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A tetraploid F 2 progeny segregating for resistance to black spot, growth habit, and absence of prickles on the stem and petioles was used to construct genetic linkage maps of rose. The F 1 of the progeny, 90-69, was created by crossing a black spot-resistant amphidiploid, 86-7, with a susceptible tetraploid, 82-1134. The F 1 was open-pollinated to obtain 115 seedlings. AFLP and SSR markers were used to eliminate seedlings produced through cross-fertilization. The remaining progeny set of 52 F 2 plants was used to study the inheritance of 675 AFLPs, one isozyme, three morphological and six SSR markers. AFLP markers were developed with three combinations of restriction enzymes, EcoRI/MseI, KpnI/MseI and PstI/MseI. Most of the markers appear to be in simplex or single-dose and segregated 3:1 in the progeny. One linkage map was constructed for each parent using only the single-dose markers. The map of 86-7 consists of 171 markers assigned to 15 linkage groups and covering more than 902 cM of the genome. The map of 82-1134 consists of 167 markers assigned to 14 linkage groups and covering more than 682 cM of the genome. In the AFLP analysis, EcoRI/MseI generated nearly twice as many markers per run than PstI/MseI. Markers developed with three restriction enzyme combinations showed a mixed distribution throughout the maps. A gene controlling the prickles on the petiole was located at the end of linkage group 7 on the map of 86-7. A gene for malate dehydrogenase locus 2 was located in the middle of linkage group 4 on the map of 86-7. These first-generation maps provide initial tools for markerassisted selection and gene introgression for the improvement of modern tetraploid roses.

New microsatellites markers [simple sequence repeat (SSR)] have been isolated from rose and integrated into an existing amplified fragment-length polymorphism genetic map. This new map was used to identify quantitative trait locus (QTL) controlling date of flowering and number of petals. From a rose bud expressed sequence tag (EST) database of 2,556 unigenes and a rose genomic library, 44 EST-SSRs and 20 genomic-SSR markers were developed, respectively. These new rose SSRs were used to expand genetic maps of the rose interspecific F 1 progeny. In addition, SSRs from other Rosaceae genera were also tested in the mapping progeny. Genetic maps for the two parents of the progeny were constructed using pseudo-testcross mapping strategy. The maps consist of seven linkage groups of 105 markers covering 432 cM for the maternal map and 136 markers covering 438 cM for the paternal map. Homologous relationships among linkage groups between the maternal and paternal maps were established using SSR markers. Loci controlling flowering traits were localised on genetic maps as a major gene and QTL for the number of petals and a QTL for the blooming date. New SSR markers developed in this study will provide tools for the establishment of a consensus linkage map for roses that combine traits and markers in various rose genetic maps.

The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare’s serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation.

Microsatellite or simple sequence repeat (SSR) markers were developed from Rosa wichurana Crépin to combine two previously constructed tetraploid rose (Rosa hybrida L.) genetic maps. To isolate SSR-containing sequences from rose a small-insert genomic library was constructed from diploid Rosa wichurana and screened with several SSR probes. Specific primers were designed for 43 unique SSR regions, of which 30 primer pairs gave rise to clear PCR products. Seventeen SSR primer pairs (57%) produced polymorphism in the tetraploid rose 90-69 mapping family. These markers were incorporated into existing maps of the parents 86-7 and 82-1134, which were constructed primarily with AFLP markers. The current map of the male parent, amphidiploid 86-7, consists of 286 markers assigned to 14 linkage groups and covering 770 cm. The map of the female tetraploid parent, 82-1134, consists of 256 markers assigned to 20 linkage groups and covering 920 cm. Nineteen rose SSR loci were mapped on the 86-7 map and 11 on the 82-1134 map. Several homeologous linkage groups within maps were identified based on SSR markers. In addition, some of the SSR markers provided anchoring points between the two parental maps. SSR markers were also useful for joining small linkage groups. Based on shared SSR markers, consensus orders for four rose linkage groups between parental maps were generated. Microsatellite markers developed in this study will provide valuable tools for many aspects of rose research including future consolidation of diploid and tetraploid rose genetic linkage maps, genetic, phylogenetic and population analyses, cultivar identification, and marker-assisted selection.

Human kallikrein 8 (KLK8) is a member of the human kallikrein gene family of serine proteases, and its protein, hK8, has recently been suggested to serve as a new ovarian cancer marker. To gain insights into the physiological role of hK8, the active recombinant enzyme was obtained in a pure state for biochemical and enzymatic characterizations. hK8 had trypsinlike activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type IV, fibrinogen, and high-molecular-weight kininogen. hK8 also converted human single-chain tissue-type plasminogen activator (65 kDa) to its two-chain form (32 and 33 kDa) by specifically cleaving the peptide bond Arg 275 -Ile 276 . This conversion resulted in a drastic increase in the activity of the activator toward the fluorogenic substrate Pyr-Gly-Arg-MCA and plasminogen in the absence of fibrin. Our findings suggest that hK8 may be implicated in ECM protein degradation in the area surrounding hK8-producing cells.

Damask roses are grown in several European and Asiatic countries for rose oil production. Twenty-six oil-bearing Rosa damascena Mill. accessions and 13 garden Damask roses were assayed by molecular markers. Microsatellite genotyping demonstrated that R. damascena Mill. accessions from Bulgaria, Iran, and India and old European Damask rose varieties possess identical microsatellite profiles, suggesting a common origin. At the same time, the data indicated that modern industrial oil rose cultivation is based on a very narrow genepool and that oil rose collections contain many genetically identical accessions. The study of long-term vegetative propagation of the Damask roses also reveals high somatic stability for the microsatellite loci analyzed.

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