Optimized protocol to isolate high quality genomic DNA from different tissues of a palm species

Abstract

The application of molecular techniques to tackle ecological and evolutionary questions requires genomic DNA in good quality and quantity. The quality of the isolated DNA, however, can be influenced by the tissue type and the way the sample was conserved and manipulated. Therefore, customizing protocols to improve the DNA isolation and locus amplification is crucial. We optimized a cheap and manual protocol of DNA extraction and microsatellites amplification using five different tissues of a palm species of the brazilian Atlantic Forest. We successfully extracted DNA from all five tissue types. Leaf, stem, and endocarp of non-dispersed seeds presented the highest rates of successful DNA extraction and microsatellite amplification; whereas root, endocarp of dispersed seeds, and embryo showed the lowest quality and quantity. Based on these results, we discussed the implications of using different tissues for studies about seed dispersal, pollination, and population genetics.

Extant fruit‐eating birds promote genetically diverse seed rain, but disperse to fewer sites in defaunated tropical forests

“…Overall, we extracted DNA from 1,330 endocarps following the protocol described by Lucas et al. (2019). All endocarps were genotyped using seven highly polymorphic microsatellite loci (EE3, EE23, EE25, EE45, EE47, EE52 and EE54; Gaiotto et al., 2001), following the PCR protocol described by Lucas et al.…”

“…All endocarps were genotyped using seven highly polymorphic microsatellite loci (EE3, EE23, EE25, EE45, EE47, EE52 and EE54; Gaiotto et al., 2001), following the PCR protocol described by Lucas et al. (2019). DNA fragments were sized in ABI Prism 3100 automated DNA sequencer (Applied Biosystems) using GeneScan Rox 500 size standard (Applied Biosystems), and scored using GeneMapper v.4.1 software (Applied Biosystems).…”

“…We identified maternal progenies (i.e. seeds from the same mother tree) by matching the multilocus genotypes of dispersed endocarps using the r package allelematch v 2.5 (Galpern et al., 2012), allowing one allele mismatching to account for the possibility of genotyping errors that are expected when genotyping low‐quality DNA sources, such as dispersed endocarp tissues (García et al., 2009; Lucas et al., 2019). Therefore, multilocus genotypes that shared at least 13 alleles out of 14 were considered identical and were assigned to a single maternal source.…”

Comparative analysis of the DNA isolated from thyme leaves using different methods

“…For example, essential oil accumulation in leaf blades influence the DNA quality (Bulavin et al, 2019b), phenolic compounds and terpenes can reduce the yield and purity of extracted DNA due to its degradation (Kanauja et al, 1999;Vega-Vela, Chacón-Sánchez, 2011). These undesirable phenomena are eliminated by adding PVP in the range 1-5% (Hills, van Staden, 2002;Sá et al, 2011;Arruda et al, 2017;Lucas et al, 2019) to the CTAB extraction buffer and further overnight rehydration of the PVP. Our results also demonstrated that DNAs isolated from four thyme cultivars with the CTAB method + 2% PVP had robust values of A 260/280 ratio despite the presence of essential oils and phenols in the plant tissues.…”

Discrimination of common Iris species from Egypt based on their genetic and metabolic profiling

“…Therefore, finding genetic markers and metabolic markers of the different plant species could be interesting and serve in plant taxonomy. However, the isolation of DNA in good quantity and quality is a critical step towards the development of the genetic markers 24–26 . Extracting the genomic DNA in good state can be influenced by the tissue type and also by how the sample is manipulated by the researcher 26,27 .…”