Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. 'Grande Naine' (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.
A genotyping by sequencing (GBS) approach was used to analyze the organization of genetic diversity in V. pubescens and V. chilensis. GBS identified 4675 and 4451 SNPs/INDELs in two papaya species. The cultivated orchards of V. pubescens exhibited scarce genetic diversity and low but significant genetic differentiation. The neutrality test yielded a negative and significant result, suggesting that V. pubescens suffered a selective sweep or a rapid expansion after a bottleneck during domestication. In contrast, V. chilensis exhibited a high level of genetic diversity. The genetic differentiation among the populations was slight, but it was possible to distinguish the two genetic groups. The neutrality test indicated no evidence that natural selection and genetic drift affect the natural population of V. chilensis. Using the Carica papaya genome as a reference, we identified critical SNPs/INDELs associated with putative genes. Most of the identified genes are related to stress responses (salt and nematode) and vegetative and reproductive development. These results will be helpful for future breeding and conservation programs of the Caricaceae family.
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