Secuenciación de alta calidad del genoma de Posidonia oceanica
In September 2021, Fundación IBEROSTAR and Fundación General del CSIC signed an agreement to sequence the genome of 14 individuals of Posidonia oceanica collected across the Mediterranean basin, and to construct a phylogeographic analyses based on the genetic diversity (SNPs) across genomes.
The aim of this extension is to add samples of Posidonia oceanica collected from other 8 Mediterranean localities, particularly from Mallorca since its large coast was underrepresented over smaller Balearic Island: Cabrera, Artà (Colònia St Pere), Formentor (Cala Figuera), PortoPetro, Port d’Andratx, Murcia, Banyuls (France) and Cyprus (or Greece) as Eastern Mediterranean outgroup. If the latter was not collected another locality from Mallorca would be added.
We also propose a new aim: to study genetic diversity across individuals of Posidonia oceanica in replanted meadows at the Bay of Pollença. We want to assess whether genetic diversity across replanted individuals is similar to that from individuals of the donor meadows, which are geographically close. Since the number of analyses samples would be high, 96 samples, it would be unfeasible to sequence the whole genome due to extremely high costs. Hence, we propose to deplete the amount of nuclear DNA from whole DNA extractions from P. oceanica samples, and hence enriching for chloroplastic and mitochondrial sequences. The main reproduction in P. oceanica is asexual by plantlets, so the genetic diversity from chloroplastic and mitochondrial sequences are very similar to that estimated from nuclear sequences since there is no recombination. Therefore, the reduced plastome and mitogenome (about 0.5 Mb in total), can be used as a proxy of the nuclear genome (3,200 Mb) at a reduced sequencing cost. The depletion of nuclear sequences will be performed with the NEBNext® Microbiome DNA Enrichment Kit (New England Biolabs) that retain nuclear DNA because is higly methylated (CpG islands) in plants, so microbiome, plastic and mitochondrial DNA is in the flow-through since they present little or null methylation. Moreover, recovered plastic and mitochondrial DNA will be sequenced in a Nanopore System that can read large DNA fragments (> 0,1 Mb) and hence recover full plastomes and mitogenomes in few fragments. Since we except that sequencing error is smaller than genetic diversity across individuals, hence we can pool many individuals in the same sample to reduce costs. This would allow to sequence 96 individuals in a single Nanopore sequencing run and just perform 8 DNA extractions of 12 specimens each. This approach also reduced the amount of leave collected from each individual that is a handicap in replanted plants since they have few leaves, which are short.