High resolution physical mapping of single gene fragments on pachytene chromosome 4 and 7 of Rosa

Background: Rosaceae is a family containing many economically important fruit and ornamental species. Although fluorescence in situ hybridization (FISH)-based physical mapping of plant genomes is a valuable tool for map-based cloning, comparative genomics and evolutionary studies, no studies using high resolution physical mapping have been performed in this family. Previously we proved that physical mapping of single-copy genes as small as 1.1 kb is possible on mitotic metaphase chromosomes of Rosa wichurana using Tyramide-FISH. In this study we aimed to further improve the physical map of Rosa wichurana by applying high resolution FISH to pachytene chromosomes. Results: Using high resolution Tyramide-FISH and multicolor Tyramide-FISH, 7 genes (1.7-3 kb) were successfully mapped on pachytene chromosomes 4 and 7 of Rosa wichurana. Additionally, by using multicolor Tyramide-FISH three closely located genes were simultaneously visualized on chromosome 7. A detailed map of heterochromatine/euchromatine patterns of chromosome 4 and 7 was developed with indication of the physical position of these 7 genes. Comparison of the gene order between Rosa wichurana and Fragaria vesca revealed a poor collinearity for chromosome 7, but a perfect collinearity for chromosome 4. Conclusions: High resolution physical mapping of short probes on pachytene chromosomes of Rosa wichurana was successfully performed for the first time. Application of Tyramide-FISH on pachytene chromosomes allowed the mapping resolution to be increased up to 20 times compared to mitotic metaphase chromosomes. High resolution Tyramide-FISH and multicolor Tyramide-FISH might become useful tools for further physical mapping of single-copy genes and for the integration of physical and genetic maps of Rosa wichurana and other members of the Rosaceae

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Effect of recombinant human vascular endothelial growth factor on testis tissue xenotransplants from prepubertal boys: a three-case study

Research question Does recombinant human vascular endothelial growth factor (VEGF-165) improve the efficiency of human immature testis tissue (ITT) xenotransplantation? Design ITT fragments from three prepubertal boys were cultured for 5 days with VEGF-165 or without (control) before xenotransplantation into the testes of immunodeficient mice. Xenotransplants were recovered at 4 and 9 months after transplantation, and vascularization, seminiferous tubule integrity, number of spermatogonia and germ cell differentiation were evaluated by histology and immunohistochemistry. Results Transplants from donor 1 and donor 2 treated with VEGF demonstrated higher vascular surface (P = 0.004) and vessel density (P = 0.011) overall and contained more intact seminiferous tubules (P = 0.039) with time, compared with controls. The number of spermatogonia was increased over time (P < 0.001) irrespective of treatment and donor, whereas, for the VEGF-treated transplants, the increase was even higher over time (P = 0.020). At 9 months, spermatocytes were present in the xenotransplants, irrespective of treatment. No transplants could be recovered from donor 3, who had already received treatment with cyclosporine for aplastic anaemia before biopsy. Conclusions In-vitro pre-treatment of human prepubertal testis tissue with VEGF improved transplant vascularization in two out of three cases, resulting in improved seminiferous tubule integrity and spermatogonial survival during xenotransplantation. Although further studies are warranted, we suggest VEGF be considered as a factor for improving the efficiency of immature testis tissue transplantation in the future