DNA extraction from wild and in vitro plants of Hedeoma multiflora Benth.

DIAZ GABUTTI MARIA SOLEDAD; VERDES PATRICIA; KURINA SANZ MARCELA; MAGALLANES NOGUERA CYNTHIA

Congreso; XL Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2022

Hedeoma multiflora Benth. (Lamiaceae) known as "peperina de las lomas" is a native and medicinal herb well-known due to its flavoring, digestive, cytotoxic, antioxidant and antirheumatic properties. Its glandular hairs secret essential oils mainly composed by the monoterpenes such as pulegone, isomenthone and menthone. Since the survival of H. multiflora natural populations is in danger, we consider in vitro culture as an alternative for its conservation. There are no genomic data about specific molecular markers to correlate chemotypes and genotypes neither for wild nor for in vitro plants. Restriction site associated DNA sequencing (RADseq) and its derived protocols, such asthe double digest RADseq (ddRADseq), offer a flexible strategy for an efficient sampling of the plant genome. However, this technique requires genomic DNA of high quality and quantity. The aim of this work was to select a proper extraction protocol of genomic DNA (gDNA) for both, wild and in vitro H. multiflora specimens. Fresh young leaves of wild plants from three different sampling sites (S1: near of El Morro 33° 17 25” S 65°28 07” W; S2: La Esquina, El Morro 33°11 03” S 65° 22 08” W and S3: Dique Boca del Río, Merlo 32° 58 37.8” S 65°02 50.4” W) and from two micropropagated lines were collected and ground with liquid nitrogen in mortar. Based on Doyle protocol we defined three different treatments by using increasing concentrations (2%, 3% and 4%) of cetyl trimethyl ammonium bromide (CTAB) to extract gDNA. Treatment 1 (CTAB 2%) yielded 150 – 600 ng/ul of gDNA from wild plants and 60 – 90 ng/ul from in vitro plants although 260/280 ratio was a bit low (1 to 1.3) and contamination with RNA was observed in wild plant samples. Treatment 2 (CTAB 3%) shows comparable amounts of intact gDNA nevertheless the 260/280 ratio was better (1.3 to1.5) and no contamination with RNA was evident. While the application of treatment 3 (CTAB 4%) resulted in similar amounts of gDNA with an improved 260/280 ratio for in vitro plants (1.7 to1.8), albeit the gel showed DNA degradation and RNA contamination. Therefore, treatment 2 was selected to obtain gDNA in adequate quantity and quality from both, wild and in vitro plants of H. multiflora Benth to use in the ddRADseq technique. This optimized protocol will allow to go from the DNA sample to genotyping data to study the genetic stability of micropropagated plants.