Development of novel simple sequence repeat markers from a genomic sequence survey database and their application for diversity assessment in Jatropha curcas germplasm from Guatemala.

The last few years have seen a significant increase in the number of large-scale sequencing projects generating whole genome databases. These sequence databases can be surveyed (genome sequence survey) for tandem repeats as an alternative means to develop microsatellites for monitoring and selecting natural populations and cultivars of Jatropha curcas. A total of 100 tandem repeats were revealed from mining 368 genomic surveyed sequences available in the Kazusa DNA Research Institute database. Twenty microsatellite sequences were successfully amplified, resulting in repeatable and scorable polymerase chain reaction products. Genotyping of J. curcas accessions from the Guatemalan population revealed 18 polymorphic loci. The average number of alleles per locus was 6.9, and allelic sizes ranged from 94 to 299 bp. Expected and observed heterozygosities ranged from 0.118 to 0.906 and from 0.082 to 0.794, respectively. Polymorphic information content values ranged from 0.114 (JcSSR-34) to 0.886 (JcSSR-33) with an average of 0.627. Analysis with Micro-Checker indicated few null alleles for locus JcSSR-37 in Guatemalan populations, which may be a possible cause of its deviation from Hardy-Weinberg equilibrium, even after Bonferroni's correction. No loci showed significant linkage disequilibrium. These microsatellite loci are expected to be valuable molecular markers in J. curcas because they show high levels of polymorphism and heterozygosity.

In vitro cytotoxic effects of 5-Fluorouracil on isolated murine ovarian preantral follicles.

5 fluorouracil (5FU), an antineoplastic drug, is often utilized in the therapeutic regimen for several types of cancer, including the hepatoblastoma in children. The effects of 5FU on the population of ovarian preantral follicles, which is the largest oocyte reservoir, is still poorly understood. The integrity of the ovarian preantral follicle pool is important for lifelong fertility. The better understanding of such effects may favor intervention strategies to protect fertility in 5FU-treated children and women coping with cancer. To analyze the effects of 5FU on isolated murine secondary follicles in vitro, ovaries were collected from young mice (28-30 days old), and secondary follicles were isolated and cultured for 12 days in basic culture medium, with or without 5FU at concentrations of 0.3 mM, 1 mM, 3 mM, 10 mM, and 30 mM. In the in vitro study, we analyzed the percentage of morphologically normal follicles, antrum formation, follicular diameter, and hormone production. On day 12, oocytes were recovered for in vitro maturation. 5FU treatment did not alter the percentage of morphologically normal follicles. On day 12, only 1, 10, and 30 mM 5FU significantly reduced the percentage of antrum. From day 4 onwards, 5FU treatments significantly reduced follicle diameter. The meiosis resumption rate was significantly lower in all 5FU treatments. 5FU concentrations ≥3 mM reduced estradiol levels. In conclusion, 5FU does not affect follicular morphology. However, 5FU deleteriously affects follicular growth, estradiol production, and oocyte maturation in isolated ovarian follicles.