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Ddradseq) in Stetsonia Coryne (Cactaceae

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Ddradseq) in Stetsonia Coryne (Cactaceae An Acad Bras Cienc (2021) 93(Suppl. 3): e20201778 DOI 10.1590/0001-3765202120201778 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal CELLULAR AND MOLECULAR BIOLOGY Development of novel SSR molecular markers Running title: DEVELOPMENT using a Next-Generation Sequencing approach OF SSR MARKERS IN Stetsonia coryne (ddRADseq) in Stetsonia coryne (Cactaceae) Academy Section: Cellular and ANGELA VERÓNICA GUTIÉRREZ, CARLA VALERIA FILIPPI, NATALIA CRISTINA Molecular Biology AGUIRRE, ANDREA FABIANA PUEBLA, CINTIA VANESA ACUÑA, GISEL MARÍA TABOADA & FRANCISCO PABLO ORTEGA-BAES e20201778 Abstract: The Cactaceae family is native to the American continent with several centers of diversity. In South America, one of these centers is the Central Andes and many species are considered to be threatened or vulnerable according to the International Union for 93 Conservation of Nature (IUCN). Stetsonia coryne is an emblematic giant columnar cacti (Suppl. 3) of the Chaco phytogeographic province. It has an extensive geographical distribution 93(Suppl. 3) in many countries of the continent. However, to date there are no specifi c molecular markers for this species, neither reports of population genetic variability studies, such DOI 10.1590/0001-3765202120201778 as for many cactus species. The lack of information is fundamentally due to the lack of molecular markers that allow these studies. In this work, by applying a Genotyping by Sequencing (GBS) technique, we developed polymorphic SSR markers for the Stetsonia coryne and evaluated their transferability to phylogenetically close species, in order to account for a robust panel of molecular markers for multispecies-studies within Cactaceae. Key words: Cactaceae, Stetsonia coryne, GBS, ddRADSeq, Microsatellite. INTRODUCTION in land use and climate are the most important threats for these species (Ortega-Baes et al. The Cactaceae family, native to the American 2010). A recent global assessment of the family continent shows a high diversity of species in has indicated that approximately 31 % of its México and Southwestern United States, while species present some degree of extinction risk in South America three main diversity centers (Goettsch et al. 2015) and many are considered have been recognized: 1). The Central Andes to be threatened or vulnerable according to the region, 2). East Brazil, 3). West and South Brazil, International Union for Conservation of Nature Paraguay, Uruguay (Ortega-Baes et al. 2010). Cacti (IUCN). are a group with special conservation interest In the Central Andes, Northwest Argentina as ornamental plants, in the food and medical is one of the main centers of cactus diversity industry, and as a source of wood (Casas & in the world (Ortega-Baes & Godínez-Alvarez Barbera 2002, Inglese et al. 2002, Nerd et al. 2002, 2006, Ortega-Baes et al. 2010). Studies of genetic Nefzaoui & Salem 2002, Saenz Hernandez et al. structure in South America, however, are mainly 2002, Wright et al. 2007, Ortega-Baes et al. 2010). concentrated in areas of Venezuela and Brazil Consequently, they have strong pressure for (Moraes et al. 2005, Figueredo et al. 2010, Fava et illegal collection, which along with the change al. 2016, Fernandes et al. 2016, Khan et al. 2018), An Acad Bras Cienc (2021) 93(Suppl. 3) ANGELA VERÓNICA GUTIÉRREZ et al. DEVELOPMENT OF SSR MARKERS IN Stetsonia coryne some studies in central zone of Chile (Ossa et highly distributed throughout the genomes of al. 2016, Larridon et al. 2018) and a single report eukaryotes (Li et al. 2002). Even though SSR assessed the genetic diversity of Echinopsis markers were firstly introduced in the late terscheckii, a species with Argentinean 1990s, they are still highly used for applications distribution (Quipildor et al. 2017, 2018). In other involving genome mapping, forensics, parentage words, the study of the genetic variability of identification, population studies, conservation this family in Argentina is just beginning. This is genetics and phylogeography (Hodel et al. 2016). mainly due to the absence of molecular markers Indeed, SSR markers are the most commonly that allow its study (Guerrero et al. 2019). For used molecular markers to study the genetic this reason, an exhaustive study of the genetic variability of natural plant populations, where structure of different species of this family sampling a high number of individuals, even is necessary to evaluate the possible effect with a small number of markers, is preferred of these factors on the genetic variability of over sampling a small number of individuals these species. This would allow the proposal of with a high number of markers (Bonatelli et conservation actions and priority areas with the al. 2015). The attractiveness of SSR markers greater genetic variability of this family. Of the relies on their abundance in the genome, high more than 2,000 cacti described, only around 28 levels of polymorphism, high reproducibility, wild species have been genetically analyzed to codominance and cost effectiveness (Li et al. date (Cornejo-Romero et al. 2013). 2002). Moreover, because SSR are PCR-based Stetsonia coryne (Salm Dick Britton & Rose) markers, they can be amplified using low is an emblematic giant columnar cactus (2n= concentration of DNA, even of low quality, and 22, Sosa-Pivatto et al. 2014) appreciated for its accordingly they are the markers of choice in use in ornamental, live fences, for its edible studies involving ancient DNA and forensics fruits and as a source of wood for handicrafts (Zalapa et al. 2012, Hodel et al. 2016). Another (Arenas & Scarpa 1998, Anderson 2001, Arenas important characteristic of these markers & Scarpa 2007). This species its endemic of the is the transferability between plant species Chaco phytogeographic province, which has of the same genus or family (Zalapa et al. an extensive geographical distribution that 2012, Bombonato et al. 2019). Even though includes northern Argentina, southeastern the success of amplification is inverse to the Bolivia, and Paraguay. The Gran Chaco region is evolutionary distance between species, SSR one of the areas with the highest deforestation allow multispecies studies (Barbará et al. 2007, rates globally (Gasparri & Grau 2009). Bombonato et al. 2019). Particularly, in the northwest of Argentina, this Until recently, the main drawback of these phytogeographic province has experienced the technique was the high cost associated with greatest expansion of the agricultural frontier initial marker development, which required over the last 25 years (Volante et al. 2016). a large process of cloning and sequencing of Despite the wide distribution of the species, the SSR loci (Guo et al. 2007, Davey et al. 2011). and its importance, to date there are no specific In this regard, SSR markers have been mostly molecular markers for it, so there are no reports developed for model organisms, or species of of studies of population genetic variability. agronomic interest (Bonatelli et al. 2015), and Microsatellites, or Single Sequence Repeats then transferred to related species. Nowadays, (SSRs), are repeated motifs of 1–6 nucleotides, this disadvantage has been overcome by the An Acad Bras Cienc (2021) 93(Suppl. 3) e20201778 2 | 15 ANGELA VERÓNICA GUTIÉRREZ et al. DEVELOPMENT OF SSR MARKERS IN Stetsonia coryne development of Next Generation Sequencing of molecular markers for multispecies-studies technologies (NGS), which facilitates the time within Cactaceae. and costs associated with the generation of genomic libraries (Davey et al. 2011). NGS derived genotyping strategies, as genotyping- MATERIALS AND METHODS by-sequencing (GBS, Elshire et al. 2011) and Plant material and DNA extraction double digest Restriction Associated DNA Ten individuals of Stetsonia coryne were collected sequencing (ddRADseq, Peterson et al. 2014) in the northwestern of Argentina, from two among others, have been widely used in recent populations, La Unión (Salta Province) and Dean years. In particular, ddRADseq is a strategy Funes (Córdoba Province). The individuals were characterized by the use of two digestion sampled from geographically distant locations enzymes, in which there is a reduction in the to maximize the variability between them. complexity of the genome, and it is possible Genomic DNA (gDNA) extraction was carried out to discover markers and genotype at the same from the parenchyma of the stem using a CTAB time. Even though they were firstly developed protocol (Bornet & Branchard 2001). The DNA for SNP marker identification, their use for SSR quality was verified by Nanodrop (Thermo Fisher identification has been also reported (Qin et al. Scientific, Waltham MA, USA) and 1 % agarose gel 2017, Aguirre et al. 2019) and allowed the study electrophoresis analysis. The DNA samples were of the biodiversity of numerous plant species quantified in Qubit 2.0 fluorometer (Thermo for which no specific markers were available Fisher Scientific) and the two samples with the (Deschamps et al. 2012, Egan et al. 2012, Zalapa highest DNA concentrations and best quality et al. 2012, Bonatelli et al. 2015, Qin et al. 2017). were selected for the ddRADseq protocol. Here it is important to note that, although both Additionally, the transferability of the SSR and SNP markers share some favorable generated SSRs was evaluated in three features, such as their codominant nature, individuals of each one of four selected high abundance
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