 310 amino acids. Identification of the cDNA was accomplished by expression of active enzyme in Escherichia coli harboring the cDNA and by the presence of a partial amino acid sequence identical to that found in the purified enzyme. Northern blot analysis showed the expression of S6PDH gene in apple seedlings.

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A. Levi, C. E. Thomas, A. Davis, O.U.K. Reddy, Y. Xu, X. Zhang, S. King, A. Hernandez, G. Gusmini, and T. Wehner

Genetic linkage map is being constructed for watermelon based on a testcross population and an F2 population. The testcross map comprises 262 markers (RAPD, ISSR, AFLP, SSR and ASRP markers) and covers 1,350 cM. The map comprises 11 large linkage groups (50.7–155.2 cM), 5 medium-size linkage groups (37.5–46.2 cM), and 16 small linkage groups (4.2–31.4 cM). Most AFLP markers are clustered on two linkage regions, while all other marker types are randomly dispersed on the genome. Many of the markers in this study are skewed from the classical (Mendelian) segregation ratio of1:1 in the testcross or the 3:1 ratio in the F2 population. Although the skewed segregation, marker order appeared to be consistent in linkage groups of the testcross and F2 population. A cDNA library was constructed using RNA isolated from watermelon flesh 1 week (rapid cell division stage), 2 weeks (cell growth and storage deposition stage, 4 weeks (maturation stage), and 5 weeks (postmaturation stage) post pollination. Over 1,020 cDNA clones were sequenced, and were analyzed using the Basic Local Alignment Search Tool (BLAST). The sequenced cDNA clones were designated as expressed sequenced tag (EST) markers and will be used in mapping analysis of watermelon genome.

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K.G. Raghothma, Umesh Muchhal, Chunming Liu, and U. Mukatira

Phosphate deficiency is wide spread in the nature. The deficiency results in several morphological and biochemical changes in plants. Some of these changes have been shown to involve altered gene expression. We have isolated two full--length cDNAs (AtPT1 and AtPT2), showing significant amino acid sequence similarity with the high-affinity phosphate transporters of yeast, Neurospora and the mycorrhizal fungi Glomus versiforme, from a phosphate-starved Arabidopsis root library. The transcripts of both genes are highly induced under Pi starvation and they are expressed in roots. Using Arabidopsis cDNAs as probes, we have isolated several tomato root cDNA clones representing the two different genes. The expression characteristics of the tomato isoforms of the putative high affinity phosphate transporter genes will be discussed. The northern blots of RNA isolated from phosphate-deficient and phosphate-sufficient roots of tomato indicated that both genes are strongly induced in response to Pi starvation in roots. Furthermore, by the method of differential display of mRNA, we have cloned and characterized a full-length cDNA representing a Pi starvation induced gene (TPSI1) from tomato. The gene is expressed as a specific response to Pi starvation in roots and leaves. The TPSI1 is an intron-less gene represented by a single copy in the tomato genome. The structure, expression, and functional significance of these genes will be discussed. This research has been supported, in part, by USDA grant 94-37100-0834 to KGR.

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Ann C. Smigocki and Freddi A. Hammerschlag

Immature `Redhaven' peach [Prunus persica (L.) Batsch] embryos were infected with a shooty mutant strain of Agrobacterium tumefaciens, tms328::Tn5, which carries an octopine-type Ti plasmid with a functional cytokinin gene and a mutated auxin gene. Shoots were regenerated from embryo-derived callus that was initiated on MS medium lacking phytohormones. Shoots exhibited increased frequency of branching and were more difficult to root than the noninfected. Transcripts of the tms328::Tn5-cytokinin gene were detected using northern analyses of total plant RNA. Polymerase chain reaction of genomic DNA and cDNA resulted in amplification of DNA fragments specific for the cytokinin gene, as determined by restriction enzyme and Southern analyses. The concentrations of the cytokinins zeatin and zeatin riboside in the leaves of regenerated plants were on the average 51-fold higher than in leaves taken from nontransformed plants. None of the shoots or callus tissues were postive for octopine. The expression of the T-DNA encoded cytokinin gene promotes growth of peach cells in the absence of phytohormones, thus serving as a marker for transformation. In addition, this gene appears to promote morphogenesis without an auxin inductive step.

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Jody A. Goodrich

This research tested the hypothesis that 60Co ionizing irradiation degrades polysomes to monosomes, a process that reduces growth of Pisum sativum seedlings. Dry and imbibed seeds and 5-day-old seedlings were exposed to 1.8, 3.6, 7.2, 14.4, or 28.8 krad of 60Co irradiation. Immediately after irradiation treatments, dry and imbibed seeds were planted, and later seedlings were harvested and analyzed. Five 1-cm root-tip samples from 5-day-old seedlings were crushed and layered onto 15% to 60% sucrose gradients and centrifuged for 55 min. The samples were processed through an ISCO ultraviolet chart maker. The monosome and polysome weights were read and analyzed. The monosome content was greater in the irradiated 5-day-old seedlings than in seedlings from dry and imbibed seeds. The growth of 5-day-old irradiated seedlings and seedlings from imbibed seeds was less than that of seedlings from dry seeds. The reduced growth of the irradiated seedlings suggests damage to the polysomes. When protein synthesis in plant cells is altered, perhaps through RNA decoding mechanisms, growth may be partially or completely arrested. Using sensitive plants to establish the injurious effects of ionizing irradiation on living organisms can educate and alert society to the detrimental effects of overexposure to irradiation such as that caused by nuclear accidents.

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Amnon Levi, C.E. Thomas, Angela Davis, O.U.K. Reddy, Y. Xu, X. Zhang, A. Hernandez, G. Gusmini, Todd C. Wehner, J. King, and S.R. King

A genetic linkage map was constructed for watermelon based on a testcross population and an F2 population. The testcross map includes 312 markers (RAPD, ISSR, AFLP, SSR, and ASRP). This map covered a genetic distance of 1385 cM, and identified 11 large (50.7-155.2 cm), five intermediate (37.5-46.2 cm), and 16 small linkage groups (4.2-31.4 cm). Most AFLP markers are clustered in two linkage regions, while all other markers are randomly dispersed throughout the genome. Many of the markers in this study were skewed from the classical (Mendelian) segregation ratio of 1:1 in the testcross or 3:1 in the F2 population. The order of the markers within linkage groups was similar in the testcross and F2 populations. Additionally, a cDNA library was constructed using RNA isolated from watermelon flesh 1 week (rapid cell division stage), 2 weeks (cell growth and storage deposition stage), 4 weeks (maturation stage), and 5 weeks (mature fruit) after pollination. More than 1020 cDNA clones were sequenced, and analyzed using the basic local alignment search Tool (BLAST). The sequenced cDNA clones were designated as expressed sequenced tag (EST). The ESTs were searched for simple sequence repeats. About 7% of the ESTs contained SSR motifs. The ESTs containing SSRs are being used to design PCR primers and the putative markers are being tested for polymorphism among the parental lines of the mapping populations. Polymorphic markers will then be mapped using the mapping populations.

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Richard L. Harkess and Robert E. Lyons

Histological and histochemical examination of floral initiation was conducted to determine the pattern of flowering in Rudbeckia hirta, a long-day (LD) plant. Plants were grown under 8-hour short days (SDs) until they had 14 to 16 expanded leaves. Half of the group of plants was moved to LD conditions consisting of natural daylength plus a 4-hour night interruption. Rudbeckia hirta had a pattern of differentiation in flowering similar to that reported in species requiring one inductive day for initiation. Rudbeckia hirta required 8 LDs for evocation and 18 LDs for completion of initiation. Involucral bracts initiated after 18 LDs, after which the receptacle enlarged and was capped by a meristematic mantle of cells signaling the start of development. Floret primordia did not initiate, even after 20 LDs. Increases in pyronin staining were observed in actively dividing cells of the procambium, leaf primordium, and corpus of the vegetative meristems. After 8 LDs, the pith rib meristem stained darkly, a result indicating the arrival of the floral stimulus. An increase in pyronin staining was also observed in the meristematic mantle covering the receptacle after 18 LDs, a result indicating increased RNA levels.

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Hyo-Won Seo, Jung-Yoon Yi, Young-Il Hahm, Hyun-Mook Cho, and Kuen Woo Park

Three potato (Solanum tuberosum L.) cultivars `Superior', `Irish Cobbler', and `Jopung' were transformed by co-cultivation with tuber discs and disarmed Agrobacterium tumefaciens LBA4404 carrying modified vector pBI121, that contained PLRV coat protein (CP) gene and controlled by CaMV35S promoter. Putative transformants were selected and their genomic DNA and RNA transcripts were analyzed for the confirmation of genetic stability by RT-PCR, PCR, southern, and northern blot. The growth characteristics and viral resistance of progenies of transgenic potato plants were investigated. Twelve lines among the different seven-times manipulated transgenic lines were grown in greenhouse and isolates trial field. PLRV coat protein gene was stably inherited in `Superior', but not in `Jopung'. `Jopung' was less stable than `Irish Cobbler' and `Superior' at genetic stability of PLRV CP gene. And some of these transgenic lines were highly resisted in PLRV multiplication. The yield of transformants was reduced in `Irish Cobbler' but not in `Superior'. Possible explanations for these types of resistance are gene silencing and positional effects of transformed PLRV CP genes and that had cultivar specificity. We consider the appearance of escaped transformants in `Jopung' for emergence of chimeric explants from early selection stage.

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Antonio Figueira, Jules Janick, and Peter Goldsbrough

RAPD markers were used to examine genetic similarity in cacao. DNA from 30 cacao cultivars amplified using 15 arbitrary oligonucleotide primers, produced a total of 112 fragments, of which 88% were polymorphic. A phenogram was developed which illustrates the genetic relationships among the cacao cultivars representing the four major geographic groups of cacao (Criollo, Trinitario, Forastero Lower Amazonian, and Forastero Upper Amazonian). The phenogram indicated a general separation of the four groups into three clusters. Criollos and Trinitarios (supposedly hybrids between Forastero and Criollos types) appeared in a single cluster. Lower Amazonian cultivars (mainly selections made in Bahia, Brazil) appeared in a separate cluster. The third cluster consisted of the Upper Amazonian cultivars, which were originally collected from the region believed to be the center of origin of this crop. This cluster displayed the furthest genetic distance from the others. Crosses between Upper Amazon germplasm and local selections have shown heterosis in clonal crosses, which has been exploited in all genetic improvement programs for cacao. We propose that genetic distances based on RAPD markers can be potentially used as a criterion to select parents capable of producing superior hybrids and populations. Genetic relationships can also be useful to define germplasm collections and conservation strategies. Studies are underway to compare phenograms derived from RAPD markers and ribosomal RNA gene polymorphisms.

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Bindu Chawla, Robert Bernatzki, and Michael Marcotrigiano

Lycopersicon peruvianum is a wild species of tomato that exhibits gametophytic self-incompatibility (S), wherein the SI response is controlled by the genotype of the pollen. Cultivated tomato (L. esculentum) is a self-compatible species. Assisted by phenotypic markers, periclinal graft chimeras between these two species have been obtained. Fruit set analysis following breeding demonstrated that the available five chimeras (PPE, PEE, PEP, EPP, and EEP) are able to accept pollen from L. peruvianum, suggesting that there is a failure of the SI response. SI response is known to be dependent on S-locus associated proteins. These proteins are present in the style, which is mainly derived from the L1 and L2 layers of meristem. RNA analysis of the style tissue using a cloned S-locus cDNA as a probe showed that, except for EEP, all chimeras expressed the S-allele. This was also confirmed by SDS-PAGE analysis of stylar proteins that were present in variable amounts depending on the periclinal combination. Thus, the breakdown of SI is not associated with the lack of expression of the S-locus. Further work is being conducted to understand the nature of this breakdown.