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Development of SSR Loci in Prosopis Tamarugo Phillipi and Assessment

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Development of SSR Loci in Prosopis Tamarugo Phillipi and Assessment Forest Systems 29 (1), e012, 10 pages (2020) eISSN: 2171-9845 https://doi.org/10.5424/fs/2020292-16706 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) RESEARCH ARTICLE OPEN ACCESS Development of SSR loci in Prosopis tamarugo Phillipi and assessment of their transferability to species of the Strombocarpa section Roberto Contreras (Contreras, R.)1*, Felipe S. Carevic (Carevic, F.S.)2, Vincenzo Porcile (Porcile, V.)1 and Mariana Arias (Arias, M.)1 1Centro Regional de Investigación y Desarrollo Sustentable de Atacama (CRIDESAT), Universidad de Atacama, Copayapu 485, Copiapó, Chile. 2Laboratorio de Ecología Vegetal, Facultad de Recursos Naturales Renovables. Universidad Arturo Prat, Campus Huayquique, Iquique, Chile. Abstract Aim of study: Phreatophyte species of the Prosopis genus are very important to natural ecosystems in Africa, South America and Asia due to their uses as food and seed sources and in agroforestry. In this research, through next-generation sequencing, we sought to search for and develop SSR markers in Prosopis tamarugo, in addition to assessing their transferability to other species in the Strombocarpa section. Area of study: The study was carried out in species of the Strombocarpa section collected in the “Pampa del Tamarugal”, located in the Atacama Desert (Chile); which is considered the driest and oldest desert on Earth. Material and methods: The next-generation sequencing for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in P. tamarugo and their transferability in Prosopis burkartii and Prosopis strombulifera was used. Main results: A total of ~90.000 microsatellite loci in P. tamarugo were found, and a set of 43 primer pairs was used for validating SSR locus amplification. We found a large difference in the percentage of amplified SSR markers between species of the Strombocarpa and Algarobia sections. Research highlights: The present study provides for the first time 24 polymorphic SSR markers for species in the Strombocarpa section, which could be a useful tool for estimating genetic structure, developing breeding programs, quantifying genetic diversity and performing population studies. Keywords: Strombocarpa section; Prosopis tamarugo; Atacama Desert; microsatellites; NGS. Authors’ contributions: Conceived and designed the experiments, funding acquisition, and coordinating the research project: RC. Performed the experiments: RC, VP, MA. Analyzed the data: RC. Contributed reagents/materials/analysis tools: RC. Wrote the paper and critical revision of the manuscript: RC, FSC. Citation: Contreras, R., Carevic, F.S., Porcile, V., Arias, M. (2020). Development of SSR loci in Prosopis tamarugo Phillipi and as- sessment of their transferability to species of the Strombocarpa section. Forest Systems, Volume 29, Issue 2, e012. https://doi.org/10.5424/ fs/2020292-16706. Received: 17 Mar 2020 Accepted: 10 Aug 2020 Copyright © 2020 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 Interna- tional (CC-by 4.0) License. Funding agencies/institutions Project / Grant Universidad de Atacama DIUDA 19/18, 22380 Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Roberto Contreras: [email protected] Introduction emend. Burkart, Prosopis alba Griseb, Prosopis flexuosa DC (Algarobia section species), and species of the Strom- The Atacama Desert of northern Chile is the driest and bocarpa section such as Prosopis tamarugo Phillipi, Pro- oldest desert on Earth, as revealed by geological and mi- sopis burkartii Muñoz and Prosopis strombulifera (Lam.) neralogical evidence (Hartley et al., 2005; Clarke, 2006; Benth (Burkart, 1976; Calderón et al., 2015; McRostie Sun et al., 2018), and is characterized by extreme envi- et al., 2017; Garrido et al., 2018). However, the endemic ronmental conditions such as extremely low relative hu- species P. tamarugo is one of the most interesting because midity, high concentrations of salt in the soil, low average it lives in the most extreme area of the Atacama Desert, annual rainfall and high UV radiation (Azua-Bustos et between the parallels 19°33’S and 21°50’S (Pampa del al., 2012). Despite this hostile environment, several spe- Tamarugal), at an average altitude of 1,100 m above sea cies survive, such as Prosopis chilensis (Molina) Stuntz level (Burkart, 1976; Altamirano, 2006). P. tamarugo is 2 Roberto Contreras, Felipe S. Carevic, Vincenzo Porcile and Mariana Arias adapted to high temperatures and solar radiation (Lehner present study, using NGS, we identified and developed et al., 2001; Chávez et al., 2013). Besides, the species is SSR markers in P. tamarugo, and assessed their transfe- able to perform osmotic adjustment (Time et al., 2018), rability to other species within the Strombocarpa section. access groundwater via its dynamic root system and su- Furthermore, we expected to obtain a large number of perficial lateral roots, and tolerate water stress while SSR sequences in Prosopis species due to the high gene- maintaining high stomatal conductance (Aravena & Ace- tic variability found in the Strombocarpa section. These vedo, 1985; Calderón et al., 2015; Carevic et al., 2017). findings will provide a basis for improving the understan- Nevertheless, P. tamarugo has been categorized as an en- ding of the genetic of Prosopis Strombocarpa species in dangered species; therefore, additional research is needed northern Chile. concerning the various aspects of the genetic diversity so that it can be used in a more prominent role in future conservation planning and management (Carevic et al., Material and Methods 2012; Decuyper et al., 2016). The fruits and leaves of this species are also important, as they are used as fodder for Material, DNA isolation and Sequencing goats and sheep; and the wood is used for fuel, housing construction and furniture manufacturing; in addition, an In 2019, fresh leaves of six individuals of P. tamaru- anthropogenic context, P. tamarugo facilitated the sett- go, six individuals of P. burkartii and six individuals of lement of the indigenous population in the area (Barros, P. strombulifera were collected in Tamarugal Province 2010; MMA, 2019). Therefore, P. tamarugo is an impor- (Tarapacá Region, Chile) and Loa Province. Fresh lea- tant forest genetic resource in livestock, anthropogenic ves of species of the Algarobia section, such as P. flexuo- and ecosystem contexts; however, to date, few genetic stu- sa, P. chilensis and P. alba, were also collected (Table dies have focused on the diversity and genetic variability 1). Taxonomic identification of the species was carried of this species. out according to the descriptions reported by Burkart Microsatellites (SSR, short sequence repeats) are co- (1976). During recollection, samples were kept at 4 °C; dominant markers of short sequences (from 1 to 6 nucleo- afterward, they were stored at -80 °C in the laboratory. tide bases) repeated in tandem (González, 2003). Com- Table 1 shows the geographical location and registration pared with other DNA markers, these markers exhibit a number of the samples, which were deposited in the De- high rate of polymorphism, making them a good alterna- partamento de Silvicultura y Conservación de la Natura- tive for diversity studies (Contreras et al., 2019a), bottle- leza herbarium of the Universidad de Chile (EIF, Index neck detection, gene flow, hybridization, and population Herbariorum Code). structure analysis (González, 2003, Porth & El-Kassaby, DNA was isolated from the leaves via the modified 2014) and ploidy identification (Contreras et al., 2017). cetyl-trimethylammonium bromide (CTAB) protocol des- To date, SSR loci have been discovered in several Pro- cribed by Contreras et al. (2019a,b). The quality and con- sopis species, such as P. chilensis, P. flexuosa (Mottura centration of the extracted genomic DNA from samples et al., 2005, Bessega et al., 2013), Prosopis alba Griseb. were verified by the use of a Colibri microvolume spec- (Bessega et al., 2013), Prosopis rubriflora E. Hassler and trophotometer (Titertek-Berthold, Pforzheim, Germany). Prosopis ruscifolia Griseb. (Alves et al., 2014), on which The ratio of absorbance at 260/280 nm was used to as- various studies of diversity and genetic differentiation of sess the DNA purity, which was ~1.7, and the 260/230 populations have been performed (Mottura et al., 2005, ratio was used as a secondary measure of DNA purity, Bessega et al., 2013). However, despite the large number which ranged from 2.0 and 2.2 (Demeke & Jenkins, 2010; of SSRs in species of the Algarobia section, these markers Aleksic et al., 2012). The DNA extracted from P. tama- are not sufficiently transferable to species of the Strom- rugo was quantified with a QubitTM 3.0 fluorometer and bocarpa section; moreover, there is not enough genomic a QubitTM dsDNA HS Assay Kit (Life Technologies, San information to be able to develop specific SSR primers Diego, CA) according to the manual provided by the ma- in these species, including P. tamarugo. Next-generation nufacturer. DNA samples from P. tamarugo were stored sequencing (NGS) has allowed the efficient identification at -80 °C, and DNA integrity was verified with an Agilent of large numbers of SSR markers (Bastías et al., 2016). 2100 Bioanalyzer (Agilent Technologies, San Diego, CA) Several studies of Fabaceae species, such as in Dalber- prior to sequencing. Sequencing libraries were generated gia
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